Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10-to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.Adeno-associated virus type 2 (AAV-2) is a parvovirus belonging to the Dependovirus genus (16). AAV-2 has a linear, single-stranded DNA genome containing 4,679 nucleotides that encodes two genes called rep and cap. The rep gene encodes four proteins that play roles in viral DNA replication, DNA packaging, and the regulation of transcription and splicing. The cap gene encodes three proteins (VP1, VP2, and VP3) that package viral DNA and determine viral tropism. The external surface of the capsid is also the target for neutralizing antibodies. To date, about 180 different AAV capsids have been identified. The three capsid proteins are produced from the same translational reading frame by means of alternative splicing and translation initiation sites.The C termini, each of which is typically about 530 amino acids long, are identical and represent VP3. VP3 is the most abundant capsid protein, representing about 80% of the protein in an assembled capsid. VP2 is typically about 65 amino acids longer than VP3 at the amino terminus and is dispensable for most AAV-2 functions (51). VP1 is typically about 135 amino acids longer than VP2 at the amino terminus. This N-terminal domain is located inside the capsid (26) and encodes the phospholipase A2, which is required f...

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose-and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 ؋ 10 12 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.

A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.

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