# US 11,306,291 B2 · AAV vectors produced by insect cells comprising REP52 and REP78 coding sequences with differential codon biases > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,291 B2 - Document ID: US11306291B2 - Status: Granted - Kind: B2 - Application number: 16/216,775 - Filed: 2018-12-11 - Published: 2019-07-04 - Granted: 2022-04-19 - Earliest claimed: 2007-07-26 - Assignees: uniQure IP B.V. - Applicants: uniQure IP B.V. - Inventors: Andrew Christian Bakker; Wilhelmus Theodorus Johannes Maria Christiaan Hermens - Web page: https://patentgrep.com/patent/US11306291B2 ## CPC - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C07K 14/005: from viruses - C12N 15/86: Viral vectors - C12N 2710/14044: Chimeric viral vector comprising heterologous viral elements for production of another viral vector - C12N 2710/14143: viral genome or elements thereof as genetic vector - C12N 2750/14121: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2750/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2750/14143: viral genome or elements thereof as genetic vector - C12N 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles - C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host - C12N 2800/50: Vectors for producing vectors - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to production of proteins in insect cells whereby repeated coding sequences are used in baculoviral vectors. In particular the invention relates to the production of parvoviral vectors that may be used in gene therapy and to improvements in expression of the viral rep proteins that increase the productivity of parvoviral vectors. ## Claims ### Claim 1 A first baculovirus vector comprising: (i) a first nucleotide sequence encoding a first amino acid sequence of an AAV Rep52 protein selected from the group consisting of: (a) a sequence of at least 85% identity with SEQ ID NO: 10; (b) a nucleotide sequence complementary to the full length sequence of (a); and (c) a nucleotide sequence that differs from the sequence of (a) due to degeneracy of the genetic code; (ii) a second nucleotide sequence encoding a second amino acid sequence of an AAV Rep78 protein; and a further one or more baculoviral vectors comprising: (iii) a third nucleotide sequence comprising two AAV inverted terminal repeat (ITR) sequences and a nucleotide sequence encoding a gene product of interest located between the two AAV ITR sequences; and (iv) a fourth nucleotide sequence comprising AAV capsid protein-coding sequences operably linked to expression control sequences for expression in an insect cell; wherein the first and the second amino acid sequences share at least 90% sequence identity in a region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein, and wherein a portion of the first nucleotide sequence and a portion of the second nucleotide sequence that encode the region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein each comprise one or more contiguous stretches of at least 300 nucleotides that are less than 90% identical. ### Claim 2 (depends on 1) The baculovirus vector of claim 1, wherein the first nucleotide sequence is selected from the group consisting of a sequence of at least 85% identity with SEQ ID NO:10; and (ii) nucleotide sequence complementary to the full length sequence of (a). ### Claim 3 (depends on 1) The baculovirus vector of claim 1, wherein: (i) the first nucleotide sequence is operably linked to a polyhedron (polH) promoter and the second nucleotide sequence is operably linked to a p10 promoter or a deltaIE-1 promoter; or (ii) the first nucleotide sequence is operably linked to a p10 promoter and the second nucleotide sequence is operably linked to a polyhedron (polH) or deltaIE-1 promoter. ### Claim 4 (depends on 1) The baculovirus vector of claim 1, wherein the first nucleotide sequence has the sequence SEQ ID NO:10. ### Claim 5 (depends on 4) The baculovirus vector of claim 4, wherein the second nucleotide sequence is operably linked to a deltaIE-1 promoter. ### Claim 6 (depends on 1) The baculovirus vector of claim 1, wherein the translation initiation codon of the nucleotide sequence encoding the AAV Rep78 protein is a suboptimal initiation codon selected from the group consisting of ACG, CTG, GTG and TTG. ### Claim 7 (depends on 6) The baculovirus vector of claim 6, wherein all ATG codons that occur between the translation start codons of the AAV Rep78 protein and the translation start codon of the AAV Rep52 protein in the second nucleotide sequence are mutated. ### Claim 8 (depends on 7) The baculovirus vector of claim 7, wherein the first nucleotide sequence has the sequence of SEQ ID NO:10. ### Claim 9 (depends on 7) The baculovirus vector of claim 7, wherein the second nucleotide sequence is operably linked to a p10 promoter or to a polH promoter. ### Claim 10 (depends on 9) The baculovirus vector of claim 9, wherein the second nucleotide sequence is operably linked to a p10 promoter. ### Claim 11 (depends on 10) The baculovirus vector of claim 10, wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype. ### Claim 12 (depends on 9) The baculovirus vector of claim 9, wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype. ### Claim 13 (depends on 1) A method of obtaining a recombinant adeno-associated virus (AAV) viron comprising: (a) transforming or transfecting an insect cell with the baculovirus vectors of claim 1; (b) culturing the insect cell under conditions such that recombinant AAV virions are produced; and, (c) recovering the recombinant AAV virions. ### Claim 14 A first baculovirus vector comprising: (i) a first nucleotide sequence encoding a first amino acid sequence of an AAV Rep52 protein comprising SEQ ID NO: 10; (ii) a second nucleotide sequence encoding a second amino acid sequence of an AAV Rep78 protein; and a further one or more baculoviral vectors comprising: (iii) a third nucleotide sequence comprising two AAV inverted terminal repeat (ITR) sequences and a nucleotide sequence encoding a gene product of interest located between the two AAV ITR sequences; and (iv) a fourth nucleotide sequence comprising AAV capsid protein-coding sequences operably linked to expression control sequences for expression in an insect cell; wherein the Rep proteins encoded by the first and second nucleotide sequences are of the same AAV serotype; and wherein the translation initiation codon before the second nucleotide sequence encoding the AAV Rep78 protein is a suboptimal initiation codon selected from the group consisting of ACG, CTG, GTG and TTG. ### Claim 15 (depends on 14) The baculovirus vector of claim 14, wherein the first and the second amino acid sequences share at least 90% sequence identity in a region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein. ### Claim 16 (depends on 14) The baculovirus vector of claim 14, wherein a portion of the first nucleotide sequence and a portion of the second nucleotide sequence that encode the region from the second amino acid residue to the C-terminal residue of the AAV Rep52 protein each comprise one or more contiguous stretches of at least 300 nucleotides that are less than 90% identical. ### Claim 17 (depends on 14) The baculovirus vector of claim 14, wherein: (i) the first nucleotide sequence is operably linked to a polyhedron (polH) promoter and the second nucleotide sequence is operably linked to a p10 promoter or a deltaIE-1 promoter; or (ii) the first nucleotide sequence is operably linked to a p10 promoter and the second nucleotide sequence is operably linked to a polyhedron (polH) or deltaIE-1 promoter. ### Claim 18 (depends on 14) The baculovirus vector of claim 14, wherein the second nucleotide sequence is operably linked to a p10 promoter or to a polH promoter. ### Claim 19 (depends on 14) A method of obtaining a recombinant adeno-associated virus (AAV) viron comprising: (a) transforming or transfecting an insect cell with the baculovirus vectors of claim 14; (b) culturing the insect cell under conditions such that recombinant AAV virions are produced; and, (c) recovering the recombinant AAV virions.