IX production in ssAAV1 treated mice. At 4 weeks post injection, muscle transduced with ssAAV1 maintained hF. IX expression concomitant with continued CD8 T cell infiltrates, whereas mice that received scAAV1 had really couple of transduced skeletal muscle cells remaining, and CD8 T cell infiltration had subsided. Mice having a nonsense mutation fail to mount an immune response against F. IX irrespective of the AAV genome With all the indication that scAAV vectors may induce a stronger CD8 T cell response to hF. IX, we subsequent sought to identify whether they could induce a response in hemophilic mice using a mutation that results in non functional hF. IX expression. We had previously esta blished hemophilic mice carrying F9 missense mutations or even a nonsense mutation. When injected i. m. with AAV2 CMV hF.
IX vector, none with the mice of either of those lines showed a CD8 T cell response to F. IX, having said that, mice with a late quit codon mutation created antibodies against hF. IX, indicating that Ruxolitinib clinical trial these mice were not fully tolerant to hF. IX. Hence, we chose the LS line of hemophilic mice to test irrespective of whether i. m. administration of an scAAV1 vector could break CD8 T cell tolerance to hF. IX. One week right after gene transfer with either sc or ssAAV1 vectors, circulating hF. IX was detected at levels similar to these reported above for HB null mutation mice. At two and four weeks post injection, hF. IX expression improved and persisted, with expression levels in ssAAV1 treated mice about three fold greater than scAAV1 injected mice soon after four weeks. None of the LS mice deve loped antibodies inhibitors against hF.
IX more than selelck kinase inhibitor the course on the experiment. Just after four weeks, spleno cytes have been when again harvested to measure the CD8 T cell responses to hF. IX by ELISPOT. As with all the humoral immune response, there was no evidence of splenic hF. IX specific CD8 T cells in LS mice treated with either vector. The predicament inside the muscle itself reflected what had been observed systemi cally. Mice injected with either ss or scAAV1 showed related transduction of skeletal muscle devoid of proof of infiltrating CD8 T cells. In summary, use of scAAV vector didn’t increase the threat for humoral or cellular immune responses for the hF. IX transgene pro duct within the context in the LS nonsense mutation. Since LS mice displayed higher hF.
IX expression levels from ssAAV1 vectors in comparison to scAAV1 inside the absence of an immune response, we wanted to verify the functionality of your self complementary vector on an other background. Therefore, RAG deficient C57BL six mice that lack B and T cells have been injected intramuscularly with 1011 vg of either vector. In these mice, circulating hF. IX levels have been substantially greater in animals treated with scAAV1, suggesting that the inversion in expression levels observed inside the LS mice may perhaps be a strain particular effect.