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EP-4735468-A1 - IMPROVED ADENO-ASSOCIATED VIRUS VECTORS

EP4735468A1EP 4735468 A1EP4735468 A1EP 4735468A1EP-4735468-A1

Abstract

The present invention relates AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for increased titer, along with improved packing during recombinant AAV vector particle production. Titers improved several fold, without a similar increase in residual packaged hcDNA, along with maintaining percent full capsid ratio. In addition viral proteins expression and ratios remained unchanged with the variants.

Inventors

  • KONDRAGUNTA, Bhargavi
  • WANG, Ruigong
  • PAUL, Siddhartha

Assignees

  • R.P. Scherer Technologies, LLC

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. An adeno-associated virus (AAV) genome comprising a variant membrane associated accessory protein (MAAP) nucleic acid sequence having a mutation selected from the group consisting of a stop codon substitution at a codon position corresponding to amino acid positions W19, C20, S27, S57 and S88, an amino acid substitution at position LI, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof, wherein the codon positions correspond to the amino acid positions in consensus MAAP amino acid sequence SEQ ID NO:1.
  2. 2. The AAV genome of claim 1, wherein the genome comprises a substitution mutation in at least one of amino acid positions LI, C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  3. 3. The AAV genome of any one of claims 1 to 2, wherein the genome comprises a substitution mutation at amino acid position C20 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  4. 4. The AAV genome of any one of claims 1 to 2, wherein the genome comprises a substitution mutation at amino acid position C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88.
  5. 5. The AAV genome of any one of claims 1 to 2, wherein the genome comprises a substitution mutation at amino acid position LI and a stop codon substitution at a position selected from the group consisting of amino acid positions W19, C20, S27, S57 and S88.
  6. 6. The AAV genome of any one of claims 1 to 5, wherein the substitution mutation at amino acid position LI is an LIP substitution mutation.
  7. 7. The AAV genome of any one of claims 1 to 6, wherein the genome comprises a substitution mutation at one of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88.
  8. 8. The AAV genome of any one of claims 1 to 6, wherein the genome comprises a substitution mutation at both of amino acid positions C20 and C21 and a stop codon substitution at an amino acid position selected from the group consisting of S27, S57 and S88.
  9. 9. The AAV genome of any one of claims 1 to 8, wherein the substitution mutation at amino acid position C20 is selected from the group consisting of C20S, C20Y and C20F substitution mutations.
  10. 10. The AAV genome of any one of claims 1 to 8, wherein the substitution mutation at amino acid position C21 is a C21S substitution mutation.
  11. 11. The AAV genome of any one of claims 1 to 10, wherein the stop codon substitution is at amino acid position W19.
  12. 12. The AAV genome of any one of claims 1 to 10, wherein the stop codon substitution is at amino acid position C20.
  13. 13. The AAV genome of any one of claims 1 to 10, wherein the stop codon substitution is at amino acid position S27.
  14. 14. The AAV genome of any one of claims 1 to 10, wherein the stop codon substitution is at amino acid position S57.
  15. 15. The AAV genome of any one of claims 1 to 10, wherein the stop codon substitution is at amino acid position S88.
  16. 16. The AAV genome of any one of claims 1 to 15, wherein the AAV genome is selected from the group consisting of a naturally-occurring serotype and a non-naturally-occurring serotype.
  17. 17. The AAV genome of claim 16, wherein the AAV genome is a non-naturally-occurring serotype.
  18. 18. The AAV genome of claim 16, wherein the genome is a naturally-occurring serotype.
  19. 19. The AAV genome of claim 18, wherein the AAV genome comprises a serotype 6 genome.
  20. 20. The AAV genome of claim 18, wherein the AAV genome comprises a serotype 2 genome.

Description

IMPROVED ADENO-ASSOCIATED VIRUS VECTORS Cross-Reference to Related Applications [0001 ] The present application claims priority to U.S. Provisional Application No. 63/524,265, filed June 30, 2023, which is incorporated herein by reference in its entirety. Sequence Listing [0002] The text of the computer readable sequence listing filed herewith, titled “CATA_41926_601_SequenceListing.xml”, created June 27, 2024, having a file size of 168,432 bytes, is hereby incorporated by reference in its entirety. Field [0003] The present invention relates to AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for increased titer during recombinant AAV vector particle production. Background [0004] Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations. AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. These include four Rep gene encoded proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression genes gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1 : 1 : 10 (VP1 : VP2: VP3) The aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly. While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11. Another protein named membrane- associated accessory protein (MAAP), has been recently identified by Ogden et. al., Science 366,1139- 1143 (2019) which is encoded from an alternative ORF within the AAV Cap gene. [0005] A variety of AAV genomes and production systems for producing recombinant AAV vector particles for use in gene therapies are known in the art. However, there is a need for systems which provide for higher titer and thus more efficient production of recombinant AAV vector particles. Summary [0006] The present invention relates to AAV genomes, vectors and nucleic acids containing variant capsid protein sequences that allow for increased titer, without an increase in encapsidated residual host cell DNA during recombinant AAV vector particle production. [0007] Accordingly, in some preferred embodiments, the present invention provides an adeno-associated virus (AAV) genome, comprising a variant membrane associated accessory protein (MAAP) nucleic acid sequence having a mutation selected from the group consisting of a stop codon substitution at a codon position corresponding to positions W19, C20, S27, S57 and S88, an amino acid substitution at position LI, an amino acid substitution at position C20, an amino acid substitution at position C21, and combinations thereof, wherein the positions correspond to consensus sequence SEQ ID NO:1. [0008] In some preferred embodiments, the genome comprises a substitution mutation in at least one of amino acid positions LI, C20 and C21 and/or a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88. In some preferred embodiments, the genome comprises a substitution mutation at amino acid position C20 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88. In some preferred embodiments, the genome comprises a substitution mutation at amino acid position C21 and a stop codon substitution at an amino acid position selected from the group consisting of W19, C20, S27, S57 and S88. In some preferred embodiments, the genome comprises a substitution mutation at amino acid position LI and a stop codon substitution at a position selected from the group consisting of amino acid positions W19, C20, S27, S57 and S88. In some preferred embodiments,