METHODS AND COMPOSITIONS FOR MODIFYING ASSEMBLY-ACTIVATING PROTEIN (AAP)-DEPENDENCE OF VIRUSES

• US 20190330278A1
• Filed: 05/10/2019
• Published: 10/31/2019
• Est. Priority Date: 05/10/2017
• Status: Active Grant

• Current Assignee: Massachusetts Eye And Ear Infirmary (Foundation of The Massachusetts Eye and EAR Infirmary Incorporated), The Schepens Eye Research Institute Inc (Mass General Brigham, Inc.)
• Original Assignee: Massachusetts Eye And Ear Infirmary (Foundation of The Massachusetts Eye and EAR Infirmary Incorporated), The Schepens Eye Research Institute Inc (Mass General Brigham, Inc.)
• Inventors: Vandenberghe, Luk H., Maurer, Anna Claire
• Granted Patent: US 11,034,732 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/015 Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2750/14152 relating to complementing cells and packaging systems for producing virus or viral particles

Abstract

Methods and compositions are provided that can be used to modify the assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV).

Claims

1. An adeno-associated virus (AAV) capsid polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

3.

2. The AAV capsid polypeptide of claim 1, having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:

3.

3. The AAV capsid polypeptide of claim 1, having the amino acid sequence of SEQ ID NO:

3.

4. The AAV capsid polypeptide of claim 1, encoded by the nucleic acid sequence of SEQ ID NO:

4.

5. A virus particle comprising the adeno-associated virus (AAV) capsid polypeptide of claim 1.

6. The virus particle of claim 5, further comprising a transgene.

14. A packaging cell comprising a nucleic acid molecule encoding an adeno-associated virus (AAV) capsid polypeptide of claim 1, wherein the AAV capsid polypeptide has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

3.

15. The packaging cell of claim 14, wherein the packaging cell lacks an assembly activating protein (AAP).

16. A method of reducing assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV), the method comprising providing an AAV having a capsid polypeptide of claim 1 that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

3.

17. A method of relieving, at least partially, an assembly activating protein (AAP)-dependence of an adeno-associated virus (AAV), the method comprising incorporating a capsid polypeptide of claim 1 into the AAV.

18. A method of engineering an adeno-associated virus (AAV) to reduce its dependence on assembly activating protein (AAP), the method comprising engineering an AAV that comprises a capsid polypeptide of claim 1.

19. The method of claim 18, further comprising culturing the AAV in the absence of the AAP.

20. The method of claim 18, further comprising sequencing the engineered AAV.

21. The method of claim 18, further comprising comparing AAP-dependence of the engineered AAV relative to a non-engineered or wild type AAV.

22. The method of claim 18, further comprising aligning the engineered AAV with a non-engineered or wild type AAV.

7. A nucleic acid molecule comprising a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:

4 and encoding an adeno-associated virus (AAV) capsid polypeptide.

8. The nucleic acid molecule of claim 7, having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:

4.

9. The nucleic acid molecule of claim 7, having the nucleic acid sequence of SEQ ID NO:

4.

10. The nucleic acid molecule of claim 7, encoding the amino acid sequence of SEQ ID NO:

3.

11. A vector comprising the nucleic acid molecule of claim 7.

12. A host cell comprising the nucleic acid molecule of claim 7.

13. The host cell of claim 12, wherein the host cell is a packaging cell.

23. An AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO:

1 and further comprising the amino acid residues at the indicated positions shown in Table 1 for AAP-dependence.