The potential for leukemia caused by retroviral vector integration has become a significant concern for hematopoietic stem cell gene therapy. We analyzed the distribution of vector integrants in pigtailed macaque and baboon repopulating cells for the two most commonly used retroviral vector systems, human immunodeficiency virus (HIV)-based lentiviral vectors and murine leukemia virus (MLV)-based gammaretroviral vectors, to help define their relative genotoxicity. All animals had polyclonal engraftment with no apparent adverse effects from transplantation with gene-modified cells. In all, 380 MLV and 235 HIV unique vector integration sites were analyzed and had distinct distribution patterns in relation to genes and CpG islands as observed in previous in vitro studies. Both vector types were found more frequently in and near proto-oncogenes in repopulating cells than in a random dataset. Analysis of functional classes of genes with integrants within 100 kilobases (kb) of their transcription start sites showed an over-representation of genes involved in growth or survival near both lentiviral and gammaretroviral integrants. Microarray analysis showed that both gammaretroviral and lentiviral vectors were found close to genes with high expression levels in primitive cells enriched for hematopoietic stem cells. These data help define the relative risk of insertional mutagenesis with MLV-, HIV-, and simian immunodeficiency virus (SIV)-based vectors in a highly relevant primate model.
Recent studies in the NOD/SCID model have shown improved engraftment of SCID-repopulating cells and higher levels of engraftment in the secondary transplantation when cells were administered by intramarrow (IM) versus intravenous (IV) injection suggesting that direct injection into the marrow cavity may be beneficial for stem cell engraftment in a clinical setting. To study whether IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model, we compared IM vs IV injection in our competitive repopulation assay in baboons. Enriched CD34+ cells were split into 2 equal fractions and transduced with either a GFP- or YFP-expressing vector. Pretransplant transduction efficiencies and expansion of CD 34+ cells were similar in both fractions. One fraction was then infused into the marrow cavity of the right femur and the other fraction was given intravenously. Three baboons received gene-modified CD34+ enriched autologous bone marrow cells after myeloablative radiation. Peripheral blood granulocyte marking levels showed peaks at 2–3 weeks after transplantation and decreased thereafter. In all three monkeys, marking levels of IM injected cells (GFP) were lower than marking levels of IV injected cells (YFP) early after transplantation up to 7 weeks. However, in two of the three monkeys, GFP marking increased steadily after 2 months resulting in higher marking levels from IM injected cells. The trend sustained up to the last follow-up of nine months after transplantation, marking levels being 25.5% and 7.4% from IM and IV injected cells, respectively, in M00228. This pattern was recapitulated in the marking of bone marrow cells of the two animals. GFP (IM) and YFP (IV) marking levels of bone marrow cells from non-injected bone were 24.2% and 33.9%, respectively, at 1 month, 7.9% and 4.6% at 3 months, 19.1% and 12.6% at 6 months after transplantation in M00228. In addition, the GFP marking of the bone marrow cells from the injected bone was higher than that of the BM cells from non-injected bone while YFP marking level was similar. In conclusion, our data suggest that direct intramarrow injection of CD34+ cells may lead to improved engraftment of long-term repopulating cells. Clonal analysis is currently under way to determine the clonal pattern of the differentially marked repopulating cells.
In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of methylguanine methyltransferase (MGMT-P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. For some applications such as genetic diseases or anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells while for malignant diseases hematopoietic stem cell (HSC) chemo-protection may be necessary during chemotherapy dose escalation. Thus we have explored the use of gammaretrovirally expressed MGMT(P140K) mutant in three baboons. Animals received CD34−enriched cells transduced with a GALV-pseudotyped retroviral vector expressing a bicistronic message containing P140K and GFP. Two of the animals were part of a competitive repopulation assay in which one half of the cells were gene-modified with a GALV-pseudotyped vector expressing only YFP. After stable engraftment all three baboons were treated with various regimens of O6-benzylguanine (O6BG) and temozolomide (TMZ) or BCNU. Following treatment with O6BG/TMZ the selection was transient for ‘protected’ cells gene-modified with MGMT(P140K)-GFP, and the expected negative selection of ‘unprotected’ gene-modified cells (YFP transgene alone) was subtle. Conversely, positive selection of MGMT(P140K)-GFP gene-modified cells and negative selection of YFP gene-modified cells was dramatic and sustained following treatment with only a single dose of O6BG/BCNU. The increase in gene-marking (up to ~85%) is stable following selection out to 22 months. Importantly, selection of hematopoietic cells was polyclonal and no evidence of insertional mutagenesis has been detected. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. We suspect that the delivery/absorption of TMZ in non-human primates is a contributing factor to transient selection because in animals with low levels (<1%) of MGMT(P140K) gene-modified cells no pronounced or sustained drop in white blood cell or platelet counts was observed following O6BG/TMZ. This is the case even up to TMZ dose levels of 700 mg/m2 that is above dose limiting toxicity in humans. In summary, MGMT selection is efficient and well tolerated in monkeys and we believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy. Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow). Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow).
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