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US 8,709,778 B2Granted
Method of adenoviral vector synthesis
AssigneeXavier Danthinne
InventorXavier Danthinne
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Abstract
This invention provides methods for adenoviral vector synthesis. The present invention further provides methods for binding adenovirus terminal protein obtained from virus to linear DNA. The present invention further provides a recombinant adenovirus from which the adenovirus terminal protein can be purified with an inverted terminal repeat DNA sequence.
Claims17
A method of making a recombinant adenovirus vector, the method comprising the steps:
- (a) insertion of one or a group of endonuclease recognition sites into the genome of a helper adenovirus immediately downstream from adenovirus inverted terminal repeats;
- (b) amplification of the helper adenovirus, wherein the helper adenovirus contains adenovirus terminal protein bound to adenoviral DNA, wherein the adenoviral DNA is an inverted terminal repeat DNA sequence, wherein the adenovirus terminal protein bound to the inverted terminal repeat DNA sequence forms an adenovirus terminal protein-inverted terminal repeat DNA sequence complex;
- (c) purification of the adenovirus terminal protein-inverted terminal repeat DNA sequence complex from the helper adenovirus;
- and,
- (d) in vitro DNA ligation of the inverted terminal repeat DNA sequence of the adenovirus terminal protein-inverted terminal repeat DNA sequence complex to heterologous linear nucleic acid sequence by purified DNA ligase, wherein the heterologous linear nucleic acid sequence contains an adenovirus packaging signal, and wherein the adenovirus terminal protein-inverted terminal repeat DNA sequence complex ligated to said heterologous linear nucleic acid sequence is used to make recombinant adenovirus vector.
2 more elements
A recombinant adenovirus from which the adenovirus terminal protein can be purified, which comprises:
- a) an inverted terminal repeat DNA sequence;
- b) at least one endonuclease site, wherein the endonuclease site is capable of producing identical, non-palindromic sticky ends adjacent to inverted terminal repeat DNA sequences;
- c) a packaging signal;
- d) a pair of Lox sites or a pair of Frt sites flanking the packaging signal;
- e) a deletion in the E1 region;
- and
- f) a stuffer DNA fragment in the E3 or E4 region;
- wherein the stuffer fragment increases the size of the viral genome to about 36 kilobases,
- wherein the recombinant adenovirus is a helper-independent adenovirus,
- and wherein the helper-independent virus can be deleted for at least one viral gene.
7 more elements
Dependencies are inferred from the claim text.