Stable transfection of rat preproinsulin II gene into rat hematopoietic stem cells via recombinant adeno-associated virus

We investigated the ability of recombinant adeno-associated virus (rAAV), t o mediate the transfer of rat preproinsulin II (rl(2)) gene into rat hemato poietic stem cells in vitro and expression of rI(2) following intra-venous (i.v.) injection of infected stem cells into syngeneic rats. The pLP-1 reco mbinant plasmid containing rI(2) was engineered as follows: rI(2) with RSV- promoter was released from PBC12BI (ATCC), purified, and inserted into BamH 1 site of rAAV vector plasmid pWP-19. Plasmid pLP-1, together with pAAV\AD (Somatix Corp.), was used to co-transfect cell line 293 (ATCC). The rAAV ge nome was rescued using helper adenovirus and packaged into mature rAAV viri ons (vLP-1). Bone-marrow from female Wistar-Furth rats was enriched for ste m cells by using plastic adherence and negative selection with monoclonal a nti-rat CD3 and CD45RA to deplete T and B cells. The remaining cells were e xposed to vLP-1 (moi=50:1) for 2 hours. Transfection was confirmed by PCR o f neomycin resistance gene (neo(R)) after 8 days in culture. For in vivo st udies, ten million exposed stem cells were injected i.v. into syngeneic rat s (n=3). The results represent 3 identical experiments. Expression of neo(R ) and rI(2) was analyzed by RT-PCR. At week 1, neo(R) and rI(2) were expres sed in liver, spleen, thymus, peripheral blood lymphocytes and bone marrow. At week 2, neo(R) was expressed in spleen and brain, while at week 6, thym us, lymph nodes, bone-marrow, liver, spleen, and brain expressed neo(R). rI (2) was not detected after week 1. In summary, we showed that rAAV was effi cient for transferring neoR and rI(2) into rat hematopoietic stem cells.