A method for manipulating novel hybrid AAV capsids through hypervariable domain swapping.

Abstract

The present invention relates to a method for preparing a recombinant hybrid adeno-associated virus (AAV) capsid protein with improved tropism, and the recombinant hybrid AAV capsid protein obtainable by the method. The present invention also relates to derived expression vectors, modified cells, and hybrid capsid AAV vector particles packaging a gene of interest, and their use in tissue-targeted gene therapy for treating various diseases.

Description

Technical Field

[0001] The present invention relates to a method for preparing a recombinant hybrid adeno-associated virus (AAV) capsid protein having an improved tropism for muscle and / or the central nervous system, and to a recombinant hybrid AAV capsid protein obtainable by the method. The invention also relates to a derived expression vector, a modified cell, and a hybrid capsid AAV vector particle packaging a gene of interest, and to its use in tissue-targeted gene therapy for treating various diseases, particularly muscle and / or central nervous system diseases.

Background Art

[0002] Recombinant AAV (rAAV) vectors are a leading platform for gene therapy in a wide range of organs for the treatment of various human diseases. The exponential increase in clinical trials using rAAV reflects the great potential of this system and its high versatility (Valdmanis PN et al., Hum. Gene Ther., 2017, 28, pp. 361-372; Wang D et al., Nat. Rev. Drug Discov., 2019, 18, pp. 358-378).

[0003] AAV is a nonpathogenic virus belonging to the genus Dependoparvovirus within the family Parvoviridae. AAV is a non-enveloped virus consisting of a capsid approximately 26 nm in diameter and a 4.7 kb single-stranded DNA genome. The genome contains two genes, rep and cap, which are flanked by two palindromic regions called reverse-terminal repeats (ITRs), which serve as the origin of viral replication and packaging signals. The cap gene encodes three structural proteins, VP1, VP2, and VP3, which constitute the AAV capsid through alternative splicing and translation from different start codons. VP1, VP2, and VP3 share the same C-terminus, which is all of VP3. When AAV2 is used as a reference, VP1 has a 735-amino acid sequence (GenBank accession number YP_680426.1; accessed August 13, 2018); VP2 (598 amino acids) starts at threonine 138 (T138); and VP3 (533 amino acids) starts at methionine 203 (M203). The rep gene encodes four proteins required for viral replication: Rep78, Rep68, Rep52, and Rep40. The recombinant AAV vector capsid-encapsulates an ITR-adjacent rAAV genome in which a therapeutic gene expression cassette replaces the AAV protein-coding sequence.

[0004] The development of effective AAV platforms is the result of a collaborative approach between capsid and vector genome design. In this context, the capsid plays a crucial role in tissue targeting through its interaction with cell receptors and subsequent downstream internalization events. Tissue affinity and transduction efficiency are directly related to the sequence and conformation of the looped-out domain of the VP protein constituting the capsid. Notably, the amino acid variability of VP sequences from different AAV serotypes clusters in 12 hypervariable regions (HVRs) that primarily correspond to the looped-out domain (Gao G et al., Proc Natl Acad Sci US A., 2003, 100, pp. 6081-6086).

[0005] Strategies for developing new capsids can be categorized into four main approaches: natural drug discovery, rational design, directed evolution, and in silico drug discovery (Wang D et al., Nat. Rev. Drug Discov. 2019, 100, pp. 6081-6086). Natural drug discovery consists of isolating wild-type AAVs that naturally infect animals, including humans and non-human primates. Notably, AAVs isolated from human sources, such as AAV9, are the most promising serotypes (Gao G et al., J Virol., 2004, 78, pp. 6381-6388).

[0006] Rational design strategies primarily involve grafting peptides that confer new properties to the capsid, such as increasing receptor binding or inhibiting immunological recognition (Chen YH et al., Nat. Med., 2009, 15, pp. 1215-1218; Asokan A et al., Nat. Biotechnol., 2010, 28, pp. 79-82).

[0007] The directional evolution approach simulates natural evolution. Essentially, by using error-prone PCR or capsid shuffling strategies, a library of randomized capsids is generated, and this library is subjected to selective pressure to select capsids with specific characteristics (Wang D et al., Nat. Rev. Drug Discov., 2019, 18, pp. 358-378). Finally, with advances in high-throughput sequencing, bioinformatics has met the field of capsid development, and this approach has been named in silico drug discovery. Bioinformatics tools can be used to predict capsid regions that better tolerate manipulation, or to infer evolutionary intermediates of known capsids, an approach exemplified by the discovery of the ancestral capsid Anc80 (Marsic, D. et al., Mol. Ther., 2014, 22, pp. 1900-1909; Zinn E et al., Cell Rep., 2015, 12, pp. 1056-1068).

[0008] However, each approach has its own unique limitations that can affect the transduction efficiency of rAAV (Wang D et al., Nat. Rev. Drug Discov., 2019, 18, pp. 358-378). Firstly, AAV infection is endemic in human populations, and therefore rAAV must face existing capsid immunity, which is especially true when using capsids isolated from human sources (Boutin et al., Human Gene Therapy, 2010, Jun;21(6):704-712. doi:10.1089 / hum.2009.182). While rational design approaches can help overcome this problem, insufficient knowledge regarding the stability, AAV receptor binding, internal migration, and cell trafficking of modified capsids imposes significant limitations on this strategy. Additionally, the selection of animal models is crucial for properly selecting novel capsids that have the best performance for gene therapy applications in humans. This is especially true when using a directional evolutionary approach where capsid selection is deeply ingrained in the model system.

[0009] To improve AAV vectors used in gene therapy, there is a need for novel AAV capsid manipulation strategies that at least partially overcome the limitations of existing approaches. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2019 / 193119 [Non-patent literature]

[0011] [Non-Patent Document 1] Valdmanis PN et al., Hum. Gene Ther., 2017, 28, pp. 361-372. [Non-Patent Document 2] Wang D et al., Nat. Rev. Drug Discov., 2019, 18, pp. 358-378. [Non-Patent Document 3] Gao G et al., Proc Natl Acad Sci US A., 2003, 100, pp. 6081-6086. [Non-Patent Document 4] Wang D et al., Nat. Rev. Drug Discov. 2019, 100, pp. 6081-6086. [Non-Patent Document 5] Gao G et al., J Virol., 2004, 78, pp. 6381-6388 [Non-Patent Document 6] Chen YH et al., Nat. Med., 2009, 15, pp. 1215-1218. [Non-Patent Document 7] Asokan A et al., Nat. Biotechnol., 2010, 28, pp. 79-82. [Non-Patent Document 8] Marsic, D. et al., Mol. Ther., 2014, 22, pp. 1900-1909 [Non-Patent Document 9] Zinn Eら, Cell Rep., 2015, 12, pages 1056~1068 [Non-licensed Document 10] Boutin, Human Gene Therapy, 2010, Jun;21(6):704~12 pages. doi:10.1089 / hum.2009.182 [Non-licensed Document 11] Meliani, Hum Gene Ther Methods. 2015 Apr;26(2):45-53. doi:10.1089 / hgtb.2015.037 [Non-licensed Document 12] La Bella Tら, Gut, 2020, 69, pages 737~747.doi:10.1136 / gutjnk-2019-318281 [Non-licensed Document 13] Lingら, 2016 Jul 18, Hum Gene Ther Methods. [Non-licensed Document 14] Vercauterenら, 2016, Mol. Ther. Vol. 24(6), 1042 pages [Non-licensed Document 15] Rosario, 2016, Mol Ther Methods Clin Dev. 3, 16026 pages [Non-licensed Document 16] Michelfelder S.ら, PLoS One. 2009; 4(4): e5122 [Non-licensed Document 17] Girod, Nat. Med., 1999, 5, pages 1052~1056 [Non-licensed Document 18] Grifman, Molecular Therapy, 2001, 3, pages 964~975 [Non-licensed Document 19] Rabinowitz, Virology, 1999, pages 265, 274~285 [Non-licensed Document 20] Wuら, J. Virol., 2000, 74, pages 8635~8647 [Non-licensed Document 21] Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102: 1045-1054 [Non-Patent Document 22] McCarty et al., Gene Therapy, 2003, Dec., 10(26), 2112-2118 [Non-Patent Document 23] Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and Translational Neurology, 2018, 5, 1574-1587 [Non-Patent Document 24] Rohr et al., J Virol Methods., 2002, 106, 81-8. doi:10.1016 / s0166-0934(02)00138-... [Summary of the Invention] [Means for Solving the Problems]

[0012] The inventors have shown that combinations of hypervariable regions (HVRs) from different AAV serotypes can result in a mixed profile of the parental capsid that outperforms the original efficacy of the parental capsid. In particular, the inventors obtained hybrid AAV capsids that advantageously improve affinity compared to at least the parental acceptor capsid while maintaining the low seroprevalence of the acceptor capsid. This is surprising because the sequences and conformations of the 12 HVRs are directly involved in both the affinity and seroprevalence of the AAV capsid, and these molecular determinants remain not fully elucidated. Thus, it is unexpected to improve affinity without impairing seroprevalence.

[0013] Therefore, the present invention is a method for preparing a recombinant hybrid adeno-associated virus (AAV) capsid protein having improved affinity for muscle and / or the central nervous system, comprising a) A step of providing at least two recombinant AAV capsid proteins, an acceptor AAV capsid protein, and at least one donor AAV capsid protein from different AAV serotypes; wherein the donor AAV capsid serotype is AAV13 or hybrid AAV2 / 13; b) A step of replacing at least one hypervariable region (HVR) sequence selected from the HVR1-HVR10 and HVR12 sequences of the acceptor AAV capsid protein with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein to obtain a recombinant hybrid AAV capsid protein having improved affinity to muscle and / or the central nervous system compared to at least the parent acceptor AAV capsid protein. This includes methods.

[0014] In some embodiments of the method according to the present invention, the acceptor AAV capsid serotype has a low seroprevalence, and the donor AAV capsid serotype has a higher seroprevalence than the acceptor AAV capsid serotype. In some preferred embodiments of the method according to the present invention, the hybrid AAV capsid protein has a seroprevalence equivalent to that of the acceptor AAV capsid protein.

[0015] In some preferred embodiments of the method according to the present invention, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAVrh10, AAV-LK03, AAVrh74, AAV9.rh74, and AAV9.rh74-P1, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and sequences of SEQ ID NOs. 2 to 30.

[0016] In some embodiments of the method according to the present invention, the HVR sequence of the donor AAV capsid protein and / or acceptor AAV capsid protein is selected from the group consisting of the HVR1 sequence at positions 134-165, the HVR2 sequence at positions 176-192, the HVR3 sequence at positions 259-278, the HVR4 sequence at positions 379-395, the HVR5 sequence at positions 446-484, the HVR6 sequence at positions 490-500, the HVR7 sequence at positions 501-512, the HVR8 sequence at positions 514-529, the HVR9 sequence at positions 531-570, the HVR10 sequence at positions 576-613, and the HVR12 sequence at positions 705-736; the indicated position is determined by alignment with SEQ ID NO: 1.

[0017] In some embodiments of the method according to the present invention, step b) includes replacing fewer than eight HVR sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein; preferably, step b) includes replacing up to six HVR sequences of the acceptor AAV capsid protein, preferably up to four HVR sequences, with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

[0018] In some preferred embodiments of the method according to the present invention, step b) includes replacing at least the HVR5 sequence of the acceptor AAV capsid protein with a different HVR5 sequence from the donor AAV capsid protein; preferably the HVR5 sequence from the donor AAV capsid protein includes a sequence selected from the group consisting of SEQ ID NOs: 175 to 186; preferably step b) includes replacing the HVR5 sequence of the acceptor AAV capsid protein alone or in combination with one or more or all of HVR6, HVR7, HVR8, HVR9 and HVR10; preferably step b) includes replacing the HVR5 sequence of the acceptor AAV capsid protein alone or in combination with one or more or all of HVR6, HVR7 and HVR8.

[0019] In some embodiments of the method according to the present invention, step b) includes replacing all of the HVR5-HVR10 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein; preferably, step b) includes replacing all of the HVR5-HVR8 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

[0020] In some embodiments of the method according to the present invention, step b) includes replacing one of the HVR1-HVR10 and HVR12 sequences of the acceptor AAV capsid protein with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein; preferably, step b) includes replacing the HVR3, HVR5, HVR9, HVR10, or HVR12 sequence of the acceptor AAV capsid protein with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein. In some more preferred embodiments, step b) includes replacing the HVR5 of the acceptor AAV capsid protein with a different HVR5 sequence from the donor AAV capsid protein.

[0021] Another aspect of the present invention relates to recombinant hybrid AAV capsid proteins having improved affinity, which can be obtained by the methods described herein.

[0022] In some specific embodiments, the recombinant hybrid AAV capsid protein comprises an amino acid sequence selected from the group consisting of sequences SEQ ID NOs. 33-43, 45, 47-58 and 60-73 and sequences having at least 85% identity with those sequences, and the amino acid sequence variant has no mutations in at least one or all of the HVR sequences from the donor AAV capsid protein.

[0023] Another aspect of the present invention relates to a recombinant plasmid comprising a polynucleotide encoding a recombinant hybrid AAV capsid protein according to the present disclosure in an expressible form; the polynucleotide is preferably selected from the nucleotide sequences of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 102, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and ultimately further encodes an AAV replicase protein in an expressible form.

[0024] Another aspect of the present invention relates to cells stably transformed with recombinant plasmids according to the present disclosure.

[0025] Another aspect of the present invention relates to AAV vector particles packaging a gene of interest, comprising at least one hybrid recombinant AAV capsid protein according to the present disclosure; preferably, the gene of interest is selected from the group consisting of therapeutic genes; genes encoding therapeutic proteins or peptides, such as therapeutic antibodies or antibody fragments and genome editing enzymes; and genes encoding therapeutic RNA, such as interfering RNA, guide RNA for genome editing and antisense RNA having the ability to exon skip.

[0026] Another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of AAV vector particles according to the present disclosure or cells stably transduced by the AAV vector particles. The present invention also encompasses the AAV vector particles, cells, or pharmaceutical compositions of the present disclosure as pharmaceuticals, particularly for use in muscle and / or CNS diseases, preferably genetic neuromuscular diseases. [Modes for carrying out the invention]

[0027] Preparation method for hybrid AAV capsids Therefore, the present invention relates to a method for preparing recombinant hybrid adeno-associated virus (AAV) capsid proteins having improved affinity, particularly to muscle and / or CNS, a) A step of providing at least two recombinant AAV capsid proteins, an acceptor AAV capsid protein, and at least one donor AAV capsid protein from different AAV serotypes; b) A step of replacing at least one hypervariable region (HVR) sequence of an acceptor AAV capsid protein with a different HVR sequence from the corresponding HVR of a donor AAV capsid protein to obtain a recombinant hybrid AAV capsid protein having improved affinity, particularly to muscle and / or CNS, compared to at least the parent acceptor AAV capsid protein. This includes methods.

[0028] As used herein, “AAV serotype” or “AAV capsid serotype” refers to an AAV capsid having a distinct hypervariable region (HVR) amino acid sequence compared to an AAV capsid of another serotype. Different AAV serotypes have amino acid variations in their HVR sequences. The term AAV serotype encompasses any natural or artificial AAV capsid serotype, including AAV capsid variants isolated from primates (human or non-human) or non-primate species, as well as AAV capsid variants manipulated by various techniques known in the art, such as rational design, directional evolution, and in silico drug discovery. As used herein, the term AAV serotype refers to a functional AAV capsid that can be transduced into cells, tissues, or organs, particularly cells, tissues, or organs of interest (target cells, tissues, or organs), to form recombinant AAV virus particles that express the transgene in said cells, tissues, or organs, particularly target cells, tissues, or organs.

[0029] As used herein, “hypervariable region or HVR” refers to one of HVR1 to HVR12 of the AAV capsid. According to the narrow definition of HVR, HVR1 is at positions 146–153; HVR2 is at positions 183–187; HVR3 is at positions 263–267; HVR4 is at positions 384–386; HVR5 is at positions 453–477; HVR6 is at positions 493–498; HVR7 is at positions 503–507; HVR8 is at positions 517–525; HVR9 is at positions 536–559; HVR10 is at positions 584–597; HVR11 is at positions 661–670; and HVR12 is at positions 708–722; the indicated position is determined by alignment with SEQ ID NO: 1 (VP1 of AAV8 or AAV8 capsid). After sequence alignment of any other AAV capsid sequence of any other serotype with SEQ ID NO: 1 using standard protein sequence alignment programs well known in the art, such as BLAST, FASTA, CLUSTALW, and MEGA, those skilled in the art can easily obtain the corresponding locations of the hypervariable region in the other AAV capsid serotype. For example, using MEGA software (version X) with the ClustalW alignment algorithm in default parameters, HVR1-HVR12 correspond to positions 146-152, 182-186, 262-264, 381-383, 450-474, 490-495, 500-504, 514-522, 533-556, 581-594, 658-667, and 705-719, respectively, of the capsid of SEQ ID NO: 2 (named #704).

[0030] The position of the HVR sequence from the donor or acceptor AAV capsid may differ by only a few amino acids from the position indicated above (HVR reference sequence). Depending on the initial size of the HVR and the distance between different HVRs, both HVR sequences (the replacement sequence from the acceptor capsid and the replacement sequence from the donor capsid) consist of at least 2 to approximately 70 amino acids. For example, the HVR sequence from the donor or acceptor AAV capsid may have a deletion of 1 amino acid at one end of an HVR sequence of up to 5 amino acids; a deletion of up to 2 amino acids (1 or 2 amino acids) at one or both ends of an HVR sequence of 6 to 10 amino acids; or a deletion of up to 5 amino acids (1, 2, 3, 4, or 5 amino acids) at one or both ends of an HVR sequence of 11 to 25 amino acids. Alternatively, the HVR sequence from the donor or acceptor AAV capsid may have an additional sequence from the N or C terminus of the HVR sequence, for example, up to 10, 20, 30, 40, or 50 amino acids from the N or C terminus of the HVR sequence. Preferably, the amino acid deletion or addition at one or both ends of the HVR sequence is accompanied by a sequence of amino acids from the donor or acceptor AAV capsid sequence.

[0031] As used herein, the term “affinity” refers to the ability of an AAV capsid protein present in recombinant AAV virus particles to transduce certain types of cells, tissues, or organs (e.g., cell or tissue affinity). The affinity of the recombinant hybrid AAV capsid protein (or hybrid AAV capsid) according to the present invention to certain types of cells, tissues, or organs may be determined by measuring the ability of an AAV vector particle (hybrid capsid serotype AAV vector particle) containing the hybrid AAV capsid protein to transduce or express a transgene in the aforementioned types of cells, tissues, or organs using a standard assay well known in the art, such as those disclosed in the examples of this application. For example, vector transduction or transgene expression is determined by topical or systemic administration of hybrid capsid serotype AAV vector particles in animal models, such as mouse models well known in the art and those disclosed in the examples of this application. Parental AAV vector serotypes containing donor or acceptor capsids are used for comparison. Vector transduction may be determined by measuring the vector genome copy number per diploid genome using a standard assay well known in the art, such as a real-time PCR assay. Transgene expression is advantageously measured by a standard assay well known in the art, such as an in vivo or in vitro quantitative bioluminescence or fluorescence assay, using a reporter gene, such as luciferase or a fluorescent protein (GFP or others).

[0032] A hybrid AAV capsid protein is a functional AAV capsid that can transduce cells, tissues, or organs, particularly target cells, tissues, or organs, to form recombinant AAV virus particles that express an transgene in said cells, tissues, or organs, particularly target cells, tissues, or organs. Furthermore, a hybrid AAV capsid protein has improved affinity compared to its parent AAV capsid protein. A hybrid AAV capsid protein with improved affinity may have increased affinity to at least one target cell, tissue, or organ and / or decreased affinity (or detargeting) to at least one off-target cell, tissue, or organ compared to at least the parent acceptor AAV capsid. Increased affinity specifically refers to an increased transgene expression level of at least 1.5 times, preferably 2, 3, 4, 5 times, or more, in at least one target cell, tissue, or organ compared to the parent AAV capsid protein. Detargeting specifically refers to a transgene expression level that is at least 3-fold, preferably 5-10-fold or greater, in at least one off-target cell, tissue, or organ compared to a non-detargeted parental AAV capsid protein. The transgene expression level achieved using the hybrid AAV capsid protein in target cells, tissues, or organs is advantageously at least the same magnitude (less than 1.5 times lower; i.e., equivalent) as that of a reference AAV serotype, e.g., AAV9, for muscle and CNS tissues. As a result of its improved affinity, the hybrid AAV capsid protein according to the present invention has improved in vivo distribution. This means that it significantly better targets defined tissues (target tissues), groups of tissues (e.g., skeletal muscle and heart), or groups of organs (target tissues or organs) without increasing the targeting of other (non-target) tissues (e.g., improved specificity), and / or it targets specific tissues (non-target or off-target tissues or organs) with lower efficacy to reduce toxicity that is normally undesirable (tissue detargeting, e.g., liver detargeting).

[0033] As used herein, the term “muscle” refers to cardiac muscle (i.e., heart) and skeletal muscle. The term “muscle cell” refers to myoblasts, myotubes, myoblasts, and / or satellite cells. Skeletal muscle is classified into different groups based on their anatomical location in the body. The affinity of the hybrid AAV capsid according to the present invention to different muscle groups may be measured in the mouse tibia (TA), extensor digitorum longus (EDL), quadriceps (Qua), gastrocnemius (Ga), soleus (Sol), triceps, biceps, and / or diaphragm; in particular, in the mouse extensor digitorum longus (EDL), soleus (Sol), quadriceps (Qua), triceps, and diaphragm or soleus (Sol), quadriceps (Qua), triceps, and diaphragm muscles.

[0034] As used herein, the term “central nervous system or CNS” means the brain, spinal cord, retina, optic nerve, and / or olfactory nerve and epithelium. As used herein, the term “CNS cells” means any cells of the CNS, including neurons and glial cells (oligodendrocytes, astrocytes, ependymal cells, microglia).

[0035] As used herein, “serological prevalence” refers to human serological prevalence, which means the level of anti-AAV antibodies present in a human population that bind to AAV capsid serotypes and are expressed as serum antibodies or immunoglobulins. The serological prevalence of AAV capsids is measured using a cohort of human serum and a standard assay that is well known in the art and disclosed, for example, (Meliani et al., Hum Gene Ther Methods. 2015 Apr;26(2):45-53. doi:10.1089 / hgtb.2015.037). The assay may be an ELISA assay disclosed in the examples of this application. The serological prevalence of an AAV capsid serotype (or serotype) may be defined as the percentage of individuals having an ELISA titer of IgG specific to that serotype higher than 10 μg / mL. A low-prevalent serotype may be defined as a serotype in which less than approximately 30% of individuals are seropositive, corresponding to a seroprevalence similar to or lower than that of the AAV8 capsid (SEQ ID NO: 1), which is considered a low-seroprevalence reference. A high-seroprevalent AAV capsid serotype refers to an AAV capsid serotype with a seroprevalence higher than 50%. A seroprevalence equivalent to that of the acceptor AAV capsid refers to a seroprevalence of approximately 30%. Alternatively, seroprevalence may be defined as the dilution (OD50) at which a 50% reduction in the OD signal is observed using a dose-response curve. The OD50 of the AAV capsid being tested is compared to that of a reference AAV capsid with a known seroprevalence.

[0036] Unless otherwise explicitly indicated by the context, “a,” “an,” and “the” refer to multiple objects. Thus, the terms “a” (or “an”), “one or more,” or “at least one” are interchangeable herein; unless otherwise specified, “or” means “and / or.”

[0037] The term "identity" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. If the positions in both sequences being compared are occupied by the same base or the same amino acid residue, then each molecule is identical at that position. The percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. Generally, a comparison is performed when the two sequences are aligned to give maximum identity. Identity may be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pile-up program or a sequence comparison algorithm, such as BLAST, FASTA, or CLUSTALW.

[0038] Acceptor and donor AAV capsids may be from any different natural or artificial AAV serotypes. At least 13 different AAV serotypes (AAV1-13) have been identified in humans and non-human primates and have been classified into various clades and clones based on phylogenetic analysis of VP1 sequences of various primate AAV isolates: AAV1 and AAV6 belong to clade A; AAV2 to clade B; AAV2-AAV3 hybrids to clade C; AAV7 to clade D; AAV8 to clade E; and AAV9 to clade F, while AAV3, AAV4, and AAV5 are disclosed as clones (Gao et al., J. Virol., 2004, 78, pp. 6381-6388). AAV2 variant serotypes and AAV2 / 13 hybrid capsids have been isolated in human liver (La Bella et al., Gut, 2020, pp. 69, 737-747; doi:10.1136 / gutjnk-2019-318281; SEQ ID NOs. 2-30 in the attached sequence listing). Other AAV serotypes have been identified in non-primate species, such as pigs, cattle, birds, and goats. Porcine AAV includes, in particular, AAVpo1, po2.1, and po4-6. Various AAV capsid variants are also named "synthetic AAV serotypes," or new AAV serotypes have been manipulated, particularly by directional gene evolution or in silico drug discovery, including, without limitation, eight AAV serotypes (AAV2, 4, 5, 8, 9, avian, bovine, and goat), AAV-Anc80, AAV2i8, AAV-LK03, and others, which are hybrid capsids from recombinant AAV2-derived serotypes DJ, DJ8, and PHP.B.

[0039] In some embodiments, the acceptor AAV capsid protein is an AAV serotype used in gene therapy, also referred to as the "conventional AAV serotype," such as AAV1, AAV2, AAV2 variants (e.g., quadruple mutant capsid-optimized AAV2 containing an engineered capsid with the Y44+500+730F+T491V change; disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods), AAV3, and AAV3 variants (e.g., AAV3-ST variant containing an engineered AAV3 capsid with two amino acid changes, S663V+T492V; disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. AAV-3B and AAV-3B variants (disclosed on page 1042 of 24(6)), AAV-3B and AAV-3B variants, AAV4, AAV5, AAV6 and AAV6 variants (e.g., AAV6 variants including the triple-mutated AAV6 capsid Y731F / Y705F / T492V form; disclosed on page 16026 of Rosario et al., 2016, Mol Ther Methods Clin Dev. 3), AAV7, AAV8, AAV9, AAV2G9, AAV10, e.g., AAVcy10 and AAVrh10, AAVrh32,33, AAVrh39, AAVrh43, AAVrh74, AAV-DJ, AAVanc80, AAV-LK03, AAV.PHP, e.g., AAV-PHP.B, AAV-PHP.EB, AAV2i8, clade F These are derived from AAVHSCs, such as AAVHSC7, AAVHSC15 and AAVHSC17, AAV9.rh74 and AAV9.rh74-P1 (International Publication No. 2019 / 193119), porcine AAVs, such as AAVpo1, AAVpo2.1, AAVpo4 and AAVpo6, and tyrosine, lysine, and serine capsid variants of AAV serotypes.

[0040] In certain embodiments, the acceptor AAV capsid protein is from an AAV serotype selected from the group consisting of AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAVrh32,33, AAVrh39, AAVrh43, AAVrh74, AAV9.rh74, AAV9.rh74-P1, AAV-DJ, AAVAnc80, AAV2i8, AAV-LK03, and AAV.PHP. AAV4 capsid (GenBank accession number NC_001829.1); AAV5 capsid (GenBank accession number NC_006152.1; accessed August 13, 2018); AAV7 capsid (GenBank accession number NC_006260.1); AAV9 capsid (GenBank accession number AY530579.1; accessed June 24, 2004);; AAVrh10 capsid (G enBank access number AY243015.1; Access date May 14, 2003); AAV-LK03 (amino acid sequence of SEQ ID NO: 166), AAVrh74 (amino acid sequence of SEQ ID NO: 160; CDS of SEQ ID NO: 161), AAV9.rh74 (amino acid sequence of SEQ ID NO: 162; CDS of SEQ ID NO: 163), AAV9.rh74-P1 (amino acid sequence of SEQ ID NO: 164; CDS of SEQ ID NO: 165).

[0041] In certain embodiments, the donor AAV capsid protein is from a newly isolated native AAV variant serotype, such as an AAV2 / 13 hybrid serotype, particularly isolated from human tissue, such as liver tissue; more preferably selected from the group consisting of sequences SEQ ID NOs: 2-30. In some preferred embodiments, the donor AAV capsid protein is selected from the group consisting of sequences SEQ ID NOs: 2-10, 18, 20-22, 29, and 30; even more preferably selected from the group consisting of sequences SEQ ID NOs: 2, 10, 20, 21, and 30.

[0042] In certain embodiments, the donor AAV capsid protein is derived from an AAV serotype used in gene therapy. The donor AAV capsid protein may be AAV13. The AAV13 capsid gene (coding sequence or CDS) sequence corresponds to positions 1948–4149 of the AAV13 genome sequence in GenBank accession number EU285562.1 (accessed September 23); the AAV13 capsid protein (major coat protein or VP1) amino acid sequence corresponds to GenBank accession number ABZ10812.1 (accessed September 23, 2008) or Sequence ID No. 202.

[0043] In some preferred embodiments, the acceptor AAV capsid serotype has a low seroprevalence, and the donor AAV capsid serotype has a higher seroprevalence than the acceptor AAV capsid serotype. Examples of acceptor AAV capsid serotypes having a low seroprevalence include, without limitation, AAV8, AAV9, AAV5, AAV-LK03, AAVrh10, AAVrh74, AAV9.rh74, and AAV9.rh74-P1. In some more preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10. In some preferred embodiments, the donor AAV capsid serotype is selected from AAV13 and hybrid AAV2 / 13. In some more preferred embodiments, the donor AAV capsid serotype is selected from the group consisting of AAV13 and sequences of SEQ ID NOs: 2-30; preferably AAV13 and sequences of SEQ ID NOs: 2-10, 18, 20-22, 29 and 30; even more preferably AAV13 and sequences of SEQ ID NOs: 2, 10, 20, 21 and 30.

[0044] Step b) involves substituting 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) HVR sequences of acceptor capsid serotypes selected from HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, HVR7, HVR8, HVR9, HVR10, and HVR12 with different HVR sequences from the corresponding HVRs in the donor AAV capsid protein.

[0045] In some embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replacement HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and HVR7 sequence at positions 687-738. The HVR12 sequence is selected from the group consisting of HVR1 sequences (preferably HVR1 sequences at positions 134-165, HVR2 sequences at positions 176-192, HVR3 sequences at positions 259-278, HVR4 sequences at positions 379-395, HVR5 sequences at positions 446-484, HVR6 sequences at positions 490-500, HVR7 sequences at positions 501-512, HVR8 sequences at positions 514-529, HVR9 sequences at positions 531-570, HVR10 sequences at positions 576-613, and HVR12 sequences at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid). The HVR11 sequence that is not replaced in the method according to the present invention corresponds to the sequence at positions 621-687; preferably the sequence at positions 630-682; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid). These positions correspond to a broad definition of the HVR sequence.

[0046] In some embodiments, step b) includes replacing fewer than eight HVR sequences in the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein contains fewer than eight HVR sequences from the donor AAV capsid protein. In some preferred embodiments, step b) includes replacing up to six HVR sequences, preferably up to four HVR sequences, in the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein contains up to six HVR sequences, preferably up to four HVR sequences, from the donor AAV capsid protein. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and sequences of SEQ ID NOs. 2 to 30.In some preferred embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replacement HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and 687-738 The sequence is selected from the group consisting of an HVR12 sequence at position 134-165, preferably an HVR1 sequence at positions 176-192, an HVR2 sequence at positions 259-278, an HVR4 sequence at positions 379-395, an HVR5 sequence at positions 446-484, an HVR6 sequence at positions 490-500, an HVR7 sequence at positions 501-512, an HVR8 sequence at positions 514-529, an HVR9 sequence at positions 531-570, an HVR10 sequence at positions 576-613, and an HVR12 sequence at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid). In some embodiments, step b) includes replacing one or more or all of the HVR5-HVR10 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein includes one or more or all of the HVR5-HVR10 sequences from the donor AAV capsid protein. In some preferred embodiments, step b) includes replacing one or more or all of the HVR5-HVR8 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein includes one or more or all of the HVR5-HVR8 sequences from the donor AAV capsid protein. In some more preferred embodiments, step b) includes replacing at least the HVR5 sequence of the acceptor AAV capsid protein with a different HVR5 sequence from the corresponding HVR of the donor AAV capsid protein.HVR5 may be replaced alone or together with one, more or all of the acceptor AAV capsid proteins HVR6-HVR10. For example, step b) may include a step of replacing HVR5, HVR5-HVR8, HVR5-HVR9, or HVR5-HVR10. HVR5 is preferably replaced alone or together with one, more or all of the acceptor AAV capsid proteins HVR6-HVR8. For example, step b) may include a step of replacing HVR5 or HVR5-HVR8. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and the sequences of SEQ ID NOs. 2-30; preferably AAV13 and the sequences of SEQ ID NOs. 2-10, 18, 20-22, 29, and 30; more preferably AAV13 and the sequences of SEQ ID NOs. 2, 10, 20, 21, and 30. In some preferred embodiments, one or more HVR5-HVR10 sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replacement HVR sequence) are: HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; and 5 A sequence is selected from the group consisting of HVR10 sequences at positions 76-613; more preferably HVR5 sequences at positions 446-484; HVR6 sequences at positions 490-500; HVR7 sequences at positions 501-512; HVR8 sequences at positions 514-529; HVR9 sequences at positions 531-570; and HVR10 sequences at positions 576-613; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid).

[0047] In some specific embodiments, step b) is to select the HVR5-HVR8 sequence of the acceptor AAV capsid protein from any one of AAV13 and SEQ ID NOs. 2-30; preferably AAV13, #704 (SEQ ID NO 2); #1704 (SEQ ID NO 10); #3086 (SEQ ID NO 20); #1024 (SEQ ID NO 22); #508 (SEQ ID NO 9); #3142 (SEQ ID NO 21); #2320 (SEQ ID NO 29); #1010 (SEQ ID NO 6); M258 (SEQ ID NO 30); #1570 (SEQ ID NO 18); #1602 (SEQ ID NO 5); #667 (SEQ ID NO 7); #129 (SEQ ID NO 3); and #767 (SEQ ID NO 8); more preferably selected from AAV13, #704 (SEQ ID NO 2) and M258 (SEQ ID NO 30). The process includes replacing the donor AAV capsid with the HVR5-HVR8 sequences of the donor AAV capsid serotype; preferably, the HVR5 sequence of the donor AAV capsid protein (replacement HVR5 sequence) and / or the acceptor AAV capsid protein (replacement HVR5 sequence) is at positions 446-485; the HVR6 sequence is at positions 485-502; the HVR7 sequence is at positions 499-516; and the HVR8 sequence is at positions 509-531; more preferably, the HVR5 sequence is at positions 446-484; the HVR6 sequence is at positions 490-500; the HVR7 sequence is at positions 501-516; and the HVR8 sequence is at positions 514-529; the indicated position is determined by alignment with Sequence ID No. 1 (VP1 of AAV8 or AAV8 capsid). In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10.

[0048] In some embodiments, step b) includes replacing one of the HVR1-HVR10 and HVR12 of the acceptor AAV capsid protein with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein includes one HVR sequence from the donor AAV capsid protein. In some specific embodiments, step b) includes replacing HVR5, HVR6, HVR7, or HVR8 of the acceptor AAV capsid with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein includes the HVR5, HVR6, HVR7, or HVR8 sequence from the donor AAV capsid protein. In some preferred embodiments, step b) includes replacing one of HVR1, HVR3, HVR5, HVR6, HVR7, HVR8, HVR9, HVR10, and HVR12 of the acceptor AAV capsid; preferably one of HVR3, HVR5, HVR9, HVR10, or HVR12, with a different HVR sequence from the corresponding HVR of the donor AAV capsid protein. In some preferred embodiments, step b) includes replacing HVR5 of the acceptor AAV capsid with a different HVR5 sequence from the donor AAV capsid protein, for example, the recombinant hybrid AAV capsid protein contains an HVR5 sequence from the donor AAV capsid protein. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, AAV5, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and the sequences of SEQ ID NOs. 2-30; preferably AAV13 and the sequences of SEQ ID NOs. 2-10, 18, 20-22, 29, and 30; more preferably selected from the group consisting of the sequences of SEQ ID NOs. 2, 10, 20, 21, and 30.In some other preferred embodiments, step b) includes replacing one of HVR1, HVR3, HVR6, HVR7, HVR8, HVR9, HVR10, and HVR12 of AAV8; preferably HVR3, HVR9, HVR10, or HVR12 of AAV8 with a different HVR sequence from the corresponding HVR of a donor AAV capsid protein selected from the group consisting of AAV13 and the sequences of SEQ ID NOs: 2-30; preferably SEQ ID NO: 2. In some preferred embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replaced HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and H at positions 687-738. A VR12 sequence; more preferably selected from the group consisting of HVR1 sequences at positions 134-165, HVR2 sequences at positions 176-192, HVR3 sequences at positions 259-278, HVR4 sequences at positions 379-395, HVR5 sequences at positions 446-484, HVR6 sequences at positions 490-500, HVR7 sequences at positions 501-512, HVR8 sequences at positions 514-529, HVR9 sequences at positions 531-570, HVR10 sequences at positions 576-613, and HVR12 sequences at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid).

[0049] In some specific embodiments, HVR5 is from a donor AAV capsid serotype selected from the group consisting of AAV13; #704 (SEQ ID NO: 2); #1704 (SEQ ID NO: 10); #3086 (SEQ ID NO: 20); #508 (SEQ ID NO: 9); #3142 (SEQ ID NO: 21); #M258 (SEQ ID NO: 30); #1570 (SEQ ID NO: 18); #2731 (SEQ ID NO: 4); #1602 (SEQ ID NO: 5); #667 (SEQ ID NO: 7); #129 (SEQ ID NO: 3); and #767 (SEQ ID NO: 8); preferably HVR5 is from an AAV capsid serotype selected from the group consisting of sequences SEQ ID NOs: 2, 10, 20, 21, and 30. The HVR5 sequence is advantageously from positions 446 to 485; preferably from positions 446 to 484; the indicated position is determined by alignment with SEQ ID NO: 1 (VP1 of AAV8 or AAV8 capsid). In some preferred embodiments, HVR5 includes a sequence selected from the group consisting of SEQ ID NOs: 175-186; preferably SEQ ID NOs: 175-179. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10.

[0050] In some preferred embodiments, the hybrid AAV capsid protein has increased affinity for muscle and / or the central nervous system compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein. In some specific embodiments, the hybrid AAV capsid protein has increased affinity for the kidney compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein. In some specific embodiments, the hybrid AAV capsid protein has increased affinity for the heart and / or skeletal muscle compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein. Hybrid AAV capsid proteins have an advantageously increased affinity for different skeletal muscle groups; in particular, hybrid AAV capsid proteins have an increased affinity for at least two skeletal muscle groups in mice selected from the group consisting of the extensor digitorum longus (EDL), soleus (Sol), quadriceps (Qua), triceps and diaphragm, or the soleus (Sol), quadriceps (Qua), triceps and diaphragm. In some specific embodiments, hybrid AAV capsid proteins have a decreased affinity for off-target tissues, advantageously the liver.

[0051] In some preferred embodiments, the hybrid AAV capsid protein has a serum morbidity comparable to that of the acceptor AAV capsid protein. In some more preferred embodiments, the hybrid AAV capsid protein has increased affinity for muscle and / or the central nervous system compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein, and a serum morbidity comparable to that of the acceptor AAV capsid protein.

[0052] In some preferred embodiments, the acceptor AAV capsid serotype has a low seroprevalence, the donor AAV capsid serotype has a higher seroprevalence than the acceptor, and the hybrid AAV capsid protein has a seroprevalence comparable to that of the acceptor AAV capsid protein. In some more preferred embodiments, the hybrid AAV capsid protein has increased affinity in the muscle and / or central nervous system compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid proteins.

[0053] In some preferred embodiments, the acceptor AAV capsid protein is from an AAV serotype selected from the group consisting of AAV8 and AAV9, and more preferably AAV8.

[0054] In some embodiments, the hybrid AAV capsid protein is a hybrid between two AAV capsid serotypes, preferably between an acceptor AAV capsid serotype having a lower seroprevalence and a donor AAV capsid serotype having a higher seroprevalence than the acceptor AAV capsid serotype.

[0055] In some other embodiments, the hybrid AAV capsid protein is a hybrid between two or more AAV capsid serotypes, preferably between an acceptor AAV capsid serotype with a lower seroprevalence and a donor AAV capsid serotype with a higher seroprevalence than the acceptor AAV capsid serotype.

[0056] In some embodiments, the method further comprises the steps of (c) assaying the affinity of the hybrid AAV capsid protein obtained in step (b) by comparison with at least its parental acceptor capsid protein, and (d) selecting the hybrid AAV capsid protein having improved affinity compared to at least its parental acceptor capsid protein. In some preferred embodiments, the method further comprises the steps of (e) assaying the serum prevalence of the hybrid AAV capsid protein and (f) selecting the hybrid AAV capsid protein having a serum prevalence equivalent to that of the acceptor AAV capsid.

[0057] In some embodiments, the method further includes the step of inserting a cell-targeting peptide, in particular a peptide known not to alter the serum prevalence of the capsid, into the hybrid AAV capsid protein obtained in step (b). In some specific embodiments, the cell-targeting peptide includes an RGD motif. Incorporation of the RGD sequence into the viral capsid allows the vector to target integrins widely expressed on several cell types (Michelfelder S. et al., PLoS One. 2009; 4(4): e5122). In particular, insertion of the peptide RGDLGLS into HVR10 of the AAV capsid leads to enhanced muscle targeting without any effect on the serum prevalence of the capsid (International Publication No. 2019 / 193119). In some preferred embodiments, the peptide consists of up to 30 amino acids and includes or comprises one of the following: RGDLGLS (SEQ ID NO: 167), LRGDGLS (SEQ ID NO: 168), LGRGDLS (SEQ ID NO: 169), LGLRGDS (SEQ ID NO: 170), LGLSRGD (SEQ ID NO: 171), and RGDMSRE (SEQ ID NO: 172); preferably SEQ ID NO: 167. Sequences containing the RGD motif may be flanked by up to five or more amino acids at their N and / or C-terminuses, for example, by GQSG (SEQ ID NO: 173) and AQAA (SEQ ID NO: 174) at the N and C-terminuses of the peptide, respectively. One or more peptides containing the RGD motif may be inserted into an exposed site on the surface of the AAV capsid. Sites on the AAV capsid that are exposed on the capsid surface and tolerate peptide insertion, i.e., do not affect the assembly and packaging of the viral capsid, are well known in the art, for example, encompassing the AAV capsid surface loop or antigenic loop (Girod et al., Nat. Med., 1999, 5, pp. 1052-1056; Grifman et al., Molecular Therapy, 2001, 3, pp. 964-975); other sites are disclosed in Rabinowitz et al., Virology, 1999, pp. 265, 274-285; and Wu et al., J. Virol., 2000, 74, pp. 8635-8647.In some specific embodiments, the cell-targeting peptide is inserted into HVR, particularly HVR3, HVR4, HVR5, or HVR10; preferably HVR10. In particular, peptides containing the RGD motif are inserted around position 261, 383, 449, 575, or 590, preferably around position 449 or 590, more preferably around position 590, according to the numbering in SEQ ID NO: 162 (AAV9.rh74). The position is indicated by referring to SEQ ID NO: 255; those skilled in the art can easily find the corresponding position in another sequence after alignment with SEQ ID NO: 162. The insertion site is advantageously between positions 587 and 592 or 588 and 593, preferably between positions 587 and 592, according to the numbering in SEQ ID NO: 162. The insertion of the peptide may or may not result in the deletion of some or all residues from the insertion site. The peptide advantageously replaces all residues from positions 587–592 or 588–593 of the AAV capsid protein, preferably all residues from positions 587–592, according to the numbering in SEQ ID NO: 162.

[0058] In some embodiments, the method is a high-throughput method in which steps (a) and (b) are performed simultaneously to prepare different hybrid AAV capsid proteins, e.g., different hybrid AAV capsid proteins derived from the same acceptor and / or donor AAV capsid protein. The high-throughput method may also include additional steps (c) to (d) and / or (e) to (f) as defined above.

[0059] Hybrid AAV capsid The present invention also relates to recombinant hybrid AAV capsid proteins having improved tissue affinity, which can be obtained or may be obtained by the methods disclosed herein.

[0060] Recombinant hybrid AAV capsid proteins may be derived from any different natural or artificial AAV serotypes used as acceptor and donor AAV capsid serotypes, e.g., those described in particular in this disclosure. A recombinant hybrid AAV capsid protein, which is a hybrid between an acceptor AAV capsid serotype and a donor AAV capsid serotype, comprises 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) HVR sequences (substitute HVR sequences) from a donor AAV capsid protein selected from HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, HVR7, HVR8, HVR9, HVR10, and HVR12, which replace the corresponding HVR sequence (HVR sequence to be replaced) of the acceptor capsid serotype; the substitute HVR sequences have, by definition, amino acid sequences different from the amino acid sequence of the HVR sequence to be replaced.

[0061] In some embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replaced HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and H at positions 687-738. VR12 sequence; preferably selected from the group consisting of HVR1 sequence at positions 134-165, HVR2 sequence at positions 176-192, HVR3 sequence at positions 259-278, HVR4 sequence at positions 379-395, HVR5 sequence at positions 446-484, HVR6 sequence at positions 490-500, HVR7 sequence at positions 501-512, HVR8 sequence at positions 514-529, HVR9 sequence at positions 531-570, HVR10 sequence at positions 576-613, and HVR12 sequence at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of AAV8 capsid).

[0062] In some preferred embodiments, the recombinant hybrid AAV capsid protein is a hybrid between an acceptor AAV capsid serotype having a low seroprevalence and a donor AAV capsid serotype having a higher seroprevalence than the acceptor AAV capsid serotype. In some specific embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, AAV5, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and the sequence of SEQ ID NOs: 2-30; preferably AAV13 and the sequence of SEQ ID NOs: 2-10, 18, 20-22, 29, and 30; more preferably AAV13 and the sequence of SEQ ID NOs: 2, 10, 20, 21, and 30. In some preferred embodiments, the donor AAV capsid serotype is SEQ ID NO: 2.

[0063] In some embodiments, the recombinant hybrid AAV capsid protein contains fewer than eight HVR sequences from the donor AAV capsid protein. In some preferred embodiments, the recombinant hybrid AAV capsid protein contains up to six; preferably up to four HVR sequences from the donor AAV capsid protein. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, AAV5, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and sequences of SEQ ID NOs: 2-30; preferably AAV13 and sequences of SEQ ID NOs: 2-10, 18, 20-22, 29, and 30; more preferably AAV13 and sequences of SEQ ID NOs: 2, 10, 20, 21, and 30. In some preferred embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and / or acceptor AAV capsid protein (replacement HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and 687-738 The sequence is selected from the group consisting of an HVR12 sequence at position 134-165, preferably an HVR1 sequence at positions 176-192, an HVR2 sequence at positions 259-278, an HVR4 sequence at positions 379-395, an HVR5 sequence at positions 446-484, an HVR6 sequence at positions 490-500, an HVR7 sequence at positions 501-512, an HVR8 sequence at positions 514-529, an HVR9 sequence at positions 531-570, an HVR10 sequence at positions 576-613, and an HVR12 sequence at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid).

[0064] In some embodiments, the recombinant hybrid AAV capsid protein contains one or more HVR5-HVR10 sequences from the donor AAV capsid protein. In some preferred embodiments, the recombinant hybrid AAV capsid protein contains one or more HVR5-HVR8 sequences from the donor AAV capsid protein. In some preferred embodiments, the recombinant hybrid AAV capsid protein contains at least an HVR5 sequence from the donor AAV capsid protein. The recombinant hybrid AAV capsid protein may contain HVR5 from the donor capsid serotype alone or in combination with one or more or all of HVR6-HVR10; preferably, HVR5 from the donor capsid serotype alone or in combination with one or more or all of HVR6-HVR8. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and the sequences of SEQ ID NOs. 2-30; preferably AAV13 and the sequences of SEQ ID NOs. 2-10, 18, 20-22, 29, and 30; more preferably AAV13 and the sequences of SEQ ID NOs. 2, 10, 20, 21, and 30.In some preferred embodiments, one or more HVR5-HVR10 sequences of the donor AAV capsid protein (substitute HVR sequence) and / or acceptor AAV capsid protein (HVR sequence to be replaced) are: HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; more preferably HVR5 sequence at positions 446-484 A sequence is selected from the group consisting of: HVR6 sequences at positions 490-500; HVR7 sequences at positions 501-512; HVR8 sequences at positions 514-529; HVR9 sequences at positions 531-570; and HVR10 sequences at positions 576-613; more preferably HVR5 sequences at positions 446-484; HVR6 sequences at positions 490-500; HVR7 sequences at positions 501-516; and HVR8 sequences at positions 514-529; and the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid). In some preferred embodiments, the recombinant hybrid AAV capsid protein comprises or consists of sequences selected from the group consisting of SEQ ID NOs: 33-36, 47-58 and 60-73; preferably SEQ ID NOs: 35, 36, 47, 48, 50, 51, 58, 67 and 73; and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0065] In some specific embodiments, the recombinant hybrid AAV capsid protein includes AAV13 and one of SEQ ID NOs: 2-30; preferably AAV13, #704 (SEQ ID NO: 2); #1704 (SEQ ID NO: 10); #3086 (SEQ ID NO: 20); #1024 (SEQ ID NO: 22); #508 (SEQ ID NO: 9); #3142 (SEQ ID NO: 21); #2320 (SEQ ID NO: 29); #1010 (SEQ ID NO: 6); M258 (SEQ ID NO: 30); #1570 (SEQ ID NO: 18); #1602 (SEQ ID NO: 5); #667 (SEQ ID NO: 7); #129 (SEQ ID NO: 3); and #767 (SEQ ID NO: 8); and more preferably an AAV serotype (donor AAV capsid serotype) HVR5-HVR8 sequence selected from the group consisting of AAV13, #704 (SEQ ID NO: 2) and M258 (SEQ ID NO: 30). Preferably, the HVR5 sequence of the donor AAV capsid protein (substitute HVR5 sequence) and / or acceptor AAV capsid protein (replaced HVR5 sequence) is at positions 446-485; the HVR6 sequence is at positions 485-502; the HVR7 sequence is at positions 499-516; and the HVR8 sequence is at positions 509-531; more preferably, the HVR5 sequence is at positions 446-484; the HVR6 sequence is at positions 490-500; the HVR7 sequence is at positions 501-512; and the HVR8 sequence is at positions 514-529; the indicated position is determined by alignment with SEQ ID NO: 1 (VP1 of AAV8 or AAV8 capsid). In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, AAV5, and AAVrh10. In some preferred embodiments, the recombinant hybrid AAV capsid protein comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 35, 58, 60-72; preferably SEQ ID NOs: 35, 58, 67; and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0066] In some embodiments, the recombinant hybrid AAV capsid protein contains one HVR sequence (HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, HVR7, HVR8, HVR9, HVR10, or HVR12) from the donor AAV capsid protein. In some specific embodiments, the recombinant hybrid AAV capsid protein contains one of the HVR5, HVR6, HVR7, or HVR8 sequences from the donor AAV capsid protein. In some preferred embodiments, the recombinant hybrid AAV capsid protein contains the HVR5 sequence from the donor AAV capsid protein. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10, and / or the donor AAV capsid serotype is selected from the group consisting of AAV13 and the sequences of SEQ ID NOs. 2-30; preferably AAV13 and the sequences of SEQ ID NOs. 2-10, 18, 20-22, 29, and 30; even more preferably, the sequences of SEQ ID NOs. 2, 10, 20, 21, and 30. In some other preferred embodiments, the recombinant hybrid AAV capsid protein is derived from an AAV8 acceptor capsid and comprises one of the HVR1, HVR3, HVR6, HVR7, HVR8, HVR9, HVR10, or HVR12 sequences from a donor AAV capsid serotype selected from the group consisting of AAV8 acceptor capsid, AAV13, and the sequences of SEQ ID NOs: 2-30; preferably SEQ ID NO: 2; more preferably, the recombinant hybrid AAV capsid protein comprises one of the HVR3, HVR9, HVR10, or HVR12 sequences from a donor AAV capsid serotype selected from the group consisting of AAV8 acceptor capsid, AAV13, and the sequences of SEQ ID NOs: 2-30; preferably SEQ ID NO: 2.In some preferred embodiments, the HVR sequences of the donor AAV capsid protein (replacement HVR sequence) and the acceptor AAV capsid protein (replacement HVR sequence) are: HVR1 sequence at positions 134-165; HVR2 sequence at positions 176-192; HVR3 sequence at positions 259-278; HVR4 sequence at positions 379-395; HVR5 sequence at positions 446-485; HVR6 sequence at positions 485-502; HVR7 sequence at positions 499-516; HVR8 sequence at positions 509-531; HVR9 sequence at positions 531-570; HVR10 sequence at positions 576-613; and HVR7 sequence at positions 687-738. The HVR12 sequence is selected from the group consisting of HVR1 sequences (preferably HVR1 sequences at positions 134-165, HVR2 sequences at positions 176-192, HVR3 sequences at positions 259-278, HVR4 sequences at positions 379-395, HVR5 sequences at positions 446-484, HVR6 sequences at positions 490-500, HVR7 sequences at positions 501-512, HVR8 sequences at positions 514-529, HVR9 sequences at positions 531-570, HVR10 sequences at positions 576-613, and HVR12 sequences at positions 705-736; the indicated position is determined by alignment with sequence number 1 (AAV8 or VP1 of the AAV8 capsid). In some preferred embodiments, the recombinant hybrid AAV capsid protein comprises or consists of sequences selected from the group consisting of SEQ ID NOs: 36-43, 45, 47-57, 73; preferably SEQ ID NOs: 36, 38, 42, 43, 45, 47, 48, 50, 51, 73, and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0067] In some specific embodiments, HVR5 is from an AAV capsid serotype selected from the group consisting of #704 (sequence number 2); #1704 (sequence number 10); #3086 (sequence number 20); #508 (sequence number 9); #3142 (sequence number 21); #M258 (sequence number 30); #1570 (sequence number 18); #2731 (sequence number 4); #1602 (sequence number 5); #667 (sequence number 7); #129 (sequence number 3); and #767 (sequence number 8).

[0068] The HVR5 sequences of the donor AAV capsid protein (substitute HVR5 sequence) and / or acceptor AAV capsid protein (substitute HVR5 sequence) are advantageously from positions 446 to 485; preferably from positions 446 to 484; the indicated position is determined by alignment with SEQ ID NO: 1 (VP1 of AAV8 or AAV8 capsid). In some preferred embodiments, HVR5 includes a sequence selected from the group consisting of SEQ ID NOs: 175 to 186; preferably SEQ ID NOs: 175 to 179. In some preferred embodiments, the acceptor AAV capsid serotype is selected from the group consisting of AAV8, AAV9, AAV5, AAV-LK03, AAVrh74, AAV9.rh74, AAV9.rh74-P1, and AAVrh10. In some preferred embodiments, the recombinant hybrid AAV capsid protein comprises or consists of sequences selected from the group consisting of Sequence ID No. 36, 47-57, 73; preferably Sequence ID No. 36, 47, 48, 50, 51, 73;3 and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0069] In some preferred embodiments, the acceptor AAV capsid protein is from an AAV serotype selected from the group consisting of AAV8 and AAV9, and more preferably AAV8.

[0070] In some embodiments, the hybrid AAV capsid protein is a hybrid between two AAV capsid serotypes, preferably between an acceptor AAV capsid serotype having a lower seroprevalence and a donor AAV capsid serotype having a higher seroprevalence than the acceptor AAV capsid serotype.

[0071] In some other embodiments, the hybrid AAV capsid protein is a hybrid between two or more AAV capsid serotypes, preferably between an acceptor AAV capsid serotype with a lower seroprevalence and a donor AAV capsid serotype with a higher seroprevalence than the acceptor AAV capsid serotype.

[0072] In some preferred embodiments, the hybrid AAV capsid protein has increased affinity to muscle and / or the central nervous system compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein. In some specific embodiments, the hybrid AAV capsid protein has increased affinity to the kidney compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid protein. In some specific embodiments, the hybrid AAV capsid protein has increased affinity to the heart and / or skeletal muscle. The hybrid AAV capsid protein advantageously has increased affinity to different skeletal muscle groups; in particular, the hybrid AAV capsid protein has increased affinity to at least two skeletal muscle groups in mice selected from the group consisting of the extensor digitorum longus (EDL), soleus (Sol), quadriceps (Qua), triceps and diaphragm, or the soleus (Sol), quadriceps (Qua), triceps and diaphragm. In some specific embodiments, the hybrid AAV capsid protein has reduced affinity to off-target tissues, advantageously the liver. In certain embodiments, the hybrid AAV capsid protein having increased affinity to muscle and / or the central nervous system compared to acceptor and donor AAV capsid proteins includes or comprises sequences selected from the group consisting of sequences 33-43, 45, 47-58, 60-73; preferably sequences 33-36, 38, 42, 43, 45, 47, 48, 50, 51, 58, 67, 73; sequences 33-36 and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0073] In some preferred embodiments, the hybrid AAV capsid protein has a serum morbidity equivalent to that of the acceptor AAV capsid protein. In some more preferred embodiments, the hybrid AAV capsid protein is derived from an acceptor AAV capsid with a lower serum morbidity and a donor AAV capsid protein with a higher serum morbidity than the acceptor AAV capsid. In some more preferred embodiments, the hybrid AAV capsid protein has increased affinity to muscle and / or the central nervous system compared to the acceptor AAV capsid protein or the acceptor and donor AAV capsid proteins, and has a serum morbidity equivalent to that of the acceptor AAV capsid protein. In certain embodiments, a hybrid AAV capsid protein having increased affinity to muscle and / or the central nervous system and a serum morbidity equivalent to that of the acceptor AAV capsid protein compared to the acceptor and donor AAV capsid protein includes or comprises sequences selected from the group consisting of SEQ ID NOs: 35-43, 45, 47-58, 60-73; preferably SEQ ID NOs: 35, 36, 38, 42, 43, 45, 47, 48, 50, 51, 58, 67, 73; and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0074] Uses for the production of polynucleotides, vectors, and AAV vectors. Another aspect of the present invention is a polynucleotide encoding a recombinant hybrid AAV capsid protein in an expressible form. The polynucleotide may be DNA, RNA, or synthetic or semi-synthetic nucleic acid.

[0075] In some embodiments, the polynucleotide encodes a recombinant hybrid AAV capsid protein having a sequence selected from the group consisting of sequences SEQ ID NOs: 33-43, 45, 47-58, 60-73; preferably sequences SEQ ID NOs: 35, 36, 38, 42, 43, 45, 47, 48, 50, 51, 58, 67, 73; and sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences; more preferably, the amino acid sequence variant has no mutations in at least the HVR sequence or any of the HVR sequences from the donor AAV capsid protein.

[0076] In some preferred embodiments, the polynucleotides are sequence numbers 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 102, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 142, 144, 146, 148 The polynucleotide comprises or comprises a sequence selected from the group consisting of the sequences 150, 152, 154, 156, 158; preferably 82, 84, 88, 96, 98, 102, 106, 108, 112, 114, 128, 146, 158, and sequences having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with the aforementioned sequences. The polynucleotide is a functional polynucleotide sequence, meaning that the sequence of the polynucleotide encodes a recombinant hybrid AAV capsid protein.

[0077] In some embodiments, the polynucleotide further encodes an AAV replicase (Rep) protein, preferably Rep from AAV2, in an expressible form.

[0078] Polynucleotides are advantageously inserted into recombinant vectors, which non-limitingly encompass linear or circular DNA or RNA molecules consisting of chromosomes, non-chromosomes, synthetic or semi-synthetic nucleic acids, such as viral vectors, plasmids, or RNA vectors. Numerous vectors are known to insert the nucleic acid molecule of interest for introduction into and maintenance within a eukaryotic host cell; the selection of a suitable vector depends on the intended use of the vector (e.g., replication of the sequence of interest, expression of the sequence, maintenance of the sequence in an extrachromosomal form, or other integration into the host's chromosomal material) and also on the properties of the host cell.

[0079] In some embodiments, the vector is a plasmid.

[0080] The recombinant vector for use in the present invention is an expression vector comprising appropriate means for the expression of a hybrid AAV capsid protein and, preferably, also an AAV Rep protein. Typically, each coding sequence (hybrid AAV Cap and AAV Rep) is inserted into separate expression cassettes, either in the same vector or separately. Each expression cassette contains a coding sequence (open reading frame or ORF) functionally linked to a regulatory sequence, such as a promoter, promoter / enhancer, intron, start codon (ATG), stop codon, or transcription termination signal, enabling the expression of the corresponding protein in AAV-producing cells. Alternatively, the hybrid AAV Cap and AAV Rep proteins may be expressed from distinct expression cassettes using an internal ribosome entry site (IRES) inserted between the two coding sequences or the viral 2A peptide. Additionally, the codon sequences encoding the hybrid AAV Cap, and AAV Rep if present, are advantageously optimized for expression in AAV-producing cells, particularly human-producing cells.

[0081] Another aspect of the present invention is cells stably transformed with a recombinant vector for the expression of a hybrid AAV capsid protein and, preferably, also an AAV Rep protein. The cells stably express the hybrid AAV capsid and the AAV Rep protein (producing cell line). The producing cells are advantageously human cells.

[0082] The vector, preferably a recombinant plasmid, and the producing cell line are useful for producing a hybrid AAV vector containing the hybrid AAV capsid protein of the present invention using standard AAV production methods well known in the art (review article in Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102: pp. 1045-1054).

[0083] In short, after co-transfection of a plasmid containing a recombinant AAV vector genome containing the target gene inserted into the expression cassette, flanked by an AAV ITR, into a cell line stably expressing a hybrid AAV capsid and AAV Rep protein, in the presence of sufficient helper functions to enable packaging of the rAAV vector genome into AAV capsid particles, the cells are incubated for a sufficient time to enable the production of AAV vector particles, the cells are then harvested and lysed, and the AAV vector particles are purified by standard purification methods, such as affinity chromatography or ultracentrifugation with an iodixanol or cesium chloride density gradient.

[0084] AAV particles, cells Another aspect of the present invention is an AAV particle containing the hybrid recombinant AAV capsid protein of the present invention. Preferably, the AAV particle is a recombinant AAV (rAAV) vector particle, which is also named a hybrid capsid serotype rAAV vector particle or a hybrid serotype rAAV vector particle. The AAV vector particle is suitable for gene therapy directed to target tissues or cells in an individual, particularly muscle, and / or CNS cells or tissues or other cells or tissues. The rAAV vector particle packages the gene of interest. The genome of the rAAV vector may be either a single-stranded genome or a self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003, Dec., 10(26), pp. 2112-2118). A self-complementary vector is generated by deleting a terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome, tend to package DNA dimers. The AAV genomes are flanked by ITRs. In certain embodiments, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from a different serotype of AAV. In some preferred embodiments, the pseudotype vector genome is derived from AAV2. rAAV vector particles can be obtained using the method for producing recombinant AAV vector particles of the present invention.

[0085] The term "target gene" refers to a gene that is useful for a specific application, such as diagnosis, reporting, modification, therapy, and genome editing, without limitation.

[0086] For example, the target gene may be a therapeutic gene, a reporter gene, or a genome editing enzyme.

[0087] "Genes for therapeutic purposes," "genes for therapeutic purposes," or "genes for therapeutic purposes" refer to genes that encode therapeutic genes or therapeutic proteins, peptides, or RNA.

[0088] The gene of interest is any nucleic acid sequence capable of modifying a target gene or target cellular pathway in cells of a target organ, particularly muscle and / or CNS, or other target organ of interest. For example, the gene may modify the expression, sequence, or regulation of a target gene or cellular pathway. In some embodiments, the gene of interest is a functional version of a gene or a fragment thereof. Such functional versions of a gene include wild-type genes, variant genes, e.g., variants belonging to the same family and others, or cleaved versions that at least partially preserve the functionality of the encoded protein. Functional versions of genes are useful for replacement or additive gene therapy to replace a deficient or non-functional gene in a patient. In other embodiments, the gene of interest is a gene that inactivates a dominant allele causing an autosomal dominant genetic disorder. Fragments of genes are useful as recombinant templates for use in combination with genome editing enzymes.

[0089] Alternatively, the gene of interest may encode a protein of interest for a specific application (e.g., an antibody or antibody fragment, a genome editing enzyme) or RNA. In some embodiments, the protein is a therapeutic protein comprising a therapeutic antibody or antibody fragment, or a genome editing enzyme. In some embodiments, the RNA is therapeutic RNA.

[0090] In some embodiments, the sequence of the gene of interest is optimized for expression in the treated individual, preferably a human individual. Sequence optimization may include several changes in the nucleic acid sequence, which include codon optimization, increased GC content, decreased number of CpG islands, decreased number of selective open reading frames (ARFs), and / or decreased number of splice donor and splice acceptor sites.

[0091] The gene of interest is a functional gene capable of producing a protein, peptide, or RNA encoded in disease target cells, particularly muscle cells and / or CNS or other target cells of interest. In some embodiments, the gene of interest is a human gene. The AAV viral vector contains the gene of interest in a form expressible in cells of a target organ, particularly muscle cells including cardiomyocytes and skeletal muscle cells, and / or cells of the CNS or other target cells of interest. In particular, the gene of interest is operably ligated to a suitable regulatory sequence for the expression of the transgene in target cells, tissues, or organs of an individual. Such sequences, well known in the art, include, in particular promoters, and further regulatory sequences having the ability to further control the expression of the transgene, such as, without limitation, enhancers, terminators, introns, silencers, particularly tissue-specific silencers, and microRNAs. The gene of interest is operably ligated to a ubiquitous, tissue-specific, or inducible promoter that is functional in cells of a target organ, particularly muscle and / or CNS. The gene of interest may be inserted into an expression cassette further comprising additional regulatory sequences disclosed above.

[0092] Examples of ubiquitous promoters include the CAG promoter, phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / promoter (CMV), SV40 initial promoter, retroviral Roussarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, β-actin promoter, and EF1 promoter. Muscle-specific promoters include, without limitation, the desmin (Des) promoter, muscle creatine kinase (MCK) promoter, CK6 promoter, alpha-myosin heavy chain (alpha-MHC) promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, synthetic muscle-specific SpC5-12 promoter, and human skeletal actin (HSA) promoter. Promoters for CNS expression include promoters that drive ubiquitous expression and promoters that drive expression in neurons. Representative promoters that drive ubiquitous expression include, without limitation, the CAG promoter (cytomegalovirus enhancer / chicken beta-actin promoter, encompassing the first exon and first intron of the chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene); the PGK (phosphoglycerate kinase 1) promoter; the β-actin promoter; the EF1a promoter; and the CMV promoter. Representative promoters that drive expression in neurons include, without limitation, the promoter of calcitonin gene-related peptide (CGRP), which is known as a motor neuron-derived factor. Other neuron-selective promoters include the promoters of choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synapsin, Hb9, and ubiquitous promoters encompassing the neuron-restrictive silencer element (NRSE). Representative promoters that drive selective expression in glial cells include the promoter of glial fibrillary acid protein (GFAP).

[0093] The RNA is advantageously complementary to the target DNA or RNA sequence, or binds to the target protein. For example, the RNA is interfering RNA, such as shRNA, microRNA, guide RNA (gRNA) for use in combination with Cas enzymes or similar enzymes for genome editing, or antisense RNA with exon-skipping ability, such as modified nuclear small RNA (snRNA) or long non-coding RNA. Interfering RNA or microRNA may be used to regulate the expression of target genes involved in muscle diseases. Guide RNA in combination with Cas enzymes or similar enzymes for genome editing may be used to modify the sequence of a target gene, particularly to correct the sequence of a mutant / deficient gene, or to modify the expression of a target gene involved in a disease, particularly neuromuscular disease. Antisense RNA with exon-skipping ability is used, in particular, to correct the reading frame and restore the expression of a defective gene with an interfered reading frame. In some embodiments, the RNA is therapeutic RNA.

[0094] The genome editing enzymes according to the present invention are any enzyme or enzyme complex having the ability to modify a target gene or target cellular pathway, particularly in muscle cells. For example, the genome editing enzyme may modify the expression, sequence, or regulation of a target gene or cellular pathway. Advantageously, the genome editing enzymes are artificial nucleases, such as, without limitation, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Cas enzymes from the clustered regularly interspaced palindromic repeats (CRISPR)-Cas system, and similar enzymes. Genome editing enzymes, particularly artificial nucleases, such as Cas enzymes and similar enzymes, may be functional nucleases used for site-specific genome editing applications, including, but not limited to, gene correction, gene substitution, gene knock-in, gene knock-out, mutagenesis, chromosomal translocation, and chromosomal deletion, by generating double-strand breaks (DSBs) or single-strand DNA breaks in a target genomic locus (nickase, e.g., Cas9 (D10A)). For site-specific genome editing applications, genome editing enzymes, particularly artificial nucleases, such as Cas enzymes and similar enzymes, may be used in combination with homologous recombination (HR) matrices or templates (also named DNA donor templates) that modify the target genomic locus by homologous recombination induced by double-strand breaks (DSBs). In particular, HR templates may introduce a desired transgene into a target genomic locus or repair mutations in a target genomic locus, preferably in an abnormal or deficient gene, that cause muscle or central nervous system (CNS) disorders, such as neuromuscular diseases. Alternatively, genome editing enzymes, such as Cas enzymes and similar enzymes, may be engineered to be nuclease-deficient and used as DNA-binding proteins for various genome engineering applications, including but not limited to transcriptional activation, transcriptional repression, epigenetic modification, genome imaging, and DNA or RNA pulldown.

[0095] The present invention also relates to isolated cells, particularly cells from an individual, that are stably transduced with the rAAV vector particles of the present invention. The individual is, advantageously, a patient to be treated. In some embodiments, the cells are muscle and / or CNS cells, precursor cells or pluripotent stem cells, such as induced pluripotent stem cells (iPS cells), embryonic stem cells, fetal stem cells and adult stem cells, according to the present disclosure.

[0096] Pharmaceutical compositions and therapeutic uses Another aspect of the present invention is a pharmaceutical composition comprising at least an activator selected from the AAV vector particles or cells of the present invention, and a pharmaceutically acceptable carrier.

[0097] The nucleic rAAV vector particles, cells, and derived pharmaceutical compositions of the present invention may be used to treat diseases by gene therapy, particularly targeted gene therapy directed to muscle and / or CNS cells or tissues. The cells and derived pharmaceutical compositions of the present invention may be used to treat diseases by cell therapy, particularly targeted cell therapy directed to muscle and / or CNS cells or other target cells of interest.

[0098] As used herein, “gene therapy” refers to the treatment of an individual involving the delivery of a target nucleic acid to the individual’s cells for the purpose of treating a disease. Nucleic acid delivery is generally achieved using a delivery medium, also known as a vector. The rAAV vector particles of the present invention may be used to deliver genes to a patient’s cells.

[0099] As used herein, “cell therapy” refers to the process by which cells stably transduced with the rAAV vector particles of the present invention are delivered to an individual in need by any suitable means, for example, intravenous injection (infusion) or injection (implantation or transplantation) into the tissue of interest. In certain embodiments, cell therapy includes the steps of collecting cells from an individual, transducing the cells of the individual using the rAAV vector particles of the present invention, and administering the stably transduced cells back to the patient. As used herein, “cells” refers to isolated cells, natural or artificial cell aggregates, bioartificial cell scaffolds, and bioartificial organs or tissues.

[0100] Gene therapy may be performed by gene transfer, gene editing, exon skipping, RNA interference, trans-splicing, or any other genetic modification of any coding or regulatory sequence in a cell, including those contained in the nucleus, mitochondria, or as viral sequences contained in symbiotic nucleic acids, for example, without limitation, within the cell.

[0101] The two main types of gene therapy are as follows: - Therapies aimed at providing functional replacement genes for deficient / abnormal genes: This is replacement or addition gene therapy; - Therapies aimed at gene or genome editing: In such cases, the goal is to provide cells with the necessary tools to correct the sequence or modify the expression or regulation of a deletion / abnormal gene, resulting in the expression of a functional gene or the suppression (inactivation) of an abnormal gene: This is gene editing therapy.

[0102] In gene therapy, the target gene may be a functional version of a gene that is deficient or mutated in the patient, for example, in the case of a genetic disorder. In such cases, the target gene restores the expression of the functional gene. Therefore, by gene editing or gene substitution, the exact version of this gene is delivered into target cells, particularly the muscle and / or CNS cells of the affected patient or other target cells, which can contribute to an effective therapy for the disease.

[0103] Gene or genome editing involves editing one or more target genes, for example. - Therapeutic RNA as defined above, such as interfering RNA, such as shRNA or microRNA, guide RNA (gRNA) for use in combination with Cas enzymes or similar enzymes, or antisense RNA capable of exon skipping, such as genes encoding modified nuclear small RNA (snRNA); and - A gene encoding a genome editing enzyme as defined above, e.g., an artificial nuclease, e.g., a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), Cas enzyme, or similar enzyme; or a combination of such genes, and also a fragment of a functional version of a gene for use as a recombinant template as defined above. Use this.

[0104] Gene therapy is used to treat a variety of hereditary (genetic) or acquired diseases or disorders affecting the structure or function of target tissues, particularly muscles and / or CNS, including skeletal muscle or cardiac muscle, brain, or spinal cord. Diseases may be caused by trauma, infection, degeneration, structural or metabolic disorders, tumors, autoimmune disorders, stroke, or other causes. Non-limiting examples of diseases that can be treated with gene therapy include neuromuscular hereditary disorders, such as muscle hereditary disorders; cancer; neurodegenerative diseases; and autoimmune diseases.

[0105] In some embodiments, the target gene for gene therapy (additional gene therapy or gene editing) is a gene that causes neuromuscular disease. Neuromuscular hereditary disorders include, in particular, muscular dystrophy, congenital muscular dystrophy, congenital myopathy, distal myopathy, other myopathy, myotonic syndrome, ion channel muscle disease, malignant hyperthermia, metabolic myopathy, hereditary cardiomyopathy, congenital myasthenic syndrome, motor neuron disease, hereditary paraplegia, hereditary sensorimotoreneuropathy, and other neuromuscular disorders. In some preferred embodiments, the target gene for gene therapy (additional gene therapy or gene editing) is a gene that causes a neuromuscular disease selected from the group including Duchenne muscular dystrophy (DMD gene), limb-girdle muscular dystrophy (LGMD) (CAPN3, DYSF, FKRP, ANO5 genes, and others), spinal muscular atrophy (SMN1 gene), myotubular myopathy (MTM1 gene), Pompe disease (GAA gene), and glycogen storage disease III (GSD3) (AGL gene).

[0106] Dystrophinopathy is a spectrum of X-linked muscle diseases caused by pathogenic variants in the DMD gene, which encodes the protein dystrophin. Dystrophinopathy includes Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy.

[0107] Limb-girdle muscular dystrophy (LGMD) is a group of disorders that are clinically similar to DMD but occur in both sexes as a result of autosomal recessive and autosomal dominant inheritance. Limb-girdle dystrophy is caused by mutations in genes encoding sarcoglycans and other proteins associated with the muscle cell membrane that interact with dystrophin. The term LGMD1 refers to a genotype that exhibits dominant inheritance (autosomal dominant), while LGMD2 refers to a type with autosomal recessive inheritance. Pathogenic variants have been reported at more than 50 loci (LGMD1A~LGMD1G; LGMD2A~LGMD2W). Calpainopathy (LGMD2A) is caused by mutations in the CAPN3 gene, of which more than 450 pathogenic variants have been described.The contributing genes to the LGMD phenotype are anoctamin 5 (ANO5), blood vessel epicardial substance (BVES), calpain 3 (CAPN3), caveolin 3 (CAV3), CDP-L-ribitol pyrophosphorylase A (CRPPA), dystroglycan 1 (DAG1), desmin (DES), DNAJ heat shock protein family (Hsp40) homolog, subfamily B, member 6 (DNAJB6), dysferrin (DYSF), fukutin-related protein (FKRP), fukutin (FKT), GDP-mannose pyrophosphorylase B (GMPPB), heterogeneous nuclear ribonucleoprotein D-like (HNRNPDL), and LIM zinc finger domain containing 2. LIMS2), lamin A:C (LMNA), myotilin (MYOT), plectin (PLEC), protein O-glucosyltransferase 1 (PLOGLUT1), protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta-1,2-) (POMGNT1), protein O-mannose kinase (POMK), protein O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2 (POMT2), sarcoglycan alpha (SGCA), sarcoglycan beta (SGCB), sarcoglycan delta (SGCD), sarcoglycan gamma (SGCG), titin-cap (TCAP), transportin 3 (TNPO3), torsine 1A interacting protein (TOR1AIP1), trafficking protein particle complex 11 (TRAPPC11), tripartite motif containing 32 (TRIM 32) and includes titin (TTN).The major contributing genes to the LGMD phenotype include CAPN3, DYSF, FKRP, and ANO5 (Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and Translational Neurology, 2018, 5, pp. 1574-1587).

[0108] Spinal muscular atrophy is a genetic disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, characterized by weakness and wasting (atrophy) of the muscles used for movement.

[0109] X-linked myotubular myopathy is a genetic disorder caused by mutations in the myotubularin (MTM1) gene that affects the muscles used for movement (skeletal muscles) and occurs almost exclusively in males. This condition is characterized by muscle weakness (myopathy) and decreased muscle tone (hypotonia).

[0110] Pompe disease is a genetic disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Mutations in the GAA gene prevent acid alpha-glucosidase from effectively breaking down glycogen, which causes this sugar to accumulate in lysosomes at toxic levels. This accumulation damages organs and tissues throughout the body, particularly muscles, leading to the progressive signs and symptoms of Pompe disease.

[0111] Glycogen storage disease III (GSD3) is an autosomal recessive metabolic disorder caused by homozygous or compound heterozygous mutations in the amylo-alpha-1,6-glucosidase, 4-alpha-glucanotransferase (AGL) gene, which encodes glycogen debranching enzymes and is associated with the accumulation of abnormal glycogen with short outer chains. Clinically, patients with GSD III present with hepatomegaly, hypoglycemia, and growth retardation in infancy or early childhood. Muscle weakness in those with GSD IIIa is minimal in childhood but can become more severe in adulthood; some patients develop cardiomyopathy.

[0112] Substitution or addition gene therapy may be used to treat cancer, particularly rhabdomyosarcoma. The target gene in cancer may regulate the cell cycle or metabolism and migration of tumor cells, or induce tumor cell death. For example, inducible caspase-9 may be expressed in muscle cells to trigger cell death, preferably in combination therapy to induce a persistent anti-tumor immune response.

[0113] Gene editing may be used to modify gene expression in target cells, particularly muscle and / or CNS cells, or to disrupt the viral cycle in such cells, in cases of autoimmune disease or cancer. In such cases, the gene of interest is preferably selected from a guide RNA (gRNA), a site-specific endonuclease (TALEN, meganuclease, zinc finger nuclease, Cas nuclease), a DNA template, and RNAi components, such as those encoding shRNA and microRNA. A tool, such as CRISPR / Cas9, may be used for this purpose.

[0114] In some embodiments, gene therapy is used to treat diseases affecting other tissues by expressing therapeutic genes in target tissues, particularly muscle and / or CNS tissues. This is particularly useful in patients with concurrent liver damage, such as hepatitis, to avoid the expression of therapeutic genes in the liver. The therapeutic genes preferably encode therapeutic proteins, peptides, or antibodies that are secreted from muscle cells into the bloodstream and there can be delivered to other target tissues, such as the liver. Examples of therapeutic genes include, but are not limited to, factor VIII, factor IX, and GAA genes.

[0115] In various embodiments of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of rAAV vector particles or cells. In the context of the present invention, therapeutically effective amount means a dose sufficient to reverse, alleviate or inhibit the progression of a disorder or condition to which such terms apply, or to reverse, alleviate or inhibit the progression of one or more symptoms of a disorder or condition to which such terms apply. The terms “effective dose” or “effective dosage” are defined as a dose sufficient to achieve, or at least partially achieve, the desired effect.

[0116] The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual being considered, such as sex, age, and weight, other drugs being administered concurrently, and other factors recognized by those skilled in the art.

[0117] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or medium.

[0118] A "pharmaceutically acceptable carrier" refers to a medium that, when administered appropriately to a mammal, particularly a human, does not produce harmful, allergic, or other adverse reactions. A pharmaceutically acceptable carrier or excipient refers to any kind of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0119] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable medium for an injectable formulation. These may be, in particular, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), or, depending on the circumstances, dry, especially freeze-dried, compositions that enable the formation of an injectable solution by the addition of sterile water or physiological saline.

[0120] Suitable pharmaceutical forms for injectable applications include sterile aqueous solutions or suspensions. The solution or suspension may contain additives that are compatible with the viral vector and do not prevent the viral vector particles from entering target cells. In all cases, the form must be sterile and fluid enough to allow easy passage through an injection needle. It must be stable under production and storage conditions and protected from microbial contamination, such as bacteria and fungi. Examples of suitable solutions include buffers, such as phosphate-buffered saline (PBS) or Ringer's lactate.

[0121] The present invention also provides a method for treating a disease by expressing a therapeutic gene in a target tissue, particularly muscle and / or CNS tissue, the method comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition to a patient.

[0122] Another aspect of the present invention relates to rAAV vector particles, cells, and pharmaceutical compositions according to the present disclosure as pharmaceuticals, particularly for use in the treatment of muscle or CNS disorders, especially neuromuscular genetic disorders.

[0123] The present invention also provides a method for treating muscle or CNS disorders, comprising the step of administering to a patient a therapeutically effective amount of the above-described pharmaceutical composition, comprising at least an activator selected from the AAV vector particles or cells of the present invention and a pharmaceutically acceptable carrier.

[0124] Further aspects of the present invention relate to the use of rAAV vector particles and cells according to the present disclosure in the production of pharmaceuticals for the treatment of muscle or CNS disorders, particularly neuromuscular genetic disorders.

[0125] Another aspect of the present invention relates to the use of rAAV vector particles or cells of the present disclosure for the treatment of muscle or CNS disorders, particularly neuromuscular genetic disorders.

[0126] Further aspects of the present invention relate to pharmaceutical compositions for the treatment of muscle or CNS disorders, particularly neuromuscular genetic disorders, comprising AAV vector particles or cells of the present disclosure as an active ingredient.

[0127] Further aspects of the present invention relate to pharmaceuticals comprising AAV vector particles or cells of the present disclosure for the treatment of muscle or CNS disorders, particularly neuromuscular genetic disorders.

[0128] As used herein, the terms “patient” or “individual” include human and other mammalian subjects receiving either prophylactic or therapeutic treatment. Preferably, the patient or individual according to the present invention is human.

[0129] "Treatment" or "to treat" is defined as, as used herein, the application or administration of a therapeutic agent or combination of therapeutic agents to a patient for the purpose of curing, restoring, reducing, alleviating, modifying, treating, relieving, improving or influencing a disease or any symptom of a disease, or the application or administration of such therapeutic agent to isolated tissue or cell lines from a patient having a disease, particularly a muscular or CNS disorder. In particular, the terms "to treat" or "to treat" mean reducing or mitigating at least one adverse clinical symptom associated with a disease.

[0130] The terms “treatment” or “to treat” are also used herein in the context of administering therapeutic agents prophylactically.

[0131] The pharmaceutical compositions of the present invention are generally administered in a dose and duration effective to induce a therapeutic effect in a patient, according to known procedures. The pharmaceutical compositions may be administered by any convenient route, which may include, for example, in a non-limiting manner, by infusion or bolus injection, or by absorption through the epithelium or mucocutaneous tissue (e.g., oral mucosa, rectal and intestinal mucosa). Administration may be systemic, topical, or systemic in combination with topical administration; systemic includes parenteral and oral administration, and topical includes topical and topical areas. Systemic administration is preferably parenteral, e.g., subcutaneous (SC), intramuscular (IM), intravascular, e.g., intravenous (IV) or intraarterial; intraperitoneal (IP); intradermal (ID), epidural, or other. Parenteral administration is advantageously by injection or perfusion. Topical administration is preferably intracerebral, ventricular, cisterna magna, and / or intrathecal administration. Administration may be, for example, by injection or perfusion. In some preferred embodiments, administration is parenteral, preferably intravascular, for example, intravenous (IV) or intraarterial. In some other preferred embodiments, administration is intracerebral, ventricular, cisterna magna, and / or intrathecal, either alone or parenterally, preferably in combination with intravascular administration. In some other preferred embodiments, administration is parenteral, preferably intravascular, either alone or in combination with intracerebral, ventricular, cisterna magna, and / or intrathecal administration.

[0132] The various embodiments of this disclosure are combinable with respect to each other, and this disclosure encompasses various combinations of the embodiments of this disclosure.

[0133] The implementation of this invention will, unless otherwise indicated, utilize prior art within the technical scope of the present art, which is adequately described in the literature.

[0134] The present invention is illustrated hereby with reference to the accompanying drawings, using the following non-limiting embodiments. [Brief explanation of the drawing]

[0135] [Figure 1]Representation of AAV Hybrids and Parent Capsids Above, a schematic representation of the VP1 amino acid sequence of AAV8 with the localization of 12 HVRs (black boxes). The number of each HVR is indicated on the corresponding box. The amino acid coordinates indicate the position used for HVR substitution. Below, schematic representations of AAV8, #704, and 6 hybrid capsids. The VP1 amino acid sequences were multiplexed using the ClustalW algorithm and compared with the AAV8 sequence (black). Gray indicates amino acid variations specific to the #704 capsid and those present in the variants. [Figure 2] Specific tissue targeting of hybrid capsids. Luciferase activity of control and novel hybrid capsids. Each column represents the mean activity in at least three mice, expressed as a multiplicative change compared to AAV8. Standard deviation is shown. Statistical analysis of multiplicative changes was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of the control (* compared to #704, compared to #AAV8). *, # = p<0.05; **, ## = p<0.01; ***, ### = p<0.001. [Figure 3] Serum prevalence of novel hybrid capsids: Levels of anti-AAV capsid antibodies in hybrid and parental capsids, assessed by ELISA in a cohort of 46 human serum samples. For each capsid, serum was categorized into three groups according to the level of anti-AAV IgG. Statistical analysis was performed using the χ² test along with Monte Carlo simulations. [Figure 4]The presence of anti-AAV capsid antibodies against the novel hybrid capsids. The presence of anti-AAV capsid antibodies was evaluated by ELISA in a pool of human IVIg. The antibody levels in the parental capsids were compared to A) mutant 1, B) mutant 2, C) mutant 3, D) mutant 4, and E) mutant 5. The x-axis represents serial dilutions of IVIg, and the y-axis represents the normalized OD value for the presence of anti-AAV antibodies. The OD50 of each capsid is shown in the graph. F) OD50 of parental and hybrid capsids obtained as a result of two independent experiments. Standard deviations are shown. Statistical analysis was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of the control (* compared with AAV8, # compared with #704). *, # = p<0.05; **, ## = p<0.01; ***, ### = p<0.001. [Figure 5] Specific tissue targeting of mutants with a single HVR substitution. Luciferase activity of AAV8 and novel hybrid capsids in six different organs. Each column represents the mean activity in at least three mice, expressed as a multiplicative change compared to AAV8. Standard deviation is shown. Statistical analysis of multiplicative changes was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of AAV8. *=p<0.05;**=p<0.01;***=p<0.001. [Figure 6]Anti-AAV capsid antibodies in mutants with a single HVR substitution. Presence of anti-AAV capsid antibodies evaluated by ELISA in a pool of human IVIg. Antibody levels in parental capsids are compared to A) AAV8-mut.HVR1, B) AAV8-mut.HVR3, C) AAV8-mut.HVR6, D) AAV8-mut.HVR7, E) AAV8-mut.HVR8, F) AAV8-mut.HVR9, G) AAV8-mut.HVR10, H) AAV8-mut.HVR11, and I) AAV8-mut.HVR12. The x-axis represents the dilution of IVIg, and the y-axis represents the normalized OD value for the presence of anti-AAV antibodies. The OD50 of each capsid is shown in the graph. J) OD50 of parental and hybrid capsids obtained as a result of two independent experiments. Standard deviation is shown. Statistical analysis was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of the control (* compared with AAV8, # compared with #704). *, # = p < 0.05; **, ## = p < 0.01; ***, ### = p < 0.001. [Figure 7] Specific tissue targeting of mutants with different HVR5 substitutions. Luciferase activity of AAV8 and novel hybrid capsids in six different organs. Each column represents the mean activity in at least three mice, expressed as a multiplicative change compared to AAV8. Standard deviation is shown. Statistical analysis of multiplicative changes was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of AAV8. *=p<0.05;**=p<0.01;***=p<0.001. [Figure 8]Specific tissue targeting of mutants with different HVR5-8 combinations. Luciferase activity of AAV8 and novel hybrid capsids in five different organs. Each column represents the mean activity in at least three mice, expressed as a multiplicative change compared to AAV8. Standard deviation is shown. Statistical analysis of multiplicative changes was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of AAV8. *=p<0.05;**=p<0.01;***=p<0.001. [Figure 9] Specific tissue targeting of AAV9 variants. Luciferase activity of AAV9 and AAV9-R5-704 capsids in six different organs. Each column represents the mean activity in at least three mice, expressed as a multiplicative change compared to AAV9. Standard deviation is shown. Statistical analysis of multiplicative changes was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of PBS. *=p<0.05;**=p<0.01;***=p<0.001. [Figure 10] Levels of anti-AAV capsid antibodies in AAV9 hybrid capsids. Presence of anti-AAV capsid antibodies assessed by ELISA in a pool of human IVIg. A) The antibody level of the parental capsid is compared to that of AAV9-R5-704. The x-axis represents serial dilutions of IVIg, and the y-axis represents the normalized OD value for the presence of anti-AAV antibodies. The OD50 of each capsid is shown in the graph. B) OD50 of parental and hybrid capsids obtained as a result of two independent experiments. Standard deviations are shown. Statistical analysis was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of the control (* compared with AAV8, # compared with #704). *, # = p<0.05; **, ## = p<0.01; ***, ### = p<0.001. [Examples]

[0136] Materials and methods 1. Plasmid construction for novel hybrid capsids Capsid sequences were synthesized (GENEWIZ) to construct plasmids containing the AAV2 Rep sequence and a novel hybrid Cap gene. The fragments were inserted into plasmid pAAV2 containing the AAV2 Rep and AAV2 Cap, replacing the AAV2 Cap with the corresponding novel Cap sequence.

[0137] 2.AAV manufacturing HEK293T cells were grown in suspension in 50 mL of serum-free medium. The cells were transfected with three plasmids: i) a transgene plasmid containing the AAV2 ITR adjacent to the expression cassette, ii) a helper plasmid pXX6 containing the adenovirus sequence necessary for AAV production, and iii) plasmids containing the AAV Rep and Cap genes that define the AAV serotype. Two days after transfection, the cells were lysed to release AAV particles.

[0138] The viral lysate was purified by affinity chromatography. The viral genome was quantified by TaqMan real-time PCR assay using primers and probes corresponding to the ITR of the AAV vector genome (Rohr et al., J Virol Methods., 2002, 106, pp. 81-88. doi:10.1016 / s0166-0934(02)00138-6).

[0139] 3. In vivo research All mouse studies were conducted in accordance with French and European legislation on animal care and experimentation (2010 / 63 / EU) and approved by the local agency's ethics committee (protocol number 2016-002C). AAV vectors were administered intravenously via the tail vein to 6-week-old male C57Bl6 / J mice. Lactation offspring injected with PBS were used as controls. Fifteen days after vector injection, tissues were collected and homogenized in DNAse / RNAse-free water using Fastprep tubes (6.5 m / s; 60 sec).

[0140] 4. Luciferase activity The expression of the reporter gene used as the transgene was measured using a luciferase assay. The tissue lysate was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was diluted in lysis buffer in a white, opaque 96-well plate. Luciferase activity was measured by sequential injection of assay buffer containing ATP and luciferin using an EnSpire (PerkinElmer).

[0141] Protein quantification for each sample was performed using a BCA assay to normalize RLU (relative luminescence units) relative to protein content. The final results were expressed as RLU per 1 mg of protein and normalized as a multiplier change compared to the AAV8 control.

[0142] 5. Serum prevalence of capsids ELISA was performed to assess the presence of anti-AAV capsid antibodies (Ab) in a cohort of human serum prepared from 1000–1500 donor serums per batch and in a commercial pool of human intravenous immunoglobulin (IVIg). AAV capsids were coated at 1 × 10⁻⁹ vg / well on Maxisorp™ plates (Nunc) and incubated overnight at 4°C. The plates were washed three times with PBS containing 6% milk and incubated at room temperature for 2 hours. The plates were washed three times with PBS containing 0.05% Tween (PBS-T) and incubated with serum diluents at 37°C for 1 hour. Each serum sample was analyzed using four logarithmic dilutions (1:10–1:10000), while the IVIg pool was analyzed using eight semi-logarithmic dilutions (1:10–1:316000). The plates were washed three times with PBS-T and incubated with HRP-conjugated goat anti-human IgG (1:10000 dilution) at 37°C for 1 hour. The plates were washed three times with PBS-T and TMB substrate was added. The reaction was stopped with H2SO4 and the optical density (OD) of the plates was read at 492 nm. For the analysis of human serum, the level of anti-AAV capsid IgG in each tested serum was determined using the IVIg standard curve. The results are expressed as μg of anti-AAV capsid IgG per 1 ml of serum. Serum with an ELISA IgG titer lower than 10 μg / ml was considered seronegative. For the analysis of IVIg samples, the OD value of each capsid was expressed as a percentage of the signal and analyzed on Prism. Using a dose-response curve model, the IVIg dilution (OD50) at which a 50% reduction in the OD signal was observed was determined. The OD50 of the hybrid capsid was compared to the OD50 of the parent capsid.

[0143] (Example 1) Manufacturing and in vivo testing of hybrid capsids from AAV8 with hypervariable regions from other AAV serotypes. The capsid designs described herein are based on a combination of two selected parental capsids: a well-known AAV8 serotype and a newly isolated AAV2 / 13 sequence's hypervariable region (HVR). The objective of the rational shuffling strategy is to transfer capsid properties from the donor capsid to the acceptor capsid without altering the acceptor capsid's seroprevalence. The VP1 sequence from AAV2 / 13 was obtained by aligning all AAV2 / 13 sequences isolated from human liver (La Bella T et al., Gut, 2020, pp. 69, 737-747. doi:10.1136 / gutjnl-2019-318281), and the resulting amino acid consensus sequence is equal to sequence #704 isolated in humans. The consensus AAV2 / 13 sequence is referred to as #704 (SEQ ID NO: 2) below. The AAV8 capsid corresponds to SEQ ID NO: 1.

[0144] The inventors have developed six hybrid capsids corresponding to the variable number of HVRs (Figure 1): - AAV8-704 is composed of AAV8 (amino acids 1-446) and #704 (amino acids 447-739). This hybrid contains HVRs 1-4 of AAV8 and 5-12 of #704. AAV8-704 has the amino acid sequence of SEQ ID NO: 31 and is encoded by the polynucleotide of SEQ ID NO: 74. - Mutant 1 consists of AAV8 amino acids 1-446, #704 amino acids 447-687, and #704 amino acids 688-739. This hybrid includes HVR1, 2, 3, 4, and 12 of AAV8, as well as #5-11 of #704. Mutant 1 has the amino acid sequence of SEQ ID NO: 32 and is encoded by the polynucleotide of SEQ ID NO: 76. - Mutant 2 consists of AAV8 amino acids 1-446, #704 amino acids 447-613, and #704 amino acids 614-739. This hybrid includes HVRs 1, 2, 3, 4, 11, and 12 of AAV8, as well as #5-10 of #704. Mutant 2 has the amino acid sequence of SEQ ID NO: 33 and is encoded by the polynucleotide of SEQ ID NO: 78. - Mutant 3 consists of AAV8 amino acids 1-446, #704 amino acids 447-570, and AAV8 amino acids 571-739. This hybrid includes HVRs 1, 2, 3, 4, 10, 11, and 12 of AAV8, as well as #5-9 of #704. Mutant 3 has the amino acid sequence of SEQ ID NO: 34 and is encoded by the polynucleotide of SEQ ID NO: 80. - Mutant 4 consists of AAV8 amino acids 1-446, #704 amino acids 447-531, and AAV8 amino acids 532-739. This hybrid includes HVRs 1, 2, 3, 4, 9, 10, 11, and 12 of AAV8, as well as #5-8 of #704. Mutant 4 has the amino acid sequence of SEQ ID NO: 35 and is encoded by the polynucleotide of SEQ ID NO: 82. - Mutant 5 consists of AAV8 amino acids 1-446, #704 amino acids 447-485, and AAV8 amino acids 486-739. This hybrid contains all HVRs of AAV8 except for HVR5 from #704. Mutant 5 has the amino acid sequence of SEQ ID NO: 36 and is encoded by the polynucleotide of SEQ ID NO: 84.

[0145] Capsid production, in vivo distribution, and seroprevalence Recombinant AAV vectors were prepared by cloning the aforementioned mutant Cap gene into a plasmid suitable for AAV vector production. A transgene expression cassette expressing a luciferase reporter gene, flanked by an AAV2 ITR, was capsid-encapsulated in the thus derived AAV vector. Vectors were prepared using triple transfection of HEK293 cells, followed by immunoaffinity column purification. With the exception of AAV8-704, which was excluded from the following in vivo analysis, all capsid sequences were efficiently prepared as AAV vectors.

[0146] [Table 1]

[0147] The vector was applied to wild-type C57Bl6 / J mice at a rate of 1 × 10⁻¹⁶. 11Different vectors were tested via intravenous injection at vg / mouse doses. Fifteen days post-injection, the animals were sacrificed, and the expression levels of the transgenes were measured in isolated tissues (liver, spleen, quadriceps, triceps, diaphragm, heart, kidney, brain, soleus muscle, and spinal cord). Results were expressed as RLU (relative luminescence units) per mg of protein and normalized as a multiplier change compared to the AAV8 control (Table 2 and Figure 2).

[0148] [Table 2]

[0149] In the liver (Figure 2A), the luciferase activity of AAV8 was significantly higher than that of all other tested capsids. The parental capsid #704 completely detargeted the liver. Luciferase activity in the liver of mice injected with mutant capsids increased exponentially with capsids containing more HVR from AAV8, reaching the highest level in mutant #5 with HVR5 of capsid #704.

[0150] In all tested muscle samples (Figures 2B-2F), all mutant capsids except mutant 1 outperformed the parent capsid #704 and showed higher efficiency against AAV8.

[0151] Increased transduction levels were also observed in the spinal cord of all mutants, particularly mutants 2 and 4, compared to the parental capsid (Figure 2G), while very low luciferase activity was observed in #704 and mutant 1.

[0152] In the brain (Figure 2H), the luciferase activity of AAV8, #704, and mutant 1 was comparable to that of PBS-injected mice. Interestingly, mutants 2, 3, 4, and 5 were able to target the brain with higher efficiency than AAV9, AAV8, and #704 (Table 2).

[0153] Finally, in contrast to #704 and mutant 1, mutant 5 was able to target the kidney along with higher luciferase expression than AAV8 (Figure 2I).

[0154] In summary, these results indicate that novel AAV mutant capsids exhibit increased affinity for muscle and CNS compared to their parental capsids, suggesting that the combination of hypervariable regions from AAV8 and wild-type #704 could be a promising strategy for the development of novel capsids.

[0155] The inventors attempted to identify the minimum number of HVR regions that can be modified in the capsid without affecting the capsid's serum prevalence. The serum prevalence of the hybrid capsid was tested in parallel with two parent capsids, AAV8 and #704. ELISA was performed to assess the presence of anti-AAV capsid antibodies (Ab) in a cohort of 46 human serum samples. As expected, from the human origin of this capsid, the number of seropositive individuals was highest for wild-type #704 (n=25; Figure 3). A decrease in the number of seropositive individuals was observed for all variants. In particular, variants 4 and 5 showed significantly lower seropositive samples than the parent capsid #704 (n=10 and n=13, respectively). Overall, the frequency of seropositive individuals gradually decreased in hybrid capsids containing fewer #704 HVRs than AAV8, reaching 22% and 28% for variants 4 and 5, respectively. Considering that the parental AAV8 capsid showed 30% serological positivity in the tested cohort, these data suggest that modifications to HVR5, 6, 7, and 8 do not alter the capsid's immunogenicity profile. Similar results were also confirmed by analyzing the levels of anti-AAV capsid antibodies in a pool of human IVIg (Figure 4). Levels of anti-AAV antibodies against the hybrid and parental capsids were compared using OD50, defined as the IVIg dilution at which a 50% reduction in OD signaling is observed. OD50s of 400 and 3370 were obtained for AAV8 and #704, respectively. Variants 1, 2, and 3 showed significantly higher OD50s than the acceptor capsid (Figures 4A, 4B, 4C, and 4F), while variants 4 and 5 were identical to the AAV8 profile with no significant differences in OD50 (Figures 4D, 4E, and 4F). All mutants showed significantly lower OD50 than the donor capsid (Figure 4F).

[0156] These results therefore demonstrate that rational shuffling can be used as a method for combining the capsid properties of multiple parent capsids. Furthermore, these results together demonstrate that rational shuffling can be used as a method for transferring capsid properties from donor capsids to acceptor capsids without altering the acceptor capsid serum prevalence.

[0157] (Example 2) Production of hybrid capsids from AAV8 with a single HVR substitution, in vitro and in vivo testing. To better characterize the properties of the 12 HVRs from #704, the HVRs of AAV8 are replaced one by one with the corresponding HVRs of the wild-type #704 capsid. The amino acid sequences of HVR2 and 4 of #704 are identical to those of AAV8, and therefore 10 AAV8 capsids with a single HVR substitution are analyzed: - AAV8-mut.HVR1 (sequence number 37) is encoded by the polynucleotide of sequence number 86; - AAV8-mut.HVR3 (sequence number 38) is encoded by the polynucleotide of sequence number 88; - AAV8-mut.HVR6 (sequence number 39), encoded by the polynucleotide of sequence number 90; - AAV8-mut.HVR7 (sequence number 40) is encoded by the polynucleotide in sequence number 92; - AAV8-mut.HVR8 (sequence number 41) is encoded by the polynucleotide of sequence number 94; - AAV8-mut.HVR9 (sequence number 42) is encoded by the polynucleotide in sequence number 96; - AAV8-mut.HVR10 (sequence number 43) is encoded by the polynucleotide of sequence number 98; - AAV8-mut.HVR11 (sequence number 44) encoded by the polynucleotide of sequence number 100; and - AAV8-mut.HVR12 (sequence number 45) encoded by the polynucleotide of sequence number 102.

[0158] Recombinant AAV vectors are prepared by cloning a modified Cap gene into a plasmid suitable for vector production. A transgene expression cassette expressing a luciferase reporter gene, flanked by an AAV2 ITR, is capsid-encapsulated in the thus derived AAV vector. Vectors are prepared using triple transfection of HEK293 cells, followed by immunoaffinity column purification. Vectors are tested in vitro in cell lines and primary cells obtained from commercial suppliers. In parallel, vectors are tested in wild-type C57Bl6 / J mice at a dose of 1 × 10⁶. 11 The different vectors were tested by intravenous injection at vg / mouse doses. On day 15 after injection, the animals were sacrificed, and the level of transgene expression was measured in isolated tissues. The serum prevalence of the mutant capsid was tested by ELISA as shown in Example 1.

[0159] In all tested muscle, brain, and spinal cord cells, all mutant capsids except AAV8-mut.HVR11 showed higher efficiency than AAV8 (Figure 5). In particular, the luciferase activity of AAV8-mut.HVR3 and 12 was significantly higher than AAV8 in at least one muscle cell. Regarding the CNS, AAV8-mut.HVR3, AAV8-mut.HVR9, AAV8-mut.HVR10, and AAV8-mut.HVR12 were significantly more efficient than AAV8 in the spinal cord and / or brain. All mutants showed significantly lower serum prevalence than donor capsid #704 and comparable serum prevalence to acceptor capsid AAV8 (Figure 6). While levels of anti-AAV antibodies were significantly lower in AAV8-mut.HVR6 than in the acceptor capsid (OD50: 145 and 400 in the mutant and AAV8, respectively), AAV8-mut.HVR12 showed a lower serum prevalence (OD50: 631), but was still significantly higher than AAV8. These results suggest that single HVR substitution can improve acceptor affinity without altering its serum prevalence.

[0160] (Example 3) Production of hybrid capsids from AAV8 with different HVR5s from wild-type AAV, in vitro and in vivo testing. Recently isolated wild-type capsids in human liver (La Bella T et al., Gut, 2020, pp. 69, 737-747. doi:10.1136 / gutjnl-2019-318281) represent the variability of AAV in the context of natural infection. These 59 capsids are characterized by unique amino acid variations, also accompanied by HVR5. Alignment of wild-type AAV capsids from two different genotypes, AAV2 and AAV2 / 13, AAV13 (GenBank accession number ABZ10812.1) and AAV2 (GenBank accession number YP_680426.1) shows four from the AAV2 serotype (wild-type AAV2; wild-type capsids #2102, #1343, #3013), AA This allowed for the identification of 19 unique HVR5 sequences, including 14 from the V2 / 13 serotype (wild-type capsids #1704, #3086, #1591, #3142, #985, #M258, #1570, #2806, #2731, #1602, #667, #129, #217, #767) and one from the AAV13 serotype (wild-type capsid #508). Similar to mutant 5 in Example 1, a new AAV8 mutant containing 12 different HVR5 substitutions was generated to characterize the new AAV mutant.

[0161] A variant 5-AAV2 (sequence number 46) encoded by the polynucleotide of sequence number 104, containing HVR5 of sequence number 187, encoded by the polynucleotide of sequence number 201.

[0162] Mutant 5-AAV13: - A variant 5-#508 (sequence number 49) encoded by the polynucleotide of sequence number 110, containing the HVR5 of sequence number 186 encoded by the polynucleotide of sequence number 200. Sequence number 186 is the HVR5 sequence of AAV13.

[0163] Mutant 5-AAV2 / 13: - A variant 5-#1704 (sequence number 47) encoded by the polynucleotide of sequence number 106, containing HVR5 of sequence number 176, encoded by the polynucleotide of sequence number 190. - A variant 5-#3086 (sequence number 48) encoded by the polynucleotide of sequence number 108, containing HVR5 of sequence number 177, encoded by the polynucleotide of sequence number 191. - A variant 5-#3142 (sequence number 50) encoded by the polynucleotide of sequence number 112, containing HVR5 of sequence number 178, which is encoded by the polynucleotide of sequence number 192. - A variant 5-#M258 (sequence number 51) encoded by the polynucleotide of sequence number 114, containing HVR5 of sequence number 179, which is encoded by the polynucleotide of sequence number 193. - A variant 5-#1570 (sequence number 52) encoded by the polynucleotide of sequence number 116, containing HVR5 of sequence number 180, encoded by the polynucleotide of sequence number 194. - A variant 5-#2731 (sequence number 53) encoded by the polynucleotide of sequence number 118, containing HVR5 of sequence number 181, encoded by the polynucleotide of sequence number 195. - A variant 5-#1602 (sequence number 54) encoded by the polynucleotide of sequence number 120, containing HVR5 of sequence number 182, encoded by the polynucleotide of sequence number 196. - A variant 5-#667 (sequence number 55) encoded by the polynucleotide of sequence number 122, containing HVR5 of sequence number 183, encoded by the polynucleotide of sequence number 197. - A variant 5-#129 (sequence number 56) encoded by the polynucleotide of sequence number 124, containing HVR5 of sequence number 184, encoded by the polynucleotide of sequence number 198. - A variant 5-#767 (sequence number 57) encoded by the polynucleotide of sequence number 126, containing HVR5 of sequence number 185, encoded by the polynucleotide of sequence number 199.

[0164] Mutant 5 (Example 1) contains HVR5 from #704 as the sequence of SEQ ID NO: 175, encoded by the polynucleotide of SEQ ID NO: 189.

[0165] HVR5 from #704 (sequence number 175) is present in other wild-type capsids of hybrid serotype 2 / 13 (#1010 (sequence number 6); #2112, #1350, #668, #367, #1020, #1158, #2107 (sequence numbers 11-17), #714 (sequence number 19), #790, #976, #1286, #163, #685, #442, #2320 (sequence numbers 22-29)).

[0166] HVR5 (sequence number 186) from AAV13 is present in wild-type capsids #1024 (sequence number 22) and #508 (sequence number 9).

[0167] Recombinant AAV vectors are prepared by cloning a modified Cap gene into a plasmid suitable for vector production. A transgene expression cassette expressing a luciferase reporter gene, flanked by an AAV2 ITR, is capsid-encapsulated in the thus derived AAV vector. Vectors are prepared using triple transfection of HEK293 cells, followed by immunoaffinity column purification. Vectors are tested in vitro in cell lines and primary cells obtained from commercial suppliers. In parallel, vectors are tested in wild-type C57Bl6 / J mice at a dose of 1 × 10⁶. 11 The different vectors were tested by intravenous injection at vg / mouse doses. On day 15 after injection, the animals were sacrificed, and the level of transgene expression was measured in isolated tissues. The serum prevalence of the mutant capsid was tested by ELISA as shown in Example 1.

[0168] All mutant capsids with HVR5 of AAV13 (mutant 5-#508) or hybrid AAV2 / 13 serotypes showed higher efficiency than AAV8 in one or more of the muscle, brain, and spinal cord. In particular, the luciferase activity of Mut5-#1704, Mut5-#3086, and Mut5-#M258 was significantly higher than AAV8 in at least one muscle. Mut5-#1704, Mut5-#3086, and Mut5-#3142 were significantly more efficient than AAV8 in spinal cord targeting. In contrast, mutant capsids with HVR5 of AAV2 serotype showed no improvement compared to AAV8 in all tested muscle, brain, and spinal cord (Figure 7).

[0169] These results suggest that AAV13 and AAV2 / 13 serotypes can be used as donor capsids for HVR5 replacement in AAV8 using rational shuffling.

[0170] (Example 4) Production of hybrid capsids from AAV8 with different HVRs (HVRs 5-8) from wild-type AAV, in vitro and in vivo testing. Similar to mutant 4 in Example 1, we designed a novel AAV8 mutant containing naturally occurring combinations of HVR5, 6, 7, and 8 in the wild-type AAV capsid. By multiple alignment of wild-type capsids recently isolated in human liver (La Bella T et al., Gut, 2020, 69, pp. 737-747, doi:10.1136 / gutjnl-2019-318281), AAV13 (GenBank accession number ABZ10812.1), and AAV2 (GenBank accession number YP_680426.1), we were able to identify 27 unique combinations of HVR5, 6, 7, and 8, one of which is derived from the AAV13 serotype (wild-type AAV13), A This includes seven from the AV2 serotype (wild-type AAV2; #2497, #2102, #2087, #1449, #1343, #3013) and nineteen from the AAV2 / 13 serotype (#1704, #3086, #1024, #1591, #508, #3142, #2320, #1010, #985, #M258, #1570, #2806, #2731, #2112, #1602, #667, #129, #217, #767). Fifteen combinations were included in the AAV8 capsid to characterize the properties of new AAV variants. - The variant 4-AAV13 (sequence number 58) encoded by the polynucleotide of sequence number 128; - The variant 4-AAV2 (sequence number 59) encoded by the polynucleotide of sequence number 130; - Mutant 4-AAV2 / 13 serotype: - The variant 4-1704 (sequence number 60) encoded by the polynucleotide of sequence number 132; - Variant 4-3086 (sequence number 61) encoded by the polynucleotide of sequence number 134; - The variant 4-1024 (sequence number 62) encoded by the polynucleotide of sequence number 136; - Variant 4-508 (sequence number 63) encoded by the polynucleotide of sequence number 138; - The variant 4-3142 (sequence number 64) encoded by the polynucleotide of sequence number 140; - The variant 4-2320 (sequence number 65) encoded by the polynucleotide of sequence number 142; - The variant 4-1010 (sequence number 66) encoded by the polynucleotide of sequence number 144; - The variant 4-M258 (sequence number 67) encoded by the polynucleotide of sequence number 146; - Variant 4-1570 (sequence number 68) encoded by the polynucleotide of sequence number 148; - The variant 4-1602 (sequence number 69) encoded by the polynucleotide of sequence number 150; - Variant 4-667 (sequence number 70) encoded by the polynucleotide of sequence number 152; - Variant 4-129 (sequence number 71) encoded by the polynucleotide of sequence number 154; - A variant 4-767 (sequence number 72) encoded by the polynucleotide of sequence number 156.

[0171] Variant 4 (Example 1) contains HVR5, HVR6, HVR7, and HVR8 from capsid #704 (SEQ ID NO: 2). HVR5, HVR6, HVR7, and HVR8 from capsid #704 are present in other wild-type capsids of hybrid serotype 2 / 13 (#2112, #1350, #668, #367, #1020, #1158, #2107 (SEQ ID NOs: 11-17), #714 (SEQ ID NO: 19), #790, #976, #1286, #163, #685, #442 (SEQ ID NOs: 22-28)).

[0172] Recombinant AAV vectors are prepared by cloning a modified Cap gene into a plasmid suitable for vector production. A transgene expression cassette expressing a luciferase reporter gene, flanked by an AAV2 ITR, is capsid-encapsulated in the thus derived AAV vector. Vectors are prepared using triple transfection of HEK293 cells, followed by immunoaffinity column purification. Vectors are tested in vitro in cell lines and primary cells obtained from commercial suppliers. In parallel, vectors are tested in wild-type C57Bl6 / J mice at a dose of 1 × 10⁶. 11 The different vectors were tested by intravenous injection at vg / mouse doses. On day 15 after injection, the animals were sacrificed, and the level of transgene expression was measured in isolated tissues. The serum prevalence of the mutant capsid was tested by ELISA as shown in Example 1.

[0173] Most mutant capsids with HVR5–HVR8 of the AAV2 / 13 serotype showed higher efficiency than AAV8 in muscle, brain, and / or spinal cord. Mut4-AAV13 and mut4-#M258 showed significantly higher luciferase activity than AAV8 in the soleus muscle and spinal cord, respectively. In contrast, mutant capsids with HVR5 of the AAV2 serotype showed no improvement compared to AAV8 in all tested muscle and brain (Figure 8).

[0174] These results suggest that the replacement of HVR5-8 of AAV8 with AAV13 or AAV2 / 13 serotypes as donor capsids can enhance the muscle and / or CNS targeting of acceptor capsids.

[0175] (Example 5) Manufacturing, in vitro, and in vivo studies of wild-type HVR5 in different reference capsids. HVR5 #704 will be cloned in AAV9 (GenBank accession number: AY530579.1), a different AAV reference capsid already used in gene therapy. AAV9-R5-704 (sequence number 73) will be encoded by the polynucleotide of sequence number 158.

[0176] Recombinant AAV vectors are prepared by cloning a modified Cap gene into a plasmid suitable for vector production. A transgene expression cassette expressing a luciferase reporter gene, flanked by an AAV2 ITR, is capsid-encapsulated in the thus induced AAV vector. Vectors are prepared using triple transfection of HEK293 cells, followed by immunoaffinity column purification. Vectors are tested in vitro in cell lines and primary cells obtained from commercial suppliers. In parallel, vectors are tested in wild-type C57Bl6 / J mice at a dose of 1 × 10⁶. 11 The different vectors were tested by intravenous injection at vg / mouse doses. On day 15 after injection, the animals were sacrificed, and the level of transgene expression was measured in isolated tissues. The serum prevalence of the mutant capsid was tested by ELISA as shown in Example 1.

[0177] The mutant capsid AAV9-R5-704 showed higher efficiency than AAV8 in muscle, brain, and spinal cord (Figure 9). The mutant capsid AAV9-R5-704 had a significantly lower serum prevalence than donor capsid #704 and was comparable to acceptor capsid AAV9 (Figure 10).

[0178] These results indicate that HVR5 replacement using rational shuffling is a valuable method for improving muscle and / or CNS targeting of other acceptor capsids such as AAV9.

Claims

1. A method for preparing recombinant hybrid adeno-associated virus (AAV) capsid proteins having improved affinity for muscles and / or the central nervous system, a) A step of providing at least two recombinant AAV capsid proteins, an acceptor AAV capsid protein, and at least one donor AAV capsid protein from different AAV serotypes, wherein the donor AAV capsid serotype is AAV13 or hybrid AAV2 / 13; b) A step of replacing at least the hypervariable region HVR5 sequence of the acceptor AAV capsid protein with a different HVR5 sequence from the corresponding HVR5 of the donor AAV capsid protein to obtain a recombinant hybrid AAV capsid protein having improved affinity to muscle and / or the central nervous system compared to at least the parent acceptor AAV capsid protein. Includes, A method for selecting an acceptor AAV capsid serotype from the group consisting of AAV8 and AAV9.

2. The method according to claim 1, wherein the acceptor AAV capsid serotype has a low seroprevalence, and the donor AAV capsid serotype has a higher seroprevalence than the acceptor AAV capsid serotype.

3. The method according to claim 2, wherein the donor AAV capsid protein is selected from the group consisting of sequences 2 to 30.

4. The method according to any one of claims 1 to 3, wherein the hybrid AAV capsid protein has a serum prevalence equivalent to that of the acceptor AAV capsid protein.

5. The method according to any one of claims 1 to 4, wherein the HVR5 sequence of the donor AAV capsid protein and / or acceptor AAV capsid protein is an HVR5 sequence at positions 446 to 484, and the indicated position is determined by alignment with SEQ ID NO:

1.

6. The method according to claim 5, wherein the hypervariable region (HVR) sequence of the donor AAV capsid protein and / or acceptor AAV capsid protein is selected from the group consisting of the HVR1 sequence at positions 134-165, the HVR2 sequence at positions 176-192, the HVR3 sequence at positions 259-278, the HVR4 sequence at positions 379-395, the HVR6 sequence at positions 490-500, the HVR7 sequence at positions 501-512, the HVR8 sequence at positions 514-529, the HVR9 sequence at positions 531-570, the HVR10 sequence at positions 576-613, and the HVR12 sequence at positions 705-736; and the indicated position is determined by alignment with SEQ ID NO:

1.

7. The method according to any one of claims 1 to 6, wherein step b) includes replacing fewer than eight HVR sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

8. The method according to claim 7, wherein step b) includes replacing up to six HVR sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

9. The method according to claim 7, wherein step b) includes replacing up to four HVR sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

10. The method according to any one of claims 1 to 9, wherein the HVR5 sequence from the donor AAV capsid protein includes a sequence selected from the group consisting of SEQ ID NOs: 175 to 186.

11. The method according to any one of claims 1 to 10, wherein step b) includes replacing the HVR5 sequence of the acceptor AAV capsid protein alone or in combination with one or more or all of HVR6, HVR7, HVR8, HVR9 and HVR10.

12. The method according to any one of claims 1 to 11, wherein step b) includes replacing the HVR5 sequence of the acceptor AAV capsid protein alone or in combination with one or more or all of HVR6, HVR7, and HVR8.

13. The method according to any one of claims 1 to 11, wherein step b) includes replacing all of the HVR5 to HVR10 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

14. The method according to any one of claims 1 to 12, wherein step b) includes replacing all of the HVR5 to HVR8 sequences of the acceptor AAV capsid protein with different HVR sequences from the corresponding HVRs of the donor AAV capsid protein.

15. A recombinant hybrid AAV capsid protein having improved affinity, comprising an amino acid sequence selected from the group consisting of sequences SEQ ID NOs. 33-43, 45, 47-58, and 60-73, and variant sequences having at least 90% identity with any one of the aforementioned sequences, wherein the variant sequence has no mutations in at least the HVR sequence from the donor AAV capsid protein or in all of the HVR sequences.

16. A recombinant plasmid comprising a polynucleotide that encodes the recombinant hybrid AAV capsid protein described in claim 15 in an expressible form, and ultimately further encodes the AAV replicase protein in an expressible form.

17. The recombinant plasmid according to claim 16, comprising a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 102, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, and 158.

18. Cells stably transformed with the recombinant plasmid described in claim 16 or 17.

19. AAV vector particles packaging a gene of interest, comprising at least one hybrid recombinant AAV capsid protein according to claim 15.

20. The AAV vector particle according to claim 19, wherein the target gene is selected from the group consisting of a therapeutic gene; a gene encoding a therapeutic protein or peptide; and a gene encoding a therapeutic RNA.

21. A pharmaceutical composition comprising a therapeutically effective amount of AAV vector particles according to claim 19 or 20, or cells stably transduced by the AAV vector particles.

22. A pharmaceutical product comprising rAAV vector particles, cells, or a pharmaceutical composition as described in any one of claims 18 to 21.

23. rAAV vector particles, cells, or pharmaceutical compositions according to any one of claims 19 to 21, for use in the treatment of muscular and / or central nervous system disorders.

24. The rAAV vector particles, cells, and pharmaceutical composition according to claim 23, wherein the disease is a neuromuscular genetic disorder.

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