CN113121653B - Novel adeno-associated virus capsid protein specific to muscle and retina - Google Patents

Abstract

The present invention relates to novel AAV capsid proteins, nucleic acid molecules encoding the capsid proteins, vectors comprising the capsid proteins, and medicaments comprising the vectors. The AAV vector of the present invention has excellent muscle and retina tissue targeting specificity, and may be used as the carrier for the delivery of therapeutic gene in treating relevant diseases.

Description

Novel adeno-associated virus capsid protein specific to muscle and retina

Technical Field

The present disclosure belongs to the field of gene therapy technology. In particular, the present disclosure relates to novel adeno-associated virus capsid proteins specific for muscle and retina, nucleic acid molecules encoding the capsid proteins, vectors comprising the capsid proteins, and medicaments comprising the vectors.

Background

In recent years, gene therapy has been vigorously developed. As a vector having a very promising effect on therapeutic gene delivery, adeno-associated virus (AAV) has high transduction efficiency, long-term therapeutic effect and low pathogenicity in various organ tissues, and these properties make AAV have significant advantages in the field of gene therapy (reference 1). However, wild-type AAV serotypes typically infect a wide spectrum of multiple tissues/organs in mammals. Thus, the current use of AAV still suffers from the following problems: wild type AAV has broad tissue targeting, resulting in gene delivery to off-target tissues, which exacerbates adverse reactions.

To date, AAV vectors have been developed that target different organs (e.g., the eye or skeletal muscle). For example, AAV 2-based gene therapy drug "luxurna" produced by Spark Therapeutics has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of Leber Congenital Amaurosis (LCA) with RPE65 mutation by the end of 2017. In 5 months 2019, the drug "Zolgensma" based on AAV9 was approved by the FDA for the treatment of spinal muscular atrophy, a neuromuscular disease caused by SMN gene mutations. Gene therapy using AAV as a vector has been used in many clinical trials, for example, to treat various ocular and muscular diseases (e.g., age-related macular degeneration, X-linked retinal schizophrenia, and duchenne's muscular dystrophy) using AAV targeted to the eye or muscle as a vector, and to achieve excellent therapeutic effects.

Studies have shown that tissue tropism and cellular transformation efficiency of AAV vectors are largely determined by their capsid. Different capsids determine different AAV's having different tissue tropism and transformation efficiency. To improve the tissue specificity of AAV vectors and to mitigate immune responses, AAV capsids can be engineered using methods such as DNA shuffling (DNA shuffling), error-prone PCR, and site-directed mutagenesis. In general, site-directed mutagenesis techniques can be used to insert 7-20 amino acid polypeptides into the capsid protein of AAV. For example, aavphp.b obtained by inserting 7 amino acids after the 588 th amino acid of AAV9 can improve blood brain barrier permeability (reference 2).

Therefore, in order to achieve better therapeutic effects, it is desirable to appropriately engineer AAV capsid proteins to obtain novel adeno-associated viral vectors with organ specificity.

Reference documents:

1.Li et al.,Nat Rev Genet(2020)21:255-272

2.Deverman et al.,Nat Biotechnol(2016)34:204-209

disclosure of Invention

Through extensive research, the inventors found that novel AAV (e.g., AAV5) capsids targeted to the retina and muscle can be obtained by inserting an oligopeptide "PLPSPSRL" in the variable region of the AAV (e.g., AAV5) capsid protein (e.g., after N573 or Q574). The novel AAV vector packaged by the novel AAV capsid has good muscle and retina tissue targeting specificity, has lower toxic and side effects and better safety potential, and can be applied to prevention, diagnosis and treatment of muscle or eye related diseases.

Thus, the present disclosure provides novel AAV capsid proteins that can be transduced in muscle cells and retinal cells in vivo or in vitro. The novel AAV capsid protein can be used for producing novel AAV vectors, thereby developing related research aiming at the novel AAV vectors or being used for treating diseases.

In a first aspect, the present disclosure provides a novel adeno-associated virus (AAV) capsid protein constructed by inserting the oligopeptide plpspspspsrl into the variable region of the AAV capsid protein.

The adeno-associated virus (AAV) can be selected from any AAV serotype. Examples of AAV serotypes include native AAV (e.g., native AAV, AAV-DJ, AAVrh8, AAVrh, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV) and other artificially engineered AAV (e.g., artificially engineered AAV, AAV-DJ, AAVrh, avian vrh, bovine AAV, canine AAV, equine AAV, and ovine AAV), preferably AAV.

In one embodiment, the variable regions are selected from the group consisting of VRII, VRIII, VRIV, VRV, VRVI, VRVII and VRVIII, preferably VRVIII. In a preferred embodiment, the oligopeptide plpspspspsrl is inserted after N573 or Q574 of the AAV capsid protein, preferably after Q574 of the AAV capsid protein, more preferably after Q574 of the AAV5 capsid protein.

In a preferred embodiment, the amino acid sequence of the novel AAV capsid protein described above is identical to SEQ ID NO: 1, preferably the amino acid sequence of the novel AAV capsid protein has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1 has at least 96%, 97%, 98%, 99% or 100% identity. In a more preferred embodiment, the novel AAV capsid protein described above comprises SEQ ID NO: 1. In a more preferred embodiment, the amino acid sequence of the novel AAV capsid protein is as set forth in SEQ ID NO: 1 is shown.

In a second aspect, the present disclosure provides a nucleic acid molecule encoding the novel AAV capsid protein described above.

In a preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is identical to SEQ ID NO: 2, preferably the nucleotide sequence of the above nucleic acid molecule has at least 95% identity with the nucleotide sequence of SEQ ID NO: 2 has at least 96%, 97%, 98%, 99% or 100% identity.

In a preferred embodiment, the nucleic acid molecule comprises SEQ ID NO: 2. In a more preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is as set forth in SEQ ID NO: 2, respectively.

In a third aspect, the present disclosure provides a novel AAV vector comprising the novel AAV capsid protein described above.

In a preferred embodiment, the novel AAV vector described above further comprises a heterologous polynucleotide comprising a nucleotide sequence encoding a therapeutic protein. In a preferred embodiment, the novel AAV vector described above further comprises a viral genome, which may be a native AAV genome or a recombinant viral genome comprising a heterologous nucleic acid, which may encode a reporter protein, a native protein, a recombinant protein, an antigen, an antibody, and/or a poly-oligonucleotide element (shRNA, miRNA) for use in nucleotide interference (RNAi) therapy, and the like.

In a preferred embodiment, the heterologous nucleic acid encodes one or more mammalian proteins, or sequences that are components of an RNAi (e.g., shRNA, siRNA, antisense oligonucleotides). In another preferred embodiment, the heterologous nucleic acid encodes a protein sequence of a certain antibody, antigen, synthetic protein or polypeptide.

In a fourth aspect, the present disclosure provides the use of the novel AAV vector described above in the preparation of a medicament for the treatment of an ocular or muscular disease.

In a fifth aspect, the present disclosure provides a medicament comprising the novel AAV vector described above and an agent that can render the viral vector pharmaceutical, for use in treating an ocular or muscular disease.

In one embodiment, the agents that can render a viral vector druggable include salts, organics, and surfactants.

In a sixth aspect, the present disclosure provides a method of treating an ocular disease comprising administering to a subject in need thereof a therapeutically effective amount of the above-described medicament.

In a seventh aspect, the present disclosure provides a method of treating a muscle disease, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described medicament.

In one embodiment, the above-mentioned medicament is administered by systemic route or local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration and intralesional administration, preferably topical administration to the eye, in particular administration as eye drops, intraocular injection or intravitreal injection into the eye.

Drawings

Figure 1A shows a screening method for candidate AAV capsid proteins.

FIG. 1B is a schematic representation of AAVz 1. AAVz1 was obtained by inserting the oligopeptide "plpspspspsrl" after Q574 of AAV5 capsid protein, the amino acid sequence of which is shown in fig. 1B.

Figure 2A shows pictures of AAV (AAVz1, AAV5, AAV8, and AAV9) infected C2C12 myoblasts and other cells (C28/I2, Huh7, and HEK293 cells). And (3) upper layer: GFP fluorescence; the lower layer: bright field; scale bar 100 μm.

FIG. 2B shows the quantitative results of the specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in C2C12 myoblasts, using the ratio of GFP-positive C2C12 cells to GFP-positive C28/I2 cells as a statistical indicator. P <0.001, n-6 well cells/group. And (4) one-way analysis of variance.

Fig. 2C shows the quantitative results of the specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in C2C12 myoblasts, with the ratio of GFP-positive C2C12 cells to GFP-positive Huh7 cells as a statistical indicator. P <0.001, n-6 well cells/group. And (4) one-way analysis of variance.

Fig. 2D shows the quantitative results of the specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in C2C12 myoblasts, with the ratio of GFP-positive C2C12 cells to GFP-positive HEK293 cells as a statistical indicator. P <0.001, n-6 well cells/group. And (4) one-way analysis of variance.

Fig. 3A shows transduction of AAV (AAVz1, AAV5, AAV8, and AAV9) in the liver, heart, lung, spleen of mice. Scale bar 100 μm.

Fig. 3B shows transduction of AAV (AAVz1, AAV5, AAV8, and AAV9) in Gastrocnemius (GA), Tibialis Anterior (TA), and Soleus (SO) in mice. Scale bar 100 μm.

Fig. 3C shows the quantitative results of specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in muscle cells, as a statistical index, the ratio of GFP-positive muscle cells (average of GA, TA and SO) to GFP-positive hepatocytes, which is based on the number of nuclei (DAPI) statistics. P <0.001, n-5 mice per group. And (4) one-way analysis of variance.

Fig. 3D shows the quantitative results of specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in muscle cells, as a statistical indicator, the ratio of GFP-positive muscle cells (average of GA, TA and SO) to GFP-positive cardiomyocytes, based on the number of nuclei (DAPI) statistics. P <0.001, n-5 mice per group. And (4) one-way analysis of variance.

Fig. 3E shows the quantitative results of specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in muscle cells, as a statistical indicator of the ratio of GFP-positive muscle cells (average of GA, TA and SO) to GFP-positive lung cells based on the number of nuclei (DAPI) statistics. P <0.001, n-5 mice per group. And (4) one-way analysis of variance.

Fig. 3F shows the quantitative results of specific transduction of AAV (AAVz1, AAV5, AAV8 and AAV9) in muscle cells, as a statistical indicator, the ratio of GFP-positive muscle cells (average of GA, TA and SO) to GFP-positive splenocytes based on the number of nuclei (DAPI) statistics. P <0.001, n-5 mice per group. And (4) one-way analysis of variance.

Fig. 4A shows retinal transduction by AAV (AAVz1, AAV5, AAV8, and AAV 9).

Figure 4B shows a retinal section of a mouse. GFP signal reaching the photosensitive layer is indicated by the white arrow. GCL: ganglion cell layer. IPL: an inner plexiform layer. INL: an inner core layer. OPL: an outer plexiform layer. ONL: an outer core layer. RPE: the retinal pigment epithelium.

Figure 4C shows the quantification of mean GFP fluorescence intensity for different AAV groups relative to AAV5 (normalized to 1). n-4 eyes/group, # p <0.001, one-way anova.

FIG. 5 shows the amino acid sequence of the AAVz1 capsid protein (SEQ ID NO: 1).

FIG. 6 shows the nucleic acid sequence (SEQ ID NO: 2) encoding the AAVz1 capsid protein.

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 to which this disclosure belongs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism that has or is susceptible to a condition that can be prevented or treated by administration of a medicament of the present disclosure, and include humans and non-human animals.

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, pheasants, and game birds such as chickens, turkeys, and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

Herein, the term "treatment" includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) ameliorating the disease, i.e., causing regression of at least one of the conditions, diseases or disorders or clinical or subclinical symptoms thereof.

As used herein, the term "therapeutically effective amount" refers to the dose that produces the therapeutic effect to which it is administered. For example, a therapeutically effective amount of a drug suitable for treating an ocular disease can be an amount that is capable of preventing or ameliorating one or more symptoms associated with the ocular disease.

As used herein, the term "amelioration" refers to an improvement in a symptom associated with a disease, and may refer to an improvement in at least one parameter that measures or quantifies the symptom.

Herein, the term "preventing" a condition, disease or disorder includes: preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a condition, disease or disorder developing in a subject who may be suffering from or susceptible to the condition, disease or disorder but who has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

Herein, the term "topical administration" or "topical route" refers to an administration having a local effect.

As used herein, the term "vector" refers to a macromolecule or series of macromolecules encapsulating a polynucleotide that facilitates delivery of the polynucleotide to a target cell in vitro or in vivo. Classes of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered is sometimes referred to as a "transgene," and includes, but is not limited to, coding sequences for certain proteins or synthetic polypeptides that can enhance, inhibit, attenuate, protect, trigger, or prevent certain biological functions and physiological functions, coding sequences of interest in vaccine development (e.g., polynucleotides that express proteins, polypeptides, or peptides suitable for eliciting an immune response in a mammal), coding sequences for RNAi components (e.g., shRNA, siRNA, antisense oligonucleotides), or optional markers.

In this context, the term "immune response" refers to the process in which host tissues and cells participate after encountering an immunogen, such as an AAV capsid protein and a transgene. It involves the proliferation, migration and differentiation of immunocompetent cells (e.g., T lymphocytes, B cells, monocytes, macrophages) in lymphoid reticulum, blood, spleen or other related tissues, resulting in the production of antibodies or the development of cell-mediated reactivity. In other words, the host induces an active immune response upon exposure to an immunogen by infection or vaccination. Active immunization is obtained by "transferring preformed substances (e.g., antibodies, transfer factors, thymic grafts, interleukin-2)" from an immunized or non-immunized host, whereas passive immunization is not.

As used herein, the term "mosaic" AAV capsid nucleic acid coding sequence or capsid protein refers to an AAV capsid sequence that has been artificially designed and engineered by methods of DNA shuffling, error-prone PCR, and site-directed mutagenesis.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process by which a heterologous polynucleotide is delivered to a host cell for transcription and translation to produce a polypeptide product, including the introduction of the heterologous polynucleotide into the host cell using a recombinant virus.

As used herein, the term "gene delivery" refers to the introduction of a heterologous polynucleotide into a cell for gene delivery, including targeting, binding, uptake, transport, replicon integration, and expression.

As used herein, the term "gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce the RNA or protein product of the gene.

As used herein, the term "infection" refers to the process by which a virus or viral particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of replication of the virus in a host cell.

In this context, the term "targeted" means that the virus preferentially enters some cell or tissue and then further expresses the viral genome or a sequence carried by the recombinant transgene in the cell. It is known to those skilled in the art that transcription of a heterologous nucleic acid sequence from the viral genome cannot be initiated without cis-and trans-acting factors (e.g., inducible promoters or other regulatory nucleic acid sequences).

Herein, the term "polypeptide" refers to a polymer of at least 20 amino acids linked by peptide bonds. The terms "polypeptide" and "protein" are used synonymously herein to refer to a polymer consisting of more than 20 amino acids. The term also includes synthetic amino acid polymers.

As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs.

In this context, the term "recombinant" in relation to a polynucleotide means that the polynucleotide is a synthetic product, different from the natural polynucleotide, obtained by multiple cloning steps. Recombinant viruses are viral particles comprising recombinant polynucleotides.

In this context, the nucleic acid sequence is in single stranded form, in the 5 '-3' direction from left to right. The nucleic acid sequences and amino acid sequences referred to herein are referred to in the form recommended by the IUPACIUB Biochemical nomenclature Commission. The amino acid sequence adopts single letter symbols or three letter symbols.

In one embodiment, the present disclosure provides a novel AAV5 capsid protein constructed by inserting the oligopeptide plpspspsrl after Q574 of AAV5 capsid protein. As an example of a novel AAV5 capsid protein, the amino acid sequence of the AAVz1 capsid protein is set forth in SEQ ID NO: 1, the nucleotide sequence of the nucleic acid molecule encoding AAVz1 is shown in SEQ ID NO: 2, respectively.

As known to those skilled in the art, AAV capsid proteins contain VP1, VP2, and VP3 proteins, and VP2 and VP3 proteins undergo transcription and translation processes at the start codon inside the VP1 protein, i.e., the VP1 sequence comprises VP2 and VP3 sequences. The present disclosure provides the amino acid sequence of VP1 protein of AAVz1 capsid (SEQ ID NO: 1).

In one embodiment, the present disclosure provides another novel AAV capsid protein constructed by inserting into an AAV capsid protein an oligopeptide having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or 100%) identity to "plpspspsrl. In one embodiment, the AAV capsid protein may be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., capsid proteins of native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV) and other artificially engineered AAV capsid proteins (e.g., capsid proteins of artificially engineered AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV). The genomic sequences, ITR sequences, Rep and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, such as the GenBank database.

Conservative substitutions of amino acids are known in the art. In one embodiment, the AAV capsid proteins of the present disclosure may have conservative substitutions of amino acids in the same group as: a) glycine and alanine; b) valine, isoleucine, leucine and proline; c) aspartic acid and glutamic acid; d) asparagine and glutamine; e) serine, threonine lysine, arginine, and histidine; f) phenylalanine, tryptophan, and tyrosine; g) methionine and cysteine. In some embodiments, non-conservative substitutions between the different sets of amino acids are also permissible.

In one embodiment, an AAV vector of the present disclosure may be loaded with a heterologous polynucleotide for delivery of the gene into a target cell. Thus, the AAV vectors of the present disclosure can be used to deliver nucleic acids to cells in vitro or in vivo.

In one embodiment, the heterologous polynucleotide delivered by the AAV vector encodes a polypeptide that acts as a reporter (i.e., a reporter protein). The reporter protein is used to indicate cells successfully infected with AAV. These reporter proteins include, but are not limited to, green fluorescent protein, beta-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.

In one embodiment, the heterologous polynucleotide delivered to the target cell by the AAV vector encodes a native protein for therapeutic use, which native protein is codon-optimized or not codon-optimized. Such native proteins include, but are not limited to: proteins useful in the treatment of various muscle diseases, for example, Cystic Fibrosis Transmembrane Regulator (CFTR), dystrophin (including some truncated forms, known as micromodulin or micromodulin), mini-agglutinin, integrin- β 1, laminin- β 2, myosin- α, myosin- β, sarcomere, synuclein, gonadotropin, mini-lecithin, Lamin a/C, four half LIM domain protein 1(FHL1), follistatin, SOD1, SOD2, full-length or dominant negative myostatin; angiogenic factors (e.g., VEGF, angiopoietin 1 or 2) and angiogenesis inhibitors (e.g., endostatin and angiostatin); anti-inflammatory polypeptides (e.g., anti-inflammatory interleukins 4, 10, 11 and 13, full-length and dominant mutant Ikappa B, Pinch and ILK genes); coagulation factors (e.g., coagulation factor VIII, coagulation factor IX, coagulation factor X); spectrin, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, leptin, and leptin receptor; neurotrophic factors (e.g., BDNF, GDNF, NGF, semaphores, SLIT1, SLIT2, SLIT3, FGF7, FGF10, and FGF22) and corresponding neurotrophin receptors; LDL receptors, lipoprotein lipase, adrenergic receptor-alpha and beta, B-globulin, C-globulin; insulin-like growth factors, such as IGF-1 and IGF-2; adenosine deaminase and the bone morphogenic protein superfamily (e.g., BMP1, BMP2, BMP4, BMP6, BMP7, TGF- β, RANKL); and molecules involved in maintaining vision, retinal layer structure, retinal cell (e.g., photoreceptor cells and retinal pigment epithelium) function, such as RPE65, RPGR, Bestrophin-1, CNGA3, CNGB3, retinochisin, ABCA4, and retinal-specific ABC transporters.

In one embodiment, the heterologous polynucleotide delivered to the target cell by the AAV vector encodes a synthetic polypeptide, including, but not limited to, Aflibercept, various recombinant interleukins (e.g., interleukin-1 and interleukin-18), TNF-alpha antagonistic soluble receptors, activin type II soluble receptor, anti-VEGF antibody, anti-sclerostin antibody, anti-RANKL antibody, anti-C5 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CGRP antibody, anti-HER 2 antibody, anti-EGFR antibody, antibody to pro-inflammatory cytokines, and receptors thereof.

In one embodiment, the heterologous polynucleotide delivered by the AAV vector may consist of RNAi components (e.g., siRNA, shRNA, snRNA, microRNA, ribozymes, antisense oligonucleotides, and antisense polynucleotides) that can knock down any endogenous gene that is activated in an aberrant manner or a heterologous gene that invades the host cell, e.g., a viral or bacterial polynucleotide known in the art. The RNAi moiety typically has 60-100% identity in sequence to its target gene and results in a reduction of the corresponding protein product by at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%).

In one embodiment, the heterologous polynucleotide delivered by the AAV vector comprises regulatory sequences, such as transcription/translation control signals, origins of replication, polyadenylation signals, Internal Ribosome Entry Sites (IRES) or 2A signals (e.g., P2A, T2A, F2A), promoters and enhancers (e.g., CMV promoter or other hybrid CMV promoters with vertebrate β -actin, β -globin or β -globin regulatory elements, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter). The use of promoters and enhancers depends on their tissue-specific expression profile. The promoter/enhancer may also be induced by chemicals or hormones (such as doxycycline or tamoxifen), depending on the need to trigger gene expression at a desired time point. In addition, promoters/enhancers may be natural or synthetic sequences, i.e., prokaryotic or eukaryotic sequences.

In one embodiment, the inducible regulatory element for gene expression may be a tissue-specific or tissue-tropic promoter/enhancer element, including but not limited to: skeletal muscle specific promoters, such as the MCK, HSA, myogenin promoters; and promoters specific for various types of ocular cells, such as ganglion cell-specific promoters (e.g., Tuj1 promoter), astrocyte and Muller cell-specific promoters (e.g., GFAP promoter), and retinal pigment epithelium-specific promoters (e.g., RPE65 promoter).

In one embodiment, the AAV vector of the present disclosure comprises an AAV capsid protein and a viral genome, which may be a native AAV genome or a recombinant vector of a heterologous polynucleotide for therapeutic purposes. In one embodiment, the heterologous polynucleotide encodes a mammalian protein or RNAi component (e.g., shRNA, siRNA, antisense oligonucleotide). In one embodiment, the heterologous polynucleotide encodes the amino acid sequence of certain antibodies, antigens, synthetic proteins or polypeptides.

In one embodiment, the translation products of the viral genome enhance, inhibit, attenuate, protect, trigger, or prevent one or more endogenous signaling pathways involved in metabolic regulation and health maintenance in a mammal.

In one embodiment, AAV viral particles of the present disclosure can be administered to a host cell in vitro, and the cell implanted into a subject. Thus, the heterologous nucleic acid packaged in the virus is introduced into the subject via the cell for transcription and/or translation, resulting in a protein or RNA product that is secreted from the cell into the subject or that modulates the biological activity of the host cell, thereby effecting a therapeutic effect.

In one embodiment, an AAV vector of the present disclosure is formulated for administration to a human or other mammal in a pharmaceutical formulation (e.g., injection, tablet, capsule, powder, eye drop). The pharmaceutical preparation may further comprise other ingredients, such as pharmaceutical adjuvants, water-soluble or organic solvents (such as water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (such as sodium chloride, potassium chloride, phosphate, acetate, bicarbonate, Tris-HCl and Tris-acetate), and dissolution retarding agents (such asSuch as paraffin), surfactants, antimicrobial agents, liposomes, lipid complexes, immunosuppressants (e.g., cortisone, prednisone, cyclosporine), non-steroidal anti-inflammatory drugs (NSAIDs, e.g., aspirin, ibuprofen, acetaminophen) microspheres, hard matrices, semi-solid carriers, nanospheres or nanoparticles. The titer of AAV particles in a pharmaceutical formulation can be 10⁵-10¹⁴vg/mL. In addition, AAV can be delivered in single or multiple doses by inhalation, systemic or local (e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal) administration.

In one embodiment, the present disclosure provides a medicament comprising an AAV vector of the present disclosure and an agent (e.g., a salt, an organic substance, and a surfactant) that can render the AAV vector druggable. The medicaments are useful for transducing cells in vitro or mammals (e.g., rodents, primates, and humans) in vivo, thereby treating a variety of diseases, such as ocular and muscle diseases.

Herein, the ocular disease is selected from: inherited dystrophies of the retina, glaucoma, glaucomatous neuropathy, age-related macular degeneration, refractive error, dry eye, inherited dystrophies of ocular inflammation, uveitis, orbital inflammation, cataracts, allergic conjunctivitis, diabetic retinopathy, macular edema, corneal edema, keratoconus, proliferative vitreoretinopathy (fibrosis), periretinal fibrosis, central serous chorioretinopathy, vitreoretinopathy, vitreous macular traction, and vitreous hemorrhage. In one embodiment, the ocular disease involves a deterioration of eye and/or visual function.

In one embodiment, treating an ocular disorder refers to improving visual acuity, contrast vision, color vision, and visual field of the patient receiving the treatment.

Herein, the muscle disease may be a muscle disease caused by a decrease in muscle function, muscle wasting or muscle degeneration, and may be selected from: duchenne Muscular Dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Limb Girdle Muscular Dystrophy (LGMD), myasthenia gravis, congenital myasthenia syndrome, sarcopenia, cachexia, Amyotrophic Lateral Sclerosis (ALS), myotonic dystrophy types I and II.

In one embodiment, treating a muscle disease refers to inhibiting or delaying the onset of muscle disease, increasing muscle mass, improving muscle strength, or improving muscle function.

In one embodiment, the disclosure relates to a method of producing an AAV vector from a cell. The cells support efficient transfection of plasmids encoding AAV Rep/Cap proteins, helper genes, and recombinant vectors encoding native viral genomes or heterologous proteins. AAV vectors of the disclosure can be produced from HEK293 cells using three plasmid transfection methods well known to those skilled in the art. For example, AAV vectors of the present disclosure are produced by co-transfecting an orthodromic plasmid encoding recombinant proteins such as GFP, an AAV Rep/Cap plasmid, a phepper plasmid into HEK293 cells.

Standard methods well known to those skilled in the art can be used to produce polypeptides, antibodies or antigen-binding fragments; altering the nucleic acid sequence; producing a transformed cell; constructing a recombinant AAV vector; engineering a capsid protein; packaging a vector expressing the AAV Rep and Cap sequences; transient transfection and stable transfection packaging cells.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1: engineering and screening for AAV

As shown in fig. 1A: first, engineered AAV libraries and helper plasmids were transfected into HEK293 cells. Subsequently, HEK293 cell lysates containing AAV were added to cultured C2C12 myoblasts along with Ad5 (adenovirus type 5). Cell lysates were enriched and C2C12 cells were infected 4-5 times repeatedly. Viral genomic bands of candidate AAV capsids were enriched by PCR on C2C12 cell lysates and sequenced. By screening, the serotype sequences with high enrichment are selected, and a serotype mutant AAVz1 is obtained (FIG. 1B).

The AAVz1 particles were purified by AAVX (thermo scientific) affinity chromatography plus iodixanol ultracentrifugation and concentrated to 200. mu.l for further experiments to determine the tissue targeting of the vector.

Example 2: validation of C2C12 transduction specificity of mutant AAVz1

To investigate transduction specificity, cells were infected with AAV carrying the GFP gene (AAVz1, AAV5, AAV8 and AAV9) at a multiplicity of infection (MOI) of 1X 10⁵vg/cell. Pictures were taken 72h after AAV treatment.

As shown in fig. 2A, wild-type AAV8 or AAV9 showed significant transduction in C2C12 myoblasts (myocyte progenitor cells), C28/I2 (human chondrocyte line) cells, and Huh7 (human hepatoma cell line in hepatocyte lineage) cells, and wild-type AAV5 was almost non-infectious to all cells. In contrast, AAVz1 showed stable and efficient GFP expression in C2C12 myoblasts and transduced C28/I2, Huh7 and HEK293 cells only less.

In addition, the infection specificity of mutant AAVz1 for C2C12 was further quantified by calculating the ratio of GFP-positive C2C12 cells to GFP-positive C28/I2, Huh7 and HEK293 cells. The results show that AAVz1 has 3-4 times higher C2C12 transduction specificity than AAV5, 8 and 9 (fig. 2B to fig. 2D).

Example 3: validation of muscle targeting of mutant AAVz1

C57BL/6 mice were injected 1X 10 by tail vein injection¹³vg/kg of AAV (AAVz1, AAV5, AAV8 and AAV9) virus particles carrying a GFP gene. After 4 weeks of intravenous virus injection, liver, heart, lung, spleen were isolated for immunostaining (fig. 3A). Injection of AAV (AAVz1, AAV5, AAV8 and AAV9) virus (5X 10) carrying GFP gene into hindlimb muscle of C57BL/6 mouse¹⁰vg/mouse). 4 weeks after virus injection, Gastrocnemius (GA), Tibialis Anterior (TA) and Soleus (SO) were isolated for immunostaining (FIG. 3B).

In addition, the specificity of mutant AAVz1 for muscle cells was further quantified by calculating the ratio of GFP-positive muscle cells (average of GA, TA and SO) to GFP-positive hepatocytes, heart cells, lung cells and spleen cells. The results showed that in the AAVz1 group, the transduction specificity of muscle versus liver, lung, heart and spleen was significantly higher than that of the wild-type AAV (AAV5, 8, 9) group (fig. 3C to 3F,. xp < 0.001).

The above results demonstrate that AAVz1 has superior muscle targeting specificity over AAV5, 8, 9.

Example 4: validation of retinal targeting of mutant AAVz1

At 3X 10⁹Vg/eye dose AAV particles carrying the GFP gene (wild type AAV and AAVz1) were injected intravitreally into C57BL/6 mice. Pictures of GFP signal were taken 3 weeks after injection (fig. 4A). Retinas of mice were infected with AAV (AAVz1, AAV5, AAV8, and AAV9) and stained for GFP, DAPI, and the cone cell marker S-opsin.

The results show that AAVz1 is able to diffuse into the photosensitive layer in the retina despite intravitreal administration, whereas AAV5, 8, 9 is almost absent (fig. 4B). Furthermore, as shown in fig. 4C, the retinal GFP fluorescence levels of AAVz1 were about 4-fold higher than those of AAV5, 8, 9 (fig. 4C, p <0.001, n 4), indicating that AAVz1 has better retinal targeting than wild-type AAV5, 8, 9.

The above experimental results show that the AAVz1 vector of the present disclosure, in which a synthetic oligopeptide "plpspspsrl" was inserted after Q574 of AAV5 capsid protein, improved transduction in retina and muscle of mice compared to wild-type AAV5, AAV8, and AAV 9. Moreover, the transduction efficiency of the AAVz1 vectors of the disclosure in other tissues (e.g., heart, lung, spleen) is significantly lower than that of wild-type AAV (AAV5, AAV8 and AAV9), indicating that the AAVz1 vectors of the disclosure have better targeting to muscle and retinal tissues. Thus, both the infection rate and the incidence of potential adverse effects of off-target tissues are reduced when the AAVz1 of the present disclosure is used clinically as a therapeutic vector as compared to wild-type AAV5, 8, 9.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Sequence listing

<110> Shanghai Xin-Zhi-pharmaceutical science and technology Co., Ltd

<120> novel adeno-associated virus capsid protein specific to muscle and retina

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 732

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of AAVz1 capsid protein

<400> 1

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

225 230 235 240

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

245 250 255

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

305 310 315 320

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp

325 330 335

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

340 345 350

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

355 360 365

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

370 375 380

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

385 390 395 400

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

405 410 415

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

420 425 430

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

435 440 445

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

450 455 460

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

465 470 475 480

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

485 490 495

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

500 505 510

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

515 520 525

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

530 535 540

Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg

545 550 555 560

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Pro Leu

565 570 575

Pro Ser Pro Ser Arg Leu Ser Ser Thr Thr Ala Pro Ala Thr Gly Thr

580 585 590

Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met Glu Arg Asp

595 600 605

Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu Thr Gly Ala

610 615 620

His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu Lys His Pro

625 630 635 640

Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly Asn Ile Thr

645 650 655

Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln Tyr Ser Thr

660 665 670

Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys Glu Asn Ser

675 680 685

Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr Asn Asp Pro

690 695 700

Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr Arg Thr Thr

705 710 715 720

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730

<210> 2

<211> 2199

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleic acid sequence encoding AAVz1 capsid protein

<400> 2

atgtcttttg ttgatcaccc tccagattgg ttggaagaag ttggtgaagg tcttcgcgag 60

tttttgggcc ttgaagcggg cccaccgaaa ccaaaaccca atcagcagca tcaagatcaa 120

gcccgtggtc ttgtgctgcc tggttataac tatctcggac ccggaaacgg tctcgatcga 180

ggagagcctg tcaacagggc agacgaggtc gcgcgagagc acgacatctc gtacaacgag 240

cagcttgagg cgggagacaa cccctacctc aagtacaacc acgcggacgc cgagtttcag 300

gagaagctcg ccgacgacac atccttcggg ggaaacctcg gaaaggcagt ctttcaggcc 360

aagaaaaggg ttctcgaacc ttttggcctg gttgaagagg gtgctaagac ggcccctacc 420

ggaaagcgga tagacgacca ctttccaaaa agaaagaagg ctcggaccga agaggactcc 480

aagccttcca cctcgtcaga cgccgaagct ggacccagcg gatcccagca gctgcaaatc 540

ccagcccaac cagcctcaag tttgggagct gatacaatgt ctgcgggagg tggcggccca 600

ttgggcgaca ataaccaagg tgccgatgga gtgggcaatg cctcgggaga ttggcattgc 660

gattccacgt ggatggggga cagagtcgtc accaagtcca cccgaacctg ggtgctgccc 720

agctacaaca accaccagta ccgagagatc aaaagcggct ccgtcgacgg aagcaacgcc 780

aacgcctact ttggatacag caccccctgg gggtactttg actttaaccg cttccacagc 840

cactggagcc cccgagactg gcaaagactc atcaacaact actggggctt cagaccccgg 900

tccctcagag tcaaaatctt caacattcaa gtcaaagagg tcacggtgca ggactccacc 960

accaccatcg ccaacaacct cacctccacc gtccaagtgt ttacggacga cgactaccag 1020

ctgccctacg tcgtcggcaa cgggaccgag ggatgcctgc cggccttccc tccgcaggtc 1080

tttacgctgc cgcagtacgg ttacgcgacg ctgaaccgcg acaacacaga aaatcccacc 1140

gagaggagca gcttcttctg cctagagtac tttcccagca agatgctgag aacgggcaac 1200

aactttgagt ttacctacaa ctttgaggag gtgcccttcc actccagctt cgctcccagt 1260

cagaacctct tcaagctggc caacccgctg gtggaccagt acttgtaccg cttcgtgagc 1320

acaaataaca ctggcggagt ccagttcaac aagaacctgg ccgggagata cgccaacacc 1380

tacaaaaact ggttcccggg gcccatgggc cgaacccagg gctggaacct gggctccggg 1440

gtcaaccgcg ccagtgtcag cgccttcgcc acgaccaata ggatggagct cgagggcgcg 1500

agttaccagg tgcccccgca gccgaacggc atgaccaaca acctccaggg cagcaacacc 1560

tatgccctgg agaacactat gatcttcaac agccagccgg cgaacccggg caccaccgcc 1620

acgtacctcg agggcaacat gctcatcacc agcgagagcg agacgcagcc ggtgaaccgc 1680

gtggcgtaca acgtcggcgg gcagatggcc accaacaacc agccacttcc gtcaccatcg 1740

cgcctcagct ccaccactgc ccccgcgacc ggcacgtaca acctccagga aatcgtgccc 1800

ggcagcgtgt ggatggagag ggacgtgtac ctccaaggac ccatctgggc caagatccca 1860

gagacggggg cgcactttca cccctctccg gccatgggcg gattcggact caaacaccca 1920

ccgcccatga tgctcatcaa gaacacgcct gtgcccggaa atatcaccag cttctcggac 1980

gtgcccgtca gcagcttcat cacccagtac agcaccgggc aggtcaccgt ggagatggag 2040

tgggagctca agaaggaaaa ctccaagagg tggaacccag agatccagta cacaaacaac 2100

tacaacgacc cccagtttgt ggactttgcc ccggacagca ccggggaata cagaaccacc 2160

agacctatcg gaacccgata ccttacccga cccctttaa 2199

Claims (14)

1. An AAV capsid protein, wherein the amino acid sequence of said AAV capsid protein is set forth in SEQ ID NO: 1 is shown.
2. 2. A nucleic acid molecule, wherein the nucleic acid molecule encodes the AAV capsid protein of claim 1.
3. 3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is identical to the nucleotide sequence of SEQ ID NO: 2 has at least 95% identity.
4. 4. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is identical to the nucleotide sequence of SEQ ID NO: 2 has 96%, 97%, 98% or 99% identity.
5. 5. The nucleic acid molecule of any one of claims 2 to 4, wherein the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 2, respectively.
6. An AAV vector, wherein said AAV vector comprises the AAV capsid protein of claim 1.
7. 7. The AAV vector of claim 6, wherein the AAV vector further comprises a heterologous polynucleotide comprising a nucleotide sequence encoding a therapeutic protein.
8. 8. Use of an AAV vector according to claim 6 or 7 in the manufacture of a medicament for the treatment of an ocular or muscular disease.
9. 9. A medicament comprising the AAV vector of claim 6 or 7 and an agent that can render the viral vector pharmaceutical for use in treating an ocular or muscle disease.
10. 10. The medicament of claim 9, wherein the medicament is administered by a systemic route or a local route.
11. 11. The medicament of claim 9, wherein the medicament is administered intravenously, intramuscularly, subcutaneously, orally, topically, intraperitoneally, or intralesionally.
12. 12. The medicament of claim 9, wherein the medicament is administered topically to the eye.
13. 13. The medicament of claim 12, wherein the medicament is administered as eye drops, intraocular injection, or intravitreal injection into the eye.
14. 14. The medicament of any one of claims 9 to 13, wherein the agent capable of rendering the viral vector pharmaceutically comprises a salt, an organic substance and a surfactant.

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