Adeno-associated virus compositions with increased cardiac enrichment

Abstract

Described herein are compositions and kits comprising recombinant adeno-associated viruses (rAAVs) with increased transduction enrichment in the heart. The rAAV compositions described herein encapsidate transgenes, such as therapeutic nucleic acids. Gene therapy using rAAVs is described. Methods of treating cardiovascular-related diseases and conditions are also described. The rAAVs have broad transduction to the heart and vasculature and may include the amino acid sequences set forth in any one of Tables 1-2, FIG. 1, and / or Formulas I-VII.

Description

[Background technology]

[0001] background Recombinant adeno-associated viruses (rAAV) have been widely used as vectors for gene delivery in therapeutic applications due to their ability to transduce both dividing and non-dividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These features make them attractive for application in therapeutic applications such as gene therapy. However, there is a need to significantly improve the performance of existing AAV serotypes to be selectively and efficiently expressed in different cell types upon systemic delivery to a subject. This need is particularly acute when AAV must be expressed in cardiac and vascular tissues. Summary of the Invention

[0002] Summary of the Invention Disclosed herein are rAAVs with engineered transduction into the capsid structure via recursive rounds of selection in non-human primates (NHPs) that result in variants with increased transduction as measured in the heart.

[0003] The present invention provides rAAV with broad transduction of the heart and vasculature.

[0004] The present invention provides, in one aspect, a peptide insert sequence comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1, and / or Formulas I-VIII.

[0005] Another aspect of the invention is an AAV capsid protein having a peptide insertion comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1 and / or Formulas I-VIII, characterized by increased cardiac transduction in a subject.

[0006] Another aspect of the invention is an AAV capsid protein having a peptide insertion comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1 and / or Formulas I-VIII, characterized by increased enrichment in cardiac tissue of a subject.

[0007] Moreover, the disclosure includes a pharmaceutical composition comprising an rAAV having a peptide insertion comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1, and / or Formulas I-VII, and a pharma- ceutically acceptable excipient.

[0008] The aspects disclosed herein provide a method for treating disease or condition in a subject, comprising administering a pharmaceutical preparation comprising a therapeutically effective amount of the AAV capsid protein or AAV capsid of the present disclosure.In some embodiments, the disease or condition is a disease or condition of the heart and cardiovascular tissues of the subject.Relatedly, the present invention includes the use of rAAV in the manufacture of a medicament for treating or preventing disease or medical condition.

[0009] Other aspects of the present invention will become apparent from the detailed description and claims that follow.

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief description of the drawings]

[0011] [Figure 1-1] FIG. 1 shows the insertion and substitution amino acid sequences of AAV capsid protein encoding amino acid sequences found in non-human primate heart tissue after two rounds of evaluation of the engineered AAV library. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14] Same as above. [Figure 1-15] Same as above. [Figure 1-16] Same as above. [Figure 1-17] Same as above. [Figure 1-18] Same as above. [Figure 1-19] Same as above. [Figure 1-20] Same as above. [Figure 1-21] Same as above. [Figure 1-22] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Disclosure In one aspect, the present disclosure provides an rAAV that has high expression levels in the heart.

[0013] In one aspect, the disclosure provides an rAAV having a peptide insertion and or substitution comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1, and / or Formulas I-VIII.

[0014] Embodiments disclosed herein include an amino acid sequence of Formula I X 1 -GGX 2 -X 3 -X 4 -X 5( I) (In the formula, X1 is an amino acid selected from G, I, K, L and V; X 2 is an amino acid selected from E, G, H, L, N, S and Q; X 3 is an amino acid selected from A, F, I, L, P, T and Y; 4 is an amino acid selected from F, L, S and Y; 5 is an amino acid selected from E, H, I, K, P, S and T. The present invention provides an AAV capsid comprising an AAV capsid protein comprising:

[0015] In some embodiments, the AAV capsid protein is X 1 is I, K, or L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 1 is K. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 1 is I.

[0016] In some embodiments, the AAV capsid protein is X 2 is L, H, or Q. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 2 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 2 is H.

[0017] In some embodiments, the AAV capsid protein is X 3 is I, P, or Y. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 3 is P. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, 3 is Y.

[0018] In some embodiments, the AAV capsid protein is X 4 is F or L.

[0019] In some embodiments, the AAV capsid protein is X 5 is I or H.

[0020] In some embodiments, the sequence as set forth in FIG. 1 is selected from KGGLPFI, IGGHYLH, LGGQILI, LGGGFSE, IGGNLLK, KGGETSS, VGGSTYP, and GGGLAST.

[0021] In some embodiments, the sequence as set forth in Figure 1 is selected from AQKGGLPFIAQ, AQIGGHYLHAQ, AQLGGQILIAQ, AQLGGGFSEAQ, AQIGGNLLKAQ, AQKGGETSSAQ, AQVGGSTYPAQ, and AQGGGLASTAQ.

[0022] Embodiments disclosed herein include an amino acid sequence of Formula II RSX 6 -X 7 -X 8 -X 9 -X 10( II) (In the formula, X 6 is an amino acid selected from E, G, I, N, Q and T; X 7 is an amino acid selected from L, H, Q, S and T; X 8 is an amino acid selected from D, E, H, P and S; X 9 is an amino acid selected from A, F, L, R, S and T; X 10 is an amino acid selected from E, I, M, S and V The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0023] In some embodiments, the AAV capsid protein is X10 is I, V, or M. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 10 is M.

[0024] In some embodiments, the AAV capsid protein is X 9 is F or L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 9 is F.

[0025] In some embodiments, the AAV capsid protein is X 8 is P or H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 8 is P. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 8 is H.

[0026] In some embodiments, the AAV capsid protein is X 7 is H or L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 7 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 7 is L.

[0027] In some embodiments, the AAV capsid protein is X 6 is E or G. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 6 is E. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula II, 7 is G.

[0028] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from RSGHPFV, RSGLHLM, RSELHFI, RSQTSTE, RSISDRV, RSTTDAS, and RSNQESV.

[0029] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from AQRSGHPFVAQ, AQRSGLHLMAQ, AQRSELHFIAQ, AQRSQTSTEAQ, AQRSISDRVAQ, AQRSTTDASAQ, and AQRSNQESVAQ.

[0030] Embodiments disclosed herein include an amino acid sequence of formula III RX 11 -GX 12 -X 13 -X 14 -X 15( III) (In the formula, X 11 is an amino acid selected from A, D, E, I, N, V ​​and S; 12 is an amino acid selected from E, H, L, M, S and V; X 13 is an amino acid selected from F, H, L, P, V and Y; 14 is an amino acid selected from F, L and T; 15 is an amino acid selected from D, H, K, L, M, Q, S and V. The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0031] In some embodiments, the AAV capsid protein is X 11 is an amino acid selected from A, D, N and S. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 11 is A. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 11 is D. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 11is N. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 11 is S.

[0032] In some embodiments, the AAV capsid protein is X 12 is an amino acid selected from E, H, L and V. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 12 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 12 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 12 is E. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 12 is V.

[0033] In some embodiments, the AAV capsid protein is X 13 is an amino acid selected from F, H, P and Y.

[0034] In some embodiments, the AAV capsid protein is X 14 is F or L.

[0035] In some embodiments, the AAV capsid protein is X 15 is an amino acid selected from K, L, M and V. In some embodiments, the AAV capsid protein comprises an amino acid sequence of formula II, 15 is V. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 15 is I. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 15is K. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula III, 15 is L.

[0036] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from RSGHPFV, RNGEHFK, RDGHYLL, RNGEFFH, REGMPFL, RAGVHFV, RSGLHLM, RIGVHTS, RIGSVTQ, and RVGSLTD.

[0037] Embodiments disclosed herein include an amino acid sequence of Formula IV X 16 -X 17 -GHX 18 -X 19 -X 20( IV) (In the formula, X 16 is an amino acid selected from I, R and L; X 17 is an amino acid selected from D, G, Q and S; 18 is an amino acid selected from I, P and Y; 19 is an amino acid selected from F, I, L, S and Y; 20 is an amino acid selected from H, L and V The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0038] In some embodiments, the AAV capsid protein is X 16 is an amino acid selected from I, L and R.

[0039] In some embodiments, the AAV capsid protein is X 17 is selected from G, Q and S.

[0040] In some embodiments, the AAV capsid protein is X 19is an amino acid selected from F, I, L, S and Y.

[0041] In some embodiments, the AAV capsid protein is X 20 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula IV, 20 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula IV, 20 is V.

[0042] In some embodiments, the AAV capsid protein is X 18 is I. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula IV, 18 is P. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula IV, 18 is Y.

[0043] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from RSGHPFV, RDGHYLL, LQGHILL, and IGGHYLH.

[0044] In some embodiments, the insert sequence as set forth in Figure 1 is selected from AQRSGHPFVAQ, AQRDGHYLLAQ, AQLQGHILLAQ, and AQIGGHYLHAQ.

[0045] Embodiments disclosed herein include an amino acid sequence of formula V RX 21 -X 22 -X 23 -HFX 24( V) (In the formula, X 21 is an amino acid selected from A, N and S, and X 22 is an amino acid selected from D, E and G; X 23 is an amino acid selected from E, L and V; X24 is an amino acid selected from I, K, Q and V The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0046] In some embodiments, the AAV capsid protein is X 21 is A. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, wherein X 21 is N. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 21 is S.

[0047] In some embodiments, the AAV capsid protein is X 22 is D. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 22 is G. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 22 is E.

[0048] In some embodiments, the AAV capsid protein is X 23 is E. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 23 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 23 is V.

[0049] In some embodiments, the AAV capsid protein is X 24 is Q. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 24 is K. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula V, 24 is V.

[0050] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from RADLHFQ, RAGVHFV, RNGEHFK, RSELHFI.

[0051] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from AQRADLHFQAQ, AQRAGVHFVAQ, AQRNGEHFKAQ, and AQRSELHFIAQ.

[0052] Embodiments disclosed herein include an amino acid sequence of Formula VI X 25 -X 26 -X 27 -X 28 -X 29 -L(VI) (In the formula, X 25 is an amino acid selected from A, L, R and Y; 26 is an amino acid selected from Q, E, D, T, K and F; 27 is an amino acid selected from G, Q and A; X 28 is an amino acid selected from H, G, T, V and D; 29 is an amino acid selected from I, S, T and Y. The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0053] In some embodiments, the AAV capsid protein is X 25 is L or R.

[0054] In some embodiments, the AAV capsid protein is X 26 is Q, E or D.

[0055] In some embodiments, the AAV capsid protein is X 27 is G or Q.

[0056] In some embodiments, the AAV capsid protein is X 28 is H or G.

[0057] In some embodiments, the AAV capsid protein is X 29 is I or Y.

[0058] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from LQGHILL, REQGYLL, RDGHYLL, YTGTSLL, AKAVTLL, and AFGDSLL.

[0059] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from AQLQGHILLAQ, AQREQGYLLAQ, AQRDGHYLLAQ, AQYTGTSLLAQ, AQAKAVTLLAQ, and AQAFGDSLLAQ.

[0060] Embodiments disclosed herein include an amino acid sequence of Formula VII X 30 -X 31 -X 32 -X 33 -X 34 -SS(VII) (In the formula, X 30 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, S, T, V and Y; X 31 is an amino acid selected from F, G, H, I, K, L, M, P, Q, R, S, T and V; X 32 is an amino acid selected from A, G, H, L, P, Q, S and V; X 33 is an amino acid selected from E, G, H, I, L, P, Q, R, S, T and V; X 34 is an amino acid selected from D, G, I, L, M, N, R, S, T and V. The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0061] In some embodiments, the AAV capsid protein is X 30 is V, Q or H.

[0062] In some embodiments, the AAV capsid protein is X 31 is F, L or M.

[0063] In some embodiments, the AAV capsid protein is X 32 is S, A or Q.

[0064] In some embodiments, the AAV capsid protein is X 33 is G, H or S.

[0065] In some embodiments, the AAV capsid protein is X 34 is G, V or M.

[0066] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from VFSGVSS, QLQHGSS, HMASMSS, SSLTLSS, SVPSSSS, ITPSVSS, MTVQVSS, SHLSVSS, YPHQTSS, PQGGSSS, FRSSLSS, ISVSISS, ETQRGSS, IHPPSSS, TSAITSS, FKSQSSS, ASHSTSS, AVMRDSS, KGGETSS, VVSVTSS, LISIRSS, NSLGLSS, and HLAQNSS.

[0067] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from AQVFSGVSSAQ, AQQLQHGSSAQ, AQHMASMSSAQ, AQSSLTLSSAQ, AQSVPSSSSAQ, AQITPSVSSAQ, AQMTVQVSSAQ, AQSHLSVSSAQ, AQYPHQTSSAQ, AQPQGGSSSAQ, AQFRSSLSSAQ, AQISVSISSAQ, AQETQRGSSAQ, AQIHPPSSSAQ, AQTSAITSSAQ, AQFKSQSSSAQ, AQASHSTSSAQ, AQAVMRDSSAQ, AQKGGETSSAQ, AQVVSVTSSAQ, AQLISIRSSAQ, AQNSLGLSSAQ, and AQHLAQNSSAQ.

[0068] Embodiments disclosed herein include an amino acid sequence of Formula VIII DLX 35 -X 36 -X 37 -X 38 -X 39( VIII) (In the formula, X 35 is an amino acid selected from G, H, Q, S and T; 36 is an amino acid selected from K, N, P, R and S; X 37 is an amino acid selected from H, L, R and S; X 38 is an amino acid selected from D, K, R, S, T and V; X 39 is an amino acid selected from A, D, E, L and V The present invention provides an AAV capsid having greater expression in the heart, comprising an AAV capsid protein comprising:

[0069] In some embodiments, the AAV capsid protein is X 35 is H or S.

[0070] In some embodiments, the AAV capsid protein is X 36 is K or R.

[0071] In some embodiments, the AAV capsid protein is X 37 is L or S.

[0072] In some embodiments, the AAV capsid protein is X 38 is S or V.

[0073] In some embodiments, the AAV capsid protein is X 39 is A or L.

[0074] In some embodiments, the insert sequence as set forth in FIG. 1 is selected from DLSRLS, DLHKSVA, DLGNLKV, DLTPHKD, DLQRSDL, and DLGSRRE.

[0075] In some embodiments, the insertion sequence as set forth in FIG. 1 is selected from AQDLSRLSLAQ, AQDLHKSVAAQ, AQDLGNLKVAQ, AQDLTPHKDAQ, AQDLQRSDLAQ, and AQDLGSRREAQ.

[0076] In some embodiments, the insert sequence is represented by a peptide sequence listed in Tables 1-2. Table 1 [Table 1-1] [Table 1-2] Table 2. [Table 2]

[0077] In some embodiments, the inserted amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Tables 1-2, Figure 1, and / or Formulas I-VII. In some embodiments, the inserted amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Tables 1-2, Figure 1, and / or Formulas I-VII.

[0078] Also disclosed herein are methods and kits for making therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising the rAAV particles, for the treatment of diseases or conditions affecting the heart.

[0079] The present specification discloses an engineered AAV capsid that has increased viral transduction in the heart.The AAV capsid can encapsidate a viral vector that has a heterologous nucleic acid, for example, encoding a therapeutic gene expression product.The transduction of heterologous nucleic acid in the heart can be achieved upon systemic delivery of the AAV capsid of the present disclosure to a subject, which encapsidates heterologous nucleic acid.The AAV capsid disclosed herein is advantageous for many applications of gene therapy to treat human diseases, including but not limited to cardiovascular disorders.

[0080] Also provided herein is a recombinant AAV vector comprising a nucleic acid sequence encoding the AAV capsid protein of the present disclosure. For example, the viral vector of the present disclosure comprises a nucleic acid sequence comprising the AAV viral Cap (capsid) encoding VP1, VP2, and VP3, at least one of which is modified to create the AAV capsid protein of the present disclosure. The provided recombinant AAV vector can be derived from an AAV serotype (for example, AAV9) or a variant AAV serotype comprising the insertion of the present invention.

[0081] AAV Capsid Provided herein are modified adeno-associated (AAV) virus capsid compositions useful for integrating a transgene into a target cell or environment (in a subject when administered systemically to the subject).

[0082] rAAV contains an AAV capsid that can be engineered to encapsidate heterologous nucleic acids (e.g., therapeutic nucleic acids, gene editing machinery). The AAV capsid is composed of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid. VP1 covers the entirety of the VP2 protein in addition to an N-terminal region of about 137 amino acids (VP1u), and VP2 covers the entirety of VP3 in addition to an N-terminal region of about 65 amino acids (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0083] Without wishing to be bound by theory, it is understood that parent AAV capsid sequence comprises VP1 region.In certain embodiments, parent AAV capsid sequence comprises VP1, VP2 and / or VP3 region, or any combination thereof.Parent VP1 sequence can be considered synonymous with parent AAV capsid sequence.

[0084] The VP3 structure of AAV contains a highly conserved region common to all serotypes, a core eight-stranded β-barrel motif (βB-βI) and a small α-helix (αA). The loop region inserted between the β-strands consists of the unique HI loop between β-strands H and I, the DE loop between β-strands D and E, and nine variable regions (VRs) that form the top of the loops. These VRs, such as the AA588 loop, are found on the capsid surface and may be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity.

[0085] In some embodiments, the rAAV variants of the invention comprise an AAV capsid protein having a peptide insertion at residues corresponding to amino acids 588-589 of the AAV9 native sequence of SEQ ID NO:1.

[0086] AAV capsids contain AAV capsid proteins (examples include VP1, VP2, and VP3), each of which has an insertion such as in the 588 loop of the parent AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains the site of heparan sulfate binding for AAV2 and is suitable for peptide display. The only known receptor for AAV9 is the N-linked terminal galactose and AAV receptor (AAVR), although many indications point out that there are others. Modifications of the AAV9 588 loop to confer increased transgene transduction in a target in vivo environment are presented herein.

[0087] The present invention provides, in certain aspects, a peptide insertion at the AAV 588 loop comprising or consisting of an amino acid sequence set forth in any one of Tables 1-2, FIG. 1 and / or Formulas I-VII.

[0088] Disclosed herein is an AAV capsid comprising an AAV capsid protein having an insertion in the 588 loop that confers higher transduction to cardiac cell types (for example).In particular, the AAV capsid protein disclosed herein allows rAAV-mediated transduction of heterologous nucleic acid (for example, transgene) in the heart of a subject.The AAV capsid of the present disclosure can be formulated as a pharmaceutical composition.In addition, the AAV capsid can be isolated and purified to be used for various applications.

[0089] In some embodiments, the rAAV capsid of the present disclosure is produced using the methods disclosed herein.In some cases, the rAAV capsid is chimeric.In some cases, the rAAV or variant AAV protein is compared with the parent AAV capsid or capsid protein that it contains, and confers increased localization of the rAAV in target tissue.

[0090] AAV Capsid Protein Recombinant AAV (rAAV) capsids comprising AAV capsid proteins engineered with modified capsid proteins (e.g., VP1, VP2, VP3) are disclosed herein. In some embodiments, the rAAV capsid proteins of the present disclosure are produced using the methods disclosed herein. In some embodiments, the AAV capsid proteins are used in methods for delivering therapeutic nucleic acids (e.g., transgenes) to a subject. In some cases, the rAAV capsid proteins have the desired AAV expression, making them particularly suitable for certain therapeutic applications, e.g., for treating diseases or disorders in a subject, such as those disclosed herein.

[0091] The rAAV capsid protein is engineered for optimized expression in the heart of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid protein is engineered to contain an insertion as provided in Tables 1-2, FIG. 1, and / or Formulas I-VII. The rAAV capsid protein containing an insertion as provided in Tables 1-2, FIG. 1, and / or Formulas I-VII is engineered to achieve efficient transduction of the encapsidated transgene. In particular, the rAAV capsid protein has increased expression in the heart of the subject.

[0092] The engineered AAV capsid proteins described herein, in some cases, have an insertion of an amino acid heterologous to the parent AAV capsid protein at an amino acid position within the 588 loop. In some embodiments, the amino acid is not endogenous to the parent AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid at the same or equivalent amino acid position as the replacement insertion in a different AAV capsid protein.

[0093] Typically, the insertion comprises a 5, 6, or 7 amino acid sequence (a 5-mer, 6-mer, or 7-mer, respectively) inserted or substituted into the 588 loop of the parent AAV capsid protein. Embodiments provided herein provide amino acid insertions comprising a 7 amino acid polymer (7-mer) inserted at AA588-589, which may additionally comprise substitutions of one or two amino acids at amino acid positions adjacent to the 7-mer sequence (e.g., AA587-588 and / or AA589-590) to create an 11 amino acid polymer (11-mer) at the 588 loop of the parent AAV capsid protein. The 7-mers described herein are advantageously generated using polymerase chain reaction (PCR) with degenerate primers, where each of the 7 amino acids is encoded by the deoxyribose nucleic acid (DNA) sequence NNK. "N" is any of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method of generating random heptamer amino acid sequences allows for 1.28 billion possible combinations at the protein level.

[0094] The rAAV capsid proteins of the present disclosure include an amino acid insertion in the amino acid sequence of the AAV capsid protein. The AAV capsid from which the engineered AAV capsid proteins of the present disclosure are made is referred to as the "parent" AAV capsid. The complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, and the complete genome of AAV-6 is provided in GenBank Accession No. NC_001829. 1862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), the AAV-11 genome is provided in Virology, 330(2):375-383 (2004), a portion of the AAV-12 genome is provided in Genbank Accession No. DQ813647, and a portion of the AAV-13 genome is provided in Genbank Accession No. EU285562.

[0095] In some cases, the parent AAV is derived from an AAV having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. The AAV capsid protein "derived" from another may be a variant AAV capsid protein. The variant may, for example, include a heterologous amino acid in the amino acid sequence of the AAV capsid protein. The heterologous amino acid may not naturally occur in the AAV capsid protein. The heterologous amino acid may naturally occur in a different AAV capsid protein. In some cases, the parent AAV capsid is described in U.S. Patent Application Publication No. 2020 / 0165576 and U.S. Provisional Patent Application Publication No. 62 / 832,826 and PCT / US20 / 20778, the contents of each of which are incorporated herein.

[0096] In some cases, the parent AAV is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of the AAV9 VP1 capsid protein (>tr|Q6JC40|Q6JC40_9VIRU capsid protein VP1 OS=adeno-associated virus 9 OX=235455 GN=cap PE=1 SV=1) comprises SEQ ID NO: 1. [ka] [ka] In some cases, the parent AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:1.

[0097] AAV capsid proteins from native AAV serotypes, such as AAV9, that have a tropism that includes the liver, can activate the innate immune response and, in some cases, cause a severe inflammatory response in subjects, leading to multiple organ failure. By improving the transduction of native AAV serotypes to target in vivo tissues (e.g., the heart), the rAAV particles of the present disclosure can reduce undesirable side effects.

[0098] In some cases, the parent AAV capsid protein includes the entire VP1 region (e.g., amino acids 1-736) provided in SEQ ID NO: 1. In some cases, the parent AAV capsid protein includes amino acids 217-736 in SEQ ID NO: 1, which is a common region found in the VP1, VP2 and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein includes amino acids 64-736 in SEQ ID NO: 1, which is a common region found in VP1 and VP2. The sequences of the parent AAV capsid proteins are 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200-736, 210-736, 220-736, 230-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, 450-736, 460-736, 470-736, 480-736, 490-736, 500-736, 510-736, 520-736, 530-736, 540-736, 550-736, 560-7 The rAAV variants may comprise amino acids selected from 0-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, and 450-736. In some embodiments, the rAAV variants comprise an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO:1. In some cases, the amino acid insertion is at the three-fold axis of symmetry of the corresponding parent AAV capsid protein.

[0099] Insertions of amino acid sequences into AAV capsid proteins are disclosed herein. Where the sequence numbering designation "588-589" is noted for AAV9, e.g., AAV VP1, the present invention also includes insertions in similar locations in other AAV serotypes. As used herein, "AA588-589" indicates that the insertion of an amino acid (or amino acid sequence) is immediately after the amino acid (AA) at position 588 and immediately before the AA at position 589 in the amino acid sequence of the parent AAV VP capsid protein (VP1 numbering). Amino acids 587-591 contain a motif containing "AQAQA" as set forth in SEQ ID NO:1. Exemplary AAV capsid protein sequences are provided in Table 3. For example, KGGLPFI is inserted at AA588-589 in the AAV9 capsid amino acid sequence to provide variant A. It is contemplated that the sequences disclosed herein (Tables 1-2, FIG. 1 and Formulas I-VII) may be inserted at AA588-589, or at AA587-590 (replacing amino acids AA587-590) of the amino acid sequence of the parent AAV9 capsid protein, variants thereof, or the equivalent amino acid positions of a parent AAV of a different serotype (e.g., AAV1, AAV2, AAV3, etc.). Table 3. Exemplary AAV capsid protein sequences [Table 3-1] [Table 3-2] [Table 3-3]

[0100] The insertions described herein may, in some cases, include a 7-mer insertion at AA 588-589. In addition to substitutions with any amino acid at amino acid positions 587-590 [AQAQ], it is contemplated that any of the 7-mer insertions disclosed herein may constitute an 11-mer.

[0101] Disclosed herein is an AAV capsid protein having the above insertion in the parent AAV capsid protein, which confers increased transduction in the heart in a subject even when delivered systemically.One of the many advantages of the AAV capsid proteins described herein is their ability to target tissues and cells in the heart.

[0102] In some cases, the AAV capsid protein comprises an insertion of at least or about 7, 8, 9, 10 or 11 amino acids of the amino acid sequence of Tables 1-2, Figure 1 and Formulas I-VII at amino acid positions 588-589 or 587-590 of the parent AAV9 capsid protein (SEQ ID NO:1). In some cases, the AAV capsid protein has increased viral transduction enrichment in the heart.

[0103] The rAAV capsid proteins of the present disclosure may also have an amino acid sequence substitution at amino acid positions 452-458 of the parent AAV9 capsid protein or variant thereof, as described in WO2020068990. In some embodiments, the amino acid sequence substitution comprises KDNTPGR at amino acid positions 452-458 of the parent AAV9 capsid protein. In some embodiments, the amino acid sequence substitution comprises DGAATKN at amino acid positions 452-458 of the parent AAV9 capsid protein.

[0104] The rAAV capsid protein described herein can be isolated and purified.AAV can be isolated and purified by standard methods in the art, such as by column chromatography, iodixanol gradient, or cesium chloride gradient.Methods for purifying AAV from helper virus are known in the art, and can include, for example, the methods disclosed in Clark et al., Hum.Gene Ther.,10(6):1031-1039(1999), Schenpp and Clark, Methods Mol.Med.69:427-443(2002), U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0105] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, may in some cases be formulated into pharmaceutical preparations which further comprise a pharma- ceutically acceptable carrier.

[0106] rAAV capsid protein can be conjugated to nanoparticle, second molecule, or viral capsid protein.In some cases, nanoparticle or viral capsid protein encapsidates therapeutic nucleic acid as described herein.In some cases, the second molecule is a therapeutic agent, such as a small molecule, an antibody, an antigen-binding fragment, a peptide, or a protein, as described herein.

[0107] "Percent identity" is the percent of symbols that actually match. Percent similarity is the percent of symbols that are similar. Symbols across a gap are ignored. Similarity is scored if the score matrix value for a pair of symbols exceeds or is equal to a similarity threshold of 0.50. The scoring matrix used in version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0108] The sequence identity / similarity values ​​provided herein may refer to values ​​obtained using the BLAST+2.5.0 suite of programs using default settings (blast.ncbi.nlm.nih.gov) (Camacho, C. et al. (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0109] As those skilled in the art can understand, BLAST search assumes that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequences, which may be homopolymer tracts, short periodic repeats, or regions enriched with one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even if other regions of the proteins are not completely similar. Several low-complexity filter programs can be used to reduce such low-complexity alignments. For example, SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Ci-ayerie and States (1993) Comput. Chem. 17:191-201) low-complexity filters can be used alone or in combination.

[0110] The terms "substantial identity" and "substantially identical" refer to a polypeptide or nucleic acid having between 55-100% sequence identity to a reference sequence, at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity, or any percentage value within the range of 55-100% sequence identity to a reference sequence. The sequence identity percentage may occur over a specified comparison window. Optimal alignment may be confirmed or performed using the Needleman and Wunsch homology alignment algorithm described above.

[0111] For example, an insertion sequence may include, but is not limited to, sequences that are not exactly the same as the sequences disclosed herein, but have, in addition to the substitutions explicitly set forth for the various sequences listed herein, additional substitutions of amino acid residues that do not substantially impair the activity or properties of the sequences described herein, such as those predicted by homology software, such as the BLOSUM62 matrix.

[0112] AAV particles The rAAV particles having the insert sequence described herein have increased transduction enrichment in the heart. In some cases, the increased transduction enrichment comprises 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increase or more. In some cases, the increased transduction enrichment is at least 1-fold. In some cases, the increased transduction enrichment is at least 2-fold. In some cases, the increased transduction enrichment is at least 4-fold.

[0113] The rAAV particles having the insert sequence described herein have increased expression enrichment in the heart.Detecting whether rAAV has more or less specificity for the target in vivo environment includes measuring the level of gene expression product (for example, RNA or protein) expressed from the heterologous nucleic acid encapsidated by rAAV in tissue samples obtained from subject.Preferred methods for measuring the expression of gene expression product include next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0114] The increased expression in the heart is represented by the enrichment values ​​provided in FIG.

[0115] heterologous nucleic acid Therapeutic nucleic acids useful for treating or preventing a disease or condition, or a symptom of a disease or condition, are disclosed herein. In some embodiments, the therapeutic nucleic acid encodes a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.), and gene editing components for use in treating, preventing, and / or ameliorating a disease or disorder, or a symptom of a disease or disorder. In some cases, the therapeutic nucleic acid is placed in the subject's organism, cell, tissue, or organ by rAAV, such as those disclosed herein.

[0116] Disclosed herein are rAAVs, each comprising a viral vector (eg, a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector comprises two inverted terminal repeat (ITR) sequences, each of about 145 bases, flanking the transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid, and in some cases, a promoter in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter can initiate transcription of the therapeutic nucleic acid in the nucleus of the target cell. The ITR sequence can be from any AAV serotype. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some cases, the ITR is from AAV2. In some cases, the ITR is from AAV9.

[0117] Disclosed herein are transgenes that may comprise any number of nucleotides. In some cases, the transgene may comprise less than about 100 nucleotides. In some cases, the transgene may comprise at least about 100 nucleotides. In some cases, the transgene may comprise at least about 200 nucleotides. In some cases, the transgene may comprise at least about 300 nucleotides. In some cases, the transgene may comprise at least about 400 nucleotides. In some cases, the transgene may comprise at least about 500 nucleotides. In some cases, the transgene may comprise at least about 1000 nucleotides. In some cases, the transgene may comprise at least about 5000 nucleotides. In some cases, the transgene may comprise between about 500 and about 5000 nucleotides. In some cases, the transgene comprises about 5000 nucleotides. In any of the cases disclosed herein, the transgene may comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene may be single-stranded. In some cases, the transgene may be double-stranded.

[0118] The transgene is disclosed herein for use in regulating the expression or activity of target gene or its gene expression product.In some cases, the transgene is encapsidated by the rAAV capsid protein of the rAAV particle described herein.In some cases, the rAAV particle is delivered to a subject to treat the disease or symptoms disclosed herein in the subject.In some cases, the delivery is systemic.

[0119] The transgenes disclosed herein are useful for expressing endogenous genes at levels similar to those of healthy or normal individuals. This is particularly useful for treating diseases or conditions associated with under- or lack of expression of gene expression products. In some embodiments, the transgenes disclosed herein are useful for over-expressing endogenous genes such that the expression level of the endogenous gene exceeds that of a healthy or normal individual. Additionally, the transgenes can be used to express exogenous genes (e.g., active agents such as antibodies, peptides, nucleic acids, or gene editing components). In some embodiments, the therapeutic gene expression product can alter, enhance, increase, or induce the activity of one or more endogenous biological processes in the cell. In some embodiments, the transgenes disclosed herein are useful for reducing the expression of endogenous genes, e.g., dominant negative genes. In some embodiments, the therapeutic gene expression product can alter, inhibit, reduce, prevent, eliminate, or impair the activity of one or more endogenous biological processes in the cell. In some embodiments, an increase in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one embodiment, the protein product of the targeted gene can be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some embodiments, a decrease in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one embodiment, the protein product of the targeted gene can be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0120] When an endogenous sequence (part of an endogenous or transgene) is expressed together with a transgene, the endogenous sequence may be a full-length sequence (wild-type or mutant) or a partial sequence. The endogenous sequence may be functional. Non-limiting examples of the functions of these full-length or partial sequences include extending the serum half-life of the polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0121] A transgene may be inserted into an endogenous gene such that all, part, or none of the endogenous gene is expressed. For example, a transgene as described herein may be inserted into an endogenous locus such that a part of the endogenous sequence (the N-terminus and / or C-terminus of the transgene) or none of the endogenous sequence is expressed, for example as a fusion with the transgene. In other cases, a transgene (e.g., with or without additional coding sequences of the endogenous gene) is integrated into any endogenous locus, e.g., a safe harbor locus. For example, a frataxin (FXN) transgene may be inserted into the endogenous FXN gene. A transgene may be inserted into any gene, e.g., a gene as described herein.

[0122] At least one advantage of the present disclosure is that virtually any therapeutic nucleic acid can be used to express any therapeutic gene expression product. In some cases, the therapeutic gene expression product is a therapeutic protein or peptide (e.g., an antibody, an antigen-binding fragment, a peptide, or a protein). In one embodiment, the protein encoded by the therapeutic nucleic acid is between 50 and 2000 amino acids in length. In some embodiments, the encoded protein is between 50 and 16000 amino acids in length. In some embodiments, the encoded protein is between 50 and 1500 amino acids in length. In some embodiments, the encoded protein is between 50 and 1000 amino acids in length. In some embodiments, the encoded protein is between 50 and 800 amino acids in length. In some embodiments, the encoded protein is between 50 and 600 amino acids in length. In some embodiments, the encoded protein is between 50 and 400 amino acids in length. In some embodiments, the encoded protein is between 50 and 200 amino acids in length. In some embodiments, the encoded protein is between 50 and 100 amino acids in length. In some embodiments, the encoded peptide is between 4-50 amino acids in length. In some embodiments, the encoded protein is a tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some embodiments, the encoded protein comprises a peptide of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. In some embodiments, the encoded protein comprises a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide of 50 amino acids or less, such as 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0123] Non-limiting examples of therapeutic proteins or peptides include adrenergic agents, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietic agents, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartate antagonists, plexins, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphoring, semaphorin receptors, serotonin transport proteins, serotonin uptake inhibitors, serotonin receptors, serpins, serpin receptors, and tumor suppressors. In certain embodiments, the therapeutic protein or peptide is selected from the group consisting of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropins, interferon-gamma (IFN), insulin-like growth factor 1 (IFG-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), transforming growth factor (TGF), transforming growth factor (TGF-1), and the like. The inhibitor is selected from transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, somatotropin, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10 viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.

[0124] The therapeutic gene expression product may include a gene editing component. Non-limiting examples of gene editing components include CRISPR / Cas requisites, artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), and transcription factor-like effector nucleases (TALENs). In a non-limiting example, a subject with Huntington's disease is identified. The subject is then systemically administered a first amount of rAAV that encapsidates a viral vector encoding a ZFN engineered to suppress transcription of the huntingtin (HTT) gene. The rAAV comprises a modified AAV capsid protein that includes an amino acid sequence provided in any one of Tables 1-2, FIG. 1, and Formulas I-VII, allowing for proper targeting of the ZFN to the nervous system while reducing expression in off-target organs such as the liver. If necessary, the subject is administered a second or third dose of rAAV until a therapeutically effective amount of the ZFN is expressed in the subject's nervous system.

[0125] Therapeutic nucleic acids may include sequences encoding non-protein-coding genes, such as antisense RNA, RNAi, shRNA, and microRNA (miRNA), miRNA sponges or decoys, conditional gene deletion, recombinase delivery for conditional (recombinase-dependent) expression, including those required for gene editing components described herein. Non-protein-coding genes may also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (IncRNA). In some cases, non-protein-coding genes can regulate the expression or activity of target genes or gene expression products. For example, RNAs described herein can be used to inhibit gene expression in the cardiovascular system. In some cases, inhibition of gene expression refers to at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% inhibition. In some cases, the protein product of the targeted gene can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene can be either a wild type gene or a gene with at least one mutation. The targeted protein can be either a wild type protein or a protein with at least one mutation.

[0126] The therapeutic nucleic acid can regulate the expression or activity of a gene or gene expression product expressed from a gene that is involved in heart disease or disorder.For example, the therapeutic nucleic acid is, in some cases, a modified version of a gene or gene described herein.In some cases, the gene or gene expression product is inhibited.In some cases, the gene or gene expression product is enhanced.

[0127] In another example, the therapeutic nucleic acid comprises an effector gene expression product, such as a gene editing component, specific for targeting a gene therein. Non-limiting examples of genes include target genes or gene expression products selected from MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin, and FXN.

[0128] In some cases, the gene or gene expression product is inhibited. In some cases, the gene or gene expression product is enhanced.

[0129] AAV vectors The embodiments disclosed herein include plasmid vectors that contain the nucleic acid sequences encoding the AAV capsid and AAV capsid proteins described herein.The AAV vectors described herein are useful for the assembly of rAAV and the viral packaging of heterologous nucleic acid.In addition, AAV vectors can encode transgenes that contain heterologous nucleic acid.

[0130] The AAV vector may, in some cases, contain a transgene encoding a heterologous gene expression product (e.g., a therapeutic gene expression product, a recombinant capsid protein, etc.). The transgene is in cis with two inverted terminal repeats (ITRs) flanking the transgene. The transgene may contain a therapeutic nucleic acid encoding a therapeutic gene expression product. Due to the limited packaging capacity of rAAV (approximately 5 kB), in some cases, a longer transgene may be split between two AAV vectors (the first one with a 3' splice donor and the second one with a 5' splice acceptor). Upon co-infection of cells, concatemers form, which are spliced ​​together to express the full-length transgene.

[0131] A transgene is generally inserted such that its expression is driven by the endogenous promoter at the integration site, i.e., the promoter that drives the expression of the endogenous gene into which the transgene is inserted.In some cases, the transgene includes a promoter and / or enhancer, such as a constitutive promoter or an inducible or tissue / cell-specific promoter.As a non-limiting example, the promoter can be a CMV promoter, a CMV-β-actin-intron-β-globin hybrid promoter (CAG), a CBA promoter, a FRDA or FXN promoter, a UBC promoter, a GUSB promoter, an NSE promoter, a synapsin promoter, a MeCP2 promoter, a GFAP promoter, a H1 promoter, a U6 promoter, a NFL promoter, a NFH promoter, a SCN8A promoter, or a PGK promoter. As non-limiting examples, the promoter may be a tissue-specific expression element, including, but not limited to, human elongation factor 1 α-subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC). The transgene may include tissue-specific expression elements for neurons, such as, but not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk, and EAAT2 promoters. The transgene may include tissue-specific expression elements for astrocytes, such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may include tissue-specific expression elements for oligodendrocytes, such as, but not limited to, the myelin basic protein (MBP) promoter.

[0132] In some embodiments, the promoter is less than 1 kb. In some embodiments, the length may be 0, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. The promoter may provide expression of the therapeutic gene expression product over a period of time in a targeted tissue, such as, but not limited to, the heart.Expression of therapeutic gene products was measured at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days , 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years The term may be for 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, or more than 65 years. Expression of the payload can be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years, or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50-55 years, or 55-60 years, or 60-65 years.

[0133] AAV vectors can contain the genome of helper virus. Helper virus proteins are necessary for the assembly of recombinant AAV (rAAV) and the packaging of transgenes containing heterologous nucleic acid into rAAV. Helper virus genes are adenovirus genes E4, E2a and VA, which support AAV replication when expressed in cells. In some embodiments, AAV vectors contain E2. In some embodiments, AAV vectors contain E4. In some embodiments, AAV vectors contain VA. In some cases, AAV vectors contain one or any combination of helper virus proteins.

[0134] Target genes or gene expression products for use in transgenes may be selected from MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin, and FXN.

[0135] The AAV vector may comprise a viral genome comprising a nucleic acid encoding a recombinant AAV (rAAV) capsid protein described herein. The viral genome may comprise a Rep gene encoding a replication (Rep) protein and a Cap gene encoding an AAP protein in a first open reading frame (ORF1) or a capsid (Cap) protein in a second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some cases, the Cap gene is modified to encode a modified AAV capsid protein described herein. The wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three of VP1-VP3 are modified. The AAV vector can contain nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.

[0136] In some cases, the AAV9 VP1 gene provided in Table 4 is modified to include any one of the variants listed in Table 1, Table 2, Table 3, or Figure 1. The AAV vectors described herein can be used to generate variant AAV capsids by the methods described herein. Table 4. Nucleic acid sequence of VP1 capsid protein [Table 4-1] [Table 4-2]

[0137] How to make rAAV Disclosed herein are methods for making AAV capsids, including AAV capsid proteins and viral vectors encoding therapeutic nucleic acids. AAV capsid proteins are made by introducing into a cell (e.g., an immortalized stem cell) a first vector containing a transgene cassette (the transgene cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell) flanked by inverted terminal repeat (ITR) sequences from a parent AAV virus, a second vector encoding an AAV genome (encoding the AAV Rep gene, as well as a modified Cap gene for the variant to be made) with AAV capsid proteins, and a third vector encoding helper virus proteins required for assembly of the AAV capsid structure and packaging of the transgene into the modified AAV capsid structure. The assembled AAV capsids can be isolated and purified from the cells using suitable methods known in the art. Tables 1-2 provide DNA sequences for use in the methods described herein.

[0138] Also provided herein is a transgene that is contained in a recombinant AAV (rAAV) vector and is encapsidated by the AAV capsid protein of the present disclosure.The transgene disclosed herein is delivered to a subject for various purposes, such as to treat a disease or condition in the subject.The transgene can be a gene editing component that regulates the activity or expression of a target gene or gene expression product.Alternatively, the transgene is a gene that encodes a therapeutic gene expression product that is effective in regulating the activity or expression of itself or another target gene or gene expression product.

[0139] The embodiments disclosed herein provide a method for producing rAAV virus or virus particles, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector comprising a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parent AAV virus (the transgene cassette has a promoter sequence driving the transcription of a heterologous nucleic acid in the nucleus of a target cell); (ii) a second vector encoding an AAV genome having an AAV capsid protein of the present invention; and (iii) a vector encoding helper virus proteins required for assembly of an AAV capsid structure and packaging of a transgene into the modified AAV capsid structure; (b) expressing in the cell the AAV capsid proteins described herein; (c) assembling an AAV particle comprising the AAV capsid proteins disclosed herein; and (d) packaging the AAV particle. In some cases, the cell is mammalian. In some cases, the cell is immortalized. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In some cases, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293). In some cases, the 5'ITR and the 3'ITR are from an AAV2 serotype. In some cases, the 5'ITR and the 3'ITR are from an AAV5 serotype. In some cases, the 5'ITR and the 3'ITR are from an AAV9 serotype. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are in trans. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are in cis. In some cases, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence are in trans.

[0140] The Cap gene disclosed herein comprises any one of the sequences from Table 1 or Table 2, or FIG.

[0141] In some cases, the method includes packaging a first nucleic acid sequence encoding a therapeutic gene expression product such that it is encapsidated by a modified AAV capsid protein, hi some embodiments, the rAAV particles are isolated, concentrated, and purified using a suitable virus purification method, such as those described herein.

[0142] In some cases, the rAAV of the present disclosure is produced using the method described in Challis, RC et al., Nat. Protoc. 14, 379 (2019). Briefly, triple transfection of HEK293T cells (ATCC) is performed using polyethyleneimine (PEI), and virus is collected from both cell lysate and medium after 120 hours and purified with iodixanol. In a non-limiting example, rAAV is produced by triple transfection of precursor cells (e.g., HEK293T) cells using a standard transfection protocol (e.g., PEI). Viral particles are collected from the medium after a period of time (e.g., 72 hours after transfection) and from the cells and medium at a later time point (e.g., 120 hours after transfection). Virus present in the medium is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500 mM sodium chloride, and the precipitated virus is added to a lysate prepared from the collected cells. Virus is purified on an iodixanol (Optiprep, Sigma) step gradient (15%, 25%, 40% and 60%). Virus is concentrated and formulated in PBS. Viral titers are determined by measuring the number of DNaseI-resistant vector genome copies (VG) using qPCR and a linearized genome plasmid as a control.

[0143] The cell may be selected from human, primate, murine, feline, canine, porcine, ovine, bovine, equine, epinephrine, caprine, and wolf host cells. In some cases, the cell is a progenitor or precursor cell, such as a stem cell. In some cases, the stem cell is a mesenchymal cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a fibroblast, or another tissue-specific stem cell. The cell may be immortalized. In some cases, the immortalized cell is a HEK293 cell. In some cases, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in conjunction with any transgenic strain that expresses a recombinase in a target cell type of interest to develop an AAV capsid that more efficiently transduces that target cell population.

[0144] Treatment Method Disclosed herein are methods of treating a disease or condition, or a symptom of a disease or condition, in a subject, comprising administering to the subject a therapeutically effective amount of one or more compositions (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) disclosed herein. In some embodiments, the composition is a rAAV capsid protein described herein. In some embodiments, the composition is an isolated or purified rAAV capsid protein described herein. In some embodiments, the rAAV particles encapsidate an AAV vector containing a transgene (e.g., a therapeutic nucleic acid). In some embodiments, the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising rAAV particles and a pharma- ceutically acceptable carrier. In some embodiments, one or more compositions are administered alone (e.g., stand-alone therapy) to a subject. In some embodiments, the composition is a first-line treatment for a disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line treatment for a disease or condition.

[0145] Recombinant adeno-associated virus (rAAV)-mediated gene delivery exploits the AAV mechanism of viral transduction for nuclear expression of episomal heterologous nucleic acids (e.g., transgenes, therapeutic nucleic acids). Upon delivery to the host's in vivo environment, rAAV (1) binds or attaches to cell surface receptors of target cells, (2) endocytose, (3) transports to the nucleus, (4) uncoats the virus to release the encapsidated heterologous nucleic acid, (5) converts the heterologous nucleic acid from single-stranded DNA to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribes the episomal heterologous nucleic acid in the host cell's nucleus ("transduction"). rAAV engineered to have increased transduction enrichment (transcription of episomal heterologous nucleic acids in host cells) is desirable for gene therapy applications.

[0146] Aspects disclosed herein provide a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an rAAV of the present disclosure or a pharmaceutical formulation of the present disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, the administration is intracranially, intraventricular, intracerebroventricular, intravenously, intraarterially, intranasally, intrathecally, intracisternally, or subcutaneously.

[0147] Provided herein is a method for treating a disease or condition associated with abnormal expression or activity of a target gene or its gene expression product, comprising regulating the expression or activity of the target gene or gene expression product in a subject by administering an rAAV that encapsidates a heterologous nucleic acid of the present disclosure. In some cases, the expression or activity of the target gene or gene expression product is reduced compared to the expression or activity in a normal (non-disease) individual, and administering the rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product. In some cases, the expression or activity of the gene or gene expression product is increased compared to the expression or activity of a normal individual, and administering the rAAV to the subject is sufficient to decrease the expression or activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer's disease, in some cases caused by a gain of function of presenilin 1 and / or presenilin 2 (encoded by the genes PSEN1 and PSEN2, respectively), is administered an rAAV disclosed herein that encapsidates a therapeutic nucleic acid that is a silencing RNA (siRNA) or other RNAi that has a loss-of-function effect on PSEN1 mRNA.

[0148] Also provided is a method for preventing a disease or condition disclosed herein in a subject, comprising administering to the subject a therapeutically effective amount of a rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product as described herein.The rAAV vector can be encapsidated into a modified capsid protein or rAAV viral particle as described herein.In some cases, the therapeutic gene expression product is effective for regulating the activity or expression of a target gene or gene expression product.

[0149] Disclosed herein is a method for treating disease or symptoms in a subject by administering a composition comprising the rAAV disclosed herein.The advantage of the rAAV disclosed herein is that it can be used to treat virtually any disease or symptoms that benefit from transgene therapy, including but not limited to lysosomal storage diseases, including Friedreich's ataxia, Pompe disease and Danon disease, chronic heart failure, congestive heart failure, cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy and arrhythmogenic cardiomyopathy, and arrhythmia.

[0150] In some cases, the disease or condition is localized to a particular in vivo environment in the subject, for example, the heart. The compositions of the present disclosure are particularly useful for treating the diseases or conditions described herein, since they specifically or more efficiently target the in vivo environment and deliver engineered therapeutic nucleic acids that regulate the activity or expression of target gene expression products involved in the etiology or pathology of the disease or condition.

[0151] Provided herein are methods of treating a disease or condition, or a symptom of a disease or condition, in a subject, comprising: (a) diagnosing a subject having a disease or condition that affects a target in vivo environment; and (b) treating the disease or condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., an rAAV particle, an AAV vector, a pharmaceutical composition), where the composition has been engineered to have increased specificity for the target in vivo environment.

[0152] Disclosed herein are methods of treating a disease or condition, or a symptom of a disease or condition, affecting a target in a subject, comprising: (a) administering to the subject a composition (e.g., rAAV particles, AAV vectors, pharmaceutical compositions); and (b) expressing a therapeutic nucleic acid in a target in vivo environment in the subject that has increased transduction enrichment.

[0153] In some embodiments, the method further comprises reducing or eliminating delivery of the heterologous nucleic acid in off-target in vivo environments, such as the liver, In some embodiments, the delivery is characterized by increased enrichment of transduction (e.g., of the heterologous nucleic acid) in the heart.

[0154] In some embodiments, a method of treating a disease or condition affecting the heart comprises administering rAAV particles to the heart in a subject, the rAAV particles comprising a rAAV capsid protein comprising an insertion of about 5, 6, or 7 amino acids of the amino acid sequence provided in Tables 1-2, FIG. 1, and Formulas I-VII at amino acid positions 588-589 of the parent AAV capsid protein. In some embodiments, a method of treating a disease or condition affecting the heart comprises administering rAAV particles to the heart in a subject, the rAAV particles comprising a rAAV capsid protein comprising an insertion of about 5, 6, or 7 amino acids of the amino acid sequence as well as one or more substitutions at amino acids [AQAQ] found at amino acid positions 587-590 as provided in Tables 1-2, FIG. 1, and Formulas I-VII. In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein (e.g., as provided in SEQ ID NO: 1).

[0155] Also provided are methods of regulating a target gene expression product, the methods comprising administering a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein to a subject in need thereof. For example, the methods provided herein comprise administering to a subject an rAAV having an rAAV capsid protein that encapsidates a viral vector that includes a heterologous nucleic acid that regulates the expression or activity of a target gene expression product.

[0156] The term "normal individual" refers to an individual not affected by a disease or condition characterized by altered expression or activity of a gene or its gene expression product.

[0157] In some examples, the method is useful for treating a degenerative muscle disease or condition (e.g., familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease with resulting ischemic cardiomyopathy). In some examples, the subject method is useful for treating an individual with a heart or cardiovascular disease or disorder, such as cardiovascular disease, aneurysm, angina pectoris, arrhythmia, atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery, arterial disease dilation, diastolic dysfunction, endocarditis, high blood pressure (hypertension), cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease with resulting ischemic cardiomyopathy, mitral valve prolapse, myocardial infarction (heart attack), or venous thromboembolism. In some examples, the subject is suffering from or at risk of cardiomyopathy.

[0158] In some embodiments, the cardiac disease or condition is selected from lysosomal storage diseases, including Friedreich's ataxia, Pompe disease, and Danon disease, chronic heart failure, congestive heart failure, cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic cardiomyopathy, and arrhythmias.

[0159] In some embodiments, the pharmaceutical formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some cases, the therapeutic gene expression product is effective to regulate the activity or expression of a target gene or gene expression product selected from MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin, and FXN.

[0160] In some cases, the therapeutic gene expression product comprises a gene editing component. In some cases, the gene editing component is selected from an artificial site-specific RNA endonuclease (ASRE), a zinc finger endonuclease (ZFN), a transcription factor-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas enzyme, and a CRISPR / Cas guide RNA.

[0161] In some cases, expression of the gene or expression or activity of the gene expression product is inhibited by administration of the composition to a subject. In some cases, expression of the gene or expression or activity of the gene expression product is enhanced by administration of the composition to a subject.

[0162] Formulation, Dosage, and Route of Administration Disclosed herein are methods comprising delivering rAAV particles that encapsidate a heterologous nucleic acid to the heart in a subject, the rAAV particles comprising (i) increased transduction of the heterologous nucleic acid in the heart, wherein the rAAV particles have an rAAV capsid protein comprising an insertion of 5, 6, or 7 amino acids of the amino acid sequence provided in Tables 1-2, FIG. 1, and Formulas I-VII at amino acid positions 588-589 of the parent AAV capsid protein, as well as one or more substitutions at amino acids [AQAQ] found at amino acid positions 587-590 as provided in Tables 1-2, FIG. 1, and Formulas I-VII.

[0163] In general, the methods disclosed herein include administering the therapeutic rAAV composition by systemic administration. In some cases, the methods include administering the therapeutic rAAV composition by intravenous ("iv") administration. The therapeutic rAAV composition can be administered by additional routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intraventricular administration, intrathecal administration, intracisternal administration, or any other suitable parenteral administration. The route, dosage, time point, and duration of administration of the therapeutic agent may be adjusted. In some embodiments, administration of the therapeutic agent is before or after the onset of either or both acute and chronic symptoms of the disease or condition. Other routes of delivery to the heart include, but are not limited to, intracranial administration, lateral ventricular administration, and intravascular administration.

[0164] The effective dose and administration of the pharmaceutical composition for preventing or treating a disease or condition disclosed herein is defined by an observed beneficial response associated with the disease or condition, or a symptom of the disease or condition. A beneficial response includes preventing, alleviating, arresting, or curing the disease or condition, or a symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity of a biomarker, transcriptome risk profile, or gut microbiota in a subject. "Improvement," as used herein, refers to a shift in the presence, level, or activity to that observed in a normal individual (e.g., an individual not suffering from the disease or condition). In cases where the therapeutic rAAV composition is not therapeutically effective or does not provide sufficient relief of the disease or condition, or a symptom of the disease or condition, the dosage and / or route of administration may be altered, or additional agents may be administered to the subject along with the therapeutic rAAV composition. In some embodiments, once a patient is initiated on a regimen of a therapeutic rAAV composition, the patient is also weaned off the second treatment regimen (e.g., dose tapering).

[0165] In some cases, the dose of the pharmaceutical composition is at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 In some cases, the concentration of infectious particles is greater than or equal to 2×10 7 , 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16 , or 2 × 10 17 In some cases, the concentration of infectious particles is 3×10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , or 3 × 10 17 In some cases, the concentration of infectious particles is 4×10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 11 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 , or 4 × 10 17 In some cases, the concentration of infectious particles is 5×10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11, 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 , or 5 × 10 17 In some cases, the concentration of infectious particles is 6×10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11 , 6×10 12 , 6×10 13 , 6×10 14 , 6×10 15 , 6×10 16 , or 6 × 10 17 In some cases, the concentration of infectious particles is 7×10 7 , 7×10 8 , 7×10 9 , 7×10 10 , 7×10 11 , 7×10 12 , 7×10 13 , 7×10 14 , 7×10 15 , 7×10 16 , or 7 × 10 17 In some cases, the concentration of infectious particles is 8×10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , or 8 × 10 17 In some cases, the concentration of infectious particles is 9×10 7 , 9×10 8 , 9×10 9 , 9×10 10 , 9×10 11 , 9×10 12 , 9×10 13 , 9×10 14 , 9×10 15 , 9×10 16 , or 9×1017 It is.

[0166] In some embodiments, the present specification discloses the formulation of pharma- ceutically acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable administration and treatment regimens for using the specific compositions described herein in various treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition may be adjusted to obtain a suitable dosage at any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0167] In some embodiments, pharmaceutical forms of rAAV-based virus compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars or sodium chloride.

[0168] In some cases, for administration of injectable aqueous solutions, the solution may be appropriately buffered as necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any case determine the appropriate dose for each individual subject. Moreover, for human administration, preparations should meet the sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

[0169] Sterile injectable solutions containing the rAAV compositions disclosed herein are disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in a suitable solvent together with some of the other components listed above as necessary, followed by filtration sterilization.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary components from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying techniques that obtain a powder of active ingredient + any additional desired components from its previously sterile filtered solution.Injectable solutions can be advantageous for systemic administration, for example, by intravenous or intrathecal administration.

[0170] The appropriate dose and dosage to be administered to a subject will be determined by factors including, but not limited to, the particular therapeutic rAAV composition, the symptoms of the disease and their severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0171] The amount of rAAV composition and the time of administration of such composition will be within the scope of a person skilled in the art who has the benefit of the present teachings.However, it is highly likely that the administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, for example, a single injection of a sufficient number of infectious particles to provide therapeutic benefit to the patient undergoing such treatment.This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, eliminating the need for multiple or chronic administrations.

[0172] In certain embodiments, data obtained from cell culture assays and animal studies are used in formulating therapeutically effective daily dose ranges and / or therapeutically effective unit doses for use in mammals, including humans. In certain embodiments, the dose ranges and / or unit doses vary within this range depending on the dosage form employed and the route of administration utilized.

[0173] Combination therapy Therapeutic rAAV can be used alone or in combination with additional therapeutic agents (together, "therapeutic agents"). In some cases, therapeutic rAAV as used herein is administered alone. Therapeutic agents can be administered together or sequentially in combination therapy. The combination therapy can be administered on the same day, or one or more days, weeks, months, or years apart.

[0174] The additional therapeutic agent may include a small molecule. The additional therapeutic agent may include an antibody or antigen-binding fragment. The additional therapeutic agent may include a lipid nanoparticle-based therapy, an antisense oligonucleotide therapy, as well as other viral therapies.

[0175] The additional therapeutic agent may include cell-based therapy. Exemplary cell-based therapies include, but are not limited to, immune effector cell therapy, chimeric antigen receptor T cell (CAR-T) therapy, natural killer cell therapy, and chimeric antigen receptor natural killer (NK) cell therapy. Either NK cells, or CAR-NK cells, or a combination of both NK cells and CAR-NK cells, may be used in combination with the methods disclosed herein. In some embodiments, the NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSCs), umbilical cord blood, or cell lines. The NK cells and CAR-NK cells may include cytokine receptors and suicide genes. The cell-based therapy may include stem cell therapy. The stem cell therapy may be embryonic or somatic stem cells. The stem cells may be isolated from a donor (allogeneic) or from the subject (autologous). The stem cells can be expanded adipose-derived stem cells (eASCs), hematopoietic stem cells (HSCs), mesenchymal stem (stromal) cells (MSCs), or induced pluripotent stem cells (iPSCs) derived from cells of the subject.

[0176] kit The present specification discloses a kit that comprises the composition disclosed herein.The present specification also discloses a kit for treating or preventing heart disease or condition.In some cases, the disease or condition is cancer, pathogen infection, lung disease or condition, neurological disease, muscular disease, or immune disorder, such as those described herein.

[0177] In one embodiment, the kit may include a therapeutic or prophylactic composition comprising an effective amount of a rAAV particle that encapsidates a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., a therapeutic nucleic acid) and a composition of recombinant AAV (rAAV) capsid proteins of the present disclosure. In another embodiment, the kit may include a therapeutic or prophylactic composition comprising an effective amount of a cell modified by rAAV ("modified cell") described herein (which expresses a therapeutic nucleic acid) in a unit dosage form. In some embodiments, the kit includes a sterile container that can contain the therapeutic composition, which can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceutical products.

[0178] Optionally, the kit further comprises a cell. Optionally, the cell is mammalian. Optionally, the cell is immortalized. Optionally, the immortalized cell is an embryonic stem cell. Optionally, the embryonic stem cell is a human embryonic stem cell. Optionally, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293). Optionally, the kit further comprises an AAV vector comprising a heterologous nucleic acid encoding a therapeutic gene expression product. Optionally, the AAV vector is episomal.

[0179] In some cases, the rAAV is provided with instructions for administering the rAAV to a subject having or at risk of developing a disease or condition, such as a heart condition. The instructions may generally include information regarding the use of the composition for the treatment or prevention of the disease or condition.

[0180] In some cases, the instructions include at least one of the following: a description of the therapeutic rAAV composition, dosage schedules and administration for treating or preventing a disease or condition disclosed herein, cautions, warnings, indications, non-indications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or references. The instructions may be printed directly on the container (if present) or may be printed as a label affixed to the container or as a separate sheet, pamphlet, card, or folder provided in or with the container. In some cases, the instructions provide instructions for administering the rAAV to the subject alone. In some cases, the instructions provide that the rAAV is formulated for systemic delivery.

[0181] definition The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0182] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to convention, for a given value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean an acceptable error range of the particular value.

[0183] As used herein, "consisting essentially of" when used to define compositions and methods shall mean excluding other elements of essential importance to the combination for the purpose described. Thus, a composition consisting essentially of elements as defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure, such as compositions for treating skin disorders such as acne, eczema, psoriasis, and rosacea.

[0184] The terms "homologous," "homology," or "percent homology" are used herein to generally mean an amino acid sequence or a nucleic acid sequence that has the same or similar sequence as a reference sequence. Percent sequence homology may be determined using the latest version of BLAST as of the filing date of this application.

[0185] The term "increased" or "increase" is generally used herein to mean an increase of a statically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% or any increase between 10-100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more compared to a reference level.

[0186] The term "reduced" or "reduction" is generally used herein to mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reduction" means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% compared to a reference level (e.g., nonexistent or undetectable levels compared to a reference level), or any reduction between 10-100%. In the context of a marker or symptom, these terms refer to a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more than 40%, preferably down to a level that is accepted as within the normal range for an individual without a given disease.

[0187] The term "subject" is any living organism. In some cases, the organism is a mammal. Non-limiting examples of mammals include any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. In one aspect, the mammal is a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, e.g., a rodent such as a rat or mouse. In one embodiment, a "non-human primate" is a mammal, e.g., a monkey. In some cases, the subject is a patient, which as used herein may refer to a subject diagnosed with a particular disease or disorder.

[0188] The term "gene," as used herein, refers to a segment of nucleic acid that encodes a particular protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally together with associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

[0189] The term "adeno-associated virus" or "AAV" as used herein refers to adeno-associated virus or its derivatives.Non-limiting examples of AAV include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.In some cases, AAV is described as "primate AAV", which refers to the AAV that infects primates. Similarly, AAV can infect bovine animals (e.g., "bovine AAV"). In some cases, the AAV is wild-type or naturally occurring. In some cases, the AAV is recombinant.

[0190] The term "AAV capsid" as used herein refers to the capsid protein or peptide of adeno-associated virus.In some cases, the AAV capsid protein is configured to encapsidate genetic information (e.g., transgene, therapeutic nucleic acid, viral genome).In some cases, the AAV capsid of the present disclosure is a modified AAV capsid compared to the corresponding parent AAV capsid protein.

[0191] The term "tropism" as used herein refers to the quality or characteristic of AAV capsid, which may include the specificity and / or the increase or decrease of enrichment for expressing encapsidated genetic information in an in vivo environment compared to a second in vivo environment.In some cases, the in vivo environment is a cell type.In some cases, the in vivo environment is an organ or organ system.

[0192] The term "AAV vector" as used herein refers to a nucleic acid polymer that encodes genetic information related to a virus.AAV vector can be a recombinant AAV vector (rAAV), which refers to an AAV vector that is produced using recombinant genetic methods.In some cases, the rAAV vector comprises at least one heterologous polynucleotide (e.g., a polynucleotide other than the wild-type or naturally occurring AAV genome, such as a transgene).

[0193] As used herein, the term "AAV particle" refers to an AAV virus, virion, AAV capsid protein or a component thereof. In some cases, the AAV particle is modified relative to the parent AAV particle.

[0194] The term "gene product" of "gene expression product" refers to an expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein, or RNA, including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA).

[0195] The term "heterologous" as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[0196] The term "endogenous" as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is naturally present in or associated with an organism or a particular cell within an organism.

[0197] As used herein, the terms "treat", "treating" and "treatment" refer to alleviating or suppressing a disorder, disease or condition, or one or more symptoms associated with a disorder, disease or condition; or alleviating or eradicating the cause of the disorder, disease or condition itself. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis.

[0198] The term "therapeutically effective amount" refers to an amount of a compound or therapy that, when administered, is sufficient to prevent or alleviate to some extent the onset of one or more of a disorder, symptoms of a disease, or symptoms of a disease; or an amount of a compound that is sufficient to elicit the biological or medical response in a cell, tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician.

[0199] The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component may be "pharmaceutical acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and may be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005, Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds. The Pharmaceutical Press and the American Pharmaceutical Association: 2005, and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007, Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0200] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. A pharmaceutical composition can facilitate administration of a compound to an organism. Multiple techniques for administering a compound exist in the art, including, but not limited to, systemic administration.

[0201] Non-limiting examples of "samples" include any material from which nucleic acid and / or protein can be obtained. As non-limiting examples, this includes whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph, fecal extracts, buccal swabs, cells or other bodily fluids or tissues (including but not limited to tissues obtained by surgical biopsy or surgical resection). Alternatively, samples can be obtained through cell lines from primary patients or archived patient samples in the form of archived samples or freshly frozen samples.

[0202] The term "in vivo" is used to describe events that take place inside a subject's body.

[0203] The term "in vitro" is used to describe events that occur contained in a container for holding laboratory reagents such that the material is separated from the biological source from which it is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0204] The term "heart" or "cardiovascular system" refers to tissues selected from the heart, veins, and aorta.

[0205] The term "systemic delivery" is defined as the route of administration of a pharmaceutical or other substance into the circulatory system such that the entire body is affected. Administration can occur via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally by injection, infusion, or implantation). "Circulatory system" includes the blood circulatory system. Examples of systemic administration include intra-arterial, intravenous, or intrathecal injection. The terms "systemic administration" and "systemic delivery" are used interchangeably.

[0206] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0207] Example 1 Methods for identifying modified capsid proteins in cynomolgus monkeys A major concern for the therapeutic applicability of engineered adeno-associated viruses (AAVs) is how well their transduction profiles translate to human applications. Previous technical efforts have focused on in vitro or in vivo rodent screening platforms due to their ease and flexibility of use, but direct screening efforts in non-human primates (NHPs) are much more likely to identify transforming viruses. We selected the archaic NHP, the cynomolgus monkey, for our technical efforts. We focused our technical efforts on a region of the AAV9 capsid surface located at amino acid position 588, a variable region between natural AAV serotypes and one of the most exposed loops on the capsid surface that has a role in receptor binding. We created and analyzed a library of viral capsids by making random 7 amino acid insertions at this site in AAV9 for a novel tropism toward the heart of NHPs.

[0208] Plasmids.The first-round viral DNA library was generated by amplification of a section of the AAV9 capsid genome between amino acids 450-599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 588 and 589 with all possible variations. The resulting library inserts were then introduced into the rAAV-ΔCap-in-rev-RNA plasmid via Gibson assembly as previously described. The resulting capsid DNA library, rAAV-Cap-Cag-GFP11, contained a diversity of approximately 1.28 billion variants at the amino acid level. The second-round viral DNA library was generated similarly to the first round, but instead of the NNK degenerate primers inserted at 588, a synthetic oligo pool (Twist Biosicence) was used to generate only selected variants in the UBC-Cap-DNA and CAG-Cap-DNA constructs with CAP. This second-round DNA library contained a diversity of 20,000 variants at the amino acid level, with a three-codon overlap for each variant. The AAV2 / 9 REP-AAP-ΔCAP plasmid, transfected into HEK293T cells to provide the Rep genes for library virus generation, prevents the generation of wild-type AAV9 capsids during viral library generation after valid recombination events between this plasmid, which is co-transfected with the library plasmid at each stage containing the library insert.

[0209] Virus production. Recombinant AAV was generated according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple transfected with four vectors using polyethylenimine (PEI). The first vector was the rAAV-Cap-in-cis-Lox library, flanked by inverted terminal repeat (ITR) sequences from the parent AAV virus. The second vector was the AAV2 / 9 REP-AAP-ΔCAP plasmid. The third vector contains nucleic acids encoding helper virus proteins required for virus assembly and packaging of heterologous nucleic acid into a modified capsid structure. The fourth is a pUC-18 plasmid included to achieve the correct PEI / DNA ratio for optimal transfection enrichment. To reduce the chance of multiple library DNAs entering the same cell, only 10 ng of rAAV-Cap-in-cis-Lox library DNA (per 150 mm plate) was transfected. 60 hours after transfection, viral particles are collected from the cells and medium. The virus present in the medium is concentrated by precipitation with 8% polyethylene glycol and 500 mM sodium chloride, and the precipitated virus is added to a lysate prepared from the collected cells. The virus is purified on an iodixanol (Optiprep, Sigma) step gradient (15%, 25%, 40% and 60%). The virus is concentrated and formulated in PBS. The virus titer is determined by measuring the number of DNaseI-resistant vector genome copies (VG) using qPCR and a linearized genome plasmid as a control.

[0210] Animals. Cynomolgus monkey procedures were approved by Envol Biomedical's IACUC committee. Cynomolgus monkeys were born and raised in the Envol Biomedical colony and housed in family groups under standard conditions. They fed ad libitum and received enrichment as part of Envol Biomedical's NHP primate enrichment program. For AAV infusion, animals were screened for endogenous neutralizing antibodies (Nab). None of the screened animals showed a detectable blocking reaction at a 1:10 dilution of serum. They were then housed for several days and allowed to acclimate to the new room before injection. A total of four juvenile monkeys were used for screening, two for the first-round library and two for the second-round library. Animals were restrained and test substances were administered via intravenous infusion for 10 min. Activity and behavior were monitored daily throughout their lives.

[0211] DNA / RNA recovery and sequencing. Round 1 and round 2 viral libraries were cultured at 2.5 × 10 13rAAV was injected into cynomolgus monkeys at a dose of 10 ... Viral genomes were enriched by 25 cycles of PCR amplification with primers flanking the insertion site at 588–589 of the capsid genome using 50% of the total extracted viral DNA as template. After Zymo DNA purification, samples were diluted 1:10–1:1000 depending on tissue type, and each dilution was further amplified around the library variable region using 10 cycles of PCR. Samples were then amplified for an additional 10 cycles using custom primers with Illumina Indices. Amplification products were run on a 2% low melting agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 210 bp band.

[0212] For the second round library only, packaged viral library DNA was isolated from the injected viral library by digestion of the viral capsid and purification of the contained ssDNA. These viral genomes were amplified by two PCR amplification steps and purified after gel electrophoresis, as was the viral DNA extracted from tissues, to add adapters and indexes for Illumina next generation sequencing. This viral library DNA, together with the viral DNA extracted from tissues, was sent for deep sequencing using the Illumina NextSeq 2000 system.

[0213] NGS data alignment and processing. Raw fastq files from the NGS runs were processed using custom-written scripts (Capsida CapSeq Tools). For the first-round libraries, the pipeline for processing these datasets involved filtering to remove low-quality reads, utilizing a quality score for each sequence, and eliminating bias from PCR-induced mutations or high GC content. The filtered datasets were then aligned by an exact string match algorithm and trimmed to improve alignment quality. Read counts for each sequence were pulled and displayed per tissue, at which point all sequences found in the heart were compiled for the formation of second-round libraries.

[0214] For the second round library, the read counts per tissue were tabulated similarly. A read count of 1 was then added to each sequence to remove values ​​of 0, all heart regions for each sequence were summed together, and the read sequences for each codon overlap of a given 7-mer amino acid sequence were summed together to give a single value for each peptide insertion. Finally, the data was normalized by log10 counts per million (Cpm). Enrichment values ​​were calculated using the normalized cpm of heart versus cpm of virus and transformed to log10.

[0215] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0216] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An AAV capsid protein comprising a sequence as provided in Tables 1-2 or FIG.

2. Insert sequence of formula I X １ -G-G-X ２ -X ３ -X ４ -X ５（ I) (In the formula, X １ is an amino acid selected from G, I, K, L and V; X ２ is an amino acid selected from E, G, H, L, N, S and Q; ３ is an amino acid selected from A, F, I, L, P, T and Y; ４ is an amino acid selected from F, L, S and Y; ５ is an amino acid selected from E, H, I, K, P, S and T. An AAV capsid comprising:

3. X １ The AAV capsid protein of claim 2, wherein is I, K or L.

4. X １ The AAV capsid protein of claim 2, wherein is K.

5. X １ The AAV capsid protein of claim 2, wherein is I.

6. X ２ The AAV capsid protein of claim 2, wherein is L, H or Q.

7. X ２ The AAV capsid protein of claim 2, wherein is L.

8. X ２ The AAV capsid protein of claim 2, wherein is H.

9. X ３ The AAV capsid protein of claim 2, wherein is I, P or Y.

10. X ４ The AAV capsid protein of claim 2, wherein is F or L.

11. X ５ The AAV capsid protein of claim 2, wherein is I or H.

12. 3. The AAV capsid protein of claim 2, wherein the sequence is selected from KGGLPFI, IGGHYLH, LGGQILI, LGGGFSE, IGGNLLK, KGGETSS, VGGSTYP, and GGGLAST.

13. Insert sequence of formula II _____ ６ ︹ ７ ︹ ８ ︹ ９ ︹ １０（ ..) (In the formula, X ６ is an amino acid selected from E, G, I, N, Q and T; ７ is an amino acid selected from L, H, Q, S and T; ８ is an amino acid selected from D, E, H, P and S; ９ is an amino acid selected from A, F, L, R, S and T; X １０ is an amino acid selected from E, I, M, S and V. The AAV capsid protein comprising:

14. X １０ is I, V or M.

15. X １０ The AAV capsid protein of claim 13, wherein is M.

16. X ９ is F or L.

17. X ８ is P or H. The AAV capsid protein of claim 13 .

18. X ７ The AAV capsid protein of claim 13, wherein is H or L.

19. X ６ is E or G. The AAV capsid protein of claim 13 .

20. 14. The AAV capsid protein of claim 13, wherein the insertion sequence is selected from RSGHPFV, RSGLHLM, RSELHFI, RSQTSTE, RSISDRV, RSTTDAS, and RSNQESV.

21. Insert sequence of formula III R-X １１ -G-X １２ -X １３ -X １４ -X １５（ III) (In the formula, X １１ is an amino acid selected from A, D, E, I, N, V ​​and S; １２ is an amino acid selected from E, H, L, M, S and V; X １３ is an amino acid selected from F, H, L, P, V and Y; １４ is an amino acid selected from F, L and T; １５ is an amino acid selected from D, H, K, L, M, Q, S and V. The AAV capsid protein comprising:

22. X １１ is an amino acid selected from A, D, N and S.

23. X １２ is an amino acid selected from E, H, L, and V.

24. X １３ is an amino acid selected from F, H, P, and Y.

25. X １４ is F or L. The AAV capsid protein of claim 21 .

26. X １５ is an amino acid selected from K, L, M and V.

27. 22. The AAV capsid protein of claim 21, wherein the insertion sequence is selected from RSGHPFV, RNGEHFK, RDGHYLL, RNGEFFH, REGMPFL, RAGVHFV, RSGLHLM, RIGVHTS, RIGSVTQ, and RVGSLTD.

28. Insert sequence of formula IV X １６ -X １７ -G-H-X １８ -X １９ -X ２０（ (U) (In the formula, X １６ is an amino acid selected from I, R and L; X １７ is an amino acid selected from D, G, Q and S; １８ is an amino acid selected from I, P and Y; １９ is F or L, and X ２０ is an amino acid selected from H, L and V The AAV capsid protein comprising:

29. X １６ is L or R.

30. X １７ is an amino acid selected from P, Q and S.

31. X １９ is F or L.

32. X ２０ The AAV capsid protein of claim 28, wherein is L.

33. X ２０ is V.

34. X １８ is I or Y.

35. 29. The AAV capsid protein of claim 28, wherein the insertion sequence as set forth in FIG. 1 is selected from RSGHPFV, RDGHYLL, LQGHILL, and IGGHYLH.

36. Insert sequence of formula V R-X ２１ -X ２２ -X ２３ -H-F-X ２４（ V) (In the formula, X ２１ is an amino acid selected from A, N and S; X ２２ is an amino acid selected from D, E and G; ２３ is an amino acid selected from E, L and V; X ２４ is an amino acid selected from I, K, Q and V. AAV capsid proteins, including AAV capsid proteins comprising:

37. X ２１ The AAV capsid protein of claim 36, wherein is A.

38. X ２２ The AAV capsid protein of claim 36, wherein is D.

39. X ２３ The AAV capsid protein of claim 36, wherein is E.

40. X ２３ The AAV capsid protein of claim 36, wherein is L.

41. X ２４ The AAV capsid protein of claim 36, wherein is Q.

42. 37. The AAV capsid protein of claim 36, wherein the insertion sequence is selected from RADLHFQ, RAGVHFV, RNGEHFK, and RSELHFI.

43. Insert sequence of formula VI X ２５ -X ２６ -X ２７ -X ２８ -X ２９ -L (V) (In the formula, X ２５ is an amino acid selected from A, L, R and Y; ２６ is an amino acid selected from Q, E, D, T, K and F; ２７ is an amino acid selected from G, Q and A; ２８ is an amino acid selected from H, G, T, V and D; X ２９ is an amino acid selected from I, S, T and Y. AAV capsid proteins, including AAV capsid proteins comprising:

44. X ２５ is L or R.

45. X ２６ is an amino acid selected from Q, E and D.

46. X ２７ is G or Q.

47. X ２８ is H or G.

48. X ２９ is I or Y.

49. 44. The AAV capsid protein of claim 43, wherein the insertion sequence is selected from LQGHILL, REQGYLL, RDGHYLL, YTGTSLL, AKAVTLL, and AFGDSLL.

50. Insert sequence of formula VII X ３０ -X ３１ -X ３２ -X ３３ -X ３４ -S-S(VII) (In the formula, X ３０ is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, S, T, V and Y; X ３１ is an amino acid selected from F, G, H, I, K, L, M, P, Q, R, S, T and V; X ３２ is an amino acid selected from A, G, H, L, M, P, Q, S and V; X ３３ is an amino acid selected from E, G, H, I, P, Q, R, S, T and V; X ３４ is an amino acid selected from D, G, I, L, M, N, R, S, T and V. The AAV capsid protein comprising:

51. X ３０ is V, Q or H.

52. X ３１ is F, L or M.

53. X ３２ is S, A or Q.

54. X ３３ is G, H or S.

55. X ３４ is G, V or M.

56. 51. The AAV capsid protein of claim 50, wherein the sequence is selected from VFSGVSS, QLQHGSS, HMASMSS, SSLTLSS, SVPSSSS, ITPSVSS, MTVQVSS, SHLSVSS, YPHQTSS, PQGGSS, FRSSLSS, ISVSISS, ETQRGSS, IHPPSSS, TSAITSS, FKSQSSS, ASHSTSS, AVMRDSS, KGGETSS, VVSVTSS, LISIRSS, NSLGLSS, and HLAQNSS.

57. Insert sequence of formula VIII ︤️ ３５ ︹ ３６ ︹ ３７ ︹ ３８ ︹ ３９（ _==) (In the formula, X ３５ is an amino acid selected from G, H, Q, S and T; ３６ is an amino acid selected from K, N, P, R and S; X ３７ is an amino acid selected from H, L, R and S; ３８ is an amino acid selected from D, K, R, S, T and V; X ３９ is an amino acid selected from A, D, E, L and V. The AAV capsid protein comprising:

58. X ３５ is H or S.

59. X ３６ is K or R.

60. X ３７ is L or S.

61. X ３８ is S or V.

62. X ３９ is A or L.

63. 58. The AAV capsid protein of claim 57, wherein the sequence is selected from DLSRLSL, DLHKSVA, DLGNLKV, DLTPHKD, DLQRSDL, and DLGSRRE.

64. The AAV capsid protein of any one of claims 1 to 63, wherein the AAV is AAV9.

65. 65. The AAV capsid protein of any one of claims 1 to 64, wherein the AAV is provided in SEQ ID NO:

1.

66. 66. The AAV capsid protein of any one of claims 1 to 65, wherein the insertion is between amino acid 588 and amino acid 589.

67. The AAV capsid protein of any one of claims 1 to 66, wherein 60 copies of the AAV capsid protein are assembled into the AAV capsid.

68. 68. An AAV capsid protein according to any one of claims 1 to 67, which is present in VP1, VP2, and VP3 of the AAV capsid.

69. 69. The AAV capsid protein of any one of claims 1 to 68, characterized by at least one increased transduction enrichment when measured in the cardiovascular system in a subject when systemically delivered to the subject.

70. 70. The AAV capsid protein of any one of claims 1 to 69, further comprising an amino acid substitution comprising A589N or Q590P.

71. An AAV capsid comprising an AAV capsid protein according to any one of claims 1 to 70.

72. 72. The AAV capsid of claim 71, which is chimeric.

73. 73. An AAV capsid according to any one of claims 71 to 72, which is isolated and purified.

74. 74. The AAV capsid of any one of claims 71 to 73, formulated as a pharmaceutical preparation for systemic administration to treat a cardiac disease or condition, said pharmaceutical preparation further comprising a pharma- ceutically acceptable carrier.

75. The AAV capsid protein of any one of claims 1 to 70, wherein the capsid protein is expressed in the cardiovascular system.

76. The AAV capsid protein of claim 75, wherein the cardiovascular system comprises a cardiac cell.

77. The AAV capsid of any one of claims 75 to 76, wherein the cardiovascular system comprises a tissue selected from the heart.

78. A recombinant vector comprising a nucleic acid encoding an AAV capsid protein according to any one of claims 1 to 70.

79. a) a first vector comprising the recombinant vector of claim 78; b) a second vector encoding helper virus proteins; and c) a third vector comprising a therapeutic nucleic acid encoding a therapeutic gene expression product; Including the kit.

80. 71. A method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising the AAV capsid protein of any one of claims 1 to 70.

81. 81. The method of claim 80, wherein the disease or condition is a cardiac disease or condition in the subject.

82. A method for producing recombinant AAV particles from the AAV capsid of any one of claims 1 to 70, comprising: a. below: i. a first nucleic acid sequence encoding a therapeutic gene expression product; ii. a second nucleic acid sequence encoding a recombinant viral genome comprising a capsid (Cap) gene, the second nucleic acid sequence being modified to express the AAV capsid of any one of claims 1 to 70; and iii. A third nucleic acid sequence encoding an AAV helper virus genome. introducing into a cell a nucleic acid comprising b. assembling the recombinant AAV particles, the recombinant AAV particles comprising the AAV capsid that encapsidates a first nucleic acid; A method comprising:

83. 71. An AAV capsid protein according to any of claims 1 to 70, characterized by increased transduction enrichment when measured in tissues in a subject when systemically delivered to said subject.

84. The AAV capsid protein of any one of claims 1 to 70, wherein the tissue is heart or heart tissue.

85. An AAV particle comprising an AAV capsid protein according to any one of claims 1 to 70 and a viral genome.

86. A peptide comprising an amino acid sequence selected from DLSRLSL, KLDPLLM, TPKHLLI, PDQRRTD, RHDPLVM, SHFSGRN, VSAVRDR, VGVPVVL, LLPSNYL, QLQITKE, LQVTSVL, and VFSGVSS.

87. A recombinant adeno-associated virus (rAAV) comprising a capsid comprising any one of KGGLPFI, DLSRLSL, RSGHPFV, RADLHFQ, LQGHILL, KLDPLLM, IGGHYLH, RAGVHFV, LGGQILI, TPKHLLI, RNGEHFK, KDSIPFR, PDQRRTD, RHDPLVM, DNYTRHT, RSGLHLM, and TNSVRVV.

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