Adenovirus helper-free recombinant AAV Virion production
First Claim
1. A nucleic acid molecule with (a) an adenovirus region coding for the E2A 72 kD protein, (b) an adenovirus VA RNA coding region, and (c) an adenovirus E4 ORF6 coding region, wherein said nucleic acid molecule, when expressed together with an adenovirus E1 coding region, provides accessory functions which support efficient recombinant AAV (rAAV) virion production in a mammalian cell.
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Abstract
Accessory functions capable of supporting efficient recombinant AAV (rAAV) virion production in a suitable host cell are provided. The accessory functions are in the form of one or more vectors that are capable of being transferred between cells. Methods of producing rAAV virions are also provided. The methods can be practiced to produce commercially significant levels of rAAV particles without also generating significant levels of infectious helper virus or other contaminating by-products.
37 Citations
10 Claims
- 1. A nucleic acid molecule with (a) an adenovirus region coding for the E2A 72 kD protein, (b) an adenovirus VA RNA coding region, and (c) an adenovirus E4 ORF6 coding region, wherein said nucleic acid molecule, when expressed together with an adenovirus E1 coding region, provides accessory functions which support efficient recombinant AAV (rAAV) virion production in a mammalian cell.
- 6. A cell line comprising a nucleic acid molecule which comprises (a) an adenovirus region coding for the E2A 72 kD protein, (b) an adenovirus VA RNA coding region, and (c) an adenovirus E4 ORF6 coding region, wherein said nucleic acid molecule, when expressed together with an adenovirus E1 coding region, provides accessory functions which support efficient recombinant AAV (rAAV) virion production.
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10. A method of producing recombinant AAV (rAAV) virions, comprising:
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(a) introducing an AAV vector into a mammalian host cell; (b) introducing an AAV helper construct into the host cell, said helper construct comprising AAV coding regions that are expressed in the host cell to complement AAV helper functions missing from said AAV vector; (c) introducing a nucleic acid molecule into the host cell, said nucleic acid molecule comprising (i) an adenovirus region coding for the E2A 72 kD protein, (ii) an adenovirus VA RNA coding region, and (iii) an adenovirus E4 ORF6 coding region and further wherein said nucleic acid molecule, when expressed together with an adenovirus E1 coding region, provides accessory functions for supporting efficient rAAV virion production in the host cell; and (d) culturing the host cell to produce rAAV virions.
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Specification