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.     

Efficient gene transfer by hybrid retroviral vectors to murine spermatogenic cells.

Using murine spermatogenic cell lines GC-1 spg and GC-2 spd(ts) as target cells, an attempt was made to design a retroviral vector that would transduce genes efficiently. Promoter activities of various retroviral long terminal repeats (LTRs) were examined by using chloramphenicol acetyltransferase (CAT) as a reporter. The U3 region of spleen focus-forming virus (SFFVp) showed higher enhancer activity than that of Moloney murine leukemia virus (Mo-MuLV) in both cell lines. The U3 region of myeloproliferative sarcoma virus (MPSV) showed higher activity only in GC-1 spg cells. Expression was suppressed by the repressor element of the primer-binding site (PBS) of the Moloney-related virus. The efficiency of transduction of the multidrug-resistance gene (mdr-1) by an Mo-MuLV-based vector was compared with hybrid vectors consisting of the murine embryonic stem cell virus (MESV) PBS and the LTR of either SFFVp or MPSV. Rhodamine efflux assays and colchicine-resistant colony-forming assays demonstrated higher gene expression by the hybrid vectors. Amphotropic and ecotropic receptors were found to be expressed and functional in both cell lines. Thus, these hybrid vectors represent a powerful tool by which to transfer genes into spermatogenic cells.     

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell 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)     

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.     

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.     

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.     

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.     

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus–based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.     

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.     

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

Gene transfer to the early-stage embryonic brain using the ultrasound image-guided gene delivery (UIGD) technique has proven to be valuable for investigating brain development. Thus far, this technology has been restricted to the study of embryonic neurogenesis. When this technique is designed to be employed for the study in adult animals, a long-term stable gene expression will be required. We attempted to develop a retroviral vector suitable for expressing exogenous genes in the brains of postnatal and adult mice in the context of the UIGD technique. Retroviral vectors containing four different long terminal repeats (LTRs) (each from Moloney murine leukemia virus (MoMLV), murine stem cell virus (MSCV), myeloproliferative sarcoma virus (MPSV) and spleen focus-forming virus (SFFV)) were compared using the well-known CE vector having the EF1α internal promoter as a control. The MS vector containing MSCV LTR produced a higher viral titer and a higher level of gene expression than other vectors including CE. The MS vector drove the gene expression in cultured neural stem cells for 3 weeks. Furthermore, the MS vector could efficiently deliver the gene to the mouse central nervous system, as transgene expression was found in various regions of the brains and spinal cords as well as in all major neural cell types. The data from an in vivo luciferase imaging analysis showed that the gene expression from the MS vector was sustainable for almost 3 months. Our data suggested that the MS vector would be suitable to construct mice containing the transgene expressed in the brain or spinal cord in a quick and cost-effective manner.