Stable Integration of Recombinant Adeno-Associated Virus Vector Genomes After Transduction of Murine Hematopoietic Stem Cells

Abstract

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes

“…60 Importantly, integration sites in the mouse genome have not been found within coding regions of known genes, including oncogenes, and infection in mice or humans with AAV has not been linked to any pathological abnormalities. 36,37 Our data indicate that AAV-mediated expression of GLP-1 in beta-cells following single intraperitoneal administration can mitigate the onset of diabetes in a model of type-1 diabetes. Targeted production of GLP-1 in beta-cells on its own, or coupled with a therapy designed to reduce autoimmune destruction, such as beta-cell expression of interleukin-4 as described by Rehman et al, 29 may represent a novel therapeutic strategy for type-1 diabetes.…”

“…AAV vectors represent an innovative gene therapy tool, having reduced immunogenicity compared with that of adenoviruses, 36 while being capable of directing long-term expression of transgenes both in humans and in mice. 37-39 AAV8 appears not to be neutralized by antisera directed against other AAV serotypes, and only low levels of neutralizing antibodies against AAV8 have been detected in normal human subjects, 26,40 suggesting that immune responses that have been observed following AAV2-mediated gene therapy 41 may be minimized following AAV8 administration.…”

“…Thus, long-term, stable expression of GLP-1 could be expected in human beta-cells following AAV administration. In mice, integration of AAV is less well-defined; 37 however, long-term expression of transgenes delivered through AAV vectors has been observed, 28 possibly through extra-chromosomal maintenance of the viral vector, or through random integration into the genome as can occur in the absence of Rep protein expression. 60 Importantly, integration sites in the mouse genome have not been found within coding regions of known genes, including oncogenes, and infection in mice or humans with AAV has not been linked to any pathological abnormalities.…”

Gene Transfer Properties and Structural Modeling of Human Stem Cell-derived AAV

“…We and others have previously demonstrated integration of AAV vector sequences in CD34 + cells by fluorescent in situ hybridization, Southern blots, and sequence analysis. 25,[35][36][37] Deep sequencing studies are in progress to further evaluate the status of the long-term surviving AAV vector genomes.…”

Enhanced Long-Term Transduction and Multilineage Engraftment of Human Hematopoietic Stem Cells Transduced with Tyrosine-Modified Recombinant Adeno-Associated Virus Serotype 2

“…þ HSCs (Fisher-Adams et al, 1996;Chatterjee et al, 1999;Santat et al, 2005;Han et al, 2008) and efficient transduction of primitive, pluripotent, self-renewing human HSCs capable of supporting primary and secondary multilineage engraftment in immune-deficient nonobese diabetic/severe combined immunodeficiency mice (Santat et al, 2005). Subsequently, we showed that transduction of primitive HSCs capable of supporting serial engraftment was attributable to the propensity of rAAV to efficiently transduce primitive, quiescent CD34 þ CD38 -cells residing in G 0 (Paz et al, 2007).…”