A new direction for gene therapy: intrathymic T cell-specific lentiviral gene transfer

Reports of neoplasia related to insertional activation of protooncogenes by retroviral vectors have raised serious safety concerns in the field of gene therapy. Modification of current approaches is urgently required to minimize the deleterious consequences of insertional mutagenesis. In this issue of the JCI, Adjali and colleagues report on their treatment of SCID mice lacking the 70-kDa protein tyrosine kinase, ZAP-70, with direct intrathymic injection of a ZAP-70-expressing T cell-specific lentiviral vector, which resulted in T cell reconstitution. Using lentiviral vectors and in situ gene transfer may represent a safer approach than using retroviral vectors for ex vivo gene transfer into HSCs, avoiding 3 factors potentially linked to leukemogenesis, namely HSC targets, ex vivo transduction and expansion, and standard Moloney leukemia virus-based retroviral vectors.     

A comparison of targeting performance of oncoretroviral versus lentiviral vectors on human keratinocytes

The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant regeneration. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene integration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epidermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-generation of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. However, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentiviral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern.     

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I- transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.     

Quantitative regulation of class switch recombination by switch region transcription

Hematogenous precursors repopulate the thymus of normal adult mice, but it is not known whether this process is continuous or intermittent. Here, two approaches were used to demonstrate that the importation of prothymocytes in adult life is a gated phenomenon. In the first, age-dependent receptivity to thymic chimerism was studied in nonirradiated Ly 5 congenic mice by quantitative intrathymic and intravenous bone marrow (BM) adoptive transfer assays. In the second, the kinetics of importation of blood-borne prothymocytes was determined by timed separation of parabiotic mice. The results showed that >60% of 3-18-wk-old mice developed thymic chimerism after intrathymic injection of BM cells, and that the levels of chimerism (range, 5-90% donor-origin cells) varied cyclically (periodicity, 3 to 5 wk). In contrast, only 11-14% of intravenously injected recipients became chimeric, and chimerism occurred intermittently (receptive period approximately 1 wk; refractory period approximately 3 wk). In the intravenously injected mice, chimerism occurred simultaneously in both thymic lobes; gate opening occurred only after most intrathymic niches for prothymocytes had emptied; and the ensuing wave of thymocytopoiesis encompassed two periods of gating. These kinetics were confirmed in parabiotic mice, and in cohorts of mice in whom gating was synchronized by an initial intrathymic injection of BM cells. In addition, a protocol was developed by which sequential intravenous injections of BM cells over a 3 to 4 wk period routinely induces thymic chimerism in the apparent absence of stem cell chimerism. Hence, the results not only provide a new paradigm for the regulation of prothymocyte importation during adult life, but may also have applied implications for the selective induction of thymocytopoiesis in nonmyeloablated hosts.     

Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors.

Safe and efficient genetic modification of liver cells could enable new therapies for a variety of hepatic and systemic diseases. Lentiviral vectors are promising tools for in vivo gene delivery. Previous data suggested that recruitment into the cell cycle was required for transduction of hepatocytes in vivo. We developed an improved vector design that enhanced nuclear translocation in target cells and significantly improved gene transfer performance. Using the new vector and a panel of internal promoters, we showed that rat hepatocytes were transduced ex vivo to high frequency without requirement for proliferation. On intravenous administration of vector into adult severe combined immunodeficient (SCID) mice, we found high levels (up to 30%) of transduction of parenchymal and nonparenchymal cells of the liver, integration of the vector genome in liver DNA and stable expression of the marker green fluorescent protein (GFP)-encoding gene without signs of toxicity. Coadministration of vectors and 5'-bromo-2'-deoxyuridine in vivo proved that cell cycling was not required for efficient transduction of hepatocytes. In addition to the liver, the spleen and the bone marrow were transduced effectively by systemic delivery of vector. GFP expression was observed in all these organs when driven by the cytomegalovirus promoter and by the phosphoglycerate kinase gene promoter. Using the promoter of the albumin gene, we could restrict expression to hepatocytes. By a single vector injection into the bloodstream of SCID mice, we achieved therapeutic-range levels of the human clotting factor IX, stable in the plasma for up to 1 year (the longest time tested), indicating the potential efficacy of improved lentiviral vectors for the gene therapy of hemophilias and other diseases.     

Studies of thymocytopoiesis in rats and mice. I. Kinetics of appearance of thymocytes using a direct intrathymic adoptive transfer assay for thymocyte precursors

We describe a quantitative intrathymic (i.t.) adoptive transfer system for detecting thymocyte precursor cells in rats and mice. In this system, the generation of donor-origin thymocytes is analyzed on the FACS after the injection of test cells directly into the thymus of sublethally irradiated, histocompatible, RT-7 (rat) or Ly-1 (mouse) alloantigen-disparate recipients. Like the standard i.v. adoptive transfer assays for prothymocytes, the i.t. transfer assay is time, dose, and irradiation dependent. However, unlike the i.v. assays, the i.t. assay is highly sensitive, independent of cell migration, and specific for T-lineage precursor cells. Thus, the i.t. system requires between 25- and 50-fold fewer precursor cells than do the i.v. systems to generate a given number of donor-origin thymocytes; it detects nonmigratory as well as migratory subsets of precursor cells; it detects prethymic and intrathymic precursor cells with equal facility; and it produces a discrete, self-limited wave of donor-origin thymocytes and peripheral T cells. Moreover, neither hemopoietic nor lymphopoietic stem cell chimerism occurs at extrathymic sites. Comparison of the kinetics of thymocytopoiesis in the i.t. and i.v. transfer systems suggest that the seeding efficiency of prothymocytes in the i.v. assay approximates 0.04; the lag phase of the time-response curve is not due to a delay in the entry of prothymocytes into the thymus; and the relative amount of thymocyte precursor activity in various lymphohemopoietic tissues is highest in bone marrow, lowest (or absent) in lymph node, and intermediate in spleen, blood, and thymus. Moreover, the occurrence of saturation kinetics in the dose-response curve of the i.t. system supports the hypothesis that a finite number of microenvironmental niches for prothymocytes may exist in the thymus. These initial observations will require confirmation and extension in future studies. However, based on the present findings and related observations, we anticipate that the i.t. adoptive transfer system will contribute importantly to the definitive analysis of both normal and abnormal thymocytopoiesis.     

Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency

Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.     

Development of an enhanced B-specific lentiviral vector expressing BTK: a tool for gene therapy of XLA

Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved from xenografted NOD/SCID/gammac(null) mice. Using this lentiviral vector, we further demonstrated the possibility to express a functional human BTK protein in long-term human CD34+ cell B-lymphoid progeny. This newly designed lentiviral vector fulfils one of the pre-requisites for the development of efficient and safe gene therapy for X-linked agammaglobulinaemia, the most common primary humoral immunodeficiency disorder.     

慢病毒介导的B区缺失的人凝血因子Ⅷ在NOD/SCID小鼠中的持续表达(英文)

近年来,基因治疗作为一种新的治疗方法给血友病A的治疗提供了新的思路。本研究旨在探讨在体外和NOD/SCID小鼠中应用慢病毒载体介导的血友病A基因疗法的可能性。构建含有B区缺失的人凝血因子Ⅷ(BDDhFⅧ)基因和IRES-eGFP编码序列的慢病毒表达载体pXZ9/BDDFⅧ。通过3质粒共转染293FT包装细胞,包装后感染293FT,HLF,Chang-liver和人骨髓间充质干细胞。在感染后分别通过酶联免疫吸附试验(ELISA),一期法,逆转录-聚合酶链反应(RT-PCR)和聚合酶链反应(PCR)法检测凝血因子Ⅷ(FⅧ)活性,FⅧ抗原,FⅧ的mRNA转录和基因整合情况。超速离心收集病毒颗粒,并通过门静脉注射感染NOD/SCID小鼠。ELISA分析小鼠血浆FⅧ抗原,荧光显微镜观察绿色荧光蛋白的表达,转导后1个月RT-PCR分析小鼠肝脏人FⅧ的转录情况。结果表明:成功制备高浓度的重组慢病毒,并能在体外高效转导靶细胞。感染后72 h能检测到高水平的FⅧ活性和FⅧ抗原。RT-PCR和PCR法能敏感检测到人FⅧ基因转录和整合至感染后的细胞中。在所有接受重组慢病毒颗粒注射后的NOD/SCID小鼠肝脏中均能检测到人FⅧ基因的转录,同时重组慢病毒也能在体内高效转导小鼠肝细胞。在感染后72 h小鼠血浆中人FⅧ水平为(49±6)mU,1周后为(54±8)mU,1个月后为(23±4)mU。结论:携带BDDhFⅧ基因的慢病毒颗粒在体内外能高效转导靶细胞,且所有被转导的靶细胞都能有效的分泌人FⅧ。经过门静脉注射慢病毒颗粒的NOD/SCID小鼠可以持续表达人FⅧ。     

Simplified retroviral vector gcsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in nonobese diabetic/severe combined immunodeficient mice

Despite efforts toward improvements in retrovirus-mediated gene transfer, stable high-level expression of a therapeutic gene in human hematopoietic stem cells remains a great challenge. We have evaluated the efficiency of different viral long terminal repeats (LTRs) in long-term expression of a transgene in vivo, using severe combined immunodeficiency (SCID)-repopulating cell assays. Vectors used were variants of the simplified retroviral vector GCsap with the different LTRs of Moloney murine leukemia virus (MLV), myeloproliferative sarcoma virus (MPSV), and murine stem cell virus (MSCV). The enhanced green fluorescent protein (EGFP) gene was used as a marker to assess levels of transduction efficiency. CD34+ cells isolated from human cord blood were transduced by exposure to virus-containing supernatants on fibronectin fragments and in the presence of stem cell factor, interleukin 6, Flt-3 ligand, and thrombopoietin, and then transplanted into nonobese diabetic/SCID mice. Engraftment of human cells highly expressing EGFP, with differentiation along multiple cell lineages, was demonstrated for up to 18 weeks posttransplant, although the three different vectors showed different transduction frequencies (MLV, <0.1-33.2%; MPSV, <0.1-22.8%; MSCV, 0.3-51.7%). Of importance is that high-level transduction frequencies in human progenitor cells were also confirmed by colony-forming cell assays using bone marrow from transplanted mice, in which EGFP-expressing, highly proliferative potential colonies were observed by fluorescence microscopy. In these mice the vector carrying the MSCV LTR generated more EGFP-expressing human cells than did either of the other two constructs, indicating that GCsap carrying the MSCV LTR may be an efficient tool for stem cell gene therapy.     

Successful engraftment of human postnatal thymus in severe combined immune deficient (SCID) mice: differential engraftment of thymic components with irradiation versus anti-asialo GM-1 immunosuppressive regimens

To develop a model of human thymus growth in vivo, we have implanted postnatal human thymus under the renal capsule of severe combined immune deficient (SCID) mice and assayed for graft survival and graft characteristics 1-3 mo after engraftment. Three groups of SCID mice were engrafted with postnatal human thymus: untreated SCID mice, SCID mice pretreated with 400 cGy of gamma irradiation 1-5 d before engraftment, and SCID mice treated with intraperitoneal anti-asialo GM-1 antiserum every 4-5 d during engraftment. In the untreated group of SCID mice, only 37% of grafts survived and consisted of human thymic microenvironment components and human immature thymocytes. Irradiation of SCID mice before engraftment improved survival of human thymic grafts to 83%, but these grafts were largely devoid of thymocytes and contained only thymic microenvironment components with large numbers of thymic macrophages. Treatment of SCID mice with anti-asialo GM-1 antiserum throughout the engraftment period also promoted human thymus engraftment (70%) and induced SCID B cell Ig production (SCID[Ig+]) in 38% of animals. In SCID(Ig-) anti-asialo GM-1-treated mice, the human thymic grafts were similar in content to those in untreated SCID mice. However, in anti-asialo GM-1-treated animals with grafts that became SCID(Ig+), all animals were found to have mouse-human chimeric grafts in that the human thymic microenvironment (human fibroblasts, thymic epithelium, vessels) was colonized by murine T cells. These data demonstrate that human postnatal thymus will grow as xenografts in SCID mice, and that the components of human thymus that engraft are dependent on the immunosuppressive regimen used in recipient mice. A striking finding in this study was the induction of T and B lymphopoiesis in SCID mice by abrogation of NK cell activity with in vivo anti-asialo GM-1 treatment. These data strongly suggest that asialo GM-1+ NK cells and/or macrophages play a role in mediation of suppression of lymphopoiesis in SCID mice.     

稳定转染增强型绿色荧光蛋白大鼠骨髓间充质干细胞系的建立

背景：利用间充质干细胞或含有治疗因子的干细胞进行有选择性的杀伤肿瘤细胞是一种有前途的治疗方法。目的：建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。方法：通过脂质体介导慢病毒质粒pVector-EGFP、pHelper、Envelope共转染293T细胞完成载体病毒构建,以实时荧光定量PCR检测慢病毒滴度;取对数生长期的SD大鼠骨髓间充质干细胞,以复感染指数MOI值0,5,10,15,20加入携带报告基因增强型绿色荧光蛋白的慢病毒载体稀释液,72h后观察各组增强型绿色荧光蛋白的表达效率及阳性转染率。结果与结论：携带增强型绿色荧光蛋白的慢病毒载体系统转染293T细胞能够正确表达,滴度为1×108TU/mL。包装好的病毒颗粒转染大鼠骨髓间充质干细胞二三天后,各孔均有增强型绿色荧光蛋白的表达。MOI值从0增至10,细胞的阳性表达率逐渐提高（P〈0.05）,MOI值为10的组能获得〉70%的转染率,但MOI值从10增至20,转染率变化不明显。说明以MOI值为10的滴度将慢病毒载体可将外源基因高效转入大鼠骨髓间充质干细胞内,建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。     

Lentiviral vectors mediate efficient and stable gene transfer in adult neural stem cells in vivo

Modulation of adult neurogenesis may offer new therapeutic strategies for various brain disorders. In the adult mammalian brain the subventricular zone (SVZ) of the lateral ventricle is a region of continuous neurogenesis. Lentiviral vectors stably integrate into dividing and nondividing cells, in contrast to retroviral vectors, which integrate only into dividing cells. We compared their potential for gene transfer into both quiescent and slowly dividing stem cells as well as into more rapidly dividing progenitor cells. In contrast to retroviral vectors, stereotactic injection of lentiviral vectors into the SVZ of adult mice resulted in efficient and long-term marker gene expression in cells with characteristics of both immature type B cells and migrating precursor cells. After migration along the rostral migratory stream and differentiation, the number of enhanced green fluorescent protein (eGFP)-expressing granular and periglomerular interneurons increased over time in the ipsilateral olfactory bulb. Moreover, the number of eGFP-labeled neuronal progenitor cells in the SVZ increased over time. By intraventricular injection of lentiviral vectors we could restrict gene transfer to ependymal cells and type B astroglial-like stem cells. In conclusion, lentiviral vectors surpass retroviral vectors in efficient long-term in vivo marking of subventricular zone stem cells for basic research and therapeutic applications.     

Shuttle of lentiviral vectors via transplanted cells in vivo

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.     

Correction of Murine SCID-X1 by Lentiviral Gene Therapy Using a Codon-optimized IL2RG Gene and Minimal Pretransplant Conditioning

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg^−/− mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.     

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.     

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.