# US 8,163,543 B2 · AAV vectors produced in insect cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,163,543 B2 - Document ID: US8163543B2 - Status: Granted - Kind: B2 - Application number: 12/091,022 - Filed: 2006-10-19 - Published: 2012-02-02 - Granted: 2012-04-24 - Earliest claimed: 2005-10-20 - Assignees: Amsterdam Molecular Therapeutics B.V. - Inventors: Masashi Urabe; Keiya Ozawa; Saskia Jacoba Petronella Haast; Wilhelmus Theodorus Johannes Maria Christiaan Hermens - Web page: https://patentgrep.com/patent/US8163543B2 ## CPC - C12N 15/86: Viral vectors - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C12N 2710/14144: Chimeric viral vector comprising heterologous viral elements for production of another viral vector - 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 2750/14151: Methods of production or purification of viral material - 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) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present invention relates to the production of adeno-associated viral vectors in insect cells. The insect cells therefore comprise a first nucleotide sequence encoding the adeno-associated virus (AAV) capsid proteins, whereby the initiation codon for translation of the AAV VP1 capsid protein is a non-ATG, suboptimal initiation codon. The insect cell further comprises a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence; a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell; and, a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell. The invention further relates to adeno-associated viral vectors with an altered ratio of the viral capsid proteins that provides improved infectivity of the viral particles. ## Claims ### Claim 1 A nucleic acid construct comprising a nucleotide sequence encoding adeno-associated virus (AAV) VP1, VP2, and VP3 capsid proteins, wherein the initiation codon for translation of the AAV VP1 capsid protein is CTG, TTG, or GTG and wherein the nucleotide sequence is operably linked to an expression control sequence for expression in an insect cell. ### Claim 2 (depends on 1) A nucleic acid construct according to claim 1, wherein the initiation codon is CTG. ### Claim 3 (depends on 1) A nucleic acid construct according to claim 1, wherein the expression control sequence comprises a nine nucleotide sequence of SEQ ID NO:7 or a sequence substantially homologous to SEQ ID NO:7 upstream of the initiation codon. ### Claim 4 (depends on 1) A nucleic acid construct according to claim 1, wherein the expression control sequence comprises a Kozak consensus sequence around the initiation codon, which Kozak consensus sequence is GCCRCCNNNG (SEQ ID NO:8), wherein R is a purine and wherein NNN is the initiation codon. ### Claim 5 (depends on 1) A nucleic acid construct according to claim 1, wherein the nucleotide sequence encoding said AAV VP1 capsid protein comprises at least one modification selected from the group consisting of G at position 12, an A at position 21, and a C at position 24. ### Claim 6 (depends on 1) A nucleic acid construct according to claim 1, wherein the nucleotide sequence is operably linked to a polyhedron promoter. ### Claim 7 (depends on 1) A nucleic acid construct according to claim 1 that is in the form of a baculoviral vector capable of productive transformation or transfection of an insect cell. ### Claim 8 (depends on 1) An insect cell comprising the nucleic acid construct according to claim 1. ### Claim 9 (depends on 8) An insect cell according to claim 8, that further comprises: (a) a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence; (b) a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell; and, (c) a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell. ### Claim 10 (depends on 9) An insect cell according to claim 9, that comprises: (a) a first nucleic acid construct comprising, (i) a first nucleotide sequence encoding AAV VP1, VP2, and VP3 capsid proteins, wherein the initiation codon for translation of the VP1 capsid protein is CTG, TTG, or GTG and wherein the nucleotide sequence is operably linked to an expression control sequence for expression in an insect cell; (ii) the third nucleotide sequence as defined in claim 9(b); and (iii) the fourth nucleotide sequence as defined in claim 9(c); and, (b) a second nucleic acid construct comprising the second nucleotide sequence as defined in claim 9(a). ### Claim 11 (depends on 10) An insect cell according to claim 10, wherein the first and/or second nucleic acid construct is in the form of a baculoviral vector capable of productive transformation or transfection of an insect cell. ### Claim 12 (depends on 9) An insect cell according to claim 9, wherein the second nucleotide sequence further comprises at least one nucleotide sequence encoding a gene product of interest for expression in a mammalian cell, which coding sequence becomes incorporated into the genome of an AAV produced in the insect cell. ### Claim 13 (depends on 12) An insect cell according to claim 12, wherein the second nucleotide sequence comprises two AAV ITR nucleotide sequences between which is located the at least one sequence encoding the product of interest. ### Claim 14 (depends on 9) The insect cell according to claim 9, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, and the fourth nucleotide sequence are stably integrated in the genome of the insect cell. ### Claim 15 An AAV virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest, which sequence is not a native AAV nucleotide sequence, and wherein VP1 capsid protein is present in the virion in at least 110% of the amount of capsid protein VP2 and at least 11% of the amount of capsid protein VP3, the amount of said capsid proteins being determined using an antibody that recognizes an epitope that is common to each of VP1, VP2 and VP3. ### Claim 16 An AAV virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest which sequence is not a native AAV nucleotide sequence, and wherein the virion comprises a VP1 capsid protein comprising leucine or valine at amino acid position 1. ### Claim 17 (depends on 15) An AAV virion according to claim 15, wherein the VP1 capsid protein comprises leucine or valine at amino acid position 1. ### Claim 18 (depends on 8) A method for producing an AAV virion in an insect cell, comprising the steps of: (a) culturing the insect cell according to claim 8 under conditions such that the AAV virion is produced; and, (b) recovering the AAV virion. ### Claim 19 (depends on 18) A method according to claim 18, further comprising the step of affinity-purifying the AAV virion using an anti-AAV antibody. ### Claim 20 (depends on 19) A method according to claim 19, wherein the anti-AAV antibody is a single chain cameloid antibody or a fragment thereof. ### Claim 21 (depends on 19) The method according to claim 19, wherein the antibody is immobilized.