We have previously demonstrated that our standard HSC transplant procedure for high risk or relapsed AIDS related lymphoma (ARL) resulted in very low mortality and long term survival(22
). We have also suggested a role for RNA based gene therapy as a potential therapeutic approach to controlling HIV infection(28
). Based on these results, we designed and conducted a clinical trial to assess the safety and feasibility of HPC based lentiviral gene therapy for HIV in the context of treatment for ARL. Secondary objectives included monitoring gene marking and RNA expression in the peripheral blood and marrow of treated patients and disease outcome. We describe here the results of a five patient study that establishes compelling evidence for the feasibility of the approach.
Unlike the lentivirus-based stem cell transplantation in adrenoleukodystrophy patients (30
) in which only transduction processed HPC-A cells were infused after complete myeloablation, our study included an unmanipulated HPC-A graft. This was done to comply with regulatory requirements related to the then unknown toxicity and engraftment potential of the transduced cells. While we were successful in mobilizing enough peripheral blood stem cells in this patient population to prepare both unmanipulated and gene modified products for infusion in four of five patients, the infusion of the un-manipulated HPC-A product one day after the gene modified product contributed to a reduction in the frequency of gene modified progenitor cells in vivo
. An additional possible explanation for the low level of marking of the more immature cells is the lack of entry into cell cycle during transduction. Although there is evidence that lentiviral vectors do not need cells to enter cycle to be transfected(31
), other reports indicate that cytokines that induce cell proliferation enhance transduction of engrafting cells (32
). Process development studies to optimize CD34+ cell transduction with minimal loss of hematopoietic potential are currently under investigation in our laboratories.
Importantly, there was no short term toxicity associated with the infusion of the genetically modified HPC-A product, and observed toxicities were procedure-related events consistent with standard autologous HCT. It has been noted, however, that siRNA can be toxic to cells in vitro(35
), and therefore, a major concern for this study was whether expression of siRNA would be observed in the mature cell compartment of treated individuals. We demonstrated with both in vitro and in vivo studies that there was no overt hematopoietic toxicity associated with the lentiviral transduction process. Specifically, the persistent levels of shRNA expression observed in UPN0304 and UPN306 up to 24 and 18 months respectively strongly supports a conclusion that constitutive expression of the tat/rev siRNA is not toxic to peripheral blood cells. In addition, given the genetic marking of T cells, B cells, and granulocytes, there is no evidence for lineage specific toxicity, consistent with in vitro analysis and pre-clinical studies with this vector(13
Interestingly, patients UPN0306 and UPN0304 showed increased levels of gene marking immediately following a viremia (UPN0306 at day 0, UPN0304 at 15 months). Although we can not formally attribute the increase in gene modified cell frequency to viremia, the selective expansion of disease resistant cells in the face of viral selective pressure has been observed in a previous HIV gene therapy trial(19
). While it is not possible at this time to predict what percentage of gene-marked cells would be required for clinical benefit in AIDS, a long term goal of this approach is the selection of genetically modified cells. If HIV could be a selection factor, then treatment interruption of HAART could become a component of gene transfer approaches. Alternatively, the inclusion of a selectable genetic marker in the anti-viral construct may allow for prospective chemotherapeutic selection of disease resistant cells. Support for this strategy comes from studies in large animal models that have demonstrated substantial increases in the percentage of gene marked cells in peripheral blood following transplant of gene modified HSC and drug selection (37
In conclusion, we have developed methods for the isolation, genetic modification and infusion of CD34+ cells that support clinical investigation of stem cell gene therapy strategies for HIV. The sustained expression of siRNA and ribozyme for up to 24 months post-infusion marks an initial milestone in development of a genetic therapy for HIV infection using stem cells. Development of improved transduction processes and revising transplant procedures to preferentially infuse only transduced cells are likely to lead to higher levels of engrafted genetically modified cells. This would provide a setting for delivery and/or selection of therapeutic levels of HIV-resistant cells.