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US 8,642,314 B2Granted
Optimization of expression of parvoviral rep and cap proteins in insect cells
AssigneeAmsterdam Molecular Therapeutics (AMT) B.V.
InventorsAndrew Christian Bakker, Wilhelmus Theodorus Johannes Maria Christiaan Hermens, Yvet Noordman
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Abstract
The present invention relates to the improved production of recombinant parvoviral virions in insect cells. In particular, the invention relates to an improved process for the production of recombinant parvoviral virions in insect cells, wherein the full/empty parvoviral virion ratio is increased. The invention also 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.
Claims14
A method for the production of a recombinant parvoviral virion, which comprises culturing an insect cell comprising one or more nucleic acid constructs which comprise:
- (a) a nucleotide sequence comprising a transgene that is flanked by at least one parvoviral inverted terminal repeat (ITR) nucleotide sequence;
- (b) a first expression cassette comprising a nucleotide sequence encoding at least one of parvoviral Rep proteins Rep78 and Rep68, which nucleotide sequence is operably linked to a first promoter that is capable of driving expression of the at least one of Rep78 and Rep68 proteins in the insect cell;
- and
- (c) a second expression cassette comprising a nucleotide sequence encoding a parvoviral capsid protein which is operably linked to a second promoter that is capable of driving expression of the capsid protein in the insect cell;
- under conditions conducive to the expression of the Rep protein and the capsid protein;
- wherein
- (i) the first and second expression cassettes are present on a single nucleic acid construct and are present in equimolar amounts in the insect cell,
- (ii) the ratio of the Rep78 and/or Rep68 protein expression to the capsid protein expression is regulated by one or more of the following:
- (A) the first promoter is stronger than the second promoter;
- (B) the presence of more and/or stronger enhancer elements in the first expression cassette as compared to the second expression cassette;
- (C) the nucleotide sequence encoding the Rep78 and/or Rep68 protein has a higher codon adaptation index as compared to the nucleotide sequence encoding the capsid protein;
- (D) temperature optimization of the Rep78 and/or Rep68 protein;
- and/or
- (E) a variant Rep78 and/or Rep68 protein with an alteration in the amino acid sequence as compared to the sequence of corresponding wild-type Rep78 and/or Rep68 protein, wherein the sequence alteration results in increased Rep78 and/or Rep68 protein function assessed as increased adeno-associated virus (AAV) production in the insect cell,
- resulting in expression of the Rep78 and/or Rep68 protein that is greater than expression of the capsid protein.
12 more elements
A kit comprising
- (a) a first nucleic acid construct comprising:
- (i) a first expression cassette comprising a nucleotide sequence encoding at least one of a parvoviral Rep protein Rep78 and Rep68 which is operably linked to a first promoter that is capable of driving expression of the Rep78 or Rep68 protein in a host insect cell;
- and
- (ii) a second expression cassette comprising a nucleotide sequence encoding a parvoviral capsid protein which is operably linked to a second promoter that is capable of driving expression of the capsid protein in the insect cell;
- wherein
- (A) the first promoter is or stronger than the second promoter;
- (B) the first expression cassette comprises more and/or stronger enhancer elements as compared to the second expression cassette;
- (C) the nucleotide sequence encoding the Rep78 and/or Rep68 protein has a higher codon adaptation index as compared to the nucleotide sequence encoding the capsid protein;
- (D) the Rep78 and/or Rep68 protein is temperature optimized;
- and/or
- (E) the Rep78 and/or Rep68 protein is a variant with an alteration in the amino acid sequence as compared to the sequence of corresponding wild-type Rep78 and/or Rep68 protein, wherein the sequence alteration results in increased Rep78 and/or Rep68 protein function assessed as increased adeno-associated virus (AAV) production in the insect cell,
- such that, when expressed in the insect cell, expression of the Rep78 and/or Rep68 protein is greater than expression of the capsid protein,
- and
- (b) a second nucleic acid construct comprising a nucleotide sequence encoding a multiple cloning site for a transgene, which site is flanked by at least one parvoviral ITR nucleotide sequence, and which transgene is operably linked to a promoter capable of driving its expression in a host insect cell.
11 more elements
An insect cell comprising one or more nucleic acid constructs, which comprise:
- (a) a nucleotide sequence comprising a transgene that is flanked by at least one parvoviral ITR nucleotide sequence;
- (b) a first expression cassette comprising a nucleotide sequence encoding at least one of parvoviral Rep proteins Rep78 and Rep68, which nucleotide sequence is operably linked to a first promoter that is capable of driving expression of the at least one of Rep78 and Rep68 proteins in the insect cell;
- and
- (c) a second expression cassette comprising a nucleotide sequence encoding a parvoviral capsid protein which is operably linked to a second promoter that is capable of driving expression of the capsid protein in the insect cell;
- wherein
- (i) the first and second expression cassettes are present on a single nucleic acid construct and are present in equimolar amounts in the cell;
- and
- (ii) the ratio of Rep78 and/or Rep68 protein expression to capsid protein expression in said cell is regulated by one or more of the following:
- (A) the first promoter is or stronger than the second promoter;
- (B) the presence of more and/or stronger enhancer elements in the first expression cassette as compared to the second expression cassette;
- (C) the nucleotide sequence encoding the Rep78 and/or Rep68 protein has a higher codon adaptation index as compared to the nucleotide sequence encoding the capsid protein;
- (D) temperature optimization of the Rep78 and/or Rep68 protein;
- and/or
- (E) a variant Rep78 and/or Rep68 protein with an alteration in the amino acid sequence as compared to the sequence of corresponding wild-type Rep78 and/or Rep68 protein, wherein the sequence alteration results in increased Rep78 and/or Rep68 protein function assessed as increased AAV production in the cell
- such that expression of Rep78 and/or Rep68 protein is greater than expression of the capsid protein.
12 more elements
Dependencies are inferred from the claim text.