A Comparative Study Of Retroviral And Lentiviral Vector-induced Genotoxicity And The Application In Induced Pluripotent Stem Cells(iPSCs)

Part I:A comparative study of retroviral and lentiviral vector-induced genotoxicityBackground:Retroviral vectors (RV) are widely used in human gene therapy trials. However, random vector integration can activate adjacent cellular genes. Study of genotoxicity has so far been limited to tumorigenesis through genetic selection. Other types of genotoxicity that permanently alter cell phenotypes receive much less attention. As RV and lentiviral vectors (LV) are increasingly used to modify stem cells, these phenotypic changes will have a long-term effect on the progeny derived from these stem cells. It is therefore important to study vector-induced genotoxicity in the absence of genetic selection.Objectives:To determine the effect of vector integration on host gene expression.Methods:Transduced Jurkat clones were isolated and vector integration site mapped by ligation-mediated PCR. The BLAT program was used to map sequences to the human genome (UCSC Human Genome Project Working Draft). Effect of vector integration on host gene expression was determined by Real time-PCR.Results:(1) RV integration into the human genome prefers retrovirus integration sites (RIS) previously mapped in rodent cells whereas LV integration does not show such a preference. (2) RV preferentially integrates near a transcription start site whereas LV does not, suggesting that the mechanism for vector integration between these two systems is different. (3) Insertion of the cHS4 insulator sequence alters the integration pattern of LV toward the transcription start site. (4) In contrast to LV, RV integration leads to host gene activation at high frequencies. This profound effect suggests that LV is a safer vector to use in gene and cell-based therapy. However, RV may serve to induce insertional mutagenesis in host cells, allowing the identification of genes important for the regulation of cell proliferation and differentiation. RV demonstrated preferential integration near a group of genes termed retroviral common integration sites (CIS) whereas LV exhibited no such preference. RV activated host gene expression more frequently than LV. However, gene activation was not correlated with how far the integration site was from the host gene promoter.Conclusions:RV prefers to integrate near CIS enriched for cell cycle and apoptosis genes. Since gene activation by RV did not depend on the distance between the promoter and the integration site, the three-dimensional architecture of the host gene might affect the RV enhancer to activate host gene transcription. These results also suggest that the process of selecting integration site or the mechanism of integration is different between RV and LV.Partâ…¡:The application of viral vector in somatic cell reprogrammingBackground:Although the insertional mutagenesis in retroviral vector (RV) is more frequently than in lentiviral vector (LV), there are two advantages by using RV. First is the high efficiency of somatic cell reprogramming. Second, the MLV promoter in RV will be silenced during stem cell stage, which is helpful during the differentiation of iPSCs to lineage specific cells. Spinal muscular atrophy (SMA) is an inherited disease of single gene mutation, adapting to investigat the molecular mechanism of SMA and gene correction therapy. Development of induced pluripotent stem (iPS) cells provides an unprecedented opportunity to generate motor neurons.Objectives:(1) To generate normal iPSCs derived from normal human skin fibroblast cells by transduced with four key transcription factors deliveried by RV. (2) To establish SMA iPS cells, and investigate the feasibility of disease-specific iPSCs as mechanism research model. (2) To modify and optimize the reprogramming process.Methods:Fibroblasts from normal human and SMA patient were transduced with the four transcription factors (Oct3/4, Sox2, Klf4 and C-myc) by retroviral vectors respectively. iPS cell colonies were isolated four weeks after transduction and analyzed for the expression of ES cell-specific markers, including TRA-1-80, hBRIX, hDNMT, NODAL, TDGF1, GDF3, NANOG, REX13, hTERT, DPPA4, Oct3/4, Sox2, Klf4 and C-myc. Formation of embryoid bodies (EBs) and differentiation into three germ layers were carried out to confirm the pluripotent potential of these iPS cell lines. In addition, reprogramming process was optimized by combination of LV and transponse et al.Results:(1) iPS cell lines (from normal or patient fibroblast cells) with self renewal and pluripotent potential were established. (2) There is no significant diffience of reprogramming efficiency between normal and SMA patient fibroblast cells. (3) The transgene contained by RV was silenced and the endogenous key transcription factors were reactivated. (4) RV transduced normal or SMA-iPSCs showed no differentiation block. (5) When combination of LV and transponse, the reprogramming is successful, but the efficiency is poor.Conclusion:SMA disease specific iPSCs can be generated by the same way and same efficiency as normal iPSCs. Reprogramming comdition can be optimized by combinated both viral technique and non-viral technique, which will contribute to get safer and effective iPSCs.Part III:The application of viral vector in gene correction of induced pluripotent stem cellsBackground:Gene correction and mouse embryonic stem cells techeniques got together and developed faster during these two decades. Many gene knockout and knockin mouse models were developed by using homologous recombination (HR). Gene therapy in vitro were tested and clinic trial were designed. But due to the ethical problem and genotoxiticity issue of gene therapy, researches and application of gene correction in human monogenic disease is growing slowly.Objectives:(1) To test the feasibility of gene correction on patient's fibroblast cells. (2) To test the feasibility of gene correction on disease specific SMA-iPSCs. (3) Compare the cell phenotype and neural lineage differentiation between SMA-iPSCs and SMA-iPSCs post correction.Methods:(1) Successfully constructure the LV vector contained wild-type SMN gene and Neo drug resistant gene (LV-SMN-Neo). (2) Somatic cells (patient's skin fibroblast cells) were transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. (3) Transduced cells expressed SMN protein and the specific "gem bodies" in nuclears were increased. (4) SMA-iPSCs was transduced with LV-SMN-Neo vector and positive clones were selected by G418 antibiotics. Positive clones expressed SMN protein and specific "gem bodies" in nuclears were increased. (5) During neural lineage differentiation study, we found motor neuron derived from normal iPSCs showed normal mortor neuron formation and neuron specific gene expression (Tuj-1, HB9). But the neuron derived from SMA-iPSCs showed sparse neuron formation and weak expression of neuron specific gene. Neurason formation was much less than normal iPSCs. (6) SMA-iPSCs corrected by LV-SMN-Neo vector were improved in their neural differentiation but still worse than normal iPSCs.Results:It is feasible to correct gene mutation by LV vector in both somatic cell and iPSCs level. LV transduced positive clones could improve the cell phenotype and lineage specific differentiation. Confirming the presence of abnormalities not only will validate the strategy of using iPS cell to study disease pathogenesis but may also provide a platform for drug validation and screening. The availability of SMA motor neurons will also provide an opportunity to study the cellular and molecular mechanisms of SMA pathogenesis.