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CN-122029183-A - Recombinant AAV with improved tropism and specificity

CN122029183ACN 122029183 ACN122029183 ACN 122029183ACN-122029183-A

Abstract

The present disclosure provides a modified AAV capsid protein comprising a targeting peptide in Variable Region VIII (VRVIII). The modified AAV capsid proteins can form rAAV with preferred chemotaxis, specificity, or in vivo or in vitro biodistribution. The rAAV of the present disclosure can be used for gene therapy targeting specific tissues.

Inventors

  • C. F. Albright
  • CAO XIAOHONG
  • HUANG LINYA

Assignees

  • 艾菲尼亚治疗公司

Dates

Publication Date
20260512
Application Date
20231019
Priority Date
20221019

Claims (20)

  1. 1. A modified adeno-associated virus (AAV) capsid protein comprising Targeting peptides within variable region VIII (VR VIII), Wherein the targeting peptide has the sequence of X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 and X 1 、X 2 、X 3 、X 4 、X 5 、X 6 、X 7 、X 8 and X 9 are each independently selected from any amino acid residue.
  2. 2. The modified AAV capsid protein of claim 1, wherein (I) X 1 is independently selected from proline (P) and glycine (G); (ii) X 2 is independently selected from lysine (L), threonine (T), serine (S), alanine (a), valine (V), and isoleucine (I); (iii) X 3 is independently selected from asparagine (N), glutamine (Q), and proline (P); (iv) X 4 is independently selected from glycine (G) and alanine (a); (v) X 5 is independently selected from alanine (a), threonine (T), serine (S), valine (V), and glycine (G); (vi) X 6 is independently selected from valine (V), leucine (L), alanine (a), isoleucine (I), glycine (G), serine (S) and threonine (T); (vii) X 7 is independently selected from histidine (H), arginine (R), and lysine (K); (viii) X 8 is independently selected from leucine (L) and valine (V), and (Ix) X 9 is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K), and phenylalanine (F).
  3. 3. The modified AAV capsid protein according to claim 1 or 2, wherein the targeting peptide within VR VIII has a sequence selected from the group consisting of SEQ ID NOs 160-55819 and 55857-55859.
  4. 4. A modified AAV capsid protein according to any one of claims 1-3, wherein the targeting peptide has the sequence of PX 2 X 3 GAVX 7 LY (SEQ ID NO: 2), and X 2 、X 3 and X 7 are independently selected from any amino acid residue.
  5. 5. A modified AAV capsid protein according to claim 3, wherein (I) X 2 is independently selected from lysine (L), isoleucine (I), valine (V) and alanine (a); (ii) X 3 is asparagine (N) or glutamine (Q), and (Iii) X 7 is independently selected from histidine (H), arginine (R) and lysine (K).
  6. 6. The modified AAV capsid protein of claim 4 or 5, wherein the targeting peptide is: (i)PLQGAVHLY(SEQ ID NO:3); (ii)PLQGAVRLY(SEQ ID NO:4); (iii)PLQGAVKLY(SEQ ID NO:5); (iv)PINGAVHLY(SEQ ID NO:6); (v)PVNGAVHLY(SEQ ID NO:7); (vi) PANGAVHLY (SEQ ID NO: 8), or (Vii)PLNGAVHLY(SEQ ID NO:9)。
  7. 7. The modified AAV capsid protein of any one of claims 1-6, wherein the targeting peptide is inserted between S586 and a589 of an AAV9 capsid protein, thereby replacing a587 and Q588 of the AAV9 capsid protein.
  8. 8. The modified AAV capsid protein of any one of claims 1-7, having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an AAV9 capsid protein.
  9. 9. The modified AAV capsid protein of any one of claims 1-6, wherein the targeting peptide is inserted (I) Between S586 and T589 of the Anc80L65 capsid protein, thereby replacing a587 and N588 of said Anc80L65 capsid protein; (ii) Between Q585 and N588 of the Anc80L65 capsid protein, thereby replacing S586 and a587 of said Anc80L65 capsid protein; (iii) Between L584 and a587 of the Anc80L65 capsid protein, thereby replacing Q585 and S586 of said Anc80L65 capsid protein; (iv) Between A587 and A590 of the Anc80L65 capsid protein, thereby replacing N588 and T589 of said Anc80L65 capsid protein, or (V) Between S586 and a587 of the Anc80L65 capsid protein.
  10. 10. The modified AAV capsid protein according to claim 9, wherein the targeting peptide comprises PLNGAVHLY (SEQ ID NO: 9).
  11. 11. The modified AAV capsid protein of claim 9 or 10, having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an Anc80L65 capsid protein.
  12. 12. The modified AAV capsid protein of claim 1 or 2, wherein the targeting peptide has the sequence of PX 2 X 3 GX 5 X 6 X 7 LY (SEQ ID NO: 10), and X 2 、X 3 、X 5 、X 6 and X 7 are independently selected from any amino acid residue.
  13. 13. The modified AAV capsid protein of claim 12, wherein (I) X 2 is independently selected from leucine (L), threonine (T) or serine (S); (ii) X 3 is independently selected from asparagine (N) and glutamine (Q); (iii) X 5 is independently selected from alanine (a) and threonine (T); (iv) X 6 is independently selected from valine (V) and leucine (L), and (V) x 7 is independently selected from histidine (H), arginine (R) and lysine (K).
  14. 14. The modified AAV capsid protein of claim 12 or 13, wherein the targeting peptide is: (i)PTNGTVRLY(SEQ ID NO:11); (ii)PTNGTVHLY(SEQ ID NO:12); (iii)PTNGTVKLY(SEQ ID NO:13); (iv)PSNGTLRLY(SEQ ID NO:14); (v)PSNGTLHLY(SEQ ID NO:15); (vi)PSNGTLKLY(SEQ ID NO:16); (vii)PTNGTLRLY(SEQ ID NO:17); (viii) PTNGTLHLY (SEQ ID NO: 18), or (ix)PTNGTLKLY(SEQ ID NO:19)。
  15. 15. The modified AAV capsid protein of claim 1 or 2, wherein the targeting peptide has the sequence of PX 2 X 3 GAVX 7 X 8 X 9 (SEQ ID NO: 20), and X 2 、X 3 、X 5 、X 6 and X 7 are independently selected from any amino acid residue.
  16. 16. The modified AAV capsid protein of claim 15, wherein (I) X 2 is independently selected from leucine (L), threonine (T) or serine (S); (ii) X 3 is independently selected from asparagine (N) and glutamine (Q); (iii) X 7 is independently selected from histidine (H) and threonine (T); (iv) X 8 is independently selected from valine (V) and leucine (L), and (V) X 9 is independently selected from tyrosine (Y) and arginine (R).
  17. 17. The modified AAV capsid protein of claim 15 or 16, wherein the targeting peptide is: (i)PTQGAVTVR(SEQ ID NO:21); (ii)PLQGAVTVR(SEQ ID NO:22); (iii)PLQGAVHVR(SEQ ID NO:23); (iv)PLQGAVHVY(SEQ ID NO:24); (v)PSQGAVTLR(SEQ ID NO:25); (vi)PLQGAVTLR(SEQ ID NO:26); (vii) PLQGAVHLR (SEQ ID NO: 27), or (viii)PTQGAVTLR(SEQ ID NO:28)。
  18. 18. The modified AAV capsid protein of any one of claims 1-17, wherein the targeting peptide does not comprise PLNGAVHLY (SEQ ID NO: 9).
  19. 19. The modified AAV capsid protein of any one of claims 1-17, wherein the targeting peptide comprises PLNGSVHLY (SEQ ID NO: 3603) or PLNGTVHLY (SEQ ID NO: 1232).
  20. 20. The modified AAV capsid protein of any one of claims 12-19, wherein the targeting peptide is inserted between S586 and a589 of an AAV9 capsid protein, thereby replacing a587 and Q588 of the AAV9 capsid protein.

Description

Recombinant AAV with improved tropism and specificity 1. Cross-reference to related applications The present application claims the benefit of U.S. provisional application number 63/380,170 filed on day 10 and day 19 of 2022, U.S. provisional application number 63/482,874 filed on day 2 of 2023, and U.S. provisional application number 63/502,871 filed on day 5 and day 17 of 2023, each of which are hereby incorporated by reference in their entirety. 2. Sequence listing The present application contains a sequence listing of 55,921 sequences, which has been submitted by PATENT CENTER and is incorporated herein by reference in its entirety. The XML copy created at 10/16 of 2023 is named 53023wo_crf_sequencing. The application also incorporates by reference in its entirety the appendices entitled "appendix A" and "appendix B" and the sequences described therein. Appendix A and appendix B are filed concurrently. Background Adeno-associated virus (AAV) has become the carrier system of choice for in vivo gene therapy. More and more recombinant AAV (rAAV) designed to deliver therapeutic nucleic acids have been developed and tested in non-human primates and humans, and two rAAV gene therapy products have recently been approved by the FDA for commercialization. Although AAV vectors are safer and less inflammatory than other viruses, toxicity has occurred after administration of high doses of rAAV for gene therapy. Thus, topical application of rAAV to target tissues or organs has been used to improve targeting and reduce systemic toxicity. In addition, various natural AAV variants and synthetic AAV variants have been tested to develop AAV vectors with the desired tropism and specificity. In general, capsids are considered to be the major determinants of infectivity and host-vector related properties (such as adaptive immune response, tropism, specificity, potency and biodistribution). Indeed, several of these properties are known to differ between the native serotype and the engineered AAV variant. Over the last decade, novel synthetic AAV variants have been developed by using a variety of capsid engineering techniques, one of which is the insertion of small peptides into the exposed loop of the capsid protein, termed variable region VIII (VR VIII). In some cases, insertion of the novel peptide into a wild-type capsid alters the tropism of the variant. However, to date, little is known about how changes in AAV capsids alter their biological properties, and AAV vectors with the desired tropism and specificity for therapeutic targets, such as the Central Nervous System (CNS), have not been obtained. Species-specific differences in AAV tropism, such as between mice and non-human primate (NHP), make it difficult to develop AAV vectors with the desired tropism in humans. Treatment of diseases of the CNS remains a tricky problem. Currently, therapeutic agents for CNS disorders are limited because many do not cross the blood brain barrier when delivered intravenously or are not widely distributed when delivered directly to the brain. Thus, there is a need for an AAV vector with a preferential and specific trend toward the CNS for the treatment of such CNS diseases. Disclosure of Invention The present disclosure provides a modified AAV capsid protein that can form a rAAV with preferred tropism and specificity for a therapeutic target. In some embodiments, a modified adeno-associated virus (AAV) capsid protein having such preferred tropism comprises (i) a targeting peptide at a site within variable region VIII (VR VIII), wherein the targeting peptide has the sequence of X 1X2X3X4X5X6X7X8X9, wherein X 1、X2、X3、X4、X5、X6、X7、X8 and X 9 are each independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprising the targeting peptide having the sequence of X 1X2X3X4X5X6X7X8X9 at a site within variable region VIII (VR VIII) comprises a deletion of amino acid residues (e.g., a587 and Q588 in the AAV9 capsid). The applicant has previously demonstrated that a single injection of a rationally designed synthetic vector Anc80L65 (the vector described in WO2015/054653, the vector of WO2015/054653 being incorporated herein by reference in its entirety) into the CSF of an adult cynomolgus monkey results in efficient transduction of a broad region of the CNS and is surprisingly superior to AAV 9's ability to target the cortex and deep brain nuclei (PCT application No. PCT/US 2022/024462, which is incorporated herein by reference in its entirety). Applicants now report a modified AAV capsid protein (e.g., anc80L65, AAV9, and other AAV capsid proteins) comprising a targeting peptide within variable region VIII (VR VIII) that provides a synergistic effect for the specific targeting of rAAV to a target tissue (e.g., CNS). Thus, a modified AAV capsid protein of the present disclosure (e.g., a modified AAV capsid protein comprising a targeting peptide described herein inserted at an insertion site described