Deleted adenovirus vectors and methods of making and administering the same

A propagation-defective adenovirus comprising an adenovirus genome comprising a heterologous nucleotide sequence encoding a lysosomal acid α-glucosidase and one or more deletions in one or more regions selected from the group consisting of:

• (a) the polymerase region, wherein said deletion(s) essentially prevents the expression of a functional polymerase from said deleted region,
• (b) the preterminal protein region, wherein said deletion(s) essentially prevents the expression of a functional preterminal protein from said deleted region,
• (c) the 100K region, wherein said deletion(s) essentially prevents the expression of a functional 100K protein from said deleted region;
• and
• (d) the IVa2 region, wherein said deletion(s) essentially prevents the expression of a functional IVa2 protein from said deleted region.

A method of producing a propagation-defective adenovirus particle comprising one or more deletion(s) in the adenovirus genome, comprising:

• introducing an adenovirus into a mammalian cell, wherein the introduced adenovirus comprises an adenovirus genome comprising one or more deletions in one or more regions selected from the group consisting of:
• (a) the 100K region, wherein the deletion(s) essentially prevents the expression of a functional 100K protein from the deleted region,
• (b) the IVa2 region, wherein the deletion(s) essentially prevents the expression of a functional IVa2 protein from the deleted region, and
• (c) and the preterminal protein region, wherein the deletion(s) essentially prevents the expression of a functional preterminal protein from the deleted region;
• wherein the mammalian cell transcomplements the function(s) deleted from the adenovirus genome;
• and
• collecting the propagation-defective adenovirus particle.

A method of producing a propagation-defective adenovirus particle comprising one or more deletions in the polymerase region and one or more deletions in the E1 region, comprising:

• introducing an adenovirus into a mammalian cell, wherein the introduced adenovirus comprises an adenovirus genome comprising:
• (a) one or more deletions in the polymerase region, wherein the deletion(s) essentially prevents the expression of a functional polymerase from the deleted region,
• (b) one or more deletions in the E1 region, wherein the deletion(s) essentially prevents the expression of one or more functional E1 proteins from said deleted region, and
• further wherein the mammalian cell transcomplements the deleted functions in the adenovirus genome;
• and
• collecting the propagation-defective adenovirus particle.

A method of producing a propagation-defective adenovirus particle, comprising:

• introducing a bacterial plasmid comprising an adenovirus genome into a bacterial cell, wherein said adenovirus genome comprises one or more deletions in one or more regions selected from the group consisting of:
• (a) the polymerase region, wherein said deletion(s) essentially prevents the expression of a functional polymerase from said deleted region,
• (b) the preterminal protein region, wherein said deletion(s) essentially prevents the expression of a functional preterminal protein from said deleted region,
• (c) the 100K region, wherein said deletion(s) essentially prevents the expression of a functional 100K protein from said deleted region, and
• (d) the IVa2 region, wherein said deletion(s) essentially prevents the expression of a functional IVa2 protein from said deleted region;
• amplifying the bacterial plasmid in the bacterial cell;
• recovering the amplified bacterial plasmid from the bacterial cell;
• linearizing the recovered bacterial plasmid;
• introducing the linearized plasmid into a mammalian cell that transcomplements the deleted functions in the adenovirus genome;
• and
• collecting the propagation-defective adenovirus particle.

A method of treating a subject with lysosomal acid α-glucosidase deficiency, comprising administering to the subject a therapeutically-effective amount of a propagation-defective adenovirus comprising an adenovirus genome comprising a heterologous nucleotide sequence that encodes a lysosomal acid α-glucosidase and one or more deletions in one or more regions selected from the group consisting of:

• (a) the 100K region, wherein the deletion(s) essentially prevents the expression of a functional 100K protein from the deleted region,
• (b) the IVa2 region, wherein the deletion(s) essentially prevents the expression of a functional IVa2 protein from the deleted region,
• (c) the preterminal protein region, wherein the deletion(s) essentially prevents the expression of a functional preterminal protein from the deleted region, and
• (d) adenovirus polymerase region, wherein the deletion(s) essentially prevents the expression of a functional polymerase protein from the adenovirus genome.

A method of producing a gutted adenovirus containing a minichromosome, comprising:

• introducing into a mammalian cell expressing a functional 100K protein:
• a plasmid comprising an adenovirus inverted terminal repeat (ITR), an adenovirus packaging sequence, and a heterologous nucleotide sequence, and
• a helper adenovirus comprising an adenovirus genome comprising one or more deletions in the adenovirus 100K region, wherein the deletion(s) prevents the expression of a functional protein from the deleted region;
• collecting the gutted adenovirus containing the minichromosome from the mammalian cell.

A method of producing a gutted aduenovius containing a minichromosome, comprising:

• introducing into a mammalian cell expressing a functional IVa2 protein:
• a plasmid comprising an adenovirus inverted terminal repeat (ITR), an adenovirus packaging sequence, and a heterologous nucleotide sequence, and
• a helper adenovirus comprising an adenovirus genome comprising at one or more deletions in the adenovirus IVa2 region, wherein the deletion(s) prevents the expression of a functional protein,
• collecting the gutted adenovirus containing the minichromosome from the mammalian cell.