Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector.

One factor limiting the ability to modify human repopulating hematopoietic cells genetically with retroviral vectors is the relatively low expression of the cognate viral receptor. We have tested sequential transduction of human hematopoietic cells with an adenoviral vector encoding the ecotropic retroviral receptor followed by transduction with an ecotropic retroviral vector. Adenoviral transduction of K562 erythroleukemia cells was highly efficiently with >95% of cells expressing the ecotropic receptor at a multiplicity of infection (MOI) of 103with a correspondingly high transduction with a retroviral vector. Ecotropic receptor expression in CD34+ cells following transduction with adenoviral vectors was increased by at least two-fold (from 20 to 48%) by replacing the RSV promoter with the CMV E1a promoter, resulting in a parallel increase in retroviral transduction efficiency. Replacing the head portion of the fiber protein in conventional adenoviral vectors (serotype 5) with the corresponding portion from an adenoviral 3 serotype resulted in ecotropic receptor expression in 60% of CD34+ cells at an MOI of 104 and a retroviral transduction of 60% of hematopoietic clonogenic progenitors. The sequential transduction strategy also resulted in efficient transduction of the primitive CD34+CD38- subset suggesting that it may hold promise for genetic modification of human hematopoietic stem cells.     

Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors

Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3'LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and progenitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.     

Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.     

增强型绿色荧光蛋白逆转录病毒载体的构造和表达

逆转录病毒载体被广泛用作对造血细胞进行基因转移的工具,转导方法的改进有赖于应用可快速分析并被高效选择的基因标志,为此,我们克隆了增强型绿色荧光蛋白（ＥＧＦＰ）基因,并构建可表达ＥＧＦＰ的逆转录病毒载体ＬＧＳＮ,通过脂质体转梁和交互感染方法建立高滴度的逆转录产病毒细胞,用以分析对造血细胞标记ＥＧＦＰ基因的可行性,流式细胞术和荧光显微镜检测发现,ＥＧＦＰ病毒转录的ＧＰ＋ｅｖｎＡｍ１２细胞和Ｋ５６２细胞均可发出稳定的绿色荧光信号,阳性率可分别达９７％和８６％,聚合酶链反应分析显示ＬＧＳＮ转子细胞内有原病毒的整合,上述结果提示,作为新一代选择性标志,ＥＧＦＰ是适于研究对造血细胞基因转移与报告基因,对促进人类基因治疗的研究有重要意义。     

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.     

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34(+) cord blood-derived dendritic cells

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral- mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects

The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.     

Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors

Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38- cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38- cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38- cells. Replacing the SV40 polyadenylation signal with the human beta-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-beta-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells. Gene Therapy (2000) 7, 2132-2138.     

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。     

Enhanced transgene expression in quiescent and activated human CD8+ T cells

The level of expression of retroviral vector-encoded proteins in T cells, decreasing during periods of quiescence, could be an obstacle to their clinical utility. To identify promoter systems that could increase the strength and persistence of transgene expression in primary human CD8(+) T cells, we designed a panel of Moloney retroviral vectors to express a destabilized enhanced green fluorescent protein (d4EGFP) reporter protein (t(1/2) = 4 hr). We found that the promoters phosphoglycerate kinase (Pgk), beta-actin, and long terminal repeat (LTR) produced the highest levels of d4EGFP expression in proliferating T cells, but that expression dramatically declined in quiescent cells with all promoters. To improve gene expression, we examined the effect of the beta-interferon (IFN) scaffold attachment region (SAR). This SAR augmented expression from mammalian promoters in cycling T cells, but had a small effect on maintenance of expression in resting T cells. However, when the SAR was combined with the LTR promoter, it significantly enhanced expression in resting and cycling cells. These data support use of the IFN-beta SAR with the LTR in Moloney retroviral vectors to help sustain gene expression in resting primary human CD8(+) T cells and to enhance gene expression in activated T cells.     

Effect of scaffold attachment region on transgene expression in retrovirus vector-transduced primary T cells and macrophages.

The scaffold attachment region of the human interferon beta gene (IFN-SAR) inserted into a retroviral vector improved transgene expression in human primary CD4+ and CD8+ T cells, and in primary monocytemacrophages. In T cells, expression of the Maloney murine leukemia virus (Mo-MuLV)-based retroviral vectors was high in activated cells but low in resting cells. Addition of the IFN-SAR sequence enhanced vector expression 2- to 10-fold, and the effect was particularly pronounced in resting T cells. In CD33+CD14+CD4+ monocyte-macrophages derived from transduced hematopoietic stem/progenitor cells (HSPCs) in vitro, the IFN-SAR enhanced vector expression three- to sixfold. We have used the IFN-SAR-containing vectors to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) rev gene. Compared with a standard retroviral vector, the IFN-SAR-containing vector was significantly (p < 0.01) more potent at inhibiting HIV-1 replication in infected CD4+ peripheral blood lymphocytes. In monocytes, however, addition of the IFN-SAR did not significantly improve antiviral efficacy. To understand better the reason for the strong effect of the SAR on antiviral efficacy in T cells we have studied the expression of HIV, Mo-MuLV, and Mo-MuLV + SAR vectors in resting and activated cells. While the expression of all three vectors was lower in resting compared with activated cells, the kinetics of the decrease in expression were fastest for the Mo-MuLV vector, followed by the HIV vector and then the Mo-MuLV + SAR vector. Thus, higher level expression of the Mo-MuLV + SAR vector relative to wild-type HIV at all stages of T cell activation is the most likely explanation for the strong antiviral efficacy. Overall, this study demonstrates the utility of the IFN-SAR sequence for achieving high-level retroviral vector expression in lymphoid and myeloid hematopoietic cells.     

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.     

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.     

Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/107particles) and adenovirus. Transduction of CD34+ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated, quiescent CD34+ cells were resistant to transduction (less than 4%), but transduction increased to 23 +/- 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor alpha (51 +/- 4%) at a multiplicity of infection of 106. rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34+ and CD34+, CD38- subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 +/- 7% of CD34+ progenitors and 21 +/- 6% of CD34+, CD38- progenitors became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient. Gene Therapy (2000) 7, 183-195.     

Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulin-like growth factor receptor antisense.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.     

Toward gene therapy for Gaucher disease

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient engraftment of in utero transplanted mice with retrovirally transduced hematopoietic stem cells

Using an experimental mouse model, we have investigated the kinetics of hematopoietic reconstitution of recipients transplanted during fetal development with fresh and transduced hematopoietic stem cells (HSCs). Total bone marrow (BM) and purified Lin(-)Sca-1(+) cells, either fresh or transduced ex vivo with enhanced green fluorescent protein (EGFP)-encoding retroviral vectors, were in utero transplanted (IUT) into fetal mice. Data obtained 2 months after transplantation showed a similar proportion of engrafted animals, regardless of the fact that samples were purified or not on HSCs, and subjected or not to ex vivo transduction with retroviral vectors. The transplantation of grafts enriched in HSCs, either fresh or transduced, always improved the levels of donor chimerism of IUT mice in comparison with results obtained in mice transplanted with unpurified BM grafts (6.8 and 7.3% versus 1.15% median values, respectively). Significantly, engrafted recipients that were transplanted with the transduced graft always contained transduced EGFP(+) cells in peripheral blood (around 5% of donor cells were EGFP(+) at 2 months post-transplantation). This proportion was essentially maintained at longer times post-transplantation, as well as in secondary recipients transplanted with the BM of IUT mice. Our study describes for the first time a significant and stable engraftment of unconditioned mice subjected to IUT with HSCs transduced with retroviral vectors.     

Importance of murine study design for testing toxicity of retroviral vectors in support of phase I trials.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.