Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.

For all selection pathways, genomic DNA containing cap gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells in vitro or the target tissue in vivo. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, in vitro selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, in vivo/ex vivo selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown ex vivo for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, in vivo selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.