Modified viral vectors and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-2020325181-A1
Application number	US-202016911171-A
Country	US
Kind code	A1
Filing date	Jun 24, 2020
Priority date	Dec 29, 2017
Publication date	Oct 15, 2020
Grant date	—

Abstract

The present invention relates to production of parvoviral vectors to produce adeno-associated virus (AAV) for gene therapy. In particular the invention relates to improvements in parvoviral vectors that increase the packaging capacity, production efficiency, and infectivity of AAV virions that is necessary for large scale manufacturing of AAV for clinical purposes.

First claim

1 . An insect cell expressing adeno-associated virus (AAV) VP1 protein and AAV VP3 protein, wherein the insect cell does not express AAV VP2 protein.

2 . The insect cell according to claim 1 , comprising:
(a) a nucleotide sequence encoding AAV VP1 protein and AAV VP3 protein; and/or (b) a nucleotide sequence encoding AAV VP1 protein and a nucleotide sequence encoding AAV VP3 protein.

3 . The insect cell according to claim 2 , wherein the nucleotide sequence as defined in (a) of claim 2 comprises:
(a) an open reading frame encoding AAV VP1 protein; and
(b) an open reading frame encoding AAV VP3 protein.

4 . The insect cell according to claim 1 , comprising a nucleotide sequence encoding adeno-associated virus (AAV) VP2 protein with an inactivated VP2 initiation codon.

5 . The insect cell according to claim 1 , further comprising:
(a) a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence and at least one nucleotide sequence encoding a gene product of interest; (b) a third nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell; and optionally, (c) a fourth nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell.

6 . The insect cell according to claim 5 , wherein the insect cell comprises:
(a) a first nucleic acid construct comprising (i) nucleotide sequences for AAV VP1 protein and AAV VP3 protein, (ii) the third nucleotide sequence and, optionally, (iii) the fourth nucleotide sequence; and, (b) a second nucleic acid construct comprising the second nucleotide sequence.

7 . The insect cell according to claim 6 , wherein the second nucleic acid construct is an insect cell-compatible vector.

8 . The insect cell according to claim 7 , wherein the vector is a baculoviral vector.

9 . A method for producing an AAV in an insect cell, comprising:
(a) culturing an insect cell according to claim 5 under conditions to produce AAV; and optionally, (b) recovering the AAV.

10 . A nucleic acid construct, comprising:
(a) a nucleotide sequence comprising an open reading frame encoding adeno-associated virus (AAV) VP1 protein; and (b) a nucleotide sequence comprising an open reading frame encoding AAV VP3 protein, wherein the open reading frames encoding the AAV VP1 protein and the AAV VP3 protein are operably linked to expression control sequences for expression in an insect cell, and wherein AAV VP2 protein cannot be expressed from the nucleic acid construct.

11 . The nucleic acid construct according to claim 10 , wherein the open reading frame encoding AAV VP3 protein overlaps with the open reading frame encoding AAV VP1 protein.

12 . The nucleic acid construct according to claim 10 , wherein the open reading frame encoding AAV VP3 protein and the open reading frame encoding AAV VP1 protein are transcribed from separate nucleotide sequences.

13 . An AAV virion produced in an insect cell, the AAV virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest, wherein the at least one nucleotide sequence is not a native AAV nucleotide sequence, and wherein the AAV virion comprises AAV VP1 protein and AAV VP3 protein and does not comprise AAV VP2 protein.

14 . The AAV virion according to claim 13 , wherein the at least one nucleotide sequence encoding a gene product of interest is located between two AAV ITR nucleotide sequences.

15 . A pharmaceutical composition, comprising the AAV virion according to claim 13 .

16 . A method of treating a disease, comprising administering to a subject in need thereof a pharmaceutical composition according to claim 15 .

17 . The method according to claim 16 , wherein the disease is selected from the group consisting of Congestive heart failure, intermittent porphyria (AIP), age-related macular degeneration, Alzheimer's disease, arthritis, Batten disease, Canavan disease, Citrullinemia type 1, Crigler Najjar, congestive heart failure, cystic fibrosis, Duchene muscular dystrophy, dyslipidemia, glycogen storage disease type I (GSD-I), hemophilia A, hemophilia B, hereditary emphysema, homozygous familial hypercholesterolemia (HoFH), Huntington's disease (HD), Leber's congenital amaurosis, methylmalonic academia, ornithine transcarbamylase deficiency (OTC), Parkinson's disease, phenylketonuria (PKU), spinal muscular atrophy, paralysis, Wilson disease, epilepsy, Pompe disease, amyotrophic lateral sclerosis (ALS), Tay-Sachs disease, hyperoxaluria 9PH-1), spinocerebellar ataxia type 1 (SCA-1), SCA-3, u-dystrophin, Gaucher's types II or III, arrhythmogenic right ventricular cardiomyopathy (ARVC), Fabry disease, familial Mediterranean fever (FMF), proprionic acidemia, fragile X syndrome, Rett syndrome, Niemann-Pick, and Krabbe disease.

Assignees

• Uniqure Ip Bv

Inventors

• Du Plessis David Johannes Francois

Classifications

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N2750/14123

  Virus like particles [VLP] · CPC title

• C07K14/005 Primary

  from viruses · CPC title

• C12N2750/14122

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