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Viral Vector Facility

AAV serotypes DJ, DJ/8 and DJ/9 now available through VVF

In an effort to identify new AAV capsids that transduce more efficiently and more specifically hepatocytes and that simultaneously evade humoral immune response, cap ORFs isolated from serotype 2, 4, 5, 8, 9, avian, bovine and caprine AAVs were partially fragmented by DNase I (fragment sizes between 0.2 and 1 kbp) and reassembled by primerless PCR into full-length cap ORFs, a technique called DNA family shuffling (Grimm et al., 2008). The chimeric cap ORFs created by DNA familiy shuffling were prepared for insertion into a wild-type recipient ITR-rep plasmid by a second PCR-amplification, that was used to produde a library of AAV encoding cap ORFs.

The library was exposed to purified human intravenous immunoglobulins prior to be added in vitro to primary human hepatocytes or human hepatoma cell lines for amplification. Sequential immunoglobuling exposure and reamplification resulted in various pools of AAVs, of which AAV-DJ (D: Dirk, J: Joyce; the two first authors of the above publication) was the only clone of pool C. The capsid of AAV-DJ is a chimera composed of AAV serotypes 2, 8 and 9 and has shown superior transduction efficiency in vitro and in vivo not restricted to liver cells or the liver.

In order to reduce the heparin binding of AAV-DJ and thereby gain for example more efficient brain transduction after intravenous delivery, two conserved arginines within the AAV serotype 2 heparin binding domain (HBD) located in the AAV-DJ capsid were mutated in such a way, that the amino acid sequence either is in agreement with that of AAV serotype 8 (R586Q and R587T) or serotype 9 (R586S and R587A), resulting in AAV-DJ/8 and AAV-DJ/9, respectively.