Minimal adenoviral vector

A method of generating a recombinant adenoviral vector consisting of the steps of, in combination,

• transfecting a cell stably transfected with a DNA molecule comprising the Ad E1 gene sequence, said sequence having no overlapping sequence with the genome of an E1-deleted helper Ad genome, with an isolated DNA molecule comprising an adenoviral inverted terminal repeat (ITR), a packaging signal, a transcriptional control region, an effector or reporter gene, and either a genomic integration sequence or an episomal maintenance sequence, all operatively associated for generating an infectious, replication-defective recombinant adenoviral vector wherein the remaining portion of said DNA molecule does not encode an adenoviral protein;
• infecting said cell with an Ad helper virus comprising an Ad helper genome;
• and,
• preparing a cell-free lysate of the cell;
• wherein said cell-free lysate comprises infective, replication-impaired recombinant adenoviral vector particles comprising said DNA molecule.

A method of generating a non-human, transgenic animal comprising the steps of, in combination,

• transfecting an embryonic stem cell with a DNA molecule comprising the AAVS1 sequence and a drug selection marker gene;
• selecting an embryonic stem cell comprising said DNA molecule within its genome by growth in the presence of a drug to which expression of said drug selection marker gene confers resistance;
• microinjecting said embryonic stem cell into a blastocyst;
• implanting said blastocyst into a foster mother;
• breeding the resulting offspring;
• assaying said offspring for the presence of the AAVS1 sequence;
• and
• breeding to homozygosity those offspring in which the AAVS1 sequence has been detected;
• whereby a transgenic animal having the AAVS1 sequence incorporated into its genome results.

A method of testing in vivo delivery of a viral vector comprising an AAV-ITR to a non-human, transgenic animal having the AAVS1 sequence incorporated into its genome comprising, in combination, the steps of:

• injecting intravenously or into the portal vein a vector of said animal comprising a DNA molecule having an AAV-ITR sequence and encoding a reporter or effector gene in a physiologically acceptable buffer;
• obtaining multiple blood samples from said animal;
• measuring the level of gene expression in said blood sample to determine the duration of transgene expression;
• and,
• isolating genomic DNA from liver tissue of said animal;
• and,
• performing polymerase chain reaction (PCR) analysis of said tissue using AAVS1-specific primers to detect integration of said vector into the genome of said animal.

A method of generating a non-human, transgenic animal tolerized to human FVIII comprising the steps of, in combination,

• transfecting an embryonic stem cell with a DNA molecule comprising a nucleic acid encoding human FVIII and a drug selection marker gene;
• selecting an embryonic stem cell comprising said DNA molecule within its genome by growing said cell in the presence of a drug to which expression of the protein encoded by said drug selection marker gene confers resistance;
• microinjecting said embryonic stem cell into a blastocyst;
• implanting said blastocyst into a foster mother;
• breeding the resulting offspring;
• assaying said offspring for the presence of said human FVIII gene within the genome of said offspring;
• and
• breeding to homozygosity those offspring in which said human FVIII gene has been detected;
• whereby a non-human, transgenic animal tolerized to human FVI results.

A method of generating a non-human, transgenic animal tolerized to green fluorescent protein comprising the steps of, in combination,

• transfecting an embryonic stem cell with a DNA molecule comprising a green fluorescent protein gene under the transcriptional control of a promoter selected from the group consisting of a synthetic, tissue-specific or developmentally-regulated promoter;
• selecting an embryonic stem cell comprising said DNA molecule within its genome by growth in the presence of a drug to which expression of the protein product of said drug selection marker gene confers resistance;
• microinjecting said embryonic stem cell into a blastocyst;
• implanting said blastocyst into a foster mother;
• breeding the resulting offspring;
• assaying said offspring for the presence of the green fluorescent protein gene sequence;
• and
• breeding to homozygosity those offspring in which the green fluorescent protein gene sequence has been detected within the genome of said offspring;
• whereby a transgenic animal tolerized to the green fluorescent protein is generated.

A method of testing in vivo delivery of a vector comprising an AAV-ITR and a green fluorescent protein gene to a non-human, transgenic, green-fluorescent protein tolerized animal comprising the AAVS1 sequence within its genome comprising, in combination, the steps of:

• injecting said vector into said animal;
• isolating a blood sample from said animal and analyzing said blood sample for the presence of green fluorescent protein;
• isolating liver tissue from said animal;
• analyzing said liver tissue for the presence of nucleic acid encoding green fluorescent protein gene sequence by polymerase chain reaction (PCR) analysis;
• preparing frozen sections of said liver tissue and analyzing said frozen sections for the presence of green fluorescent protein using a fluorescent microscopic technique;
• wherein the presence of green fluorescent protein is detected within said transgenic animal.