TW202424195A - Antiviral nucleic acids and compositions - Google Patents

Abstract

The present invention relates generally to compositions and methods for inhibiting the replication of coronaviruses and treating diseases caused by coronavirus infection. More specifically, the invention provides nucleic acids capable of inhibiting coronavirus (e.g. SARS-CoV-2) replication and their use in treating patients infected by the virus.

Description

Antiviral nucleic acids and compositions

The present invention generally relates to compositions and methods for inhibiting coronavirus replication and treating diseases caused by coronavirus infection. More specifically, the present invention provides nucleic acids capable of inhibiting coronavirus (e.g., SARS-CoV-2) replication and the use of such nucleic acids for treating patients infected by the virus.

SARS-CoV-2 is a positive-sense, single-stranded RNA virus that is contagious in humans and other animals. It is the cause of the 2019 coronavirus disease (COVID-19), which has severely affected humans worldwide. As of September 2022, conservative estimates indicate that SARS-CoV-2 has infected more than 610 million people since the start of the current pandemic, with more than 6.5 million deaths from COVID-19 (see WHO Coronavirus (COVID-19) Dashboard).

In humans, SARS-CoV-2 infection results in a range of outcomes, with many experiencing very mild or barely discernible symptoms, while others manifest moderate to severe illness, with an estimated mortality rate of approximately 1% worldwide. While considerable resources and effort have been devoted to preventive measures through vaccine development, it is well known that therapeutic measures are critical to reducing morbidity and mortality. In particular, antiviral agents suitable for administration to patients during the early stages of the disease offer the opportunity to reduce viral replication and thereby suppress the onset of severe disease, especially in high-risk categories (e.g., immunocompromised persons, the elderly, etc.).

Effective therapeutic treatments are needed to combat COVID-19 disease, including those that can inhibit viral replication during the early stages of COVID-19 disease.

The present invention meets the needs in the field of therapeutic treatment of COVID-19 disease by providing nucleic acids capable of inhibiting the expression of SARS-CoV-2.

Various aspects of the present invention relate to nucleic acids capable of inhibiting the replication of coronaviruses (such as SARS-CoV-2). Such nucleic acids and compositions comprising the same can be used to treat diseases caused by coronavirus infections (including COVID-19).

In some embodiments, a nucleic acid is provided that comprises 15 to 30 nucleotides and is capable of specifically hybridizing with the SARS-CoV-2 sequence defined in SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or is capable of hybridizing with a fragment of the SARS-CoV-2 sequence. The nucleic acid may comprise or consist of: 15 to 25 nucleotides; 15 to 23 nucleotides; 15 to 22 nucleotides; 15 to 21 nucleotides; 15 to 20 nucleotides; 15 to 19 nucleotides; 19 nucleotides; 20 nucleotides; 21 nucleotides, 22 nucleotides, or 23 nucleotides.

In other embodiments, a nucleic acid is provided that has at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity with the following sequences: A sequence defined in SEQ ID NO: 2 or 24, wherein the nucleic acid is capable of specifically hybridizing with the SARS-CoV-2 sequence defined in SEQ ID NO: 23; or A sequence defined in SEQ ID NO: 4 or 26, wherein the nucleic acid is capable of specifically hybridizing with the SARS-CoV-2 sequence defined in SEQ ID NO: 25; or A sequence defined in SEQ ID NO: 6 or 28, wherein the nucleic acid is capable of specifically hybridizing with the SARS-CoV-2 sequence defined in SEQ ID NO: 27.

In other embodiments, fragments of the nucleic acids of the invention are provided, wherein the fragments are 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length.

In other embodiments, a double-stranded nucleic acid for inhibiting SARS-CoV-2 expression is provided, comprising a sense strand and an antisense strand, wherein: The sense strand comprises a sequence defined in SEQ ID NO: 1 or 23, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 2 or 24, or a variant or fragment thereof; or The sense strand comprises a sequence defined in SEQ ID NO: 3 or 25, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 4 or 26, or a variant or fragment thereof; or The sense strand comprises a sequence defined in SEQ ID NO: 5 or 27, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 6 or 28, or a variant or fragment thereof.

In other embodiments, the nucleic acid of the present invention may include: 2'-deoxy-2'-fluoro modified nucleotides; 2'-deoxy modified nucleotides; locked nucleotides; abasic nucleotides; 2'-amine modified nucleotides; 2'-alkyl modified nucleotides; morpholino nucleotides; nucleotides containing non-natural bases; 2'-O-methyl modified nucleotides; 2'O-methoxyethoxy modified nucleotides; 2' fluoro modified nucleotides; 5- Methyl-modified cytidine; pseudouridine; nucleotides containing a 5'-phosphorothioate group and a terminal nucleotide linked to a cholesterol derivative or dodecyl bis(dodecyl)amide; nucleotides containing phosphoamidate, phosphodiamidate, phosphothioate, phosphorodithioate, phosphophosphocarboxylate, phosphoacetic acid, phosphoformic acid, methylphosphonate, borophosphonate or O-methylphosphoamidate; or nucleotides containing a deoxyribose sugar.

In other embodiments, the nucleic acids of the present invention may be conjugated to a ligand.

In other embodiments, the nucleic acid of the present invention may be RNA, antisense RNA or siRNA.

Other aspects of the invention provide cells comprising the nucleic acids described herein, vectors comprising nucleic acid sequences encoding RNA, antisense RNA or siRNA described herein, lipid nanoparticles comprising the nucleic acids and/or vectors described herein, and pharmaceutical compositions comprising the nucleic acids, vectors and/or lipid nanoparticles described herein.

In some embodiments, the lipid nanoparticles of the present invention may comprise any one or more of the following: non-cationic lipids, cationic lipids, and bound lipids for preventing nanoparticle aggregation.

In other embodiments, the pharmaceutical composition may be a liquid for intravenous administration or an aerosol for intranasal administration.

Other aspects of the invention provide methods for inhibiting coronavirus replication in cells, methods for treating coronavirus infection in an individual, and methods for treating COVID-19 disease in an individual.

In some embodiments, a method for inhibiting coronavirus replication in a cell is provided, the method comprising administering to the cell a nucleic acid, vector, lipid nanoparticle or pharmaceutical composition described herein, thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting the replication of the coronavirus.

In other embodiments, a method for treating a coronavirus infection in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition described herein, thereby inhibiting coronavirus replication and treating the infection.

In other embodiments, a method for treating COVID-19 disease in an individual is provided, the method comprising administering to the individual a therapeutically effective amount of a nucleic acid, vector, lipid nanoparticle, or pharmaceutical composition described herein, thereby inhibiting the replication of the coronavirus and treating the COVID-19 disease.

In some embodiments, the coronavirus is SARS-CoV-2.

Other aspects of the present invention provide methods for preparing pharmaceutical preparations and medical uses of the various nucleic acids, vectors, lipid nanoparticles and pharmaceutical compositions described herein.

In some embodiments, a use of a nucleic acid, a vector, a lipid nanoparticle, or a pharmaceutical composition described herein is provided for preparing a medicament for inhibiting coronavirus replication in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual.

In other embodiments, the nucleic acid, vector, lipid nanoparticle or pharmaceutical composition described herein is provided for inhibiting the replication of coronavirus in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual.

In some embodiments, the coronavirus is SARS-CoV-2.

Definitions As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells unless otherwise specified.

As used herein, the term "comprising" means "including" in a non-exhaustive sense. Variations of the word "comprising", such as "comprise" and "comprises", have correspondingly modified meanings. Thus, for example, a composition "comprising" a given component A may consist exclusively of component A, or may include one or more additional components, such as component B. Similarly, a pharmaceutical composition "comprising" a given nucleic acid may include one or more additional components, such as pharmaceutically acceptable excipients, diluents and/or carriers.

As used herein, the term "about" when used in reference to a recited numerical value includes the recited numerical value and values within plus or minus ten percent of the recited numerical value.

As used herein, the term "SARS-CoV-2" refers to severe acute respiratory syndrome-related coronavirus 2.

As used herein, the term "nucleic acid" refers to nucleotides and nucleotide polymers. The nucleotides covered include ribonucleotides, deoxyribonucleotides, modified forms thereof, and combinations thereof. Polymers of nucleotides may be in single-stranded or double-stranded form, including single-stranded and double-stranded RNA, single-stranded and double-stranded DNA, and double-stranded RNA/DNA hybrids. Non-limiting examples of nucleic acids include all forms of RNA, such as, for example, siRNA, mRNA, miRNA, shRNA antisense RNA, guide RNA, and dicer matrix RNA and siRNA. All such forms of RNA can functionally target and inhibit viruses. Other non-limiting examples of "nucleic acids" include all forms of DNA, such as, for example, genomic DNA, complementary DNA (cDNA), microcircle DNA, and plastid DNA. Also included within the scope of the term "nucleic acid" are nucleic acids containing nucleotide analogs, modified backbone linkages or residues, and the like, which have physical structures related to DNA or RNA molecules or residues, and which are capable of forming hydrogen bonds with DNA or RNA residues or their analogs (i.e., they are capable of hybridizing with DNA or RNA residues or their analogs to form base pairs), and thus have similar binding properties to their structurally related ribonucleotides or deoxyribonucleotide residues (i.e., alkalinucleotides), and which can be metabolized in a manner similar to alkalinucleotides. Non-limiting examples of nucleic acid analogs include methylated, iodinated, brominated, or biotinylated residues and residues with altered sugar components or phosphodiester backbones. Nucleic acid analogs encompassed also include, but are not limited to, nucleic acid analogs with modified nucleotide bases (e.g., pseudouridine, 2'fluoro, 2'O-methyl, 5-methylcytidine, 2'O-methoxyethoxy) and nucleic acid analogs with peptide nucleic acid backbones and linkages. Other analog nucleic acids include nucleic acids with modified sugars (e.g., deoxyribose), non-ionic backbones, cationic backbones, and non-ribose backbones (e.g., locked nucleic acids or phosphodiamidinomorpholino oligonucleotides).

As used herein, the term "RNA interference" refers to a mechanism of action introduced into mammalian cells that can specifically interrupt the production of proteins in the cell in a sequence-specific and potent manner. In this process, RNA molecules participate in the sequence-specific inhibition of gene expression by double-stranded RNA via translational or transcriptional inhibition. For example, RNA interference can be initiated by the introduction of small interfering RNAs (siRNAs, double-stranded RNAs of 15-30 bp), which specifically target mRNAs via sequence complementation, resulting in the subsequent degradation of the mRNAs. In addition, small hairpin RNAs (shRNAs) can also inhibit transcripts and gene expression.

As used herein, an "antisense" nucleic acid or sequence is a nucleic acid or sequence that complements at least one component of a specific target nucleic acid, such complementation facilitates hybridization with the target nucleic acid (e.g., under physiological conditions), thereby inhibiting a biological activity involving the target nucleic acid, non-limiting examples include inhibition of translation of the target nucleic acid, altered transcript splicing, and the like. Antisense nucleic acids will hybridize specifically with a nucleic acid target, meaning that the antisense nucleic acid has a greater tendency to hybridize with its nucleic acid target, e.g., under physiological conditions, than with other non-target nucleic acids. Antisense nucleic acids may include those nucleic acids comprising nucleotide analogs, modified backbone linkages or residues, and the like.

As used herein, the terms "siRNA" and "small interfering RNA" refer to single-stranded or double-stranded RNA (ribonucleic acid) that, when present in a cell containing a target nucleic acid, is capable of inhibiting the expression of a given target nucleic acid by hybridizing with the target nucleic acid and thereby inhibiting its standard biological activity (e.g., its translation into protein). The target nucleic acid may be single-stranded or double-stranded RNA, single-stranded or double-stranded DNA, and non-limiting examples include messenger RNA (mRNA) and promoter sequences. Single-stranded or double-stranded siRNAs may typically be 15 to 50 nucleotides in length. siRNAs may functionally target and inhibit viruses.

As used herein, the terms "shRNA" and "short hairpin RNA" refer to artificial RNA molecules with tight hairpin loops that can be used to silence target gene expression via RNA interference (RNAi). shRNA comprises a stem region of paired antisense and sense strands connected by unpaired nucleotides that form a loop, similar to endogenous microRNAs. The skilled artisan will appreciate that once shRNA is delivered to a cell, it can be loaded into the RNA-induced silencing complex (RISC), which degrades following the sense strand. The antisense (guide) strand directs RISC to mRNAs with complementary sequences for degradation. Thus, shRNA can functionally target and inhibit viruses and is functionally equivalent to siRNA.

As used herein, the term "loop" or "loop region" refers to the sequence that joins two complementary nucleic acid strands. In certain embodiments, the loop region is 4 to 20 nucleotides in length, such as 15 to 19 nucleotides in length. 0% to 50% of the loop region may be complementary to another part of the loop region. The skilled person will fully understand that the nucleotide sequence of the loop region may vary and may be, for example, (5'-GCAA-3'), (5'-GCGC-3') or (5'-TTGC-3') or other sequences. As used herein, the term "hybridise" or "hybridisation" refers to the joining of nucleic acid strands by complementary base pairing, and includes situations where the joining is based on partial complementation or based on complete complementation. As is known to those of ordinary skill in the art, the degree of hybridization between two nucleic acid strands can be affected by parameters such as temperature, salt concentration, and the like, and the conditions under which two complementary or partially complementary nucleic acid strands will hybridize in a particular manner and avoid hybridization with other potential binding partners can be readily determined using standard optimization.

As used herein, the term "variant" refers to substantially similar nucleic acid or polypeptide sequences. In general, sequence variants have a common qualitative biological activity. In addition, such sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the full length of the reference sequence or with a specified region of the reference sequence. The term "variant" also includes homologs, which are usually polypeptides or nucleic acids from different species, but share substantially the same biological function or activity as the corresponding polypeptide or nucleic acid disclosed herein.

As used herein, the percentage of "sequence identity" is understood to be derived from a comparison of two sequences, wherein the sequences are aligned to obtain the maximum correlation between the sequences. This may include inserting "gaps" in one or both sequences to enhance the degree of alignment. The percentage of sequence identity may then be determined over the length of each sequence compared or a portion thereof. For example, a nucleotide sequence ("target sequence") having at least 95% "sequence identity" with another nucleotide sequence ("query sequence") is intended to mean that the target sequence is identical to the query sequence, except that the target sequence may include up to five nucleotide changes per 100 nucleotides of the query sequence. In other words, to obtain a nucleotide sequence having at least 95% sequence identity with the query sequence, up to 5% (i.e., 5 out of 100) of the nucleotides in the target sequence may be inserted into another nucleotide or replaced or deleted by another nucleotide. The percentage of sequence identity between two sequences can be determined by comparing the two best aligned sequences within a comparison window. A portion of the sequence in the comparison window may, for example, comprise deletions or additions (i.e., gaps) compared to a reference sequence that does not comprise deletions or additions (e.g., a reference sequence derived from another species) in order to optimally align the two sequences, or vice versa. The percentage of sequence identity can then be calculated by the following steps: determining the number of positions of identical nucleotides present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. In the case of two or more nucleic acid sequences, percent sequence identity refers to a specified percentage of identical nucleotides in a specified region (or, when not specified, in the entire sequence) when compared and aligned for maximum correspondence within a comparison window or specified region, as measured using one of the following sequence alignment algorithms or by manual alignment and visual inspection. For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. When a sequence comparison algorithm is used, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Sequence alignment methods for comparison are known in the art. The best pairing of sequences to determine sequence identity can be achieved in a learned manner using known computer programs, including, but not limited to, CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); ALIGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using default parameters. Another method for determining the best overall match between a query sequence and a target sequence (also known as a global sequence alignment) can be determined using the FASTDB computer program based on the algorithm of Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)).

As used herein, the term "fragment" with respect to a nucleic acid refers to a component of that nucleic acid. Typically, a fragment has the same qualitative biological activity as a nucleic acid, which may include, for example, hybridization with another target nucleic acid, resulting in reduced expression, inhibition, and the like of the target nucleic acid. A fragment may be derived from a nucleic acid of the invention or alternatively may be synthesized by some other means (e.g., chemical synthesis).

As used herein, the term "isolated" in the context of a nucleic acid or other biological entity is understood to mean that the isolated nucleic acid or other biological entity is at least partially free, and in some cases free or substantially free, of nucleic acids, proteins, lipids, carbohydrates, or other substances that normally accompany the isolated nucleic acid or other biological entity as it is found in its natural/native state. An "isolated" nucleic acid or other biological entity may be purified so as to be partially or completely separated from other biological entities that normally accompany the isolated nucleic acid or other biological entity as it is found in its natural/native state.

As used herein, the terms "treat", "treating", "treatment" and similar terms refer to the reduction or improvement of a disorder, disease and/or symptoms associated therewith. It should be understood that, although not excluded, treatment of a disorder or condition does not necessarily require that the disorder, condition or symptoms associated therewith be completely eliminated. However, it is contemplated that the disorder, condition or symptoms associated therewith should be improved compared to before the start of treatment.

As used herein, the term "subject" includes any animal of economic, social or research interest, including bovine, equine, ovine, primate, avian and rodent species. Thus, an "individual" may be a mammal, such as, for example, a human or a non-human mammal.

As used herein, a "therapeutically effective amount" of a given composition or analog administered to a subject is understood to be an amount sufficient to partially or completely improve the condition or disease being treated, including, for example, a reduction in symptoms of the condition or disease (such as by reducing the severity or frequency of symptoms) or elimination of symptoms. For example, a therapeutically effective amount may result in a reduction of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or 100% of a given symptom. The actual "therapeutically effective amount" will depend on the specific composition administered, the condition or disease being treated, the age and health of the subject being treated, etc. These parameters are routine parameters used for evaluation and can be easily determined by one of ordinary skill in the art.

The following embodiments convey exemplary embodiments of the present invention in sufficient detail to enable those skilled in the art to practice the present invention. The features or limitations of the various embodiments described do not necessarily limit other embodiments of the present invention or the entire present invention. Therefore, the following embodiments do not limit the scope of the present invention, which is defined solely by the scope of the patent application.

It will be appreciated by those skilled in the art that many variations and/or modifications may be made to the invention disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the embodiments of the invention should be considered in all aspects as illustrative and not restrictive.

Inhibitory Nucleic Acids The present invention provides nucleic acids designed to inhibit the expression of a target gene and/or inhibit the biological activity of a non-translated target sequence (eg, a regulatory sequence).

Without limitation, the genes and regulatory sequences targeted by the inhibitory nucleic acids described herein can be derived from pathogens that cause a disease or condition after infecting a host organism. For example, the pathogen can be a virus that can infect mammalian subjects, including humans.

The virus may be an RNA virus, such as, for example, a coronavirus. Non-limiting examples include: alpha coronaviruses, including human coronaviruses HCoV-229E and HCoV-NL63; and beta coronaviruses, including human coronaviruses SARS-CoV-1 (causing severe acute respiratory syndrome), SARS-CoV-2 (causing COVID-19), MERS-CoV (causing Middle East respiratory syndrome), HCoV-HKU1, and HCoV-OC43.

The inhibitory nucleic acids described herein can inhibit the expression of viral proteins, including coronavirus proteins. By way of non-limiting example, the inhibitory nucleic acids can inhibit the expression of structural coronavirus proteins (e.g., spike proteins, membrane proteins, envelope proteins, or nucleocapsid proteins). Proteins can be necessary for viral replication, such as, for example, helicase proteins. Additionally or alternatively, the inhibitory nucleic acids can inhibit the expression of non-structural coronavirus proteins. Additionally or alternatively, the inhibitory nucleic acids can inhibit the expression of ORF1ab and/or polymerase genes.

The inhibitory nucleic acids described herein can inhibit the biological activity of the non-translated segments of the viral genome. For example, the inhibitory nucleic acids can inhibit the biological activity of the stem loop structure in the 5' or 3' non-translated region (i.e., 5'UTR or 3'UTR) of the coronavirus (e.g., stem loop 5 (SL5) of the coronavirus 5'UTR).

The inhibitory nucleic acids described herein may be single-stranded or double-stranded (including but not limited to hairpin structures).

The inhibitory nucleic acids described herein can be between 15 and 30 nucleotides, 15 and 25 nucleotides, 15 and 20 nucleotides, 18 and 25 nucleotides, or 25 and 30 nucleotides, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

The double-stranded inhibitory nucleic acid may be provided in a form where the strands of the duplex are of the same length or of different lengths. The double-stranded inhibitory nucleic acid may thus comprise one or more single-stranded overhangs having at least 1, 2, 3 or 4 nucleotides, 1 to 2 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 2 to 5 nucleotides, 1 to 10 nucleotides, 2 to 10 nucleotides or 5 to 10 nucleotides. The overhang may be provided at the 5' and/or 3' end of the sense strand, the 5' and/or 3' end of the antisense strand, the 5' end of the sense strand and the 5' end of the antisense strand or the 3' end of the sense strand and the 3' end of the antisense strand.

The inhibitory nucleic acids described herein (e.g., variants of inhibitory nucleic acids) may contain one or more mismatches with a target sequence (e.g., 1, 2, 3, 4, or 5 mismatched nucleotides, or less than 5, less than 4, or less than 3 mismatched nucleotides). The mismatches may be located 5' and/or 3' to the central portion of the nucleic acid, including, for example, within 6, 5, 4, 3, or 2 nucleotides of its 3' and/or 5' ends. For example, in the case of a nucleic acid sequence defined by any one of SEQ ID NOs: 1 to 20 or 23 to 42, the mismatches with its respective target sequence may not be present in the central 9, 10, 11, 14, 15, 16, 17, or 18 nucleotides of SEQ ID NOs: 1 to 20 or 23 to 42. Based on the methods described herein and common knowledge in this art, one of ordinary skill can readily determine whether a given variant of an inhibitory nucleic acid described herein that contains nucleotides that are mismatched with its target sequence can still effectively inhibit the expression of a target gene and/or the biological activity of a non-translated target sequence (e.g., a regulatory sequence). In addition, one of ordinary skill will appreciate that there are no substantial differences in potency between siRNAs formed from different overhangs, and that the composition of the overhangs does not appear to play an important role in target mRNA recognition and cleavage. An siRNA may have a dTdT overhang, but may also have a UU or AA overhang or another overhang, such as an overhang that is complementary to the mRNA sequence, or may have no overhang.

The inhibitory nucleic acids described herein may comprise one or more modifications, such as, for example, modified backbones, substitutions of internucleoside bonds, substitutions of sugar moieties, nucleobases, and the like. Typically, and although not required, modifications improve one or more performance parameters of the nucleic acid (e.g., stability, hybridization strength with the target sequence, providing simpler/more cost-effective manufacturing). Those of ordinary skill in the art are familiar with the various nucleic acid modifications available, their benefits, and how to introduce them into a reference sequence.

The inhibitory nucleic acids described herein can be made using standard methods known in the art, including, for example, those described in " Current protocols in nucleic acid chemistry " , Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, the entire contents of which are incorporated herein by cross-reference. Suitable and non-limiting chemical synthesis methods for generating unmodified nucleic acids include procedures utilizing common nucleic acids, such as phosphoamido at the 3' end and a dimethoxytrityl at the 5' end (described, for example, in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433). Taking inhibitory nucleic acids (e.g., siRNA) as a specific example, non-limiting methods include those involving synthesis, removal of protecting groups, and analysis as described, for example, in U.S. Patent Nos. 6,649,751, 6,673,918, 6,686,463, 6,989,442, and 6,995,259. Alternatively, the inhibitory nucleic acids can be synthesized separately and combined after synthesis, for example by ligation (see Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204; International PCT Publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Moore et al., 1992, Science 256, 9923) or by hybridization after synthesis and/or removal of protecting groups. They can also be synthesized as described in U.S. Patent Nos. 5,889,136, 6,008,400 and 6,111,086.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 1 or 23 or a variant or fragment of a sequence defined in SEQ ID NO: 1 or 23. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 1. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 1 or 23.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 2 or 24 or a variant or fragment of a sequence defined in SEQ ID NO: 2 or 24. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 2 or 24. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 2 or 24.

In certain aspects of the invention, an inhibitory nucleic acid is provided, the nucleic acid comprising or consisting of a sequence defined in SEQ ID NO: 3 or 25 or a variant or fragment of a sequence defined in SEQ ID NO: 3 or 25. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 3 or 25. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 3 or 25.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 4 or 26 or a variant or fragment of a sequence defined in SEQ ID NO: 4 or 26. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 4 or 26. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 4 or 26.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 5 or 27 or a variant or fragment of a sequence defined in SEQ ID NO: 5 or 27. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 5 or 27. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 5 or 27.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 6 or 28 or a variant or fragment of a sequence defined in SEQ ID NO: 6 or 28. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 6 or 28. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 6 or 28.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 7 or 29 or a variant or fragment of a sequence defined in SEQ ID NO: 7 or 29. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 7 or 29. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 7 or 29.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 8 or 30 or a variant or fragment of a sequence defined in SEQ ID NO: 8 or 30. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 8 or 30. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 8 or 30.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 9 or 31 or a variant or fragment of a sequence defined in SEQ ID NO: 9 or 31. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 9 or 31. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 9 or 31.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 10 or 32 or a variant or fragment of a sequence defined in SEQ ID NO: 10 or 32. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 10 or 32. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 10 or 32.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 11 or 33 or a variant or fragment of a sequence defined in SEQ ID NO: 11 or 33. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 11 or 33. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 11 or 33.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 12 or 34 or a variant or fragment of a sequence defined in SEQ ID NO: 12 or 34. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 12 or 34. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 12 or 34.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence as defined in SEQ ID NO: 13 or 35, or a variant or fragment of a sequence as defined in SEQ ID NO: 13 or 35. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 13 or 35. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence as defined in SEQ ID NO: 13 or 35.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 14 or 36 or a variant or fragment of a sequence defined in SEQ ID NO: 14 or 36. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to the sequence defined in SEQ ID NO: 14 or 36. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of the sequence defined in SEQ ID NO: 14 or 36.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 15 or 37 or a variant or fragment of a sequence defined in SEQ ID NO: 15 or 37. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 15 or 37. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 15 or 37.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 16 or 38 or a variant or fragment of a sequence defined in SEQ ID NO: 16 or 38. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 16 or 38. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 16 or 38.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 17 or 39 or a variant or fragment of a sequence defined in SEQ ID NO: 17 or 39. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 17 or 39. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 17 or 39.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 18 or 40 or a variant or fragment of a sequence defined in SEQ ID NO: 18 or 40. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 18 or 40. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 18 or 40.

In certain aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence defined in SEQ ID NO: 19 or 41 or a variant or fragment of a sequence defined in SEQ ID NO: 19 or 41. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence defined in SEQ ID NO: 19 or 41. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence defined in SEQ ID NO: 19 or 41.

In other aspects of the invention, an inhibitory nucleic acid is provided that comprises or consists of a sequence as defined in SEQ ID NO: 20 or 42, or a variant or fragment of a sequence as defined in SEQ ID NO: 20 or 42. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 20 or 42. Where an overhang is specified, the variant may include a different overhang. The fragments may comprise or consist of a 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragment of a sequence as defined in SEQ ID NO: 20 or 42.

In other aspects of the invention, an inhibitory nucleic acid is provided, which comprises or consists of a first nucleic acid strand and a second nucleic acid strand that are hybridized by complementary base pairing. In some embodiments, the first strand and the second strand may be in a hairpin structure. In other embodiments, the first strand and the second strand are not in a hairpin structure (i.e., they each terminate separately at their 5' end and 3' end). Either or both strands may have an unhybridized nucleotide overhang (e.g., 1, 2, 3, 4, or 5 unhybridized nucleotides) at their 3' end. Alternatively, there may be no overhang sequence on either strand. The 5' end of either or both strands may be phosphorylated and/or the 3' end of either or both strands may be hydroxylated.

The first strand may comprise or consist of a sequence as defined in SEQ ID NO: 1 or 23, or a variant or fragment of a sequence as defined in SEQ ID NO: 1 or 23. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 1 or 23. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 1 or 23. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 2 or 24, or a variant or fragment of a sequence as defined in SEQ ID NO: 2 or 24. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 2 or 24. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 2 or 24.

Alternatively, the first strand may comprise or consist of a sequence defined in SEQ ID NO: 3, or a variant or fragment of a sequence defined in SEQ ID NO: 3 or 25. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence defined in SEQ ID NO: 3 or 25. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence defined in SEQ ID NO: 3 or 25. The second strand may comprise or consist of a sequence defined in SEQ ID NO: 4 or 26, or a variant or fragment of a sequence defined in SEQ ID NO: 4 or 26. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 4 or 26. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 4 or 26.

Alternatively, the first strand may comprise or consist of a sequence defined in SEQ ID NO: 5, or a variant or fragment of a sequence defined in SEQ ID NO: 5 or 27. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence defined in SEQ ID NO: 5 or 27. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence defined in SEQ ID NO: 5 or 27. The second strand may comprise or consist of a sequence defined in SEQ ID NO: 6 or 28, or a variant or fragment of a sequence defined in SEQ ID NO: 6 or 28. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 6 or 28. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 6 or 28.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 7 or 29, or a variant or fragment of a sequence as defined in SEQ ID NO: 7 or 29. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 7 or 29. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 7 or 29. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 8 or 30, or a variant or fragment of a sequence as defined in SEQ ID NO: 8 or 30. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 8 or 30. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 8 or 30.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 9 or 31, or a variant or fragment of a sequence as defined in SEQ ID NO: 9 or 31. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 9 or 31. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 9 or 31. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 10 or 32, or a variant or fragment of a sequence as defined in SEQ ID NO: 10 or 32. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 10 or 32. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 10 or 32.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 11 or 33, or a variant or fragment of a sequence as defined in SEQ ID NO: 11 or 33. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 11 or 33. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 11 or 33. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 12 or 34, or a variant or fragment of a sequence as defined in SEQ ID NO: 12 or 34. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 12 or 34. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 12 or 34.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 13 or 35, or a variant or fragment of a sequence as defined in SEQ ID NO: 13 or 35. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 13 or 35. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 13 or 35. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 14 or 36, or a variant or fragment of a sequence as defined in SEQ ID NO: 14 or 36. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 14 or 36. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 14 or 36.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 15 or 37, or a variant or fragment of a sequence as defined in SEQ ID NO: 15 or 37. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 15 or 37. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 15 or 37. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 16 or 38, or a variant or fragment of a sequence as defined in SEQ ID NO: 16 or 38. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 16 or 38. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 16 or 38.

Alternatively, the first strand may comprise or consist of a sequence defined in SEQ ID NO: 17 or 39, or a variant or fragment of a sequence defined in SEQ ID NO: 17 or 39. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence defined in SEQ ID NO: 17 or 39. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence defined in SEQ ID NO: 17 or 39. The second strand may comprise or consist of a sequence defined in SEQ ID NO: 18 or 40, or a variant or fragment of a sequence defined in SEQ ID NO: 18 or 40. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 18 or 40. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 18 or 40.

Alternatively, the first strand may comprise or consist of a sequence as defined in SEQ ID NO: 19 or 41, or a variant or fragment of a sequence as defined in SEQ ID NO: 19 or 41. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to a sequence as defined in SEQ ID NO: 19 or 41. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of a sequence as defined in SEQ ID NO: 19 or 41. The second strand may comprise or consist of a sequence as defined in SEQ ID NO: 20 or 42, or a variant or fragment of a sequence as defined in SEQ ID NO: 20 or 42. The variant may comprise or consist of a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity to the sequence defined in SEQ ID NO: 20 or 42. Where overhangs are specified, the variant may include different overhangs. The fragments may comprise or consist of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotide fragments of the sequence defined in SEQ ID NO: 20 or 42.

Pharmaceutical Compositions, Dosages and Routes of Administration The inhibitory nucleic acids of the present invention can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions can be prepared using methods known to those skilled in the art. Non-limiting examples of suitable methods are described in Gennaro et al. (eds.), (1990), "Remington's Pharmaceutical Sciences ", Mack Publishing Co., Easton, Pennsylvania, USA.

The pharmaceutical composition may include a pharmaceutically acceptable carrier, excipient and/or diluent. As used herein, a "pharmaceutically acceptable" carrier, excipient and/or diluent is a substance that does not produce adverse reactions when administered to a specific recipient (such as a human or non-human animal). Pharmaceutically acceptable carriers, excipients and diluents are generally also compatible with the other ingredients of the composition. Non-limiting examples of suitable excipients, diluents and carriers can be found in " Handbook of Pharmaceutical Excipients ", 4th edition, (2003) Rowe et al. (eds.), The Pharmaceutical Press, London, American Pharmaceutical Association, Washington. Non-limiting examples of pharmaceutically acceptable carriers, excipients and diluents include demineralized or distilled water; physiological saline solution; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil or coconut oil; silicone oils including silicones such as methyl silicone, phenyl silicone and methylphenyl silicone; volatile silicones; mineral oils such as liquid wax, soft wax or squalane; cellulose derivatives such as methylcellulose. cellulose, ethylcellulose, sodium carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols such as ethanol or isopropanol; lower aryl alkanols; lower polyalkylene glycols or lower alkanediols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerol; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; tragacanth gum or gum arabic; and petroleum jelly. Typically, one or more carriers will form 10% to 99.9% by weight of the composition.

The pharmaceutical composition may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as eye drops, in the form of an aerosol suitable for administration by inhalation (e.g., intranasal inhalation or oral inhalation), or in a form suitable for parenteral administration (i.e., intradermal, subcutaneous, intramuscular or intravenous injection). Injectable solutions or suspensions may employ non-toxic parenterally acceptable diluents or vehicles, such as, for example, Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol and 1,2-propylene glycol.

The pharmaceutical composition may include any suitable surfactant, such as anionic, cationic or non-ionic surfactants, such as sorbitan esters or their polyethylene oxide derivatives. It may also include suspending agents, such as natural rubber, cellulose derivatives or inorganic materials, such as siliceous silica, and other ingredients, such as wool wax.

In one embodiment, an inhibitory nucleic acid described herein is administered without a delivery system covalently or non-covalently bound to the molecule, ie, as naked siRNA.

In one embodiment, the inhibitory nucleic acid described herein is a protected pharmaceutical composition, for example, protected by encapsulation or binding to a ligand. The pharmaceutical composition can be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multi-layered aqueous liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The pharmaceutical composition in the form of liposomes may contain stabilizers, preservatives, excipients and the like. Preferred lipids are natural and synthetic phospholipids and phospholipid acylcholine (lecithin). Methods for forming liposomes are known in the art and specific references thereto are: Prescott, ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

The pharmaceutical composition can be formulated as lipid nanoparticles. Any suitable liquid nanoparticle delivery system known to those skilled in the art can be used. For example, the inhibitory nucleic acids described herein can be formulated using a lipid nanoparticle composition comprising, for example, cationic lipid/cholesterol/PEG-DMG/DSPC in a ratio of 40/48/2/10, or, for example, cationic lipid/cholesterol/PEG-C-DMA/DSPC in a ratio of 40/48/2/10. The cationic lipid can be, for example, CLinDMA or DLinDMA. The PEG can be, for example, PEG-DMG. Other suitable and non-limiting lipid nanoparticle delivery systems include those described in the examples of this application; Idris et al. 2021, " A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19 " , Molecular Therapy, Vol. 29, No. 7, pp. 2219-2226; Wu et al. 2008, " Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use " , Pharmaceutical Research, Vol. 26, No. 3, pp. 512-522; and U.S. Patent Nos. 7514099, 9061063, 10369226, 11071784, and 11382979.

The pharmaceutical composition can be administered in the form of exosomes. As used herein, the term "exosome" refers to a cell-derived small (diameter between 20 and 300 nm, preferably between 40 and 200 nm) vesicle that includes a membrane encapsulating an internal space, and which is produced from the cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides and further contain inhibitory nucleic acids described herein as being effectively loaded. Exosomes can be derived from production cells and separated from production cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes can be directly loaded with exogenous nucleic acids or drugs by electroporation, liposome transfection, sonication, and contact with calcium chloride. Alternatively, purified exosomes can be loaded ex vivo by, for example, electroporation.

The exosomes of the present invention can be produced by cells grown in vitro or by body fluids of an individual. When the exosomes are produced by cell culture in vitro, various production cells can be used, such as HEK293 cells, Chinese hamster ovary (CHO) cells or mesenchymal stem cells (MSC).

Pharmaceutical compositions can also be formulated by incorporating the inhibitory nucleic acids described herein into adenovirus or adeno-associated virus (AAV), formulated with cell penetrating peptides, lentiviral vectors, polymers, dendrimers, or prepared as siRNA bioconjugates, such as GalNAc-siRNA conjugate delivery platforms.

If cytosomes or vectors are used as vehicles to deliver siRNA, then candidate siRNAs are delivered as shRNAs. Both siRNAs and shRNAs can target and inhibit viruses and are functionally equivalent. When candidate siRNAs are delivered as shRNAs, they are derived from the cellular system and packaged into cytosomes or vectors (AAV or lentiviral vectors), as described above.

The shRNA can be provided in an expression cassette containing a promoter linked in series to the siRNA described herein. In embodiments, the promoter is a polII or polIII promoter, such as a U6 promoter (e.g., a mouse U6 promoter) or an H1 promoter. In embodiments, the expression cassette further contains a marker gene. In embodiments, the promoter is a polII promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a polIII promoter. In embodiments, the promoter is a U6 or H1 promoter.

Also provided are vectors containing the expression cassettes described herein. Examples of suitable vectors include adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based virus vectors. In one embodiment, the vector is an adeno-associated virus (AAV) vector.

The shRNA molecule comprises a paired RNA sequence and a loop portion located between the paired RNA sequences to form a hairpin. The length of the loop can vary. In some embodiments, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In certain embodiments, the length of the loop is 18 nucleotides. The hairpin structure may also contain a 3' and/or 5' protrusion portion. In some embodiments, the protrusion is a 3' and/or 5' protrusion of 0, 1, 2, 3, 4, or 5 nucleotides in length. The skilled artisan will fully appreciate that the nucleotide sequence of the loop region may vary and may be, for example, (5'-GCAA-3'), (5'-GCGC-3') or (5'-TTGC-3') or other sequences.

The dosage of the pharmaceutical compositions described herein may be sufficient to inhibit the expression of the target gene or the biological activity of the non-translated target sequence (e.g., regulatory sequence) in the cell, tissue, or organism being treated. The specific dosage of the inhibitory nucleic acid described herein administered to a given individual will depend on factors such as the route of administration and the individual's physical characteristics (including health status). For example, the appropriate dosage of a given pharmaceutical composition comprising the inhibitory nucleic acid described herein may depend on a variety of factors, including (but not limited to) the individual's physical characteristics (e.g., age, weight, sex), the progression of a given coronavirus infection (i.e., pathological state), and other factors that are readily recognized by those skilled in the art. Various general considerations that may be taken into account in determining the appropriate dosage are described, for example, in Gennaro et al. (eds.), (1990), " Remington's Pharmaceutical Sciences " , Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al. (eds.), (1990), " Goodman And Gilman's: The Pharmacological Bases of Therapeutics " , Pergamon Press. Non-limiting examples of suitable dosages of the inhibitory nucleic acids described herein include dosages in the following ranges: 0.01 to 200 mg/kg of recipient body weight/day, 1 to 50 mg/kg of recipient body weight/day, 1 to 40 mg/kg of recipient body weight/day, 1 to 30 mg/kg of recipient body weight/day, 1 to 20 mg/kg of recipient body weight/day, 1 to 10 mg/kg of recipient body weight/day, 1 to 5 mg/kg of recipient body weight/day, 1 to 3 mg/kg of recipient body weight/day, 1 to 2 mg/kg of recipient body weight/day, 0.1 to 1 mg/kg of recipient body weight/day, 0.1 to 0.9 mg/kg of recipient body weight/day , 0.1 to 0.8 mg/kg body weight/day, 0.1 to 0.7 mg/kg body weight/day, 0.1 to 0.6 mg/kg body weight/day, 0.1 to 0.5 mg/kg body weight/day, 0.1 to 0.4 mg/kg body weight/day, 0.1 to 0.3 mg/kg body weight/day, 0.1 to 0.2 mg/kg body weight/day, 0.01 to 0.1 mg/kg body weight/day, 0.01 to 0.05 mg/kg body weight/day, 0.01 to 0.02 mg/kg body weight/day and 0.005 to 0.01 mg/kg body weight/day.

One of ordinary skill in the art will be able to determine by routine experimentation an effective non-toxic amount of the pharmaceutical compositions and/or inhibitory nucleic acids described herein, which may be included in a single dose or a range of doses to achieve the desired therapeutic result.

Typically, in therapeutic applications, treatment will continue for the duration of the infection, disease condition or condition. Furthermore, it will be apparent to one of ordinary skill in the art that the optimal amounts and intervals of individual doses will be determined by the nature and extent of the infection, disease condition or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimal conditions can also be determined using learned techniques.

In many cases, several or more administrations of the pharmaceutical compositions described herein are required. For example, the pharmaceutical compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Administration may be at intervals of about one week to about twelve weeks, and in certain embodiments at intervals of about one week to about four weeks. In the case of repeated exposure to a specific pathogen targeted by the pharmaceutical compositions described herein, periodic re-administration may be required.

It will also be apparent to one of ordinary skill in the art that the optimal course of treatment can be determined using known course of treatment determination tests.

Suitable techniques for introducing the inhibitory nucleic acids described herein into cells, tissues and organisms include various carrier systems, vectors and reagents. Non-limiting examples include lipid nanoparticles (LNPs), micelles, nucleic acid-lipid particles, liposome complexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus-like particles (VLPs) and mixtures thereof.

The pharmaceutical compositions of the present invention may be administered in any suitable manner, such as, for example, intravenously, buccally, parenterally, intranasally, orally, sublingually or topically. Thus, administration may be topical, pulmonary (e.g., by inhalation or insufflation of an aerosol or powder (including with a nebulizer)), intranasal, intratracheal, epidermal, transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intracerebral parenchymal, intrathecal or intraventricular) administration.

In one embodiment, the pharmaceutical composition is adapted for intranasal administration. When the pharmaceutical composition of the present invention is delivered intranasally, it can achieve significant antiviral targeting effects in the lungs and nasal cavity of mice infected with SARS-COV-2.

In one embodiment, the pharmaceutical composition of the present invention is formulated as a direct-acting nasal spray. In one embodiment, the nasal spray can be self-administered at a designated care station.

Methods of Treatment In general, the inhibitory nucleic acids described herein are shown to inhibit, reduce or prevent coronavirus replication.

Thus, various aspects of the invention provide methods for inhibiting coronavirus replication in a subject in need thereof, methods for treating coronavirus infection, and methods for treating conditions and diseases caused by coronavirus infection.

In some embodiments, a method of inhibiting coronavirus replication in a cell is provided, the method comprising administering to the cell an inhibitory nucleic acid described herein (which may be included in a vector, lipid nanoparticle, or pharmaceutical composition), thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting coronavirus replication.

In other embodiments, a method of treating a coronavirus infection in an individual is provided, the method comprising administering an inhibitory nucleic acid described herein (which may be included in a vector, lipid nanoparticle, or pharmaceutical composition), thereby inhibiting coronavirus replication and treating the infection.

In other embodiments, a method of treating COVID-19 disease in an individual is provided, the method comprising administering to the individual a therapeutically effective amount of an inhibitory nucleic acid described herein (which may be included in a carrier, lipid nanoparticle, or pharmaceutical composition), thereby inhibiting coronavirus replication and treating the COVID-19 disease.

In other embodiments, the use of the inhibitory nucleic acids described herein (which may be included in a carrier, lipid nanoparticle, or pharmaceutical composition) is provided for the preparation of a medicament for inhibiting coronavirus replication in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual.

In other embodiments, an inhibitory nucleic acid described herein (which may be included in a vector, lipid nanoparticle, or pharmaceutical composition) is provided for inhibiting coronavirus replication in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual.

In one embodiment, the administration is intranasal. When the pharmaceutical composition of the present invention is delivered intranasally, significant antiviral targeting is achieved in the lungs and nasal cavity of mice infected with SARS-COV-2. Although not wishing to be bound by theory, it is believed that anti-SARS-CoV-2 siRNA antivirals delivered intranasally can improve viral replication in the nasal cavity and prevent aerosol transmission of respiratory viruses. In one embodiment, the encapsulated siRNA is delivered intranasally.

In one embodiment, administration is by means of a direct acting nasal spray. In one embodiment, the nasal spray can be self-administered at a point-of-care station.

For many respiratory viruses, including SARS-CoV-2, the nasal cavity is a major target of respiratory viral replication early in the disease and a key source of virus spread via aerosols. RNAi is cost-effective, scalable, and can be easily programmed to target any viral RNA. This approach could bring a new, cost-effective, programmable RNA platform technology and self-administered IN delivery platform, and could also provide first-in-class RNA medicines for any emerging RNA respiratory virus of concern.

The subject may be any animal of economic, social or research interest, including bovine, equine, ovine, primate, poultry and rodent species. Thus, the subject may be a mammal, such as, for example, a human or a non-human mammal (e.g., a pig, cat, dog, cow, horse or sheep). The subject may be a laboratory animal (e.g., a rodent, such as a mouse, rat or guinea pig; a rabbit and the like), a bird (e.g., poultry), a fish or a crustacean.

The treatment method can be used for individuals infected with alphacoronaviruses (such as HCoV-229E and HCoV-NL63) or betacoronaviruses (such as SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-HKU1 or HCoV-OC43).

The treatment methods may be used to treat individuals diagnosed with conditions or diseases caused by coronavirus infection, including (but not limited to) severe acute respiratory syndrome, coronavirus disease 2019 (COVID-19), and Middle East respiratory syndrome.

Individuals identified in need of treatment by the methods described herein may include individuals identified by coronavirus infection testing and/or individuals identified by symptom assessment.

Individuals positive for coronavirus infection can be identified using standard techniques known in the art, including, for example, CT scanning, PCR-based methods (e.g., cycle threshold (CT) value, qRT-PCR), sequencing, CRISPR, ELISA, LFA, RT-LAMP, colloidal gold immunolateral flow chromatography.

Symptoms of coronavirus infection vary and are difficult to detect in many cases. In addition, there are overlapping symptoms with symptoms caused by other independent sources. Therefore, in some embodiments, individual symptoms can be assessed for consistency with coronavirus infection, followed by diagnostic testing to confirm or deny symptoms caused by coronavirus infection. Non-limiting examples of symptoms caused by coronavirus infection include (but are not limited to) fever, cough, fatigue, loss of taste or smell, sore throat, headache, muscle or joint pain, nausea or loss of appetite, diarrhea, vomiting, difficulty breathing or shortness of breath, loss of speech or mobility, confusion and/or chest pain.

The efficacy of the treatment methods described herein can be assessed using standard methods known to those generally familiar with the art. For example, PCR-based techniques (e.g., qRTPCR) used to assess coronavirus load during or after treatment can be used to assess the effectiveness of the treatment methods described herein. Common techniques capable of assessing the efficacy of the treatment methods described herein include in vitro plaque assays and the use of in vivo mouse models. Additionally or alternatively, techniques for diagnosing coronavirus infection (including CRISPR, ELISA, LFA, RT-LAMP, colloidal gold immunolateral flow chromatography) can be used to assess the efficacy of treatment, especially when adapted to provide quantitative data.

Examples The present invention will now be described with reference to specific examples, but such examples should not be construed as limiting the present invention in any way.

Example 1 : siRNA targeting SARS-CoV Materials and Methods (a) siRNA design Several candidate siRNAs were developed using an algorithm (https://weinbergmorrislab.wixsite.com/weinbergmorrislab/tgs-algorithm-format; see also Ackley et al. 2013, " An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells " , Molecular Therapy-Nucleic Acids (2013) 2, e104) that selectively finds siRNA seed sequences based on the purine tract in the target RNA. Regions in the viral genome that are highly conserved and easily targeted by siRNA and contain 4-6 bp purine tracts were selected. These non-chemically modified siRNAs were designed to target various conserved sites in the SARS-CoV-2 genome, including the conserved stem of the 5' non-translated region (5'UTR), the helicase region, and the conserved region in the RNA-dependent RNA polymerase (RdRp) gene.

(b) Interferon Screening THP-1 DUAL cells (a well-established standard for measuring immune stimulation) were transfected with the indicated siRNA using Fugene 6 for 24 hours, followed by quantification of (A) IRF and (B) NFkB gene reporter expression. 2'3'-cGAMP (20 μg/ml) and LPS (100 ng/ml) were used as positive controls for stimulation of the IRF and NFkB pathways, respectively.

(c) Plaque Assay The ability of various siRNAs to inhibit SARS-CoV-2 (Vic-1; Wuhan) was assessed using a plaque assay. VeroE6 cells were untreated (virus), untreated (Lipo+virus), or pretreated with 30 nM target siRNA complexed with Lipofectamine 2000 for 24 h prior to infection with 250 PFU of Wuhan (ancestral) SARS-CoV-2. The best new candidate siRNAs were also infected with 250 PFU of α, β, κ, δ, or ο variant SARS-CoV-2. After treatment, viral plaques were counted 4 days later. Data were collected from triplicate treatments.

(d) qRTPCR screening VeroE6 cells were pretreated without (Lipo only) or with siRNA complexed with Lipofectamine 2000 for 24 h before infection with 250 PFU of SARS-CoV-2. The siRNA combinations were selected, mixed at an equimolar ratio to a final concentration of 30 nM, and viral copy numbers were determined by droplet digital PCR targeting the N gene at 4 dpi. Data represent the range of the mean of duplicate treatments.

(e) Dose-response evaluation of delta variant inhibition Viral genes and phylogenetically conserved regions (targeting candidate siRNAs) were designed and selected for screening against SARS-CoV-2 infected cells. VeroE6 cells were pretreated without (Lipo+virus) or with seven dilutions of target siRNA complexed with Lipofectamine 2000 (30, 20, 10, 5, 2.5, 1, or 0.1 nM) for 24 h prior to infection with 250 PFU of delta variant SARS-CoV-2. Viral plaques were counted 4 days after treatment. Data were collected from triplicate treatments.

(f) Lipid nanoparticle (LNP) delivery of siRNA K18hACE2 mice infected with 5×10 ⁴ live delta SARS-CoV-2 variants received prophylactic intravenous (retro-orbital) treatment of siRNA-LNP (1 mg/kg) daily (-1 to 2 dpi). Pulmonary viral tissue counts/g at 3 dpi are shown. Each point represents data from one mouse. The previously discovered siRNA Hel2 that inhibits SARS-COV-2 was compared with the new siRNA siHelUP2.

Results There are several methods for preparing siRNA, such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. In chemical synthesis, commercial suppliers provide siRNA with dTdT 3' overhangs. As an alternative, the overhangs can be UU or other overhangs. The skilled artisan will appreciate that there is no substantial difference in potency between siRNAs formed with dTdT or UU overhangs, and that the composition of the overhangs does not appear to play an important role in target mRNA recognition and cleavage. The overhangs are not part of the target site in the mRNA, but can be used for other purposes, such as Ago-2 and cleavage activity and loading into the complex.

The candidate siRNAs developed are shown in Table 1. The siRNAs in Table 1 have dTdT overhangs and are loaded into LNPs in this form, but may also have UU overhangs or another overhang, such as one that is complementary to the mRNA sequence, or no overhang may be present. Table 1. siRNAs targeting various loci in the SARS-CoV-2 virus. siRNAs include dTdT overhangs. The best performing siRNAs are highlighted in bold/underlined. siRNA Sense RNA sequence (5'-3') Antisense RNA sequence (5'-3') SL5_si1 (siCoV_9) AGAUGGAGAGCCUUGUCCCtt (SEQ ID NO: 1) GGGACAAGGCUCUCCAUCUtt (SEQ ID NO: 2) siHELUp1 (siCoV_4) CGUGGUAAGAGAAUUCCUUtt (SEQ ID NO: 3) AAGGAAUUCUCUUACCACGtt (SEQ ID NO: 4) siHELUp2 (siCoV_1) GAGAAAAGCUGUCUUUAUUtt (SEQ ID NO: 5) AAUAAAGACAGCUUUUCUCtt (SEQ ID NO: 6) siUC7TGS-1 (siCoV_10) CUUACUAAAGGACCUCAUGtt (SEQ ID NO: 7) CAUGAGGUCCUUUAGUAAGtt (SEQ ID NO: 8) siUC7_TGS-2 (siCoV_8) AUUAUCAAAACAAUGUUUUtt (SEQ ID NO: 9) AAAACAUUGUUUUGAUAAUtt (SEQ ID NO: 10) siUC7_TGS-3 (siCoV_7) AUGUCUGAAGCAAAAUGUUtt (SEQ ID NO: 11) AACAUUUGCUUCAGACAUtt (SEQ ID NO: 12) siHEL_TGS-1 (siCoV_6) UUUUGGGACUACCAACUCAtt (SEQ ID NO: 13) UGAGUUGGUAGUCCCAAAAtt (SEQ ID NO: 14) siHEL_TGS-2 (siCoV_5) CCUCAAAGAUUUGGGACUtt (SEQ ID NO: 15) AGUCCCAAAAUCUUUGAGGtt (SEQ ID NO: 16) siHELdwn1 (siCoV_3) UGACAUUGUCAUAUUCACUtt (SEQ ID NO: 17) AGUGAAUAUGACAUAGUCAtt (SEQ ID NO: 18) siHELdwn2 (siCoV_2) ACCACUGAAACAGCUCACUtt (SEQ ID NO: 19) AGUGAGCUGUUUCAGUGGUtt (SEQ ID NO: 20) N367 (Comparison) CUGACCUUUGGAUGGUGCUtt (SEQ ID NO: 21) AGCACCAUCCAAAGGUCAGtt (SEQ ID NO: 22) Table 2. Target sites of siRNAs of Table 1. dTdT overhangs are not shown. siRNA Sense RNA sequence (5'-3') Antisense RNA sequence (5'-3') SL5_si1 (siCoV_9) AGAUGGAGAGCCUUGUCCC (SEQ ID NO: 23) GGGACAAGGCUCUCCAUCU (SEQ ID NO: 24) siHELUp1 (siCoV_4) CGUGGUAAGAGAAUUCCUU (SEQ ID NO: 25) AAGGAAUUCCUUACCACG (SEQ ID NO: 26) siHELUp2 (siCoV_1) GAGAAAAGCUGUCUUUAUU (SEQ ID NO: 27) AAUAAAGACAGCUUUUCUC (SEQ ID NO: 28) siUC7TGS-1 (siCoV_10) CUUACUAAAGGACCUCAUG (SEQ ID NO: 29) CAUGAGGUCCUUUAGUAAG (SEQ ID NO: 30) siUC7_TGS-2 (siCoV_8) AUUAUCAAAACAAUGUUUU (SEQ ID NO: 31) AAAACAUUGUUUUGAUAAU (SEQ ID NO: 32) siUC7_TGS-3 (siCoV_7) AUGUCUGAAGCAAAAUGUU (SEQ ID NO: 33) AACAUUUGCUUCAGACAU (SEQ ID NO: 34) siHEL_TGS-1 (siCoV_6) UUUUGGGACUACCAACUCA (SEQ ID NO: 35) UGAGUUGGUAGUCCCAAAA (SEQ ID NO: 36) siHEL_TGS-2 (siCoV_5) CCUCAAAGAUUUGGGACU (SEQ ID NO: 37) AGUCCCAAAAUCUUUGAGG (SEQ ID NO: 38) siHELdwn1 (siCoV_3) UGACUAUGUCAUAUUCACU (SEQ ID NO: 39) AGUGAAUAUGACAUAGUCA (SEQ ID NO: 40) siHELdwn2 (siCoV_2) ACCACUGAAACAGCUCACU (SEQ ID NO: 41) AGUGAGCUGUUUCAGUGGU (SEQ ID NO: 42) N367 (Comparison) CUGACCUUUGGAUGGUGCU (SEQ ID NO: 43) AGCACCAUCCAAAGGUCAG (SEQ ID NO: 44)

Sequences mentioned for comparison purposes are shown in Tables 3 and 4. Table 3. siRNAs prepared by Idris et al. (2021) and included for comparison. siRNAs include dTdT overhangs. siRNA Sense RNA sequence (5'-3') Antisense RNA sequence (5'-3') UC7_as1 (siCoV_1as) GACAUAAAAACAUUGUUUUtt (SEQ ID NO: 45) AAAACAAUGUUUUAUGUCtt (SEQ ID NO: 46) UC7_as2 (siCoV_2as) AAACAUUGUUUUGAUAAUAatt (SEQ ID NO: 47) UAUUAUCAAAACAAUGUUUtt (SEQ ID NO: 48) UC7_as3 (siCoV_3as) GUAUGUUGAGAGCAAAAUUtt (SEQ ID NO: 49) AAUUUUGCUCUCAACAAUACtt (SEQ ID NO: 50) UC7_as4 (siCoV_4as) UAAUCAUCACCCUGUUUAAtt (SEQ ID NO: 51) UUAAACAGGGUGAUGAUUAtt (SEQ ID NO: 52) UC7_as5 (siCoV_5as) AUUUUGCUUCAGACAUAAAtt (SEQ ID NO: 53) UUUAUGUCUGAAGCAAAAUtt (SEQ ID NO: 54) UTR5_as (siCoV_6as) CUACAAGAGAUCGAAAGUUtt (SEQ ID NO: 55) AACUUUCGAUCUUGUAGtt (SEQ ID NO: 56) Hel4_as (siCoV_7as) CUGUUUCAGUGGUUUGAGUtt (SEQ ID NO: 57) ACUCAAACCACUGAAACAGtt (SEQ ID NO: 58) Hel5_as (siCoV_8as) GGUUUGAGUGAAUAUGACatt (SEQ ID NO: 59) UGUCAUAUUCACUCAAACCtt (SEQ ID NO: 60) Table 4. Target sites of siRNAs prepared by Idris et al. (2021) and included for comparison. dTdT overhangs are not shown. siRNA Sense RNA sequence (5'-3') Antisense RNA sequence (5'-3') UC7_as1 (siCoV_1as) GACAUAAAAACAUUGUUUU (SEQ ID NO: 61) AAAACAAUGUUUUAUGUC (SEQ ID NO: 62) UC7_as2 (siCoV_2as) AAACAUUGUUUUGAUAAUA (SEQ ID NO: 63) UAUUAUCAAAACAAUGUUU (SEQ ID NO: 64) UC7_as3 (siCoV_3as) GUAUGUUGAGAGCAAAAUU (SEQ ID NO: 65) AAUUUUGCUCUCAACAUAC (SEQ ID NO: 66) UC7_as4 (siCoV_4as) UAAUCAUCACCCUGUUUAA (SEQ ID NO: 67) UUAAACAGGGUGAUGAUUA (SEQ ID NO: 68) UC7_as5 (siCoV_5as) AUUUUGCUUCAGACAUAAA (SEQ ID NO: 69) UUUAUGUCUGAAGCAAAAU (SEQ ID NO: 70) UTR5_as (siCoV_6as) CUACAAGAGAUCGAAAGUU (SEQ ID NO: 71) AACUUUCGAUCUCUUGUAG (SEQ ID NO: 72) Hel4_as (siCoV_7as) CUGUUUCAGUGGUUUGAGU (SEQ ID NO: 73) ACUCAAACCACUGAAACAG (SEQ ID NO: 74) Hel5_as (siCoV_8as) GGUUUGAGUAUAUGACA (SEQ ID NO: 75) UGUCAUAUUCACUCAAACC (SEQ ID NO: 76)

Regions in the viral genome that are highly conserved and easily targeted by siRNA and contain a 4-6bp purine tract were selected and screened for any interferon activation ( Figure 1A- Figure 1B ). None of the siRNAs induced any significant interferon activation, indicating that they were non-immunogenic. Subsequently, to determine the ability of various siRNAs to inhibit SARS-CoV-2, Vero cells were transfected and subsequently infected with SARS-COV-2. Several candidates appeared to potently inhibit SARS-COV-2, including siHelUP2ps, siHELdwn3s, SL5-si1S, which were determined by plaque assay ( Figure 2 ) and qRT-PCR ( Figure 3 ).

The effectiveness of these new algorithm candidate siRNAs was then compared with previously published siRNAs that were shown to potently inhibit the Wuhan SARS-COV-2 variant (see Idris et al., 2021, supra) ( Figure 4 ). The best candidate siRNAs appear to be conserved for specific regions, and previous studies have also found potent siRNA candidates for these specific regions, including helicase loci (e.g., siHel2), RNA-dependent RNA polymerases (e.g., siUC7), and 5'UTRs (e.g., siUTR3) (see Idris et al., 2021, supra) ( Figure 4 )

There are several variants of SARS-COV-2, including α, β, κ, and δ. The best candidate siRNAs, including those previously disclosed, were screened for effectiveness against various viral variants. All tested siRNAs, including algorithmically generated siRNAs, were found to be effective in inhibiting various viral variants ( Figure 5 ). The SARS-COV-2 virus undergoes negative RNA strand synthesis during the viral life cycle. The effectiveness of siRNA targeting this step of the viral life cycle was studied, and it was observed that after targeting the negative strand of the δ variant, viral expression was gradually inhibited to a certain extent ( Figure 6 ). To determine the ability of combinations of siRNAs (sense and negative strand targeting siRNAs) to inhibit SARS-COV-2 and whether such combinations have additional benefits, single and double treated cells challenged with the virus were compared, and it was observed that the combination of siRNAs did not greatly enhance or attenuate the observed inhibition ( Figure 7 ). Subsequently, the top three candidate siRNAs were tested for dose response against in vitro inhibition of the delta variant ( Figure 8 ). The top three siRNAs were also evaluated for their siRNA target sites in the new o variant of SARS-CoV-2, and it was observed that all siRNAs contained 100% sequence targeting homology with the new o variant. Finally, the best candidate siRNA, siHelUP2, was selected and compared with siHEL2, which was previously reported to inhibit the δ SARS-CoV-2 virus in vivo (see Idris et al., 2021, supra). Notably, siHelUP2 was able to inhibit SARS-CoV-2 in vivo just as well as siHel2 ( Figure 9 ), indicating that siHelUP2 is also a good candidate siRNA for broad inhibition of SARS-CoV-2.

siHELUp2 (siCoV_1), which targets a conserved site in the helicase gene region of SARS-CoV-2, was subsequently screened for efficacy against various SARS-CoV-2 VOCs and found to be equipotent in all tested VOCs, including the o variant ( FIG. 10 ). Following bioinformatic assessment of the siRNA target site of siHELUp2 in the o variant, we confirmed that this siRNA showed 100% sequence targeting homology with this variant, further emphasizing the ultraconservative design of this siRNA. Compared to siControl, siHELUp2 showed an IC ₅₀ of 3.99 nM (IC ₅₀ =1.3×10 ¹² nM) for the dose response against SARS-CoV-2 ( FIG. 11 ). SARS-CoV-2 undergoes negative RNA strand synthesis during its viral life cycle.

Overall, these data highlight that the algorithm-defined siRNAs are effective in inhibiting all variants of SARS-COV-2, and that the top three candidate siRNAs all work equally well and can be used in LNP-based delivery methods as therapeutics for COVID-19.

Example 2 : Intranasal Administration Materials and Methods Mice were infected intranasally (IN) with 10 ⁵ plaque forming units (PFU) (20 μL total volume) of live δ SARS-CoV-2 VOC using the IN instillation technique under isoflurane anesthesia. Subsequently, mice were treated with siRNA complexed with sLNPs or dmLNPs administered retro-orbitally (IV) (100 μL total volume) or IN (20 μL total volume) while under isoflurane anesthesia. For siRNA administration on the day of infection (day 0 post-infection (dpi)), siRNA was administered 2 hours before mice were infected with SARS-CoV-2. Mice were monitored daily for weight and clinical scoring. We employed the same preventive treatment strategy as previously employed using LNP-based siRNA delivery in K18-hACE2 mice infected with SARS-CoV-2 (Supramaniam et al.) ( FIG. 12A ).

It was observed that IV-delivered siHELUp2 (siCoV-1) inhibited SARS-CoV-2 in the lungs of infected mice when formulated in "stealth LNPs" (sLNPs) ( Figure 12B ). We then formulated siHELUp2 in dmLNPs, a lung-targeted LNP that has been shown to biodistribute and retain siRNA in the nasal cavity (Supramaniam et al.). Notably, IN-delivered siHELUp2-LNPs significantly reduced not only lung viral load, but also nasal viral load. This is the first demonstration of an anti-SARS-CoV-2 siRNA targeting both the lower and upper respiratory tracts. Overall, these data highlight that the algorithm-defined siRNA, siHELUp2, is effective in inhibiting all variants of SARS-COV-2 and that it can be used for LNP-based delivery to reduce viral load in the lungs and nasal cavities of mice.

Example 3 - shRNA If cytosomes or vectors are used as vehicles to deliver siRNA, candidate siRNAs are delivered as shRNAs. Both siRNAs and shRNAs can target and inhibit viruses and are functionally equivalent. When candidate siRNAs are delivered as shRNAs, they are derived from the cellular system and packaged into cytosomes or vectors (AAV or lentiviral vectors). They do not have dtdT overhangs, but only have the sequences shown in Table 2.

The shRNA is provided in an expression cassette containing a promoter linked in series to the siRNA. A dual shRNA expression cassette ( FIG. 13 ) can be used to express siHelUP2 and siHelUP1 packaged into EVs. Both the H1 promoter and the U6 promoter are shown in FIG. 13 , but each promoter and shRNA can be expressed alone or, as in this case, in combination. The skilled person will fully appreciate that the nucleotide sequence of the loop region is (5'-GCAA-3'), but it can also be (5'-GCGC-3') or (5'-TTGC-3') or other sequences.

The sequence of the insert fragment shown in FIG. 13 is as follows: 5'-gaaaaaaGAGAAAAGCTGTCTTTATT GCAA ttgc AAGGAATTCTCTTACCACGtttttt-3' (SEQ ID NO: 77)

References The following references are incorporated herein by reference: Wang H, Paulson KR, Pease SA, Watson S, Comfort H, Zheng P, et al. Estimating excess mortality due to the COVID-19 pandemic: a systematic analysis of COVID-19-related mortality, 2020–21. The Lancet. 2022;399(10334):1513-36. Meganck RM, Baric RS. Developing therapeutic approaches for twenty-first-century emerging infectious viral diseases. Nature Medicine. 2021;27(3):401-10. Cao Z, Gao W, Bao H, Feng H, Mei S, Chen P, et al. VV116 versus Nirmatrelvir-Ritonavir for Oral Treatment of Covid-19. New England Journal of Medicine. 2022;388(5):406-17. Fischer W, Eron JJ, Holman W, Cohen MS, Fang L, Szewczyk LJ, et al. Molnupiravir, an Oral Antiviral Treatment for COVID-19. medRxiv. 2021. Takashita E, Kinoshita N, Yamayoshi S, Sakai-Tagawa Y, Fujisaki S, Ito M, et al. Efficacy of Antibodies and Antiviral Drugs against Covid-19 Omicron Variant. N Engl J Med. 2022;386(10):995-8. Idris A, Davis A, Supramaniam A, Acharya D, Kelly G, Tayyar Y, et al. A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19. Mol Ther. Volume 29, Issue 7, 2219-2226. 2021. Zhang X, Goel V, Robbie GJ. Pharmacokinetics of Patisiran, the First Approved RNA Interference Therapy in Patients With Hereditary Transthyretin-Mediated Amyloidosis. The Journal of Clinical Pharmacology. 2020;60(5):573-85. To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020;20(5):565-74. Chang YC, Yang CF, Chen YF, Yang CC, Chou YL, Chou HW, et al. A siRNA targets and inhibits a broad range of SARS-CoV-2 infections including Delta variant. EMBO Molecular Medicine. n/a(n/a):e15298. Supramaniam A, Tayyar Y, Clarke DTW, Kelly G, Acharya D, Morris KV, et al. Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection. Journal of Microbiology, Immunology and Infection. 2023. Amarilla AA, Modhiran N, Setoh YX, Peng NYG, Sng JDJ, Liang B, et al. An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2. Front Microbiol. 2021;12:625136. Khaitov M, Nikonova A, Shilovskiy I, Kozhikhova K, Kofiadi I, Vishnyakova L, et al. Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation. Allergy. 2021;76(9):2840-54. Bowden-Reid E, Ledger S, Zhang Y, Di Giallonardo F, Aggarwal A, Stella AO, et al. Novel siRNA therapeutics demonstrate multi-variant efficacy against SARS-CoV-2. Antiviral Research. 2023;217:105677. Ackley A, Lenox A, Stapleton K, Knowling S, Lu T, Sabir KS, et al. An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells. Molecular Therapy - Nucleic Acids. 2013;2. McCaskill JL, Marsh GA, Monaghan P, Wang LF, Doran T, McMillan NA. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation. PLoS One. 2013;8(5):e64360. Brutlag et al. Comp. App. Biosci. 6:237-245 (1990). Usman et al., 1987, J. Am. Chem. Soc., 109, 7845. Scaringe et al., 1990, Nucleic Acids Res., 18, 5433. Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951. Bellon et al., 1997, Bioconjugate Chem. 8, 204. Shabarova et al., 1991, Nucleic Acids Research 19, 4247. Moore et al., 1992, Science 256, 9923. Wu et al. 2008, " Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use " , Pharmaceutical Research, Vol. 26, No. 3, 512-522.

Preferred embodiments of the invention will now be described by way of example only and with reference to the accompanying drawings, in which: FIG. 1A and FIG . 1B show interferon screening results for new candidate siRNAs targeting SAR-COV-2. Error bars represent the SEM of three replicates. FIG. 2 provides plaque assay screening and evaluation results for new candidate siRNAs for inhibition of SARS-CoV-2 in vitro. Data are shown as standard error of the mean of three replicates, and ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.001 were considered statistically significant when compared to N3675 (control) as determined by one-way ANOVA analysis (Dunnett post hoc test). Figure 3 shows the results of qRTPCR screening of candidate new siRNAs. Figure 4 provides the results of a plaque assay screening and evaluation of new candidate siRNAs for in vitro inhibition of SARS-CoV-2. Data shown are % plaque inhibition relative to the average plaque count for each treatment for the virus alone. Figure 5 provides the results of another plaque assay screening and evaluation of selected candidate siRNAs for in vitro inhibition of α, β, κ, δ or ο variant SARS-CoV-2 VOCs. Data were collected from triplicate treatments and presented as standard error of the mean of triplicate treatments, and were considered statistically significant when compared to N3675 (control) as determined by one-way ANOVA analysis (Dunnett post hoc test). Figure 6 shows the results of additional plaque assay screening and evaluation of new candidate negative strand targeting siRNAs for inhibition of SARS-CoV-2 delta variants in vitro. Data were collected from triplicate treatments and presented as standard error of the mean of triplicate treatments, and were considered statistically significant at ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.001 compared to N3675 (control) as determined by one-way ANOVA analysis (Dunnett post hoc test). Figure 7 provides the results of additional plaque assay screening and evaluation of new candidate negative and positive targeting siRNA combinations for in vitro inhibition of SARS-CoV-2 Wuhan and delta variants. Data were collected from triplicate treatments and are shown as standard error of the mean of triplicate treatments, and were considered statistically significant when compared to N3675 (control) as determined by one-way ANOVA analysis (Dunnett post hoc test). % inhibition of viral plaque lysis by siRNA is also shown. Figures 8(A) to 8(D) provide the results of the dose response evaluation of the first three candidate siRNAs used for in vitro inhibition of delta variants. Data were collected from triplicate treatments and are shown as standard error of the mean of triplicate treatments reflected on each bar graph. FIG. 9 provides A) a timeline of viral infection from day -1 to day 3 followed by treatment, where a daily dose of 1 mg/kg siRNA was administered on each day from day 0 and all mice were killed on day 3; and B) in vivo evaluation of HelUP2 siRNA delivered by stealth LNPs. Pulmonary viral tissue counts/g at 3 dpi are shown. Each point represents data from one mouse. ** p<0.005, one-way ANOVA. FIG. 10 shows the results of an experiment in which Vero E6 cells were transfected with siControl and siCoV_1 (30 nM) complexed with lipofectamine 2000 for 24 hours and then infected with 250 plaque forming units (PFU) of δ SARS-CoV-2 VOC. Infectious viral plaques were counted 4 days post infection (dpi). Data are presented as mean plaque inhibition percentage relative to virus alone (control) and represent standard error (SEM) of the mean of triplicate treatments. One-way ANOVA (Dunnett post hoc test) was performed and compared to siControl. Figure 11 shows the results of an experiment in which Vero E6 cells were transfected with increasing concentrations (0.1-30 nM) of siControl and siCoV-1 complexed with lipofectamine 2000 for 24 hours and then infected with 250 PFU of δ SARS-CoV-2 VOC. Infectious virus plaques were counted at 4 dpi. Data are presented as mean plaque counts and standard error (SEM) of the mean of triplicate treatments. One-way ANOVA (Dunnett post hoc test) was performed and compared to siControl. Figure 12 provides A) a schematic overview of the in vivo assay and siRNA treatment protocol and B) K18h-ACE2 mice infected with 1×10 ⁵ live δ SARS-CoV-2 VOCs received daily intravenous (IV) (retro-orbital, sLNP) or intranasal (IN, dmLNP) treatment of siRNA-LNP (1 mg/kg) every day (-1 to 2 dpi). The control siRNA used here was siN367 (siControl). Tissue viral tissue counts/g at 3 dpi are shown. Each point represents data from one mouse and the bar represents the mean. One-way ANOVA (Dunnett post hoc test) was performed on siControl. Mice were weighed daily and data points represent mean percent weight gain and error bars represent SEM. Two-way ANOVA was performed on siControl at each time point. Figure 13 is a schematic diagram depicting a dual shRNA expression cassette for expressing SARS-COV-2 shRNA to be packaged in EVs. Here both the H1 and U6 promoters are shown, but the U6 or H1 promoter and shRNA can be expressed individually or, as in this case, in combination.

TW202424195A_112139588_SEQL.xml

Claims (26)

An isolated nucleic acid comprising 15 to 30 nucleotides and capable of specifically hybridizing with the SARS-CoV-2 sequence defined in SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or capable of hybridizing with a fragment of the SARS-CoV-2 sequence. The isolated nucleic acid of claim 1, comprising or consisting of: 15 to 25 nucleotides; 15 to 23 nucleotides; 15 to 22 nucleotides; 15 to 21 nucleotides; 15 to 20 nucleotides; 15 to 19 nucleotides; 19 nucleotides; 20 nucleotides; 21 nucleotides, 22 nucleotides or 23 nucleotides. An isolated nucleic acid having at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity with the following sequences: A sequence defined in SEQ ID NO: 2 or 24, wherein the nucleic acid is capable of specific hybridization with the SARS-CoV-2 sequence defined in SEQ ID NO: 23; or A sequence defined in SEQ ID NO: 4 or 26, wherein the nucleic acid is capable of specific hybridization with the SARS-CoV-2 sequence defined in SEQ ID NO: 25; or A sequence defined in SEQ ID NO: 6 or 28, wherein the nucleic acid is capable of specific hybridization with the SARS-CoV-2 sequence defined in SEQ ID NO: 27. An isolated fragment of the isolated nucleic acid of any one of claims 1 to 3, wherein the fragment is 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. A separated double-stranded nucleic acid for inhibiting the expression of SARS-CoV-2, comprising a sense strand and an antisense strand, wherein: The sense strand comprises a sequence defined in SEQ ID NO: 1 or 23, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 2 or 24, or a variant or fragment thereof; or The sense strand comprises a sequence defined in SEQ ID NO: 3 or 25, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 4 or 26, or a variant or fragment thereof; or The sense strand comprises a sequence defined in SEQ ID NO: 5 or 27, or a variant or fragment thereof, and the antisense strand comprises a sequence defined in SEQ ID NO: 6 or 28, or a variant or fragment thereof. The isolated nucleic acid of any one of claims 1 to 3, the isolated fragment of claim 4, or the isolated double-stranded nucleic acid of claim 5, which comprises: 2'-deoxy-2'-fluoro modified nucleotides; 2'-deoxy modified nucleotides; locked nucleotides; abasic nucleotides; 2'-amine modified nucleotides; 2'-alkyl modified nucleotides; morpholino nucleotides; nucleotides containing non-natural bases; 2'-O-methyl modified nucleotides; 2'O-methoxyethoxy modified nucleotides; Nucleotides; 2' fluorine-modified nucleotides; 5-methyl-modified cytidine; pseudouridine; nucleotides containing a 5'-phosphorothioate group and a terminal nucleotide linked to a cholesterol derivative or dodecyl bis(dodecyl)amide; nucleotides containing phosphoamidate, phosphodiamidate, phosphothioate, phosphorodithioate, phosphophosphocarboxylic acid, phosphocarboxylate, phosphoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate or O-methylphosphoamidate; or nucleotides containing a deoxyribose sugar. The separated nucleic acid of any one of claims 1 to 3 or 6, the separated fragment of claim 4 or claim 6, or the separated double-stranded nucleic acid of claim 5 or claim 6, which is bound to a ligand. A cell comprising the isolated nucleic acid of any one of claims 1 to 3, 6 or 7, the isolated fragment of any one of claims 4, 6 or 7, or the isolated double-stranded nucleic acid of any one of claims 5 to 7. The isolated nucleic acid of any one of claims 1 to 3 or 6 to 8, the isolated fragment of any one of claims 4, 6, 7 or 8, or the isolated double-stranded nucleic acid of any one of claims 5 to 8, which is RNA, antisense RNA or siRNA. A vector comprising a nucleotide sequence encoding the RNA, antisense RNA or siRNA of claim 9. A lipid nanoparticle comprising any one or more of the following: The separated nucleic acid of any one of claims 1 to 3, 6, 7 or 9; The separated fragment of any one of claims 4 or 6, 7 or 9; The separated double-stranded nucleic acid of any one of claims 5 to 7 or 9; The carrier of claim 10. The lipid nanoparticle of claim 11, wherein the lipid comprises any one or more of the following: non-cationic lipids, cationic lipids, and bound lipids for preventing aggregation of the nanoparticles. A pharmaceutical composition comprising any one or more of the following: The separated nucleic acid of any one of claims 1 to 3, 6, 7 or 9; The separated fragment of any one of claims 4, 6, 7 or 9; The separated double-stranded nucleic acid of any one of claims 5 to 7 or 9; The carrier of claim 10; The lipid nanoparticle of claim 11 or claim 12; And a pharmaceutically acceptable excipient, carrier or diluent. A pharmaceutical composition as claimed in claim 13, wherein the isolated nucleic acid as claimed in any one of claims 1 to 3, 6, 7 or 9, the isolated fragment as claimed in any one of claims 4, 6, 7 or 9, or the isolated double-stranded nucleic acid as claimed in any one of claims 5 to 7 or 9 is protected. The pharmaceutical composition of claim 14, comprising liposomes, AAV or exosomes. The pharmaceutical composition of claim 14, comprising the lipid nanoparticles of claim 11 or claim 12. The pharmaceutical composition of claim 13 comprises naked siRNA. The pharmaceutical composition of claim 13 comprises shRNA. A pharmaceutical composition according to any one of claims 13 to 18, which is a liquid for intravenous administration. A pharmaceutical composition according to any one of claims 13 to 18, which is an aerosol for intranasal administration. A method for inhibiting coronavirus replication in a cell, the method comprising administering to the cell a nucleic acid as described in any one of claims 1 to 3, 6, 7 or 9, a fragment as described in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid as described in any one of claims 5 to 7 or 9, a vector as described in claim 10, a lipid nanoparticle as described in claim 11 or claim 12, or a pharmaceutical composition as described in any one of claims 13 to 20, thereby causing degradation of coronavirus mRNA molecules in the cell and inhibiting the replication of the coronavirus. A method for treating a coronavirus infection in an individual, the method comprising administering to the individual a therapeutically effective amount of a nucleic acid as described in any one of claims 1 to 3, 6, 7 or 9, a fragment as described in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid as described in any one of claims 5 to 7 or 9, a vector as described in claim 10, a lipid nanoparticle as described in claim 11 or claim 12, or a pharmaceutical composition as described in any one of claims 13 to 20, thereby inhibiting the replication of the coronavirus and treating the infection. A method for treating COVID-19 disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a nucleic acid as described in any one of claims 1 to 3, 6, 7 or 9, a fragment as described in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid as described in any one of claims 5 to 7 or 9, a vector as described in claim 10, a lipid nanoparticle as described in claim 11 or claim 12, or a pharmaceutical composition as described in any one of claims 13 to 20, thereby inhibiting the replication of the coronavirus and treating the COVID-19 disease. A use of a nucleic acid as claimed in any one of claims 1 to 3, 6, 7 or 9, a fragment as claimed in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid as claimed in any one of claims 5 to 7 or 9, a vector as claimed in claim 10, a lipid nanoparticle as claimed in claim 11 or claim 12, or a pharmaceutical composition as claimed in any one of claims 13 to 20, for preparing a medicament for inhibiting the replication of coronavirus in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual. A nucleic acid as in any one of claims 1 to 3, 6, 7 or 9, a fragment as in any one of claims 4, 6, 7 or 9, a double-stranded nucleic acid as in any one of claims 5 to 7 or 9, a vector as in claim 10, a lipid nanoparticle as in claim 11 or claim 12, or a pharmaceutical composition as in any one of claims 13 to 20, for inhibiting the replication of coronavirus in cells, treating coronavirus infection in an individual, or treating COVID-19 disease in an individual. A method as in any one of claims 21 to 23, a use as in claim 24, or a nucleic acid, a double-stranded nucleic acid, a vector, a lipid nanoparticle or a pharmaceutical composition as in claim 25, wherein the coronavirus is SARS-CoV-2.