Transduktionseffizienz, Langzeitexpression und Integrationsspezifität von HSV/AAV-Hybridvektoren in primären humanen hämatopoetischen und mesenchymalen Stammzellen
Final Report Abstract
Due to their self renewal and differentiation capacity genetically modified haematopoietic stem cells (hHSC) have proven their potential in clinical gene therapy applications. In diseases with pathology related to a dysfunctional gene product not confined to the haematopoietic lineage, mesenchymal stem cells (hMSC) have also evolved as an attractive delivery system. Yet to ensure long-term transgene expression for gene-therapy, integration into the host genome is required bearing the risk of insertional mutagenesis. As a vector integrating into a defined genomic locus on chromosome 19, designated the AAVS1-site, Adeno-associated virus (AAV) holds promise for human gene therapy, primarily due to its lack of pathogenicity and low risk of insertional mutagenesis. For directed gene-transfer, Herpesvirus (HSV)/AAV hybrid vectors combine the unique features of each of the parental virus in an ideal manner. Thus the large packaging capacity of HSV amplicon vectors allows for assembly of the essential AAV elements for site-specific integration and the transgene on one vector. In addition, up to 10 copies of the transgene cassette or alternatively large size inserts such as genomic transgene sequences incorporating regulatory elements may be packaged and effectively delivered into a variety of target cells due to the pleiotropic nature of HSV. Employing such a AAV/HSV hybrid vector, this is the first documentation of high efficiency gene tranfer and successfully targeted transgene integration spanning the entire AAVS1 locus in primary human hHSC and hMSC. Long term transgene expression following HSV/AAV infection of human cells has so far been shown only for human neoplastic cell lines or otherwise immortalized cells. Still reports on efficacious in vivo expression of large transgenes in murine fibroblasts bearing the human AAVS1 locus with restoration of gene function in murine models of monogenetic disorders underscore the potential of HSV/AAV hybrid vectors for corrective gene therapy. Our detailed sequence analysis of viral/genomic junctions at the AAVS1-site provides further insight into the complex integration pattern following infection with HSV/AAV hybrid vectors. In hHSC and hMSC cells sustained intermediate level transgene expression during the first 2-3 weeks with a subsequent decline to low level long term transgene expression indicates that early post transduction, episomal copies contribute to transgene expression until finally lost after several rounds of cellular divisions with the exception of those cells harbouring integrated vectors. Frequency of stable transgene expressing cells might thus need to be further optimized by in vitro or in vivo selection of positively transduced progenitor cells for corrective gene transfer approaches. In the absence of such selection and based on the substantiated integration events in hMSC as well as their migratory capacity to sites of tissue damage, inflammation and tumours, a number of therapeutic scenarios can be envisaged in which high levels of a therapeutic molecule are required in the acute treatment phase followed by a therapeutic window in which continuous low level supplementation is sufficient. In view of applications in tissue repair it is noteworthy that the differentiation capacity of HSV/AAV-infected hMSC is maintained.
