49. In Vivo Gene Therapy of Mobilized Hematopoietic Stem Cells With AD5/35 Vectors Encoding a Sleeping Beauty Transposase Integration Machinery

Abstract

Current protocols for hematopoietic stem cell (HSC) gene therapy involve the collection of HSCs from donors/patients, in vitro culture, transduction with retrovirus vectors, and retransplantation into myelo-conditioned patients. Besides its technical complexity, disadvantages of this approach include the necessity for culture in the presence of multiple cytokines which can affect the pluripotency of HSCs and their engraftment potential. Furthermore, the requirement for myeloablative regimens in patients with non-malignant disorders creates additional risks. We therefore explored the potential for in vivo transduction and genome editing of HSCs. Previously, we and others reported that human HSCs express high levels of CD46, a complement regulatory protein, and can therefore be efficiently transduced by CD46-targeting adenovirus vectors such as Ad5/35 vectors.In a CD46-transgenic mouse model we showed that HSCs could be flushed from the bone marrow using a mobilizing regimen of GCSF and the CXCR4 antagonist AMD3100. These mobilized HSCs could then be transduced with intravenously injected, GFP-encoding Ad5/35 vectors. Upon transduction, mobilized HSCs relocalized back to the bone marrow, expressed the transgene and were clonogenically active in colony forming unit assays. When we followed transgene expressing HSCs in vivo over a course of 2 weeks we saw a loss of transgene expression over time. This is most likely due to leaky viral gene expression of the first generation Ad vector used in these studies. To avoid these cytotoxic effects, we employed a Helper-dependent Ad (HDAd) vector that is devoid of all viral genes. We compared the transduction performance of the HDAd to the first generation vector both in the CD46-transgenic mouse model as well as in a humanized NOG mouse model.Adenoviral vectors do not integrate into the genome of the host cell and we have therefore only been able to follow transduced cells for limited periods of time. In order to be able to track transduced HSCs and their progeny for extended periods of time and to assess their long-term repopulating potential we are developing a set of HDAd vectors that allow for genomic integration of the transgene using a sleeping beauty transposon approach. In in vitro transduction studies we were able to show that the HDAd-encoded integration machinery can be used to insert a GFP cassette into the genome of HSCs, that the transduced HSCs remain clonogenic and that the transgene is passed on to progeny cells. Preliminary in vivo transduction studies in mobilized CD46-tg mice show GFP expression in up to 2.5% of bone marrow-derived lineage-depleted cells at 4 weeks after vector injection, indicating stable in vivo transduction of HSCs.