Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles

Abstract

We have cloned and characterized the full-length genome of adeno-associated virus type 4 (AAV4). The genome of AAV4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to AAV2 and AAV3. Within the inverted terminal repeat (ITR), several base changes were identified with respect to AAV2. However, these changes did not affect the ability of this region to fold into a hairpin structure. Within the ITR, the terminal resolution site and Rep binding sites were conserved; however, the Rep binding site was expanded from three GAGC repeats to four. The Rep gene product of AAV4 shows greater than 90% homology to the Rep products of serotypes 2 and 3, with none of the changes occurring in regions which had previously been shown to affect the known functions of Rep68 or Rep78. Most of the differences in the capsid proteins lie in regions which are thought to be on the exterior surface of the viral capsid. It is these unique regions which are most likely to be responsible for the lack of cross-reacting antibodies and the altered tissue tropism compared to AAV2. The results of our studies, performed with a recombinant version of AAV4 carrying a lacZ reporter gene, suggest that AAV4 can transduce human, monkey, and rat cells. Furthermore, comparison of transduction efficiencies in a number of cell lines, competition cotransduction experiments, and the effect of trypsin on transduction efficiency all suggest that the cellular receptor for AAV4 is distinct from that of AAV2.

Adeno‐associated virus (AAV) serotypes 2, 4 and 5 display similar transduction profiles and penetrate solid tumor tissue in models of human glioma

“…For recombinant AAV4 vector production, the transfection was performed using 32 µg/plate of the Ad 5‐derived helper plasmid pSR449B 40, 20 µg/plate of AAVRnLacZ, or an expression vector containing the GFP gene and 20 µg/plate of pSV40oriAAV 4–2 containing AAV4 rep and cap sequences 21. To generate AAV5 vectors, 32 µg/plate of pSR449B, 20 µg/plate of AAV5‐nlacZ 20, also expressing the nuclear‐targeted β‐galactosidase gene or the nuclear‐targeted green fluorescent protein (nGFP) gene, and 20 µg/plate of the Rep‐Cap helper plasmid 41 was delivered to the cells.…”

“…A family of distinct AAV serotypes has been identified (AAV1 to 8) 20–24. Although the genomic organization of each serotype is similar, the AAV serotypes differ in their coding sequences.…”

Insertional Mutagenesis of AAV2 Capsid and the Production of Recombinant Virus

“…In addition, the wild-type viable insertion mutants provide a unique opportunity to test insert size limitation as well as ligand-specific retargeting of modified virions (Rabinowitz and Samulski, manuscript in preparation). Recently, the sequence and viral tropism of AAV serotypes 4 and 5 have been characterized (Chiorini et al, 1999(Chiorini et al, , 1997. These viruses do not appear to utilize heparan sulfate as a receptor for infection, and therefore domain swapping between serotypes should provide a unique opportunity to identify amino acids specific for viral binding to target cells (Rabinowitz and Samulski, manuscript in preparation).…”

Postsurgical Assessment and Long-term Safety of Recombinant Adeno-Associated Virus–Mediated Gene Transfer Into the Retinas of Dogs and Primates

“…More recently, 7 other rAAV serotypes (rAAV-1, -3, -4, -5, -6, -7, and -8) have been isolated and cloned. [3][4][5][6][7][8] Recombinant AAV vectors are among the most efficient vehicles for treatment of retinal diseases because the tropisms and transduction patterns of these vectors lead to efficient and stable gene transfer in retinal pigment epithelial (RPE), photoreceptor, and ganglion cells. [9][10][11][12][13][14][15][16][17][18] As a consequence, an increasing number of strategies for the molecular treatment of retinal disease relies on rAAV vectors.…”