Bobbie Thomasson

Methylguanine methyltransferase–mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.

Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs.

The optimal stem cell source for stem cell gene therapy has not been defined. Most gene transfer studies have used peripheral blood or marrow repopulating cells collected after administration of granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF). For clinical applications, however, growth factor administration may not be feasible. Thus, in the current study we used a competitive repopulation assay in the dog to directly compare transduction efficiency of steady-state marrow, G-CSF/SCF-primed marrow, and G-CSF/SCF-mobilized peripheral blood. Cells from all three sources were transduced, cryopreserved, and thawed together before infusion into myeloablated dogs. Gene marking in hematopoietic repopulating cells was assessed by polymerase chain reaction. While primed marrow resulted in the highest long-term marking levels, steady-state marrow was transduced at least as efficiently as mobilized peripheral blood in all three dogs. These results suggest that steady-state marrow may be an appropriate source for genetic modification of hematopoietic stem cells.

704. Ex vivo and in vivo expansion of HoxB4-Transduced CD34+ cells from dogs, baboons, macaques and humans

Successful ex vivo and in vivo expansion of transgene modified hematopoietic stem cells (HSC) have great application potential for clinical gene therapy. Overexpression of the human HoxB4 gene induces ex vivo expansion and self-renewal of murine HSC. Since mouse and human differ substantially, we have started to study the effect of HoxB4 on HSC in a large animal model. Here we investigated if human HoxB4 enhances ex vivo expansion and in vivo selection of CD34+ cells from mobilized dog peripheral blood (PB), cytokine primed baboon and macaque bone marrow (BM), human cord blood (CB) and mobilized human PB. CD34+ cells were transduced with Phoenix-GALV or RD114-pseudotype MSCV-HoxB4-ires-GFP or the control vector, MSCV-ires-YFP. After 4 weeks culture, the percentage of HoxB4-overexpressing cells significantly increased for dog, baboon and macaque cells. HoxB4 also imparts a noteworthy effect on expansion of progenitors. HoxB4 expression resulted in an up to 2-fold expansion for human PB, 4-fold expansion for human CB, 5 to 7-fold expansion for baboon and macaque, and 28-fold expansion for dog cells in CFUs when compared with YFP-transduced cells. Furthermore, in vivo competitive repopulation experiments in NOD/SCID mouse model were performed to test the effects of HoxB4 on HSC. The engraftment level for GFP+ cells was 6-fold higher for PB cells, and 10-fold higher for CB cells compared with YFP+ cells, indicating a significant growth advantage of HoxB4-transduced cells. To examine whether HoxB4 expression promotes expansion of HSC in large animal model, myeloblated dogs were transplanted with HoxB4- and YFP- transduced cells. Preliminary data show that the ratio of GFP+ cells to YFP+ cells increased from 0.9 at day 0 to ratio of 9 at day 20 post-transplant, demonstrating a remarkable in vivo selection of HoxB4-transduced cells in clinically relevant model. To explore the mechanisms that underlie the apparently more pronounced effects on cells from dog, macaque and baboon compared to human cells, we monitored the HoxB4 expression in these cells at the RNA level by real-time RT-PCR and at the protein level by western blot. Comparable level of HoxB4 and YFP transcripts was detected in dog and mouse transduced cells, while copies of HoxB4 transcripts were only 10–30% those of YFP transcripts for human and baboon transduced cells. Likewise, HoxB4 protein expression in human and baboon cells decreased continuously with time, in parallel with the differentiation of these cells. However, protein expression in transduced mouse and dog cells was stable, which was associated with inhibition of cell differentiation. We also found that HoxB4 was tyrosine phosphorylated in transduced mouse and dog cells. Our results suggest that the higher level of HoxB4 protein expressed inhibit the differentiation and promote self-renewal of HSC, while lower level confers a less pronounced effects on self-renewal. Further insights into the regulation of transduced HoxB4 in human cells might pave the road to its successful application in clinical gene therapy.

1. Molecular Long-Term Follow Up in Non-Human Primates That Received Retrovirally Transduced Stem Cells: Despite Proto-Oncogene Insertion No Leukemia or Hematopoietic Abnormalities

Replication defective retroviral gene transfer vectors have been used as an effective method for transduction of hematopoietic stem cells (HSC). Recent advances allowed for gene transfer levels that made gene therapy applicable to use in clinical trials and successfully cured immunodeficient children. Unfortunately two patients in the trial developed T cell leukemia that was found to be associated with insertional mutagenesis caused by retroviral integration in the LMO2 gene (Hacein-Bey-Abina S., et al., Science, 302: 415). This unexpected result has caused a critical evaluation of retrovirus integration in clinically relevant large animal models to assess the likelihood of malignant transformation occurring in gene therapy trials in patients with different diseases and different transgenes. Here we describe the genomic location of more than 100 viral integrations after transplantation of lentivirally or oncoretrovirally transduced stem cells in non-human primates. CD34-enriched cells from G-CSF/SCF primed marrow or mobilized peripheral blood were transduced with oncoretroviral or lentiviral vectors and transplanted into the animals and peripheral blood DNA was prepared for retroviral integration analysis. Integration sites were cloned out of the bulk genomic DNA background by linear-amplification mediated PCR (LAM-PCR). A valid sequence was only scored as an actual integration site if it contained LTR sequence, linker sequence, and matched with at least 90% identity to the July 2003 assembly of the human genome as scored by the BLAT genome analyzing software. For both oncoretroviral and lentiviral integration sites analyzed, 40% mapped into a coding region for a RefSeq gene and that is approximately two times the frequency that was calculated in a computer simulation (Wu X., et al., Science, 300: 1749). This dramatic shift in distribution of integration sites to RefSeq genes indicates a non-random nature to retroviral insertion. Several sites are of interest in that the RefSeq genes are potential proto-oncogenes: melanoma differentiation associate protein-5 (MDA5), neuroblastoma-amplified protein, and RAB37 a member of RAS oncogene family. A variety of genomic locations have insertions, but no gene or region of the genome has been shown to have retroviral integrations in more than one animal. Importantly, all of these animals have been followed from about one to three years and are healthy with no leukemia or other hematologic abnormalities. This evaluation and other recent evidence (Dave U., et al., Science, 303: 333) suggest that retroviral integration into an oncogene is not sufficient for malignant transformation but requires other additional genetic alterations. This analysis and others like it are critical pre-clinical data to address the inherent risks of gene therapy and the effect that retroviral integration has on hematopoietic development.