WO2023160317A1 - Promoter sequence of specific promoting gene in mammalian muscle and uses thereof - Google Patents

Abstract

Provided is a chimeric promoter capable of specific expression in muscle tissue, comprising a muscle-specific transcription factor recognition binding site, a muscle-specific cis-regulatory module and a muscle-specific promoter. Also provided are an expression cassette, an expression vector and a virion comprising the chimeric promoter, and the therapeutic uses thereof.

Description

Promoter sequence and application of specific promoter gene in mammalian muscle

This application claims priority to Chinese Patent Application No. 202210162371.4 filed with the China Patent Office on February 22, 2022, which is hereby incorporated by reference in its entirety.

technical field

The present invention relates to promoters, especially chimeric promoters capable of specifically promoting gene expression in mammalian muscle tissue. The invention also relates to medical applications of the chimeric promoter.

Background technique

Inherited muscle disorders often result in high morbidity and mortality due to skeletal muscle and cardiac dysfunction, and currently lack effective treatments. Gene therapy, an effective and promising approach to treating genetic diseases, is under rapid development and offers hope for many genetic diseases, including muscle disorders. Viral vectors, including lentivirus, adeno-associated virus and other vectors, are common tools for delivering genes in gene therapy. However, there are still some challenges in achieving efficient gene delivery in muscle cells. Enhancing the tissue-specific expression of viral vector drugs by developing muscle tissue-specific promoters, reducing the non-specific expression of delivered genes, achieving controlled release of viral vector drugs and reducing the amount of virus used is one of the ways to improve the application of viral vectors in gene therapy for inherited muscle diseases. an effective method.

Human Desmin gene encodes an intermediate filament structural protein, which is synthesized in cardiac muscle, skeletal muscle, smooth muscle and other muscle cells. The specific expression of Desmin gene in muscle cells is mainly strictly regulated by elements in the promoter region. Desmin gene promoter as a muscle tissue-specific promoter has been reported in many articles, including -973--693 and -228-0 truncations have higher expression efficiency ^[1] . However, its delivery efficiency still has shortcomings. The CRM4 cis-enhancing element, reported by Sarcar S et al., has good specificity in mouse muscle cells ^[2] . Myod1 transcription factor (Myoblast determination protein 1) acts as a transcriptional activator that promotes the transcription of muscle-specific target genes and plays a role in muscle differentiation. However, there is no report on combining them to obtain a promoter with higher activity and specificity.

Contents of the invention

This article discloses an artificially optimized promoter sequence capable of specifically initiating gene transcription in mammalian muscle and its application. The promoter was optimized for human Desmin gene promoter by adding CRM4 cis-enhancer element, Myod1 transcription factor recognition sequence and SV40 intron sequence. Through experiments in mice, the optimized human Desmin gene promoter has greatly improved its tissue specificity and expression ability, and has high application value.

In one aspect, provided herein are muscle-specific chimeric promoters comprising:

1) a first transcriptional regulatory element, the first transcriptional regulatory element is a muscle-specific transcription factor recognition binding site;

2) a second transcriptional regulatory element that is a muscle-specific cis-regulatory module; and

3) The third transcriptional regulatory element, the third transcriptional regulatory element is a muscle-specific promoter.

In some embodiments, the number of the first transcriptional regulatory elements in the muscle-specific chimeric promoter is two or more.

In some embodiments, two or more of the first transcriptional regulatory elements are connected in series or separated by the second transcriptional regulatory element and/or the third transcriptional regulatory element.

In some embodiments, the number of the first transcriptional regulatory element is two, which are respectively located on both sides of the second transcriptional regulatory element or the third transcriptional regulatory element.

In some embodiments, the muscle-specific chimeric promoter further includes a fourth transcriptional regulatory element, and the fourth transcriptional regulatory element is an intron sequence.

In some embodiments, the fourth transcriptional regulatory element is an SV40 intron sequence.

In some embodiments, the muscle-specific transcription factor is a member of the Myod family of proteins.

In some embodiments, the muscle-specific transcription factor is Myod1 transcription factor.

In some embodiments, the first transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 1 or a functional variant having at least 90% sequence identity with SEQ ID NO: 1.

In some embodiments, the second transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 2 or a functional variant having at least 90% sequence identity with SEQ ID NO: 2.

In some embodiments, the third transcriptional regulatory element comprises a desmin gene promoter or a functional fragment thereof.

In some embodiments, the third transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 3 or a functional variant thereof having at least 90% sequence identity to SEQ ID NO: 3.

In some embodiments, the fourth transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO:4 or a functional variant having at least 90% sequence identity with SEQ ID NO:4.

In some embodiments, the muscle-specific chimeric promoter includes the first transcriptional regulatory element, the second transcriptional regulatory element, the first transcriptional regulatory element, the a third transcriptional regulatory element and said fourth transcriptional regulatory element.

In some embodiments, the muscle-specific chimeric promoter comprises the nucleotide sequence set forth in SEQ ID NO:5 or a functional variant having at least 90% sequence identity to SEQ ID NO:5.

In another aspect, provided herein is a gene expression cassette comprising the muscle-specific chimeric promoter described above.

In another aspect, an expression vector is provided herein, which includes the above-mentioned muscle-specific chimeric promoter or the above-mentioned gene expression cassette.

In some embodiments, the expression vector described above 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 the muscle-specific chimeric promoter, gene expression cassette or expression vector described above.

In another aspect, this paper provides a pharmaceutical composition, which includes the above-mentioned gene expression cassette, expression vector or host cell and a pharmaceutically acceptable carrier.

In another aspect, this paper provides the use of the above-mentioned gene expression cassette, expression vector, host cell or pharmaceutical composition in the preparation of medicines for treating muscle-related diseases.

In another aspect, this paper provides a method for treating muscle-related disorders, comprising administering an effective amount of the above-mentioned gene expression cassette, expression vector, host cell or pharmaceutical composition to a subject in need.

The present invention combines the human Desmin gene promoter truncation body through the CRM4 cis-enhancing element, Myod1 transcription factor and SV40 intron sequence to improve its muscle tissue specificity and expression ability.

Description of drawings

Figure 1 shows mouse intravital imaging fluorescence maps using different promoters. Group A: pAAV-human desmin-luciferase-p2A-maxGFP; Group B: pAAV-CRM4-human desmin-luciferase-p2A-maxGFP; Group C: pAAV-Myod1-CRM4-human desmin-luciferase-p2A-maxGFP. Exposure time 100ms.

Figure 2 shows the results of luciferase gene expression in different tissues measured by qPCR using different promoters. Group A: pAAV-human desmin-luciferase-p2A-maxGFP; Group B: pAAV-CRM4-human desmin-luciferase-p2A-maxGFP; Group C: pAAV-Myod1-CRM4-human desmin-luciferase-p2A-maxGFP, each group Muscle tissue was used as a quantitative reference.

Figure 3 shows the results of the expression efficiency of luciferase in different groups of muscle tissues determined by qPCR when using different promoters. Group A: pAAV-human desmin-luciferase-p2A-maxGFP; Group B: pAAV-CRM4-humandesmin-luciferase-p2A-maxGFP; Group C: pAAV-Myod1-CRM4-human desmin-luciferase-p2A-maxGFP. Group A was used as the reference group.

Detailed ways

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

"Promoter" is a DNA sequence that RNA polymerase recognizes, binds, and initiates transcription. It contains conserved sequences required for RNA polymerase specific binding and transcription initiation, and most of them are located upstream of the transcription initiation point of structural genes. The promoter itself is not transcribed. Examples of promoters include, but are not limited to, CMV, EF1A, CAG, CBh, SFFV promoters.

"Chimeric promoter", which may also be referred to as "combined promoter" or "composite promoter", means that, in addition to the promoter sequence, it also includes at least one transcriptional regulatory element, and the transcriptional regulatory element is not related to the promoter. Natively present in the transcriptional regulatory sequence of the same gene. For example, the promoter is naturally present in the transcriptional regulatory sequence of a first gene, and another transcriptional regulatory element (such as a transcription factor recognition binding site) is naturally present in the transcriptional regulatory sequence of a second gene, when the two transcriptional regulatory elements When they are artificially located in the same DNA molecule and control the transcription of the same gene, they can be considered to constitute a chimeric promoter.

"Muscle-specific" when referring to a chimeric promoter or other transcriptional regulatory element means that the chimeric promoter or other transcriptional regulatory element preferentially drives or enhances an operably linked gene of interest in muscle tissue or muscle cells expression. "Muscle-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 to some extent in another tissue, only that its expression is relative to that in another tissue. table of muscle tissue much lower. For example, a muscle-specific chimeric promoter can drive the expression of a gene of interest in muscle tissue and liver tissue, but the expression level of the gene of interest in muscle tissue is more than 2 times, or more than 5 times the expression level in liver tissue, or 10 times more, or 20 times more, or even 50 times more or more.

A "transcriptional regulatory element" refers to a nucleotide segment capable of driving (eg, a promoter) or enhancing (eg, 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 target gene, and they may exist continuously in the same DNA molecule, or exist in the same DNA molecule with intervals.

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

"Transcription factor recognition and binding site" refers to a nucleotide sequence on a DNA molecule that transcription factors can recognize and bind to. After the transcription factor binds to it, it helps to form a transcription initiation complex with other proteins (such as RNA polymerase) to initiate the transcription process.

"Cis-regulatory module (CRM)" refers to a nucleotide sequence on a DNA molecule that provides recognition and binding sites for the simultaneous binding of multiple transcription factors. The cis-regulatory module is described in more detail below.

"Functional variants" or "functional fragments" refer to protein or nucleic acid variants obtained after including minor changes (such as amino acid or nucleotide deletions, additions or substitutions) on the basis of the original sequence (such as a native sequence), which All or at least part of the functionality of the original sequence is still preserved. For example, a functional variant may retain 50%, 60%, 70%, 80%, 90%, 100% of a certain activity of the original sequence or even have a higher activity than the original sequence.

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 unnatural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides comprised in a nucleic acid molecule or polynucleotide. For DNA molecules, since the double strands are complementary, when referring to the sequence of one of the strands herein, those skilled in the art will realize that the complementary strand or double-stranded DNA molecule including its complementary strand has been mentioned at the same time.

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

A "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293, and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. A host cell includes progeny of a single host cell, and progeny Not necessarily identical (in terms of morphology or genomic DNA complementation) to the original mother cell, possibly due to natural, accidental, or deliberate mutation. Host cells also include cells transfected in vivo with nucleic acid molecules or expression vectors provided herein.

"Target gene" refers to a polynucleotide sequence encoding RNA or protein product, which can be introduced into cells or individuals according to the desired purpose, and can be expressed under suitable conditions. A gene of interest may encode a product of interest, such as a therapeutic or diagnostic product of interest. A gene of therapeutic interest may be used after it is introduced into a cell, tissue or organ and expressed to produce a desired therapeutic outcome. Therapy can be achieved in a number of ways, including by expressing the protein in cells that do not express the protein, by expressing the protein in cells that express a mutated version of the protein, by expressing the protein that is toxic to the target cell that expresses it (strategies used eg to kill unwanted cells such as cancer cells), by expressing antisense RNA to induce gene repression or exon skipping, or by expressing silencing RNA, such as shRNA, with the purpose of inhibiting protein expression. The gene of interest may also encode a nuclease for genomic targeting, such as a CRISPR-associated endonuclease or a transcription activator-like effector nuclease (TALEN). Alternatively, the gene of interest can also be a guide RNA or a set of guide RNAs used with the CRISPR/Cas9 system.

The term "treatment" includes curative, palliative or prophylactic effects. Accordingly, therapeutic and preventive treatment include ameliorating the symptoms of a disorder or preventing or otherwise reducing the risk of developing a particular symptom. 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 as reducing the severity or onset of a particular condition. "Prophylactic" also includes preventing the recurrence of a particular condition in patients previously diagnosed with the condition. "Therapeutic" can also mean 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 a particular embodiment, the mammal may be an individual, such as a human patient, of a muscle-related disorder.

chimeric promoter

The inventors designed transcriptional regulatory elements, referred to herein as "chimeric promoters", to drive or enhance expression of a gene of interest in muscle tissue.

The chimeric promoter comprises: one or more muscle-specific transcription factor recognition binding sites; a muscle-specific cis-regulatory module; and a muscle-specific promoter.

In the case of two or more muscle-specific transcription factor recognition binding sites, these recognition binding sites are directly connected in tandem or separated by linker sequences. Direct tandem linkage means that the first nucleotide of the latter binding recognition site immediately follows the last nucleotide of the upstream binding recognition site. In the case of linkage by a linker, there is an additional nucleotide sequence between the last nucleotide of the upstream binding recognition site and the first nucleotide of the subsequent downstream binding recognition site. For example, the linker sequence may be between 1 and 50 nucleotides in length, such as 1 to 40 nucleotides, such as 1 to 30 nucleotides, such as 1 to 20 nucleotides, such as 1 to 20 nucleotides in length. 10 nucleotides. In some embodiments, the design of chimeric promoters can take into account the size constraints of the vector to be used, thus such linker sequences, if present, are preferably short sequences. Representative short linker sequences consist of less than 15 nucleotides, especially less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 cores A nucleic acid sequence composed of nucleotides, such as a linker sequence of 1 nucleotide.

In some embodiments, the muscle-specific transcription factor recognition binding site is a recognition binding site of a Myod protein family member. Myod protein family members may, for example, include Myod1, Myf5, MyoG, and Myf6 transcription factors son. Preferably, the muscle-specific transcription factor recognition binding site is the recognition binding site of transcription factor Myod1. More specifically, the muscle-specific transcription factor recognition binding site includes the nucleotide sequence shown in SEQ ID NO:1. In addition, it can be expected that individual nucleotide changes (addition, substitution or deletion) of the nucleotide sequence may still have Myod1 binding ability (although the binding affinity may be weakened), and the functional variants after these changes should also be included in the scope of the present invention.

The muscle-specific cis-regulatory modules (CRMs) in the chimeric promoters provided herein include clusters of binding sites for transcription factors including E2A, CEBP, LRF, Myod, SREBP, and any combination thereof, such as CEBP, E2A and LRF; E2A, LRF and Myod; LRF and Myod; or CEBP, E2A and LRF. Preferably, the muscle-specific cis-regulatory module comprises E2A, CEBP, LRF, Myod and SREBP recognition binding sites. More specifically, the muscle-specific cis-regulatory module includes the nucleotide sequence shown in SEQ ID NO:2. In addition, it is contemplated that individual nucleotides (e.g., no more than 50, 20, 10, 5, 4, 3, 2 or 1 nucleotide) of the nucleotide sequence are altered ( addition, replacement or deletion), it is also possible to obtain muscle-specific cis-regulatory modules that still have partial transcription factor binding ability (although binding affinity may be weakened), and these altered functional variants should also be included in the scope of the present invention Inside. In some embodiments, the functional variant is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the nucleotide sequence shown in SEQ ID NO:2 %, 97%, 98%, 99% or even higher sequence identity.

Another transcriptional regulatory element in the chimeric promoters provided herein is a muscle-specific promoter, such as a natural, truncated or artificial muscle-specific promoter. In some embodiments, the muscle-specific promoter has a length of less than 2600 nucleotides, especially less than 2000 nucleotides, and has muscle tissue promoter activity when operably linked to a gene of interest. In specific embodiments, the muscle selective promoter has a length of less than 1500 nucleotides, less than 1100 nucleotides, less than 600 nucleotides. Examples of muscle-specific promoters include desmin gene promoter, MLC-2v gene promoter, CK6 promoter, CK8 promoter, Actal gene promoter, MCK gene promoter and the like. Preferably, the muscle-specific promoter is a desmin gene promoter or a variant thereof, such as a truncated desmin gene promoter. In some specific embodiments, the muscle-specific promoter comprises the nucleotide sequence shown in SEQ ID NO:3. In addition, it is contemplated that individual nucleotides (e.g., no more than 50, 20, 10, 5, 4, 3, 2 or 1 nucleotide) of the nucleotide sequence are altered ( addition, replacement or deletion), it is also possible to obtain functional variants that still have muscle-specific promoter activity, and these modified functional variants should also be included within the scope of the present invention. In some embodiments, the functional variant is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the nucleotide sequence shown in SEQ ID NO:3 %, 97%, 98%, 99% or even higher sequence identity.

The chimeric promoters provided herein may also include intron sequences, such as the minute virus of mice (MVM) intron and the SV40 intron. In some embodiments, a chimeric promoter provided herein can include the SV40 intron sequence set forth in SEQ ID NO:4. In other embodiments, the chimeric promoters provided herein may include functional variants of the SV40 intron sequence set forth in SEQ ID NO:4. Generally, these functional variants have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher sequence identity.

Where two or more muscle-specific transcription factor (eg Myod1) recognition binding sites are included in the chimeric promoter provided herein, these muscle-specific transcription factor recognition binding sites may be located in muscle-specific cis The regulatory module is flanked (ie, upstream and downstream thereof), either by a muscle-specific promoter, or by an interconnected muscle-specific cis-regulatory module and a muscle-specific promoter.

The transcriptional regulatory elements of the chimeric promoters provided herein can be connected directly or through a linker sequence. For example, the first regulatory element (i.e. muscle-specific transcription factor recognition binding site) can be directly linked to the second regulatory element (i.e. CRM) directly or via a nucleotide linker, i.e. the last nucleotide of the first regulatory element There are no other nucleotides or nucleotide sequences between the first nucleotide of the second regulatory element. In the case of linkage by a nucleotide linker, between the last nucleotide of the first regulatory element and the first nucleotide of the second regulatory element, there is an additional nucleotide sequence (ie a nucleotide linker). For example, the length of the nucleotide linker may be between 1 and 1500 nucleotides, such as 1 to 1000 nucleotides (such as 101, 300, 500 or 1000 nucleotides), such as 1 to 500 nucleotides Nucleotides, such as 1 to 300 nucleotides, such as 1 to 100 nucleotides, such as 1 to 50 nucleotides, such as 1 to 40 nucleotides, such as 1 to 30 nucleotides, such as 1 to 20 nucleotides, such as 1 to 10 nucleotides. Such nucleotide linkers, if present, are preferably short, taking into account the usual size constraints of carrier molecules. Representative short linkers include those consisting of less than 15 nucleotides, especially less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2 nuclei A nucleic acid sequence composed of nucleotides, such as a nucleotide linker of 1 nucleotide.

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites);

- a second regulatory element (i.e. CRM);

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof); and optionally

- a fourth regulatory element (intron sequence).

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- a second regulatory element (i.e. CRM);

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites);

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof); and optionally

- a fourth regulatory element (intron sequence).

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- a second regulatory element (i.e. CRM);

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof);

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites); and optionally

- a fourth regulatory element (intron sequence).

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites);

- a second regulatory element (i.e. CRM);

- one or more first regulatory elements;

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof); and optionally

- a fourth regulatory element (intron sequence).

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- a second regulatory element (i.e. CRM);

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites);

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof)

- one or more first regulatory elements; and optionally

- a fourth regulatory element (intron sequence).

In a specific embodiment, provided herein is a chimeric promoter comprising, from 5' to 3' in order:

- one or more first regulatory elements (i.e. muscle-specific transcription factor recognition binding sites);

- a second regulatory element (i.e. CRM);

- a third regulatory element (i.e. a muscle-specific promoter or a truncated form thereof)

- one or more first regulatory elements; and optionally

- a fourth regulatory element (intron sequence).

In all embodiments of the chimeric promoters disclosed herein, a nucleotide linker may be included between each transcriptional regulatory element. These nucleotide linkers may be located between first transcriptional regulatory elements or between different transcriptional regulatory elements. The length of the nucleotide linker is as described above. In general, nucleotide linkers with shorter sequences are preferred.

In a specific example, the chimeric promoter provided herein comprises the nucleotide sequence shown in SEQ ID NO:5, or comprises at least 80% sequence identity with SEQ ID NO:5, for example with SEQ ID NO:5 have a sequence identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% and function as a muscle-selective promoter activity sex variant sequence.

expression cassette

The chimeric promoters provided herein can be introduced into expression cassettes designed to provide expression of a gene of interest in a tissue of interest, such as muscle tissue.

Accordingly, the expression cassettes provided herein include the chimeric promoters described above and the gene of interest.

In certain embodiments, the expression cassettes provided herein comprise, in order from 5' to 3':

- a chimeric promoter provided herein;

- the gene of interest; and

- polyadenylation signal.

In certain variations of this embodiment, an intron may be introduced between the chimeric promoter provided herein and the gene of interest. Alternatively, the intron may be located within the gene of interest. In a specific example, the intron may be an SV40 intron, for example comprising the nucleotide sequence shown in SEQ ID NO:4.

From the teachings disclosed herein and general knowledge in the fields of molecular biology and gene therapy, one skilled in the art may also consider incorporating other transcriptional regulatory elements into the chimeric promoters disclosed herein, such as introducing other enhancer sequences ( For example MCK enhancer or functional variant thereof) and intronic sequences.

Genes of interest that may be introduced in expression may include any gene of interest, especially therapeutic gene sequences associated with muscular disorders. These therapeutic genes are expected to be useful in the treatment of the following diseases: muscular dystrophy (such as congenital muscular dystrophy), amyotrophic lateral sclerosis, inflammatory myopathy, muscle metabolism disease (such as glycogen metabolic myopathy ), myotonia congenita, and other neuromuscular disorders.

Vectors, Cells and Pharmaceutical Compositions

The expression cassettes provided herein can be introduced into vectors. Therefore, the present invention also relates to a vector comprising the above-mentioned expression cassette. The vector used in the present invention is a vector suitable for RNA/protein expression, especially 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 the expression cassette of the invention.

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

In another embodiment, the vector is a viral vector suitable for gene therapy. In such cases, additional sequences suitable for the production of highly efficient viral vectors may be added to the expression cassettes provided herein, as is well known in the art. In certain embodiments, the viral vector may be derived from an adenovirus, a retrovirus, or a lentivirus (eg, an integration-deficient lentivirus). Where the viral vector is derived from a retrovirus or lentivirus, the other sequences may be retroviral or lentiviral LTR sequences flanking the expression cassette. In another specific embodiment, said viral vector is a parvoviral vector, such as an AAV vector, such as an AAV vector suitable for transducing muscle. In this embodiment, said other sequences are AAV ITR sequences flanking said expression cassette.

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

Among the serotypes of AAV isolated and well characterized from humans or non-human primates (NHP), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other currently used AAV serotypes include AAV-1, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, etc. In addition, other non-naturally 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 tuning stability and particle lifetime, for Efficient degradation for precise delivery to the nucleus.

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 are readily available in a variety of different vector systems and host cells.

The invention also relates to an isolated cell, such as a muscle cell, transformed with a nucleic acid sequence of the invention or an expression cassette of the invention. The cells of the present invention can be injected into the tissue of interest or the blood stream of a subject in need , delivered to the object. 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 returning 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 aforementioned expression cassettes, vectors or cells, and a pharmaceutically acceptable carrier.

Referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" as used refers to solid or liquid diluents, fillers, antioxidants, stabilizers, etc. drug without undue adverse side effects, while being suitable for maintaining the viability of the drug or active agent located therein. Depending on the route of administration, various 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 buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc.

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

The pharmaceutical composition provided herein can be made into clinically acceptable dosage forms such as powder and injection. The pharmaceutical composition of the present invention may be administered to a subject by any suitable route, for example, orally, intravenously, intramuscularly, subcutaneously, subperitoneally, rectally, sublingually, or by inhalation, transdermally, etc. route of administration.

In a preferred embodiment, the pharmaceutical composition is formulated according to conventional procedures and is 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 also contain a solubilizer 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, sheep, Goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals and mammalian pets. The subject can be male or female and can be of any appropriate age, including infant, infant, adolescent, adult and geriatric subjects. In some instances, a subject refers to an individual in need of treatment for a disease or condition. In some examples, a subject receiving treatment can be a patient who has, or is at risk of developing, a disorder associated with the treatment. In certain instances, the subject is a human, such as a human patient. The term is often used interchangeably with "patient", "subject", "subject" and the like.

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

therapeutic application

The chimeric promoters, expression cassettes or vectors provided herein can be used to express a gene of interest in muscle or myocytes. Therefore, in some embodiments, the present invention relates to the use of the above-mentioned expression cassette, vector, cell or pharmaceutical composition in the preparation of a medicament for treating muscle or muscle cell-related diseases.

The expression cassettes and vectors provided herein can also be used in gene therapy. Therefore, in some embodiments, the present invention relates to a method for treating muscle or muscle cell-related diseases, comprising administering an effective amount of the above-mentioned expression cassette, vector, cell or pharmaceutical composition to a subject in need.

In some embodiments, provided herein is a method for expressing a gene of interest in muscle cells, which comprises introducing the expression cassette or vector provided herein into the muscle cells, 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 muscle cells.

In some embodiments, this paper provides a method for expressing a gene of interest in muscle tissue, which includes introducing the expression cassette or expression vector provided herein into the muscle tissue, and expressing the gene of interest. Preferably, the muscle tissue is skeletal muscle.

In certain embodiments, it may be desirable to administer pharmaceutical compositions of the present invention or the like topically to an area in need of treatment, such as localized muscle tissue. This can be achieved, for example, with implants, including porous, non-porous or gel-like materials.

The dosage administered to a subject in need thereof will vary with 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 obtain a therapeutic effect. Based on these and other factors, one skilled in the art can readily determine the required dosage based on the knowledge of the art. In the case of administration with an AAV vector, a typical dose of the vector is at least 1 x ¹⁰⁸ copies of the vector genome per kilogram of body weight (vg/kg), e.g. at least 1 x ¹⁰⁹ vg/kg, at least 1 x ¹⁰¹⁰ vg/kg , at least 1×10 ¹¹ vg/kg, at least 1×10 ¹² vg/kg, at least 1×10 ¹³ vg/kg, at least 1×10 ¹⁴ vg/kg, or at least 1×10 ¹⁵ vg/kg. Of course, doctors can also choose other doses beyond this range according to the individual conditions of the subjects.

The beneficial effect of the invention is that: by adding CRM4 cis-enhancing element, Myod1 transcription factor recognition binding site and SV40 intron sequence, the human desmin gene promoter truncation is optimized. Compared with the unoptimized human desmin gene promoter truncated body, the optimized promoter has greatly improved its tissue specificity and expression ability in tissues and heart tissues through mouse in vivo experiments.

The present invention is further illustrated below through specific examples.

Example 1

An artificially optimized promoter sequence capable of specifically promoting genes in mammalian muscle is obtained by artificially optimizing the human Desmin gene. The specific sequence and the sequences of each transcriptional regulatory element are as follows.

The vector is constructed as follows:

Example 1 plasmid construction:

(1) The target plasmid was constructed by molecular tools such as the online gene synthesis website DNAWorks (v3.2.4) and seamless cloning. Primers were biosynthesized by Jinweizhi. Synthesize the following plasmids:

(2) First construct pAAV-CRM4-human desmin-luciferase-p2A-maxGFP plasmid. The primers are used to synthesize the CRM4-human desmin promoter. The simple steps are as follows: Dilute the synthesized primers to 100uM, add 0.1μl to the reaction system, and use the high-guarantee enzyme PrimeSTAR to amplify.

The reaction system is as follows:

Amplification system:

Take 1 μl of the above PCR product as the template for the second amplification reaction, add primers F/R at both ends, and carry out the second PCR reaction. The reaction system and conditions are the same as above. The second PCR reaction product was recovered by gel, and the recovered product was named Fragment 1. At the same time, using primers Intron-F/EcoRI-R, using our company's plasmid EA0211 as a template (plasmid EA0211 contains ITR sequences at both ends, luciferase-p2A-maxGFP sequences, and a CAG promoter composed of CMV Ehancer, CB Promoter, and SV40intron) to amplify SV40 intron+luciferase-p2A-maxGFP fragment, and gel recovery, the recovered product was named fragment 2. The vector EA0211 was digested with MluI+EcoRI, and a 3356bp fragment was recovered, and the recovered product was named fragment 3.

(3) Use Exnase Multis ligase for seamless cloning, and connect the vector fragment with the PCR product. The connection system is as follows:

React at 37°C for 30 minutes.

(4) Transfect E.coli DH5ɑ, smear it on an ampicillin plate, pick the bacteria the next day, and send the positive clones to Guangzhou Jinweizhi Company for sequencing. The plasmid with the correct sequencing result is named pAAV-CRM4-human desmin-luciferase- p2A-maxGFP.

(5) Using the primers human-desmin-F/human-desmin-R in the same way, and using the pAAV-CRM4-human desmin-luciferase-p2A-maxGFP plasmid as a template, a PCR reaction was performed to amplify the human-desmin promoter fragment, which was named Fragment 4 (human-desmin promoter sequence). Using luciferase-F/EcoRI-R as primers and EA0211 as a template, luciferase-p2A-maxGFP was amplified and named fragment 5 (luciferase-p2A-maxGFP sequence). Fragment 4, fragment 5 and vector fragment 3 (recovered sequence from EA0211 vector digestion) were seamlessly cloned. The plasmid with correct sequencing results was named pAAV-human desmin-luciferase-p2A-maxGFP.

(6) Using the CRM4-Myod1-F/CRM4-Myod1-R primers carrying the Myod1 transcription factor recognition site -5'GGCAGCTGTTGCT3'- respectively, PCR was performed using pAAV-CRM4-human desmin-luciferase-p2A-maxGFP as a template After amplification, the recovered product was named fragment 6 (CRM4 sequence with Myod1 transcription factor recognition site). At the same time, the primers promoter-F/EcoRI-R, pAAV-CRM4-human desmin-luciferase-p2A-maxGFP were used as templates for PCR amplification, and the recovered product was named fragment 7 (human desmin-luciferase-p2A-maxGFP sequence). Fragment 6, fragment 7 and restriction vector fragment 3 were seamlessly cloned. The plasmid with correct sequencing results was named pAAV-Myod1-CRM4-human desmin-luciferase-p2A-maxGFP.

Example 2 Preparation of recombinant adeno-associated virus

In order to better verify the specificity of the optimized artificial muscle-specific promoter, the expression efficiency of luciferase protein was measured by packaging adeno-associated serotype 9 and transfecting mice for verification.

Recombinant adeno-associated virus packaging. Inoculate in a 15cm culture dish at a density of 5E+5cell/ml cells, and culture overnight for 16-18 hours. Add 15 μg pHelper, 10 μg pRep2Cap9, 7 μg pAAV-CRM4-human-desmin-luciferase-p2A-maxGFP (pAAV-human-desmin-luciferase-p2A-maxGFP or pAAV-Myod1-CRM4-human desmin-luciferase-p2A-maxGFP) per dish ). Incubate transfection with 10 μg of transfection reagent polyethyleneimine, 72 hours after transfection. Cells and supernatant were collected and centrifuged through iodixanol density gradient. Virus titers were determined by SYBRGreenIqPCR and stored in a -80°C refrigerator before use.

Example 3 Live Imaging and Tissue Sampling of Mouse Virus Injection

Twelve BALB/c mice aged 5-6 weeks were selected and randomly divided into 3 groups, 4 mice in each group, 3 experimental mice and 1 blank mouse. The virus was injected into the tail vein (with a titer of 2.5×10 ¹² GC/mL and an injection volume of 200 μL). Taking the day of virus injection as 1 day, live imaging of mice was performed 21 days later. The results are shown in Figure 1. It can be seen from the figure that the expression efficiency of the optimized Myod1-CRM4-human desmin promoter is significantly enhanced, and it is mainly expressed in muscle tissue. Mouse tissue sampling was performed on day 22 after intravital imaging. Six tissue parts of liver, muscle, heart, kidney, brain and spinal cord were collected respectively. The collected tissue samples were stored in a -80°C refrigerator.

Example 4 RNA Extraction and Reverse Transcription Quantification of Mouse Tissue Samples

Follow the instructions of the RNA extraction kit. Take 0.1-0.5g tissue sample, transfer to 1.5ml EP tube containing 1ml transzol up, add two RNA free steel beads. Grind with a vibrating homogenizer. 70HZ, oscillate for 50s, stop for 10s. Repeat 7 times. Let stand at room temperature for 5 minutes, perform centrifugation, and collect the supernatant. Chloroform was added at a ratio of 5:1, shaken vigorously for 30 seconds, and left at room temperature for 3 minutes. Low temperature centrifugation was performed at 4°C. Collect the supernatant, add an equal volume of absolute ethanol, and mix slightly. Add the resulting solution to a spin column, centrifuge, and discard the waste. Add 500μl CB9, centrifuge at room temperature, discard waste liquid, repeat twice. Add 500μl WB9, centrifuge at room temperature, discard waste liquid, repeat twice. Idle for 20s, and remove residual ethanol at room temperature. Add 50 μl of RNA free water for elution. The concentration of the extracted RNA was measured using NanoDrop. Draw 500ng for reverse transcription. SYBRGreenI qPCR was used for quantification, and the results are shown in Figure 2 and Figure 3. It can be seen from Figure 2 that the unoptimized human desmin promoter expresses 40 times more luciferase in liver tissue than in muscle tissue. The expression of the optimized human desmin promoter decreased significantly in liver tissue. It can be seen from Figure 3 that the optimized human desmin muscle tissue expression efficiency is 50 times that of the unoptimized one.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

references:

[1] Li Z, Paulin. High-level desmin expression depends on a muscle-specific enhancer [J]. Cell Biology International Reports, 1990, 14 (ABSTR. SUPPL): 50.

[2] Sarcar S, Tulalamba W, MY Rincón, et al. Next-generation muscle-directed gene therapy by in silico vector design [J]. Nature Communications, 2019, 10(1).

Claims (23)

Muscle-specific chimeric promoters, including: 1) a first transcriptional regulatory element, the first transcriptional regulatory element is a muscle-specific transcription factor recognition binding site; 2) a second transcriptional regulatory element that is a muscle-specific cis-regulatory module; and 3) The third transcriptional regulatory element, the third transcriptional regulatory element is a muscle-specific promoter. The muscle-specific chimeric promoter according to claim 1, wherein the number of the first transcriptional regulatory elements in the muscle-specific chimeric promoter is two or more. The muscle-specific chimeric promoter according to claim 2, wherein two or more of the first transcriptional regulatory elements are connected in series or replaced by the second transcriptional regulatory element and/or the third transcriptional regulatory element separated. The muscle-specific chimeric promoter according to claim 2, wherein the number of the first transcriptional regulatory elements is two, respectively located on both sides of the second transcriptional regulatory element or the third transcriptional regulatory element. The muscle-specific chimeric promoter according to claim 1 or 2, further comprising a fourth transcriptional regulatory element, said fourth transcriptional regulatory element being an intron sequence. The muscle-specific chimeric promoter of claim 5, wherein the fourth transcriptional regulatory element is an SV40 intron sequence. The muscle-specific chimeric promoter according to claim 1 or 2, wherein the muscle-specific transcription factor is a member of the Myod protein family. The muscle-specific chimeric promoter according to claim 7, wherein the muscle-specific transcription factor is Myod1 transcription factor. The muscle-specific chimeric promoter as claimed in claim 1 or 2, wherein said first transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 1 or has at least 90% sequence with SEQ ID NO: 1 Consistent functional variants. The muscle-specific chimeric promoter as claimed in claim 1 or 2, wherein said second transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 2 or has at least 90% sequence with SEQ ID NO: 2 Consistent functional variants. The muscle-specific chimeric promoter according to claim 1 or 2, wherein the third transcriptional regulatory element comprises a desmin gene promoter or a functional fragment thereof. The muscle-specific chimeric promoter according to claim 11, wherein said third transcriptional regulatory element comprises the nucleotide sequence shown in SEQ ID NO: 3 or has at least 90% sequence identity with SEQ ID NO: 3 its functional variant. The muscle-specific chimeric promoter of claim 5, wherein the fourth transcriptional regulatory element comprises The nucleotide sequence shown in SEQ ID NO:4 or a functional variant having at least 90% sequence identity with SEQ ID NO:4. The muscle-specific chimeric promoter according to claim 5, wherein the muscle-specific chimeric promoter comprises the first transcriptional regulatory element, the second transcriptional regulatory element, The first transcriptional regulatory element, the third transcriptional regulatory element, and the fourth transcriptional regulatory element. The muscle specific chimeric promoter as claimed in claim 1 or 2, wherein said muscle specific chimeric promoter comprises the nucleotide sequence described in SEQ ID NO:5 or has at least 90 with SEQ ID NO:5 Functional variants with % sequence identity. A gene expression cassette, comprising the muscle-specific chimeric promoter according to any one of claims 1-15 and a gene of interest operably linked thereto. An expression vector comprising the muscle-specific chimeric promoter according to any one of claims 1-15 or the gene expression cassette according to claim 16. The expression vector according to claim 17, which is a viral expression vector. The expression vector according to claim 17 or 18, which is an adeno-associated virus (AAV) expression vector. A host cell comprising the muscle-specific chimeric promoter according to any one of claims 1-15, the gene expression cassette according to claim 16 or the expression vector according to any one of claims 17-19. Pharmaceutical compositions, comprising: 1) the gene expression cassette described in claim 16; The expression vector according to any one of claims 17-19; or The host cell of claim 20; and 2) A pharmaceutically acceptable carrier. The gene expression cassette described in claim 16, the expression vector described in any one of claims 17-19, the host cell described in claim 20 or the pharmaceutical composition described in claim 21 are used in the preparation of medicines for the treatment of muscle-related diseases use in . The method for treating muscle-related diseases, comprising the gene expression cassette described in claim 16, the expression vector described in any one of claims 17-19, the host cell described in claim 20 or the described expression vector described in claim 21 The pharmaceutical composition is administered to subjects in need.