Aav capsid variants targeting human glioblastoma cells

Patent metadata

Field	Value
Publication number	US-2022333136-A1
Application number	US-202017633769-A
Country	US
Kind code	A1
Filing date	Aug 7, 2020
Priority date	Aug 9, 2019
Publication date	Oct 20, 2022
Grant date	—

Abstract

Described herein are AAV particles having capsid proteins with amino acid substitutions that confer increased affinity for and transduction of glioma cells. The disclosed capsid proteins may be used in conjunction with any therapeutic transgene for treatment of cancer, including in the manufacture of an AAV particle. Also described are methods of identifying AAV variants having increased affinity for and transduction of specific tumor cell types.

First claim

What is claimed is:

1 . An adeno-associated virus (AAV) particle for transducing glioblastoma cells (GBM) and/or glioma stem-like cells (GSC) comprising a capsid protein comprising the amino acid substitutions as described in any of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, or Table 9.

2 . An AAV particle comprising a capsid protein comprising substitutions at one of the following combinations of amino acid positions:
(a) Q263, S264, Y444, T450, P451, S452, T454, Q457, R459, Q461, S492, A493, D494, E499, and Y500, or (b) E531, K532, S547, E548, D553, R585, and R588;
of a wild-type AAV2 VP1 capsid sequence as set forth in SEQ ID NO: 1, or homologous amino acids of a wild-type VP1 capsid sequence of an AAV serotype other than AAV2.

3 . The AAV particle of claim 2 (a) or 2(b), further comprising substitutions at E530, E531, and K532 of a wild-type AAV2 VP1 capsid sequence as set forth in SEQ ID NO: 1, or homologous amino acids of a wild-type VP1 capsid sequence of an AAV serotype other than AAV2.

4 . The AAV particle of claim 1 or 2 , wherein the amino acid substitutions comprise E531G, K532R, S547A, E548G, T550N, V552A, D553A, E555G, K556Q, R585A, and R588A relative to the amino acid sequence of SEQ ID NO: 1.

5 . The AAV particle of claim 1 or 2 , wherein the amino acid substitutions comprise: Q263G, S264T, Y444F, T450A, P451E, S452G, T454L, T455K, Q457M, R459H, Q461L, S492D, A493G, D494E, E499D, and Y500F relative to the amino acid sequence of SEQ ID NO: 1.

6 . The AAV particle of claim 1 , wherein the amino acid substitutions comprise S264A, Y444F, T450D, P451R, S452G, T454S, Q457T, R459A, Q461S, S492D, A493G, D494E, E499D, and Y500F relative to the amino acid sequence of SEQ ID NO: 1.

7 . The AAV particle of claim 6 , further comprising Q263A.

8 . The AAV particle of any one of claims 1 - 3 , wherein the amino acid substitutions comprise E530N, E531D, K532R, S547A, E548K, K549G, D553A, I554V, R585A, and R588A relative to the amino acid sequence of SEQ ID NO: 1.

9 . The AAV particle of claim 1 or 2 , wherein the amino acid substitutions comprise Y444F, T450A, P451S, S452A, T454S, Q457T, R459T, Q461G, T491Q, S492P, A493G, E499D, Y500F, E530D, E531D, and K532R relative to the amino acid sequence of SEQ ID NO: 1.

10 . The AAV particle of claim 1 , wherein the amino acid substitutions comprise Q263E, Y444F, T450A, P451S, S452A, T454S, Q457T, R459T, Q461G, T491Q, S492P, A493G, E499D, Y500F, E530D, E531D, and K532R relative to the amino acid sequence of SEQ ID NO: 1.

11 . The AAV particle of any of claims 1 - 3 , wherein the capsid has an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 95% identity to any one of SEQ ID NOs: 4-7 and 9-28.

12 . The AAV particle of any of claims 1 - 3 and 11 , wherein the capsid has an amino acid sequence comprising any one of SEQ ID NOs: 4-7 and 9-28.

13 . The AAV particle of any one of claims 1 - 3 and 11 - 12 , wherein the capsid has an amino acid sequence comprising SEQ ID NO: 4.

14 . The AAV particle of any one of claims 1 - 13 , wherein said substitutions confer an increased transduction of a GBM or a GSC cell compared to an AAV particle comprising a wild-type capsid protein.

15 . The AAV particle of any one of claims 1 - 14 , wherein the AAV particle comprises a recombinant genome encoding at least one heterologous nucleic acid sequence.

16 . The AAV particle of claim 15 , wherein said heterologous nucleic acid sequence encodes a therapeutic peptide or protein.

17 . The AAV particle of claim 16 , wherein said therapeutic peptide is a repair enzyme, a checkpoint inhibitor, a transcription factor, a growth factor receptor a growth factor ligand, or a tumor suppressor protein.

18 . The AAV particle of any of claims 1 - 3 , wherein the capsid has an amino acid sequence having at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, or at least 95% identity to any one of SEQ ID NOs: 4-7 and 9-7010, optionally wherein the capsid has an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 4-7 and 9-28.

19 . A recombinant AAV vector, comprising a capsid and a genome, wherein said capsid comprises a VPX protein having amino acid substitutions as described in any of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, or Table 8, wherein said substitutions confer an increased transduction of GBM cells or GSC compared to an AAV particle comprising a wild-type capsid protein.

20 . A method of delivering a heterologous gene or targeting construct to a glioma cell comprising: inserting the heterologous gene or targeting contruct into the AAV particle of any one of claims 1 - 18 , and contacting the glioma cell with the AAV.

21 . A method of identifying viral variants having affinity for a tumor cell type comprising: providing a continuous intratumor graded infusion of a viral library into a tumor wherein the rate of infusion is proportional to the rate of tumor growth.

Assignees

• Univ Florida

Inventors

• Zolotukhin Sergei
• Tran David
• Kondratov Oleksandr

Classifications

• C12N15/86

  Viral vectors · CPC title

• C12N15/861 Primary

  Adenoviral vectors · CPC title

• C12N2750/14122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2750/14145

  Special targeting system for viral vectors · CPC title

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title