JPWO2022071974A5 - - Google Patents

Description

The present disclosure generally relates to compositions and methods for treating or eradicating herpes simplex virus infections. The present disclosure particularly relates to targeting herpes simplex virus genes with gene editing complexes.

(Cross reference to related applications)
This application is filed in US Provisional Application No. 63/086,648 (Patent Document 1) filed on October 2, 2020 and US Provisional Application No. 63/109,511 filed on November 4, 2020. (US Pat. No. 5,300,302), each of which is incorporated herein by reference.

Pharmacological treatment with nucleoside analogs is the mainstay of treatment for primary HSV1 infection and viral reactivation events. These agents can effectively limit the damage caused by the spread of HSV1 infection to other cells, but do not affect the establishment of latent HSV1 reactivation or future HSV1 reactivation events. Given the limitations of current treatments, there is a need in the art for compositions and methods for the treatment and prevention of both lytic and latent HSV1 infections.

U.S. Provisional Application No. 63/086,648 U.S. Provisional Application No. 63/109,511

In one aspect, the present disclosure provides compositions for treating or preventing herpesvirus infections. The composition comprises a) a CRISPR-associated (Cas) peptide or an isolated nucleic acid encoding a Cas peptide. b) an isolated guide nucleic acid, or an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is substantially complementary to a target sequence in a herpesvirus genome.
In certain embodiments, the pharmaceutical composition comprises a) a CRISPR-associated (Cas) peptide or an isolated nucleic acid encoding a Cas peptide. b) an isolated guide nucleic acid, or an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is substantially complementary to a target sequence in a herpesvirus genome.

In certain embodiments, the composition comprises an expression vector encoding a CRISPR-associated (Cas) peptide and a guide nucleic acid, the guide nucleic acid comprising a nucleotide sequence that is substantially complementary to a target sequence in a herpesvirus genome. In some embodiments, the present disclosure provides host cells containing expression vectors.

In certain embodiments, the method of treating or preventing a herpesvirus infection or herpesvirus-related disorder in a subject comprises: a) a CRISPR-associated (Cas) peptide, or an isolated nucleic acid encoding a Cas peptide. b) an isolated guide nucleic acid, or an isolated nucleic acid encoding a guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence that is substantially complementary to a target sequence in a herpesvirus genome.

In certain embodiments, the composition comprises a plurality of isolated guide nucleic acids, each guide nucleic acid comprising a nucleotide sequence that is substantially complementary to a different target sequence within the herpesvirus genome. In certain embodiments, the composition comprises one or more isolated nucleic acids, the one or more isolated nucleic acids encoding a plurality of guide nucleic acids, each guide nucleic acid targeting a different target in the herpesvirus genome. nucleotide sequence that is substantially complementary to the sequence.

In certain embodiments, the Cas peptide is Cas9 or a variant thereof. In certain embodiments, the Cas9 variants are R780A, K810A, K848A, K855A, H982A, KI003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A, and M698A selected from the group consisting of contains one or more point mutations compared to wild-type Streptococcus pyogenes Cas9 (spCas9). In some embodiments, the Cas peptide is Cpf1 or a variant thereof.

In some embodiments, isolated nucleic acids encoding Cas peptides are optimized for expression in human cells.
In some embodiments, the target sequence comprises a sequence within the ICPO domain of the herpesvirus genome. In some embodiments, the guide nucleic acid is RNA.
In some embodiments, the guide nucleic acids include crRNA and tracrRNA.
In certain embodiments, the target sequence to which the gRNA is substantially complementary is within the UL56, ICP0, ICP4, or ICP27 gene.
In certain embodiments, the HSV target sequence is within the ICP0 gene, the UL56 gene, or a combination thereof.
In certain embodiments, the gRNA is at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 91%) relative to SEQ ID NOS: 1-96, 194-212, and 356-371. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity.

In certain embodiments, the gRNA comprises a nucleic acid sequence comprising SEQ ID NOs: 1-96, 194-212, and 356-371.
In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more gRNAs comprising nucleic acid sequences comprising SEQ ID NOs: 1-96, 194-212, and 356-371.
In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of two or more gRNAs comprising nucleic acid sequences comprising SEQ ID NOs: 1-96, 194-212, and 356-371.
In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of three or more gRNAs comprising nucleic acid sequences comprising SEQ ID NOs: 1-96, 194-212, and 356-371.
In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of four or more gRNAs comprising nucleic acid sequences comprising SEQ ID NOs: 1-96, 194-212, and 356-371.

In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of 5 or 6 or 7 or 8 or 9 or more gRNAs comprising nucleic acid sequences comprising SEQ ID NOS: 1-96, 194-212, and 356-371. including.
In some embodiments, the PAM sequence is at least about 70% (eg, at least about 75%, 80%, 85%, 90%, 91%) relative to SEQ ID NOs: 97-193, 213-231, or combinations thereof. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more ) sequence identity .
In some embodiments, the PAM sequence comprises a nucleic acid sequence comprising SEQ ID NO: 97-193, 213-231, or a combination thereof.
In certain embodiments, the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3); varicella zoster virus (VZV), human herpes virus 4 ( HHV-4; Epstein -Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 ( HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)).

In certain embodiments, compositions are disclosed herein comprising: a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease, or a nucleic acid sequence encoding a CRISPR-associated endonuclease; A first guide nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, wherein the first guide nucleic acid is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome. a second guide nucleic acid, or a nucleic acid sequence encoding a second guide nucleic acid, wherein the second guide nucleic acid is a second target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target within or near the ICP27 gene of the herpesvirus genome. complementary to a nucleic acid sequence, wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

In some embodiments, the composition further comprises a fourth guide nucleic acid, or a nucleic acid sequence encoding a fourth guide nucleic acid, wherein the fourth guide nucleic acid is located within or near the ICP27 gene of the herpesvirus genome. Complementary to a fourth target nucleic acid sequence. In some embodiments, the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. In some embodiments, the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasO endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. In some embodiments, the CRISPR-associated endonuclease is optimized for expression in human cells. In some embodiments, the guide nucleic acid is RNA. In some embodiments, the guide nucleic acids include crRNA and tracrRNA.

In some embodiments, the first target nucleic acid sequence comprises at least about 90% sequence identity with any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs : 1-96 or 372- 375 . In some embodiments, the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96, 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. include. In some embodiments, the second target nucleic acid sequence is for any one of SEQ ID NOs: 1-96, 372-375, or the complement of any one of SEQ ID NOs: 1-96, 372-375 . Includes sequences that contain at least about 90% sequence identity. In some embodiments, the second target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. including.

In some embodiments, the third target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to . In some embodiments, the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body. In some embodiments, the fourth target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to . In some embodiments, the fourth target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body.

In some embodiments, the first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third The target nucleic acid sequence of comprises the sequence according to SEQ ID NO: 376 or the complement thereof. In some embodiments, the first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof; The target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or its complement, and the fourth target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377 or its complement. In some embodiments, the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV)), human herpes virus 4 ( HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7)), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)).

In certain embodiments, a composition comprising a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease or a nucleic acid sequence encoding a CRISPR-associated endonuclease is disclosed ; a first guide; a nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, the first guide nucleic acid being complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; or a nucleic acid sequence encoding a second guide nucleic acid, the second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; The third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. Here, the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

In some embodiments, the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasI endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. In some embodiments, the CRISPR-associated endonuclease is optimized for expression in human cells. In some embodiments, the guide nucleic acid is RNA. In some embodiments, the guide nucleic acids include crRNA and tracrRNA. In some embodiments, the first target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. sequences that contain at least about 90% sequence identity .

In some embodiments, the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. include. In some embodiments, the second target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to . In some embodiments, the second target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body. In some embodiments, the third target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences containing at least about 90% sequence identity to . In some embodiments, the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body.

In some embodiments, the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7 or the complement thereof, the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or the complement thereof, and a third The target nucleic acid sequence comprises the sequence according to SEQ ID NO: 377 or its complement. In some embodiments, the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV)), human herpes virus 4 (HHV-4 ; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)) .

As used herein, in certain embodiments, a CRISPR-Cas system has : a CRISPR-associated endonuclease ; at least 90% sequence identity to a first guide nucleic acid comprising a nucleic acid sequence complementary to a sequence having the identity; a second guide nucleic acid, SEQ ID NO: 376 or 377 or the complement thereof; a second guide nucleic acid comprising a nucleic acid sequence complementary to a sequence having;
In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO:7.

In some embodiments, the second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, the second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO : 377 . In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2 , and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2. 376. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2 , and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2. 377.

In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376 . comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO: 377. comprises a nucleic acid sequence that is complementary to a nucleic acid sequence with at least 90% sequence identity.

In certain embodiments, nucleic acids encoding the CRISPR-Cas systems described herein are disclosed.

In certain embodiments, disclosed herein are adeno-associated virus (AAV) vectors that include a nucleic acid encoding a CRISPR-associated endonuclease . a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; a second guide nucleic acid that is complementary to a first target nucleic acid sequence that is within or near the ICP0 gene of the herpesvirus genome; A second guide nucleic acid that is complementary to a second target nucleic acid sequence within or near the ICP0 gene of. A third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. , wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

In some embodiments, the vector further comprises a fourth guide nucleic acid that is complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. In some embodiments, the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. In some embodiments, the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasΦ endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease.

In some embodiments, the CRISPR-associated endonuclease is optimized for expression in human cells. In some embodiments, the guide nucleic acid is RNA. In some embodiments, the guide nucleic acids include crRNA and tracrRNA. In some embodiments, the first target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. sequences that contain at least about 90% sequence identity. In some embodiments, the first target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. including.

In some embodiments, the second target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. Sequences containing at least about 90% sequence identity. In some embodiments, the second target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. including. In some embodiments, the third target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences containing at least about 90% sequence identity to. In some embodiments, the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body.

In some embodiments, the fourth target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Sequences having at least about 90% sequence identity to. In some embodiments, the fourth target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body. In some embodiments, the first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third The target of the nucleic acid sequence comprises the sequence according to SEQ ID NO: 376 or the complement thereof. In some embodiments, the first target nucleic acid sequence comprises a sequence according to SEQ ID NO:2. wherein the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 376 or the complement thereof, and the fourth target nucleic acid sequence comprises the sequence according to SEQ ID NO: 376 or the complement thereof. 377 or its complement. In some embodiments, the nucleic acid further comprises a promoter.

In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a human cytomegalovirus promoter. In some embodiments, the nucleic acid further comprises an enhancer element. In some embodiments, the enhancer element is a human cytomegalovirus enhancer element. In some embodiments, the nucleic acid further comprises a 5'ITR element and a 3'ITR element . In some embodiments, the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8. In some embodiments, the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV)), human herpes virus 4 ( HHV-4 ; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV))).

In certain embodiments, disclosed herein are adeno-associated virus (AAV) vectors comprising a nucleic acid encoding a CRISPR-associated endonuclease : a first guide nucleic acid within or of the ICP0 gene of a herpesvirus genome. a first guide nucleic acid that is complementary to a first target nucleic acid sequence in the vicinity; a second guide nucleic acid that is complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; a second guide nucleic acid; a third guide nucleic acid that is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; Here, the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

In some embodiments, the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasI endonuclease. In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. In some embodiments, the CRISPR-associated endonuclease is optimized for expression in human cells. In some embodiments, the guide nucleic acid is RNA. In some embodiments, the guide nucleic acids include crRNA and tracrRNA. In some embodiments, the first target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. Sequences containing at least about 90% sequence identity. In some embodiments, the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. include.

In some embodiments, the second target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to. In some embodiments, the second target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body. In some embodiments, the third target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to . In some embodiments, the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body.

In some embodiments, the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7 or the complement thereof, the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or the complement thereof, and a third The target nucleic acid sequence of comprises the sequence according to SEQ ID NO: 377 or the complement thereof. In some embodiments, the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV)), human herpes virus 4 ( HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)).

In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a human cytomegalovirus promoter. In some embodiments, the nucleic acid further comprises an enhancer element. In some embodiments, the enhancer element is a human cytomegalovirus enhancer element . In some embodiments, the nucleic acid further comprises a 5'ITR element and a 3'ITR element . In some embodiments, the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8.

In certain embodiments, disclosed herein are methods of excising some or all of herpesvirus sequences from a cell, which methods include the compositions described herein, the CRISPR-Cas system, or providing a cell with an AAV vector described herein.

In certain embodiments, disclosed herein is a method of inhibiting or reducing herpesvirus replication in a cell, which method comprises a composition described herein, a CRISPR-Cas system described herein, or providing a cell with an AAV vector described herein. In some embodiments, the cell is within the subject . In some embodiments, the subject is a human.

FIG. 1 is a schematic diagram of the herpesvirus genome and gene editing vectors used in targeting the herpesvirus genome. FIG. 1 is a schematic diagram of the herpesvirus genome and gene editing vectors used in targeting the herpesvirus genome. FIG. 1 is a schematic diagram of the herpesvirus genome and gene editing vectors used in targeting the herpesvirus genome. FIG. 3 shows data showing delivery and expression of gene editing vectors within cells. FIG. 2 shows data demonstrating DNA excision assays in cells. FIG. 2 shows data demonstrating DNA excision assays in cells. FIG. 3 shows data showing HSV replication within cells. FIG. 3 shows data showing gRNA expression in cells. FIG. 3 shows data showing gRNA expression in cells. FIG. 3 shows data from an intracellular herpesvirus model. FIG. 2 shows data demonstrating DNA excision assays in cells. FIG. 2 shows data demonstrating DNA excision assays in cells. FIG. 3 shows data showing decreased expression of target genes in infected cells.

(definition)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein .

As used herein, each of the following terms has the meaning associated with it within this section.

The articles "a" and "an" are used herein to refer to one or more (ie, at least one) of the grammatical object of the article. As an example, " an element " means an element or elements.

As used herein, "about" refers to a measurable value, such as amount, duration, etc., of ±20%, ±10%, ±5%, ±1%, or ±0.1 from the specified value. % variations that are appropriate for carrying out the disclosed method.

The term "abnormal" when used in the context of an organism, tissue, cell, or component thereof, in which at least one observable or detectable characteristic (e.g., age, treatment, time, etc.) Refers to organisms, tissues, cells, or components thereof that differ from those organisms, tissues, cells, or components thereof that exhibit "normal " (expected) respective characteristics. Properties that are normal or expected in one cell or tissue type may be abnormal in another cell or tissue type.

As used herein, the term "comprising" , and variations thereof, in connection with a defined or described element of an article, composition, device, method, process, system, etc., means: do. Inclusive or unlimited, permitting additional elements such that the defined or described item, composition, apparatus, method, process, system, etc. It is meant to include, and that other elements may be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.

A "disease" is a health condition in which an animal cannot maintain homeostasis, and if the disease is not improved, the animal's health will continue to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but whose state of health is less favorable than it would be without the disorder. Leaving a disease untreated does not necessarily mean that the animal's health will deteriorate further.

A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which a patient experiences such symptoms, or both are reduced.

"Coding" means that a specific sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, is used in a biological process to encode a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or amino acids. refers to the unique properties that serve as templates for the synthesis of other polymers and macromolecules that have any of the defined sequences and biological properties that result from them. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually found in a sequence listing; and the non-coding strand , which is used as a template for transcription of the gene or cDNA, cDNA can be said to encode other products.

An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to produce a beneficial effect in the subject to whom the compound is administered. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver the compound.

"Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include cosmids that incorporate recombinant polynucleotides, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). Contains all known vectors.

"Homologous" refers to sequence similarity or identity between two polypeptides or two nucleic acid molecules. If both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. be . The percent homology between two sequences is a function of the number of matching or homologous positions the two sequences share divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when two sequences are aligned to obtain maximum homology.

"Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from its natural coexisting materials. Ru. An isolated nucleic acid or protein can exist in substantially purified form or can exist in a non-natural environment, such as a host cell.

In the context of this disclosure, the following abbreviations for commonly occurring nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, insofar as the nucleotide sequence encoding a protein may in some versions include introns.

Terms such as "patient,"" subject ," and "individual" are used interchangeably herein and refer to any animal or its cells described in the methods described herein, whether in vitro or in situ. Point. In certain non-limiting embodiments, the patient, subject or individual is a human.

"Parenteral" administration of a composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques. .

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Accordingly, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides are hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, polynucleotides derived from recombinant libraries or cells, by recombinant means, i.e., using conventional cloning techniques and PCR, and by synthetic means. This includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, cloning of nucleic acid sequences.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, insofar as the nucleotide sequence encoding a protein may in some versions include introns.

The term "pharmaceutically acceptable" (or "pharmacologically acceptable"), as appropriate, refers to Refers to molecular entities and compositions that do not produce a reaction. As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antimicrobial agent, isotonic agent, absorbent agent, etc. that can be used as a vehicle for a pharmaceutically acceptable substance. Included are retarders, buffers, excipients, binders, lubricants, gels, surfactants, and the like.

As used herein, the terms "peptide,""polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to, for example, short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of which there are many types. Refers to both chains. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, Includes analogs, fusion proteins, etc. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof.

The term "promoter" as used herein is defined as a DNA sequence recognized by the cellular or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence. .

As used herein, the term "promoter/control sequence" refers to a nucleic acid sequence necessary for the expression of a gene product operably linked to the promoter/control sequence. In some cases, this sequence is the core promoter sequence; in other cases, this sequence may also include enhancer sequences and other regulatory elements necessary for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of a gene product in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is one that, when operably linked to a polynucleotide encoding or specifying a gene product, produces a gene product in a cell only if an inducer that substantially corresponds to the promoter is present in the cell. This is the nucleotide sequence that causes the production of

As used in this specification and the appended claims, the term "or" is generally used in its inclusive sense, unless the content clearly dictates otherwise.

A "tissue-specific" promoter is a promoter that, when operably linked to a polynucleotide encoding or specified by a gene, allows the promoter to enter the cell substantially only if the cell is of the corresponding tissue type. A nucleotide sequence that causes the production of a gene product.

A "therapeutic" treatment is a treatment administered to a subject exhibiting pathological signs for the purpose of reducing or eliminating those signs.

As used herein, "treating a disease or disorder" means reducing the frequency with which a patient experiences symptoms of the disease or disorder. Disease and disorder are used interchangeably herein.

As used herein, the phrase "therapeutically effective amount" means to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, alleviate, reverse, etc.) a disease or condition. ) refers to an amount sufficient or effective to reduce the symptoms of such disease.

The term "treating" a disease, as used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject .

The term "variant" as used herein is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are usually limited or conservative, such that the sequences of the reference peptide and the variant are very similar overall and are identical in many regions. A variant and a reference peptide may differ in amino acid sequence by any combination of one or more substitutions, additions, deletions. A variant of a nucleic acid or peptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be made by mutagenesis techniques or direct synthesis.

A "vector" is a composition of matter that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid inside a cell. A large number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides conjugated to ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

Ranges: Throughout this disclosure, various aspects of this disclosure can be expressed in range format. It is to be understood that the description in range format is merely for convenience and brevity and is not to be construed as in any way limiting the scope of the disclosure. Accordingly, range descriptions should be considered as specifically disclosing all possible subranges and individual numerical values within that range. For example, a description of a range such as 1 to 6 may be interpreted as subranges such as 1 to 3 , 1 to 4, 1 to 5, 2 to 4 , 2 to 6, 3 to 6, and the individual numbers within that range (1 to 6). , 2, 2.7, 3, 4, 5, 5.3, 6, etc.). This applies regardless of the scope.

(detailed explanation)
(Herpes virus targeting)
Embodiments include compositions and methods for treating and preventing herpesvirus infections in a subject in need thereof. For example, in certain embodiments, the present disclosure provides compositions that specifically cleave a target sequence within the viral genome of a herpesvirus, thereby preventing or reducing the ability of the virus to replicate, and thus inhibiting the infectivity of the herpesvirus. provide something.

In certain embodiments, gene editing complexes, such as human herpes simplex virus-specific single and multiplex CRISPR-Cas systems, compromise the integrity of the viral DNA sequence, resulting in HSV fragmentation between targeted HSV regions. The genome is excised. For example, the CRISPR-Cas molecules described herein have the potential to remove large segments of the HSV genome, disabling the ability of the virus to replicate within infected cells. Accordingly, the present disclosure provides compositions and methods that target the HSV genome within infected cells for destruction of the viral genome in acute or latent HSV1 infections as novel therapeutic and preventive strategies.

In certain embodiments, compositions and methods related to targeting the HSV genome are described herein. In some embodiments, the compositions and methods include a CRISPR/Cas system for targeting the HSV genome. In some embodiments, the compositions and methods provide for excising part or all of the HSV genome. In some embodiments, the compositions and methods provide for excising some or all of the sequences within the HSV genome at 1, 2, 3, 4, 5, or 6 different genes of the HSV genome. In some embodiments, the compositions and methods provide at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, resulting in 9000 resections.

In some embodiments, methods and compositions comprising a CRISPR-associated (Cas) peptide, or a nucleic acid sequence encoding a CRISPR-associated (Cas) peptide, and a plurality of guide nucleic acids, or a nucleic acid sequence encoding a plurality of guide nucleic acids. provided herein. In some embodiments, the compositions and methods described herein include 1, 2, 3, 4, 5, 6, or more gRNAs. In some embodiments, the compositions and methods described herein include 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs. In some embodiments, the compositions and methods described herein include four or at least four different gRNAs. In certain embodiments, the one or more gRNAs target one or more different regions or sequences in the HSV genome (eg, ICP0 and ICP27).

In some embodiments, different gRNAs target different sequences within the HSV genome. In some embodiments, the different gRNAs are complementary to different target sequences within the HSV genome. In some embodiments, the target sequence is within or near the UL56, ICP0, ICP4, or ICP27 genes of the HSV genome. In certain embodiments, the gRNA that targets the UL56, ICP0, ICP4, or ICP27 gene hybridizes to a region within or near the UL56, ICP0, ICP4, or ICP27 gene. In some embodiments, the region within the UL56, ICP0, ICP4, or ICP27 gene comprises at least one nucleotide within the UL56, ICP0, ICP4, or ICP27 gene. In some embodiments, the region near the UL56, ICP0, ICP4, or ICP27 gene is 5, 10, 15, 20, 25, 30, or 35 base positions around the UL56, ICP0, ICP4, or ICP27 gene. including.

In some embodiments, the compositions and methods described herein target (e.g., hybridize or anneal to) a region within UL56, ICP0, ICP4, ICP27, or a combination thereof of the HSV genome . , or 2, 3, 4, 5, 6, or more than 6 different gRNAs complementary thereto. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the UL56 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the UL56 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the UL56 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP0 gene of the genome . In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the UL56 gene of the HSV genome, and 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP4 gene . In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the UL56 gene of the HSV genome, and 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP27 gene . In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP0 gene of the HSV genome, and 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP4 gene . In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP0 gene of the HSV genome, and 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP27 gene . In some embodiments, the compositions and methods described herein target 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP4 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that target the ICP27 gene in the genome .

In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and one gRNA that targets the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and two different gRNAs that target the ICP0 gene of the HSV genome. . In some embodiments, the compositions and methods described herein include one gRNA that targets the UL56 gene of the HSV genome and two different gRNAs that target the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and one gRNA that targets the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and two different gRNAs that target the ICP4 gene of the HSV genome. In some embodiments, the combinations and methods described herein include one gRNA that targets the UL56 gene of the HSV genome and two different gRNAs that target the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and one gRNA that targets the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the UL56 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that targets the UL56 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP0 gene of the HSV genome and one gRNA that targets the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP0 gene of the HSV genome and two different gRNAs that target the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that targets the ICP0 gene of the HSV genome and two different gRNAs that target the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP0 gene of the HSV genome and one gRNA that targets the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP0 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that targets the ICP0 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP4 gene of the HSV genome and one gRNA that targets the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that target the ICP4 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that targets the ICP4 gene of the HSV genome and two different gRNAs that target the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the UL56 gene of the HSV genome, and 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP0 gene of . In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the UL56 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP4 gene of the genome . In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the UL56 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP27 gene of the genome . In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP0 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP4 gene of the genome . In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP0 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP27 gene of the genome . In some embodiments, the compositions and methods described herein contain 1, 2, 3, 4, 5, 6, or more than 6 different gRNAs that hybridize to the ICP4 gene of the HSV genome, and Contains 1, 2, 3, 4, 5, 6 or more than 6 different gRNAs that hybridize to the ICP27 gene of the genome .

In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and one gRNA that hybridizes to the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP0 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and one gRNA that hybridizes to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and one gRNA that hybridizes to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the UL56 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP0 gene of the HSV genome and one gRNA that hybridizes to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP0 gene of the HSV genome and two different gRNAs that hybridize to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the ICP0 gene of the HSV genome and two different gRNAs that hybridize to the ICP4 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP0 gene of the HSV genome and one gRNA that hybridizes to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP0 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the ICP0 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome.

In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP4 gene of the HSV genome and one gRNA that hybridizes to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include two different gRNAs that hybridize to the ICP4 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome. In some embodiments, the compositions and methods described herein include one gRNA that hybridizes to the ICP4 gene of the HSV genome and two different gRNAs that hybridize to the ICP27 gene of the HSV genome.

Provided herein, in certain embodiments, are methods and compositions for targeting the HSV genome using four guide nucleic acids. In some embodiments, a first guide nucleic acid of the plurality of guide nucleic acids is complementary to a first target sequence within the HSV genome. In some embodiments, a second guide nucleic acid of the plurality of guide nucleic acids is complementary to a second target sequence within the HSV genome. In some embodiments, a third guide nucleic acid of the plurality of guide nucleic acids is complementary to a third target sequence within the HSV genome. In some embodiments, a fourth guide nucleic acid of the plurality of guide nucleic acids is complementary to a fourth target sequence within the HSV genome. In some embodiments, the first target sequence, second target sequence, third target sequence, and fourth target sequence are different.

In some embodiments, the ICP0 sequence targeted by the gRNA is at least or about 70%, 80% relative to any one of the sequences shown in Table 4 , SEQ ID NOs: 1-96 or 372-373. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, %, 98%, 99%, or 100% sequence identity. In some embodiments, the ICP0 sequence targeted by the gRNA is a sequence complementary to any one of SEQ ID NOs: 1-96 or 372-373 or to one sequence shown in Table 4. , at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity Contains sequences with specific characteristics. In some cases, the ICP0 sequence targeted by the gRNA has at least 95% or about 95% homology to any one of SEQ ID NOs: 1-96 or 372-373 , or the sequences set forth in Table 4. include. In some cases, the ICP0 sequence targeted by the gRNA is at least or about 95% homologous to any one of SEQ ID NOs: 1-96 or 372-373 or a sequence complementary to one sequence shown in Table 4. Contains a gender sequence. In some cases, the ICP0 sequence targeted by the gRNA is at least or about 97% homologous to any one of SEQ ID NOs: 1-96 or 372-373 or any one of the sequences shown in Table 4. Contains a gender sequence. In some cases, the ICP0 sequence targeted by the gRNA is to a sequence complementary to any one of SEQ ID NOs: 1-96 or 372-373, or any one of the sequences shown in Table 4. Contains sequences that are at least or about 97% homologous.

In some cases, the ICP0 sequence targeted by the gRNA comprises a sequence at least or about 99% homologous to any one of SEQ ID NOs: 1-96 or 372-373, or the sequences set forth in Table 4. In some cases, the ICP0 sequence targeted by the gRNA is a sequence complementary to any one of SEQ ID NOs: 1-96 or 372-373, or at least or about 99% homologous to the sequences set forth in Table 4. Contains a gender sequence. In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 1-96 or 372-373, or the sequences set forth in Table 4. In some cases, the ICP0 sequence targeted by the gRNA is at least or about 100% homologous to any one of SEQ ID NOs: 1-96 or 372-373, or a sequence complementary to the sequences set forth in Table 4. Contains an array. In some cases, the ICP0 sequence targeted by the gRNA is any one of SEQ ID NOs: 1-96, or at least or about 3, 4, 5, 6, 7, 8, 9, 10 of the sequences set forth in Table 4. , 12, 14, 16, 17, 18, 19, 20 or more than 20 nucleotides . In some cases, the ICP0 sequence targeted by the gRNA is at least or about 3, 4, 5, 1, 2, 3, 3, 3, 4, 5 of any one of SEQ ID NOs: 1-96 or 372-373, or a sequence complementary to the sequences set forth in Table 4 . Contains sequences of more than 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20 or 20 nucleotides .

In some embodiments, the ICP27 sequence targeted by the gRNA is at least or about 70%, 80%, 85%, 90% relative to any one of SEQ ID NOs: 363, 371, or 374-377. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the ICP27 sequence targeted by the gRNA is at least or about 70%, 80%, 85% complementary to any one of SEQ ID NOs: 363, 371, or 374-377. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some examples, the ICP27 sequence targeted by the gRNA comprises a sequence at least or about 95% homologous to any one of SEQ ID NOs: 363, 371, or 374-377. In some cases, the ICP27 sequence targeted by the gRNA comprises a sequence at least or about 95% homologous to a sequence complementary to any one of SEQ ID NOs: 363, 371, or 374-377. . In some examples, the ICP27 sequence targeted by the gRNA comprises a sequence at least or about 97% homologous to any one of SEQ ID NOs: 363, 371, or 374-377. In some examples, the ICP27 sequence targeted by the gRNA comprises a sequence that is at least or about 97% homologous to a sequence complementary to any one of 363, 371, or 374-377. In some examples, the ICP27 sequence targeted by the gRNA comprises a sequence at least or about 99% homologous to any one of SEQ ID NOs: 363, 371, or 374-377.

In some cases, the ICP27 sequence targeted by the gRNA comprises a sequence that is at least or about 99% homologous to a sequence complementary to any one of SEQ ID NOs: 363, 371, or 374-377. In some cases, the ICP27 sequence targeted by the gRNA comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 363, 371, or 374-377. In some cases, the ICP27 sequence targeted by the gRNA comprises a sequence that is at least or about 100% homologous to a sequence complementary to any one of SEQ ID NOs: 363, 371, or 374-377. In some examples, the ICP27 sequence targeted by the gRNA is at least or about 3, 4, 5, 6, 7, 8, 9, 10 of any one of SEQ ID NOs: 363, 371, or 374-377. Contains sequences of 12, 14, 16, 17, 18, 19, 20 or 20 or more nucleotides. In some examples, the ICP27 sequence targeted by the gRNA comprises at least or about 3, 4, 5, 6, 7 , A sequence of 8, 9, 10, 12, 14, 16, 17, 18, 19, 20 or more than 20 nucleotides .

In some embodiments, the ICP0 sequence targeted by the first gRNA is at least or about 70%, 80%, 85%, 90%, 91%, 92% of any one of SEQ ID NO: 372 or 373. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity , and the ICP27 sequence targeted by the second gRNA has the sequence any one of numbers 372 or 373 and at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , or containing sequences of 100% sequence identity.

In some embodiments, the sequence targeted by the gRNA comprises a sequence set forth in any one of SEQ ID NOs: 372-375. In some embodiments, the sequence targeted by the first gRNA is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94% relative to SEQ ID NO: 372. %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity ; the sequence targeted by the second gRNA is at least or Containing sequences of about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity the sequence targeted by the third gRNA is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, relative to SEQ ID NO: 374 ; comprises a sequence of 96%, 97%, 98%, 99% or 100% sequence identity , and the sequence targeted by the fourth gRNA has at least or about 70%, 80% sequence identity to SEQ ID NO: 375. %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity .

In some embodiments, the sequence targeted by the gRNA comprises the sequence set forth in any one of SEQ ID NO: 2, 7, 376, or 377. In some embodiments, the sequence targeted by the first gRNA is at least or about 70%, 80%, 85%, 90%, 91%, 92% relative to SEQ ID NO: 2 or its complement. %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity ; the sequence targeted by the second gRNA is SEQ ID NO: 7. or its complement and at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the sequence targeted by the third gRNA is at least or about 70%, 80%, 85%, 90%, 91%, 92%, comprises a sequence of 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity ; and the sequence targeted by the fourth gRNA is SEQ ID NO: 377 or at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its complement; Contains sequences of sequence identity .

In some embodiments, at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93% to any one of SEQ ID NOs: 97-193, or the sequences set forth in Table 4. , 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity are described herein. In some cases , the PAM sequence comprises a sequence having at least or about 95% homology to any one of SEQ ID NOs: 97-193, or the sequences set forth in Table 4. In some cases, the PAM sequence comprises a sequence having at least or about 99% homology to any one of SEQ ID NOs: 97-193, or the sequences set forth in Table 4. In some cases, the PAM sequence comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 97-193, or the sequences set forth in Table 4. In some cases, the PAM sequence comprises a sequence of 1, 2, 3, 4, 5, 6, or more than 6 nucleotides of any one of SEQ ID NOs: 97-193 or the sequences shown in Table 4 . In some embodiments, any one of SEQ ID NOs: 97-193 has at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 1-96 or 372-375 at least or about 70%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Ru.

In some embodiments, the sequence targeted by the gRNA is at least or about 70%, 80%, 85%, 90% relative to any one of SEQ ID NOs: 194-212, or the sequences set forth in Table 1. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity . In some cases, the sequence targeted by the gRNA comprises at least or about 95% of the sequence relative to any one of SEQ ID NOs: 194-212, or the sequences set forth in Table 1 . In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 97% homologous to any one of SEQ ID NOs: 194-212, or the sequences set forth in Table 1 . In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 99% homologous to any one of SEQ ID NOs: 194-212, or the sequences set forth in Table 1. In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 194-212, or the sequences set forth in Table 1. The gRNA is any one of SEQ ID NOs: 194-212, or at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, of the sequences shown in Table 1. Contains sequences of more than 19, 20 or 20 nucleotides.

In some embodiments , at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93 to any one of SEQ ID NOs: 213-231, or the sequences set forth in Table 1. Compositions and methods are described herein that include PAM sequences that include sequences that have %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity . In some cases, the PAM sequence comprises a sequence having at least or about 95% homology to any one of SEQ ID NOs: 213-231, or the sequences set forth in Table 1. In some cases, the PAM sequence comprises a sequence having at least or about 99% homology to any one of SEQ ID NOs: 213-231, or the sequences set forth in Table 1. In some cases, the PAM sequence comprises a sequence having at least or about 100% homology to any one of SEQ ID NOs: 213-231, or the sequences set forth in Table 1. In some cases , the PAM sequence comprises a sequence of 1, 2, 3, 4, 5, 6, or more than 6 nucleotides of any one of SEQ ID NOs: 213-231, or the sequences set forth in Table 1. In some embodiments, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one of SEQ ID NOs: 213-231. %, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 194-212. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, the sequence targeted by the gRNA is at least or about 70%, 80%, 85%, 90% relative to any one of SEQ ID NOs: 232-243, or the sequences set forth in Table 2. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity . In some cases, the sequence targeted by the gRNA comprises a sequence having at least or about 95% homology to any one of SEQ ID NOs: 232-243, or the sequences set forth in Table 2 . In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 97% homologous to any one of SEQ ID NOs: 232-243, or the sequences set forth in Table 2 . In some cases, the sequence targeted by the gRNA has a sequence at least or about 99% homologous to any one of SEQ ID NOs: 232-243, or any one of the sequences shown in Table 2. include. In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 232-243, or the sequences set forth in Table 2. In some examples, the sequence targeted by the gRNA is any one of SEQ ID NOs: 232-243, or at least or about 3, 4, 5, 6, 7, 8, 9, A sequence of 10, 12, 14, 16, 17, 18, 19, 20 or more than 20 nucleotides.

In some embodiments, at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93% for any one of SEQ ID NOs: 244-255, or the sequences shown in Table 2. Compositions and methods are described herein that include PAM sequences that include sequences that have %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity . In some cases, the PAM sequence comprises a sequence that has at least or about 95% homology to any one of SEQ ID NOs: 244-255, or the sequences set forth in Table 2 . In some cases, the PAM sequence comprises a sequence having at least or about 99% homology to any one of SEQ ID NOs: 244-255, or the sequences set forth in Table 2. In some cases, the PAM sequence comprises a sequence that has at least or about 100% homology to any one of SEQ ID NOs: 244-255, or the sequences set forth in Table 2. In some cases, the sequence targeted by the gRNA is a sequence of 1, 2, 3, 4, 5, 6, or more than 6 nucleotides of any one of SEQ ID NOs: 244-255 or the sequences set forth in Table 2. including . In some embodiments , at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to any one of SEQ ID NOs: 244-255. , 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 232-243 at least or about 70%, 80%, 85%, 90% , used with gRNAs targeting sequences containing sequences with , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity .

In some embodiments, the sequence targeted by the gRNA is at least or about 70%, 80%, 85%, 90% relative to any one of SEQ ID NOs: 256-305, or the sequences set forth in Table 3. , 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 100% sequence identity . In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 95% homologous to any one of SEQ ID NOs: 256-305, or the sequences set forth in Table 3 . Optionally, the sequence targeted by the gRNA comprises a sequence at least or about 97% homologous to any one of SEQ ID NOs: 256-305, or the sequences set forth in Table 3 : Sequences targeted by the gRNA include sequences having at least or about 99% homology to any one of SEQ ID NOs: 256-305, or the sequences set forth in Table 3. In some cases, the sequence targeted by the gRNA comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 256-305, or the sequences set forth in Table 3. Optionally, the sequence targeted by the gRNA is any one of SEQ ID NOs: 256-305, or at least or about 3, 4, 5, 6, 7, 8, 9, 10, or the sequences set forth in Table 3. Contains sequences of 12, 14, 16, 17, 18, 19, 20 or more than 20 nucleotides .

In some embodiments, at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93% for any one of SEQ ID NOs: 306-355, or the sequences shown in Table 3. Compositions and methods are described herein that include PAM sequences that include sequences that have %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity . In some cases, the PAM sequence comprises a sequence having at least or about 95% homology to any one of SEQ ID NOs: 306-355, or the sequences set forth in Table 3. In some cases, the PAM sequence comprises a sequence having at least or about 99% homology to any one of SEQ ID NOs: 306-355, or the sequences set forth in Table 3. In some cases, the PAM sequence comprises a sequence at least or about 100% homologous to any one of SEQ ID NOs: 306-355, or the sequences set forth in Table 3. In some cases, the PAM sequence comprises a sequence having at least or about 100% homology to any one of SEQ ID NOs: 306-355, or the sequences set forth in Table 3 . In some cases, the PAM sequence comprises a sequence of 1, 2, 3, 4, 5, 6, or more than 6 nucleotides of any one of SEQ ID NOs: 306-355, or the sequences set forth in Table 3 . In some embodiments, at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 256-305. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% sequence identity. used with gRNA targeting

In certain embodiments, compositions and methods for targeting sequences within the HSV genome are described herein. In some embodiments, the constructs or vectors are used with the compositions and methods described herein. In some embodiments, the construct or vector is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to SEQ ID NO: 380. , 97%, 98% , 99%, or 100% sequence identity . In some cases, the construct or vector includes a sequence that has at least or about 95% homology to SEQ ID NO: 380. In some cases, the construct or vector comprises a sequence at least or about 97% homologous to SEQ ID NO: 380. In some examples, the construct or vector comprises a sequence at least or about 100% homologous to SEQ ID NO: 380. In some examples, the construct or vector comprises at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 , 1200, 1400, 1600, 1800, 2000, 2200 of SEQ ID NO: 380. , 2400, 2600, 2800, 3000, 3200, 3400, 3600, 38 00, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 700 0, A sequence of 7200, 7400, 7600, 7800, 8000, 8200, 8400, or more than 8400 nucleotides. In certain embodiments, the construct or vector comprises a CRISPR-Cas enzyme sequence of SEQ ID NO: 380 and a gRNA sequence. In certain embodiments, the construct or vector has a sequence identity of 70%, 80%, 85%, 90%, 95% to the CRISPR-Cas enzyme sequence and gRNA sequence of SEQ ID NO: 380. Contains enzyme and gRNA sequences.

In certain embodiments, compositions and methods for targeting sequences within the HSV genome are described herein. In some embodiments, nucleic acid constructs or vectors are used with the compositions and methods described herein. In some embodiments, the construct or vector is at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to SEQ ID NO: 381. , 97%, and 98% homology . In some cases, the construct or vector comprises a sequence that has at least or about 95% homology to SEQ ID NO: 381. Optionally, the construct or vector comprises a sequence that is at least or about 97% homologous to SEQ ID NO: 381. In some cases, the construct or vector comprises a sequence at least or about 99% homologous to SEQ ID NO: 381. In some cases, the construct or vector comprises a sequence at least or about 100% homologous to SEQ ID NO: 381. In some cases, the construct or vector comprises at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400 of SEQ ID NO: 381. , 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides .

Furthermore, the nucleic acid comprises a sequence encoding one or more gRNAs that hybridize to one or more target sequences of the ICP0 gene and/or one or more gRNAs that hybridize to one or more target sequences of the ICP27 gene. provided. In some embodiments, the nucleic acid comprises a sequence encoding one or more gRNAs according to SEQ ID NO: 2 and/or SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sequence encoding one or more gRNAs according to SEQ ID NO: 376 and/or SEQ ID NO: 377 . In some embodiments, the nucleic acid comprises a sequence encoding one or more gRNAs according to any one of SEQ ID NOs: 2, 7, 376, and 377. In some embodiments, the nucleic acid is about 70%, 80%, 85%, 90%, 91%, 92%, 93% relative to any one of SEQ ID NOs: 2, 7, 376, and 377. , 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity .

Additionally, in certain embodiments, one or more gRNAs encode one or more gRNAs that hybridize to one or more target sequences of the ICP0 gene, and/or one or more gRNAs that hybridize to one or more target sequences of the ICP27 gene. A nucleic acid comprising a sequence is provided. In some embodiments, the nucleic acid comprises a sequence encoding one or more gRNAs according to any one of SEQ ID NOs: 1-377. In some embodiments, the nucleic acid is about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% relative to any one of SEQ ID NOs: 1-377. , 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the nucleic acid further comprises a 5'ITR element and a 3'ITR element . In some embodiments, the nucleic acid is configured to be packaged into an adeno-associated virus (AAV) vector. In some embodiments, the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. In some embodiments, the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8.

In some embodiments, the CRISPR endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasI endonuclease. In some embodiments, the CRISPR endonuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease.

In some embodiments, the present disclosure provides compositions for the treatment or prevention of herpesvirus infections in a subject in need thereof. In some embodiments, the composition includes at least one isolated guide nucleic acid that includes a nucleotide sequence that is complementary to a target region in a herpesvirus genome. In some embodiments, the composition comprises a CRISPR-associated (Cas) peptide, or a functional fragment or derivative thereof. The isolated nucleic acid guide molecule and CRISPR-associated (Cas) peptide function together to introduce one or more mutations at a target site within the herpesvirus genome, thereby inhibiting viral infectivity.

The compositions also include isolated nucleic acids encoding one or more elements of the CRISPR-Cas system. For example, in some embodiments, the composition comprises a guide nucleic acid and an isolated nucleic acid encoding at least one of a CRISPR-associated (Cas) peptide, or a functional fragment or derivative thereof.

In some embodiments, the present disclosure provides methods for the treatment or prevention of a herpesvirus infection in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising at least one of a guide nucleic acid and a CRISPR-associated (Cas) peptide, or a functional fragment or derivative thereof. In certain cases, the method comprises administering a composition comprising a guide nucleic acid and an isolated nucleic acid encoding at least one of a CRISPR-associated (Cas) peptide, or a functional fragment or derivative thereof. including. In certain embodiments, the method comprises administering a composition described herein to a subject diagnosed with a herpesvirus infection who is at risk for developing a herpesvirus infection.

In some embodiments, the method uses herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV)), human herpes virus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)) .

(herpes virus)
The herpesvirus genus includes alphaherpesviruses (such as HSV1, herpes simplex virus type 2, which causes genital herpesviruses, and varicella-zoster virus, which causes chickenpox and shingles) ; betaherpesviruses (e.g., HHV-6, which causes the sixth disease); It is divided into three genera : gammaherpesviruses (e.g., Epstein-Barr virus, which causes mononucleosis and other diseases, and HHV-8, which causes Kaposi's sarcoma); and HHV-7, which causes infantile roseola. In addition to sharing a similar life cycle, alphaherpesviruses have DNA sequences homologous to many of the viral proteins essential for viral replication and reactivation.

Herpes simplex virus type 1 (HSV1) is an encapsulated 153 kilobase (kB) double-stranded DNA virus that is a nearly ubiquitous human pathogen. Up to 60% of the US population is infected with HSV1 by the age of 40. Primary infection usually occurs in early childhood and is characterized by fever and lesions of the buccal and gingival mucosa, although subclinical infections frequently occur. Primary HSV1 infection can also occur through sexual contact, and HSV1 has been increasingly found to be the cause of genital herpesviruses. Symptoms of initial infection are usually fairly mild, but life-threatening infections can occur, especially if infected as a newborn or if an immunocompromised person comes into contact with HSV1.

After primary infection, the HSV1 genome can remain dormant for long periods of time within sensory neurons. At this stage of the viral life cycle, the HSV1 genome resides in a supercoiled episomal state within the nucleus of latently infected neurons. In this state, no viral proteins are produced and only one viral transcript, latency-associated transcript (LAT), is produced. Various stimuli, such as stress and ultraviolet light, can trigger reactivation of the virus from latently infected neurons. Reactivation can occur repeatedly, many years after the initial infection. Symptoms caused by HSV1 reactivation vary depending on the site of initial infection and the degree of reactivation. The most commonly recognized form of HSV1 reactivation is herpes labialis. These usually appear on the vermilion borders of the mouth after various stimuli such as UV exposure. Reactivation of the virus stored in the dorsal root ganglion neurons that innervate the genitals results in the form of recurrent genital herpesvirus. The orofacial and anogenital symptoms of HSV1 reactivation are characterized by an initial prodromal tingling sensation in the area, followed by the appearance of painful blisters containing infectious virus, which ulcerate and eventually is cured. Other rarer symptoms of HSV1 reactivation include Bell's palsy, delayed facial paralysis after otologic surgery, and vestibular neuritis. Reactivation of HSV1 can lead to more severe conditions such as herpesvirus encephalitis and disseminated herpesvirus.

During primary infection, a defined series of protein expressions occur.
When the virus enters the cell during primary infection, the viral capsid is released in the cytoplasm and host protein synthesis is stopped by the coat protein VHS/UL41. A second coat protein, VP16, forms a complex with host proteins Oct1 and HCF to induce immediate early HSV1 transcription. These immediate early genes induce the expression of HSV1-encoded enzymes required for viral DNA replication, such as thymidine kinase (TK) and viral DNA polymerase (UL30). As viral DNA production progresses, late viral proteins such as capsid proteins, coat proteins, and glycoproteins necessary for viral entry into cells are produced. Viral particles assemble and viral DNA is packaged into capsids. Eventually, infectious virus particles are produced, causing infection of surrounding cells and making it possible to infect others.

The sequence of early stages of viral reactivation is thought to be mirrored during the reactivation of HSV1 in latently infected neurons at later stages of reactivation. Proteins that play important roles in lytic infection, such as VP16, are thought to play a similarly important role in reactivation. As the process of HSV1 reactivation progresses, early gene expression soon occurs, followed by early and late protein production. Ultimately, the generation of infectious virus particles occurs, leading to the transmission of infection to neighboring cells and uninfected individuals.

In recent years, DNA repair mechanisms mediated by homing endonucleases (HEs) or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently site-specific double-stranded DNA breaks (DSBs) have been developed. Several systems have been developed to target endogenous genes, such as the clustered regularly interspaced short palindromic repeat (CRISPR) -associated system 9 (Cas9) protein, which utilizes the clustered regularly interspaced short palindromic repeats (CRISPR)-associated system 9 (Cas9) protein . These enzymes induce accurate and efficient genome cleavage through the DSB-mediated DNS repair mechanism. These DSB-mediated genome editing techniques allow deletion, insertion, or modification of target genes.

In recent years, ZFN and TALEN have revolutionized genome editing. The main drawbacks of ZFNs and TALENs are uncontrollable off-target effects and tedious and expensive manipulation of custom DNA-binding fusion proteins tailored to each target site, limiting their universal application and clinical safety.

RNA-guided Cas9 biotechnology induces genome editing without detecting off-target effects. This technology takes advantage of the bacterial genomic defense mechanism in which the CRISPR/Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, conjugative plasmids). Three types (I-III) of CRISPR systems have been identified. CRISPR clusters contain a spacer, a complementary sequence to the preceding mobile element. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). Cas9 belongs to the type II CRISPR/Cas system and has a strong endonuclease activity that cleaves target DNA.

Cas9 contains a unique targeting sequence of approximately 20 base pairs (bp) (called a spacer) and a transactivating small RNA (tracrRNA) that serves as a guide for the processing of pre-crRNA using ribonuclease III . guided by mature crRNA. The crRNA:tracrRNA duplex directs Cas9 to the target DNA through complementary base pairing between a spacer on the crRNA and a complementary sequence (called the protospacer) on the target DNA (tDNA). Cas9 recognizes the trinucleotide (NGG) protospacer adjacent motif (PAM) and specifies the cleavage site (third nucleotide from the PAM). crRNA and tracrRNA can be expressed separately or engineered into an artificial fused small guide RNA (gRNA) via a synthetic stem-loop (AGAAAU) to mimic the natural crRNA/tracrRNA duplex. Such gRNAs, like shRNAs, can be synthesized or transcribed in vitro for direct RNA transfection, or can be expressed from RNA expression vectors (eg, U6 or H1 promoter-driven vectors). Therefore, Cas9 gRNA technology requires expression of Cas9 protein and gRNA. This results in the formation of gene editing complexes at specific target DNA binding sites within the target genome, causing cleavage/mutation of the target DNA.

However, the present disclosure is not limited to the use of Cas9-mediated gene editing. Rather, the present disclosure encompasses the use of other CRISPR-related peptides that can be targeted to target sequences using gRNA and edited to the desired target site. For example, in some embodiments, the present disclosure utilizes Cpf 1 to edit target sites in a subject .

As described herein, in some embodiments, the present disclosure uses a novel RNA-guided CRISPR technology to target the HSV1 genome and to target the HSV1 immediate early gene, infected cell protein 0 (ICP0). Employing a genetic strategy to disable its expression by editing specific domains of .

However, the present disclosure is not limited to the prevention or treatment of HSV1. Rather, the present disclosure can be used to treat or prevent other herpesviruses. For example, because the HSV2 genome is similar to the HSV1 genome, the strategies described herein would be effective in treating or preventing HSV2.

(nuclease)
Engineered CRISPR systems typically include two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). In nature, CRISPR/CRISPR-associated (Cas) systems provide adaptive immunity to viruses and plasmids in bacteria and archaea by using CRISPR RNA (crRNA) to induce silencing of invading nucleic acids. CRISPR-Cas is an RNA-mediated adaptive defense system that relies on small RNA molecules for sequence-specific detection and silencing of foreign nucleic acids. The CRISPR/Cas system consists of the cas gene, which is organized into operons, and a CRISPR array, which is made up of genome-targeting sequences (called spacers). Provided herein is an engineered CRISPR system that detects and silences HSV DNA within cells.

As described herein, CRISPR-Cas systems generally include a guide RNA sequence that includes a nucleotide sequence that is complementary or substantially complementary to a region of a target polynucleotide and a protein that has nuclease activity. Refers to the enzyme system. CRISPR-Cas systems include Type I CRISPR-Cas systems, Type II CRISPR-Cas systems, Type III CRISPR-Cas systems, and derivatives thereof. CRISPR-Cas systems include engineered and/or programmed nuclease systems derived from the naturally occurring CRISPR-Cas system. In certain embodiments, CRISPR-Cas systems include engineered and/or mutated Cas proteins. In some embodiments, nuclease generally refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of a nucleic acid. In some embodiments, endonucleases are generally capable of cleaving phosphodiester bonds within polynucleotide chains. Nickase refers to an endonuclease that cleaves only one strand of a DNA duplex.

In some embodiments, the CRISPR/Cas system used herein can be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9, Cas10 , Cas10d. , CasF, CasG, CasH, CasX, Cas Φ , Csy 1 , Csy2, Csy3, Cse 1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc 1 , Csc2, Csa5 , Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb 1 , Csb2, Csb3, Csx 17 , Csx 14 , Csx 16 , CsaX, Csx3, Csz1 , Csx l5, Csf 1 , Csf2, Csf3, Csf4 and Cu 1966 . As a further example, in some embodiments, the CRISPR-Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas10 , Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1 , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl6, CsaX, Csx3, Csx 1 , Csx 15 , Csf 1 , Csf2, Csf3, Csf4, Cas9, Cas 12 (e.g., Cas 12 a, Cas 12 b, Cas 12 c, Cas 12 d, Cas 12 k, Cas 12 j/ Cas Φ , Cas 12 L, etc.), Cas13 (e.g., Cas 13 a, Cas 13 b (Cas 13 b-t 1 , Cas 13 b-t2, Cas 13 b-t3, etc.), Cas 13 c, Cas 13 d, etc.), Cas14, CasX, CasY, or Cas It is an engineered form of a protein. In some embodiments, the CRISPR/Cas protein or endonuclease is Cas9. In some embodiments, the CRISPR/Cas protein or endonuclease is Cas12. In certain embodiments, the Cas 12 polypeptide is Cas 12 a, Cas 12 b, Cas 12 c, Cas 12 d, Cas 12 e , Cas 12 g, Cas 12 h, Cas 12 i, Cas 12 L, or Cas 12 It is J. In some embodiments, the CRISPR/Cas protein or endonuclease is CasX. In some embodiments, the CRISPR/Cas protein or endonuclease is CasY. In some embodiments, the CRISPR/Cas protein or endonuclease is Cas Φ .

In some embodiments, the Cas9 protein is Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dasonvillei, Streptomyces pristina espialis, Streptomyces viridochromogenes, Streptomyces・Viridochromogenes, Streptosporan, Ium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomicoi, Bacillus serenityreducens , Exiguobacterium sibilicum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla. Marina, Burkholderial bacteria, Polaromonas naphthalenivorans, Polar omonas sp., Crocosphaera watsoni, Cyanoteces sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium alabaticum, Ammonifex degenii, Caldicellocilloptor Vexii, Candidatus desulfordis, Clostridium botulinum, Clostridium difficile, Fine Gordia magna, Natranaerobius thermophilus, Perotomaculum the lomopropionicum, Acidithiobacillus cardus, Acidithiobacillus ferro. Oxydance, Arrocomatium Vinosam, Marino Bacter, Nitroso Sokokas Halo Fast, Nitroso Socokas Watsoni, Chude Altar Omonas Hallopactis, Kutedo novactor Rasemfer, Metano Halobium Eventigatum, Anabena Barriveris, NODULARIA SPUMIGENA, NOSTOC SP. , Arthrospira maxima, Arthrospira platensis, Arthrospira sp. , Lyngbya sp. , Microcoleus chonoplastes, Oscillatoria sp. , Petrotoga Moblis, Thermosipho africanus, or Acaryochromis marina.

In some embodiments, the composition comprises a CRISPR-associated (Cas) protein, or a functional fragment or derivative thereof. In some embodiments, the Cas protein is an endonuclease, including but not limited to Cas9 nuclease. In some embodiments, the Cas9 protein comprises an amino acid sequence that is identical to a wild-type Streptococcus pyogenes or Staphylococcus aureus Cas9 amino acid sequence. In some embodiments, the Cas protein is derived from other streptococcal species, such as thermophilus , Pseudomonas aeruginosa, E. coli, or other sequenced bacterial genomes and archaea, or other prokaryotic microorganisms. contains the amino acid sequence of the Cas protein. Other Cas proteins useful in this disclosure are known or can be identified using methods known in the art (see, e.g., Esvelt et al., 2013, Nature Methods, 10:1116-1121). ). In some embodiments, the Cas protein comprises a modified amino acid sequence compared to its natural source. CRISPR/Cas proteins contain at least one RNA recognition domain and/or RNA binding domain. The RNA recognition domain and/or RNA binding domain interacts with guide RNA (gRNA). CRISPR/Cas proteins can also include nuclease domains (ie, DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein - protein interaction domains, dimerization domains, and other domains.

The CRISPR/Cas-like protein can be a wild-type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild-type or modified CRISPR/Cas protein. CRISPR/Cas-like proteins can be modified to increase nucleic acid binding affinity and/or specificity, alter enzymatic activity, and/or change other properties of the protein. For example, the nuclease (ie, DNase, RNase) domain of a CRISPR/Cas-like protein can be modified, deleted, or inactivated. Alternatively, CRISPR/Cas-like proteins can be cleaved to remove domains that are not essential for Cas protein function. CRISPR/Cas-like proteins can be truncated or modified to optimize the activity of the effector domain of the Cas protein.

In some embodiments, a CRISPR/Cas-like protein can be derived from a wild-type Cas protein or a fragment thereof. In some embodiments, the CRISPR/Cas-like protein is a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.) compared to the wild type or another Cas protein. . Alternatively, domains of the Cas9 protein that are not involved in RNA-induced cleavage can be removed from the protein so that the modified Cas9 protein is smaller than the wild-type Cas9 protein.

The disclosed CRISPR-Cas compositions should also be construed to include any form of protein that has substantial homology to the Cas proteins disclosed herein (e.g., Cas9, saCas9, Cas9 protein). It is. In some embodiments, a protein that is "substantially homologous" is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous to the Cas protein amino acid sequence disclosed herein. % homology, about 95% homology, or about 99% homology.

In some embodiments, the composition comprises a CRISPR-associated (Cas) peptide, or a functional fragment or derivative thereof. In certain embodiments, the Cas peptide is an endonuclease, including but not limited to Cas9 nuclease. In some embodiments, the Cas9 peptide comprises an amino acid sequence that is identical to a wild-type Streptococcus pyogene Cas9 amino acid sequence. In some embodiments, the Cas peptide is derived from other species, e.g., other streptococcal species such as Pseudomonas thermophilus, Pseudomonas aeruginosa, E. coli, or other sequenced bacterial genomes and archaea, or other prokaryotes. It may contain the amino acid sequence of a Cas protein derived from a microorganism. Other Cas peptides useful in this disclosure are known or can be identified using methods known in the art (see, e.g., Esvelt et al., 2013, Nature Methods, 10:1116-1121). ). In certain embodiments, a Cas peptide may include a modified amino acid sequence compared to its natural source. For example, in some embodiments, a wild type Streptococcus pyogenes Cas9 sequence can be modified. In certain embodiments, the amino acid sequence may be codon-optimized for efficient expression in human cells (ie, "humanized") or the species of interest. The humanization CAS9 Nukurease sequence is, for example, an expression vector listed on Genbank Access number KM099231.1 GL6691993757; KM099232.1 GL669193761; or KM099233.1 GL669193765 . It can be a CAS9 nucleese sequence encoded by either of one. Alternatively, the Cas9 nuclease sequence may be a sequence contained within a commercially available vector, such as, for example, PX330 or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease is a Genbank accession number KM099231.1 GL669193757; KM099232.1 GL669193761; Cas9 of dgene, Cambridge, MA) ; It can have an amino acid sequence that is a variant or fragment of any endonuclease sequence.

Cas9 nucleotide sequences can be modified to encode biologically active variants of Cas9, and these variants may contain, for example, one or more mutations (e.g., additions, deletions, substitutions). (or a combination of such mutations) may have an amino acid sequence that differs from wild-type Cas9. One or more substitution mutations can be substitutions (eg, conservative amino acid substitutions).

In certain embodiments, the Cas peptide is a mutant Cas9, and the mutant Cas9 has reduced off-target effects compared to wild-type Cas9. In some embodiments, the mutant Cas9 is a Streptococcus pyogenes Cas9 (SpCas9) mutant.

In some embodiments, the SpCas9 variant comprises one or more point mutations, including but not limited to R780A, K810A, K848A, K855A, H982A, KI003A, and R1060A (Slaymaker et al., 2016, Science , 351(6268):84-88). In some embodiments, the SpCas9 variant comprises a DI135E point mutation (Kleinstiver et al ., 2015, Nature, 523(7561): 481-485). In some embodiments, the SpCas9 variant comprises the N497A, R661A, Q695A, Q926A, D1135E, L169A, and Y450A point mutations (Kleinstiver et al ., 2016, Nature, doi:10.1038/naturel6526). In some embodiments, the SpCas9 variant contains one or more point mutations, including, but not limited to, M495A, M694A, and M698A. Y450 is involved in stacking of hydrophobic base pairs. N497, R661, Q695, and Q926 are involved in hydrogen bonds between residues and bases that contribute to off-target effects. N497 is involved in hydrogen bonding through the peptide backbone. L169A is involved in stacking of hydrophobic base pairs. M495A, M694A, and H698A are involved in hydrophobic base pair stacking.

In some embodiments, the SpCas9 variant comprises one or more point mutations in one or more of the following residues: R780, K810, K848, K855, H982, KI003, R1060, DI135, N497, R661 , Q695, Q926, L169, Y450, M495, M694, M698. In some embodiments, the SpCas9 variants are R780A, K810A, K848A, K855A, H982A, K1003A, R1060A, D1135E, N497A, R661A, Q695A, Q926A, L169A, Y450A, M495A, M694A, and M698A. selected from the group Contains one or more point mutations.

In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, and Q926A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and D1135E compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and L169A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and Y450A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and M495A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and M694A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, and H698A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, DI135E, and L169A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, DI135E, and Y450A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, D1135E, and M495A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, D1135E, and M694A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations N497A, R661A, Q695A, Q926A, D1135E, and M698A compared to wild-type SpCas9.

In some embodiments, the SpCas9 variant comprises R661A, Q695A, and Q926A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, and DI135E compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises R661A, Q695A, Q926A, and L169A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises R661A, Q695A, Q926A, and Y450A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises R661A, Q695A, Q926A, and M495A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, and M694A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises R661A, Q695A, Q926A, and H698A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, DI135E, and L169A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises R661A, Q695A, Q926A, D1135E, and Y450A point mutations compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, D1135E, and M495A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, D1135E, and M694A compared to wild-type SpCas9. In some embodiments, the SpCas9 variant comprises point mutations R661A, Q695A, Q926A, D1135E, and M698A compared to wild-type SpCas9.

In some embodiments, the mutant Cas9 comprises one or more mutations that alter PAM specificity (Kleinstiver et al ., 2015, Nature, 523(7561):481-485; Kleinstiver et al., 2015, Nat Biotechnol , 33(12): 1293-1298). In some embodiments, the mutant Cas9 comprises one or more mutations that alter the catalytic activity of Cas9, including but not limited to D10A of RuvC and H840A of HNH (Cong et al., 2013; Science 339 :919-823; Gasiubas et al., 2012; PNAS 109:E2579-2586; Jinek et al., 2012; Science 337:816-821).

However , the present disclosure is not limited to the use of Cas9-mediated gene editing. Rather, the present disclosure encompasses the use of other CRISPR-related peptides that can be targeted to target sequences using gRNA and edited to the desired target site. For example, in some embodiments, the present disclosure utilizes Cpfl to edit target sites in a subject . Cpfl is a single crRNA-guided class 2 CRISPR effector protein that can effectively edit target DNA sequences in human cells. Exemplary Cpfl include, but are not limited to, Acidaminococcus sp. Cpfl (AsCpfl) and Lachnospiraceae Cpfl (LbCpfl) .

This disclosure should also be construed to include any form of peptide that has substantial homology to the Cas peptides disclosed herein (eg, Cas9). Preferably, a peptide that is "substantially homologous" is about 50% homologous, more preferably about 70% homologous, and even more preferably about 80% homologous to the amino acid sequence of the Cas peptide disclosed herein. % homology, more preferably about 90% homology, even more preferably about 95% homology, even more preferably about 99% homology.

Alternatively , peptides may be made by recombinant means or by cleavage from longer polypeptides. Peptide composition can be confirmed by amino acid analysis or sequencing.

Variants of peptides according to the present disclosure include: (i) one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such amino acid residues are (ii) there are one or more modified amino acid residues, e.g., residues modified by the attachment of substituents; (iii) (iv) a fragment of the peptide, and /or (v) the peptide is an alternative splice variant of a peptide of the present disclosure; A peptide is fused to another peptide , such as the sequence used for the Sv5 epitope tag). Fragments include peptides generated by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may undergo post-translational or chemical modifications. Such variations are considered to be within the scope of those skilled in the art given the teachings herein.

As known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. be done. A variant is a peptide sequence that differs from the original sequence, preferably a peptide sequence that differs from the original sequence by less than 40% of the residues per segment of interest, more preferably a peptide that differs from the original sequence by less than 25% of the residues per segment of interest. more preferably differs by less than 10% of the residues per segment of interest, and most preferably differs from the original protein sequence in only a few residues per segment of interest while simultaneously sufficiently homologous to the sequence to preserve the functionality of the original sequence. The present disclosure includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods widely known to those skilled in the art. Identity between two amino acid sequences is preferably determined using the BLASTP algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., J. Mol. Biol. 215:403- 410 (1990)).

Peptides of the present disclosure can be post-translationally modified. For example, post-translational modifications within the scope of this disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, proteolytic processing, and the like. Some modifications or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding dog microsomal membranes or Xenopus egg extract (US Pat. No. 6,103,489) to standard translation reactions.

Peptides of the present disclosure may contain unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. Various approaches are available to introduce unnatural amino acids during protein translation.

Peptides or proteins of the present disclosure can be combined with other molecules, such as proteins, to prepare fusion proteins. This can be achieved, for example, by synthesis of N-terminal or C-terminal fusion proteins, provided that the resulting fusion protein retains the functionality of the Cas peptide.

Peptides or proteins of the present disclosure can be phosphorylated using conventional methods, such as those described in Reedijk et al. (EMBO Journal 11(4):1365, 1992) .

Cyclic derivatives of the peptides of this disclosure are also part of this disclosure. Cyclization may allow the peptide to adopt a more favorable conformation for binding to other molecules. Cyclization can be accomplished using techniques known in the art. For example, a disulfide bond may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization is also described by Ulysse, L. et al., J. Am. Chem. Soc. 1995, 117 , 8466-8467. The bond-forming moiety may be an amino acid side chain, a non-amino acid moiety, or a combination of the two. In one embodiment of the present disclosure, the cyclic peptide may include a beta turn in the correct position. Beta turns can be introduced into the peptides of the present disclosure by adding the amino acid Pro-Gly at the correct position.

It may be desirable to produce cyclic peptides that are more flexible than the peptide bond-containing cyclic peptides described above. More flexible peptides can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to deform or rotate the β sheet. This peptide is more flexible due to the length of the disulfide bonds and fewer hydrogen bonds in the beta sheet portion. The relative flexibility of cyclic peptides can be determined by molecular dynamics simulations.

The present disclosure also relates to Cas peptides fused or integrated to targeting proteins and/or peptides containing targeting domains that can direct the chimeric protein to a desired cellular component or cell type or tissue. Chimeric proteins may also include additional amino acid sequences or domains. Chimeric proteins are recombinant in the sense that the various components are derived from different sources and are not found together in nature (ie, are heterologous).

In some embodiments, the targeting domain can be a transmembrane domain, a membrane binding domain, or a sequence that causes the protein to associate with, for example, a vesicle or the nucleus. In some embodiments, the targeting domain can target the peptide to a particular cell type or tissue. For example, the targeting domain can be a cell surface ligand or an antibody directed against a cell surface antigen of the target tissue (eg, cancerous tissue). The targeting domain can target the peptides of the present disclosure to cellular components. In certain embodiments, the targeting domain targets a tumor-specific or tumor-associated antigen.

N-terminal or C-terminal fusion proteins comprising a peptide or chimeric protein of the present disclosure combined with other molecules have a desired biological function with the N-terminus or C-terminus of the peptide or chimeric protein through recombinant techniques. obtained by fusing sequences of selected proteins or selectable markers. The resulting fusion protein comprises a Cas peptide or chimeric protein fused to a selected protein or marker protein, as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

Peptides of the present disclosure can be synthesized by conventional techniques. For example, peptides of the present disclosure can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods use either solid-phase or liquid-phase synthesis techniques (e.g., for solid-phase synthesis techniques, see J.M. Stewart and J.D. Young, Solid-Phase Peptide Synthesis, 2nd ed. , Pierce Chemical Co., Rockford IL (1984) and G. Barany and R. B. Merrifield, "Peptides; Analysis and Synthesis", Biology Editor's Edition, Gross and J. Meienhofer Vol. 2 Acad. emic Press, New York, 1980, pp. 3-254; and on classical liquid phase synthesis, M. Bodansky, Principles of Peptide Synthesis , Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, eds., Peptides; Synthesis”, Biology, Volume 1).

Peptides of the present disclosure can be prepared by standard chemical or biological means of peptide synthesis. Biological methods include, but are not limited to, expression of nucleic acids encoding the peptides in host cells or in vitro translation systems.
Biological preparation of the peptides of the present disclosure involves expression of a nucleic acid encoding the desired peptide. Expression cassettes containing such coding sequences can be used to produce desired peptides. For example, subclones of the nucleic acid sequences encoding the peptides of the present disclosure are described in Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Springs Laboratories, Cold Springs Harbor, 1997, each of which is herein incorporated by reference in its entirety. New York (2012), and subcloning gene fragments as described in Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons (New York, NY) (1999 and earlier editions). can be produced using conventional molecular genetic engineering. The subclones are then expressed in bacterial cells in vitro or in vivo to produce smaller proteins or polypeptides that can be tested for specific activities.

Regarding expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. The coding sequences for desired peptides of the present disclosure can be codon-optimized based on the intended host cell codon usage to improve expression efficiency as demonstrated herein. Codon usage patterns can be found in the literature (nakamura et al., 2000, Nuc Acids Res. 28:292). Representative examples of suitable hosts include bacterial cells such as Streptococcus, Staphylococcus, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells such as yeast cells and Aspergillus cells; insects such as Drosophila S2 cells and Spodoptera Sf9 cells. cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293 and Bowes melanoma cells; and plant cells.

A large number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides conjugated to ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

Expression vectors can be introduced into host cells by physical, biological or chemical means, as discussed in detail elsewhere herein.

Analysis of peptide composition can be performed to confirm that the peptide obtained from either chemical or biological synthesis techniques is the desired peptide. Such amino acid composition analysis can be performed using high resolution mass spectrometry to determine the molecular weight of the peptide. Alternatively, or in addition, the amino acid content of a peptide can be determined by hydrolyzing the peptide in an aqueous acid solution and separating, identifying and quantifying the components of the mixture using HPLC or an amino acid analyzer. Protein sequencers, which sequentially degrade peptides and sequentially identify amino acids, can also be used to unambiguously determine the sequence of a peptide.

Peptides and chimeric proteins of the present disclosure can be prepared using inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, or formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, It can be converted into pharmaceutical salts by reaction with organic acids such as tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, toluenesulfonic acid, etc.

In certain embodiments, gene editing systems are described herein that include meganucleases. In some embodiments, the gene editing system includes a zinc finger nuclease (ZFN). In some embodiments, the gene editing system includes a transcription activator-like effector nuclease (TALEN). These gene editing systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs, TALENs, and meganucleases achieve specific DNA binding through protein-DNA interactions, whereas CRISPR- The Cas system is targeted to specific DNA sequences by short RNA guide molecules that base pair directly with the target DNA and by protein-DNA interactions . Accordingly, protein targeting or nucleic acid targeting can be used to target the HSV genome described herein.

(Guide nucleic acid)
In some embodiments, the composition comprises at least one isolated guide nucleic acid or fragment thereof, the guide nucleic acid comprising a nucleotide sequence complementary to one or more target sequences in a herpesvirus genome. . In some embodiments, the guide nucleic acid is a guide RNA (gRNA).

In some embodiments, the gRNA comprises a crRNA:tracrRNA duplex. In some embodiments, the gRNA includes a stem-loop that mimics the natural duplex between crRNA and tracrRNA. In some embodiments, the stem-loop includes a nucleotide sequence that includes AGAAAAU. For example, in some embodiments, the compositions include synthetic or chimeric guide RNAs including crRNA, stem, and tracrRNA.

In certain embodiments, the composition comprises isolated crRNA and/or isolated tracrRNA that hybridize to form a natural duplex. For example, in some embodiments, the gRNA comprises a crRNA or a pre-crRNA (pre-crRNA) that includes a targeting sequence.

In some embodiments, the gRNA comprises a nucleotide sequence that is substantially complementary to a target sequence within the herpesvirus genome. The target sequence within the herpesvirus genome can be any sequence in the coding or non-coding region where CRISPR/Cas-mediated gene editing results in mutation of the genome and inhibition of viral infectivity. In certain embodiments, the target sequence to which the gRNA is substantially complementary is within the UL56, ICP0, ICP4, or ICP27 gene.

Exemplary gRNA nucleotide sequences targeting the ICP0 gene are: c catggagccccgccccgga (SEQ ID NO: 1); gtacccgacggcccccgcgt (SEQ ID NO: 2); gacacgggcaccacacacca (SEQ ID NO: 3); tcccgcgtcaatcagcacc c (SEQ ID NO: 4); tcacttttcccctccccgac (SEQ ID NO: 5); ccttactcacacgcatctag (SEQ ID NO: 6); ctcaggccgcgaaccaagaa (SEQ ID NO: 7); gttcgcggcctgagccaggg (SEQ ID NO: 8); gctaaggggaaaaagggg (SEQ ID NO: 9); tttgactcagacgc agggcc (SEQ ID NO: 10); ttttttccccttagcccgcc (SEQ ID NO: 11); caacagacagcaaaaaatccc (SEQ ID NO: 12); tcgaacagcatgttccccac (SEQ ID NO: 13); gaccctatatacagggac (SEQ ID NO: 14); gcgggagaagagaggaagaag (SEQ ID NO: 15); cctggctgctgcgtctcgct (SEQ ID NO: 16); ccccacttcggtctccgcct (SEQ ID NO: 17); ggtg cgtccgaggaagaggc (SEQ ID NO: 18); gcgtcggagtggaacagccct (SEQ ID NO: 19); ggtctgcaaccaaaggtggt ( ggtctgtatatataaagtca (SEQ ID NO: 21); ctcccgccctccagacgcac (SEQ ID NO: 22); gtgtctctgtgtatgagtca (SEQ ID NO: 23); tgcatcccgtgcatgaaaac (SEQ ID NO: 24); gg gtaaccacgtgatgcccc (SEQ ID NO: 25); tgatgcggagaggggcggc (SEQ ID NO: 26); cgtgctgtccgcctcggagg (SEQ ID NO: 25); gaggccgccgaggacgtcag (SEQ ID NO: 28); ttacccgcggtctcggggag (SEQ ID NO: 29); ccccacccccccctagatgcgt (SEQ ID NO: 30); ctctgttgtttgcaaggggg (SEQ ID NO: 31); ga agagggaagaagaagagggt (SEQ ID NO: 32); gggggagtcgctgatcacta (SEQ ID NO: 33); gcaccctgctccccgagacc (SEQ ID NO: 34); cggaagtccagggcgccccac (SEQ ID NO: 35); gatagtgggcgtgacgccca (SEQ ID NO: 36); ggcgacccggggccctgcgt (SEQ ID NO: 37); gtctgggggtcgttcacgat (SEQ ID NO: 38); tgc atccaggttttcatgca (SEQ ID NO: 39); tcgtccgtggtgggctccgg (SEQ ID NO: 40); tccctgtatatatagtgtca (SEQ ID NO: 41) ); cacaacaaacacacaggac (SEQ ID NO: 42); gggaaaaaaggggggcgggt (SEQ ID NO: 43); ggggcgtctggcccctccgg (SEQ ID NO: 44); gggacgcgtggactgggggg (SEQ ID NO: 45); acccgg agcccaccacggac (SEQ ID NO: 46); ccctaataaaaaaaactca (SEQ ID NO: 47); tgggggcggccctcaggccg (SEQ ID NO: 48) ; ccccggccctgagtcggagg (SEQ ID NO: 49); tccctgtatatatagggtca (SEQ ID NO: 50); gccctcaccgtgtgcccccc (SEQ ID NO: 51); ccactccgacgcgggggccg (SEQ ID NO: 52); acggcct cctcggcctccat (SEQ ID NO: 53); atgttccccgtctccatgtc (SEQ ID NO: 54); cccggctcccgtgtatgagt (SEQ ID NO: 55); gcgacgtgtgcgccgtgtgc (SEQ ID NO: 56); atggcgcccggctcccgtgt (SEQ ID NO: 57); gggggcgccccgcaactgc (SEQ ID NO: 58); atgggggtcgtatgcggctg (SEQ ID NO: 59); ccctectcc tcctcctcccc (SEQ ID NO: 60); gtggggcgtgtctctgtgt (SEQ ID NO: 61); ccggggaccgcggcccgcag (SEQ ID NO: 62); gtcgcggacggaggtccct at ggcggccggttccagtgt (SEQ ID NO: 68); cggctggaggtcgcggacg (SEQ ID NO: 69); aggtggtctgggtccgtcct ( cctatgttttccctcgtc (SEQ ID NO: 71); ccggttccagtgtaagggtc (SEQ ID NO: 72); gctccggggcggggctccat (SEQ ID NO: 73); cctcggaagaggggggagaa (SEQ ID NO: 74); gac ccggtccctgtatata (SEQ ID NO: 75); ctggccgcgccccccccggcc (SEQ ID NO: 76); gggggggttggggttggggt (SEQ ID NO: 75); No. 77); ggggagggggggtcgggcg (SEQ ID NO: 78); ggggggggagaggggaactc (SEQ ID NO: 79); gcggaagaggcggccccgc (SEQ ID NO: 80); acgcgctacctgcccatctc (SEQ ID NO: 81); tgag taaggggggcctgcgt (SEQ ID NO: 82); ggaccgggggcgccatgtta (SEQ ID NO: 83); ccccgtgtttgtggggagg (SEQ ID NO: 82); atccctcgtccgtggtgggct (SEQ ID NO: 85); tctggcccctccggggggt (SEQ ID NO: 86); aggaggaggggggggagg (SEQ ID NO: 87); ccacggccgcgcgggggcgc (SEQ ID NO: 88); gctc gggggggccgggcgtg (SEQ ID NO: 89); cctccagacgcaccggagtc (SEQ ID NO: 90); cgccccctgctccccggacc (SEQ ID NO: 91); ctcggcctccatgcgggtct (SEQ ID NO: 92); gggacgggtcgccctgtt (SEQ ID NO: 93); ccctccggggggggttggggt (SEQ ID NO: 94); ggctgctgggggccgcagggc (SEQ ID NO: 95); cag ggccggggggggcgcggc (SEQ ID NO: 96).

Exemplary PAM sequences for use with gRNA are: SEQ ID NOs: 1-96;
gcgagt (SEQ ID NO: 97); cggagt (SEQ ID NO: 98); gcgggt (SEQ ID NO: 99); acgagt (SEQ ID NO: 100); acggat (SEQ ID NO: 101); gggggt (SEQ ID NO: 102); cagagt (SEQ ID NO: 103); acgagt (SEQ ID NO: 104); gcgggt (SEQ ID NO: 105); cggggt (SEQ ID NO: 106); ccggat (SEQ ID NO: 107); ctgagt (SEQ ID NO: 108); gggggt (SEQ ID NO: 109); SEQ ID NO: 111); ccgagt (SEQ ID NO: 112); cagagt (SEQ ID NO: 113); gcgggt (SEQ ID NO: 114); ctggat (SEQ ID NO: 115); ctgggt (SEQ ID NO: 116); gggggt (SEQ ID NO: 117); No. 118); gggggt (SEQ ID NO: 119); ctggat (SEQ ID NO: 120); ccgagt (SEQ ID NO: 121); ccgagt (SEQ ID NO: 122); cggagt (SEQ ID NO: 123); gggggt (SEQ ID NO: 124); cagggt (SEQ ID NO: 124); gtgagt (SEQ ID NO: 126); gcgggt (SEQ ID NO: 127); cgggat (SEQ ID NO: 128); tggggt (SEQ ID NO: 129); gcgggt (SEQ ID NO: 130); tagggt (SEQ ID NO: 131); gcgggt (SEQ ID NO: 132) ); ctgagt (SEQ ID NO: 133); cgggat (SEQ ID NO: 134); cgggat (SEQ ID NO: 135); gtgggt (SEQ ID NO: 136); cggggt (SEQ ID NO: 137); cggggt (SEQ ID NO: 138); aagaat (SEQ ID NO: 139 ); gggggt (SEQ ID NO: 140); aggggt (SEQ ID NO: 141); gaggat (SEQ ID NO: 142); ggggat (SEQ ID NO: 143); gcggggt (SEQ ID NO: 144); gggggt (SEQ ID NO: 145); gggggt (SEQ ID NO: 146) cagggt (SEQ ID NO: 147); tcgggt (SEQ ID NO: 148); gcgggt (SEQ ID NO: 149); caggat (SEQ ID NO: 150); gggggt (SEQ ID NO: 151); acggat (SEQ ID NO: 152); atgagt (SEQ ID NO: 153); cgggt (SEQ ID NO: 154); gagggt (SEQ ID NO: 155); cagggt (SEQ ID NO: 156); atgagt (SEQ ID NO: 157); ccgggt (SEQ ID NO: 158); gggggt (SEQ ID NO: 159); ggggat (SEQ ID NO: 160); gggagt (SEQ ID NO: 161); ctgggt (SEQ ID NO: 162); gggggt (SEQ ID NO: 163); aaggt (SEQ ID NO: 164); gagggt (SEQ ID NO: 165); ttggat (SEQ ID NO: 166); ccgggt (SEQ ID NO: 167); gggggt ( SEQ ID NO: 168); gggggt (SEQ ID NO: 169); aggggt (SEQ ID NO: 170); tagggt (SEQ ID NO: 171); ctgagt (SEQ ID NO: 172); tggggt (SEQ ID NO: 173); ctgggt (SEQ ID NO: 174); gtgggt (SEQ ID NO: 174); No. 175); gggggt (SEQ ID No. 176); gggggt (SEQ ID No. 177); atgagt (SEQ ID No. 178); gggggt (SEQ ID No. 179); gggggt (SEQ ID No. 180); ccgggt (SEQ ID No. 181); tggggt (SEQ ID No. 182); aggaat (SEQ ID NO: 183); gcgggt (SEQ ID NO: 184); gagggt (SEQ ID NO: 185); gggggt (SEQ ID NO: 186) ; SEQ ID NO: 187); acgggt (SEQ ID NO: 188); gggggt (SEQ ID NO: 189); gggggt (SEQ ID NO: 190); tggggt (SEQ ID NO: 191); gtggat (SEQ ID NO: 192); cagggt (SEQ ID NO: 193).

Exemplary gRNA nucleotide sequences for targeting the UL56 gene are: ccgcgctccataaacccgcg (SEQ ID NO: 194); ctggtttccggaagaaacag (SEQ ID NO: 195); cacggacaacagggcccag (SEQ ID NO: 196); gcttaccgccac aggaatac (SEQ ID NO: 197); ccctctccggaggaggttgg (SEQ ID NO: 198) ; ttgggccctgtacagctcgc (SEQ ID NO: 199); acaagaggtcccttgtgatg (SEQ ID NO: 200); caagctatcgtagggggcg (SEQ ID NO: 201); ccgaacgacgtgcgcagcgc (SEQ ID NO: 202); cacg acagtggcataggttg (SEQ ID NO: 203); acaggggcgctttaccgccac (SEQ ID NO: 204); ctgtggcggtaagcgcccct (SEQ ID NO: 205); gcgccggagttttggccctg (SEQ ID NO: 206); cccagcagagtacggtggag (SEQ ID NO: 207); cctaggaggccgccacgcgc (SEQ ID NO: 208); tacggtggaggtgggtccgt (SEQ ID NO: 209); cggag gcggcgcaacccgac (SEQ ID NO: 210); gtgtggcgccatgctgtatt (SEQ ID NO: 211); tcgggcgcgtggcggcctcc (SEQ ID NO: 212).

PAM sequences for use with SEQ ID NOS: 194-212 include:
tcgggt (SEQ ID NO: 213); gggggt (SEQ ID NO: 214); cagagt (SEQ ID NO: 215); cagaat (SEQ ID NO: 216); cggaat (SEQ ID NO: 217); gcgaat (SEQ ID NO: 218); tcgggt (SEQ ID NO: 219); ggggat (SEQ ID NO: 220); cggagt (SEQ ID NO: 221); gggggt (SEQ ID NO: 222); aggaat (SEQ ID NO: 223); gtgagt (SEQ ID NO: 224); gcgggt (SEQ ID NO: 225); gtgggt (SEQ ID NO: 226); ccgagt ( SEQ ID NO: 227); gggggt (SEQ ID NO: 228); gcgggt (SEQ ID NO: 229); tggggt (SEQ ID NO: 230); tagggt (SEQ ID NO: 231).

110 off-target gRNA sequences:
cagcactgcataaacctcg (SEQ ID NO: 232); ccgctttccgtaaacccggg (SEQ ID NO: 233); ccgcggttcctaaaaccgcg (SEQ ID NO: 234); ccgggctccctgaactcgcg (SEQ ID NO: 235); gcgg ctccataaagccccg (SEQ ID NO: 236); ccgggtccataaaccctct (SEQ ID NO: 237); ccacgctccatcaaccctcc (SEQ ID NO: 238); ccgagctccatctacccacg (SEQ ID NO: 239); ccgccctccacagacacgcg (SEQ ID NO: 240); ccgcactccatgcacgcgcg (SEQ ID NO: 241); ccgccctccagaaaagccccg (SEQ ID NO: 242); ccgcgctcccaaaagccccg (SEQ ID NO: 243) ).

PAM sequences for use with SEQ ID NOS: 232-231 include:
cagga (SEQ ID NO: 244); ccggg (SEQ ID NO: 245); gtgaa (SEQ ID NO: 246); ccggg (SEQ ID NO: 247); ctgga (SEQ ID NO: 248); gggaa (SEQ ID NO: 249); ctgaa (SEQ ID NO: 250); ccgag (SEQ ID NO: 251); cgggg (SEQ ID NO: 252); atggg (SEQ ID NO: 253); cgggg (SEQ ID NO: 254); gcggg (SEQ ID NO: 255).

417 off-target gRNA sequences:
ctcctttccagaagaaacag (SEQ ID NO: 256); ctggtttctgtaagaaacag (SEQ ID NO: 257); ctcctttctggaagaaacag (SEQ ID NO: 258); gtggtttccaaaagaaacag (SEQ ID NO: 259); taagt ttcctgaagaaacag (SEQ ID NO: 260); gttttttcctgaagaaacag (SEQ ID NO: 261); ctgtatttcagaagaaacag (SEQ ID NO: 262); atgtttcccagaagaaacag (SEQ ID NO: 263); gttgtttgaggaagaaacag (SEQ ID NO: 264); aagatttcaggaagaaacag (SEQ ID NO: 265); ctcgctacctgaagaaacag (SEQ ID NO: 266); attctttctggaagaaacag (SEQ ID NO: 267) ); ctggcttcggcaagaaacag (SEQ ID NO: 268); caggtttctggaagaatcag (SEQ ID NO: 269); ctggcttctggaagaagcag ( SEQ ID NO: 270); ctggattcctgaaggaacag (SEQ ID NO: 271); ttggtttgctgaagaaacgg (SEQ ID NO: 272); ctgtttaagggaagaaacag (SEQ ID NO: 273); gtgatttctgcaagaaacag (SEQ ID NO: 274) ); ctagcagccggaagaaacag (SEQ ID NO: 275); atagtttctgaaagaaacag (SEQ ID NO: 276); ttggtttatgaaagaaacag (SEQ ID NO: 275); cttgtatggggaagaaacag (SEQ ID NO: 278); cttttgtcaggaagaaacag (SEQ ID NO: 279); ctgccctctggaagaaacag (SEQ ID NO: 280); ctcatttctggaagaaaacaa (SEQ ID NO: 281) ); ctggttaggaagaagaaacag (SEQ ID NO: 282); ctgccttctggaagaaaacaa (SEQ ID NO: 283); ctgatttaggaaagaaacag (SEQ ID NO: cttgtttttgggagaaacag (SEQ ID NO: 285); cttgttttggggagaaacag (SEQ ID NO: 286); ctgctttgaggaagaaacag (SEQ ID NO: 287); atggtttcatgtagaaacag (SEQ ID NO: 288) ; catgtttcaggaagaatcag (SEQ ID NO: 289); ttggtttacagaaggaacag (SEQ ID NO: 290); ctggtgtcccgaagtaacag (SEQ ID NO: 291 ); ctggtttgtaaaagaaacag (SEQ ID NO: 292); ttgttttcaggaggaaacag (SEQ ID NO: 293); ctggcttcccctaagaaaacaa (SEQ ID NO: 294); caggtttgaggacgaaacag (SEQ ID NO: 295); g tggattcctgaagaaaaag (SEQ ID NO: 296); ctgcttttaggaggaaacag (SEQ ID NO: 297); cgggcttcctgaagaaagag (SEQ ID NO: 298) ctggtgcgaggaagaaacag (SEQ ID NO: 299); ctgcattccagaagaaaaaag (SEQ ID NO: 300); atggtttcctgaagaatcaa (SEQ ID NO: 301); ctgatttacagaagaaaaag (SEQ ID NO: 302); ctg ttttactgaagaaagag (SEQ ID NO: 303); gtgatttccagaagaacacag (SEQ ID NO: 304); gtggtgtctggcagaaacag (SEQ ID NO: 305).

PAM sequences for use with SEQ ID NOS: 256-305 include:
tagaa (SEQ ID NO: 306); cagga (SEQ ID NO: 307); tggga (SEQ ID NO: 308); atgag (SEQ ID NO: 309); taggg (SEQ ID NO: 310); cagg (SEQ ID NO: 311); tcgaa (SEQ ID NO: 312); aagag (SEQ ID NO: 313); aagga (SEQ ID NO: 314); aagga (SEQ ID NO: 315); gaga (SEQ ID NO: 316); cagg (SEQ ID NO: 317); gagag (SEQ ID NO: 318); aagaa (SEQ ID NO: 319); agggg (SEQ ID NO: 320); tagga (SEQ ID NO: 321); tgga (SEQ ID NO: 322); cagg (SEQ ID NO: 323); ctgaa (SEQ ID NO: 324); ttgaa (SEQ ID NO: 325); aagaa (SEQ ID NO: 326); cggag (SEQ ID NO: 327); tagaa (SEQ ID NO: 328); ctgaa (SEQ ID NO: 329); aagag (SEQ ID NO: 330); gaggg (SEQ ID NO: 331); gaga (SEQ ID NO: 332); aagaa (SEQ ID NO: 333); gaga (SEQ ID NO: 334); aagg (SEQ ID NO: 335); cagga (SEQ ID NO: 336); ttgaa (SEQ ID NO: 337); tagaa (SEQ ID NO: 338); ttggg (SEQ ID NO: 339); aagaa (SEQ ID NO: 340); cagag ( cagga (SEQ ID NO: 342); tggga (SEQ ID NO: 343); tgga (SEQ ID NO: 344); ctggg (SEQ ID NO: 345); ctggg (SEQ ID NO: 346); cagga (SEQ ID NO: 347); gaga (SEQ ID NO: 347); No. 348); gggag (SEQ ID No. 349); aagga (SEQ ID No. 350); tagaa (SEQ ID No. 351); aagga (SEQ ID No. 352); aagg (SEQ ID No. 353); aggga (SEQ ID No. 354); cagg (SEQ ID No. 354); 355).

In certain embodiments, the gRNA sequence that is substantially complementary to the target is about 10-30 nucleotides in length. In certain embodiments, the gRNA comprises a nucleotide sequence that binds to a target sequence of the HSV genome. For example, in certain embodiments, the gRNA includes a nucleotide sequence that is substantially complementary to, and thus binds to, the target sequence. For example, in certain embodiments, the gRNA is substantially complementary to a target sequence of the HSV genome selected from the group consisting of:
TCTGGGTGTTTCCCTGCGACCGAGACCTGC (SEQ ID NO: 356, "2A");
GGACAGCACGGACACGGAACTGTTCGAGACG (SEQ ID NO: 357, "2B");
GCATCCCGTGCATGAAAAC (SEQ ID NO: 358, "2C");
TGTGCAACGCCAAGCTGGTGTACCTGATAG-3' (SEQ ID NO: 359, "2D");
GCGAGTACCCGCCGGCCTGA (SEQ ID NO: 360, "1");
GCGAGCCGCGGCGCCGCGGG (SEQ ID NO: 361, "3");
TTCTACGCGCGCTATCGCGA (SEQ ID NO: 362, "ICP4 ml");
GGAGTGTTCCTCGTCGGACG (SEQ ID NO: 363, "ICP27ml")

In certain embodiments, the target sequence precedes the PAM sequence. For example, in some embodiments, the target sequence precedes the NGG PAM sequence. Exemplary target sequences + PAM sequences (PAM sequences are underlined) are:
TCTGGGTGTTTCCCTGCGACCGAGACCTGC CGG (SEQ ID NO: 364, "2A");
GGACAGCACGGACACGGAACTGTTCGAGACG GGG (SEQ ID NO: 365, "2B")
GCATCCCGTGCATGAAAAACC TGG (SEQ ID NO: 366, "2C");
TGTGCAACGCCAAGCTGGTGTACCTGATAG TGG (SEQ ID NO: 367, "2D");
GCGAGTACCCGCCGGCCTGA GGG (SEQ ID NO: 368, "1");
GCGAGCCGCGGCGCCGCGGG GGG (SEQ ID NO: 369, "3");
TTCTACGCGCGCTATCGCGA CGG (SEQ ID NO: 370, "ICP4ml");
GGAGTGTTCCTCGTCGGACG AGG (SEQ ID NO: 371, "ICP27ml");
GTACCCGACGGCCCCGCGT CGGAGT (SEQ ID NO: 372, "ICP0-M1");
CTCAGGCCGCGAACCAAAGAA CAGAGT (SEQ ID NO: 373, " ICP0 -M2");
AATCCTAGACACGCACCGCC AGGAGT (SEQ ID NO: 374, "ICP27-M1");
TCGCCAGCGTCATTAGCGGG GGGGGT (SEQ ID NO: 375, "ICP27-M2");
AATCCTAGACACGCACCGCC (SEQ ID NO: 376, "ICP27-M1");
TCGCCAGCGTCATTAGCGGG (SEQ ID NO: 377 , "ICP27-M2")

Additionally, the present disclosure encompasses isolated nucleic acids (eg, gRNAs) that have substantial homology to the nucleic acids disclosed herein. In certain embodiments, the isolated nucleic acid is at least 75%, 80%, 85%, 90%, 95%, 96%, 97% the same as the nucleotide sequence of a gRNA described elsewhere herein. , 98%, or 99% sequence homology.

A guide RNA sequence can be a sense or antisense sequence. In the CRISPR-Cas system from P. pyogenes, the target DNA is usually immediately preceding the 5'-NGG protospacer adjacent motif (PAM). Other Cas9 orthologs may have different PAM specificities. For example, S. Cas9 from thermophilus requires 5'-NNAGAA for CRISPR1, 5'-NGGNG for CRISPR3 , and 5'-NNNNNGATT for N. meningitidis). The specific sequence of the guide RNA will vary , but regardless of the sequence, a useful guide RNA sequence will be one that minimizes off-target effects while achieving high efficiency mutagenesis of herpesvirus target sequences. The specific sequence of the guide RNA will vary , but regardless of the sequence, a useful guide RNA sequence will be one that minimizes off-target effects while achieving high efficiency editing of the HSV genome. The length of the guide RNA sequence can vary from about 20 nucleotides to about 60 or more nucleotides, e.g., about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, about 60 or more nucleotide . A useful selection method is one that identifies regions of very low homology between the foreign viral genome and the host cell genome, and can be used as a target to exclude off-target human transcriptome or (rarely) untranslated genomic sites. Bioinformatics screening using sequence + NGG target selection criteria, and WGS, Sanger. Sequencing and SURVEYOR assays will identify and exclude potential off-target effects. Use algorithms such as the CRISPR Design Tool (CRISPR Genome Engineering Resources; Broad Institute) to determine the essential PAM sequences defined by the type of Cas peptide used (i.e., Cas9, Cas9 variant, Cpfl) or close target sequence can be identified.

In certain embodiments, the composition comprises a plurality of different gRNAs, each targeting a different target sequence. In certain embodiments, this multiplexing strategy provides improved effectiveness. In some embodiments, the compositions described herein utilize about 1 gRNA to about 6 gRNAs. In some embodiments, the compositions described herein utilize at least about one gRNA. In some embodiments, the compositions described herein utilize up to about 6 gRNAs. In some embodiments, the compositions described herein contain about 1 g RNA to about 2 g RNA, about 1 g RNA to about 3 g RNA, about 1 g RNA to about 4 g RNA, about 1 g RNA to about 5 g RNA, about 1 g RNA to about 6 g RNA, about 2 g RNA ~3gRNA, approximately 2gRNA to approximately 4gRNA, approximately 2gRNA to approximately 5gRNA, approximately 2gRNA to approximately 6gRNA, approximately 3gRNA to approximately 4gRNA, approximately 3gRNA to approximately 5gRNA, approximately 3gRNA to approximately 6gRNA, approximately 4gRNA to approximately 5gRNA, approximately 4gRNA to approximately 6gRNA, or about 5gRNA to about 6gRNA. In some embodiments, the compositions described herein utilize about 1 gRNA, about 2gRNA, about 3gRNA, about 4gRNA, about 5gRNA, or about 6gRNA.

In certain embodiments, RNA (eg, crRNA, tracrRNA, gRNA) can be engineered to include one or more modified nucleobases. For example, known modifications of RNA are described in, for example, Lewis, ed., Genes VI, Chapter 9 (“Interpreting the Genetic Code” (1997, Oxford University Press, New York)) and “RNA Modification and Editing”, Grosjean and Benne (1998, ASM Press, Washington, DC). Modified RNA components include: N4-methylcytidine; N4-2'-O-dimethylcytidine; N4-acetylcytidine; 5-methylcytidine; 5,2'-O-dimethylcytidine; 5 -hydroxymethylcytidine; 5-formylcytidine; 2'-O-methyl-5-formalincytidine; 3-methylcytidine; 2-thiocytidine; lysidine; 2'-O-methyluridine; 2-thiouridine; 2-thio-2 '-O-methyluridine; 3,2'-O-dimethyluridine; 3-(3-amino-3-carboxypropyl)uridine; 4-thiouridine; ribosylthymine; 5,2'-O-dimethyluridine; 5-methyl -2-thiouridine; 5-hydroxyuridine; 5-methoxyuridine; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 5-carboxymethyluridine; 5-methoxycarbonylmethyluridine; 5-methoxycarbonylmethyl-2' -O-methyluridine; 5-methoxycarbonylmethyl-2'-thiouridine; 5-carbamoylmethyluridine; 5-carbamoylmethyl-2'-O-methyluridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxy methyl)uridine methyl ester; 5-aminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-carboxymethylaminomethyluridine ; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; dihydrouridine; dihydroribosylthymine; 2'-methyladenosine; 2-methyladenosine; N6N-methyladenosine; N6,N6-dimethyladenosine; N6,2'-O-trimethyladenosine; 2-methylthio-N6N-isopentenyladenosine; N6-(cis-hydroxyisopentenyl)-adenosine; 2-methylthio-N6-(cis-hydroxyiso N6-glycinylcarbamoyl)adenosine; N6-threonylcarbamoyl adenosine; N6-methyl-N6-threonylcarbamoyl adenosine; 2-methylthio-N6-methyl-N6-threonylcarbamoyl adenosine; N6-hydroxynol Valylcarbamoyl adenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine; 2'-O-ribosyladenosine (phosphoric acid); inosine; 2'O-methylinosine; 1-methylinosine; 1;2'-O-dimethyl Inosine; 2'-O-methylguanosine; 1-methylguanosine; N2-methylguanosine; N2,N2-dimethylguanosine; N2,2'-O-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; 2 '-O-ribosylguanosine (phosphate); 7-methylguanosine; N2; 7-dimethylguanosine; N2; N2; 7-trimethylguanosine; wiosin; methylwosin; insufficiently modified hydroxywibutosin; wibutosin; butocin; peroxywibutocin; cuosin; epoxycuosin; galactosyl-queosin; mannosyl-queosin; 7-cyano-7-deazaguanosine; aracheeosin [also called 7-formamide-7-deazaguanosine]; and 7- Aminomethyl-7-deazaguanosine. Additional modified RNAs can be identified using the methods of this disclosure or other methods in the art.

In some embodiments, the gRNA is a synthetic oligonucleotide. In some embodiments, synthetic nucleotides include modified nucleotides. Modifications of the internucleoside linkers (i.e. backbones) can be used to improve stability and pharmacodynamic properties. For example, internucleoside linker modifications prevent or reduce degradation by cellular nucleases, resulting in increased gRNA pharmacokinetics and bioavailability. Generally, modified internucleoside linkers include any linker other than a phosphodiester (PO) liner that covalently joins two nucleosides. In some embodiments, the modified internucleoside linker increases the nuclease resistance of the gRNA compared to a phosphodiester linker. For naturally occurring oligonucleotides, internucleoside linkers include phosphate groups that form phosphodiester bonds between adjacent nucleosides. In some embodiments, the gRNA includes an internucleoside linker modified from one or more naturally occurring phosphodiesters . In some embodiments, the internucleoside linker of the gRNA or all of its contiguous nucleotide sequence is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage.

Modifications to ribose sugars or nucleobases can also be utilized in the present invention .
Generally, modified nucleosides include the introduction of one or more modifications of the sugar or nucleobase moiety. In some embodiments, the gRNA comprises one or more nucleosides that include a modified sugar moiety, as described, where the modified sugar moiety is similar to the ribose sugar moieties found in deoxyribose nucleic acids (DNA) and RNA. This is the modification of the sugar moiety in comparison. A number of nucleosides are available that are modified on the ribose sugar moiety, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or stability. Such modifications include modifications in which the ribose ring structure is modified. These modifications include a hexose ring (HNA), a bicyclic ring with a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g. locked nucleic acids (LNA)), or typically C2 and C3 Includes substitution with an unbonded ribose ring that lacks a carbon -carbon bond (such as UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids. Modified nucleosides also include nucleosides in which sugar moieties are replaced with non-sugar moieties, for example in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

Sugar modifications also include modifications made by changing substituents on the ribose ring to groups other than hydrogen or groups other than the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents can be introduced, for example, in the 2', 3', 4' or 5' positions. Nucleosides having modified sugar moieties also include 2' modified nucleosides such as 2' substituted nucleosides. Indeed, much passion has gone into the development of 2'-substituted nucleosides, and many 2'-substituted nucleosides have beneficial effects when incorporated into oligonucleotides, such as increased nucleoside tolerance or increased affinity. It has been found that it has the following properties. A 2' sugar-modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2'-substituted nucleoside), or a nucleoside containing a 2'-linked biradicle; 4′ biradicle crosslinking) Contains nucleosides. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'- amino- DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides . As a further example, in some embodiments, the modification at the ribose group includes modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl). selected.

In some embodiments, the gRNA includes one or more modified sugars. In some embodiments, the gRNA contains only modified sugars. In certain embodiments, the gRNA comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar includes a 2'-O-methoxyethyl group. In some embodiments, the gRNA includes both internucleoside linker modifications and nucleoside modifications.

Target specificity can be used with respect to a guide RNA or crRNA that is specific for a target polynucleotide sequence or region (e.g., the ICP0 gene or ICP27 gene of the herpesvirus genome), and a target polynucleotide (e.g., corresponding to the target), For example, it further comprises a sequence of nucleotides that can selectively anneal/ hybridize to a target (sequence or region) of target DNA . In some embodiments, the crRNA or derivative thereof includes a target-specific nucleotide region that is complementary to a region of the target DNA sequence. In some embodiments, the crRNA or derivative thereof comprises other nucleotide sequences than the target-specific nucleotide region. In some embodiments, other nucleotide sequences are derived from tracrRNA sequences.

gRNAs are generally supported by a scaffold, where the scaffold includes sequences that are substantially identical or highly conserved (e.g., do not confer target specificity) across natural species. Refers to a portion of a gRNA or crRNA molecule. The scaffold includes the tracrRNA segment and any portion of the crRNA segment other than the polynucleotide targeting guide sequence at or near the 5' end of the crRNA segment, including any unconserved sequences in native crRNA and tracrRNA. Natural parts are excluded. In some embodiments, the crRNA or tracrRNA includes a modified sequence. In certain embodiments, the crRNA or tracrRNA comprises at least 1, 2, 3, 4, 5, 10, or 15 modified bases (eg, a modified natural base sequence).

Complementary, as used herein, generally refers to a polynucleotide that includes a nucleotide sequence that is capable of selectively annealing to an identification region of a target polynucleotide under certain conditions. As used herein, the term "substantially complementary" and grammatical equivalents refers to a polynucleotide comprising a nucleotide sequence that is capable of annealing specifically to an identification region of a target polynucleotide under certain conditions. intended to mean. Annealing refers to nucleotide base-pairing interactions of one nucleic acid with another that result in the formation of duplexes, triplexes, or other higher order structures. Primary interactions are usually nucleotide base specific (A:T, A:U, G:C, etc.) through Watson-Crick and Hoogsteen type hydrogen bonds. In some embodiments, base stacking and hydrophobic interactions may also contribute to duplex stability. Conditions for annealing a polynucleotide to a complementary or substantially complementary region of a target nucleic acid are described, for example, in Nucleic Acid Hybridization, A Practical Approach , edited by Hames and Higgins, IRL Press, Washington, DC. C. (1985) and Wetmur and Davidson, Molecular Biology 31:349 (1968). Annealing conditions will depend on the particular application and can be determined routinely by one of ordinary skill in the art without undue experimentation. Hybridization generally refers to the process by which two single-stranded polynucleotides are non-covalently linked to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a "hybrid" or "duplex." In certain examples, hybridization does not require 100% sequence identity; in certain embodiments, hybridization comprises about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity. Occurs with sequence identity exceeding. In certain embodiments, sequence identity includes sequences containing insertions and/or deletions in addition to non-identical nucleobases.

Nucleic acids of the present disclosure, including RNA (eg, crRNA, tracrRNA, gRNA) or nucleic acids encoding RNA, can be produced by standard techniques. For example, polymerase chain reaction (PCR) technology can be used to obtain isolated nucleic acids comprising nucleotide sequences described herein, including nucleotide sequences encoding polypeptides described herein. PCR can be used to amplify specific sequences from DNA and RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, 2nd Edition, edited by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 2003. It is used to design oligonucleotide primers that are identical or similar in sequence to the opposite strand of the template to be amplified. A variety of PCR strategies are also available that can introduce site-specific nucleotide sequence modifications to template nucleic acids.

Isolated nucleic acids can also be chemically synthesized as a single nucleic acid (eg, using automated 3' to 5' DNA synthesis using phosphoramidite technology) or as a series of oligonucleotides. Isolated nucleic acids of the present disclosure can also be obtained, for example, by mutagenesis of DNA encoding a naturally occurring partial crRNA, tracrRNA, RNA, or DNA encoding Cas9.

In certain embodiments, isolated RNA is synthesized from expression vectors encoding RNA molecules, as described in detail elsewhere herein.

(Nucleic acids and vectors )
In some embodiments, compositions of the present disclosure include isolated nucleic acids encoding one or more elements of the CRISPR-Cas system described herein. For example, in some embodiments, the composition includes an isolated nucleic acid encoding at least one guide nucleic acid (eg, gRNA). In some embodiments, the composition comprises an isolated nucleic acid encoding a Cas peptide, or a functional fragment or derivative thereof. In some embodiments, the composition comprises an isolated nucleic acid encoding at least one guide nucleic acid (eg, gRNA) and encoding a Cas peptide, or a functional fragment or derivative thereof. In some embodiments, the composition comprises an isolated nucleic acid encoding at least one guide nucleic acid (eg, gRNA) and further comprises an isolated nucleic acid encoding a Cas peptide, or a functional fragment or derivative thereof.

In some embodiments, the composition comprises at least one isolated nucleic acid encoding a gRNA, as described elsewhere herein, wherein the gRNA is substantially linked to a target sequence of the HSV genome. are complementary. In some embodiments, the composition comprises at least one isolated nucleic acid encoding a gRNA, wherein the gRNA is at least 75%, 80%, 85%, 90% more sensitive to a target sequence described herein. %, 95%, 96%, 97%, 98% or 99% sequence homology .

In some embodiments, the composition comprises at least one isolated nucleic acid encoding a Cas peptide described elsewhere herein, or a functional fragment or derivative thereof. In some embodiments, the composition has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, at least one isolated nucleic acid encoding a Cas peptide with 98%, or 99% amino acid sequence homology .

Isolated nucleic acids can include any type of nucleic acid including, but not limited to, DNA and RNA. For example, in some embodiments, the composition comprises an isolated DNA, including, for example, an isolated cDNA encoding a gRNA of the present disclosure or a peptide of the present disclosure , or a functional fragment thereof . In some embodiments, the composition comprises isolated RNA encoding a peptide of the present disclosure, or a functional fragment thereof . Isolated nucleic acids can be synthesized using any method known in the art.

The present disclosure can include the use of vectors into which the isolated nucleic acids described herein are inserted. The art is replete with suitable vectors useful in this disclosure. Vectors include, for example, viral vectors (such as adenovirus (“Ad”), adeno-associated virus (AAV), vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes , and Other macromolecular complexes capable of mediating the delivery of polynucleotides are included. Vectors can also contain other components or functions that further modulate gene delivery and/or gene expression or provide beneficial properties to the target cells. Such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell-type or tissue-specific binding) ; affect uptake of vector nucleic acids by cells; Components that affect the localization of the polynucleotide within the cell after uptake (such as agents that mediate nuclear localization) ; and components that affect the expression of the polynucleotide . Such components may also include markers, such as detectable and/or selectable markers, that can be used to detect or select cells that have taken up and expressed the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors that have components or functions that mediate binding and uptake), or the vector can be modified to provide such functions. It can also be provided. Other vectors include those described in Chen et al ., BioTechniques, 34: 167-171 (2003); A wide variety of such vectors are known in the art and are publicly available.

Briefly summarized, expression of a natural or synthetic nucleic acid encoding an RNA and/or a peptide typically involves operably linking the RNA and/or the nucleic acid encoding the peptide, or a portion thereof, to a promoter. This is accomplished by incorporating the construct into an expression vector . The vectors used are suitable for replication and optionally integration in eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

Vectors of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Pat. In another embodiment, the present disclosure provides gene therapy vectors.

Isolated nucleic acids of the present disclosure can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Additionally, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. ing. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, a suitable vector will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01/96584, WO01/29058, and Patent No. 6,326,193) .

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject 's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

For example, vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. It also has the added advantage of being less immunogenic. In some embodiments, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for the treatment of various diseases. AAV vectors possess many characteristics that make them ideally suited for gene therapy, including lack of virulence, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. By selecting the appropriate combination of AAV serotype, promoter, and delivery method, expression of particular genes contained within the AAV vector can be specifically targeted to one or more cell types.

Further provided are nucleic acids encoding the CRISPR-Cas systems described herein. Provided herein are adeno-associated virus (AAV) vectors that include nucleic acids encoding the CRISPR-Cas systems described herein. In certain examples, AAV vectors include or are derived from components of AAV and can be used in any of a number of tissue types, such as brain, heart, lung, skeletal muscle , liver, kidney, spleen, or pancreas. Any vector suitable for infecting mammalian cells (including human cells) in vitro or in vivo is included. In certain examples, an AAV vector comprises an AAV-type viral particle (or virion) that includes a nucleic acid encoding a protein of interest (eg, the CRISPR-Cas system described herein). In some embodiments, as further described herein, the AAVs disclosed herein are derived from various serotypes, including combinations of serotypes (e.g., " pseudotyped " AAVs). or from different genomes (eg, single-stranded or self-complementary). In some embodiments, the AAV vector is a human serotype AAV vector. In such embodiments, the human serotype AAV is derived from any known serotype, eg, AAV1, AAV2, AAV4, AAV6, or AAV9. In some embodiments, the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8.

In some embodiments, the composition includes a vector derived from an adeno-associated virus (AAV). AAV vectors possess many characteristics that make them ideally suited for gene therapy, including lack of virulence, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. By selecting the appropriate combination of AAV serotype, promoter, and delivery method, expression of particular genes contained within the AAV vector can be specifically targeted to one or more cell types.
Although a variety of different AAV capsids have been described and can be used, AAVs that preferentially target the liver and/or deliver genes with high efficiency are particularly desirable. The sequence of AAV8 is available from various databases. Although the examples utilize AAV vectors with the same capsid, the capsid of the gene editing vector and the capsid of the AAV targeting vector are the same AAV capsid. Another suitable AAV is, for example, rhl0 (see WO2003/042397). Still other AAV sources include, for example, AAV9 (see, e.g., US Pat. No. 7,906,111; US Patent Application Publication No. 2011-0236353-A1), and/or hu37 (e.g., , 906,111; US Patent Application Publication No. 2011-0236353-A1) , AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8 . (U.S. Patent No. 7,790,449; U.S. Patent No. 7,282,199, WO2003/042397; WO2005/033321, WO2006/110689; U.S. Patent No. 7,790,449; U.S. Patent No. 7,282, No. 199; see U.S. Pat. No. 7,588,772). Additionally, other AAVs can optionally be selected taking into account the tissue preference of the selected AAV capsid.

In some embodiments, the AAV vectors disclosed herein include a nucleic acid encoding the CRISPR-Cas system described herein. In some embodiments, the nucleic acid also includes sequences encoding promoters, enhancers, polyadenylation signals, internal ribosome entry sites ("IRES"), protein transduction domains ("PTD"), etc. , for the expression of a protein of interest. , and in some embodiments one or more regulatory sequences that enable secretion. Thus, in some embodiments, the nucleic acid includes a promoter region operably linked to the coding sequence to cause or improve expression of the protein of interest in infected cells. Such promoters can be ubiquitous, cell- or tissue-specific, strong, weak, regulated, chimeric, etc., for example, to allow efficient and stable production of the protein in infected tissues. In certain embodiments, the promoter is homologous or heterologous to the encoded protein, but generally the promoters used in the disclosed methods are functional in human cells. Examples of regulated promoters include, but are not limited to, Teton on/off element- containing promoters, rapamycin-inducible promoters, tamoxifen-inducible promoters, and metallothionein promoters. In certain embodiments, other promoters used include promoters specific for tissues such as kidney, spleen, and pancreas.
Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, RSV promoter, SV40 promoter, etc., and cellular promoters such as the phosphoglycerate kinase (PGK) promoter and the β-actin promoter.

In some embodiments, a recombinant AAV vector comprises a nucleic acid packaged within an AAV capsid, generally comprising a 5'AAV ITR, an expression cassette described herein, and a 3'AAV ITR. As described herein, in some embodiments, the expression cassettes include regulatory elements for an open reading frame within each expression cassette, and the nucleic acid optionally includes additional regulatory elements . In some embodiments, the AAV vector comprises a full-length AAV 5' inverted repeat (ITR) and a full-length 3' ITR. An improved version of the 5'ITR, termed short AITR, has been described in which the D sequence and the terminal resolution site (trs) have been deleted. The abbreviation "sc" means self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by a recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of a second strand, the two complementary halves of the scAAV associate to form one double-stranded DNA (dsDNA) unit that is ready for replication and transcription. (For example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independent of DNA synthesis," Gene Therapy, (August 2001); See also U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683). When pseudotyped AAV is generated, the ITRs are selected from a different source than the capsid's AAV source. For example, in some embodiments, AAV2 ITRs are selected for use with AAV capsids that have particular efficiencies against selected cellular receptors, target tissues, or viral targets. In some embodiments, the ITR sequence from AAV2, or a deleted version thereof (AITR), is used for convenience and to facilitate regulatory approval (i.e., pseudotyped). In some embodiments, single-stranded AAV viral vectors are used.

Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art (e.g., U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199). No.: WO2003/042397; WO2005/033321 , WO2006 /110689, and U.S. Patent No. 7,588,772 B2, U.S. Patent No. 5,139,941, 5,741,683, 6,057 , No. 152, No. 6,204,059, No. 6,268,213, No. 6,491,907, No. 6,660,514, No. 6,660,514, No. 6,951,753; 7,094, No. 604; No. 7,172,893; No. 7,201,898; No. 7,229,823; and No. 7,439,065). In one system, a producer cell line is transiently transfected with constructs encoding the transgene flanking the ITR and constructs encoding rep and cap. In the second system, a packaging cell line stably supplying rep and cap is transfected (transiently or stably) with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenoviruses or herpesviruses, requiring the separation of rAAV from contaminating virus. More recently, AAV, i.e., adenoviruses E1, E2a, VA, and E4, or herpesviruses UL5, UL8, UL52, and UL29, and herpesvirus polymerases, to helper viruses to restore the necessary helper functions. A system has been developed that does not require infection. ) is also supplied by the system in a transformer. In these new systems, helper functions can be supplied either by transiently transfecting cells with constructs encoding the desired helper functions or by engineering cells to stably contain genes encoding helper functions. and its expression can be regulated at the transcriptional or post-transcriptional level . In yet another system, a transgene flanked by ITR and rep/cap genes is introduced into insect cells by infection with a baculovirus-based vector.

The CRISPR-Cas system, e.g. Cas9, and/or any subject RNA, e.g. guide RNA, is delivered using adeno-associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof. be able to. Cas9 and one or more guide RNAs can be packaged into one or more viral vectors. In some embodiments, the viral vector is delivered to the tissue of interest by, for example, intramuscular injection; in other cases, viral delivery is intravenous, transdermal, intranasal, oral, mucosal, or via other delivery methods. Such delivery can occur via either a single dose or multiple doses. Those skilled in the art will appreciate that the actual dose delivered herein will depend on the vector selected, the target cell, organism, or tissue, the general condition of the subject being treated, the general condition of the subject being treated , the transformation/modification sought. It is understood that this can vary greatly depending on a variety of factors , such as the extent of the disease, route of administration, method of administration, and type of transformation/modification desired .

Poxvirus vectors introduce genes into the cytoplasm of cells. Avipox viral vectors express nucleic acids only for short periods of time. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors may be suitable for some embodiments. Adenoviral vectors provide shorter term expression (eg, less than about a month) than adeno-associated viruses, but in some embodiments may exhibit much longer expression. The particular vector chosen will depend on the target cell and the condition being treated.

In certain embodiments, the vector also binds to the transgene in a manner that allows its transcription, translation, and/or expression in cells transfected with the plasmid vector or infected with viruses produced according to the present disclosure. It includes a conventional control element operably connected thereto. As used herein, "operably linked" sequences include expression control sequences that flank the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Both are included. Expression control sequences include appropriate transcription initiation, transcription termination, promoter, and enhancer sequences ; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals ; sequences that stabilize cytoplasmic mRNA ; and sequences that increase translation efficiency. (i.e., the Kozak consensus sequence) ; sequences that enhance protein stability ; and, optionally, sequences that enhance secretion of the encoded product . A large number of expression control sequences are known in the art and available, including natural, constitutive, inducible and/or tissue-specific promoters.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Usually these are located in a region 30-110 bp upstream of the start site, but it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained even when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between spa promoter elements can be increased to 50 bp before activity begins to decrease. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

Selection of an appropriate promoter is easily accomplished. In certain embodiments, high expression promoters will be used. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked to it. Rous sarcoma virus (RSV) and MMT promoters can also be used. Certain proteins can be expressed using native promoters. Other elements that can enhance expression can also be included, such as enhancers and systems that provide high levels of expression, such as the tat gene or tar element . This cassette can then be inserted into a vector, such as a plasmid vector such as pUC19, pUC118, pBR322, or other known plasmid vectors containing, for example, an E. coli origin of replication.

Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, examples include, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre- Other constitutive promoter sequences may also be used , including the early promoter, the Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, creatinine kinase promoter . Furthermore, this disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Enhancer sequences found on vectors also regulate the expression of genes contained in the vector. Enhancers normally bind protein factors to enhance transcription of genes. An enhancer may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in particular cells or tissue types. In some embodiments, vectors of the present disclosure include one or more enhancers to promote transcription of genes present within the vector.

To assess the expression of nucleic acids and/or peptides, expression vectors are introduced into cells to facilitate the identification and selection of expressing cells from the population of cells that are to be transfected or infected via the viral vector. may also include a selectable marker gene or reporter gene or both. In other embodiments, the selectable marker can be kept on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes such as neo.

Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present or expressed in the recipient organism or tissue, and whose expression is manifested by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at appropriate time points after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79 -82). Suitable expression systems are well known, may be prepared using known techniques, or may be commercially available. Generally, the construct with the smallest 5' flanking region that shows the highest level of expression of the reporter gene is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vector can be readily introduced into a host cell, eg, a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells containing vectors and/or exogenous nucleic acids are well known in the art. (See, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, eg human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, eg, US Pat. No. 5,611,112 , US Pat. No. 5,350,674 and US Pat. No. 5,585,362.

Chemical means for introducing polynucleotides into host cells include polymeric complexes, colloidal dispersions such as nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. Includes lipid-based systems that include. An exemplary colloid system used as a delivery vehicle in vitro and in vivo is liposomes (eg, artificial membrane vesicles).

If a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-bound nucleic acid is encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule that binds both the liposome and the oligonucleotide, and becomes entrapped in the liposome. form a complex with liposomes, be dispersed in a solution containing lipids, be mixed with lipids, be bound to lipids, be contained in suspension in lipids, be contained in micelles or form complexes, or otherwise It can bind to lipids in the following manner. Compositions associated with lipids, lipids/DNA, or lipids/expression vectors are not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles, or in "collapsed" structures. Moreover, they are simply dispersed in the solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets within the cytoplasm, as well as a class of compounds that include long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Missouri. Dicetyl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY). Cholesterol (“Choi”) is available from Calbiochem-Behring. Dimyristylphosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. Available from (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vehicles formed by the production of encapsulated lipid bilayers or aggregates. Liposomes are characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement before forming closed structures, trapping water and dissolved solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have a structure different from the normal vesicular structure in solution are also included. For example, lipids may have a micellar structure or simply exist as a heterogeneous collection of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

Regardless of the method used to introduce exogenous nucleic acid into a host cell, a variety of assays can be performed to confirm the presence of the recombinant nucleic acid sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR ; for example , by immunological means (ELISA and Western blots), or Assays described herein for identifying agents falling within the scope of this disclosure include "biochemical" assays, such as detecting the presence or absence of particular peptides.

In certain embodiments, the composition comprises cells genetically modified to express one or more isolated nucleic acids and/or peptides described herein. For example, cells can be transfected or transformed with one or more vectors containing isolated nucleic acid sequences encoding gRNA and/or Cas peptides. The cells may be those of the subject, or cells or cell lines with matching haplotypes. Cells can be irradiated to prevent replication. In some embodiments, the cells are human leukocyte antigen (HLA) matched cell lines, autologous cell lines, or a combination thereof. In other embodiments, the cells may be stem cells. Examples include embryonic stem cells and induced pluripotent stem cells (induced pluripotent stem cells (iPS cells)). Embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) have been established from many animal species including humans. These types of pluripotent stem cells are the most suitable for regenerative medicine, as they can differentiate into almost any organ with appropriate differentiation induction, while retaining the ability to actively divide while maintaining pluripotency. It is considered a useful cell source . Some iPS cells are less likely to pose ethical and social problems, especially since they can be established from autologous somatic cells, compared to ES cells, which are created by destroying embryos. Furthermore, since iPS cells are autologous cells, it is possible to avoid rejection, which is the biggest obstacle in regenerative medicine and transplant therapy.

(Pharmaceutical composition)
The compositions described herein are suitable for use in the various drug delivery systems described above. Additionally, to prolong the in vivo serum half-life of an administered compound, the composition may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or otherwise conventionally prolonging serum half-life. technology can be used. Various methods are available for preparing liposomes, as described, for example, in US Pat. No. 5,611,008 to Szoka et al. Each of these is incorporated herein by reference. Additionally, drugs can be administered in targeted drug delivery systems, such as liposomes coated with tissue-specific antibodies. Liposomes are selectively targeted to organs for uptake.

The present disclosure also provides pharmaceutical compositions comprising one or more of the compositions described herein. The formulations can be used in admixture with conventional excipients, ie, pharmaceutically acceptable organic or inorganic carrier substances suitable for administration to the wound or treatment site. The pharmaceutical compositions can be sterilized and, if desired, contain adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic buffering, colorants, and/or It may also be mixed with aromatic substances. They may also be combined with other active agents, such as other analgesics, if desired.

Administration of the compositions of the present disclosure can be carried out, for example, by parenteral, intravenous, intratumoral, subcutaneous, intramuscular, or intraperitoneal injection, or by infusion, or by any other acceptable systemic method. . Formulations for administration of compositions include formulations suitable for rectal, nasal, oral, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. Contains things. The formulations may conveniently be presented in unit dosage form, e.g. They include tablets and sustained release capsules, and can be prepared by any method well known in the art of pharmacy.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following : surfactants; dispersants; inert diluents. Granulating and disintegrating agents; Binders; Lubricants; Coloring agents; Preservatives; Physiologically degradable compositions such as gelatin; Aqueous vehicles and solvents; Oil vehicles and solvents; Suspending agents; Dispersing or wetting agents; Emulsifying agents; demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present disclosure are known in the art, and are described, for example, in Genaro, ed., (1985, Remingtons Pharmaceutical Sciences, Mack Publishing Co., Easton, PA). , which is incorporated herein by reference.

Compositions of the present disclosure may include a preservative from about 0.005% to 2.0% of the total weight of the composition. Preservatives are used to prevent spoilage when exposed to contaminants in the environment. Examples of preservatives useful in accordance with this disclosure include, but are not limited to, preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

In one embodiment, the composition includes an antioxidant and a chelating agent that inhibits the degradation of one or more components of the composition. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol, and ascorbic acid, with a preferred range of about 0.01% to 0.3% by weight, based on the total weight of the composition. is BHT in the range of 0.03% to 0.1% by weight. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Particularly preferred chelating agents include edetate salt in the range of about 0.01% to 0.20%, more preferably 0.02% to 0.10% by weight, based on the total weight of the composition. (eg disodium edetate) and citric acid. Chelating agents are useful to chelate metal ions in the composition that can adversely affect the shelf life of the formulation. BHT and edetate disodium, respectively, are particularly preferred antioxidants and chelating agents for some compounds, but other suitable and equivalent antioxidants and chelating agents may be substituted, as is known to those skilled in the art. You may.

Liquid suspensions can be prepared using conventional methods of suspending compositions of the disclosure in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions contain one or more additives including, but not limited to, suspending, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffering agents, salts, flavoring, coloring, and sweetening agents. Additional ingredients may also be included. The oily suspensions may further contain a thickening agent. Known suspending agents include sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Not limited. Known dispersants or wetting agents include natural phospholipids such as lecithin, condensation products of alkylene oxides with fatty acids, long-chain aliphatic alcohols, partial esters derived from fatty acids, and hexitols, or fatty acids and hexitol anhydrides. (e.g., polyoxyethylene stearate, heptadecaethylene oxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoate, ascorbic acid, and sorbic acid.

(Method of treatment)
The present disclosure provides methods of treating or preventing herpesvirus-mediated infections. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a composition comprising a guide nucleic acid and at least one of a Cas peptide, or a functional fragment or derivative thereof. . In some embodiments, the method includes administering a composition comprising a guide nucleic acid and an isolated nucleic acid encoding at least one of a Cas peptide, or a functional fragment or derivative thereof. In certain embodiments, the method comprises administering the compositions described herein to a subject who has been diagnosed with a herpesvirus infection , is at risk of developing a herpesvirus infection , has a latent herpesvirus infection, etc. Including administering something.

In certain embodiments herein, the CRISPR-Cas systems or compositions described herein are used to modify and/or edit herpesvirus sequences within the genome of a cell (e.g., a host cell). A method is provided. Generally, modifying and/or editing herpesvirus sequences within the genome of a cell (e.g., a host cell) involves CRISPR-Cas targeting one or more regions within the UL56, ICP0, ICP4, or ICP27 genes . contacting or providing the system or composition to the cell . In some embodiments, the method includes removing or excising the sequence from the genome of the cell. In some embodiments, the method comprises detecting at least or about 100 , 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 of the HSV genome. , 9000, or 9000 or more base pairs are excised.

Provided herein, in certain embodiments, are methods comprising administering a composition comprising : a) a CRISPR-associated endonuclease or a nucleic acid sequence encoding a CRISPR-associated endonuclease; b) a first guide. a nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, the first guide nucleic acid being complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; c) a first guide nucleic acid; 2, or a nucleic acid sequence encoding a second guide nucleic acid, the second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. In some embodiments, the method further comprises administering a fourth guide nucleic acid, or a nucleic acid sequence encoding a fourth guide nucleic acid, wherein the fourth guide nucleic acid is within or within the ICP27 gene of the herpesvirus genome. It is complementary to a fourth target nucleic acid sequence in its vicinity. In some embodiments, the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence.

Provided herein, in certain embodiments, are methods comprising administering a composition comprising : a) a CRISPR-associated endonuclease or a nucleic acid sequence encoding a CRISPR-associated endonuclease; b) a first guide. a nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, the first guide nucleic acid being complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; c) a first guide nucleic acid; or a nucleic acid sequence encoding a second guide nucleic acid, the second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

Provided herein, in certain embodiments, are methods comprising administering a CRISPR-Cas system comprising : a) a CRISPR-associated endonuclease b) a first guide nucleic acid comprising SEQ ID NO. a first guide nucleic acid comprising a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to or 7 or its complement ; c) a second guide nucleic acid, the second guide nucleic acid comprising: comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376 or 377 or its complement . In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO:7 . In some embodiments, the second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO: 376. In some embodiments, the second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO: 377. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. A nucleic acid sequence having at least 90% sequence identity to. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. includes a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to. In some embodiments, the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. comprises a nucleic acid sequence that is complementary to a sequence with at least 90% sequence identity.

Provided herein, in certain embodiments, are methods comprising administering an adeno-associated virus (AAV) vector comprising a nucleic acid encoding : a) a CRISPR-associated endonuclease ; b) an ICP0 of a herpesvirus genome. a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the gene; c) a second guide nucleic acid. The second guide nucleic acid is complementary to a second target nucleic acid sequence within or near the ICPO gene of the herpesvirus genome. d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. In some embodiments, the method further comprises administering a fourth guide nucleic acid that is complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome.

A particular em embodiment, defined herein, is a method comprising administering an adeno-associated virus (AAV) vector comprising a nucleic acid encoding : a) a CRISPR-associated endonuclease ; b) a herpesvirus genome. a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the ICP0 gene; c) a second guide nucleic acid. The second guide nucleic acid is complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. d) a third guide nucleic acid complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; have different target nucleic acid sequences.

In some embodiments, the first target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. sequences that contain at least about 90% sequence identity. In some embodiments, the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. include. In some embodiments, the second target nucleic acid sequence is for any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. Sequences containing at least about 90% sequence identity. In some embodiments, the second target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. include. In some embodiments, the third target nucleic acid sequence is any one of SEQ ID NO: 363 , 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; Sequences that contain at least about 90% sequence identity to . In some embodiments, the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. body, including. In some embodiments, the fourth target nucleic acid sequence is for any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of 363, 371, or 374-377. Sequences containing at least about 90% sequence identity . In some embodiments, the fourth target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377. Including the body. In some embodiments, the first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third The target of the nucleic acid sequence comprises the sequence according to SEQ ID NO: 378 or the complement thereof. In some embodiments, the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or its complement, the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 7 or its complement, and the third The target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or its complement, and the fourth target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377 or its complement.

In some embodiments, the method uses herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3; varicella zoster virus (VZV) ) , human herpes virus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)).

In some embodiments, the methods of the present disclosure can be used to treat diseases including, but not limited to, herpes labialis, genital herpesvirus, herpesviral encephalitis, chickenpox, herpes zoster, Bell's palsy, vestibular neuritis, and herpes zoster neuralgia . , used in the treatment or prevention of diseases and disorders associated with herpesvirus infections.

Subjects to which the pharmaceutical compositions of the present disclosure are contemplated include humans and other primates, non-human primates, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats , and dogs . Includes, but is not limited to, mammals. Therapeutic agents can be administered according to a metronomic regimen. As used herein, "metronomic" therapy refers to the continuous administration of low doses of a therapeutic agent.

The compositions can be administered in conjunction with (eg, before, simultaneously with, or after) one or more treatments. For example, in certain embodiments, the method uses the presently disclosed drugs in combination with additional antiherpesviral therapies, including, but not limited to, TK inhibitors, UL30 inhibitors, acyclovir, foscamet, cidofovir, and derivatives thereof. administering the composition.

Dosage, toxicity and therapeutic efficacy of the compositions of the invention are determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g. effective dose). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Cas9/gRNA compositions that exhibit high therapeutic indices are preferred. Although Cas9/gRNA compositions that exhibit toxic side effects may be used, the design of delivery systems that target such compositions to diseased tissues is hampered by the potential for damage to non-diseased cells. Care must be taken to minimize toxicity and thereby reduce side effects .

The data obtained from cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage of such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any composition used in the methods of this disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount (ie, effective dose) of a composition means an amount sufficient to produce a therapeutically (eg, clinically) desired result. The compositions can be administered from one or more times per day to one or more times per week, including every other day . Those skilled in the art will appreciate that certain factors will effectively treat a subject , including, but not limited to, the severity of the disease or disorder, previous treatments, the subject 's general health and/or age, and other factors. It will be appreciated that this may affect the dosage and timing required.

Additionally, treatment of a subject with a therapeutically effective amount of a composition of the present disclosure can include a single treatment or a series of treatments. gRNA expression cassettes can be delivered to a subject by methods known in the art. In some embodiments, Cas may be a fragment that includes the active domain of the Cas molecule, thereby reducing the size of the molecule. Therefore, Cas/gRNA molecules can be used clinically, similar to the approaches taken in current gene therapy.

In some embodiments, the method includes genetically modifying the cell to express the guide nucleic acid and/or the Cas peptide. For example, in some embodiments, the method includes contacting the cell with a guide nucleic acid and/or an isolated nucleic acid encoding a Cas peptide.

In some embodiments, for viral vector-mediated delivery, the dose comprises at least 1×10 ⁵ particles to about 1×10 ¹⁵ particles. In some embodiments, delivery is via an adenovirus, such as a single dose comprising at least 1 x ¹⁰ particles (also referred to as particle units, pu) of an adenoviral vector. In some embodiments, the dose comprises at least about 1×10 ⁶ particles (e.g., about 1×10 ⁶ to 1×10 ¹² particles), at least about 1×10 ⁷ particles, at least about 1×10 ⁸ particles (e.g., , about 1×10 ⁸ to 1× ^{10 11} particles or about 1×10 ¹² particles ), at least about 1×10 ⁹ particles (e.g., about 1×10 ⁹ to 1×10 ¹⁰ particles or about 1×10 ⁹ to 1×10 ¹² particles), or at least about 1×10 ¹⁰ particles (eg, about 1×10 ¹⁰ to 1×10 ¹² particles) . Alternatively, the dosage may include no more than about ^1x10 particles, no more than about ^1x10 particles, no more than about 1x10 particles, and no more than about ^1x10 particles, and no more than about ^1x10 particles, and no more than about 1x10 particles, and no more than about 1x10 particles. Contains no more than ¹⁰ particles (eg, greater than about 1 x 10 ⁹ particles). Thus, in some embodiments, the dose is, e.g.
about 1×10 ⁶ particle units (pu), about 2×10 ⁶ pu, about 4×10 ⁶ pu,
Approximately 1×10 ⁷ pu, approximately 2×10 ⁷ pu, approximately 4×10 ⁷ pu,
Approximately 1×10 ⁸ pu, approximately 2×10 ⁸ pu, approximately 4×10 ⁸ pu,
Approximately 1×10 ⁹ pu, approximately 2×10 ⁹ pu, approximately 4×10 ⁹ pu,
Approximately 1 × 10 ¹⁰ pu, approximately 2 × 10 ¹⁰ pu, approximately 4 × 10 ¹⁰ pu,
Approximately 1×10 ¹¹ pu, approximately 2×10 ¹¹ pu, approximately 4×10 ¹¹ pu ,
About 1×10 ¹² pu, about 2×10 ¹² pu, or about 4×10 ¹² pu of an adenoviral vector. In some embodiments, the adenovirus is delivered in multiple doses.

In some embodiments, delivery is via an AAV. A therapeutically effective dose for in vivo delivery of AAV to humans is within the range of about 20 to about 50 ml of saline containing about 1×10 ¹⁰ to about 1×10 ¹⁰ functional AAV/ml solution. It is thought that. Dosage can be adjusted to balance therapeutic effect against side effects. In some embodiments, the AAV dose generally includes about 1×10 ⁵ to 1×10 ⁵⁰ genome AAV, about 1×10 ⁸ to 1×10 ²⁰ genome AAV, about 1×10 ¹⁰ to about 1×10 ¹⁶ genomic AAV , or about 1×10 ¹¹ to about 1×10 ¹⁶ genomic AAV. In some embodiments, the human dose is about 1×10 ¹³ genomes AAV. In some embodiments, such concentrations are delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. Other effective doses can be readily established by those skilled in the art through routine experimentation to establish dose-response curves . (See, eg, US Pat. No. 8,404,658).

In some embodiments, the cells are genetically modified in vivo in the subject for whom treatment is intended. In certain embodiments, for in vivo delivery, the nucleic acid is injected directly into the subject . For example, in some embodiments, the nucleic acid is delivered to the site where the composition is needed. In vivo nucleic acid transfer techniques include transfection with viral vectors such as adenovirus, herpes simplex virus I virus, and adeno-associated virus, lipid-based systems (useful lipids for lipid-mediated gene transfer include DOTMA, DOPE, and DC-Chol). etc. ), naked DNA, and transposon-based expression systems . For an exemplary gene therapy protocol, see Anderson et al., Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein. In certain embodiments, the method involves administering RNA, eg, mRNA, directly to the subject (see, eg, Zangi et al., 2013 Nature Biotechnology, 31:898-907).

For ex vivo therapy, isolated cells are modified in an ex vivo or in vitro environment. In some embodiments, the cells are autologous to the subject for whom treatment is intended. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the subject . The modified cells can then be administered directly to a subject .

Those skilled in the art will recognize that different delivery methods may be utilized to administer isolated nucleic acids to cells. For example, (1) methods that utilize physical means such as electroporation (electricity), gene guns (physical force), and large amounts of liquid (pressure); (2) A method in which the nucleic acid or vector forms a complex with another entity such as a liposome, an aggregation protein, or a transporter molecule.

The amount of vector added per cell is likely to vary depending on the length and stability of the therapeutic gene inserted into the vector, as well as the nature of the sequence, a parameter that particularly needs to be determined empirically. It may also be modified by factors not specific to the disclosed methods (eg, costs associated with synthesis). Those skilled in the art can easily make necessary adjustments depending on the exigencies of the particular situation.

Genetically modified cells may also contain suicide genes, genes that encode products that can be used to destroy the cell. In many gene therapy situations, it is desirable not only to be able to express genes within host cells for therapeutic purposes, but also to be able to destroy host cells at will. The therapeutic agent can bind to a suicide gene whose expression is not activated in the absence of the activator compound. If killing a cell into which both a drug and a suicide gene have been introduced is desired, an activator compound is administered to the cell, thereby activating expression of the suicide gene and causing the cell to die. Examples of suicide gene/prodrug combinations that can be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir ; oxidoreductase and cycloheximide ; cytosine deaminase and 5-fluorocytosine ; thymidine kinase, thymidylate kinase (Tdk); ::Tmk) and AZT ; deoxycytidine kinase and cytosine arabinoside.

The present invention will be explained in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, this disclosure should in no way be construed as limited to the following examples, but rather to encompass any variations that become apparent as a result of the teachings provided herein. be.

(Example)
Embodiment 1 includes a composition comprising: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease or a nucleic acid sequence encoding a CRISPR-associated endonuclease; 1, or a nucleic acid sequence encoding a first guide nucleic acid, the first guide nucleic acid being complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; c) a second guide nucleic acid, or a nucleic acid sequence encoding a second guide nucleic acid, wherein the second guide nucleic acid is complementary to a second target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is a third target nucleic acid within or near the ICP27 gene of the herpesvirus genome; complementary to the sequences, wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different.

Embodiment 2 includes the composition of Embodiment 1 and further includes a fourth guide nucleic acid. The fourth guide nucleic acid comprises a fourth guide nucleic acid, or a nucleic acid sequence encoding a fourth guide nucleic acid, that is complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. . Embodiment 3 is the composition of any one of embodiments 1-2, wherein the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. including. Embodiment 4 includes the composition of any one of Embodiments 1-3, wherein the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. Embodiment 5 includes the composition of any one of embodiments 1-4, wherein the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasQ endonuclease. Embodiment 6 includes the composition of any one of embodiments 1-5, wherein the CRISPR-associated endonuclease is Cas9 nuclease. Embodiment 7 includes the composition of any one of embodiments 1-6, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Embodiment 8 includes the composition of any one of embodiments 1-7, wherein the CRISPR-associated endonuclease is optimized for expression in human cells. Embodiment 9 includes the composition of any one of Embodiments 1-8, wherein the guide nucleic acid is RNA. Embodiment 10 includes the composition of any one of embodiments 1-9, wherein the guide nucleic acid comprises crRNA and tracrRNA.

Embodiment 11 includes the composition of any one of Embodiments 1-10, wherein the first target nucleic acid sequence is any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs: 1-96 or 372-375 . Embodiment 12 comprises the composition of any one of Embodiments 1-11, wherein the first target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96, 372-375, or SEQ ID NOs: 1-11. 96, 372-375. Embodiment 13 includes the composition of any one of Embodiments 1-12, wherein the second target nucleic acid sequence is any one of SEQ ID NOs: 1-96, 372-375, or SEQ ID NOs: 1-96. or the complement of any one of 372-375 . Embodiment 14 comprises the composition of any one of Embodiments 1-13, wherein the second target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs: 1-13. 96 or the complement of any one of 372-375. Embodiment 15 includes the composition of any one of Embodiments 1-14, wherein the third target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363, 371, or the complement of any one of 374-377 .

Embodiment 16 includes the composition of any one of embodiments 1-15, wherein the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377. Embodiment 17 includes the composition of any one of Embodiments 1-16, wherein the fourth target nucleic acid sequence is at least about 90% % sequence identity, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377. Embodiment 18 comprises the composition of any one of embodiments 1-17, wherein the fourth target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377. Embodiment 19 comprises the composition of any one of embodiments 1-18, wherein the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or the complement thereof, and the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2, or the complement thereof. 376 , or the complement thereof , and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376, or the complement thereof. Embodiment 20 comprises the composition of any one of embodiments 1-19, wherein the first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, and the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2, or the complement thereof. 7 or the complement thereof , the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 376 or the complement thereof, and the fourth target nucleic acid sequence comprises the sequence according to SEQ ID NO: 377 or the complement thereof including.

Embodiment 21 includes the composition of any one of embodiments 1-20, wherein the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), or human herpes virus 3 (HHV-3). ; varicella-zoster virus (VZV)), human herpesvirus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 ( HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). Embodiment 22 includes a composition comprising: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease, or a nucleic acid sequence encoding a CRISPR-associated endonuclease; b) ) a first guide nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, wherein the first guide nucleic acid is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; c) a second guide nucleic acid, or a nucleic acid sequence encoding a second guide nucleic acid, wherein the second guide nucleic acid is linked to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is a third guide nucleic acid within or near the ICP27 gene of the herpesvirus genome; complementary to a target nucleic acid sequence; wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. Embodiment 23 includes the composition of any one of Embodiments 1-22, wherein the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease. Embodiment 24 includes the composition of any one of embodiments 1-23 , wherein the CRISPR-associated endonuclease is Cas9 endonuclease, Cas12 endonuclease, CasX endonuclease, or CasQ endonuclease. Embodiment 25 includes the composition of any one of Embodiments 1-24, wherein the CRISPR-associated endonuclease is Cas9 nuclease.

Embodiment 26 includes the composition of any one of Embodiments 1-25 , wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Embodiment 27 includes the composition of any one of embodiments 1-26, wherein the CRISPR-associated endonuclease is optimized for expression in human cells. Embodiment 28 includes the composition of any one of Embodiments 1-27, wherein the guide nucleic acid is RNA. Embodiment 29 includes the composition of any one of Embodiments 1-28, wherein the guide nucleic acid comprises crRNA and tracrRNA. Embodiment 30 includes the composition of any one of Embodiments 1-29, wherein the first target nucleic acid sequence is any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs: 1-96 or 372-375 .

Embodiment 31 comprises the composition of any one of embodiments 1-30, wherein the first target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs : 1-375. 96 or the complement of any one of 372-375 . Embodiment 32 includes the composition of any one of Embodiments 1-31, wherein the second target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363, 371 , or the complement of any one of 374-377 . Embodiment 33 includes the composition of any one of embodiments 1-32, wherein the second target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377. Embodiment 34 includes the composition of any one of Embodiments 1-33, wherein the third target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363, 371 , or the complement of any one of 374-377 . Embodiment 35 includes the composition of any one of embodiments 1-34, wherein the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377 .

Embodiment 36 comprises the composition of any one of embodiments 1-35, wherein the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7, or the complement thereof, and the second target nucleic acid sequence comprises: , a sequence according to SEQ ID NO: 376 , or the complement thereof, and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377, or the complement thereof. Embodiment 37 includes the composition of any one of embodiments 1-36, wherein the herpesvirus is herpes simplex type I (HSV1), herpes simplex virus type 2 (HSV2), or human herpesvirus 3 (HHV-3). ; varicella -zoster virus (VZV)), human herpesvirus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 ( HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV). Embodiment 38 comprises: A CRISPR-Cas system comprising: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease; b ) a first guide nucleic acid having SEQ ID NO: 2 or 7. or a first guide nucleic acid comprising a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to or to its complement; c) a second guide nucleic acid, wherein the second guide nucleic acid comprises : , comprising a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376 or 377 or its complement . Embodiment 39 is the CRISPR-Cas of any one of embodiments 1-38, wherein the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2. Including system. Embodiment 40 provides the CRISPR-Cas of any one of embodiments 1-39, wherein the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7. Including system.

Embodiment 41 includes the CRISPR-Cas system of any one of embodiments 1-40, wherein the second guide nucleic acid is a nucleic acid complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. Contains arrays. Embodiment 42 is the CRISPR-Cas of any one of embodiments 1-41, wherein the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. Including system. Embodiment 43 includes a CRISPR-Cas system according to any one of embodiments 1-42 , wherein the first guide nucleic acid is complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2. 376, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. Embodiment 44 provides that the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2 , and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. the CRISPR-Cas system of any one of embodiments 1-43. Embodiment 45 includes a CRISPR-Cas system according to any one of embodiments 1-44, wherein the first guide nucleic acid is complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7. The second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376 .

Embodiment 46 includes the CRISPR-Cas system of any one of embodiments 1-45, wherein the first guide nucleic acid is a nucleic acid complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7. The second guide nucleic acid comprises a nucleic acid sequence that is complementary to a sequence that has at least 90% sequence identity to SEQ ID NO:377 . Embodiment 47 includes a nucleic acid encoding the CRISPR-Cas system of any one of embodiments 1-46. Embodiment 48 includes an adeno-associated virus (AAV) vector comprising a nucleic acid encoding: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease; b) a herpesvirus. a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the genome; c) a second guide nucleic acid. The second guide nucleic acid is complementary to a second target nucleic acid sequence within or near the ICPO gene of the herpesvirus genome. d) a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. Embodiment 49 provides the AAV vector of any one of embodiments 1-48, further comprising a fourth guide nucleic acid that is complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. include. Embodiment 50 is the AAV vector of any one of embodiments 1-49, wherein the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. including.

Embodiment 51 includes the AAV vector of any one of embodiments 1-50, wherein the CRISPR-associated endonuclease is a type I, type II, or type III Cas endonuclease. Embodiment 52 includes the AAV vector of any one of embodiments 1-51, wherein the CRISPR-associated endonuclease is Cas9 endonuclease, Casl2 endonuclease, CasX endonuclease, or CasΦ endonuclease. Embodiment 53 includes the AAV vector of any one of embodiments 1-52, wherein the CRISPR-associated endonuclease is a Cas9 nuclease. Embodiment 54 includes the AAV vector of any one of embodiments 1-53, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Embodiment 55 includes the AAV vector of any one of embodiments 1-54, wherein the CRISPR-associated endonuclease is optimized for expression in human cells.

Embodiment 56 includes the AAV vector of any one of embodiments 1-55, wherein the guide nucleic acid is RNA. Embodiment 57 includes the AAV vector of any one of embodiments 1-56, wherein the guide nucleic acid comprises crRNA and tracrRNA. Embodiment 58 includes the AAV vector of any one of embodiments 1-57, wherein the first target nucleic acid sequence is at least about 90% comprises sequences that contain sequence identity, or comprises the complement of any one of SEQ ID NOs: 1-96 or 372-375. Embodiment 59 comprises the AAV vector of any one of embodiments 1-58, wherein the first target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 1-96 or 372-375, or SEQ ID NO : 1-58. 96 or the complement of any one of 372-375 . Embodiment 60 comprises the AAV vector of any one of embodiments 1-59, wherein the second target nucleic acid sequence is any one of SEQ ID NOs: 1-96 or 372-375, or SEQ ID NOs: 1-96 or 372-375 .

Embodiment 61 comprises the AAV vector of any one of embodiments 1-60, wherein the second target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 1-96 or 372-375, or SEQ ID NO: 1-60. 96 or the complement of any one of 372-375. Embodiment 62 comprises the AAV vector of any one of embodiments 1-61, wherein the third target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO : 363, 371, or the complement of any one of 374-377 . Embodiment 63 comprises the AAV vector of any one of embodiments 1-62, wherein the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377. Embodiment 64 includes the AAV vector of any one of embodiments 1-63, wherein the fourth target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363, 371 , or the complement of any one of 374-377 . Embodiment 65 comprises the AAV vector of any one of embodiments 1-64 , wherein the fourth target nucleic acid sequence is a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or and the complement of any one of SEQ ID NOs: 363, 371, or 374-377.

Embodiment 66 comprises the AAV vector of any one of embodiments 1-65, wherein the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or the complement thereof, and the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or the complement thereof. 7 or the complement thereof , and the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 376 or the complement thereof. Embodiment 67 comprises the AAV vector of any one of embodiments 1-66, wherein the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or the complement thereof, and the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2, or the complement thereof. 7 , the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or the complement thereof, and the fourth target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377 or the complement thereof. Embodiment 68 includes the AAV vector of any one of embodiments 1-67, and the nucleic acid further includes a promoter. Embodiment 69 includes the AAV vector of any one of Embodiments 1-68, wherein the promoter is a ubiquitous promoter. Embodiment 70 includes the AAV vector of any one of embodiments 1-69, wherein the promoter is a tissue-specific promoter.

Embodiment 71 comprises the AAV vector of any one of embodiments 1-70, wherein the promoter is a constitutive promoter. Embodiment 72 comprises the AAV vector of any one of embodiments 1-71, wherein the promoter is the human cytomegalovirus promoter. Embodiment 73 comprises the AAV vector of any one of embodiments 1-72, wherein the nucleic acid further comprises an enhancer element . Embodiment 74 comprises the AAV vector of any one of embodiments 1-73, wherein the enhancer element is a human cytomegalovirus enhancer element . Embodiment 75 comprises the AAV vector of any one of embodiments 1-74, wherein the nucleic acid further comprises a 5'ITR element and a 3'ITR element .

Embodiment 76 includes the AAV vector of any one of embodiments 1-75, wherein the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. Embodiment 77 includes the AAV vector of any one of embodiments 1-76, wherein the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11. , AAVDJ, or AAVDJ/8. Embodiment 78 includes the AAV vector of any one of embodiments 1-77, wherein the herpesvirus is herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), or human herpes virus 3 (HHV-3). ; varicella-zoster virus (VZV)) , human herpesvirus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 ( HHV-6; roseolovirus), human herpesvirus 7 (HHV-7) and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). Embodiment 79 includes an adeno-associated virus (AAV) vector comprising a nucleic acid encoding: a) a CRISPR-associated endonuclease. b) a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; c) a second guide nucleic acid. The second guide nucleic acid is complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. d) a third guide nucleic acid complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; have different target nucleic acid sequences. Embodiment 80 comprises the AAV vector of any one of embodiments 1-79, wherein the CRISPR-associated endonuclease is a Type I, Type II, or Type III Cas endonuclease.

Embodiment 81 comprises the AAV vector of any one of embodiments 1-80, wherein the CRISPR-associated endonuclease is Cas9 endonuclease, Cas12 endonuclease, CasX endonuclease, or CasΦ endonuclease. Embodiment 82 comprises the AAV vector of any one of embodiments 1-81, wherein the CRISPR-associated endonuclease is Cas9 nuclease. Embodiment 83 comprises the AAV vector of any one of embodiments 1-82, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Embodiment 84 includes the AAV vector of any one of embodiments 1-83, wherein the CRISPR-associated endonuclease is optimized for expression in human cells. Embodiment 85 includes the AAV vector of any one of embodiments 1-84, wherein the guide nucleic acid is RNA.

Embodiment 86 includes the AAV vector of any one of embodiments 1-85, wherein the guide nucleic acid includes crRNA and tracrRNA. Embodiment 87 includes the AAV vector of any one of embodiments 1-86, wherein the first target nucleic acid sequence shares at least about a close sequence identity with any one of SEQ ID NOs: 1-96 or 372-375. or the complement of any one of SEQ ID NOs: 1-96 or 372-375. Embodiment 88 comprises the AAV vector of any one of embodiments 1-87, wherein the first target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 1-96 or 372-375, or SEQ ID NO: 1-87. 96 or the complement of any one of 372-375. Embodiment 89 comprises the AAV vector of any one of embodiments 1-88, wherein the second target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363, 371 , or the complement of any one of 374-377 . Embodiment 90 comprises the AAV vector of any one of embodiments 1-89, wherein the second target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377.

Embodiment 91 comprises the AAV vector of any one of embodiments 1-90 , wherein the third target nucleic acid sequence is any one of SEQ ID NO: 363, 371, or 374-377 , or SEQ ID NO: 363, 371. , or the complement of any one of 374-377 . Embodiment 92 comprises the AAV vector of any one of embodiments 1-91, wherein the third target nucleic acid sequence is a sequence according to any one of SEQ ID NO: 363, 371, or 374-377, or SEQ ID NO: 363. , 371, or the complement of any one of 374-377. Embodiment 93 comprises the AAV vector of any one of embodiments 1-92, wherein the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7, or the complement thereof, and the second target nucleic acid sequence comprises , a sequence according to SEQ ID NO: 376 , or the complement thereof , and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377, or the complement thereof. Embodiment 94 includes the AAV vector of any one of embodiments 1-93, wherein the herpesvirus is herpes simplex type I (HSV1), herpes simplex virus type 2 (HSV2), human herpesvirus 3 (HHV-3); Varicella zoster virus (VZV )), human herpesvirus 4 (HHV-4; Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV) -6; Roseolovirus). Embodiment 95 includes the AAV vector of any one of embodiments 1-94, wherein the nucleic acid further includes a promoter.

Embodiment 96 includes the AAV vector of any one of Embodiments 1-95, wherein the promoter is a ubiquitous promoter. Embodiment 97 includes the AAV vector of any one of embodiments 1-96, wherein the promoter is a tissue-specific promoter. Embodiment 98 includes the AAV vector of any one of Embodiments 1-97, wherein the promoter is a constitutive promoter. Embodiment 99 comprises the AAV vector of any one of embodiments 1-98, wherein the promoter is the human cytomegalovirus promoter. Embodiment 100 comprises the AAV vector of any one of embodiments 1-99, wherein the nucleic acid further comprises an enhancer element .

Embodiment 101 comprises the AAV vector of any one of embodiments 1-100, wherein the enhancer element is a human cytomegalovirus enhancer element . Embodiment 102 comprises the AAV vector of any one of embodiments 1-101, wherein the nucleic acid further comprises a 5'ITR element and a 3'ITR element . Embodiment 103 includes the AAV vector of any one of embodiments 1-102, wherein the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. Embodiment 104 includes the AAV vector of any one of embodiments 1-103, wherein the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11. , AAVDJ, or AAVDJ/8 . Embodiment 105 includes a method of excising some or all of a herpesvirus sequence from a cell, the method comprising the composition of any one of embodiments 1-104, the CRISPR of any one of embodiments 1-104. -Cas system, or the AAV vector of any one of embodiments 1-104 to the cell. Embodiment 106 includes a method of inhibiting or reducing herpesvirus replication in a cell, the method comprising the composition of any one of embodiments 1-104, the CRISPR-Cas system of any one of embodiments 1-104. , or providing the AAV vector of any one of embodiments 1-104 to the cell. Embodiment 107 includes the method of any one of embodiments 1-106, wherein the cell is within the subject . Embodiment 108 includes the method of any one of Embodiments 1-107, wherein the subject is a human .

(Example)
(Example 1)

Example 2 - Inhibition of HSV-1 replication in vitro by gene editing
Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus that infects a large portion of the world's population, with a seroprevalence of 90% in asymptomatic normal individuals. Current treatments for primary HSV-1 infection and disease reactivation are non-selective, do not prevent the establishment of latent infection or reactivation of the virus, and have deleterious side effects. Environmental factors such as UV stimulation, hyperthermia, social stress, and drugs can cause reactivation of the latent HSV-1 genome, leading to disease progression. Current anti-HSV drugs can limit the spread of HSV-1 infection, but cannot inhibit the establishment of the latent period or HSV reactivation.

Considering the limitations of current treatments, new therapeutic approaches that not only effectively suppress viral replication but also eradicate latent viral genomes are needed. Therefore, as described and provided herein, CRISPR/Cas9 specifically targets the HSV-1 genome for the purpose of creating indel mutations or removing large segments of viral DNA sequences important for viral replication. The system can be used to inhibit HSV-1 replication . In particular, targeting the ICP0 and ICP27 genes of HSV-1 significantly reduces the expression levels of ICP0 and ICP27, leading to suppression of HSV-1 infection. As provided herein, the specificity of the targeting strategy for gene mutations/elimination within the HSV-1 genome (eg, ICP0 and ICP27) has been verified by genetic analysis of an in vitro cell culture model. Furthermore, expression of HSV-1-directed Cas9/gRNA in cells protected them from HSV-1 infection.

FIG. 1A shows a schematic diagram of the HSV-1 genome, ICP0 and ICP27 genes. In the described studies, the viral genes ICP0 and ICP27 genes are targeted by the CRISPR Cas9 gene editing system. The location and nucleotide composition of gRNAs containing PAM are shown (SEQ ID NOs: 372-375). For nucleotide positions, see RefSeq NC_001806.2. FIG. 1B provides a graphical representation of plasmid P31. The plasmid contains four gRNAs, two gRNAs targeting ICP0 (m1 and m2) and two gRNAs targeting ICP27 (m1 and m2) cloned into the downstream U6 promoter, and one copy of the SaCas9 gene. . P31 was packaged into AAV2 particles resulting in an AAV2-HSV construct. px601 without ICP0 and ICP27 gRNA ('px601' or 'px601saCas9') was used as an additional control.

Targeting ICP0 and ICP27 effectively inhibits HSV-1 replication . Figure 2 shows the expression of SaCas9- and ICP0 -related gRNAs and ICP27-related gRNAs and the px601 P31 of TC620 cell line infected with HSV-1 NS1 clinical strain and HSV-1 GFP Patton strain to confirm the excision activity against genes ICP0 and ICP27 . A schematic diagram of transient transfection is shown. Additionally, Western blot analysis for the detection of ICP0 and ICP27 in the clinical strains HSV-1 NS1 (left panel) and HSV-1 GFP (right panel) infected with TC620 human oligodendroglioma showed that the reduction of ICP0 and ICP27 was Proven. The TC620 human oligodendroglioma cell line was transiently transfected with plasmid P31. Expression of SaCas9 and the housekeeping GAPDH protein is also shown. Targeting ICP0 and ICP27 effectively edits ICP0 and ICP27. fig. Figure 3 shows the results obtained with ICP0 and ICP27 specific primers in TC620 cell line infected with HSV-1 NS1 (left panel) and HSV-1 GFP (right panel) after transient transfection with P31 plasmid. Data from DNA excision assays on agarose gels showing amplicons are shown. DNA sequencing identified specific gRNAs of each target gene and specific excision induced by SaCas9 (bottom panel).

Figure 4 shows data from immunofluorescence evaluation of HSV-1 GFP replication in the TC620 cell line transiently transfected with the P31 plasmid. Representative plaque assay using supernatants from HSV-1 NS1 and HSV-1 GFP infected TC620 cell lines. We show that suppression of ICP0 and ICP27 by SaCas9 and gRNA editing in infected cells results in a significant reduction in plaque number. Transient transfection of the TC620 cell line with plasmid P31 and infection with HSV-1 NS1 (right panel) and HSV-1 GFP (left panel) using a reverse transcriptase (RT) assay (Figure 5). gRNA expression was confirmed.

Inhibition of HSV-1 replication targeting ICP0 and ICP27 was further demonstrated in VERO (African green monkey kidney) cells. fig. A schematic diagram of AAV2-HSV construct transduction of VERO cell lines infected with HSV-1 NS1 clinical strain and HSV-1 GFP Patton strain to confirm the expression of SaCas9 and ICP0 -related gRNAs and ICP27-related gRNAs is shown. Excision activity against genes ICP0 and ICP27. As demonstrated by Western blot analysis for the detection of ICP0 and ICP27 in the VERO human cell line infected with clinical strains HSV-1 NS1 (left panel) and HSV-1 GFP (right panel) transduced by the AAV2-HSV construct. , ICP0 and ICP27 are effectively suppressed. Proteins expressed SaCas9 and the housekeeping GAPDH protein are also shown. Targeting ICP0 and ICP27 effectively edits ICP0 and ICP27 in VERO cells. Figure 7 shows DNA excision on an agarose gel showing amplicons obtained with ICP0 and ICP27 specific primers in VERO cell lines infected with HSV-1 NS1 and HSV-1 GFP after transduction with AAV2-HSV constructs. Data from the assay are shown. Reverse transcriptase (RT) assay to confirm gRNA expression after transduction of VERO cell line with AAV2-HSV construct (right panel). DNA sequencing identified specific gRNAs of each target gene and specific excision induced by SaCas9 (bottom). Figure 8 shows data from plaque assays using supernatants from VERO cell lines infected with HSV-1 NS1 (left panel) and HSV-1 GFP (right panel), showing that SaCas9 and gRNA of infected cells By editing cells, we show that the number of plaques was dramatically reduced as a result of ICP0 and ICP27 being suppressed.

Claims (108)

A composition comprising:
a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease, or a nucleic acid sequence encoding a CRISPR-associated endonuclease;
b) a first guide nucleic acid, or a nucleic acid sequence encoding said first guide nucleic acid, wherein said first guide nucleic acid is directed to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome. complementary to said first guide nucleic acid, or a nucleic acid sequence encoding said first guide nucleic acid;
c) a second guide nucleic acid, or a nucleic acid sequence encoding said second guide nucleic acid, wherein said second guide nucleic acid is linked to a second target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome. a second guide nucleic acid, or a nucleic acid sequence encoding said second guide nucleic acid, which is complementary; and d) a third guide nucleic acid, or a nucleic acid sequence encoding said third guide nucleic acid, The third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome, or a nucleic acid encoding the third guide nucleic acid. Array and;
, wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. further comprising a fourth guide nucleic acid, or a nucleic acid sequence encoding said fourth guide nucleic acid, wherein said fourth guide nucleic acid is complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. The composition according to claim 1, characterized in that: 3. The composition of claim 2, wherein the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. . Composition according to any one of claims 1 to 3, characterized in that the CRISPR-related endonuclease is a type I, type II or type III Cas endonuclease. Composition according to any one of claims 1 to 3, characterized in that the CRISPR-related endonuclease is Cas9 endonuclease, Cas12 endonuclease, CasX endonuclease or CasQ endonuclease. Composition according to any one of claims 1 to 3, characterized in that the CRISPR-related endonuclease is Cas9 nuclease. 7. The composition of claim 6, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Composition according to any one of claims 1 to 7, characterized in that the CRISPR-associated endonuclease is optimized for expression in human cells. The composition according to any one of claims 1 to 8, characterized in that the guide nucleic acid is RNA. The composition according to any one of claims 1 to 9, characterized in that the guide nucleic acid comprises crRNA and tracrRNA. The first target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. 2. The composition of claim 1, comprising sequences comprising sequence identity. characterized in that the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96, 372-375, or the complement of any one of SEQ ID NOs: 1-96, 372-375. The composition according to claim 1. The second target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 1-96, 372-375, or the complement of any one of SEQ ID NOs: 1-96, 372-375. 2. The composition of claim 1, comprising sequences comprising sequence identity. characterized in that the second target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96, 372-375, or a complement of any one of SEQ ID NOs: 1-96, 372-375. The composition according to claim 1. The third target nucleic acid sequence is at least about 90% of SEQ ID NO: 363, 371, or 374-377, or the complement of SEQ ID NO: 363, 371, or 374-377 % sequence identity. that the third target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; A composition according to claim 1, characterized in that: The fourth target nucleic acid sequence is at least about 90% of SEQ ID NO: 363 , 371, or 374-377, or the complement of SEQ ID NO: 363, 371, or 374-377. 3. The composition of claim 2, comprising sequences having % sequence identity. that the fourth target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; A composition according to claim 2, characterized in that: the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or its complement; the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 7 or its complement; and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 7 or its complement; 2. A composition according to claim 1, characterized in that it comprises a sequence according to 376 or its complement. the first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or its complement; the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 7 or its complement; and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 7 or its complement; 376 or its complement, and the fourth target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377 or its complement. The herpesviruses include herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpesvirus 3 (HHV-3, varicella zoster) virus (VZV), human herpesvirus 4 (HHV-4); Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). A composition comprising:
a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease, or a nucleic acid sequence encoding a CRISPR-associated endonuclease;
b) a first guide nucleic acid, or a nucleic acid sequence encoding a first guide nucleic acid, wherein the first guide nucleic acid is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; a first guide nucleic acid, or a nucleic acid sequence encoding the first guide nucleic acid;
c) a second guide nucleic acid or a nucleic acid sequence encoding a second guide nucleic acid, the second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; a second guide nucleic acid, or a nucleic acid sequence encoding the second guide nucleic acid; and d) a third guide nucleic acid, or a nucleic acid sequence encoding the third guide nucleic acid, wherein the third guide nucleic acid is a third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome;
has
A composition, wherein the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. 23. The composition of claim 22, wherein the CRISPR-associated endonuclease is a type I, type II, or type III Cas endonuclease. 23. The composition of claim 22, wherein the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasQ endonuclease. 23. The composition of claim 22, wherein the CRISPR-associated endonuclease is a Cas9 nuclease. 26. The composition of claim 25, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. Composition according to any one of claims 22 to 26, characterized in that the CRISPR-associated endonuclease is optimized for expression in human cells. Composition according to any one of claims 22 to 27, characterized in that the guide nucleic acid is RNA. Composition according to any one of claims 22 to 28, characterized in that the guide nucleic acid comprises crRNA and tracrRNA. The first target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. 23. The composition of claim 22, comprising sequences comprising sequence identity. characterized in that the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. 23. The composition according to claim 22. The second target nucleic acid sequence is at least about 90% of SEQ ID NO: 363, 371, or 374-377, or the complement of SEQ ID NO: 363, 371, or 374-377. 23. A composition according to claim 22, characterized in that the composition comprises sequences comprising % sequence identity. that said second target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 23. The composition of claim 22, characterized in that: the third target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 23. The composition of claim 22, comprising a sequence comprising sequence identity of. that said third target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 23. The composition of claim 22, characterized in that: The first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7 or the complement thereof, the second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or the complement thereof, and the third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or the complement thereof. 23. A composition according to claim 22, characterized in that it comprises a sequence according to SEQ ID NO: 377, or its complement. The herpesviruses include herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3, varicella zoster) virus (VZV), human herpes virus 4 (HHV-4); Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). The CRISPR-Cas system:
a) Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease;
b) a first guide nucleic acid comprising a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2 or 7 , or the complement thereof ; and c) SEQ ID NO: 376. or 377 , or its complement ;
A CRISPR-Cas system comprising: 39. The CRISPR-Cas system of claim 38, wherein the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO:2. 39. The CRISPR-Cas system of claim 38, wherein the first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO:7. 39. The CRISPR-Cas system of claim 38, wherein the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO:376. 39. The CRISPR-Cas system of claim 38, wherein the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO:377. The first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. 39. The CRISPR-Cas system according to claim 38, characterized in that it comprises a nucleic acid sequence complementary to a sequence having sequence identity of . said first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 2, and said second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. CRISPR-Cas system according to claim 38, characterized in that it is complementary to a sequence having a sequence identity of . The first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 376. 39. The CRISPR-Cas system according to claim 38, characterized in that it comprises a nucleic acid sequence complementary to a sequence having sequence identity of . The first guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 7, and the second guide nucleic acid comprises a nucleic acid sequence complementary to a sequence having at least 90% sequence identity to SEQ ID NO: 377. 39. The CRISPR-Cas system according to claim 38, characterized in that it comprises a nucleic acid sequence complementary to a sequence having sequence identity of . A nucleic acid encoding the CRISPR-Cas system according to any one of claims 38-46. An adeno-associated virus (AAV) vector containing a nucleic acid encoding:
a) Clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonucleases;
b) a first guide nucleic acid that is complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome;
c) a second guide nucleic acid, the second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome; and d) A third guide nucleic acid, or a nucleic acid sequence encoding a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. a third guide nucleic acid;
Here, the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. 49. Claim 48, further comprising a fourth guide nucleic acid, said fourth guide nucleic acid being complementary to a fourth target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome. AAV vector. 50. The AAV vector of claim 49, wherein the fourth target nucleic acid sequence is different from the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence. . 51. AAV vector according to any one of claims 48 to 50, characterized in that the CRISPR-related endonuclease is a type I, type II or type III Cas endonuclease. 51. AAV vector according to any one of claims 48 to 50, characterized in that the CRISPR-associated endonuclease is Cas9 endonuclease, Cas12 endonuclease, CasX endonuclease, or CasQ endonuclease. AAV vector according to any one of claims 48 to 50, characterized in that the CRISPR-associated endonuclease is Cas9 nuclease. 54. The AAV vector of claim 53, wherein the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. AAV vector according to any one of claims 48 to 54, characterized in that the CRISPR-associated endonuclease is optimized for expression in human cells. AAV vector according to any one of claims 48 to 55, characterized in that the guide nucleic acid is RNA. AAV vector according to any one of claims 48 to 56, characterized in that the guide nucleic acid comprises crRNA and tracrRNA. the first target nucleic acid sequence is at least about 90% of the sequence of any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375; 49. AAV vector according to claim 48, characterized in that it comprises sequences containing identity. characterized in that the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. The AAV vector according to claim 48. The second target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. 49. AAV vector according to claim 48, characterized in that it comprises sequences that contain sequence identity. characterized in that the second target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. The AAV vector according to claim 48. the third target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; 49. AAV vector according to claim 48, characterized in that it comprises a sequence comprising sequence identity of. that the third target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 49. The AAV vector of claim 48. the fourth target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of any one of SEQ ID NO: 363, 371, or 374-377; 50. AAV vector according to claim 49, characterized in that it comprises a sequence comprising sequence identity of. characterized in that the fourth target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377. The AAV vector according to claim 49. The first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof. 49. AAV vector according to claim 48, characterized in that it comprises the sequence according to number 376 or its complement. The first target nucleic acid sequence comprises the sequence according to SEQ ID NO: 2 or the complement thereof, the second target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof, and the third target nucleic acid sequence comprises the sequence according to SEQ ID NO: 7 or the complement thereof. 50. AAV vector according to claim 49, characterized in that it comprises a sequence according to SEQ ID NO: 376 or its complement, and said fourth target nucleic acid sequence comprises a sequence according to SEQ ID NO: 377 or its complement. AAV vector according to any one of claims 48 to 67, characterized in that the nucleic acid further comprises a promoter. 69. The AAV vector of claim 68, wherein the promoter is a ubiquitous promoter. 69. The AAV vector of claim 68, wherein the promoter is a tissue-specific promoter. 69. The AAV vector of claim 68, wherein the promoter is a constitutive promoter. 69. The AAV vector of claim 68, wherein the promoter is a human cytomegalovirus promoter. AAV vector according to any one of claims 48 to 72, characterized in that the nucleic acid further comprises an enhancer element . 96. AAV vector according to claim 95, characterized in that said enhancer element is a human cytomegalovirus enhancer element . 97. AAV vector according to any one of claims 49 to 96, characterized in that the nucleic acid further comprises a 5'ITR element and a 3'ITR element . 76. AAV vector according to any one of claims 48 to 75, characterized in that the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8 or AAV9. Claims 48-75 characterized in that the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8. The AAV vector according to any one of . The herpesviruses include herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3, varicella), herpes zoster virus (VZV), human herpes virus 4 (HHV-4); Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). An adeno-associated virus (AAV) vector containing a nucleic acid encoding:
a) Clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonucleases;
b) a first guide nucleic acid, the first guide nucleic acid being complementary to a first target nucleic acid sequence within or near the ICP0 gene of the herpesvirus genome;
c) a second guide nucleic acid, said second guide nucleic acid being complementary to a second target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome; and d) a third guide nucleic acid, wherein the third guide nucleic acid is complementary to a third target nucleic acid sequence within or near the ICP27 gene of the herpesvirus genome;
Here, the first target nucleic acid sequence, the second target nucleic acid sequence, and the third target nucleic acid sequence are different. 80. The AAV vector of claim 79, wherein the CRISPR-associated endonuclease is a type I, type II, or type III Cas endonuclease. 80. The AAV vector of claim 79, wherein the CRISPR-associated endonuclease is a Cas9 endonuclease, a Cas12 endonuclease, a CasX endonuclease, or a CasQ endonuclease. 80. AAV vector according to claim 79, characterized in that the CRISPR-associated endonuclease is a Cas9 nuclease. 83. AAV vector according to claim 82, characterized in that the Cas9 nuclease is Staphylococcus aureus Cas9 nuclease. AAV vector according to any one of claims 79 to 83, characterized in that the CRISPR-related endonuclease is optimized for expression in human cells. AAV vector according to any one of claims 79 to 84, characterized in that the guide nucleic acid is RNA. AAV vector according to any one of claims 79 to 84, characterized in that the guide nucleic acid comprises crRNA and tracrRNA. the first target nucleic acid sequence is at least about 90% of the sequence of any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375; 80. AAV vector according to claim 79, characterized in that it comprises sequences containing identity. characterized in that the first target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 1-96 or 372-375, or the complement of any one of SEQ ID NOs: 1-96 or 372-375. 80. The AAV vector of claim 79. the second target nucleic acid sequence has at least about 90% sequence identity to any one of SEQ ID NO: 363, 371, or 374-377, or the complement of SEQ ID NO: 363, 371, or 374-377; 80. The AAV vector of claim 79, comprising a sequence comprising: characterized in that the second target nucleic acid sequence comprises a sequence according to any one of SEQ ID NO: 363, 371 or 374-377, or the complement of any one of SEQ ID NO: 363, 371 or 374-377. 80. The AAV vector of claim 79. the third target nucleic acid sequence is at least about 90% complementary to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 80. AAV vector according to claim 79, characterized in that it comprises a sequence comprising sequence identity of. that the third target nucleic acid sequence comprises a sequence according to any one of SEQ ID NOs: 363, 371, or 374-377, or the complement of any one of SEQ ID NOs: 363, 371, or 374-377; 80. The AAV vector of claim 79, characterized in: said first target nucleic acid sequence comprises a sequence according to SEQ ID NO: 2 or 7 or its complement; said second target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or 376 or its complement; and said third target nucleic acid sequence comprises a sequence according to SEQ ID NO: 376 or 376 or its complement 80. AAV vector according to claim 79, characterized in that the sequence comprises the sequence according to SEQ ID NO: 377 or its complement. The herpesviruses include herpes simplex virus type I (HSV1), herpes simplex virus type 2 (HSV2), human herpes virus 3 (HHV-3, varicella), herpes zoster virus (VZV), human herpes virus 4 (HHV-4); Epstein-Barr virus (EBV)), human herpesvirus 5 (HHV-5; cytomegalovirus (CMV)), human herpesvirus 6 (HHV-6; roseolovirus), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8; Karposi's sarcoma-associated herpesvirus (KSHV)). AAV vector according to any one of claims 79 to 94, characterized in that the nucleic acid further comprises a promoter. 96. The AAV vector of claim 95, wherein the promoter is a ubiquitous promoter. 9569. The AAV vector of claim 9568, wherein the promoter is a tissue-specific promoter. 96. The AAV vector of claim 95, wherein the promoter is a constitutive promoter. 96. The AAV vector of claim 95, wherein the promoter is a human cytomegalovirus promoter. AAV vector according to any one of claims 79 to 99, characterized in that the nucleic acid further comprises an enhancer element . 101. AAV vector according to claim 100, characterized in that the enhancer element is a human cytomegalovirus enhancer element . 102. AAV vector according to any one of claims 79 to 101, characterized in that the nucleic acid further comprises a 5'ITR element and a 3'ITR element . 103. AAV vector according to any one of claims 79 to 102, characterized in that the adeno-associated virus (AAV) vector is AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9. 79- characterized in that the adeno-associated virus (AAV) vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVDJ, or AAVDJ/8. 103. AAV vector according to any one of 102. A method of excising part or all of a herpesvirus sequence from a cell, said method comprising: a composition according to any one of claims 1 to 37; a composition according to any one of claims 38 to 46; 105. A method characterized in that the method comprises the step of providing the cell with the CRISPR-Cas system of or an AAV vector according to any one of claims 48 to 104. A method of inhibiting or reducing herpesvirus replication in a cell, said method comprising: a composition according to any one of claims 1 to 37; A method characterized in that it comprises the step of providing the cell with a Cas system or an AAV vector according to any one of claims 48 to 104. 107. A method according to any one of claims 105 to 106, characterized in that the cells are within a subject . 108. The method of claim 107, wherein the subject is a human .