CN115838725A

CN115838725A - Promoter sequence of specific promoter in mammal heart and application thereof

Info

Publication number: CN115838725A
Application number: CN202211739921.0A
Authority: CN
Inventors: 卜晔; 李华鹏; 檀克勤; 陈君霖; 钟育健; 潘越; 陈欢
Original assignee: Guangzhou Packgene Biotech Co ltd
Current assignee: Guangzhou Packgene Biotech Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-03-24
Anticipated expiration: 2042-12-30
Also published as: WO2024138810A1; CN115838725B

Abstract

Provided herein are chimeric promoters capable of specific expression in mammalian heart tissue, comprising: myocardial specific promoters, trans-acting factor binding elements (Trans), transcription factor binding sites (MyoG), muscle specific transcription enhancers (2 RS5, MEF1, MEF 2-C). Also provided herein are expression cassettes, expression vectors, viral particles comprising the chimeric promoters, and therapeutic uses thereof.

Description

Promoter sequence of specific promoter in mammal heart and application thereof

Technical Field

The present invention relates to promoters, and in particular to chimeric promoters capable of specifically promoting gene expression in myocardial tissue of a mammal. The invention also relates to medical application of the chimeric promoter.

Background

Heart disease is a large disease species in the disease field, and as the population ages, mortality and hospitalization rates have long been high. There are many drugs currently used clinically for the prevention and treatment of heart disease, but many patients still develop heart failure. It is estimated that over 3000 million heart failure patients exist in the world at present, and 450 million heart failure patients exist in China. With the progress of the disease, the heart failure patients will enter the end stage of the disease, and the development of new heart failure treatment drugs is particularly important. With the research on the heart failure-related signaling pathway being more and more intensive, new molecular targets for heart failure treatment were discovered. However, many signaling pathway molecules are difficult to regulate by small molecule drugs, and by means of gene therapy, the pathological process of heart failure can be directly and specifically regulated at the molecular and cellular level. Some gene therapy programs aiming at heart failure enter a clinical test stage, achieve certain therapeutic effect and show revolutionary and breakthrough therapeutic prospects. Adeno-associated viruses (AAV), which are characterized by non-pathogenicity and replication defects, are currently the most promising delivery vector system for gene therapy. The use of the myocardial specific promoter is helpful for improving the specific expression of the gene in myocardial tissues, greatly reducing the expression of the gene in non-myocardial tissues, reducing the cytotoxicity of gene therapy and improving the treatment efficiency of diseases such as heart failure.

AAV transduction driven by the E-syn promoter has been shown to have good therapeutic efficacy in animal models of heart failure and muscular dystrophy without any physical or pharmaceutical intervention and is relatively more specific in the heart [1-2], a property that was also demonstrated in our earlier experiments. But its myocardial-specific expression level still remains to be improved. Michael A et al found that an increase in the number of transcription enhancing elements 2RS5 in this promoter contributes to the increase in the level of gene expression that it drives [2]. Trans-acting factor binding elements have been shown to bind to Trans-acting factors(s) in rat α -cardiac MHC, and are also present in mouse sites, with the targeted Trans-acting factor proteins specifically binding to myogenic transcription factors MEF1, MEF2 [3]. MEF2 is a member of the mads-box transcription enhancer family encoded by the MEF2-C gene and plays a role in the development of muscle tissues including the myocardium. MEF2 polypeptide C has transactivation and dnabinding activity, and this protein may play a role in maintaining the differentiation state of muscle cells [5-6]. MyoG has the ability to enhance the expression of a subset of genes driven by Myod, which is essential for the expression of key genes for the myogenic process, and is widely found in various muscle-associated promoters [7-8]. At present, no report has been made to verify whether a combination of these gene sequences can obtain a promoter with a higher level of myocardial-specific expression.

Disclosure of Invention

In one aspect, provided herein is a myocardium-specific chimeric promoter comprising:

1) A myocardial-specific promoter; and

2) One or more enhancers;

wherein the enhancer is selected from the group consisting of the 2RS5 enhancer, the MEF1 enhancer, and the MEF2C enhancer.

In some embodiments, the myocardium-specific promoter is an E-syn promoter; preferably, the E-syn promoter comprises SEQ ID NO:6 or a functional variant having at least 90% sequence identity to SEQ ID No. 6.

In some embodiments, the myocardium-specific chimeric promoter further comprises a Trans-acting factor binding element of Trans; preferably, the Trans-acting factor binding element comprises SEQ ID NO:1, or a functional variant having at least 90% sequence identity to SEQ id No. 1.

In some embodiments, the myocardium-specific chimeric promoter further comprises a MyoG transcription factor binding site;

preferably, the MyoG transcription factor binding site comprises SEQ ID NO:2, or a functional variant having at least 90% sequence identity to SEQ ID No. 2.

In some embodiments, the 2RS5 enhancer comprises SEQ ID NO:3, or a functional variant having at least 90% sequence identity to SEQ id No. 3.

In some embodiments, the MEF1 enhancer comprises SEQ ID NO:4, or a functional variant having at least 90% sequence identity to SEQ id No. 4.

In some embodiments, the MEF2C enhancer comprises SEQ ID NO:5, or a functional variant having at least 90% sequence identity to SEQ id No. 5.

In some embodiments, the myocardium-specific chimeric promoter, in 5 'to 3' direction, comprises, in order, the Trans-acting factor binding element, the MyoG transcription factor binding site, the 2RS5 enhancer, the E-syn promoter, and the MEF1 enhancer.

In some embodiments, the myocardium-specific chimeric promoter comprises the E-syn promoter and at least two of the MEF1 enhancers.

In some embodiments, the myocardium-specific chimeric promoter, from 5 'to 3', comprises the MEF1 enhancer, the E-syn promoter, and the MEF1 enhancer, in that order.

In another aspect, provided herein is a gene expression cassette comprising the aforementioned myocardium-specific chimeric promoter and a gene of interest operably linked thereto.

In another aspect, provided herein is an expression vector comprising a myocardium-specific chimeric promoter or gene expression cassette described above.

In some embodiments, the expression vector is a viral expression vector.

In some embodiments, the expression vector is an adeno-associated virus (AAV) expression vector.

In another aspect, provided herein is a host cell comprising a cardiac muscle-specific chimeric promoter, gene expression cassette or expression vector as described above.

In another aspect, provided herein is a pharmaceutical composition comprising: 1) The above gene expression cassette, expression vector or host cell; and 2) a pharmaceutically acceptable carrier.

In another aspect, provided herein is the use of a gene expression cassette, expression vector, host cell or pharmaceutical composition as described above in the manufacture of a medicament for the treatment of a myocardial-related disorder.

In another aspect, provided herein is a method of treating a myocardial-related disorder comprising administering to a subject in need thereof an effective amount of the gene expression cassette, expression vector, host cell, or pharmaceutical composition.

Two artificially optimized promoter sequences capable of specifically promoting gene transcription in cardiac muscle (or heart) of mammal and their application are disclosed. The first promoter optimizes the E-syn gene promoter by adding a Trans-acting factor binding element, a MyoG transcription factor binding site, a transcription enhancement element 2RS5 and an MEF1 transcription enhancement factor sequence; the second promoter was optimized for the E-syn promoter by adding 2 MEF2 transcription enhancer sequences. Through in vivo experiments of mice, the heart tissue specificity and expression level of the optimized E-syn promoter are greatly improved, and the E-syn promoter has a better application prospect.

Drawings

FIG. 1 shows fluorescence images of mice in vivo when different promoters were used. FIG. 1-1 is supine; fig. 1-2 lying on the left side; fig. 1-3 lying on the right side; fig. 1-4 are in a prone position. Group A: e-syn-luciferase-p2A-maxGFP; group B: trans-MyoG-2RS5-E-syn-MEF1-luciferase-p2A-maxGFP; group C: MEF2C-E-syn-MEF2C-luciferase-p2A-maxGFP. The exposure time is 200ms.

FIG. 2 shows the relative expression levels of the luciferase gene in different tissues (myocardium reference within group) and the relative expression levels of the luciferase gene in cardiac tissues within each group (group A reference) of different promoters at the transcriptional level as determined by qPCR using different promoters. FIG. 2-1 relative expression levels of promoter-driven Luciferase in different organ tissues as determined by group A qPCR; FIGS. 2-2B relative expression levels of promoter-driven Luciferase in different organ tissues as determined by group qPCR; FIGS. 2-3C relative expression levels of promoter-driven Luciferase in different organ tissues as determined by group qPCR; FIGS. 2-4 relative expression levels of luciferase gene in cardiac tissue in each group for the different promoters determined by qPCR.

FIG. 3 shows the results of the expression level of luciferase gene in heart and liver tissues using various promoters as determined by Western Blot at the translation level. FIG. 3-1 Western Blot results of luciferase in various groups of heart and liver tissues using different promoters; FIGS. 3-2Western Blot assay for luciferase gene expression levels in various groups of heart tissues using different promoters; FIGS. 3-3 expression levels of luciferase gene in liver tissues of each group using different promoters determined by Western Blot.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The term "or" refers to a single element of the recited list of selectable elements unless the context clearly dictates otherwise. The term "and/or" means any one, any two, any three, any more, or all of the listed selectable elements.

As used herein, the terms "comprising," "including," "having," and the like are intended to mean that elements not listed are not excluded. These terms also include the situation where only the recited elements are made.

"promoter" is a DNA sequence recognized, bound and initiated by RNA polymerase and contains conserved sequences required for RNA polymerase specific binding and transcription initiation, most of which are located upstream (5' direction) of the transcription initiation point of a structural gene, and the promoter itself is not transcribed. Examples of promoters include, but are not limited to, CMV, EF1A, CAG, CBh, SFFV, and the like.

"chimeric promoter", which may also be referred to as a "combined promoter" or "composite promoter", means that it includes, in addition to a promoter sequence, at least one transcriptional regulatory element that is not naturally present in the transcriptional regulatory sequences of the same gene as the promoter. For example, the promoter is naturally present in the transcriptional regulatory sequence of a first gene, and another transcriptional regulatory element (e.g., a transcription factor recognition binding site) is naturally present in the transcriptional regulatory sequence of a second gene, and when the two transcriptional regulatory elements are manually manipulated to be in the same DNA molecule and control transcription of the same gene, they are considered to constitute a chimeric promoter. In addition, a new chimeric promoter may be formed by further adding a transcription regulatory element to the chimeric promoter, and in this case, the chimeric promoter as a base may be directly referred to as a promoter so as to be distinguished from the new chimeric promoter.

In reference to a chimeric promoter or other transcriptional regulatory element, "myocardial-specific" means that the chimeric promoter or other transcriptional regulatory element preferentially drives or enhances expression of an operably linked gene of interest in the myocardium. "myocardial-specific" does not exclude the possibility that the chimeric promoter or other transcriptional regulatory element drives or enhances the expression of an operably linked gene of interest in another tissue to some extent, except that its expression is much lower relative to that in the myocardium. For example, a myocardium-specific chimeric promoter may drive the expression of a gene of interest in myocardium and liver tissue, but the expression level of the gene of interest in myocardium is more than 2 times, or more than 5 times, or more than 10 times, or more than the expression level in liver tissue.

"transcriptional regulatory element" refers to a nucleotide fragment capable of driving (e.g., a promoter) or enhancing (e.g., an enhancer) the expression of an operably linked gene of interest in a tissue or cell. "transcriptional regulatory sequence" refers to the sum of transcriptional regulatory elements that control the expression of a gene of interest, which may be present consecutively in the same DNA molecule or at intervals in the same DNA molecule.

"operably linked" refers to a regulatory sequence and its regulatory target connected in such a way that the regulatory sequence can exert an effect on its regulatory target. For example, a promoter is "operably linked" to a gene of interest means that the promoter can drive transcription of the gene of interest from the correct start site.

A "transcription factor recognition binding site" refers to a nucleotide sequence on a DNA molecule to which a transcription factor recognizes and binds. The transcription factor, when bound to it, helps to form a transcription initiation complex with other proteins (e.g., RNA polymerase) and initiates the transcription process.

"functional variant" or "functional fragment" refers to a protein or nucleic acid variant that includes some minor modifications (e.g., amino acid or nucleotide deletions, additions, or substitutions) on the original sequence (e.g., the native sequence), which still retains all or at least a portion of the functionality of the original sequence. For example, a functional variant may retain 50%, 60%, 70%, 80%, 90%, 100% or even have a higher activity than the original sequence.

"E-syn promoter" as used herein refers to a promoter that is capable of driving expression of a gene of interest in muscle tissue (e.g., myocardium). The E-syn promoter is a chimeric promoter, and the sequence of the E-syn promoter is shown as SEQ ID NO: and 6.

An "enhancer" or "transcription enhancer," used interchangeably herein, refers to a transcriptional regulatory sequence that enhances the activity of a promoter to which it is operably linked, either by cis or trans action. Enhancers may be located upstream, downstream or within the gene to be regulated, and may even not be necessarily close to the gene to be regulated, e.g. >10kb apart. Enhancer sequences referred to herein include the 2RS5, MEF1 and MEF2 enhancers, which sequences are set forth in SEQ ID NOs: 3. 4 and 5.

As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide. In the case of DNA molecules, where reference is made herein to the sequence of one of the strands, the skilled artisan will appreciate that reference has been made to a double stranded DNA molecule having both its complementary strand or including its complementary strand.

The term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, plasmid, or virus, etc.) that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or can replicate in a host cell. The carrier may comprise one or more of the following components: an origin of replication, one or more regulatory sequences that regulate the expression of the polynucleotide of interest (such as a promoter and/or enhancer), and/or one or more selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, e.g., β -galactose). The term "expression vector" refers to a vector for expressing a gene of interest in a host cell.

"host cell" refers to a cell that may or may not have been a recipient for a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; a plant cell; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 and CHO cells, as well as derived cells thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (in terms of morphological or genomic DNA complementation) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells also include cells transfected in vivo with a nucleic acid molecule or expression vector provided herein.

"Gene of interest" refers to a polynucleotide sequence encoding an RNA or protein product, which can be introduced into a cell or an individual according to the desired purpose, and which is capable of being expressed under suitable conditions. The gene of interest may encode a product of interest, e.g., a therapeutic or diagnostic product of interest. Therapeutic genes of interest can be used, introduced into cells, tissues or organs and expressed to produce the desired therapeutic result. Treatment may be achieved in a variety of ways, including by expressing the protein in a cell that does not express the protein, by expressing the protein in a cell that expresses a mutated version of the protein, by expressing a protein that is toxic to the target cell in which it is expressed (a strategy used, for example, to kill unwanted cells such as cancer cells), by expressing an antisense RNA to induce gene repression or exon skipping, or by expressing a silencing RNA, such as a shRNA, that is intended to inhibit protein expression. The gene of interest may also encode a nuclease for targeting the genome, such as a CRISPR-associated endonuclease or a transcription activator-like effector nuclease (TALEN). In addition, the gene of interest can also be a guide RNA or a set of guide RNAs used with the CRISPR/Cas9 system.

When referring to nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of degree of identity, typically expressed as a percentage, between two nucleotide sequences, e.g., a query sequence and a reference sequence. Typically, prior to calculating the percent identity between two nucleotide sequences, sequence alignments (alignment) and gaps (gaps), if any, are introduced. If the bases in the two sequences are the same at a certain alignment position, the two sequences are considered to be consistent or matched at the position; if the two sequences differ in base, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matched positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the length of the gaps are also taken into account. Commonly used sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of the present invention, the disclosed alignment software BLAST (available from https:// www.ncbi.nlm.nih.gov.) can be used to obtain optimal sequence alignment and to calculate sequence identity between two nucleotide sequences by using default settings.

The term "treatment" includes curative, palliative or prophylactic effects. Thus, therapeutic and prophylactic treatment includes ameliorating the symptoms of a disorder or preventing or otherwise reducing the risk of developing specific symptoms. Treatment may be provided to delay, slow or reverse the progression of the disease and/or one or more symptoms thereof. The term "prophylactic" can be considered to reduce the severity or onset of a particular disorder. "prophylactic" also includes preventing recurrence of a particular disorder in a patient who has previously been diagnosed with the disorder. "therapeutic" may also refer to reducing the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that may benefit an animal, particularly a mammal, more particularly a human subject. In particular embodiments, the mammal may be an individual of a myocardial-related disorder, such as a human patient.

Chimeric promoters

The present inventors designed transcriptional regulatory elements, referred to herein as "chimeric promoters," for driving or enhancing the expression of a gene of interest in the myocardium (or heart).

The chimeric promoter includes: a myocardial-specific promoter; and one or more enhancers; wherein the enhancer is selected from the group consisting of the 2RS5 enhancer, the MEF1 enhancer, and the MEF2C enhancer.

Where two or more enhancers are present, these enhancers are linked directly in series or are separated by a linker sequence. Direct tandem linkage means that the first nucleotide of the latter enhancer immediately follows the last nucleotide of the upstream enhancer. In the case of linkage by a linker sequence, there is an additional nucleotide sequence between the last nucleotide of the upstream enhancer and the first nucleotide of the subsequent downstream enhancer.

In some embodiments, the chimeric promoter further comprises a Trans-acting factor binding element. The trans-acting factor herein refers to a DNA binding protein, such as MEF1 or MEF2. Preferably, the Trans-acting factor binding element of the Trans is located 5' to the chimeric promoter. Trans-acting elements of the Trans can be linked to other transcriptional regulatory sequences in the chimeric promoter either directly or through linker sequences.

In some embodiments, the chimeric promoter further comprises a transcription factor binding site. The transcription factor binding site may be a recognition binding site for a member of the Myod protein family. Members of the Myod protein family may, for example, include Myod1, myf5, myoG and Myf6 transcription factors. Preferably, the transcription factor binding site is a recognition binding site for the transcription factor MyoG. More specifically, the MyoG binding site comprises SEQ ID NO: 2.

In some preferred embodiments, the chimeric promoter, in 5 'to 3' direction, comprises, in order, the Trans-acting factor binding element of Trans, the MyoG transcription factor binding site, the 2RS5 enhancer, the E-syn promoter, and the MEF1 enhancer

In some preferred embodiments, the MEF1 enhancer, the E-syn promoter, and the MEF1 enhancer are included in this order from 5 'to 3'.

In addition, it is contemplated that modifications (additions, substitutions, or deletions) of individual nucleotides of the above-described transcriptional regulatory sequences may still have the ability to specifically express the gene of interest in myocardium, and such modified functional variants should also be included in the scope of the present invention. For example, functional variants of the chimeric promoters provided herein that are myocardial-specific can be obtained by altering (adding, replacing, or deleting) individual nucleotides (e.g., no more than 50, 20, 10, 5, 4, 3, 2, or 1 nucleotide) of the above-described transcriptional regulatory sequences and testing their ability to drive expression of the gene of interest in vitro or in vivo, and such functional variants are also intended to be included within the scope of the present invention. For example, in some embodiments, a functional variant can include a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even greater sequence identity to any of the above-described transcriptional regulatory sequences.

The transcription regulatory elements can be connected directly or through a linker sequence. For example, the linker sequence may be between 1 to 50 nucleotides, such as 1 to 40 nucleotides, for example 1 to 30 nucleotides, such as 1 to 20 nucleotides, for example 1 to 10 nucleotides in length. In some embodiments, the chimeric promoter may be designed in view of the size limitations of the vector to be used, and therefore such linker sequences, if present, are preferably short sequences. Representative short linker sequences include nucleic acid sequences consisting of less than 15 nucleotides, particularly less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 nucleotides, for example a 1 nucleotide linker sequence.

Expression cassette

The chimeric promoters provided herein can be introduced into an expression cassette designed to provide for expression of a gene of interest in a tissue of interest (e.g., myocardium).

Thus, the expression cassettes provided herein include the chimeric promoters and genes of interest described above.

In a particular embodiment, the expression cassettes provided herein comprise, in order from 5 'to 3':

-a chimeric promoter as provided herein;

-a gene of interest; and

-a polyadenylation signal.

From the teachings disclosed herein and the general knowledge in the field of molecular biology and gene therapy, one skilled in the art can also consider incorporating other transcriptional regulatory elements into the chimeric promoters disclosed herein, such as the introduction of other enhancer sequences (e.g., the MCK enhancer or functional variants thereof) and intron sequences. Genes of interest that may be introduced in expression may include any gene of interest, particularly therapeutic gene sequences associated with myocardial disorders. Myocardial-related diseases may include primary and secondary myocardial disorders such as dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and indeterminate cardiomyopathy, as well as viral cardiomyopathy, myocarditis, and the like.

Carrier, cell and pharmaceutical composition

The expression cassettes provided herein can be introduced into a vector. The invention therefore also relates to a vector comprising an expression cassette as described above. The vectors used in the present invention are suitable for RNA/protein expression, in particular for gene therapy.

In some embodiments, the vector is a plasmid vector.

In other embodiments, the vector is a non-viral vector, such as a nanoparticle, lipid Nanoparticle (LNP), or liposome containing an expression cassette of the invention.

In other embodiments, the vector is a transposon-based system, allowing for integration of an expression cassette provided herein into the genome of a target cell.

In another embodiment, the vector is a viral vector suitable for gene therapy. In such cases, additional sequences suitable for producing highly efficient viral vectors can be added to the expression cassettes provided herein, as is well known in the art. In particular embodiments, the viral vector may be derived from an adenovirus, retrovirus, or lentivirus (e.g., an integration-defective lentivirus). In the case where the viral vector is derived from a retrovirus or lentivirus, the additional sequence may be a retrovirus or lentivirus LTR sequence flanking the expression cassette. In another specific embodiment, the viral vector is a parvoviral vector, such as an AAV vector, e.g., an AAV vector suitable for transducing myocardium. In this embodiment, the additional sequence is an AAV ITR sequence flanked by the expression cassettes.

In a preferred embodiment, the vector is an AAV vector. Human adeno-associated virus (AAV) is a naturally replication-defective dependent virus that integrates into the genome of infected cells to establish latent infection. AAV vectors have numerous applications as vectors for human gene therapy. Advantageous properties of the viral vector include its absence of association with any human disease, its ability to infect both dividing and non-dividing cells, and the possibility of infecting a wide range of cell lines derived from different tissues.

Among the serotypes of AAV isolated and well characterized from human or non-human primates (NHPs), human serotype 2 is the first AAV developed as a gene transfer vector. Other presently used AAV serotypes also include AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and the like. In addition, other non-native engineered variants and chimeric AAVs may also be useful.

AAV viruses can be engineered using conventional molecular biology techniques such that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for modulating stability and particle lifetime, for efficient degradation, for precise delivery to the nucleus.

The desired AAV fragments for assembly into vectors include capsid proteins, including vp1, vp2, vp3 and hypervariable regions, rep proteins, including rep 78, rep 68, rep 52 and rep 40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of different vector systems and host cells.

The invention also relates to an isolated cell, such as a cardiomyocyte, which is transformed with a nucleic acid sequence according to the invention or an expression cassette according to the invention. The cells of the invention may be delivered to a subject in need thereof by injection into a tissue of interest or into the bloodstream of the subject. In a particular embodiment, the invention relates to introducing a nucleic acid molecule or expression cassette of the invention into cells of a subject to be treated and administering said cells into which the nucleic acid or expression cassette has been introduced back to said subject.

Also provided herein are pharmaceutical compositions comprising the above expression cassettes, vectors or host cells. Such compositions comprise a therapeutically effective amount of the above-described expression cassette, vector or cell, and a pharmaceutically acceptable carrier.

With reference to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" is used to refer to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be safely administered and that are suitable for administration by a subject without undue adverse side effects while maintaining the viability of the drug or active agent located therein. Depending on the route of administration, a variety of different carriers well known in the art may be used, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and/or pyrogen-free water, among others.

The pharmaceutical compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

The pharmaceutical composition provided by the invention can be prepared into clinically acceptable dosage forms such as powder, injection and the like. The pharmaceutical compositions of the invention may be administered to a subject using any suitable route, for example, by oral, intravenous infusion, intramuscular injection, subcutaneous injection, subdermal, rectal, sublingual, or by inhalation, transdermal, etc.

In a preferred embodiment, the pharmaceutical composition is formulated according to conventional procedures suitable for intravenous or intramuscular administration. Typically, pharmaceutical compositions for intravenous or intramuscular administration are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may further comprise a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site of the subject.

As used herein, "subject" refers to an animal, such as a mammal, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. The subject may be male or female and may be any suitable age subject, including infant, juvenile, adolescent, adult and geriatric subjects. In some examples, a subject refers to an individual in need of treatment for a disease or disorder. In some examples, the subject receiving treatment may be a patient who has, or is at risk of developing, a condition associated with the treatment. In a particular example, the subject is a human, such as a human patient. The term is generally used interchangeably with "patient", "test subject", "treatment subject", and the like.

In one embodiment, the expression cassette or vector of the invention may be delivered in vesicles, particularly liposomes. In yet another embodiment, the nucleic acid sequence, expression cassette or vector of the invention may be delivered in a controlled release system.

Therapeutic applications

The chimeric promoters, expression cassettes, or vectors provided herein can be used to express a gene of interest in the myocardium. Thus, in some embodiments, the present invention relates to the use of the above-described expression cassette, vector, cell or pharmaceutical composition in the manufacture of a medicament for the treatment of a myocardial-related disorder.

The expression cassettes and vectors provided herein may also be used in gene therapy. Thus, in some embodiments, the present invention relates to a method of treating a myocardial-related disease comprising administering to a subject in need thereof an effective amount of the above-described expression cassette, vector, cell, or pharmaceutical composition.

In some embodiments, provided herein is a method of expressing a gene of interest in a muscle cell, comprising introducing an expression cassette or vector provided herein into the muscle cell and expressing the gene of interest. The method may be an in vitro, ex vivo or in vivo method for expressing a gene of interest in a muscle cell.

In some embodiments, provided herein is a method of expressing a gene of interest in cardiac muscle comprising introducing an expression cassette or expression vector provided herein into cardiac muscle and expressing the gene of interest.

In certain embodiments, it may be desirable to administer a pharmaceutical composition or the like of the present invention locally to an area in need of treatment, such as local myocardial tissue. This may be achieved, for example, with implants comprising porous, non-porous or gel-like materials.

The dosage administered to a subject in need thereof will vary depending on several factors, including but not limited to the route of administration, the particular disease being treated, the age of the subject, or the level of expression necessary to achieve a therapeutic effect. The required dosage can be readily determined by one skilled in the art based on these and other factors, based on the knowledge in the art. In the case of administration with AAV vectors, a typical dose of the vector is at least 1X 10 per kg body weight ⁸ Copies of the vector genome (vg/kg), e.g., at least 1X 10 ⁹ vg/kg, at least 1X 10 ¹⁰ vg/kg, at least 1X 10 ¹¹ vg/kg, at least 1X 10 ¹² vg/kg, at least 1X 10 ¹³ vg/kg, at least 1X 10 ¹⁴ vg/kg or at least 1X 10 ¹⁵ vg/kg. Of course, the physician may choose to fall outside this range depending on the individual condition of the subjectOther doses of (a).

The invention is further illustrated by the following specific examples.

EXAMPLE 1 construction of plasmid vector

Two artificially optimized promoter sequences capable of specifically promoting genes in cardiac muscle of mammals are obtained by artificially optimizing E-syn promoter genes, and the specific sequences and each transcription regulatory element sequence are as follows.

The vector construction process is as follows:

(1) The target plasmid is constructed by molecular tools such as online gene synthesis website DNAworks (v3.2.4) and seamless cloning. The primers were biosynthesized from Jinzhi. The following plasmids were synthesized:

(2) And constructing the plasmid pAAV-E-syn-luciferase-p 2A-maxGFP. The primers were used to synthesize the E-syn promoter by the following steps: primers were added to the reaction at a concentration of 10. Mu.M in a volume of 0.5. Mu.L and round 1 amplification was performed using the high assurance DNA polymerase PrimeSTARGXL. The reaction system is as follows:

PCR conditions were as follows:

pipette 5. Mu.L of the 1 st round PCR reaction product as template, and take 1. Mu.L of E-syn 5 'and 3' primers for 2 nd round amplification. The reaction system is as follows:

PCR conditions were as follows:

the PCR reaction product of the 2 nd round of gel recovery is named Fragment-1. The plasmid EA0211 (EA 0211 contains ITR sequences at two ends, luciferase-p2A-maxGFP sequences and CAG Promoter consisting of CMV Enhancer, CB Promoter and SV40 intron) is digested by endonuclease MluI and EcoRI, and a 3337bp vector Skeleton is recovered by glue, and the recovered product is named as Skeleton-1. Using EA0211 plasmid as template, designing primer PCR amplification and recovering SV40 intron + luciferase-p2A-maxGFP Fragment, and recovering the product named Fragment-2. The reaction system is as follows:

PCR conditions were as follows:

seamless cloning was performed using a Gibson Assembly Mix ligase premix prepared by this company, and the reaction system was as follows:

reaction conditions are as follows:

taking 10 mu L of seamless clone product to transform E.coli DH5 alpha, coating the product on an ampicillin resistant LB solid plate, culturing for 20h at 32 ℃, inoculating the product on an ampicillin resistant LB liquid culture medium, culturing for 20h at 32 ℃ and 250rpm, taking bacterial liquid for PCR identification, and carrying out restriction enzyme identification on plasmids to screen positive clones. The correct clones were sent to Guangzhou Jinwei Zhi Biotech Co., ltd for sequencing.

(3) Construction of pAAV-Trans-MyoG-2RS5-E-syn-MEF1-luciferase-p2A-maxGFP and pAAV-MEF2C-E-syn-MEF2C-luciferase-p2A-maxGFP plasmids. In a similar way, primers are designed, and the pAAV-E-syn-luciferase-p2A-maxGFP plasmid is used as a PCR template and a plasmid skeleton source to obtain the plasmid.

EXAMPLE 2 preparation of recombinant adeno-associated Virus

HEK-293T cells are transfected by 3 plasmids, the constructed promoter plasmids are packaged into serotype 9 adeno-associated viruses, and C57 mice are infected by tail vein injection to measure the expression efficiency of the luciferase protein at the transcription and translation levels so as to compare the promoter efficiency.

And (4) packaging the recombinant adeno-associated virus. HEK-293T cells are inoculated in a 15cm culture dish at the density of 5E +6cell/well cells and cultured for 24-48 h in a cell culture box at 37 ℃. After the cell density was detected to be 60-80%, 15. Mu.g pHelper, 10. Mu.g pRep2Cap9, 7. Mu.g pAAV-E-syn-luciferase-p2A-maxGFP plasmid (pAAV-Trans-MyoG-2 RS5-E-syn-MEF1-luciferase-p2A-maxGF or pAAV-MEF2C-E-syn-MEF2C-luciferase-p 2A-maxGFP) was added to each dish. And 10. Mu.g of the transfection reagent polyethyleneimine (PEI-pro) were incubated for transfection, 72 hours after transfection. Cells and supernatant were collected, virus was purified, and virus titer was determined by real-time fluorescent quantitative PCR.

Example 3 animal Virus injection, in vivo imaging and tissue sampling

Randomly selecting 3 BALB/c mice with age of 5-6 weeks and weight close to each other from each group, adaptively feeding for 1 week, and performing the above stepsTail vein injection of granule packaged adeno-associated virus with titer of 2.5X 10 ¹² GC/mL, injection volume 200. Mu.L. In vivo imaging was performed on day 21 after virus injection (day of injection is designated as day 1), and the results are shown in FIGS. 1-1 to 1-4. Obviously, the two optimized E-syn promoters showed a further increase in the level of expression of Luciferase in the mouse heart. Tissue sampling was performed on day 2 post-in vivo imaging. 6 tissue parts of Liver (Liver), heart (Heart), forelimb muscle (Fleg), hind limb muscle (Bleg), abdominal muscle (Abdo), lung (Lung) and brain (Brian) were separately soaked in RNA protective solution and stored in a refrigerator at-80 ℃.

Example 4 measurement of expression amount of transcript level of target Gene in tissue sample

(1) RNA extraction of each tissue of the mouse was performed according to the Beijing all-round gold TransZol Up Plus RNA Kit. 50-100 mg of tissue was cut and placed in an RNA free grinding tube, and 2 zirconium oxide grinding beads with a diameter of 3mm, 600. Mu.L of TransZol Up and 120. Mu.L of RNA Extraction Agent were added. Placing the grinding tube in a grinding instrument pre-cooled to-20 ℃ in advance, wherein the grinding parameters are as follows: 70HZ, shake for 50s, stop for 10s, repeat 3 times. Standing at room temperature for 5min,12000xg, centrifuging at 4 ℃ for 15min. The colorless aqueous phase was transferred to a fresh centrifuge tube, an equal volume of absolute ethanol (where precipitation may occur) was added, and the mixture was gently inverted and mixed. Centrifugation can thereafter be carried out at room temperature. The resulting solution was added to the centrifuge together with the pellet at 12000Xg, centrifuged at room temperature for 30s, and the effluent discarded (this can be done in several portions if the volume is larger than the capacity of the column). Add 500. Mu.L of CB9, 12000Xg at room temperature, centrifuge for 30s, and discard the effluent. The CB9 column chromatography was repeated 1 time. Add 500. Mu.L of WB9 (check whether absolute ethanol was added before use), centrifuge at 12000Xg at RT for 30s, and discard the effluent. Repeat WB9 column-pass 1 time. The residual ethanol was removed thoroughly by centrifugation at 12000Xg for 2min at room temperature. Placing the column in RNase-free Tube, adding 50-100 μ L RNase-free Water in the center of the column, and standing at room temperature for 1min. The RNA was eluted by centrifugation at 12000Xg for 1min at room temperature. The RNA was stored at-80 ℃.

(2) Reverse transcription of RNA. cDNA synthesis was performed with reference to the HiScript III RT Supermix for qPCR (+ gDNA wiper) reverse transcription kit of Nanjing Novozae. Removing genome DNA, and preparing the following mixed solution in an RNase-free centrifuge tube:

gently beat and mix with a pipette. 42 ℃ for 2min. The reverse transcription reaction system is as follows:

gently pipetting and mixing. 15min at 37 ℃;85 ℃ for 5s.

(3) Real-time fluorescent quantitative PCR. The transcript level of the target gene Luciferase was quantified by referring to the 2 × SYBR Green qPCR Master Mix kit of Bimake, USA. The qPCR reaction system was configured as follows:

the QPCR program settings are as follows:

as can be seen from FIGS. 2-1 to 2-4, the optimized E-syn promoter can specifically express a target gene in heart tissue compared with the original promoter, and the specific expression levels are respectively increased by 2.59 times and 3.96 times, wherein the specific expression efficiency of the promoter MEF2C-E-syn-MEF2C is remarkably higher (P < 0.05) than that of the E-syn promoter.

Example 5 measurement of expression amount of translation level of target Gene in tissue sample

(1) Extracting protein in the tissue and measuring the concentration of the protein in the tissue by a BCA method. Referring to the using method of Biyuntian RIPA lysate, 20-50 mg of tissue is cut and placed in a grinding tube, 2 zirconium oxide grinding beads with the diameter of 3mM and 300 mu L of RIPA lysate (PMSF with the final concentration of 1 mM) are added, the temperature of a freezing grinder is set to be-20 ℃, and grinding parameters are as follows: 70HZ, shake for 50s, stop for 10s, repeat 3 times. Centrifuge at 12000Xg for 10min at 4 ℃. The supernatant was transferred into a 1.5mL centrifuge tube, and the protein concentration was measured by referring to the method of the modified BCA method protein concentration measurement kit of Biotechnology engineering (Shanghai) Ltd, and stored in a refrigerator at-20 ℃.

(2) Western Bolt. According to the protein concentration, heart and liver tissues of 3 mice are selected from each group, and the protein expression levels are compared. Adjusting the concentration of each sample to be consistent, wherein the loading quantity is 20 mug, SDS-PAGE gel with the concentration of 4-12% is used, and the electrophoresis conditions are as follows: 100V,15min;125V,50min. Film transferring conditions: 300mA, constant current, 60min. Grey scale analysis was performed using ImageJ software to compare the relative expression levels of the target gene proteins. 3-1 to 3-3, the optimized E-syn promoter showed a significant increase in the Luciferase protein level (P1 =0.0382, P2= 0.0029) in the heart group compared to the original promoter, which is about 1.93-fold and 2.65-fold of the expression efficiency of the original promoter, respectively. Meanwhile, the optimized promoter has no significant influence on the expression of the Luciferase protein in the liver.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Reference documents:

[1]Tong,Zhu,Liqiao,et al.Sustained whole-body functional rescue in congestive heart failure and muscular dystrophy hamsters by systemic gene transfer.Circulation,2005；

[2]Wang B,Li J,Fu F H,et al.Construction and analysis of compact muscle-specific promoters for AAV vectors.Gene Therapy,2008,15(22):1489-1499；

[3]Gulick J,Subramaniam A,Neumann J,et al.Isolation and characterization of the mouse cardiac myosin heavy chain genes.Journal of Biological Chemistry,1991,266(14):9180-9185；

[4]Berkes C A,Bergstrom D A,Penn B H,et al.Pbx Marks Genes for Activation by MyoD Indicating a Role for a Homeodomain Protein in Establishing Myogenic Potential[J].Molecular Cell,2004,14(4):465-477；

[5]Zeng ZH,Chen HX,Liu XC,Yang Q,He GW.Functional significance of novel variants ofthe MEF2C gene promoter in congenital ventricular septal defects.Am J Med Genet A.2022Aug；188(8):2397-2405；

[6]Andres V,Cervera M,Mahdavi V.Determination of the Consensus Binding Site for MEF2Expressed in Muscle and Brain Reveals Tissue-specific Sequence Constraints.Journal ofBiological Chemistry,1995,270(40):23246-9；

[7]Kumar R,Berkes C.Cao,Y.et al.Global and gene-specific analyses show distinct roles forMyod and Myog at a common set of promoters.EMBO J.25,502-511；

[8]Singh K,Dilworth F J.Differential modulation of cell cycle progression distinguishesmembers of the myogenic regulatory factor family of transcription factors.Febs Journal,2013,280.

Claims

1. a myocardium-specific chimeric promoter comprising:

1) A myocardial-specific promoter; and

2) One or more enhancers;

2. The myocardium-specific chimeric promoter of claim 1, wherein the myocardium-specific promoter is an E-syn promoter; preferably, the E-syn promoter comprises SEQ ID NO:6 or a functional variant having at least 90% sequence identity to SEQ ID No. 6.

3. The myocardial-specific chimeric promoter of claim 1, further comprising a Trans-acting factor binding element; preferably, the Trans-acting factor binding element comprises SEQ ID NO:1, or a functional variant having at least 90% sequence identity to SEQ ID No. 1.

4. The myocardial-specific chimeric promoter of claim 1, further comprising a MyoG transcription factor binding site; preferably, the MyoG transcription factor binding site comprises SEQ ID NO:2, or a functional variant having at least 90% sequence identity to SEQ ID No. 2.

5. The myocardium-specific chimeric promoter of claim 1, wherein the 2RS5 enhancer comprises SEQ ID NO:3, or a functional variant having at least 90% sequence identity to SEQ ID No. 3.

6. The myocardium-specific chimeric promoter of claim 1, wherein the MEF1 enhancer comprises SEQ ID NO:4, or a functional variant having at least 90% sequence identity to SEQ ID No. 4.

7. The myocardial-specific chimeric promoter of claim 1, wherein the MEF2C enhancer comprises SEQ ID NO:5, or a functional variant having at least 90% sequence identity to SEQ ID No. 5.

8. The myocardial-specific chimeric promoter of claim 1, wherein the Trans-acting factor binding element, the MyoG transcription factor binding site, the 2RS5 enhancer, the E-syn promoter and the MEF1 enhancer are included in this order from 5 'to 3'.

9. The myocardium-specific chimeric promoter of claim 1, comprising the E-syn promoter and at least two of the MEF1 enhancers.

10. The myocardial-specific chimeric promoter of claim 1, wherein the MEF1 enhancer, the E-syn promoter, and the MEF1 enhancer are included in this order from 5 'to 3'.

11. A gene expression cassette comprising the myocardium-specific chimeric promoter of any one of claims 1-10 and a gene of interest operably linked thereto.

12. An expression vector comprising the myocardium-specific chimeric promoter of any one of claims 1 to 10 or the gene expression cassette of claim 11.

13. The expression vector of claim 12, which is a viral expression vector.

14. The expression vector of claim 12 or 13, which is an adeno-associated virus (AAV) expression vector.

15. A host cell comprising the myocardium-specific chimeric promoter of any one of claims 1 to 10, the gene expression cassette of claim 11, or the expression vector of any one of claims 12 to 14.

16. A pharmaceutical composition comprising:

1) A gene expression cassette according to claim 11;

the expression vector of any one of claims 12-14; or

The host cell of claim 15; and

2) A pharmaceutically acceptable carrier.

17. Use of the gene expression cassette of claim 11, the expression vector of any one of claims 12-14, the host cell of claim 15, or the pharmaceutical composition of claim 16 in the manufacture of a medicament for treating a myocardial-related disorder.

18. A method of treating a myocardial-related disorder comprising administering to a subject in need thereof an effective amount of the gene expression cassette of claim 11, the expression vector of any one of claims 12-14, the host cell of claim 15, or the pharmaceutical composition of claim 16.