Abstract
Lentiviral vectors derived from the Simian Immunodeficiency Virus (SIV) mediate relatively efficient transduction of hematopoietic stem cells (HSCs) from rhesus macaques. While integration sites associated with onco-retroviral vectors have been extensively studied in primate transplantation experiments, much less in known about lentiviral vector integration site patterns. The existing literature is limited to one report showing that SIV vectors have a distinctive genomic integration pattern compared with onco-retroviral vectors (Hematti et al 2004). Here we report our results mapping 263 integration sites for SIV vectors in an autologous rhesus macaque transplantation model. Two SIV vectors were used that expressed either MGMT-P140K alone or MGMT-P140K together with HOXB4 from an internal MSCV promoter. Two rhesus macaques were transplanted with autologous CD34+ cells, half of which were transduced with the MGMT vector and half were transduced with MGMT-HOXB4 vector. The first animal was treated with 7 courses of temozolomide and 6-BG which has resulted in selection of transduced cells in vivo, both at the level of myeloid progenitors, and to a lesser degree, in HSCs. A total of 152 integration sites were identified from this animal based on LAM-PCR. Sequence analysis showed a favored preference for integration into transcription units, which comprised 70% of all integrations, with 64% integrations occurring within introns and 6% within exons. The highest density of SIV integration sites per Mbp were on chromosomes 17 and 19 (0.17 and 0.2 respectively). At different time points during drug treatment, multiple clones contributed to hematopoiesis and 24 clones were identified repetitively. The second animal was treated with two courses of TMZ/BG and two courses of BCNU/BG resulting in selection of transduced cells in all lineages. So far, a total of 111 integration sites have been identified in this animal and a similar general integration pattern was observed as seen in the first animal. Integration into transcription units was favored (71%) with 65% occurring within introns and 6% within exons. The three most gene-dense chromosomes 17, 19 and 22 had the highest density of SIV integration sites (0.11, 0.16 and 0.18 respectively). In this animal, 10 out 111 integration sites were identified repetitively during the drug treatments. Vector integrations near previously described oncogenes were identified in both animals (19 out 152 and 11 out of 111 integration sites for each animal respectively). However, no common integration sites (CIS) into a single oncogene were observed and no abnormal hematopoietic proliferation developed in either animal. Moreover, there were no integrations seen within the MDS/Evi locus that has been previously shown to be a CIS for onco-retroviral vectors. Our study shows that the SIV integration pattern is distinctly different from that obtained with murine oncoretroviral vectors and is consistent with the previous study. The lack of integrations within the MDS1/Evi locus represents a potential safety advantage, however further study will be necessary to determine whether the overall propensity for insertional mutagenesis and transformation is decreased. We also show that multiple clones contributed to hematopoiesis before and after MGMT-mediated selection suggesting that this approach is not necessarily associated with restrictions in clonal numbers contributing to hematopoiesis.
Is normal hematopoiesis maintained solely by long-term multipotent stem cells?