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US 9,260,724 B2Granted
Optimization of expression of parvoviral rep and cap proteins in insect cells
AssigneeuniQure IP B.V.
InventorsAndrew Christian Bakker, Wilhelmus Theodorus Johannes Maria Christiaan Maria Christ Hermens, Yvet Noordman
Life of this patent
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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.
Claims25
A method for the production of a recombinant parvoviral virion which decreases the total:full virion particle ratio, wherein the method 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 parvoviral Rep proteins Rep78 and Rep52, which nucleotide sequence is operably linked to a first promoter that is capable of driving expression of the Rep78 and Rep52 proteins in the insect cell and wherein the nucleotide sequence encoding the parvoviral Rep proteins is a single open reading frame encoding Rep78 and Rep52 proteins;
- and
- (c) a second expression cassette comprising a nucleotide sequence encoding parvoviral capsid proteins which is operably linked to a second promoter that is capable of driving expression of the capsid proteins in the insect cell;
- under conditions conducive to the expression of the Rep proteins and the capsid proteins wherein expression of the Rep 78 and Rep52 proteins is greater than expression of the capsid proteins;
- 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 expression of Rep78 and Rep52 proteins to the expression of capsid proteins is regulated by one or more of the following structures or conditions:
- (A) the first promoter is stronger than the second promoter;
- (B) more and/or stronger enhancer elements are present in the first expression cassette as compared to the second expression cassette;
- (C) the nucleotide sequence encoding the Rep78 and the Rep52 proteins has a higher codon adaptation index compared to the nucleotide sequence encoding the capsid proteins;
- (D) the Rep78 and the Rep52 proteins are temperature optimized;
- (E) the Rep78 and the Rep52 proteins are variants with altered amino acid sequences as compared to the sequences of corresponding wild-type Rep78 and Rep52 proteins, wherein the altered sequences result in increased Rep78 and Rep52 protein function manifest as increased adeno-associated virus (AAV) production in the insect cell;
- and/or
- (F) the first promoter is as strong as the second promoter, one or more of structures or conditions (B)-(E) is present, and the insect cell comprises an additional nucleotide sequence encoding for an additional Rep protein,
- resulting in expression of the Rep78 and Rep52 proteins that is greater than expression of the capsid proteins,
- thereby decreasing the total:full virion particle ratio compared to a method in which the expression of the Rep78 and the Rep52 proteins is not greater than the expression of the capsid proteins.
14 more elements
A kit comprising
- (a) a first nucleic acid construct comprising:
- (i) a first expression cassette comprising a nucleotide sequence encoding parvoviral Rep proteins Rep78 and Rep52 which is operably linked to a first promoter that is capable of driving expression of the Rep78 or Rep52 protein in a host insect cell and wherein the nucleotide sequence encoding the parvoviral Rep protein is a single open reading frame encoding Rep78 and Rep52 proteins;
- and
- (ii) a second expression cassette comprising a nucleotide sequence encoding parvoviral capsid proteins which is operably linked to a second promoter that is capable of driving expression of the capsid proteins in the insect cell;
- wherein expression of Rep78 and Rep52 proteins in insect cells is regulated by one or more of the following structures or conditions:
- (A) the first promoter is 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 the Rep52 proteins has a higher codon adaptation index compared to the nucleotide sequence encoding the capsid proteins;
- (D) the Rep78 and the Rep52 proteins are temperature optimized;
- (E) the Rep78 and the Rep52 proteins are variants with altered amino acid sequences as compared to the sequences of corresponding wild-type Rep78 and Rep52 proteins, wherein the altered sequences result in increased Rep78 and Rep52 protein function manifest as increased adeno-associated virus (AAV) production in the insect cell;
- and/or
- (F) the first promoter is as strong as the second promoter and one or more of structures or conditions (B)-(E) is present;
- such that, when expressed in the insect cell, expression of the Rep78 and the Rep52 proteins is greater than expression of the capsid proteins,
- which decreases the total:full virion particle ratio compared to a method in which the expression of the Rep78 and the Rep52 proteins is not greater than the expression of the capsid proteins,
- wherein when the first promoter is as strong as the second promoter and one or more of (B)-(E) is present, then the insect cell comprises an additional nucleotide sequence encoding an additional Rep 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.
13 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 parvoviral Rep proteins Rep78 and Rep52, which nucleotide sequence is operably linked to a first promoter that is capable of driving expression of the Rep78 and Rep52 proteins in the insect cell and wherein the nucleotide sequence encoding the parvoviral Rep proteins is a single open reading frame encoding Rep78 and Rep52 proteins;
- and
- (c) a second expression cassette comprising a nucleotide sequence encoding parvoviral capsid proteins which is operably linked to a second promoter that is capable of driving expression of the capsid proteins 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 insect cell, and
- (ii) the ratio of the expression of Rep78 and Rep52 proteins to the expression of capsid proteins in said cell is regulated by one or more of the following structures or conditions:
- (A) the first promoter is stronger than the second promoter;
- (B) more and/or stronger enhancer elements are present in the first expression cassette as compared to the second expression cassette;
- (C) the nucleotide sequence encoding the Rep78 and the Rep52 proteins has a higher codon adaptation index compared to the nucleotide sequence encoding the capsid proteins;
- (D) the Rep78 and the Rep52 proteins are temperature optimized;
- (E) the Rep78 and the Rep52 proteins are variants with altered amino acid sequences as compared to the sequences of corresponding wild-type Rep78 and Rep52 proteins, wherein the altered sequences result in increased Rep78 and Rep52 protein function manifest as increased adeno-associated virus (AAV) production in the insect cell;
- and/or
- (F) the first promoter is as strong as the second promoter, one or more of structures or conditions (B)-(E) is present, and the insect cell comprises an additional nucleotide sequence encoding for an additional Rep protein,
- such that, when expressed in the insect cell, expression of the Rep78 and Rep52 proteins is greater than expression of the capsid proteins,
- which decreases the total:full virion particle ratio compared to a method in which the expression of the Rep78 and the Rep52 proteins is not greater than the expression of the capsid proteins.
13 more elements
Dependencies are inferred from the claim text.