AAV capsid for improved cardiac transduction and liver de-targeting

Abstract

Provided herein are novel AAV capsids and recombinant AAV vectors comprising the novel AAV capsids. In certain embodiments, carriers, including the novel AAV capsids, exhibit improved cardiac tissue transduction and / or reduced liver tissue targeting as compared to prior art AAV capsids.

Description

[0001] References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing ("21-9720.PCT.xml"; size: 44,339 bytes; and creation date: January 24, 2023) are hereby incorporated by reference in its entirety. Background Art

[0003] Adeno-associated virus (AAV) vectors are safe and effective gene transfer agents for several clinical indications. Recombinant AAV vectors have a vector genome lacking AAV coding sequences packaged in an AAV capsid. The AAV capsid is icosahedral in structure and contains 60 viral protein (VP) monomers (VP1, VP2, and VP3) in a 1:1:10 ratio (Xie Q et al., Proc Natl Acad Sci USA. 2002; 99(16): 10405-10). The entire VP3 protein sequence (519aa) is contained in the C-terminus of both VP1 and VP2, and the common VP3 sequence is primarily responsible for the overall capsid structure. Due to the structural flexibility of the VP1 / VP2 unique region and the low representation of VP1 and VP2 monomers in the assembled capsid relative to the VP3 monomer, VP3 is the only capsid protein resolved by x-ray crystallography (Nam HJ et al. J Virol. 2007;81(22):12260-71).

[0004] The natural tropism of AAV vectors for the liver is a major obstacle to the expansion of AAV gene therapy into clinical indications affecting non-liver peripheral organs, such as the heart. This problem is exemplified by deaths and adverse events related to hepatotoxicity in recent trials of high-dose AAV gene therapy targeting peripheral organs.

[0005] There remains a need in the art for improved AAV vectors that are targeted to selected cell and tissue types, including vectors that de-target the liver while retaining the ability to transduce peripheral organs. Summary of the invention

[0006] In one aspect, the present invention provides a recombinant adeno-associated virus (rAAV) having: an AAV clade F capsid, wherein the AAV clade F capsid includes a mutant galactose binding site, wherein when the residue positions are determined using the residue numbering of SEQ ID NO: 10 as a reference, the mutant galactose binding site includes (a) any amino acid residue (X) at position 446 (Y446X), (b) arginine at position 470 (N470R), and (c) histidine at position 503 (W503H); and a vector genome, wherein the vector genome includes a 5' inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, a regulatory sequence that directs the expression of the gene product operably linked to the coding sequence, and a 3' ITR sequence. In certain embodiments, the mutant capsid comprises: (a) H (HRH) at position 446; (b) S (SRH) at position 446; (c) V (VRH) at position 446; (d) G (GRH) at position 446; (e) F (FRH) at position 446; (f) T (TRH) at position 446; or (g) S (SRH) at position 446. In certain embodiments, the parental AAV clade F capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid, or an AAVhu96 capsid, or a mutant or variant of any of the foregoing.

[0007] In another aspect, provided herein is a method for producing an rAAV comprising a mutant capsid derived from a parental AAV capsid having liver specificity, the method comprising culturing a packaging host cell, the packaging host cell comprising: (a) a nucleic acid sequence encoding a mutant AAV clade F capsid, the nucleic acid sequence being operably linked to a regulatory control sequence directing its expression in the packaging host cell, wherein the encoded capsid protein comprises (i) any amino acid residue (X) at position 446 (Y446X), (ii) arginine at position 470 (N470R), and (iii) histidine at position 503 (W503H), wherein the amino acid residue positions are determined using SEQ ID NO:10 residue numbering as a reference; (b) a nucleic acid molecule comprising a vector genome, the vector genome comprising a 5' inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, a regulatory sequence for directing the expression of the gene product operably linked to the coding sequence, and a 3'ITR sequence; and (c) an auxiliary function, the auxiliary function is necessary to package the vector genome of (b) into the mutant clade F capsid. In certain embodiments, the parent AAV capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid, or an AAVhu96 capsid, or a mutant or variant of any of the foregoing.

[0008] In another aspect, provided herein is a rAAV produced according to the disclosed methods.

[0009] In another aspect, provided herein is a method for reducing hepatotoxicity associated with the delivery of a rAAV vector, the method comprising delivering to a subject a rAAV disclosed herein.

[0010] In an additional aspect, provided herein is a method for improving delivery of a gene product to cardiac cells or tissues, the method comprising administering to a subject a rAAV disclosed herein.

[0011] In yet another aspect, provided herein is a nucleic acid comprising a sequence encoding a mutant AAV clade F VP1 protein having a mutant galactose binding pocket, the mutant AAV clade F VP1 protein comprising (a) any amino acid residue (X) at position 446 (Y446X), (b) arginine at position 470 (N470R), and (c) histidine at position 503 (W503H), wherein the amino acid residue positions are determined using the residue numbering of SEQ ID NO: 10 as a reference, the VP1 protein coding sequence being operably linked to an expression control sequence directing its expression in a packaging host cell. In certain embodiments, the nucleic acid molecule is a plasmid. A host cell comprising the nucleic acid is also provided.

[0012] Additional aspects and advantages of these compositions and methods are further described in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A – Figure 1B Provides AAV vector library generation ( Figure 1A ) and an overview of the screen, which includes a delineation of the AAV9 capsid and the positions of amino acids that were altered in the design of the library ( Figure 1B ).

[0014] Figure 2 Plot showing the yield ranking of variants in the first round of library screening.

[0015] Figure 3 Plots showing enrichment of variants in the heart (top) or liver (bottom) in the first round of library screening.

[0016] Figure 4 Plot showing enrichment of variants in heart versus liver in the first round of library screening.

[0017] Figure 5 Plot showing enrichment of variants in heart versus liver in the first round of library screening. Results with scores > 0.25 relative to AAV9 yield are shown.

[0018] Figure 6 An overview of AAV9 variants identified in the first round of screening is shown (approximately 200 AAV9 Gal-binding variants).

[0019] Fig. 7A – Figure 7F Pie charts are provided showing the positional analysis of variants identified in heart and liver.

[0020] Figure 8 An overview of the study design for the second round of AAV vector production and screening is provided.

[0021] Fig. 9A and Fig. 9B Enrichment in the heart versus liver of mice is shown. The figure identifies a collection of variants with an XRH motif that exhibit enhanced cardiac targeting and liver detargeting.

[0022] Fig.10 Enrichment in the heart relative to the liver of non-human primates is shown.

[0023] Fig.11 Comparison of enrichment in mouse and NHP livers is shown. Most variants behave similarly in mouse and NHP livers, while AAV9 and its close relatives have lower RPMs in NHP livers than in mouse livers.

[0024] Fig. 12A and Fig. 12B A review of the methods and results of studies evaluating galactose binding affinity in vitro to characterize the relative binding strength of AAV9 variants is provided.

[0025] Fig.13A and Fig. 13B Results of galactose binding affinity studies are presented.

[0026] Fig.14A – Fig. 14C Relative transduction of the eGFP transgene in the heart after delivery of vectors with HRH capsid, SRH capsid, and VRH capsid is shown (DNA) Fig.14A )、RNA( Fig. 14B ) and protein ( Fig. 14C ) expression. Vectors with AAV9 capsid or AAV9 capsid with W503A mutation were included as controls.

[0027] Fig.15A – Fig. 15C Relative transduction of the eGFP transgene in the liver after delivery of vectors with HRH capsid, SRH capsid, and VRH capsid is shown (DNA) Fig.15A ) and RNA( Fig. 15B ) and protein ( Fig. 15C ) expression. Vectors with AAV9 capsid or AAV9 capsid with W503A mutation were included as controls. DETAILED DESCRIPTION

[0028] Provided herein is a novel adeno-associated virus (AAV) capsid protein. In certain embodiments, the capsid protein is characterized by reduced galactose binding, which can change the ability of the viral vector to transduce certain cell and tissue types. In certain embodiments, the capsid protein is a branch F capsid protein having the following motifs: Y446X, N470R and W503H, wherein the number of amino acid residues is relative to the vp1 protein of the known branch F vector (such as AAV9). Also provided is rAAV comprising the capsid protein described herein. In certain embodiments, after administration to a subject, the ability of rAAV to target cardiac tissue is improved and / or the ability to target the liver is reduced. rAAV can be used as a gene therapy product, for gene editing, as a vaccine, and other suitable uses in the form of a composition. Also provided is a composition comprising a nucleic acid encoding a capsid protein as described herein, wherein the capsid protein includes a host cell for producing rAAV.

[0029] Based on the observation that both liver detargeting and peripheral transduction depend on factors within the combinatorial space of the AAV9 galactose binding site, a directed evolution approach was utilized to identify rAAV vectors with novel capsid proteins. In both mice and non-human primates, libraries of AAV variants containing amino acid substitutions at Y446, N470, and W503 were generated and screened by multiple rounds of selection. A family of AAV capsid variants containing a common amino acid motif that all showed reduced liver transduction with increased cardiac transduction were identified. The study revealed five amino acid residues that altered galactose binding and identification of capsid proteins that altered tissue targeting in vivo. The findings showed that binding affinity was positively correlated with liver transduction in vivo, while non-tryptophan aromatic compounds were more favorable in vectors that detargeted the liver but maintained peripheral transduction.

[0030] In certain embodiments, a rAAV is provided having an AAV clade F capsid, the AAV clade F capsid comprising a mutant galactose binding site. In certain embodiments, the cardiac targeting of rAAV is enhanced and the liver targeting is reduced compared to its corresponding parental AAV clade F capsid. The galactose binding site is characterized by, when the residue numbering of SEQ ID NO:10 (AAV9vp1) is used as a reference to determine the residue position, (a) any amino acid residue (X) (Y446X) at position 446, (b) arginine (N470R) at position 470, and (c) histidine (W503H) at position 503. rAAV comprises a vector genome comprising a 5' inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, a regulatory sequence for guiding the expression of the gene product operably linked to the coding sequence, and a 3'ITR sequence. In certain embodiments, rAAV comprises a capsid protein comprising (a) H (HRH) at position 446; (b) S (SRH) at position 446; (c) V (VRH) at position 446; (d) G (GRH) at position 446; (e) F (FRH) at position 446; (f) T (TRH) at position 446; or (g) S (SRH) at position 446. In certain embodiments, the parent capsid is an AAV9 capsid. In certain embodiments, rAAV comprises a capsid protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9.

[0031] In certain embodiments, rAAV comprises additional mutations (insertions, deletions, substitutions). Such additional mutations include mutations described herein as well as mutations known in the art. In certain embodiments, additional mutations improve vector production efficiency. In certain embodiments, these additional mutations improve targeting to cells or tissues or reduce targeting to cells or tissues. In certain embodiments, additional mutations further improve, for example, targeting to cardiac cells or tissues.

[0032] In certain embodiments, a method for producing rAAV comprising a mutant capsid with liver specificity derived from a parent AAV capsid is provided. In certain embodiments, the parent AAV capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid or an AAVhu96 capsid or a mutant or variant of any of the foregoing. Therefore, the rAAV comprises a capsid protein having the following: when the residue numbering of SEQ ID NO: 10 (AAV9 vp1) is used as a reference to determine the residue position, (a) any amino acid residue (X) (Y446X) at position 446, (b) arginine (N470R) at position 470, and (c) histidine (W503H) at position 503. The method comprises culturing a packaging host cell, the packaging host cell comprising a nucleic acid sequence encoding a mutant AAV clade F capsid, the nucleic acid sequence being operably linked to a regulatory control sequence directing its expression in the packaging host cell. The packaging host cell further comprises a nucleic acid molecule comprising a vector genome, the vector genome comprising a 5' inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, a regulatory sequence for directing the expression of the gene product operably linked to the coding sequence, and a 3'ITR sequence; and an auxiliary function, the auxiliary function being necessary for packaging the vector genome into the mutant clade F capsid. In certain embodiments, the parent capsid is an AAV9 capsid. In certain embodiments, the nucleic acid sequence encoding the mutant AAV clade F capsid comprises a sequence encoding the amino acid sequence shown in any one of SEQ ID NO: 1-9.

[0033] In certain embodiments, the rAAV or rAAV produced according to the provided methods can be used to deliver gene products. In certain embodiments, the ability of rAAV to target cardiac cells and tissues is improved, particularly relative to the parental clade F capsid. In certain embodiments, the ability of rAAV to transduce the liver is reduced, particularly relative to the parental clade F capsid, and is therefore referred to as "de-targeting" the liver. De-targeting the liver may be beneficial in reducing hepatotoxicity, which may be observed after delivery of the parental clade F vector in some cases. Compositions, such as pharmaceutical formulations, are also provided, which include the described rAAV. In certain embodiments, the method includes reducing hepatotoxicity associated with the delivery of rAAV vectors, wherein rAAV with a mutated galactose binding site provided herein is administered to a subject. In certain embodiments, the method includes improving the delivery of gene products to a method for improving the delivery of gene products to cardiac cells or tissues, wherein rAAV with a mutated galactose binding site provided herein is administered to a subject. In certain embodiments, the reduction in hepatotoxicity associated with the delivery of rAAV vectors is relative to the parental AAV clade F capsid. In certain embodiments, the improved delivery of a gene product is a method for improving delivery of a gene product to cardiac cells or tissue relative to a parental AAV clade F capsid.

[0034] With respect to the following description, it is contemplated that in another embodiment, each of the compositions described herein can be used in the methods of the present invention. In addition, it is also contemplated that in another embodiment, each of the compositions described herein as being useful in the methods is itself an embodiment of the present invention.

[0035] Capsid

[0036] In certain embodiments, a rAAV is provided having a capsid comprising a mutation that alters binding to galactose. The altered binding to galactose helps to enhance targeting or retargeting of cells and tissues after in vivo delivery. The rAAV provided herein comprises a capsid having any amino acid residue (X) at position 446 (Y446X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H), wherein the number of amino acid residues is determined by reference to the AAV9 vp1 capsid protein (as provided in SEQ ID NO: 10).

[0037] In certain embodiments, the rAAV capsid comprises a mutation introduced into or present in a parental capsid. In certain embodiments, the parental capsid is a clade F AAV capsid. In certain embodiments, the parental AAV capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid, or an AAVhu96 capsid, or a mutant or variant of any of the foregoing. [AAVhu68 - see, e.g., US2020 / 0056159; PCT / US21 / 55436; SEQ ID NOs: 4 and 5 for nucleic acid sequences encoding hu68 capsid; SEQ ID NO: 6 for hu68 vp1 amino acid sequence], AAVhu95 capsid - [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 7 and 8 (hu95 vp1 encoding nucleic acid sequence) and SEQ ID NO: 9 (hu95 vp1 amino acid sequence), AAVhu96 capsid - [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 10 and 11 (hu96 encoding nucleic acid sequence) and SEQ ID NO: 12 (hu96 vp1 amino acid sequence), AAV9 - [see, e.g., U.S. Provisional Application No. 63 / 251,599, filed October 2, 201; SEQ ID NOs: 10 and 11 (hu96 encoding nucleic acid sequence) and SEQ ID NO: 12 (hu96 vp1 amino acid sequence), 7,906,111], engineered mutants and variants thereof - [see, e.g., WO2020 / 200499; WO2003 / 054197].

[0038] As used herein, the term "clade" as it relates to AAV groups refers to groups of AAVs that are phylogenetically related to one another as determined using a Neighbor-Joining algorithm based on an alignment of AAV vpl amino acid sequences with a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no greater than 0.05. Neighbor-Joining algorithms have been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York) (2000). Computer programs that can be used to implement this algorithm are provided. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, and the sequence of the AAV vpl capsid protein, one skilled in the art can readily determine whether a selected AAV is contained within one of the clades identified herein, within another clade, or outside of these clades. See, e.g., G Gao et al., Journal of Virology, 2004 Jun;78(12):6381-6388, which identifies clades A, B, C, D, E, and F, and provides nucleic acid sequences of novel AAVs, GenBank Accession Nos. AY530553 to AY530629. See also WO 2005 / 033321.

[0039] As used herein, an "AAV9 capsid" is a self-assembled AAV capsid composed of a variety of AAV9 vp proteins. The AAV9 vp protein is generally expressed as an alternative splice variant encoded by a nucleic acid sequence encoding the vp1 amino acid sequence of GenBank Accession No.: AAS99264. These splice variants produce proteins of different lengths. In certain embodiments, the "AAV9 capsid" comprises an AAV having an amino acid sequence that is 99% identical to AAS99264 or 99% identical thereto. See also WO 2019 / 168961, published on September 6, 2019, comprising Table G, which provides the deamidation pattern of AAV9. See also US7906111 and WO2005 / 033321. Where specified, "AAV9 variants" may include variants described in, for example, WO2016 / 049230, US 8,927,514, US2015 / 0344911, and US 8,734,809.

[0040] rAAVhu68 is composed of AAVhu68 capsid and vector genome. AAVhu68 capsid is a combination of a heterogeneous population of vp1, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins. As used herein, when used to refer to vp capsid proteins, the term "heterogeneous" or any grammatical variation thereof refers to a population composed of non-identical elements (e.g., vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences). See also PCT / US2018 / 019992, WO 2018 / 160582, entitled "Adeno-Associated Virus (AAV) Clade F Vector and Uses Therefor", and incorporated herein by reference in its entirety.

[0041] In certain embodiments, the rAAV comprises an AAV capsid protein comprising any amino acid residue (X) at position 446 (Y446X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H). In certain embodiments, the capsid protein is an AAV9 variant comprising a V, S, H, G, F, Y, T, or A residue at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H) (SEQ ID NO: 9). In certain embodiments, the capsid protein is an AAV9 variant comprising any amino acid residue (X) at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ IN NO: 10 (AAV9). In certain embodiments, the amino acid capsid protein is an AAV9 variant having any amino acid residue at position 446 (X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H) and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO: 10). In certain embodiments, the capsid protein is an AAVhu68 variant comprising any amino acid residue at position 446 (X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H). In certain embodiments, the capsid protein is an AAVhu68 variant comprising any amino acid residue at position 446 (X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising any amino acid residue at position 446 (X), an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO: 11).

[0042] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (VRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having a V at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO: 10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising a V at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO: 10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having a V at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the capsid protein is an AAVhu68 variant comprising a V at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0043] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:2 (SRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having S at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising S at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an S at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising an S at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0044] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:3 (HRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an H at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising an H at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an H at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising an H at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0045] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:4 (GRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having a G at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising a G at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having a G at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising a G at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0046] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence shown in SEQ ID NO: 5 (FRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having F at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO: 10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising F at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO: 10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an F at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising an F at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0047] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:6 (YRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having Y at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising Y at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having Y at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising Y at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0048] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence shown in SEQ ID NO:7 (TRH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having T at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising T at position 446, arginine at position 470 (N470R), and histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having a T at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising a T at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0049] In certain embodiments, the rAAV comprises an AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:8 (ARH). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an A at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence being at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IN NO:10. In certain embodiments, the AAV capsid protein is an AAV9 variant comprising an A at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (relative to SEQ ID NO:10). In certain embodiments, the rAAV comprises an AAV capsid protein comprising an amino acid sequence having an A at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), the amino acid sequence sharing at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 11. In certain embodiments, the amino acid capsid protein is an AAVhu68 variant comprising an A at position 446, an arginine at position 470 (N470R), and a histidine at position 503 (W503H), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (relative to SEQ ID NO: 11).

[0050]

[0051]

[0052]

[0053]

[0054] In certain embodiments, a rAAV is provided having a modified capsid protein having at least one exogenous peptide from the Nx-(T / I / V / A)-(K / R) targeting motif. In other embodiments, other viral vectors may be produced having one or more exogenous targeting peptides from the Nx-(T / I / V / A)-(K / R) motif in the exposed capsid protein (which may be the same or different, or a combination thereof) for regulating and / or altering the targeting specificity of the viral vector compared to the parental vector. In certain embodiments, a composition is provided that can be used to target endothelial cells. The composition is a mutant capsid, which includes at least one exogenous targeting peptide, and the at least one exogenous targeting peptide includes: an amino acid sequence of Nx-(T / I / V / A)-(K / R) (SEQ ID NO:47), wherein the amino acid sequence is optionally flanked by two to seven amino acids at the amino terminus and / or carboxyl terminus of the motif, and is optionally further conjugated to a nanoparticle, a second molecule or a viral capsid protein.

[0055] In certain embodiments, the targeting peptide comprises one of the following sequences with an optional linker sequence:

[0056] (a) SSNTVKLTSGH (SEQ ID NO: 14);

[0057] (b)EFSSNTVKLTS(SEQ ID NO:12);

[0058] (c) GGVLTNIARGEYMRGG (SEQ ID NO: 18);

[0059] (d)GGIEINATRAGTNLGG (SEQ ID NO: 17);

[0060] (e) GGSSNTVKLTSGHGG (SEQ ID NO: 13);

[0061] (f) IEINATRAGTNL (SEQ ID NO: 16); or

[0062] (g) SANFIKPTSY (SEQ ID NO:15).

[0063] See PCT / US2021 / 061312, filed on December 1, 2021, entitled “NOVEL COMPOSITIONS WITH TISSUE-SPECIFICTARGETING MOTIFS AND COMPOSITIONS CONTAINING SAME,” which is incorporated herein by reference in its entirety.

[0064] In certain embodiments, a rAAV is provided having a modified capsid having at least one exogenous peptide from the YG / A / R / KY / H-GNPA-T / R / H-RYFD-V / K (SEQ ID NO: 28) core targeting motif. In certain embodiments, the rAAV may have one or more exogenous targeting peptides from the YG / A / R / KY / H-GNPA-T / R / H-RYFD-V / K (SEQ ID NO: 28) core targeting motif (which may be the same or different, or a combination thereof) in the exposed capsid protein for regulating and / or changing the targeting specificity of the viral vector compared to the parental vector.

[0065] In certain embodiments, a rAAV is provided that can be used to target brain cells. The composition is a capsid protein, the capsid protein includes at least one exogenous targeting peptide, and the at least one exogenous targeting peptide includes: the core amino acid sequence of YGYGNPATRYFDV (SEQ ID NO: 20), the core amino acid sequence is optionally flanked by two to seven amino acids at the amino terminus and / or carboxyl terminus of its core sequence, and is optionally further conjugated to nanoparticles, a second molecule or a viral capsid protein. In certain embodiments, a rAAV is provided that has a capsid that can be used to target brain cells. The capsid includes at least one exogenous targeting peptide, and the at least one exogenous targeting peptide includes: the core amino acid sequence of YGYGNPATRYFDV (SEQ ID NO: 20), the core amino acid sequence is optionally flanked by two to seven amino acids at the amino terminus and / or carboxyl terminus of the core sequence, and is optionally further conjugated to nanoparticles, a second molecule or a viral capsid protein. The targeting peptide includes the following core amino acid sequence with an optional linking sequence:

[0066] (a) YGYGNPARRYFDV (SEQ ID NO: 25);

[0067] (b) YGYGNPAHRYFDV (SEQ ID NO: 26);

[0068] (c) YGYGNPATRYFDK (SEQ ID NO: 27);

[0069] (d) YAYGNPATRYFDV (SEQ ID NO: 21);

[0070] (e) YKYGNPATRYFDV (SEQ ID NO: 22);

[0071] (f) YRYGNPATRYFDV (SEQ ID NO: 23); or

[0072] (g) YGHGNPATRYFDV (SEQ ID NO: 24).

[0073] See U.S. Provisional Application No. 63 / 178,881, filed on April 23, 2021, entitled “NOVEL COMPOSITIONS WITH BRAIN-SPECIFIC TARGETING MOTIFS AND COMPOSITIONS CONTAINING SAME,” which is incorporated herein by reference in its entirety.

[0074] In certain embodiments, a rAAV is provided that has been modified to include a peptide insertion that improves targeting to muscle tissue (including skeletal tissue and / or cardiac tissue) (i.e., improves binding to receptors expressed on cardiac cells). In certain embodiments, the peptide insertion includes an RGD motif. See, e.g., PCT / EP2019 / 076958, filed on October 4, 2019, entitled "PEPTIDE-MODIFIED HYBRID RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE BETWEEN AAV9 AND AAVrh74 WITH REDUCED LIVER TROPISM AND INCREASED MUSCLETRANSDUCTION," which is incorporated herein by reference in its entirety.

[0075] The targeting peptide as described above can be inserted into the hypervariable loop (HVR) VIII at any suitable location. For example, based on the AAV9 VP1 amino acid sequence: SEQ ID NO: 10 numbering, the peptide is inserted with different lengths of linkers between amino acids 588 and 589 (QA) of the AAV9 capsid protein. See also WO 2019 / 168961, published on September 6, 2019, comprising Table G, which provides the deamidation pattern of AAV9; and WO 2020 / 160582, filed on September 7, 2018. The amino acid residue positioning is the same in AAVhu68 (SEQ ID NO: 11). However, another site can be selected in HVRVIII. Alternatively, another exposed loop HVR (e.g., HVRIV) can be selected for insertion. Comparable HVR regions can be selected in other capsids. In certain embodiments, the positioning of HVRVIII and HVRIV is determined using the algorithms and / or alignment techniques described in U.S. Pat. No. 9,737,618 B2 (column 15, lines 3-23) and U.S. Pat. No. 10,308,958 B2 (column 15, lines 46-column 16, line 6), which are incorporated herein by reference in their entirety. In certain embodiments, the targeting peptide can be inserted into the hypervariable loop HVRVIII as described in U.S. Provisional Patent Application No. 63 / 119,863, filed on December 1, 2020, which is incorporated herein by reference.

[0076] In certain embodiments, a nucleic acid is provided, which includes a sequence encoding a novel capsid protein described herein. In certain embodiments, the encoded amino acid sequence includes any one of SEQ ID NO: 1 to 9. In certain embodiments, the encoded amino acid sequence and any one of SEQ ID NO: 1 to 9 have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity. In certain embodiments, the encoded amino acid sequence is an AAV branch F capsid protein, and the AAV branch F capsid protein has any amino acid residue at position 446, and arginine (N470R) at position 470 and histidine (W503H) at position 503 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions (relative to SEQ ID NO: 10). In certain embodiments, the nucleic acid is a plasmid.

[0077] Designing a nucleotide sequence encoding an AAV capsid protein comprising DNA (genome or cDNA) or RNA (e.g., mRNA) is within the technical scope of the art. Such nucleotide sequences can be codon optimized to be expressed in a selected system (i.e., cell type) and can be designed by a variety of methods. This optimization can be performed using methods available online (e.g., GeneArt), disclosed methods, or companies providing codon optimization services (e.g., DNA2.0 companies (Menlo Park, California, (Menlo Park, CA))). For example, a codon optimization method is described in International Patent Publication No. WO 2015 / 012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. Patent Publication No. 2014 / 0032186 and U.S. Patent Publication No. 2006 / 0136184.

[0078] As used herein, "encoded amino acid sequence" refers to the amino acid predicted by translation of the known DNA codons of the referenced nucleic acid sequence into amino acids. The following table shows DNA codons and twenty common amino acids, which are shown in single letter codes (SLC) and three letter codes (3LC), respectively.

[0079] Reverse table of the standard genetic code (condensed using IUPAC nomenclature)

[0080]

[0081]

[0082] Expression cassettes and vectors

[0083] The vector genome sequence packaged into the AAV capsid and delivered to the host cell is usually composed of at least a transgene and its regulatory sequence and an AAV inverted terminal repeat (ITR). Both single-stranded AAV and self-complementary (sc) AAV are included in rAAV. Transgene is a nucleic acid coding sequence heterologous to the vector sequence, which encodes a polypeptide, a protein, a functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene products of interest. The nucleic acid coding sequence is operably connected to the regulatory component in a manner that allows transgenic transcription, translation and / or expression in the cells of the target tissue.

[0084] The AAV sequence of the vector generally includes cis-acting 5' and 3' inverted terminal repeats (see, e.g., BJ Carter, "Handbook of Parvoviruses", edited by P. Tijsser, CRC Press, pp. 155-168 (1990)). The length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, but minor modifications to these sequences to some extent are permitted. The ability to modify these ITR sequences is within the technical scope of the art. (See, e.g., the following texts: Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., Journal of Virology, 70:520-532 (1996)). Examples of such molecules used in the present invention are "cis-acting" plasmids containing transgenes, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. In one embodiment, the ITR is from an AAV different from the AAV supplying the capsid. In one embodiment, the ITR is from AAV2. However, ITRs from other AAV sources can be selected. A shortened version of the 5'ITR referred to as ΔITR has been described, in which the D sequence and the terminal resolution site (trs) are missing. In certain embodiments, the vector genome comprises a shortened AAV2 ITR of 130 base pairs, in which the external A element is missing. Without wishing to be bound by theory, it is believed that during the amplification of the vector DNA using the internal (A') element as a template, the shortened ITR is restored to a wild-type length of 145 base pairs. In other embodiments, full-length AAV 5' and 3'ITRs are used. In the case where the source of the ITR is from AAV2 and the AAV capsid is from another AAV source, the resulting vector can be referred to as pseudotyped. However, other configurations of these elements may be suitable.

[0085] In addition to the major elements of the recombinant AAV vector identified above, the vector also contains the necessary conventional control elements, which are operably linked to the transgene in a manner that allows its transcription, translation and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention.

[0086] Regulatory control elements typically contain a promoter sequence as part of an expression control sequence (e.g., positioned between a selected 5' ITR sequence and a coding sequence). Constitutive promoters, regulatable promoters [see, e.g., WO 2011 / 126808 and WO 2013 / 04943], tissue-specific promoters, or promoters responsive to physiological cues can be used in the vectors described herein.

[0087] Examples of constitutive promoters suitable for controlling expression of therapeutic products include, but are not limited to, the chicken β-actin (CB) promoter, the CB7 promoter, the human cytomegalovirus (CMV) promoter, the ubiquitin C promoter (UbC), the early and late promoters of simian virus 40 (SV40), the U6 promoter, the metallothionein promoter, the EF1α promoter, the ubiquitin promoter, the hypoxanthine phosphoribosyltransferase (HPRT) promoter, the dihydrofolate reductase (DHFR) promoter (Scharfmann et al., Journal of the American College of Chemistry, 1996). The promoters of the present invention include, but are not limited to, endothelin-I (ET-I) and Flt-I, which are specific for endothelial cells, and FoxJ1, which targets ciliated cells. Other examples of tissue-specific promoters suitable for use in the present invention include, but are not limited to, liver-specific promoters. Examples of liver-specific promoters may include, for example, thyroid hormone binding globulin (TBG), albumin, Miyatake et al., (1997) Journal of Virology, 71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Ther., 3:10029; or human α1-antitrypsin, phosphoenolpyruvate carboxykinase (PECK) or alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Ther., 7:1503-14).

[0088] In certain embodiments, the promoter is a tissue-specific (eg, neuron-specific) promoter.In certain embodiments, suitable promoters may include, but are not limited to, the following: elongation factor 1 alpha (EF1α) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); synapsin 1 promoter (see, e.g., Kügler S et al., Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47); neuron-specific enolase (NSE) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); synapsin 1 promoter (see, e.g., Kügler S et al., Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 2003 Feb;10(4):337-47); neuron-specific enolase (NSE) promoter (see, e.g., Kim DW et al., Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene. 1990 Jul 16;91(2):217-23); J et al., Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells. Endocrinology. 2004 Feb; 145(2): 613-9. Epub 2003 Oct 16); or CB6 promoter (see, e.g., Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene, Mol Biotechnol. 2016 Jan; 58(1): 30-6. Doi: 10.1007 / s12033-015-9899-5). Preferably, such promoter is of human origin.

[0089] In certain embodiments, the promoter is a cardiac promoter. In certain embodiments, the promoter is a cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (alpha-MHC) or myosin light chain 2 (MLC-2) promoter. See also Pacak, CA et al., tissue-specific promoters improve the specificity of AAV9mediated transgene expression following intravascular gene delivery in neonatal mice after intravascular gene delivery in neonatal mice, "Genetic Vaccines and Therapy" 2008, 6: 13. In certain embodiments, the expression cassette includes a promoter as a chicken cardiac troponin T promoter (also referred to as chicken TnT or chTnT).

[0090] Inducible promoters suitable for controlling the expression of therapeutic products include promoters that respond to exogenous agents (e.g., pharmacological agents) or physiological cues. These response elements include, but are not limited to, hypoxia response elements (HREs) that bind to HIF-Iα and β; metal ion response elements, such as those described by Mayo et al. (1982, Cell 29:99-108); Brister et al. (1982, Nature 296:39-42) and Searle et al. (1985, Mol. Cell. Biol. 5:1480-1489); or heat shock response elements, such as those described by Nouer et al. (In: Heat Shock Response, Nouer, L., ed., CRC, Boca Raton, Fla., pp. 167-220, 1991).

[0091] In one embodiment, the expression of the gene product is controlled by an adjustable promoter that provides strict control of the transcription of a sequence encoding the gene product (e.g., a pharmacological agent or a transcription factor activated by a pharmacological agent, or in an alternative embodiment, a physiological cue). Promoter systems that are not leaky and can be strictly controlled are preferred. Examples of adjustable promoters that can be used as ligand-dependent transcription factor complexes in the present invention include, but are not limited to, members of the nuclear receptor superfamily activated by their corresponding ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysone and their analogs and mimetics) and rTTA activated by tetracycline. In one aspect of the present invention, the gene switch is an EcR-based gene switch. Examples of such systems include, but are not limited to, systems described in U.S. Patent Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006 / 0014711, 2007 / 0161086, and International Published Application No. WO01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038; U.S. Published Patent Applications Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416; and International Published Applications Nos. WO 01 / 70816, WO 02 / 066612, WO 02 / 066613, WO 02 / 066614, WO 02 / 066615, WO 02 / 29075, and WO 2005 / 108617, each of which is incorporated herein by reference in its entirety. An example of a system regulated by a nonsteroidal ecdysone agonist is Mammalian Inducible Expression System (New England Biolabs, Ipswich, MA).

[0092] Still other promoter systems may comprise response elements, including but not limited to tetracycline (tet) response elements (as described by Gossen and Bujard (1992, Proc. Natl. Acad. Sci. USA 89:5547-551); or hormone response elements as described by Lee et al. (1981, Nature 294:228-232); Hynes et al. (1981, Proc. Natl. Acad. Sci. USA 78:2038-2042); Klock et al. (1987, Nature 329:734-736); and Israel and Kaufman (1989, Nucl. Acids Res.). Res. 17:2589-2604) and other inducible promoters known in the art. Using such promoters, the expression of soluble hACE2 constructs can be controlled, for example, by the following: Tet on / off system (Gossen et al., 1995, Science 268:1766-9; Gossen et al., 1992, Proceedings of the National Academy of Sciences of the United States of America, 89(12):5547-51); TetR-KRAB system (Urrutia R., 2003, Genome Biol., 4(10):231; Deuschle U et al., 1995, Mol Cell Biol. (4):1907-14); Mifepristone (RU486) regulatable system (gene switch; Wang et al., 2003, Genome Biol., 4(10):231; Deuschle U et al., 1995, Mol Cell Biol. (4):1907-14); Y et al., 1994, Proc. Natl. Acad. Sci. USA, 91(17):8180-4; Schillinger et al., 2005, Proc. Natl. Acad. Sci. USA A. 102(39):13789-94); and the humanized tamoxifen-dep regulatable system (Roscilli et al., 2002, Mol. Ther. 6(5):653-63).

[0093] In another aspect, the gene switch is based on heterodimerization of FK506 binding protein (FKBP) and FKBP rapamycin associated protein (FRAP) and is regulated by rapamycin or a non-immunosuppressive analog thereof. Examples of such systems include, but are not limited to, ARGENT TMTranscription technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in: U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595; U.S. Publication No. 2002 / 0173474; U.S. Publication No. 200910100535; U.S. Pat. No. 5,834,266; U.S. Pat. Patent No. 7,109,317, U.S. Patent No. 7,485,441, U.S. Patent No. 5,830,462, U.S. Patent No. 5,869,337, U.S. Patent No. 5,871,753, U.S. Patent No. 6,011,018, U.S. Patent No. 6,043,082, U.S. Patent No. 6,046,047, U.S. Patent No. 6,063,625, U.S. Patent No. 6,140,120, U.S. Patent No. 6,165,787, U.S. Patent No. No. 6,972,193, U.S. Patent No. 6,326,166, U.S. Patent No. 7,008,780, U.S. Patent No. 6,133,456, U.S. Patent No. 6,150,527, U.S. Patent No. 6,506,379, U.S. Patent No. 6,258,823, U.S. Patent No. 6,693,189, U.S. Patent No. 6,127,521, U.S. Patent No. 6,150,137, U.S. Patent No. 6,464,974, U.S. Patent No. No. 6,509,152, U.S. Patent No. 6,015,709, U.S. Patent No. 6,117,680, U.S. Patent No. 6,479,653, U.S. Patent No. 6,187,757, U.S. Patent No. 6,649,595, U.S. Patent No. 6,984,635, U.S. Patent No. 7,067,526, U.S. Patent No. 7,196,192, U.S. Patent No. 6,476,200, U.S. Patent No. 6,492,106, WO No. 94 / 18347, No. WO 96 / 20951, No. WO 96 / 06097, No. WO 97 / 31898, No. WO 96 / 41865, No. WO 98 / 02441, No. WO 95 / 33052, No. WO 99110508, No. WO No. 99110510, No. WO99 / 36553, No. WO 99 / 41258, No. WO 01114387, ARGENT TM Regulated Transcription Retroviral Kit Version 2.0 (9109102) and ARGENT TMRegulated Transcription Plasmid Kit Version 2.0 (9109 / 02), each of which is incorporated herein by reference in its entirety. The ARGENT system is designed to be induced by rapamycin and its analogs (referred to as "rapamycin analogs"). Examples of suitable rapamycins are provided in the ARGENT TM In one embodiment, the molecule is rapamycin [e.g., marketed by Pfizer as Rapamune TM [Sale]. In another embodiment, a rapamycin analog known as AP21967 [Ariyad] is used. Examples of these dimer molecules that can be used in the present invention include, but are not limited to, rapamycin, FK506, FK1012 (homodimer of FK506), rapamycin analogs ("rapamycin analog / rapalog"), which are easily prepared by chemically modifying the natural product to add a "bump" that reduces or eliminates affinity for endogenous FKBP and / or FRAP. Examples of rapamycin analogs include, but are not limited to, AP26113 (Ariad), AP1510 (Amara, JF et al., 1997, Proc. Natl. Acad. Sci. USA, 94(20):10618-23), AP22660, AP22594, AP21370, AP22594, AP23054, AP1855, AP1856, AP1701, AP1861, AP1692 and AP1889, which have engineered 'bumps' that minimize interaction with endogenous FKBP. Still other rapamycins may be selected, for example, AP23573 [Merck]. In certain embodiments, rapamycin or a suitable analog may be delivered topically to AAV-transfected cells in the nasopharynx. Such local delivery may be by intranasal injection, by bolus injection, cream or gel to locally reach the cells. See US patent application US2019 / 0216841 A1, which is incorporated herein by reference.

[0094] Other suitable enhancers include enhancers suitable for the desired target tissue indications. In one embodiment, the expression cassette includes one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be identical or different from each other. For example, an enhancer may include a CMV immediate early enhancer. This enhancer may be present in two copies positioned adjacent to each other. Alternatively, the double copies of the enhancer may be separated by one or more sequences. In still another embodiment, the expression cassette further contains an intron, for example, a chicken beta actin intron. Other suitable introns include introns known in the art, for example, introns described in WO 2011 / 126808. Examples of suitable polyA sequences include, for example, rabbit beta globulin (rBG), SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. Optionally, one or more sequences may be selected to stabilize mRNA. An example of such a sequence is a modified WPRE sequence, which can be engineered upstream of the polyA sequence and downstream of the coding sequence (see, eg, MA Zanta-Boussif et al., Gene Therapy (2009) 16:605-619).

[0095] AAV viral vectors can include multiple transgenes. In certain embodiments, transgenes can be used to correct or improve gene defects, and the gene defects can include defects in which normal genes are expressed at levels lower than normal or defects in which functional gene products are not expressed. Alternatively, transgenes can provide products for cells that are not naturally expressed in the cell type or host. The preferred type of transgenic sequence encodes a therapeutic protein or polypeptide expressed in a host cell. The present invention further includes the use of multiple transgenes. In some cases, different transgenes can be used to encode each subunit of a protein, or to encode different peptides or proteins. This is desirable when the size of the DNA encoding the protein subunit is large (for example, for immunoglobulins, platelet-derived growth factors, or dystrophin). In some cases, different transgenes can be used to encode each subunit of a protein (e.g., immunoglobulin domains, immunoglobulin heavy chains, immunoglobulin light chains). In one embodiment, cells produce multi-subunit proteins after being infected / transfected with a virus containing each subunit of different subunits. In another embodiment, different subunits of a protein can be encoded by the same transgene. This is desirable when the size of the DNA encoding each of the subunits is small (e.g., the total size of the DNA encoding the subunits and the IRES is less than five kilobases). As an alternative to IRES, the DNA can be separated by a sequence encoding a 2A peptide that self-cleaves in a post-translational event. See, e.g., ML Donnelly et al., (January 1997) Journal of General Virology (J. Gen. Virol.), 78(Pt1):13-21; S. Furler, S et al., (June 2001) Gene Therapy, 8(11):864-873; H. Klump et al., (May 2001) Gene Therapy, 8(10):811-817. This 2A peptide is significantly smaller than an IRES, which makes it very suitable for use when space is a limiting factor. More typically, when the transgene is large, consists of multiple subunits, or two transgenes are co-delivered, rAAVs carrying the desired transgenes or subunits are co-administered to allow them to be tandem in vivo to form a single vector genome. In such embodiments, the first AAV may carry an expression cassette expressing a single transgene, and the second AAV may carry an expression cassette expressing a different transgene for co-expression in a host cell. However, the transgene of choice may encode any biologically active product or other product, such as a product desired for research.

[0096] In addition to the elements for the expression cassette identified above, the vector also comprises conventional control elements that are operably linked to the coding sequence in a manner that allows the encoded product (e.g., UBE3A construct, gene replacement therapy in the Angelman mouse model; see U.S. Provisional Patent Application No. 63 / 119,860, filed December 1, 2020, which is incorporated herein by reference) to be transcribed, translated, and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the invention. Examples of other suitable transgenics are provided herein.

[0097] Expression control sequences include appropriate enhancers; transcription factors; transcription terminators; promoters; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, such as the woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product.

[0098] In one embodiment, the regulatory sequences are selected so that the size of the total rAAV vector genome is about 2.0 kilobases to about 5.5 kilobases. In one embodiment, it is desirable that the rAAV vector genome is close to the size of the natural AAV genome. Therefore, in one embodiment, the regulatory sequences are selected so that the size of the total rAAV vector genome is about 4.7 kb. In another embodiment, the size of the total rAAV vector genome is less than about 5.2 kb. The size of the vector genome can be manipulated based on the size of the regulatory sequences including promoters, enhancers, introns, poly A, etc. See Wu et al., Molecular Therapy, January 2010 18 (1): 80-6, which is incorporated herein by reference.

[0099] Therefore, in one embodiment, the intron is included in the vector. Suitable introns include chicken β-actin intron, human β-globulin IVS2 (Kelly et al., Nucleic Acids Research, 43 (9): 4721-32 (2015)); the Promega chimeric intron (Almond, B. and Schenborn, ETpCI-neo vector and pcDNA4 / HisMax vector comparison (A Comparison of pCI-neo Vector and pcDNA4 / HisMax Vector)) and hFIX intron. Various introns suitable for this article are known in the art and include, but are not limited to, introns found at bpg.utoledo.edu / ~afedorov / lab / eid.html, which are incorporated herein by reference. See also Shepelev V., Fedorov A. Advances in the Exon-Intron Database. Briefings in Bioinformatics 2006, 7: 178-185, incorporated herein by reference.

[0100] rAAV vector production

[0101] Nucleic acids for producing AAV viral vectors (e.g., rAAV) and expression cassettes comprising the nucleic acids can be carried on any suitable vector (e.g., plasmid) delivered to a packaging host cell. Plasmids that can be used in the present invention can be engineered so that they are suitable for in vitro replication and packaging in prokaryotic cells, insect cells, mammalian cells, and other cells. Suitable transfection techniques and packaging host cells are known and / or can be easily designed by those skilled in the art.

[0102] Methods for preparing AAV-based vectors (e.g., with AAV9 or another AAV capsid) are known. See, for example, U.S. Published Patent Application No. 2007 / 0036760 (February 15, 2007), which is incorporated by reference. Various techniques can be used to easily generate the sequence of any AAV capsid provided herein. Suitable production techniques are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, New York). Alternatively, peptides can also be synthesized by well-known solid phase peptide synthesis methods (Merrifield, (1962) Journal of the American Chemical Society (J.Am.Chem.Soc.), 85:2149; Stewart and Young, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969), pp. 27-62). Such methods may involve, for example, culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; a minigene consisting of at least an AAV inverted terminal repeat (ITR) and a transgene; and sufficient auxiliary functions for allowing the minigene to be packaged into an AAV capsid protein. These and other suitable production methods are within the knowledge of those skilled in the art and are not a limitation of the present invention.

[0103] Cultivation in host cells to package the AAV minigene in the AAV capsid required components can be provided to the host cells in trans form. Alternatively, any one or more of the required components (e.g., minigenes, rep sequences, cap sequences and / or auxiliary functions) can be provided by stable host cells, which have been engineered to contain one or more of the required components using methods known to those skilled in the art. Most preferably, such stable host cells will contain the required components under the control of an inducible promoter. However, the required components can be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided in the discussion of regulatory elements suitable for transgenes herein. In still another alternative, the selected stable host cell can contain the selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell can be produced, the host cell being derived from a 293 cell (which contains an E1 auxiliary function under the control of a constitutive promoter), but containing rep and / or cap proteins under the control of an inducible promoter. Those skilled in the art can also produce other still stable host cells.

[0104] The rAAV described herein is particularly suitable for gene delivery for therapeutic purposes. Further, the described composition can also be used for in vitro production of desired gene products. For in vitro production, the desired product (e.g., protein) can be obtained from the desired culture after rAAV transfection host cells containing molecules encoding the desired product and culturing cell cultures under conditions that allow expression. The expressed product can then be purified and separated as needed. The technology suitable for transfection, cell culture, purification and separation is known to those skilled in the art. The method for producing and separating the AAV suitable for use as a carrier is known in the art. See generally, e.g., Grieger and Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99: 119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10: 717-733; and references cited below, each of which is incorporated herein by reference in its entirety. For packaging of transgenes into virions, the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the expression cassette. The cap and rep genes can be supplied in trans.

[0105] In one embodiment, the expression cassette described herein is engineered into a gene element (e.g., a shuttle plasmid), and the immunoglobulin construct sequence carried thereon is transferred to a packaging host cell to produce a viral vector. In one embodiment, the selected gene element can be delivered to an AAV packaging cell by any suitable method, and the method comprises transfection, electroporation, liposome delivery, membrane fusion technology, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Stable AAV packaging cells can also be prepared. Alternatively, the expression cassette can be used to produce a viral vector other than AAV, or a mixture for producing antibodies in vitro. The method for preparing such constructs is known to nucleic acid manipulation technicians and comprises genetic engineering, recombinant engineering, and synthetic techniques. See, for example, "Molecular Cloning: Laboratory Manual", edited by Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012).

[0106] The term "AAV intermediate" or "AAV vector intermediate" refers to an assembled rAAV capsid lacking the desired genomic sequence packaged therein. These intermediates may also be referred to as "empty" capsids. Such capsids may contain no detectable genomic sequence of the expression cassette, or may contain only partially packaged genomic sequence insufficient to achieve expression of the gene product. These empty capsids are non-functional for transferring the gene of interest to a host cell.

[0107] The recombinant AAV described herein can be produced using known techniques. See, e.g., WO 2003 / 042397; WO 2005 / 033321; WO 2006 / 110689; US 7588772 B2. Such methods involve culturing a host cell containing a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette consisting of at least an AAV inverted terminal repeat (ITR) and a transgene; and sufficient auxiliary functions for allowing the expression cassette to be packaged into an AAV capsid protein. Thus, methods for producing capsids, coding sequences, and methods for producing rAAV viral vectors have been described. See, e.g., Gao et al., Proceedings of the National Academy of Sciences of the United States of America 100 (10), 6081-6086 (2003) and US 2013 / 0045186A1.

[0108] In one embodiment, the vector is prepared in a suitable cell culture (e.g., HEK 293 cells). The method for manufacturing the gene therapy vector described herein includes methods well known in the art, such as producing plasmid DNA, producing vectors, and purifying vectors for producing gene therapy vectors. In certain embodiments, the gene therapy vector is an AAV vector, and the plasmid produced is an AAV cis plasmid encoding the AAV genome and the gene of interest, an AAV trans plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may include method steps, such as starting cell culture, cell passage, cell inoculation, transfecting cells with plasmid DNA, exchanging the culture medium after transfection for serum-free culture medium, and collecting cells and culture medium containing the vector. The collected cells and culture medium containing the vector are referred to as crude cell collections herein. In another system, the gene therapy vector is introduced into insect cells by infecting with a baculovirus-based vector. For a review of these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of which are incorporated herein by reference in their entirety. Methods for making and using these and other AAV production systems are also described in the following U.S. Patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; No. 7,229,823; and No. 7,439,065. See also U.S. Provisional Patent Application No. 63 / 371,597, filed on August 16, 2022, entitled “Scalable Methods for Producing rAAV with Packaged Vector Genomes,” and U.S. Provisional Patent Application No. 63 / 371,592, filed on August 16, 2022, entitled “Scalable Methods for Downstream Purification of Recombinant Adeno-Associated Viruses,” both of which are incorporated by reference in their entireties.

[0109] Thereafter, the crude cell harvest may be subject to the method steps of the subject matter, such as concentrating the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of the microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and / or formulation and filtration to prepare large quantities of vector. Two-step affinity chromatography purification at high salt concentrations followed by anion exchange resin chromatography is used to purify the vector drug product and remove empty capsids. These methods are described in more detail in International Patent Application No. PCT / US2016 / 065970, entitled "Scalable Purification Method for AAV9," filed on December 9, 2016, which is incorporated herein by reference. The purification methods for AAV8 (International Patent Application No. PCT / US2016 / 065976 filed on December 9, 2016), and rh10 (International Patent Application No. PCT / US16 / 066013 filed on December 11, 2015, entitled "Scalable Purification Method for AAVrh10" filed on December 9, 2016), and AAV1 (International Patent Application No. PCT / US2016 / 065974 filed on December 9, 2016, and "Scalable Purification Method for AAV1" filed on December 11, 2015) are all incorporated herein by reference.

[0110] To calculate the content of empty and full particles, the vp3 band volume of the selected sample (e.g., in the examples herein, the preparation of iodixanol gradient purification, in which the number of GC = the number of particles) is plotted relative to the loaded GC particles. The resulting linear equation (y = mx + c) is used to calculate the number of particles in the band volume of the test product peak. The number of particles per 20 μL loaded (pt) is then multiplied by 50 to obtain particles (pt) / mL. Pt / mL is divided by GC / mL to obtain the ratio of particles to genome copies (pt / GC). Pt / mL-GC / mL obtains empty pt / mL. Empty pt / mL is divided by pt / mL and x 100 to obtain the percentage of empty particles.

[0111] In general, methods for determining empty capsids and AAV vector particles with packaged genomes are known in the art. See, for example, Grimm et al., Gene Therapy (1999) 6: 1322-1330; and Sommer et al., Molecular Therapy (2003) 7: 122-128. To test denatured capsids, the method comprises subjecting the treated AAV raw material to SDS-polyacrylamide gel electrophoresis (composed of any gel capable of separating the three capsid proteins, such as a gradient gel containing 3-8% triacetate in a buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). Then, an anti-AAV capsid antibody is used as a primary antibody that binds to the denatured capsid protein, preferably an anti-AAV capsid monoclonal antibody, most preferably a B1 anti-AAV-2 monoclonal antibody (Wobus et al., Journal of Virology (2000) 74: 9281-9293). Then use a secondary antibody, which is combined with the primary antibody and contains a device for detecting the combination with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting the combination is used to semi-quantitatively determine the combination between the primary antibody and the secondary antibody, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation or colorimetric changes, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, samples can be extracted from column fractions and heated in an SDS-PAGE loading buffer containing a reducing agent (e.g., DTT), and capsid proteins are resolved on a prefabricated gradient polyacrylamide gel (e.g., Novex). Silver staining can be performed using SilverXpress (Invitrogen, CA) or other suitable staining methods (i.e., SYPRO ruby ​​or Coomassie staining) according to the manufacturer's instructions. In one embodiment, the concentration of the AAV vector genome (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The sample is diluted and digested with DNase I (or another suitable nuclease) to remove extraneous DNA. After nuclease inactivation, primers and TaqMan RNA specific for the DNA sequence between the primers are used. TMThe fluorescent probe is further diluted and amplified. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level is measured on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing the same sequence as that contained in the AAV vector is used to generate a standard curve in a Q-PCR reaction. The value of the cycle threshold (Ct) obtained from the sample is used to determine the vector genome titer by normalizing it relative to the Ct value of the plasmid standard curve. Endpoint determination based on digital PCR can also be used.

[0112] In addition, another example of measuring the ratio of empty particles to full particles is known in the art. Sedimentation velocity as measured in an analytical ultracentrifuge (AUC) can detect aggregates and other minor components, as well as provide good quantification of the relative amounts of different particle types based on different sedimentation coefficients. This is an absolute method based on basic units of length and time, and does not require standard molecules as references. The carrier sample is loaded into a cell with a 2-channel charcoal-epoxy centerpiece with a 12 mm optical path length. The provided dilution buffer is loaded into the reference channel of each cell. The loaded cell is then placed into an AN-60Ti analytical rotor and loaded into a Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge equipped with both absorbance and RI detectors. After complete temperature equilibration at 20°C, the rotor is brought to a final operating speed of 12,000 rpm. Record the A value approximately every 3 minutes. 280 Scanning was performed for approximately 5.5 hours (a total of 110 scans per sample). The raw data was analyzed using the c(s) method and implemented in the analysis program SEDFIT. The resulting size distribution was plotted and the peaks were integrated. The percentage value associated with each peak represents the peak area fraction of the total area under all peaks and is based on the raw data generated at 280 nm; many laboratories use these values ​​to calculate the empty particle: full particle ratio. However, since the extinction coefficients of empty particles and full particles are different at this wavelength, the raw data can be adjusted accordingly. The ratio of the empty particle to full monomer peak before and after the extinction coefficient adjustment is used to determine the empty particle-full particle ratio.

[0113] In one aspect, an optimized q-PCR method is used, which utilizes a broad spectrum of serine proteases, such as proteinase K (such as commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay is similar to the standard assay, except that after DNase I digestion, the sample is diluted with proteinase K buffer and treated with proteinase K, and then heat inactivated. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the sample size. The proteinase K buffer can be concentrated 2 times or more. Typically, proteinase K treatment is about 0.2 mg / mL, but can vary between 0.1 mg / mL and about 1 mg / mL. The treatment step is usually performed at about 55°C for about 15 minutes, but can be performed at a lower temperature (e.g., about 37°C to about 50°C) for a longer period of time (e.g., about 20 minutes to about 30 minutes) or at a higher temperature (e.g., up to about 60°C) for a shorter period of time (e.g., about 5 minutes to 10 minutes). Similarly, heat inactivation is usually carried out at about 95°C for about 15 minutes, but the temperature can be lowered (e.g., from about 70°C to about 90°C) and the time can be extended (e.g., from about 20 minutes to about 30 minutes). The sample is then diluted (e.g., 1000 times) and subjected to TaqMan analysis as described in standard assays. Quantification can also be performed using ViroCyt or flow cytometry.

[0114] Additionally or alternatively, droplet digital PCR (ddPCR) can be used. For example, methods for determining the titer of single-stranded and self-complementary AAV vector genomes by ddPCR have been described. See, for example, M. Lock et al., Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 April; 25(2): 115-25. doi: 10.1089 / hgtb.2013.131. Epub 2014 Feb 14.

[0115] Pharmaceutical composition

[0116] Provided herein are compositions containing at least one rAAV or rAAV material and optionally a carrier, excipient, and / or preservative.

[0117] In certain embodiments, the composition may contain at least a second different rAAV or rAAV stock. This second vector or vector stock may differ from the first vector by having a different AAV capsid and / or a different vector genome. In certain embodiments, the compositions described herein may contain a different vector expressing an expression cassette described herein or another active component (e.g., an antibody construct, another biologic and / or a small molecule drug).

[0118] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition.

[0119] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present invention into suitable host cells. Specifically, the transgene delivered by the rAAV vector can be formulated for delivery or encapsulated in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc.

[0120] In one embodiment, the composition comprises a final formulation suitable for delivery to a subject, for example, an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration. Suitably, the final formulation is adjusted to a physiologically acceptable pH, for example, the pH can be in the range of pH 6 to 9, or pH 6.5 to 8.5, or pH 7 to 7.8. Since the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; and for intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pHs within the widest range and these sub-ranges may be selected for other delivery routes. Optionally, one or more surfactants are present in the formulation. In another embodiment, the composition can be transported in the form of a concentrate, which is diluted to be applied to a subject. In other embodiments, the composition can be lyophilized and reconstituted when applied.

[0121] A suitable surfactant or combination of surfactants can be selected from non-toxic nonionic surfactants. In one embodiment, a difunctional block copolymer surfactant terminated at a primary hydroxyl group is selected, such as F68 [BASF], which is also known as Poloxamer 188, has a neutral pH and an average molecular weight of 8400. Other surfactants and other poloxamers can be selected, namely a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (polyethylene glycol-15 hydroxystearate), LABRASOL (polyoxycaprylyl glyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamer), followed by three numbers: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage of the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

[0122] In certain embodiments, the formulation buffer is phosphate buffered saline (PBS) with a total salt concentration of 200 mM, 0.001% (w / v) pluronic F68 (final formulation buffer, FFB).

[0123] In certain embodiments, the composition includes a viral vector (i.e., a rAAV vector). The vector is administered in sufficient amounts to transfect cells and provide sufficient levels of gene transfer and expression to provide therapeutic benefits without excessive side effects or with medically acceptable physiological effects, which can be determined by a technician in the medical field. In certain embodiments, the vector is formulated for delivery by an intranasal delivery device. In certain embodiments, the vector is formulated for aerosol delivery devices, for example, by a nebulizer or by other suitable devices. In certain embodiments, the vector is formulated for intrathecal delivery. In certain embodiments, intrathecal delivery encompasses injection into the spinal canal (e.g., subarachnoid space). In certain embodiments, other delivery routes can be selected, for example, other suitable direct or systemic routes such as intracranial, intranasal, intracisternal, intracerebrospinal fluid delivery, i.e., Ommaya reservoir. In certain embodiments, the vector is formulated for intravenous delivery. Other conventional and pharmaceutically acceptable administration routes include, but are not limited to, direct delivery to desired organs (e.g., lungs), oral inhalation, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. In one embodiment, the carrier is administered using an intranasal atomization device ( MAD Nasal TM -MAD110) is administered intranasally. In another embodiment, the carrier is administered intranasally using a vibrating mesh nebulizer ( Solo) or MADgic TM The laryngeal mucosal nebulizer is administered in the lung in aerosolized form. If desired, the administration route can be combined. The administration and use routes for delivering rAAV vectors are also described in the following disclosed U.S. patent applications, each of which is incorporated herein by reference in its entirety: US2018 / 0155412A1, US 2018 / 0243416A1, US2014 / 0031418 A1, and US2019 / 0216841A1.

[0124] The dosage of the viral vector will depend primarily on factors such as the condition being treated, the patient's age, weight, and health, and therefore may vary between patients. For example, a therapeutically effective human dose of a viral vector is typically in the range of about 25 microliters to about 1000 microliters to about 5 mL of an aqueous suspension containing about 10 9 Up to 4x10 14 The dosage of the AAV vector of GC. The dosage will be adjusted to balance the therapeutic benefit with any side effects, and such dosage can vary depending on the therapeutic application using the recombinant vector. The expression level of the transgene can be monitored to determine the dosage frequency of the resulting viral vector (preferably an AAV vector containing a minigene). Optionally, a dosage regimen similar to that described for therapeutic purposes can be used for immunization using the composition of the present invention.

[0125] The replication-defective virus composition can be formulated in dosage units to contain about 10 9 Gc is about 10 16 GC of replication-defective virus (to treat an average subject with a body weight of 70 kg), said amount including all integer or fractional amounts within said range, and for human patients, preferably 10 12 Gc to 10 14 GC. In one embodiment, the composition is formulated to contain at least 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x10 9 , 7x 10 9 , 8x 10 9 or 9x10 9 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 10 , 2x 10 10 , 3x 10 10 , 4x 10 10 , 5x 1010 , 6x 10 10 , 7x 10 10 , 8x 10 10 or 9x 10 10 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 11 , 2x 10 11 , 3x 10 11 , 4x 10 11 , 5x10 11 , 6x 10 11 , 7x 10 11、 8x 10 11 or 9x 10 11 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 12 , 2x10 12 , 3x 10 12 , 4x 10 12 , 5x 10 12 , 6x 10 12 , 7x 10 12 , 8x 10 12 or 9x 10 12 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 13 , 2x 10 13 , 3x10 13 , 4x10 13 , 5x 10 13 , 6x 10 13 , 7x 10 13 , 8x 10 13 or 9x 10 13 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 14 , 2x 10 14 , 3x 10 14 , 4x10 14 , 5x 10 14 , 6x 10 14 , 7x 10 14 , 8x 10 14 or 9x 10 14 GC, including all integers or fractional amounts within the range. In another embodiment, the composition is formulated to contain at least 10 15 , 2x 10 15, 3x10 15 , 4x 10 15 , 5x10 15 , 6x 10 15 , 7x 10 15 , 8x 10 15 or 9x 10 15 GC, including all integers and fractions within the range. In one embodiment, for human use, the dosage range may be 10 10 to about 10 12 GC, including all integers and fractions within the range. In one embodiment, for human use, the dosage range may be 10 9 About 7x10 13 GC, including all integers and fractions within the range. In one embodiment, for human use, the dosage is 6.25 x 10 12 GC to 5.00x 10 13 In another embodiment, the dosage is about 6.25 x 10 12 GC, about 1.25x 10 13 GC, about 2.50x 10 13 GC or about 5.00x 10 13 GC. In certain embodiments, the dose is divided equally in half and applied to each nostril. In certain embodiments, for human use, the dose is between 6.25 x 10 12 GC to 5.00x10 13 The GC range was administered in two aliquots of 0.2 ml in each nostril, delivering a total volume of 0.8 ml in each subject.

[0126] These above-mentioned doses can be administered with various volumes of carrier, excipient or buffer formulations, ranging from about 25 to about 1000 microliters or more, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of the carrier, excipient or buffer is at least about 25 μL. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 75 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 225 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 275 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 325 μL. In another embodiment, the volume is about 350 μL. In another embodiment, the volume is about 375 μL. In another embodiment, the volume is about 400 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 550 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 650 μL. In another embodiment, the volume is about 700 μL. In another embodiment, the volume is between about 700 μL and about 1000 μL.

[0127] In one embodiment, the viral construct can be expressed in at least about 1 x 10 9 GC to about 1x 10 15 or about 1x10 11 Up to 5x 10 13Dose delivery of GC. Suitable volumes for delivering these dosages and concentrations can be determined by those skilled in the art. For example, a volume of about 1 μ L to 150 mL can be selected, wherein for adults, a larger volume is selected. Typically, for newborn infants, a suitable volume is about 0.5 mL to about 10 mL, and for older infants, about 0.5 mL to about 15 mL can be selected. For young children, a volume of about 0.5 mL to about 20 mL can be selected. For children, a volume of about 30 mL at most can be selected. For prepubertal teenagers and adolescents, a volume of about 50 mL at most can be selected. In still other embodiments, the patient can accept intrathecal administration of a volume selected as about 5 mL to about 15 mL or about 7.5 mL to about 10 mL. Other suitable volumes and dosages can be determined. The dosage will be adjusted to balance the therapeutic benefit with any side effects, and such dosages can vary according to the therapeutic application using a recombinant vector.

[0128] The composition according to the invention may include a pharmaceutically acceptable carrier, as defined above. Suitably, the composition described herein includes an effective amount of one or more AAVs suspended in a pharmaceutically suitable carrier and / or mixed with a suitable excipient designed to be delivered to a subject by injection, an osmotic pump, an intrathecal catheter, or delivered by another device or route. In one example, the composition is formulated for intrathecal delivery. The effective amount can be determined based on an animal model rather than a human patient.

[0129] As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to an administration route that is injected into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery can include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital / cistern and / or C1-2 puncture. For example, the material can be introduced by lumbar puncture to diffuse throughout the subarachnoid space. In another example, it can be injected into the cerebellomedullary cistern (i.e., in the cerebellomedullary cistern or ICM). In certain embodiments, intrathecal administration is performed as described in U.S. Patent Publication No. 2018-0339065A1, published on November 29, 2019, which is incorporated herein by reference in its entirety. In certain embodiments, CNS administration is performed using Ommaya reservoirs (also referred to as Ommaya devices or Ommaya systems).

[0130] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to administration of a drug directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically by suboccipital puncture or by injection directly into the cisterna magna or by a permanently placed cannula.

[0131] In one embodiment, a frozen composition is provided, which contains rAAV in a buffer solution as described herein in frozen form. Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers, or preservatives are present in the composition. Suitably, for use, the composition is thawed and titrated to the desired dose with a suitable diluent (e.g., sterile saline or buffered saline).

[0132] In one embodiment, the compositions described herein are used in the preparation of a medicament for treating a cardiac disorder or disease.

[0133] Methods and uses

[0134] In one aspect, provided herein are methods for treating a human subject diagnosed with a cardiac disease (eg, cardiomyopathy).

[0135] The method comprises administering to the subject a suspension of a vector or rAAV as described herein. In one embodiment, the method comprises administering to the subject a dose of about 1 x 10 9 Genome copies (GC) / kg is about 1×10 14 GC / kg of a suspension of rAAV as described herein in a formulation buffer. In another embodiment, rAAV is prepared at 3×10 13 GC / kg preparation.

[0136] The methods and compositions described herein can be used to treat cardiomyopathy at any stage. In certain embodiments, the patient is an infant, a toddler, or the patient is 3 to 6 years old, 3 to 12 years old, 3 to 18 years old, 3 to 20 years old. In certain embodiments, the patient is over 18 years old. In certain embodiments, the patient is about 20 to 60 years old. In certain embodiments, the patient is about 40 to 50 years old. In certain embodiments, the patient is over 60 years old.

[0137] In certain embodiments, methods and compositions can be used to treat mitochondrial cardiomyopathy associated with Barth syndrome. Barth syndrome is a rare X-linked recessive disease characterized by loss-of-function mutations in the TAZ gene (i.e., regulatable gene therapy). Barth syndrome is associated with pediatric paroxysmal cardiomyopathy (i.e., before the age of 5) with neutropenia, mild mitochondrial myopathy (skeletal muscle weakness), and mild intellectual disability. See also Sabbah, HN, Barth syndrome cardiomyopathy: targeting the mitochondria with elamipretide, Heart Failure Reviews (2021) 26: 237–253, which is incorporated herein by reference in its entirety.

[0138] In certain embodiments, the methods and compositions can be used to treat autosomal dominant forms of long QT syndrome (i.e., suitable for gene replacement or knockdown / alternative methods) caused by loss of function and partial dominant negative mutations of the KCNQ1 gene. Autosomal dominant forms of long QT syndrome are associated with syncope and sudden cardiac death that usually occur during exercise or emotional stress, and despite standard care (β-blockers, cardiac sympathectomy), many patients are still at risk and require an implantable cardioverter defibrillator (ICD). See also Huang H. et al., Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations, Sci. Adv. 2018, 4: 1-12, electronic version March 7, 2018, the document is incorporated herein by reference in its entirety.

[0139] In certain embodiments, the methods and compositions may be used to treat hypertrophic cardiomyopathy. In certain embodiments, the methods and compositions may be used to treat hypertrophic cardiomyopathy caused by loss-of-function mutations in the MYBPC3 gene (i.e., adjustable gene therapy). See also Mearini G. et al., Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice, Nature Communication, 2014, 5: 5515, electronic version December 2, 2014, the document is incorporated herein by reference in its entirety.

[0140] In certain embodiments, methods and compositions can be used to treat Long WT syndrome type 2 caused by loss-of-function mutations in the hERG (Kv11.1; also, Kv11.1 voltage-gated potassium channel) gene. See also Curran ME. et al., A Molecular Basis for Cardiac Arrhythmia: HERG Mutations Cause Long QT Syndrome, Cell, Voi. 80, 795-803, Mar. 10, 1995 and Hylten-Cavallius, L. et al., Patients With Long-QT Syndrome Caused by Impaired hERG-Encoded Kv11.1 Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated With Reactive Hypoglycemia, Circulation, 2017; 135: 1705–1719, which are incorporated herein by reference in their entirety.

[0141] In certain embodiments, the methods and compositions can be used to treat LMNA cardiomyopathy or diseases caused by loss-of-function mutations in the LMNA gene. See also Kang, S. et al., Laminopathies; Mutations on single gene and various human genetic diseases, BMB Rep. 2018; 51(7):327-337, which is incorporated herein by reference in its entirety.

[0142] In certain embodiments, the methods and compositions can be used to treat heart failure, ischemia-reperfusion injury, myocardial infarction, ventricular remodeling, or diseases associated with extracellular superoxide dismutase 3 (SOD3 or EcSOD). See also U.S. Patent Application Publication No. US20130136729A1, which is incorporated herein by reference in its entirety.

[0143] In certain embodiments, the methods and compositions can be used to treat myocardial infarction, reduced cardiac ejection fraction, or diseases associated with myc transcription factor, cyclin T1, and cyclin-dependent kinase 9 (CDK9). See also International Patent Application Publication No. WO2020 / 165603A1, which is incorporated herein by reference in its entirety.

[0144] In certain embodiments, the methods and compositions can be used to treat heart failure, or cardiac tissue damage, or degeneration, or diseases associated with cyclin A2 protein. See also International Patent Application Publication No. WO2020 / 051296A1, which is incorporated herein by reference in its entirety.

[0145] In certain embodiments, the methods and compositions can be used to treat dilated cardiomyopathy (DCM), heart failure, cardiac fibrosis, cardiac inflammation, ischemic heart disease, myocardial infarction, ischemic / reperfusion (I / R)-related injury, transverse aortic stenosis, or diseases associated with YY1 or BMP7 proteins. See also International Patent Application Publication No. WO2021 / 021021A1, which is incorporated herein by reference in its entirety.

[0146] In certain embodiments, the methods and compositions can be used to treat dilated cardiomyopathy or diseases associated with a cardiac apoptosis inhibitor with a caspase recruitment domain (cARC). See also International Patent Application Publication No. WO2021 / 016126A1, which is incorporated herein by reference in its entirety.

[0147] Symptoms of cardiomyopathy or diseases associated with mutations in the LMNA gene, KCNQ1 gene, MYBPC3 gene, TAZ gene, or hERG gene include atrioventricular (AV) block, atrial fibrillation, arrhythmias, including atrial arrhythmias (such as atrial flutter and atrial tachycardia) and ventricular arrhythmias (including sustained ventricular tachycardia and ventricular fibrillation (VF) and / or heart failure.

[0148] In certain embodiments, the methods and compositions described herein can be used to alleviate one or more symptoms of cardiomyopathy, including increased life expectancy and / or reduced progression of heart failure.

[0149] In certain embodiments, rAAV comprises a vector genome including a transgene encoding a therapeutic protein for treating cardiomyopathy and its symptoms, such as potassium voltage-gated channel subfamily Q member 1 protein (KCNQ1 gene), cardiac myosin binding protein C (MYBPC3 gene), tafazzin (TAZ), Kv11.1 voltage-gated potassium channel protein (hERG gene), and lamin A (LMNA gene).

[0150] In certain embodiments, co-therapy or co-treatment can be utilized, which includes co-administration with another active agent. In certain embodiments, co-therapy may further include administration of beta blockers, angiotensin converting enzyme (ACE) inhibitors, diuretics. The diuretic used may be acetazolamide (Diamox) or other suitable diuretics. In certain embodiments, diuretics are administered when gene therapy is administered. In certain embodiments, diuretics are administered before gene therapy is administered. In certain embodiments, diuretics are administered when the injection volume is 3mL. In certain embodiments, co-treatment may further include implantable cardioverter defibrillator (ICD), pacemaker (PM) and / or cardiac resynchronization therapy (CRT).

[0151] Optionally, immunosuppressive co-therapy can be used for subjects in need thereof. Immunosuppressants used for such co-therapy include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins. Immunosuppressants may include nitrogen mustard, nitrosourea, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, mithramycin, IL-2 receptor (CD25) or CD3 directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, opioids or TNF-α (tumor necrosis factor-α) binding agents. In certain embodiments, immunosuppressive therapy may be initiated on day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, or more before or after administration of gene therapy. Such immunosuppressive therapy can involve the administration of one, two or more drugs (e.g., glucocorticoids, prednisone (prednelisone), mycophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)). Such immunosuppressive drugs can be administered once, twice or more times to a subject in need with the same dosage or adjusted dosage. Such therapy can involve the co-administration of two or more drugs (e.g., prednisone, mycophenolate mofetil (MMF) and / or sirolimus (i.e., rapamycin)) on the same day. One or more of these drugs can continue with the same dosage or adjusted dosage after gene therapy is administered. As required, such therapy can last for about 1 week (7 days), about 60 days or longer. In certain embodiments, a scheme without tacrolimus is selected.

[0152] In one embodiment, a rAAV as described herein is administered once to a subject in need thereof. In one embodiment, a rAAV is administered more than once to a subject in need thereof.

[0153] As used herein, "patient" or "subject" refers interchangeably to male or female mammals, including humans, veterinary or farm animals, livestock or pets, and animals typically used for research. In one embodiment, the subject of these methods and compositions is a human patient. In one embodiment, the subject of these methods and compositions is a male or female human patient.

[0154] As used herein, the term "heterogeneous" or any grammatical variation thereof, when used to refer to vp capsid proteins, refers to a population consisting of non-identical elements (e.g., having vp1, vp2, or vp3 monomers (proteins) with different modified amino acid sequences). SEQ ID NO: 10 provides the encoded amino acid sequence of the AAV9 vp1 protein. SEQ ID NO: 11 provides the encoded amino acid sequence of the AAVhu68 vp1 protein. The term "heterogeneous" used in conjunction with the vp1, vp2, and vp3 proteins (alternatively referred to as isoforms) refers to differences in the amino acid sequences of the vp1, vp2, and vp3 proteins within the capsid. The AAV capsid contains subpopulations within the vp1 protein, within the vp2 protein, and within the vp3 protein that have modifications from predicted amino acid residues. These subpopulations contain at least some deamidated asparagine (N or Asn) residues. For example, certain subsets include at least one, two, three, or four highly deamidated asparagine (N) positions in asparagine-glycine (NG) pairs and optionally further include other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.

[0155] As used herein, unless otherwise specified, a "subgroup" of vp proteins refers to a group of vp proteins having at least one defined common property and consisting of at least one group member to less than all members of a reference group. For example, unless otherwise specified, a "subgroup" of vp1 proteins is at least one (1) vp1 protein and less than all vp1 proteins in an assembled AAV capsid. Unless otherwise specified, a "subgroup" of vp3 proteins can be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid. For example, vp1 proteins can be a subgroup of vp proteins; vp2 proteins can be a separate subgroup of vp proteins, and vp3 is still another subgroup of vp proteins in an assembled AAV capsid. In another example, vp1 proteins, vp2 proteins, and vp3 proteins can contain subgroups with different modifications, for example, at least one, two, three, or four highly deamidated asparagines, for example at asparagine-glycine pairs. Unless otherwise indicated, highly deamidated refers to at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidation at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position. Such percentages can be determined using 2D gels, mass spectrometry techniques or other suitable techniques.

[0156] As used herein, the "raw material" of rAAV refers to a population of rAAV produced by the same capsid coding sequence in an AAV production system. Various production systems can be selected, including but not limited to the production systems described herein. The rAAV raw material may comprise a heterogeneous population of rAAV, wherein the rAAV has been packaged in an AAV capsid expressed by a single AAV capsid coding sequence, but comprises a heterogeneous subpopulation of rAAV with a capsid having a deamidation pattern specific to the AAV type and production system. See, for example, WO 2019 / 168961, published on September 6, 2019, comprising Table G, which provides a deamidation pattern of AAV9; and WO 2020 / 160582, filed on September 7, 2018. See also, for example, WO2020 / 223231 (rh91, containing a table with a deamidation pattern) published on November 5, 2020, U.S. Provisional Patent Application No. 63 / 065,616 filed on August 14, 2020, and U.S. Provisional Patent Application No. 63 / 109734 filed on November 4, 2020.

[0157] The compositions described herein can be used in regimens involving co-administration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein can also be administered by one of these routes.

[0158] The abbreviation "sc" refers to self-complementary. "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, instead of waiting for the cell-mediated second strand synthesis, the two complementary halves of scAAV will associate to form a double-stranded DNA (dsDNA) ready for immediate replication and transcription. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", "Gene Therapy", (August 2001), Vol. 8, No. 16, pp. 1248-1254. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0159] As used herein, the term "operably linked" refers to both expression control sequences that are contiguous to a gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

[0160] The term "heterologous" when used in reference to a protein or nucleic acid indicates that the protein or nucleic acid includes two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid. For example, in one embodiment, a nucleic acid has a promoter from one gene arranged to direct expression of a coding sequence from a different gene. Thus, the promoter is heterologous with respect to the coding sequence.

[0161] "Replication-defective virus" or "viral vector" refers to a synthetic or artificial virus particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial virus particle cannot produce progeny virus but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless"-containing only transgenes of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. Therefore, this is considered safe for gene therapy because replication and infection by progeny virus particles will not occur unless the viral enzymes required for replication are present.

[0162] "Recombinant AAV" or "rAAV" is a DNase-resistant viral particle containing two elements, namely the AAV capsid and a vector genome containing at least non-AAV coding sequences packaged within the AAV capsid. In certain embodiments, the capsid contains about 60 proteins composed of vp1 protein, vp2 protein, and vp3 protein that self-assemble to form the capsid. Unless otherwise indicated, "recombinant AAV" or "rAAV" can be used interchangeably with the phrase "rAAV vector". rAAV is a "replication-defective virus" or "viral vector" because it lacks any functional AAV rep gene or functional AAV cap gene and cannot produce offspring. In certain embodiments, the only AAV sequence is the AAV inverted terminal repeat sequence (ITR), which is usually located at the 5' and 3' extreme ends of the vector genome to allow genes and regulatory sequences located between the ITRs to be packaged within the AAV capsid.

[0163] The term "nuclease-resistant" indicates that the AAV capsid has been assembled around expression cassettes designed to deliver transgenes to host cells and protect these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids that may be present during the production process.

[0164] As used herein, the term "host cell" may refer to a packaging cell line that produces rAAV and contains a plasmid comprising a nucleotide sequence encoding an AAV capsid as described herein. In an alternative, the term "host cell" may refer to a target cell in which transgene expression is desired.

[0165] As used herein, "cardiomyocytes" refers to myocardial tissue cells, including but not limited to heart cells, cardiac muscle cells (cardiomyocytes), conduction cells, fibroblasts, endothelial cells, smooth muscle cells and perivascular cells.

[0166] As used herein, "vector genome" refers to the nucleic acid sequence packaged inside the rAAV capsid forming a viral particle. Such nucleic acid sequences contain AAV reverse terminal repeats (ITRs). In the examples herein, the vector genome contains at least AAV 5'ITR, coding sequence and AAV 3'ITR from 5' to 3'. In certain embodiments, the ITR from AAV2 (AAV different from the source of the capsid) or the ITR in addition to the full-length ITR can be selected. In still other embodiments, longer or shorter AAV ITR can be selected. In certain embodiments, ITR comes from the AAV source identical to the AAV or trans-complementary AAV providing rep function during production. Further, other ITRs can be used. Further, the vector genome contains the regulatory sequence for the expression of the guiding gene product. The suitable components of the vector genome are discussed in more detail herein. The vector genome is sometimes referred to as "minigene" in this article.

[0167] In certain embodiments, the non-viral genetic elements used to prepare rAAV will be referred to as vectors (e.g., production vectors). In certain embodiments, these vectors are plasmids, but it is contemplated that other suitable genetic elements are used. This production plasmid can encode sequences expressed during rAAV production, for example, AAV capsids or rep proteins required for producing rAAV that are not packaged into rAAV. Alternatively, this production plasmid can carry a vector genome that is packaged into rAAV.

[0168] As used herein, "parental capsid" refers to a non-mutated or non-modified rAAV capsid. In certain embodiments, the parental capsid comprises any naturally occurring AAV capsid, the naturally occurring AAV capsid comprising a wild-type genome encoding a capsid protein (i.e., vp protein), wherein the capsid protein directs AAV transduction and / or tissue-specific tropism.

[0169] As used herein, the terms "target cell" and "target tissue" may refer to any cell or tissue that is intended to be transduced by a subject's AAV vector. The terms may refer to any one or more of muscle, liver, lung, airway epithelium, central nervous system, neurons, eye (ocular cells), or heart. In one embodiment, the target tissue is the heart.

[0170] As used herein, "variant capsid" or "variant AAV" or "variant AAV capsid" refers to a rAAV or capsid protein that is modified or mutated, for example, to include substitutions at positions 446, 470, and / or 503 or to include insertion of a tissue-specific targeting peptide. The parent capsid may in some cases include a capsid protein that is further modified to include substitutions at positions 446, 470, and / or 503 as described herein.

[0171] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a biologically useful nucleic acid sequence (e.g., a gene cDNA, mRNA, etc. encoding a protein, enzyme, or other useful gene product) and a regulatory sequence operably connected thereto, the regulatory sequence guiding or regulating the transcription, translation, and / or expression of the nucleic acid sequence and its gene product. Such regulatory sequences typically include, for example, one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain a regulatory sequence upstream (5') of the gene sequence, such as one or more of a promoter, an enhancer, an intron, etc., and an enhancer, or one or more of a regulatory sequence downstream (3') of the gene sequence, such as a 3' untranslated region (3'UTR) including a polyadenylation site, and other elements. In certain embodiments, the regulatory sequence is operably connected to the nucleic acid sequence of the gene product, wherein the regulatory sequence is separated from the nucleic acid sequence of the gene product by an intermediate nucleic acid sequence, i.e., a 5' untranslated region (5'UTR). In certain embodiments, expression cassettes include the nucleic acid sequence of one or more gene products. In certain embodiments, expression cassettes can be monocistronic expression cassettes or bicistronic expression cassettes. In other embodiments, term "transgenic" refers to one or more DNA sequences from an exogenous source inserted into a target cell. Typically, such expression cassettes can be used to produce viral vectors, and contain coding sequences and other expression control sequences of packaging signals of viral genomes flanked by gene products described herein, such as sequences described herein. In certain embodiments, vector genomes can contain two or more expression cassettes.

[0172] In the context of the present invention, the term "translation" relates to the ribosome process in which an mRNA chain controls the assembly of an amino acid sequence to produce a protein or peptide.

[0173] The term "expression" is used herein in its broadest sense and includes the production of RNA or RNA and protein. Expression may be transient or may be stable.

[0174] When referring to nucleic acids or fragments thereof, the term "substantial homology" or "substantial similarity" means that at least about 95% to 99% of the compared sequences have nucleotide sequence identity when optimally aligned with appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand). Preferably, the homology is over the full-length sequence, or its open reading frame, or another suitable fragment of at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0175] In the context of nucleic acid sequences, the terms "sequence identity", "percentage of sequence identity" or "identical percentage" refer to residues in two sequences that are identical when aligned to obtain maximum correspondence. The length of the expected sequence identity comparison may exceed the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides. However, the identity between smaller fragments may also be expected, for example, at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides. Similarly, for amino acid sequences, "percentage of sequence identity" may be easily determined over the full length of the protein or its fragment. Suitably, the length of the fragment is at least about 8 amino acids, and may be up to about 700 amino acids. Examples of suitable fragments are described herein.

[0176] The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. Identity is readily determined by those skilled in the art by using algorithms and computer programs known to those skilled in the art.

[0177] Typically, when referring to "identity", "homology" or "similarity" between two different adeno-associated viruses, "identity", "homology" or "similarity" is determined with reference to the "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Examples of such programs include "ClustalΩ", "Clustal W", "CAP Sequence Assembly", "MAP" and "MEME", which are accessible through Web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the vector NTI utility program is also used. There are many algorithms known in the art that can be used to measure nucleotide sequence identity, including the algorithms contained in the above-mentioned programs. As another example, the GCG version 6.1 program Fasta can be used. TM Compare polynucleotide sequences. Fasta TM The alignment and percent sequence identity of the best overlap region between the query sequence and the search sequence are provided. For example, percent sequence identity between nucleic acid sequences can be calculated using FastAna as provided in GCG Version 6.1 using its default parameters (font size 6 and NOPAM coefficients for the scoring matrix). TMto determine, the program is incorporated herein by reference. Multiple sequence alignment programs can also be used for amino acid sequences, such as "ClustalΩ", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used with the default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use another algorithm or computer program that provides at least the same identity or alignment level as provided by the reference algorithm and program. See, for example, JD Thomson et al., "A comprehensive comparison of multiple sequence alignments", 27 (13): 2682-2690 (1999).

[0178] As described above, unless otherwise stated, the term "about" when used to modify a numerical value means a variation of ±10%.

[0179] As used throughout this specification and claims, the terms "comprise" and "contain" and variations thereof, including other variations such as "comprises," "comprising," "contains," and "containing," include other components, elements, integers, steps, etc. The terms "consists of" or "consisting of" do not include other components, elements, integers, steps, etc.

[0180] It should be noted that the term "a" or "an" refers to one or more, for example, "an enhancer" should be understood to mean one or more enhancers. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0181] With respect to the description of these inventions, in another embodiment, it is contemplated that each of the compositions described herein is useful in the methods of the invention. In addition, it is also contemplated that in another embodiment, each of the compositions described herein as being useful in the methods is itself an embodiment of the invention.

[0182] Unless otherwise defined in this specification, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs and with reference to the public text, which provides a general guide for many of the terms used in this application to those skilled in the art.

[0183] Examples

[0184] The following examples are provided to illustrate various embodiments of the present invention. The examples are not intended to limit the present invention in any way.

[0185] Example 1: AAV9 capsid variants

[0186] The gold standard vector for intravenous AAV gene therapy is AAV9. The group described for the first time the major cellular receptor for AAV9, the cell surface glycan galactose. They were further able to identify an amino acid galactose binding pocket on the surface of the AAV9 capsid that contains residues D271, N272, Y446, N470, and W503 of the AAV9 VP1 protein. Alanine substitutions at any of these residues largely abolished entry and expression of the vector transgene in in vitro transduction or target cells. It has been shown that in vitro transduction defects translate into in vivo detargeting from murine liver. The findings suggest that both liver detargeting and peripheral transduction depend on factors within the combinatorial space of the AAV9 galactose binding site. Here, the identification of a novel AAV vector with improved transduction of target tissues is described.

[0187] Round 1: Directed evolution and the galactose binding pocket

[0188] The directed evolution of AAV vectors involves the generation of vector libraries. The vector library is generated by a plasmid library containing an array of AAVVP1 variants flanked by AAV ITR. Using this plasmid library for vector generation, a unique AAV vector pool is generated, each of which carries its unique VP1 coding sequence as a deliverable transgene. This vector is then used for transduction experiments; any cell transduced by the library vector expresses the unique VP1 protein of the variant. This expression is captured by next generation sequencing (NGS) after RNA extraction from the compartment of interest. In vivo injection of one of these libraries thus generates a data set enriched in organ-specific transduction patterns because it is associated with changes in the vector library. Vector variants with desired properties are identified from these data sets.

[0189] A directed evolution library approach was applied to the analysis of the galactose binding pocket of AAV9. A library of AAV vectors containing variations at three amino acids (i.e., positions 446, 470, and 503) of the binding pocket was created. Figure 1A and Figure 1B). Every combination of amino acid substitutions at each position was included in the library, resulting in a library containing approximately 7000 variants. One of these variants was a WT AAV9 vector, which served as an internal control. This library was then screened in C57Bl / 6J mice (n=4, 1e12 GC / mouse, IV injection, autopsy on day 14), and vector expression in liver, heart, muscle, brain, spleen, and kidney tissues was analyzed by NGS. The input production plasmid library and the resulting vector library were also sequenced to allow further performance indicators to be derived.

[0190] From the resulting dataset, the yield, liver enrichment, and heart enrichment of each vector were determined ( Figure 2 , Figure 3 and Figure 4 ). Values ​​were normalized relative to an AAV9 control to create a standard against which the data was filtered and variants of interest were selected. The first and most effective filter applied to these results was the yield filter. For each variant, a yield score was determined by dividing the number of NGS reads in the plasmid library by the number of reads in the vector library. This ratio was then normalized relative to AAV9, and only vectors with an AAV9-adjusted yield score >0.25 (yield greater than 25% AAV9) were considered for further analysis ( Figure 5 ). This filter removes potentially low-yield variants whose NGS reads, and thus performance metrics, may be more susceptible to noise in the sequencing data. The yield filter removed the majority of variants from the library, trimming the library from approximately 7,000 variants to approximately 200. The next performance metric used to evaluate variants was heart and liver enrichment. These metrics are calculated by dividing the NGS reads in a specific tissue by the reads in the vector library. By again normalizing these values ​​to those of AAV9, variants that perform worse than AAV9 in the heart or liver can be screened out. After applying the heart and liver filters, 163 unique vector variants were left ( Figure 6 ). All of these variants had respectable yields and had higher scores for cardiac or liver enrichment than AAV9.

[0191] Additional analysis of vector variants identified amino acid changes that resulted in altered capsid properties. First, it appears that position 503 is more restricted in amino acid composition than the other two positions. While positions 446 and 470 each have 15 amino acids represented in varying proportions, position 503 has only six. Analysis of these variants in heart and liver showed that those that were poorly enriched in liver had a higher trend for cardiac transduction. Positional analysis of these variants showed a preference for alternative aromatic amino acids, most notably at position W503 ( Fig. 7A– Figure 7F ). The findings suggest that galactose binding affinity positively correlates with liver transduction in vivo, whereas non-tryptophan aromatic compounds would be more advantageous in vectors that detarget the liver but maintain peripheral transduction.

[0192] Round 2: Directed evolution and the galactose binding pocket

[0193] To evaluate whether modifications that reduce galactose incorporation would de-target the liver and improve targeting to peripheral tissues including the heart, a second round of libraries was generated from the top hits of the first round of libraries ( Figure 8 ). This new library contains all 163 best performing variants from the first round of libraries, in addition to positive controls (AAV9) and negative controls (Y446A, N470A, and W503A). Although in the first round of libraries, each variant was encoded by only a single DNA sequence, the second round utilized alternative codon combinations so that each variant contained three unique coding sequences. This additional layer of internal redundancy increases the robustness of the sequencing results. This library was screened in both mice and NHPs and analyzed by amplicon sequencing.

[0194] An experiment similar to the above experiment was performed. The second library (about 200 variants) was IV injected into C57Bl / 6J mice and BALB-C mice (n=4 / group). Mice were killed on the 14th day after injection, and a large amount of tissue was collected for RNA extraction and subsequent NGS analysis. The analysis of this data set produced a number of useful data points. First, it allows the enrichment of variants in both liver and peripheral tissues to be sorted. Secondly, the data set was analyzed to determine the higher-order relationship between amino acid combinations (not just single amino acid substitutions) because it is related to the transduction pattern in vivo. In addition, the degree of similarity between two different strains of mice provides insights into the degree of conversion of galactose binding between different species. The top hits from this round were selected to test themselves in vivo (see Example 2).

[0195] like Fig. 9A and Fig. 9B As shown, a collection of variants that de-target the liver and enhance cardiac transduction were identified. The variants comprised an XRH motif (i.e., any amino acid (X) at position 446, an R at position 470, and an H at position 503). The top hits (i.e., VRH variants) increased cardiac transduction by approximately 2.7-fold and decreased liver transduction by 0.4x.

[0196] Although there was still a trend of cardiac enrichment in NHPs, there was an inconsistent trend of liver detargeting ( Fig.10This was not due to changes in any novel variants, but rather to differences in AAV9 liver performance between mice and NHPs. Most variants performed similarly in mouse and NHP livers, whereas AAV9 and its close relatives had lower RPMs in NHP livers than in mouse livers ( Fig.11 ). This indicated a dependency on galactose binding and prompted further testing of library variants in vitro.

[0197] To further test galactose binding of individual variants, the second round library was examined for binding to agarose beads with immobilized galactose. The vector library was allowed to bind to the galactose beads for one hour (in a tube rotator), at which time the mixture was spun down and the supernatant removed ( Fig. 12A ). qPCR and amplicon sequencing of both input and supernatant allowed calculation of the proportion of input vector detected in the unbound fraction. Vector with the W503A mutation was abundant in both the flow-through and washes, and analysis of the unbound proportion showed that most of the variants tested had very low binding to bead-bound galactose. The exceptions were AAV9 and its two close relatives, FNW and HNW ( Fig. 12B ). Fig.13A and Fig. 13B Results from control studies are presented to validate AAV9 binding relative to galactose binding in vitro.

[0198] Overview of methods used to assess galactose binding affinity:

[0199] 1. Load the column with 100 μl of bead resin

[0200] 2. Wash the column with 1.2 mL PBS

[0201] 3. Apply undiluted support to column - obtain flow-through (FT1)

[0202] 4. Wash 3x with 400 μl PBS -> PBS1-3

[0203] 5. Apply 400 μl 0.1M galactose and spin immediately -> Gal 0 min

[0204] 6. Apply 400 μl 0.1M galactose, incubate at RT for 10 minutes -> Gal 10 minutes

[0205] 7. Resuspend the resin in 400 μl PBS -> resin

[0206] 8. Measure the GC in each fraction and adjust the volume

[0207] Results identified AAV9 variants that outperformed AAV9 in both cardiac transduction and liver detargeting. Most of these variants had reduced ability to bind galactose compared to AAV9 and its close relatives. Findings suggest that these new variants have moderate galactose binding or are retargeted to different receptors. Additionally, this work revealed differences between mouse and NHP livers that may be due to the presence of vector-accessible galactose.

[0208] For further analysis, the second round library was also tested in vitro under different conditions on a range of cell lines. Neuraminidase is an enzyme that denitrogenates terminal glycans, exposing terminal galactose and strongly increasing transduction of 293 cells by AAV9. By transducing 293 cells with the second round library in the presence / absence of neuraminidase, it was further possible to correlate galactose availability with the transduction pattern of each variant. It is expected that peripherally transducing variants will have higher basal levels of 293 transduction, and the degree of increase in transduction after neuraminidase treatment will be less than that of liver transducing variants. This experiment can be performed in parallel using the CHO lec-2 cell line, which presents asialogalactose on its surface and has been shown to be susceptible to AAV9 transduction.

[0209] Example 2: Delivery of vector alone to mice

[0210] A preliminary study was performed to evaluate transduction and expression of a transgene (GFP) following administration of individual vectors with HRH, SRH, or VRH capsid variants (identified as described in Example 1). Each capsid was used to package a vector genome with an eGFP transgene operably linked to a CB7 promoter (CB7.CI.eGFP.WPRE.rBG; CI = chimeric intron; rBG = rabbit β-globin polyA). In addition, control vectors were generated with either an AAV9 capsid or an AAV9 capsid with the W503A mutation. Each vector (1 x 10 12 The mice were randomly divided into 4 groups (1000 genomic copies (GC) / mouse) and necropsied on day 14. Heart and liver tissues were harvested to assess transduction (DNA), RNA, and protein expression of the eGFP transgene. Fig.14A – Fig. 14C as well as Fig.15A – Fig. 15C Transduction and expression levels are shown for heart and liver, respectively, and include values ​​normalized to transduction and expression observed with AAV9.

[0211] All documents cited in this specification are incorporated herein by reference. The priority documents, namely, U.S. Provisional Patent Application No. 63 / 302,912 filed on January 25, 2022, U.S. Provisional Patent Application No. 63 / 375,005 filed on September 8, 2022, and U.S. Provisional Patent Application No. 63 / 386,572 filed on December 8, 2022, are incorporated herein by reference. Although the present invention has been described with reference to specific embodiments, it should be understood that modifications can be made without departing from the spirit of the present invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A recombinant adeno-associated virus (rAAV) having: an AAV clade F capsid, wherein the AAV clade F capsid comprises a mutant galactose binding site, wherein when the residue positions are determined using the residue numbering of SEQ ID NO: 10 as a reference, the mutant galactose binding site includes (a) any amino acid residue (X) at position 446 (Y446X), (b) arginine at position 470 (N470R), and (c) histidine at position 503 (W503H), and The vector genome comprises a 5' inverted terminal repeat (ITR) sequence, a coding sequence of a gene product, a regulatory sequence for directing the expression of the gene product operably linked to the coding sequence, and a 3' ITR sequence.

2. The rAAV of claim 1, wherein the mutant capsid comprises: (a) H at position 446 (HRH); (b) S (SRH) at position 446; (c) V(VRH) at position 446; (d) G at position 446 (GRH); (e) F at position 446 (FRH); (f) T at position 446 (TRH); or (g) S at position 446 (SRH).

3. The rAAV according to claim 1 or 2, wherein the parent AAV clade F capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid or an AAVhu96 capsid or a mutant or variant of any of the foregoing.

4. The rAAV of any one of claims 1 to 3, wherein the mutant clade F capsid further comprises an insertion mutation located between positions 588 and 589 in the HVR8 locus based on the numbering of the amino acid sequence of SEQ ID NO:

10.

5. The rAAV of claim 4, wherein the international mutation comprises: (a) the motif Nx-(T / I / V / A)-(K / R) (SEQ ID NO: 19), optionally wherein the motif is flanked by two to seven amino acids at its carboxyl and / or amino terminus; (b) an exogenous targeting peptide having the sequence: optional N-terminal linker-YG / A / R / KY / H-GNPA-T / R / H-RYFD-V / K (SEQ ID NO: 13)-optional C-terminal linker; between amino acids 588 and 589 of the AAV9 capsid protein based on the numbering of the following amino acid sequence: SEQ ID NO: 10; or (c) Peptides comprising the RGD motif.

6. The rAAV of claim 5, wherein the capsid further comprises a motif of (a) that is NTVK, optionally selected from: (a) SSNTVKLTSGH (SEQ ID NO: 14); (b)EFSSNTVKLTS(SEQ ID NO:12); (c) GGVLTNIARGEYMRGG (SEQ ID NO: 18); (d)GGIEINATRAGTNLGG (SEQ ID NO: 17); (e) GGSSNTVKLTSGHGG (SEQ ID NO: 13); (f) IEINATRAGTNL (SEQ ID NO: 16); or (g) SANFIKPTSY (SEQ ID NO:15).

7. The rAAV of claim 5, wherein the capsid further comprises a motif of (b) selected from the group consisting of: (a) YG / A / R / KY / H-GNPA-T / R / H-RYFD-V / K (SEQ ID NO: 28); (b) YGYGNPATRYFDV (SEQ ID NO: 20); (c) YGYGNPARRYFDV (SEQ ID NO: 25); (d) YGYGNPAHRYFDV (SEQ ID NO: 26); (e) YGYGNPATRYFDK (SEQ ID NO: 27); (f) YAYGNPATRYFDV (SEQ ID NO: 21); (g) YKYGNPATRYFDV (SEQ ID NO: 22); (h) YRYGNPATRYFDV (SEQ ID NO: 23); or (i) YGHGNPATRYFDV (SEQ ID NO:24).

8. The rAAV of any one of claims 1 to 7, wherein the regulatory sequence comprises a constitutive promoter.

9. The rAAV of any one of claims 1 to 7, wherein the regulatory sequence comprises a tissue-specific promoter.

10. The rAAV of claim 9, wherein the tissue-specific promoter is a cardiac promoter.

11. A method for producing rAAV comprising a mutant capsid derived from a parent AAV capsid having liver specificity, the method comprising culturing a packaging host cell, the packaging host cell comprising: (a) a nucleic acid sequence encoding a mutant AAV clade F capsid, the nucleic acid sequence being operably linked to a regulatory control sequence directing its expression in the packaging host cell, wherein the encoded capsid protein comprises (i) any amino acid residue (X) at position 446 (Y446X), (ii) an arginine at position 470 (N470R) and (iii) histidine at position 503 (W503H), wherein the amino acid residue position is represented using SEQ ID NO: The residue numbering of 10 was determined as a reference; (b) a nucleic acid molecule comprising a vector genome, the vector genome comprising a 5' inverted terminal repeat (ITR) sequence, a coding sequence for a gene product, a regulatory sequence operably linked to the coding sequence that directs the expression of the gene product, and a 3' ITR sequence; and (c) an auxiliary function, which is required for packaging the vector genome of (b) into the mutant evolutionary branch F capsid.

12. The method of claim 11, wherein the parent AAV capsid is an AAV9 capsid, an AAVhu68 capsid, an AAVhu31 capsid, an AAVhu32 capsid, an AAVhu95 capsid or an AAVhu96 capsid, or a mutant or variant of any of the foregoing.

13. A rAAV produced according to the method of claim 11 or 12.

14. A method for reducing liver toxicity associated with the delivery of a rAAV vector, the method comprising delivering to a subject the rAAV of any one of claims 1 to 10 or claim 13.

15. A method for improving delivery of a gene product to cardiac cells or tissues, the method comprising administering to a subject the rAAV of any one of claims 1 to 10 or claim 13.

16. The method according to claim 14 or 15, wherein the coding sequence comprises LaminA gene, Kv11.1 voltage-gated potassium channel (ERG) gene, tafazzin (TAZ) gene, potassium voltage-gated channel subfamily Q member 1 (KCNQ1) gene or cardiac myosin binding protein C (MYBPC3) gene.

17. A nucleic acid comprising a sequence encoding a mutant AAV clade F VP1 protein having a mutant galactose binding pocket, wherein the mutant AAV clade F VP1 protein comprises (a) any amino acid residue (X) at position 446 (Y446X), (b) arginine at position 470 (N470R), and (c) histidine at position 503 (W503H), wherein the amino acid residue positions are determined using the residue numbering of SEQ ID NO: 10 as a reference, and the VP1 protein coding sequence is operably linked to an expression control sequence that directs its expression in a packaging host cell.

18. The nucleic acid of claim 17, wherein the nucleic acid is a plasmid.

19. A host cell comprising the nucleic acid according to claim 17 or 18.

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