Ex Vivo Lentiviral Gene Therapy as a Cure for Murine Pompe Disease

patients who received intramuscular vector injection. This study was completed with preclinical mouse muscle and liver IS analyses derived from wild-type mice injected intramuscularly or intravenously with AAV1-LPLS447X, respectively. 1969 exact mappable AAV IS were detected in patient muscle, 1735 in mouse muscle and 892 in mouse liver, respectively, while the AAV vector integrated with a frequency of 10-4 to 10-5 into humans. Most AAV-derived LAM-PCR amplicons represented concatemeric rearrangements with large inverted terminal repeat deletions. In respect to gene coding regions, CpG-islands and palindromic regions the integration pattern was largely random. In the analyzed patient muscle, two AAV IS-hotspots were identifi ed showing homology to the mitochondrial (mt) genome. In mouse muscle, three hotspots were observed, one homolog to the mtDNA genome and two in the muscle specifi c nuclear genes Myh and Ttn. Interestingly, no prominent hotspot was detected after intravenous AAV1-LPLS447X injection into mice. Similar sequences present in both the nuclear and the mt genome have been previously described, termed nuclear mitochondrial DNA (NUMT). Therefore, we made use of direct sequencing of enriched murine mtDNA by the MiSeq technology to prove the occurrence of AAV IS in the mt genome. These data confi rmed the previously obtained data by LAM-PCR and showed further that AAV breakage might occur at each vector nucleotide position indicating that the integration frequency is underestimated by 1log. In total, these data are unique in their context and promising for the fi eld of clinical AAV gene therapy. Furthermore, AAV preference for the mtDNA differs from the elucidated AAV traffi cking route from the endosome to the nucleus and may broaden AAV vector application.