WO2023124740A1 - New-type adeno-associated virus capsid protein with high muscle affinity and use thereof - Google Patents

Abstract

Provided are a new-type adeno-associated virus (AAV) capsid protein with a high muscle affinity, a nucleic acid molecule encoding the capsid protein, an AAV vector comprising the capsid protein and the use thereof, and a drug containing the AAV vector. The new-type AAV capsid protein has a good muscle tissue affinity, and an AAV vector and a drug constructed by using the capsid protein can deliver therapeutic genes for treating various diseases.

Description

Novel adeno-associated virus capsid protein with high affinity for muscle and its application technical field

The disclosure relates to a novel adeno-associated virus (AAV) capsid protein with high affinity for muscle, a nucleic acid molecule encoding the capsid protein, an AAV vector comprising the capsid protein and its application, and a drug comprising the AAV vector.

Background technique

In recent years, gene therapy has flourished. As a very promising carrier for therapeutic gene delivery, adeno-associated virus (AAV) has obvious advantages in the field of gene therapy due to its high transduction efficiency, long-term therapeutic effect and low pathogenicity in various organs and tissues (Chengwen Li, R Jude Samulski, Engineering adeno-associated virus vectors for gene therapy, Nat Rev Genet 2020, 21(4):255-272). AAV is widely used to target various organs and tissues such as eyes, skeletal muscle, liver, and central nervous system to treat various diseases. For example, at the end of 2017, the AAV2-based gene therapy drug "Luxturna" produced by Spark Therapeutics has been approved by the US Food and Drug Administration (FDA) to treat Leber congenital amaurosis with RPE65 mutation. In May 2019, the FDA approved the AAV9-based drug Zolgensma to treat spinal muscular atrophy, a neuromuscular disease caused by mutations in the SMN gene. Duchenne muscular dystrophy is a relatively common muscular degenerative disease caused by mutations in the X-chromosome dystrophin (dystrophin) gene, represented by muscle atrophy and progressive loss of respiratory motor function. About 1/3500~5000. At present, gene therapy tools have been used in many clinical trials. These gene therapies use AAV as a carrier to treat a variety of genetic muscle diseases (such as limb-girdle muscular dystrophy and Duchenne muscular dystrophy, etc.), and have achieved remarkable therapeutic effects.

Although AAV vectors have achieved early therapeutic effects in the clinic, concerns about potential issues such as transduction efficiency, targeting, and immune response have also increased, such as the tolerance of human patients to systemic AAV therapy. Three children tragically died in a recent trial (NCT03199469) investigating AAV8 vectors expressing functional MTM1 in X-linked myotube myopathy (XLMTM) (Wilson JM, Flotte TR. Moving Forward After Two Deaths in a Gene Therapy Trial of Myotubular Myopathy. Hum Gene Ther. 2020, 31(13-14): 695-696). Although the cause of death is still under investigation, the infection efficiency of wild-type AAV for muscle tissue is relatively low relative to other tissues and organs (such as the liver), which makes AAV have a higher enrichment in other tissues and organs at therapeutic doses for muscle diseases , leading to some off-target side effects.

In addition, the immune response against AAV virus, including neutralizing antibody (Nab) against AAV capsid and delivery gene protein product, is mainly mediated by T cells such as CD4+ and CD8+ against AAV-transduced target cells Immune response, these are also the main factors that limit the application of AAV.

Studies have shown that the tissue tropism and cell transformation efficiency of AAV vectors are mainly determined by their capsids. Different capsids determine that different AAVs have different tissue tropisms and transformation efficiencies. In order to improve the tissue infection efficiency of AAV and reduce the immune response, methods such as DNA shuffling, error-prone PCR, site-directed mutagenesis, and peptide insertion or replacement can be used to modify the AAV capsid, thereby generating new AAV serum type.

Therefore, in order to achieve a better therapeutic effect, it is expected to properly modify the AAV capsid protein to obtain a new type of adeno-associated virus vector with organ (such as muscle) specificity.

Contents of the invention

In order to solve the above-mentioned technical problems, the inventors have conducted a lot of research and unexpectedly found that the N-terminal 1st to ( When positions 188-203) were replaced with amino acids at the corresponding positions of the AAV9 capsid protein (SEQ ID NO: 4), the obtained novel AAV capsid protein could efficiently deliver the gene and its protein product to muscle tissue.

Therefore, in a first aspect, the present disclosure provides an AAV capsid protein, wherein the AAV capsid protein replaces the N-terminal 1 to N positions of VP1 and VP2 of AAVHH67 capsid protein with AAV9 capsid protein The N-terminal 1 to N positions of VP1 and VP2 are constructed, the amino acid sequence of the AAVHH67 capsid protein is shown in SEQ ID NO: 2, and the amino acid sequence of the AAV9 capsid protein is shown in SEQ ID NO: 4 , the N is an integer between 188 and 203.

In one embodiment, N is 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 or 203.

In a preferred embodiment, N is 188, 193 or 203.

In one embodiment, the amino acid sequence of the above-mentioned AAV capsid protein has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.

In one embodiment, the amino acid sequence of the above-mentioned AAV capsid protein is shown in SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.

Compared with AAV9 and AAVHH67, the novel AAV capsid protein obtained by hybridizing and remodeling AAV9 and AAVHH67 capsid proteins in the present disclosure has better heart and muscle infection efficiency.

In a second aspect, the present disclosure provides a nucleic acid molecule encoding the AAV capsid protein according to the first aspect.

In one embodiment, the nucleotide sequence of the aforementioned nucleic acid molecule has at least 70% identity to the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.

In a preferred embodiment, the nucleotide sequence of the above-mentioned nucleic acid molecule has at least 75%, 80%, 85% of the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 , 90%, 95% or 99% identity.

In one embodiment, the above-mentioned nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.

In a preferred embodiment, the nucleotide sequence of the aforementioned nucleic acid molecule is shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.

In a third aspect, the present disclosure provides an AAV vector comprising the AAV capsid protein according to the first aspect and a viral genome.

In one embodiment, the viral genome is a native AAV genome or an artificially recombined viral genome.

In a preferred embodiment, the viral genome encodes more than one of reporter protein and therapeutic protein.

In a preferred embodiment, the therapeutic protein is a protein used in the treatment of heart disease or muscle disease.

In a fourth aspect, the present disclosure provides the use of the AAV vector according to the third aspect in the preparation of a medicament for treating heart disease or muscle disease.

In a preferred embodiment, the muscle disease is a muscle disease due to decreased muscle function, muscle wasting or muscle degeneration.

In a preferred embodiment, the muscle disease is selected from the group consisting of Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Walker-Warburg syndrome, limb-girdle muscular dystrophy (LGMD), X-linked myotubes Myopathy (XLMTM), Reducing body myopathy, X-linked myopathy with postural muscular atrophy (XMPMA), X-linked scapular myopathy (X-SPM), facioscapulohumeral muscular dystrophy (FSHD), myasthenia gravis (MG), congenital myasthenic syndrome (CMD), sarcopenia, cachexia, amyotrophic lateral sclerosis (ALS), and myotonia types I and II Malnutrition.

In a preferred embodiment, the heart disease is selected from the group consisting of congestive heart failure, congenital heart failure, ischemic cardiomyopathy, hypertrophic cardiomyopathy and rheumatic heart disease.

In the fifth aspect, the present disclosure provides a medicament comprising: the AAV vector according to the third aspect and an excipient.

In one embodiment, excipients include salts, organics and surfactants.

The AAV vectors and drugs disclosed in the present disclosure can efficiently and systematically deliver therapeutic proteins to various muscles in various parts of the body, and can be applied to the prevention, diagnosis and treatment of various related muscle diseases that require systemic systemic muscle delivery or for vaccines.

In a sixth aspect, the present disclosure provides a method of treating heart disease or muscle disease, comprising administering a therapeutically effective amount of the drug according to the fifth aspect to a subject in need thereof.

In one embodiment, the drug is administered by systemic or local routes, eg, intravenous, intramuscular, intracardiac, subcutaneous, oral, topical, intraperitoneal, and intralesional.

In a preferred embodiment, the drug is administered systemically or locally to the heart and/or muscle.

Description of drawings

Figure 1A shows novel AAV capsid proteins according to three embodiments of the present disclosure: AAVT69, AAVT70, AAVT71. AAVT69 replaces the N-terminal 1 to 203 of VP1 and VP2 of AAVHH67 capsid protein (SEQ ID NO: 2) with the N-terminal 1 to 203 of VP1 and VP2 of AAV9 capsid protein (SEQ ID NO: 4) AAVT70 was constructed by replacing the 1st to 193rd positions of the N-terminal VP1 and VP2 of the AAVHH67 capsid protein with the 1st to 193rd positions of the N-terminal VP1 and VP2 of the AAV9 capsid protein; AAVT71 was constructed by replacing the AAVHH67 The 1st to 188th positions of the N-terminal of VP1 and VP2 of the capsid protein were replaced with the 1st to 188th positions of the N-terminal of VP1 and VP2 of the AAV9 capsid protein.

Figure IB shows the production of AAV9, HH67 (AAVHH67), AAVT69, AAVT70 and AAVT71. Viral genome quantification was performed by qPCR. Viral capsid proteins were quantified by silver staining. n=3 batches of samples per group.

Figure 2A shows the transduction efficiency of AAV5, AAV8, AAV9, AAVT69, AAVT70 and AAVT71 in undifferentiated C2C12 myoblasts. C2C12 myoblasts were infected with the above-mentioned AAV serotype packaging the GFP gene, and the MOI was 1×10 ⁵ vg/cell. Photos were taken after AAV treatment for 48h. Upper layer: GFP fluorescence; lower layer: bright field. Scale bar = 100 μm.

Figure 2B shows the quantitative results of the percentage of GFP-positive myoblasts in Figure 2A, obtained by calculating the ratio of the GFP signal area to the total cell area. n=5 wells of cells per group, ***p<0.001, one-way analysis of variance.

Figure 2C shows the transduction efficiency of AAV5, AAV8, AAV9, AAVT69, AAVT70 and AAVT71 on myotubes after differentiation of C2C12 myoblasts. After C2C12 myoblasts were cultured in differentiation medium (DMEM+4% horse serum) for 72 hours, AAV was added at an MOI of 1×10 ⁵ vg/cell, and pictures were taken 48 hours later. Upper layer: GFP fluorescence; lower layer: bright field. Scale bar = 100 μm.

Figure 2D shows the quantitative results of the percentage of GFP-positive myotubes in Figure 2C, obtained by calculating the ratio of the area of myotubes positive for GFP signal to the total area of cells. n=5 wells of cells per group, ***p<0.001, one-way analysis of variance.

Figure 3A and Figure 3B show a schematic representation of C57BL/6 mice tail vein injection of 4×10 ¹³ vg/kg packaged GFP gene 21 days after AAV virus serotypes, systemic GFP signal (Figure 3A) and GFP in various muscles /DAPI co-staining (Figure 3B) imaging results. Scale bar 100 μm. The muscle names are as follows: gastrocnemius (GA); quadriceps (QU); longissimus dorsi (LON); sternocleidomastoid (ST); triceps (TRI); tongue (TON).

Figure 3C shows the quantitative statistical results of the infection efficiency of various muscles in Figure 3B, calculated from the ratio of the GFP signal area to the total cross-sectional area of the muscle. n=3 mice per group, **p<0.01, ***p<0.001, one-way analysis of variance.

Figure 3D shows the infectivity of AAV serotypes for class I muscles (represented by soleus muscle (SO)) and class II muscles (represented by tibialis anterior muscle (TA)).

Figure 3E shows the quantitative statistical results of infection efficiency of various muscles in Figure 3D. n=3 mice per group, ***p<0.001, one-way ANOVA.

Figure 4A shows the imaging results of GFP/DAPI co-staining in the heart of C57BL/6 mice 21 days after tail vein injection of 1×10 ¹³ and 4×10 ¹³ vg/kg of the packaged GFP gene. Scale bar = 100 μm.

Figure 4B shows the quantitative statistical results of the infection efficiency of each AAV serotype on cardiomyocytes in Figure 4A, calculated from the ratio of the GFP signal area to the cross-sectional area of the myocardium. n=3 mice per group, *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance.

Figure 4C shows the ability of each AAV serotype to deliver GFP gene to the liver, lung and kidney of mice at a dose of 1×10 ¹³ or 4×10 ¹³ vg/kg. Scale bar = 100 μm.

Figure 4D shows the quantitative statistical results of the percentage of GFP positive cells in Figure 4C. n=3 mice per group, *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance.

Figure 5A and Figure 5B show a schematic representation of mdx male mice tail vein injection of 3 × 10 ¹³ vg/kg packaged GFP gene 21 days after AAV virus serotype, systemic GFP signal (Figure 5A) and various muscle GFP/ Imaging results of DAPI co-staining (Fig. 5B). Scale bar = 200 μm. The muscle names are as follows: gastrocnemius (GA); quadriceps (QU); longissimus dorsi (LON); triceps (TRI); intercostals (INT); tongue (TON).

Figure 5C shows the quantitative statistical results of the infection efficiency of various muscles in Figure 5A, calculated from the ratio of the GFP signal area to the total cross-sectional area of the muscle. n=3 mice per group, **p<0.01, ***p<0.001, one-way analysis of variance.

Fig. 5D shows the infectivity of each AAV serotype to mdx male mouse cardiomyocytes.

Figure 5E shows the quantitative statistical results of the infection efficiency of each AAV serotype in Figure 5D. n=3 mice per group, **p<0.01, one-way ANOVA.

Figure 6 shows the amino acid sequence (SEQ ID NO: 6) of AAVT69 capsid protein.

Figure 7 shows the amino acid sequence (SEQ ID NO: 8) of AAVT70 capsid protein.

Figure 8 shows the amino acid sequence (SEQ ID NO: 10) of AAVT71 capsid protein.

Detailed ways

Unless otherwise defined, 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.

Unless otherwise indicated, nucleic acid or polynucleotide sequences listed herein are in single-stranded form, oriented 5' to 3', left to right. Nucleotides and amino acids are presented herein using the format recommended by the IUPACIUB Biochemical Nomenclature Commission, using either the single-letter code or the three-letter code for the amino acids.

Unless otherwise stated, "polynucleotide" is synonymous with "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. A polynucleotide may include modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs.

As used herein, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including but not limited to").

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the medicaments of the present disclosure, and Humans and non-human animals (eg, rodents or other mammals) are included.

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; experimental animals including rodents) animals such as mice, rats and guinea pigs; birds including domestic, wild 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.

As used herein, the term "treating" 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 a disease, ie, causing regression of at least one of a condition, disease or disorder, or clinical or subclinical symptoms thereof.

As used herein, the term "therapeutically effective amount" refers to a dose that produces the therapeutic effect for which it is administered. For example, a therapeutically effective amount of a drug useful in the treatment of a muscle disorder may be an amount capable of preventing or ameliorating one or more symptoms associated with the muscle disorder.

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

As used 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 developing condition, disease or disorder in a subject, The subject may suffer from or be susceptible to the condition, disease or disorder but has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

As used herein, the term "topical administration" or "local route" refers to administration with a local effect.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process of delivering exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which involves the transfer of exogenous A source polynucleotide is introduced into a host cell.

Herein, the term "gene delivery" refers to the introduction of exogenous polynucleotides into cells 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 of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.

As used herein, the term "infection" refers to the process by which a virus or virus 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 virus replication in a host cell .

Herein, the term "targeting" means that the virus preferentially enters some cells or tissues, and then further expresses the viral genome or the sequence carried by the recombinant transgene in the cells.

In this context, "recombinant" in relation to a polynucleotide means that the polynucleotide is a synthetic product different from the natural polynucleotide constructed through multiple cloning steps, or that two or more polynucleotide fragments are The hybrid synthesis product formed by ligation combination. A recombinant virus is a viral particle comprising a recombinant polynucleotide.

As used herein, the term "vector" refers to one or a series of macromolecules that encapsulate a polynucleotide, which facilitates the delivery of the polynucleotide to target cells in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotides to be delivered are sometimes referred to as "transgenes" and include, but are not limited to, coding sequences for certain proteins or synthetic polypeptides that enhance, inhibit, attenuate, protect, trigger or prevent certain biological and physiological functions, Coding sequences of interest in vaccine development (e.g. polynucleotides expressing proteins, polypeptides or peptides suitable for eliciting an immune response in mammals), RNAi components (e.g. shRNA, siRNA, snRNA, microRNA, ribozymes, anti Sense oligonucleotides and antisense polynucleotides that knock down any endogenous gene activated in an aberrant manner or foreign gene that invades the host cell) or alternative biomarkers. Viral or bacterial polynucleotides are known in the art. The RNAi component typically has 60-100% identity in sequence with its target gene and results in at least a 30% reduction in the corresponding protein product (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% % or 100%).

In this paper, "virion particle" refers to the functional viral unit formed by packaging the natural or artificial viral genome by the capsid protein of the virus, and its functions include infecting or transducing tissues, organs and cells, and delivering the viral genome to tissues and organs And cells and express the corresponding nucleic acid and protein products.

As used herein, the terms "polypeptide" and "protein" refer synonymously herein to polymers consisting of more than 20 amino acids. These terms also encompass synthetic or artificial amino acid polymers.

As used herein, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and acts as a cis element to mediate viral replication, packaging and integration. ITRs herein include, but are not limited to, terminal repeats from AAV types 1-11 (avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). Furthermore, the AAV terminal repeats need not have native terminal repeats, so long as the terminal repeats are available for viral replication, packaging and integration.

As used herein, the term "adeno-associated virus (AAV)" or "adeno-associated virus (AAV) serotype" includes native AAV capsid proteins (e.g., native AAV types 1-11, avian AAV, bovine AAV, canine AAV, Capsid proteins of equine AAV and ovine AAV) and other engineered AAV capsid proteins (e.g., capsid proteins of engineered AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV) . The genome 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 GenBank databases, such as GenBank(R) accession numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497 , U89790, AF043303, AF028705, AF028704, J02275, JO1901, J02275, XO1457, AF288061, AHO09962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF 513852, AY530579, AY631965, AY631966; the contents of which are incorporated herein by reference in their entirety; And for example Srivistava et al., J.Virol (1983) 45:555; Chiorini et al., J.Virol (1998) 71:6823; Chiorini et al., J.Virol (1999) 73:1309; Bantel-Schaal et al., J.Virol ( 1999) 73:939; Xiao et al., J. Virol (1999) 73:3994; Muramatsu et al., Virology (1996) 221:208; WO 00/28061; WO 99/61601; WO 98/11244; US 6156303.

It is known to those skilled in the art that the AAV capsid consists of a total of 60 viral protein (VP) molecules, a mixture of three overlapping gene products VP1, VP2 and VP3 (

TP et al., Adeno-associated virus capsid assembly is divergent and stochastic. Nat Commun 12, 1642, 2021). The VP3 sequence is shared among all VPs. VP2 is about 57 aa longer than VP3, the N-terminal region of VP2 is called the VP1/VP2 common region, and VP1 is about 137 aa longer than VP2. VP2 and VP3 proteins undergo transcription and translation at the start codon inside the VP1 protein. The VP1 sequence includes VP2 and VP3 sequences. The present disclosure provides the amino acid sequence of the VP1 protein of the AAV capsid.

Conservative substitutions of amino acids are known in the art. In one embodiment, amino acids in the AAV capsid protein of the present disclosure may be conservatively substituted in the same group: a) glycine and alanine; b) valine, isoleucine, leucine and pro 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 above-mentioned different groups of amino acids are also allowed.

In one embodiment, the recombinant AAV capsid protein of the present disclosure is replaced by AAV9 capsid protein (SEQ ID NO: 4) The 1st to 203rd positions of the N-terminal of VP1 and VP2 were constructed and named AAVT69 (SEQ ID NO: 6).

In one embodiment, the recombinant AAV capsid protein of the present disclosure is replaced by AAV9 capsid protein (SEQ ID NO: 2) by AAVHH67 capsid protein (SEQ ID NO: 4) The 1st to 193rd positions of the N-terminal of VP1 and VP2 were constructed and named AAVT70 (SEQ ID NO: 8).

In one embodiment, the recombinant AAV capsid protein of the present disclosure is replaced by AAV9 capsid protein (SEQ ID NO: 4) The 1st to 188th positions of the N-terminal of VP1 and VP2 were constructed and named AAVT71 (SEQ ID NO: 10).

The recombinant capsid protein of the present disclosure has high muscle transduction efficiency in vivo and in vitro, and can be used to package and produce AAV vectors containing natural or artificial viral genomes for research or treatment.

In one embodiment, the AAV vectors of the present disclosure can be loaded with exogenous polynucleotides for gene delivery into target cells. The AAV vectors of the present disclosure can be used to deliver nucleic acids to tissues, organs and cells in vitro or in vivo. The AAV vectors of the present disclosure preferentially perform muscle-specific gene delivery with relatively little off-target delivery to other organs.

The present inventors unexpectedly found that the AAV vector of the present disclosure achieves better heart and muscle infection efficiency than AAV9 and HH67 for wild-type mice and mdx mice, and effectively infects a wider variety of muscle types.

In one embodiment, the AAV vector of the present disclosure is significantly weaker than the wild type such as AAV9 in infecting other tissues and organs such as liver, lung and kidney, indicating that the AAV vector of the present disclosure has better specific targeting of cardiac muscle and skeletal muscle , has a high potential to be applied to muscle and heart-related diseases.

In some embodiments, the AAV vectors and medicaments of the present disclosure may be suitable for the treatment of muscle and heart related diseases, including but not limited to the following examples: Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Walker- Warburg syndrome, limb-girdle muscular dystrophy (LGMD), X-linked myotube myopathy (XLMTM), reducing body myopathy, X-linked myopathy with postural muscular atrophy (XMPMA), X chromosome Linked scapular myopathy (X-SPM), facial scapulohumeral muscular dystrophy (FSHD), myasthenia gravis (MG), congenital myasthenic syndrome (CMD), sarcopenia, cachexia, muscle atrophy Lateral sclerosis (ALS), and type I and type II myotonic dystrophy; congestive heart failure, congenital heart failure, ischemic cardiomyopathy, hypertrophic cardiomyopathy and rheumatic heart disease, etc.

In some embodiments, the AAV vectors and drugs of the present disclosure can deliver therapeutic proteins for the treatment of the above-mentioned heart and muscle diseases. The therapeutic proteins that can be delivered include but are not limited to: cystic fibrosis transmembrane regulatory protein (CFTR), Dystrophin (including truncated forms, called mini-dystrophin or microdystrophin, see e.g. Vincent et al., Nat Genet (1993) 5:130; US 2003017131; Wang et al., Proc Natl Acad Sci USA (2000) 97:13714-9; Harper et al., Nat Med (2002) 8:253-61); microlectins, integrin β1, laminin β2, sarcoglycans α and β, sarcomeres, synapses Synuclein, gonadotropins, mini-lecithin, Lamin A/C, four and a half LIM domain protein 1 (FHL1), follistatin, SOD1 or SOD2, full-length or dominant-negative myostatin; Angiogenic factors (eg, VEGF, angiopoietin 1 or 2) and angiogenesis inhibitors (eg, endostatin and angiostatin); anti-inflammatory polypeptides (eg, anti-inflammatory interleukins 4, 10, 11, and 13, whole long and dominant mutants (Ikappa B, Pinch, and ILK genes); coagulation factors (e.g., factor VIII, factor IX, factor X); spectrin, tyrosine hydroxylase, aromatic L-amino acid decarboxylase , leptin and leptin receptors; neurotrophic factors (e.g., BDNF, GDNF, NGF, Semaphore, Slit1, 2 and 3, FGF7, 10 and 22) 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 bone morphogenetic protein superfamily (such as BMP1, 2, 4, 6, 7 and TGF-β, RANKL).

In some embodiments, the AAV vectors and medicaments of the present disclosure are applicable to the treatment of diseases that require administration or gene delivery (such as delivery of vaccines, antibody drugs, soluble receptors or other nucleic acid protein drugs) into muscles, Deliverable polypeptides or proteins include, but are not limited to, Aflibercept (a recombinant VEGF soluble receptor with anti-angiogenic effects produced by Rengeron Pharmaceuticals); recombinant interleukin 1, 18 and TNF-α antagonistic soluble receptors; activin type II soluble receptors; Antibodies; antibodies or single-chain antibodies, including but not limited to anti-VEGF antibodies (such as bevacizumab, ranibizumab and Brolucizumab), anti-sclerostin antibodies (such as Romosozumab and Blosozumab), anti-RANKL antibodies (such as Denosumab), anti- Complement component C5 antibodies (such as Ravulizumab and Eculizumab), anti-PD-1 antibodies (such as Nivolumab, Pembrolizumab and Cemiplimab), PD-L1 antibodies (such as Avelumab and Atezolizumab), anti-CTLA-4 antibodies (such as Ipilimumab), anti-CGRP antibodies ( such as Fremanezumab, Galcanezumab, and Erenumab), anti-HER2 antibodies (such as Trastuzumab and Pertuzumab), anti-EGFR antibodies (such as Cetuximab, Panitumumab, and Necitumumab), antibodies against pro-inflammatory cytokines and their receptors (such as Sarilumab, Siltuximab, Tocilizumab, Canakinumab , Golimumab, Certolizumab, Adalimumab, Infliximab, Daclizumab, and Basiliximab); modified enzymes such as Cethrin (a neuroprotective drug available from BioAxone BioSciences Inc. for the treatment of spinal cord injuries); vaccine-producing antigens or antigenic fragments (e.g. Spike protein of viral disease 2019 (COVID 2019) or severe acute respiratory syndrome (SARS) coronavirus, envelope protein of hepatitis A, B, C and human immunodeficiency virus (HIV), various tumor cell immunogens , such as MAGE antigens, HER2, ErbB2, mucin antigens and estrogen receptors). Vaccines can trigger a protective immune response to prevent the onset of certain diseases. Methods for delivering antibodies or vaccines into subjects represented by intramuscular injection are known to those skilled in the art.

In some embodiments, the AAV vectors of the present disclosure are used in situations where it is desired to label specific cells, such as muscle cells, such as research experiments.

In one embodiment, the AAV vectors of the present disclosure comprise a reporter protein that is used to indicate or mark cells successfully infected by the virus.

In some embodiments, the deliverable heterologous polynucleotide comprises regulatory elements, such as transcriptional/translational control signals, origins of replication, polyadenylation signals, internal ribosomal entry sites, in addition to the sequence encoding the Therapeutic protein (IRES) or 2A signals (e.g. P2A, T2A, F2A), promoters and enhancers (e.g., e.g. CMV promoter or other with vertebrate β-actin, β-globin or β-globin regulatory elements heterogeneous CMV promoter, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter). The use of promoters and enhancers depends on their tissue-specific expression profiles. Promoters and enhancers can be induced by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific time points. Furthermore, promoters and enhancers may be natural or artificial or chimeric sequences, ie prokaryotic or eukaryotic sequences.

In some embodiments, regulatory elements may be tissue-specific or tissue-tropic promoter/enhancer elements, including but not limited to: skeletal muscle-specific promoters (e.g., MCK, HSA, myogenin promoter) and Various types of muscle cells have specific promoters.

In some embodiments, wild-type and engineered AAV virus particles are produced in HEK293 cells by three-plasmid transfection, followed by purification and enrichment processes including removal of cell debris and nucleic acid impurities, filtration concentration, ultracentrifugation, chromatographic column purification steps.

In one embodiment, the production of AAV adopts the three-plasmid transfection method well-known to those skilled in the art, and the basic steps are as follows: by transfecting a forward plasmid encoding a reporter gene protein product or a recombinant protein for therapeutic use, AAV Rep/Cap plasmid, The pHelper plasmid is co-transfected into HEK293 cells to produce the AAV vector of the present invention; in subsequent purification and enrichment steps, nuclease or chemical precipitation is used to remove nucleic acid impurities; ultracentrifugation methods include cesium chloride or iodine Sanol density gradient centrifugation; chromatography methods include but not limited to affinity chromatography, anion chromatography, cation chromatography, heparin sulfate column chromatography, etc.

Reagents, materials, and chemicals involved in the purification process for AAV production include, but are not limited to: chemicals or reagents used in cell culture (e.g., components of cell culture , glutamine, glucose, sucrose, sodium pyruvate, phenol red; antibiotics such as penicillin, kanamycin, streptomycin, tetracycline); chemicals or reagents for cell lysis, polynucleotide precipitation, or ultracentrifugation (such as Triton X-100, NP-40, sodium deoxycholate, sodium lauryl sulfate, dumiphene bromide, sodium lauryl salicylate, sodium chloride, magnesium chloride, calcium chloride, chlorine Barium chloride, magnesium sulfate, sodium hydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate, nitrate, potassium chloride, ammonium chloride, ammonium persulfate, ammonium sulfate, PEG-20, PEG-40, PEG-400, PEG-2000, PEG-6000, PEG-8000, PEG-20000, Tris-HCl, Tris-acetate, manganese chloride, phosphate, bicarbonate, cesium chloride, methanol, ethanol, glycerin , iodixanol, isopropanol, butanol, benzoate, DNase I, RNase); affinity column materials for AAV purification and collection (e.g. AAVX affinity resin, heparan sulfate proteoglycan and mucin resin , other materials related to AAV-specific antibodies); acids, bases, and organics contained in ion-exchange chromatography materials and wash buffers (such as hydrochloric acid, sulfuric acid, acetic acid, formic acid, nitric acid, urea, acetone, chloroform, acetonitrile, trifluoro Acetic acid, sodium hydroxide, potassium hydroxide, barium hydroxide, ammonium hydroxide, Tris base or other organic amines, poloxamer 188, Tween 20, Tween 40, Tween 80, guanidine hydrochloride).

Those skilled in the art can use known standard methods to produce recombinant and synthetic polypeptides or proteins thereof, produce antibodies or antigen-binding fragments, alter nucleic acid sequences, produce transformed cells, construct recombinant AAV mutants, engineer capsid proteins, package expression Vectors for AAV Rep and/or Cap sequences, and for transient or stable transfection of packaging cells. These techniques are known to those skilled in the art. See, e.g., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (Cold Spring Harbor, NY, 1989); F.M. AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

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

In one embodiment, the AAV vector or transgene expression cassette or gene delivery system of the present disclosure is made into pharmaceutical preparations (eg, injections, tablets, capsules, powders) and administered to humans or other mammals. The pharmaceutical preparation also contains other ingredients such as pharmaceutical excipients, water-soluble or organic solvents (such as water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (such as sodium chloride, chloride Potassium, Phosphate, Acetate, Bicarbonate, Tris-HCl and Tris-Acetate), Delaying Dissolving Agents (e.g. Paraffin), Surfactants, Antimicrobials, Liposomes, Lipoplexes, Immuno Inhibitors (eg, cortisone, prednisone, cyclosporine), nonsteroidal anti-inflammatory drugs (NSAIDs, eg, aspirin, ibuprofen, acetaminophen) microspheres, rigid matrices, semisolid carriers, Nanospheres or nanoparticles. Furthermore, the pharmaceutical formulations can be delivered in single or multiple doses by inhalation, systemic or topical (eg, intravenous, intramuscular, intracardiac, subcutaneous, oral, intraperitoneal, and intralesional) administration.

The present disclosure will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods without specific conditions indicated in the examples were operated according to conventional conditions known in the art, or according to the conditions suggested by the manufacturer.

Example

Example 1. Construction of AAVT69, AAVT70 and AAVT71 capsid proteins and virus yield test

Replace the amino acid sequence of the corresponding region of AAVHH67 with the N-terminal sequences of VP1 and VP2 of AAV9 (respectively amino acid 1 to 203, 1 to 193, 1 to 188), and the subsequent amino acid sequence of AAVHH67 capsid protein ( The amino acids 204 to 736, 194 to 736, and 189 to 736) of the AAVHH67 capsid protein are connected, and the new AAV serotypes obtained by hybridization and recombination are named AAVT69, AAVT70 and AAVT71 respectively (Figure 1A).

In order to study the effect of hybrid transformation on the yield of the new virus, the forward plasmid carrying the GFP gene, AAV Rep/Cap plasmid, pHelper plasmid and PEI were co-transfected into 2L suspension culture HEK293 cells at a cell density of 5×10 ⁶ cells /mL, 48-72 hours after transfection, add 0.01-1% Triton X-100, 150-600mM sodium chloride (NaCl), 0.5-3.2mM dumephen, centrifuge to remove cell residues and concentrate the volume, and the supernatant The solution was passed through the AAVX affinity chromatography column, washed with equilibrium solution (containing Tris-hydrochloric acid or Tris-acetic acid, sodium chloride, magnesium chloride, poloxamer 188, pH=7.4), and then eluted with acetic acid and urea aqueous solutions respectively. For viruses, the eluates were combined and subjected to iodixanol ultracentrifugation (65000 rpm, 2.5 hours). Extract the virus layer solution and replace it with phosphate buffer for quantitative analysis. Viral genome was quantified by qPCR and viral particle capsid protein was quantified by silver staining.

As shown in Figure 1B, the production of the new AAV serotypes was comparable to that of AAV9 and AAVHH67, and there was no significant decrease, indicating that the novel AAVT69, AAVT70 and AAVT71 capsid proteins thus constructed could normally produce virus particles.

Example 2. Determination of the infection efficiency of AAVT69, AAVT70 and AAVT71 on muscle cell lines

In order to study the transduction efficiency of new AAV serotypes to muscle cell lines, various AAV serotypes packaged with GFP gene were added to undifferentiated C2C12 myoblasts and C2C12 differentiated 72 cells at a multiplicity of infection of 1×10 ⁵ vg/cell. hours in myotube cells.

As shown in Figure 2A to Figure 2D, AAV5 infected 12.79% of C2C12 myoblasts and 41.2% of C2C12 differentiated myotube cells; the infection ratio of AAV8 and AAV9 to C2C12 myoblasts and C2C12 differentiated myotube cells At 60-70%. Unexpectedly, AAVT69, AAVT70, and AAVT71 could infect 85.33%, 82.95%, and 84.44% of C2C12 myoblasts, and 86.3%, 75.88%, and 83.74% of C2C12 differentiated myotubes, respectively, much higher than wild-type AAV5, AAV8, or AAV9.

In summary, AAVT69, AAVT70, and AAVT71 are significantly better than wild-type AAV in infecting muscle cell lines.

Example 3. Determination of the targeting of AAVT69, AAVT70 and AAVT71 to mouse skeletal muscle and cardiac muscle

In order to further study the transduction efficiency of the new AAV serotype to muscle in vivo, 4×10 ¹³ vg/kg virus particles packaged with GFP gene were injected into the tail vein of C57BL/6 mice, and GFP fluorescence was observed in the muscles of the whole body 21 days after injection. distributed. It was found that the mice injected with AAV9 serotype did not have GFP green fluorescence visible to the naked eye, but the green fluorescence of GFP was clearly visible in the legs and back muscles of mice injected with AAVT69 and AAVT71 serotypes (Figure 3A).

Next, various muscle tissues were removed for GFP and DAPI co-staining. It was found that the fluorescence intensity of GFP signal and the proportion of GFP positive signal muscle cells in gastrocnemius, quadriceps, longissimus dorsi, sternocleidomastoid and triceps of mice injected with AAVT69 and AAVT71 were significantly higher than those of AAV9 (Fig. 3B and Fig. 3C); In gastrocnemius, longissimus dorsi and sternocleidomastoid muscle, the fluorescence intensity of GFP signal and the ratio of GFP positive signal muscle cells were significantly higher than those of AAVHH67. Overall, the transduction efficiency of AAVT69 and AAVT71 to muscle tissue was significantly higher than that of AAV9 and AAVHH67.

As is well known in the art, muscles can be divided into Type I muscles (slow muscles, mainly aerobic metabolism) and Type II muscles (fast muscles, mainly glycolytic metabolism) according to blood flow, energy metabolism, contraction rate and fatigue tolerance. host). In order to study the infection efficiency of various AAV serotypes on the above two types of muscles, the tibialis anterior muscle (mainly type II muscle, accounting for >80%) and soleus muscle (mainly type I muscle, accounting for >80%) were selected , to detect the GFP fluorescence signal (Fig. 3D). The results showed that AAVT69, AAVT70 and AAVT71 could effectively transduce up to 90.05%, 84.39%, 87.97% of tibialis anterior muscle cells and 73.39%, 69.23%, 73.59% of soleus muscle cells, respectively (Fig. 3E), which was significantly better than AAV9, Also higher than AAVHH67.

In addition, it was also found that the infection efficiency of AAVT69 and AAVT71 on cardiomyocytes (heart) at different injection doses (1×10 ¹³ or 4×10 ¹³ vg/kg) was significantly better than that of AAV9 and AAVHH67 (Fig. 4A). The GFP-positive cardiomyocytes of AAVT69 and AAVT71 were 20-30 percentage points higher than those of AAV9 (Fig. 4B).

The above results indicated that the overall transduction efficiency of AAVT69, AAVT70 and AAVT71 to the whole body muscle tissue was better than that of AAV9 and AAVHH67.

In addition, the GFP gene delivery efficiency of each AAV serotype to other tissues and organs (such as liver, lung and kidney) was also studied. The results showed that the transduction efficiency of AAVT69 and AAVT71 to liver, lung and kidney was lower than that of AAV9 (Fig. 4C and Fig. 4D).

It can be seen that compared with AAV9, which is currently most commonly used for systemic muscle gene delivery, AAVT69, AAVT70, and AAVT71 not only have higher infection efficiency for skeletal muscle and cardiac muscle, but also have lower infection potential for off-target tissues such as liver, lung, and kidney. , has better muscle and heart-specific targeting, and is also expected to reduce potential adverse reactions.

Example 4. AAVT69 and AAVT71 to the muscle infection efficiency of mdx mice

In this example, the effects of AAVT69 and AAVT71 on Duchenne muscular dystrophy were studied using the mdx mouse model (a commonly used Duchenne muscular dystrophy model mouse). Inject 3×10 ¹³ vg/kg of each AAV virus particles packaged with GFP gene into the tail vein of mdx male mice, and observe the distribution of GFP fluorescence in the muscles of the whole body 14 days after the injection. The results showed that compared with AAV9 and AAVHH67, AAVT69 and AAVT71 had more obvious GFP green fluorescence signals in the muscles of the legs and back (Fig. 5A).

GFP and DAPI co-staining of various muscles (gastrocnemius, quadriceps, longissimus dorsi, triceps and intercostals) showed that the overall expression of GFP delivered by AAVT69 and AAVT71 was superior in these muscle tissues. in AAV9 and AAVHH67 (Figure 5B and Figure 5C).

In addition, it was also found that the gene delivery efficiency of T69 to mdx mouse cardiomyocytes was also significantly better than that of AAV9 and AAVHH67 (Fig. 5D and Fig. 5E).

The above results provide more conclusive evidence that AAVT69 and AAVT71 have a strong infectious ability for mdx mouse cardiac and skeletal muscle.

All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are hereby incorporated by reference in their entirety.

Although the present disclosure has been illustrated and described with reference to certain preferred embodiments of the present disclosure, those skilled in the art should understand that the above content is a further detailed description of the present disclosure in conjunction with specific embodiments, and cannot be deemed The specific implementation of the present disclosure is limited only by these descriptions. Those skilled in the art may make various changes in form and details, including several simple deduction or substitutions, without departing from the spirit and scope of the present disclosure.

Claims (14)

1. AAV capsid protein, wherein the AAV capsid protein is constructed by replacing the N-terminal 1 to N positions of VP1 and VP2 of AAVHH67 capsid protein with the N-terminal 1 to N positions of VP1 and VP2 of AAV9 capsid protein The amino acid sequence of the AAVHH67 capsid protein is shown in SEQ ID NO: 2, the amino acid sequence of the AAV9 capsid protein is shown in SEQ ID NO: 4, and the N is an integer between 188 and 203 .
2. The AAV capsid protein according to claim 1, wherein said N is 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202 or 203, Preferably, said N is 188, 193 or 203.
3. The AAV capsid protein according to claim 1 or 2, wherein the amino acid sequence of the AAV capsid protein has at least the same amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 98% identity, preferably as shown in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.
4. A nucleic acid molecule, wherein the nucleic acid molecule encodes the AAV capsid protein according to any one of claims 1 to 3.
5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence of the nucleic acid molecule has at least 70% of the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 Preferably, the nucleotide sequence of the nucleic acid molecule has at least 75%, 80%, 85% of the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 %, 90%, 95% or 99% identity.
6. The nucleic acid molecule according to claim 4 or 5, wherein said nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, preferably, said nucleic acid The nucleotide sequence of the molecule is shown in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
7. AAV vector, comprising: the AAV capsid protein and viral genome according to any one of claims 1 to 3.
8. The AAV vector according to claim 7, wherein the viral genome is a natural AAV genome or an artificially recombined viral genome; preferably, the viral genome encodes more than one of a reporter protein and a therapeutic protein.
9. The application of the AAV vector described in claim 7 or 8 in the preparation of medicines for treating heart diseases or muscle diseases.
10. The application according to claim 9, wherein the muscle disease is a muscle disease caused by decreased muscle function, muscle wasting or muscle degeneration; preferably, the muscle disease is selected from the group consisting of: Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Walker-Warburg syndrome, limb-girdle muscular dystrophy (LGMD), X-linked myotube myopathy (XLMTM), reducing body myopathy (Reducing body myopathy), X-linked muscle Postural muscular atrophy (XMPMA), X-linked scapular myopathy (X-SPM), facial scapulohumeral muscular dystrophy (FSHD), myasthenia gravis (MG), congenital myasthenic syndrome (CMD) , sarcopenia, tumor cachexia, amyotrophic lateral sclerosis (ALS), and myotonic dystrophy types I and II.
11. The use according to claim 9, wherein the heart disease is selected from the group consisting of congestive heart failure, congenital heart failure, ischemic cardiomyopathy, hypertrophic cardiomyopathy and rheumatic heart disease.
12. A medicament comprising: the AAV carrier and excipient according to claim 7 or 8.
13. A method of treating heart disease or muscle disease, comprising administering a therapeutically effective amount of the medicament of claim 12 to a subject in need thereof.
14. The method according to claim 13, wherein the drug is administered systemically or locally, such as intravenous administration, intramuscular administration, intracardiac administration, subcutaneous administration, oral administration, topical contact, intraperitoneal administration and focal Internal administration, preferably systemically or locally to the heart and/or muscle.

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