Packaging systems for human recombinant adenovirus to be used in gene therapy

A recombinant nucleic acid comprising an adapter plasmid, said adapter plasmid selected from the group consisting of pWE/Ad.AflIII-EcoRI, pAd5/CLIP, pAd5/L420-HSA, pMV/L420-H, pMV/CMV-LacZ, and pWE/Ad-H.

A recombinant nucleic acid comprising a recombinant adenovirus, wherein said recombinant adenovirus is pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI.

A recombinant nucleic acid comprising:

• an adenoviral nucleotide sequence, said adenoviral nucleotide sequence comprising a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof acting as Inverted Terminal Repeats,
• said recombinant nucleic acid lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.

A recombinant nucleic acid for incorporation into a cell comprising:

• an adenoviral nucleotide sequence comprising sufficient adenoviral sequences necessary for replication and capsid gene expression, but lacking at least the E1 region and an adenoviral encapsulating signal, wherein said adenoviral nucleotide sequence further lacks overlapping sequences allowing for homologous recombination leading to replication competent virus in the cell into which said recombinant nucleic acid is to be transferred.

A recombinant nucleic acid comprising a construct comprising pWE/Ad.Δ5′.

A recombinant nucleic acid comprising:

• an adenoviral nucleotide sequence comprising sufficient adenovirus sequences needed for replication and capsid gene expression and a complementary sequence to an upstream part of said recombinant nucleic acid, wherein said complementary sequence can base-pair with said upstream part so that it functions as a start-site for a nucleic acid polymerase, said adenoviral nucleotide sequence having a deletion of one Inverted Terminal Repeat, the E1 region, and the adenoviral encapsulating signal,
• wherein said recombinant nucleic acid has no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.

The recombinant nucleic acid according to claim 25, wherein said recombinant nucleic acid comprises a construct pWE/AAV.Δ5′.

A recombinant nucleic acid comprising:

• an adenoviral nucleotide sequence comprising a sequence for adenovirus-independent replication and sufficient adenoviral sequences necessary for replication;
• wherein said adenoviral nucleotide sequence has a deletion of at least the E1 region and encapsulating signal of adenovirus, said recombinant nucleic acid comprising no overlapping sequences which allow for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.

An adapter plasmid comprising:

• an adenoviral nucleotide sequence comprising, in operable configuration, at least one functional Inverted Terminal Repeat and one functional encapsulating signal, said adapter plasmid having no sequences which allow for homologous recombination leading to replication competent virus in a cell into which said adapter plasmid is transferred.

A replication defective adenovirus comprising:

• an adenoviral genome comprising at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, and lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said replication defective adenovirus is transferred.

A cell comprising:

• a replication defective adenovirus comprising an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said cell into which said replication defective adenovirus has been transferred.

A method of replacing a defective gene in a genome of a host cell, said method comprising:

• growing said host cell with a recombinant nucleic acid molecule derived from a replication defective adenovirus comprising a functional version or part of said defective gene under conditions wherein at least one allele of said defective gene in said host cell genome is replaced.

An isolated cell comprising:

• an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said isolated cell.

A replication-defective adenovirus comprising an adenoviral genome, wherein said adenoviral genome has a first deletion in an adenoviral E1 region and a second deletion in an adenoviral gp19K region.