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US-20260125704-A1 - rAAV capsids

US20260125704A1US 20260125704 A1US20260125704 A1US 20260125704A1US-20260125704-A1

Abstract

The present invention relates to the fields of medicine, molecular biology, and gene therapy. In particular, the invention relates to novel recombinant adeno-associated virus capsids, uses thereof and methods of manufacturing.

Inventors

  • - Anggakusuma
  • Elena Maria SANTIDRIAN YEBRA-PIMENTEL

Assignees

  • UNIQURE BIOPHARMA B.V.

Dates

Publication Date
20260507
Application Date
20250822
Priority Date
20240822

Claims (17)

  1. 1 . A recombinant adeno-associated virus (rAAV) capsid comprising at least one binding moiety, wherein the at least one binding moiety confers to the rAAV capsid a phenotype of increased interaction with carbonic anhydrase 4 (CA4) as compared to a control rAAV capsid not comprising the binding moiety.
  2. 2 . The rAAV capsid according to claim 1 , wherein the rAAV capsid presents at least one of the following phenotypes upon systemic administration: i) a phenotype of increased blood brain barrier (BBB) crossing as compared to a control rAAV capsid not comprising the binding moiety; and/or ii) a phenotype of increased transduction of cells in the central nervous system (CNS) as compared to a control rAAV capsid not comprising the binding moiety.
  3. 3 . The rAAV capsid according to claim 1 or 2 , wherein at least part of the binding moiety is inserted between two consecutive amino acids of a capsid protein.
  4. 4 . The rAAV capsid according to any one of claims 1-3 , wherein the binding moiety is comprised in a variable region (VR) of a surface loop of the rAAV capsid, preferably in at least one of the VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and/or VR-IX, preferably in the VR-IV and/or VR-VIII, more preferably in the VR-VIII.
  5. 5 . The rAAV capsid according to any one of claims 1-4 , wherein the binding moiety comprises an amino acid insert between two consecutive amino acids present within amino acids 580-595, preferably amino acids 585-590, more preferably amino acids 588 and 589 of an AAV9 capsid protein, or in an analogous position of a capsid protein of an AAV capsid serotype selected from the group comprising: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 and AAVrh10, or modified versions thereof, preferably rAAV6, or modified versions thereof.
  6. 6 . The rAAV capsid according to any one of claims 1-5 , wherein the amino acid insert consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids, preferably 5-9 amino acids, more preferably 7 amino acids.
  7. 7 . The rAAV capsid according to any one of claims 1-6 , wherein the binding moiety comprises at least 4, 5, 6, or 7 contiguous amino acids of an amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 (SEQ ID NO: 301), wherein: X 1 is selected from: L, R, V, I, S, D, A, K and P; X 2 is selected from: H, Q, A, N, E, R, M, G, P, V and W; X 3 is selected from: G, W, R, S, H, I and V; X 4 is selected from: L, I, G, F, V and N; X 5 is selected from: R, Q, S, E, P, A and T; X 6 is selected from: V, L, R, A, I, S, Q and P, and; X 7 is selected from: L, I, S, W and R; optionally wherein the amino acid at position 587 is mutated to D, and/or the amino acid at position 588 is mutated to G.
  8. 8 . The rAAV capsid according to any one of claims 1-7 , wherein: the binding moiety comprises a consensus sequence AQX 1 X 2 GX 3 X 4 X 5 L (SEQ ID NO: 302), wherein X 1 is selected from: L, R, V, I, S, and D; X 2 is selected from: H, Q, N, E, R, and M; X 3 is selected from: L and I; X 4 is selected from: R and Q, and; X 5 is selected from: V, L and R; or, wherein the binding moiety comprises a consensus sequence DGPWVNXPR (SEQ ID NO: 303), wherein X is selected from: S, and T;
  9. 9 . The rAAV capsid according to any one of claims 1-8 , wherein the binding moiety comprises or consists of an amino acid sequence selected from SEQ ID NO: 1-168, preferably selected from SEQ ID NO: 1, 2, 12, 26, 28, or 32-36.
  10. 10 . The rAAV capsid according to any one of claims 1-9 , wherein the binding moiety does not compete with brinzolamide for interaction with CA4.
  11. 11 . The rAAV capsid according to any one of claims 1-10 , wherein the rAAV capsid comprises an expression cassette flanked by at least one AAV inverted terminal repeat (ITR), wherein the expression cassette comprises a nucleic acid molecule encoding at least one gene product.
  12. 12 . A polynucleotide encoding the recombinant adeno-associated virus (rAAV) capsid according to any one of claims 1-11 .
  13. 13 . A host cell comprising a polynucleotide according to claim 12 , preferably wherein the polynucleotide is an expression vector for expression of the rAAV capsid.
  14. 14 . A composition comprising the rAAV capsid according to any one of claims 1-11 , preferably wherein the composition is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.
  15. 15 . The rAAV capsid according to any one of claims 1-11 , or the composition of claim 14 , for use as a medicament, preferably for use in the treatment of a condition of the central nervous system.
  16. 16 . The rAAV capsid for use according to claim 15 , wherein the rAAV capsid is administered systemically, preferably intravenously or intraarterially.
  17. 17 . A method of manufacturing an rAAV capsid according to any one of claims 1-11 , the method comprising the steps of: I) culturing a host cell comprising: i) an expression vector for expression of an rAAV capsid according to any one of claims 1-11 , and; ii) an expression vector encoding an expression cassette flanked by at least one AAV inverted terminal repeat (ITR), comprising a nucleic acid molecule encoding at least one gene product; said host cell preferably further comprising a nucleotide sequence encoding a parvoviral replication (Rep) protein under conditions such that the rAAV capsid is produced; and, II) recovery of the rAAV capsid, preferably wherein recovery of the rAAV capsid comprises at least one of: affinity-purification of the rAAV capsid using an immobilized anti-AAV antibody, preferably a single chain camelid antibody or a fragment thereof, or filtration using a filter having a nominal pore size of 30-70 nm.

Description

FIELD OF THE INVENTION The present invention relates to the fields of medicine, molecular biology, and gene therapy. In particular, the invention relates to novel recombinant adeno-associated virus capsids, uses thereof and methods of manufacturing the novel recombinant adeno-associated virus capsids. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (sequencelisting.txt; Size: 334.460 bytes; and Date of Creation: Aug. 22, 2025) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Recombinant adeno-associated virus (rAAV) has emerged as a preferred platform for gene therapy. Yet, the development of novel rAAV-based therapeutic strategies has faced several hurdles. Specifically, developing novel rAAV capsids that can transduce cells in the central nervous system (CNS) upon systemic administration is one of the challenges in the field of rAAV-based gene therapy of CNS diseases. The inefficiency of blood-brain barrier (BBB) crossing limits the CNS application of first generation rAAV-based capsids due to the narrow therapeutic window when administered systemically. Traditional methods, such as intraparenchymal injection and intrathecal infusion, are invasive and may provide limited coverage. To address these challenges, research is being conducted on capsid engineering to develop novel rAAV capsids that can efficiently cross the BBB and achieve widespread CNS transduction via less invasive routes of administration, such as systemic delivery. A major milestone in this field is the engineering of rAAV capsids via peptide insertion, which has led to the development of e.g. PHP.B rAAV capsid variants—the first to demonstrate efficacy in transducing the brain of C57BL/6 mice following systemic injection (Deverman et al., 2016, Nat Biotechnol., 34(2): 204-209). However, these engineered rAAV capsid variants did not perform consistently across different mouse strains or in non-human primates (NHPs). Consequently, various research groups have employed different rAAV capsid engineering platforms to create engineered capsids with peptide insertions capable of crossing the BBB beyond rodent models and efficiently transducing the brain (Lopez-Gordo et al., Viruses 2024, 16(3), 442). There remains, however, a need for further improved rAAV capsids that can cross the BBB and efficiently transduce cells in the CNS upon systemic administration. It is an object of the invention to provide engineered rAAV capsids with the capacity to cross the BBB. SUMMARY OF THE INVENTION In a first aspect, there is provided a recombinant adeno-associated virus (rAAV) capsid comprising at least one binding moiety, wherein the at least one binding moiety confers to the rAAV capsid a phenotype of increased interaction with carbonic anhydrase 4 (CA4) as compared to a control rAAV capsid not comprising the binding moiety. In one embodiment, the rAAV capsid presents at least one of the following phenotypes upon systemic administration: i) a phenotype of increased blood-brain barrier (BBB) crossing as compared to a control rAAV capsid not comprising the binding moiety; and/or ii) a phenotype of increased transduction of cells in the central nervous system (CNS) as compared to a control rAAV capsid not comprising the binding moiety. In one embodiment, the binding moiety comprises an amino acid insert between two consecutive amino acids present within amino acids 580-595, preferably amino acids 585-590, more preferably amino acids 588 and 589 of an AAV9 capsid protein, or in an analogous position of a capsid protein of an AAV capsid serotype selected from the group comprising: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 and AAVrh10, or modified versions thereof, preferably AAV6, or modified versions thereof. In one embodiment, the binding moiety comprises at least 4, 5, 6, or 7 contiguous amino acids of an amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 301), wherein X1 is selected from: L, R, V, I, S, D, A, K and P; X2 is selected from: H, Q, A, N, E, R, M, G, P, V and W; X3 is selected from: G, W, R, S, H, I and V; X4 is selected from: L, I, G, F, V and N; X5 is selected from: R, Q, S, E, P, A and T; X6 is selected from: V, L, R, A, I, S, Q and P, and; X7 is selected from: L, I, S, W and R; optionally wherein the amino acid at position 587 is mutated to D, and/or the amino acid at position 588 is mutated to G. In one embodiment, the binding moiety does not compete with brinzolamide for interaction with CA4. In one embodiment, the rAAV capsid comprises an expression cassette flanked by at least one AAV inverted terminal repeat (ITR), wherein the expression cassette comprises a nucleic acid molecule encoding at least one gene product. In a second aspect, there is provided a polynucleotide encoding an rAAV capsid as described herein. In a third aspect, there is provided a host cell comprising a polynucleotide for expression of the rAAV capsid as