Moloney Murine Leukemia Virus Vector Research Articles

Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration.

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

Bromodomain and extra-terminal (BET) proteins target Moloney murine leukemia virus integration to transcription start sites

A hallmark of retroviral replication is stable integration of the viral genome in the host cell DNA. This characteristic makes retroviral-derived vector particles attractive vehicles for gene therapy. However, retroviral integration is not a random process. Lentiviruses preferentially integrate in the body of active transcription units, while gammaretroviruses, including Moloney Murine Leukemia Virus (MLV), favour transcription start sites and CpG islands. In clinical trials using gammaretroviral vectors for gene therapy, leukemogenesis has been associated with integration of vectors near oncogene transcription start sites. We found that the bromodomain and extra-terminal (BET) proteins (BRD2, BRD3 and BRD4) interact with MLV integrase and direct integration towards transcription start regions. BET proteins specifically bind and co-localize with the gammaretrovirus integrase protein in the nucleus of the cell. The interaction is gammaretroviral-specific and mediated by the integrase C-terminal domain and the BET extraterminal (ET) domain as determined by co-immunoprecipitation assays and in an Alphascreen assay using recombinant proteins. Interfering with chromatin interaction of BET proteins via specific bromodomain inhibitors JQ1 and l-BET decreases MLV virus replication and MLV vector transduction 5-to 10-fold, while HIV vector transduction is not affected. Analysis of viral DNA intermediates by quantitative PCR revealed a block at the integration step. In addition, bromodomain inhibitors do not have an effect on the late steps of viral replication. MLV integration site distribution analysis revealed a strong correlation with the BET protein chromatin binding profile. Finally, expression of an artificial fusion protein that merges the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration into the body of actively transcribed genes, paralleling the Human Immunodeficiency Virus (HIV) integration pattern. Our results explain the molecular mechanism behind gammaretroviral integration site targeting and suggest methods for engineering gammaretroviral vectors with a safer integration site profile.

Deletion of the LTR Enhancer/Promoter Has No Impact on the Integration Profile of MLV Vectors in Human Hematopoietic Progenitors

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34+ human hematopoietic stem/progenitor cells (HSPCs). We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant) and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile.

Analysis of Risk and Mechanism of Insertional Oncogenesis After Gene Transfer Into Hematopoietic Progenitors with Integrating Viral Vectors

AbstractAbstract 2049Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the integration of the viral vectors increased transcription of cancer-related genes and thereby contributed to development of leukemias. The use of gamma-retroviral vectors containing full-length, long terminal repeats (LTRs) with strong promoter and enhancer activity has been well documented to have the potential of resulting in activation of expression of genes neighboring the vector insertion site. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance.In preparation for gene therapy approaches for the Wiskott-Aldrich syndrome (WAS), we used an in vitro assay of murine bone marrow (BM) cell immortalization to compare the consequences of hematopoietic stem cell transduction by three different kinds of viral vectors, including Moloney murine leukemia virus (MMLV), lentivirus (LV), and foamy virus (FV) constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested five different MMLV LTR forms: unmodified (full-MMLV), deleted of most of the two 75-bp repeats associated with the viral enhancer (delE1), deleted of all the two 75-bp repeats and negative control region (NCR) (delE2), deleted of the viral promoter sequences (delP), and with full deletion of enhancer and promoter sequences (delEP). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) or the WAS endogenous promoter (WASp). In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors.To date, full-MMLV and delE1 transduction of 123 and 132 cultures, respectively, has given rise to 48 and 43 immortalized lines (39.0% and 32.5% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. In contrast, transduction of 114 and 62 cultures with LV and FV vectors, respectively, resulted in no immortalized lines.In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 56 and 72 cultures, respectively, has given rise to 24 and 26 immortalized lines (43% and 36% immortalization rate). Again, the difference in immortalization rate between full-MMLV and delE1 was not statistically significant. In contrast, delE2, delP and delEP transduction of 24 cultures each has given rise to 2, 5 and 3 immortalized lines (8.3%, 21% and 13% immortalized ratio, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p<0.01), while there was no significant difference between the full-MMLV and delP vectors.These preliminary results confirm that gamma-retroviral vectors are prone to causing immortalization of hematopoietic cells and indicate that deletion of viral enhancer and/or promoter sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

Effects of the ligand sequence modifications on the retargeted transduction by the retroviral vector having a ligand-chimeric Env protein

There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1alpha) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 10(4) c.f.u. ml(-1). Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 10(6) c.f.u. ml(-1). The SDF-1alpha ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3-51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.

A Rapid Method for Comparing the Oncogenic Potential of Retroviral Vectors.

Retroviral gene transfer has successfully cured patients with X-linked severe combined immunodeficiency (X-SCID). Unfortunately, three of the treated patients developed leukemia at least in part due to insertional activation of a neighboring gene. Further advances of this exciting therapeutic modality will require safer viral vectors. Genotoxicity experiments in animals, although an important preclinical study, are not practical for screening large numbers of vectors. We have developed a rapid plasmid-based assay to compare downstream gene activation by retroviral vectors. We constructed a plasmid, pACT, that contains a minimal CMV promoter, an internal ribosome entry site (IRES) and a luciferase reported gene followed by a polyadenylation site. Four different proviral constructs containing identical internal PGK-EGFP expression cassettes were inserted upstream of the minimal promoter and nucleofected (Amaxa Biosystems) into K562 cells. The relative luciferase activation of each provirus was determined and compared to cells nucleofected with the basic pACT plasmid. The relative luciferase activation by a plasmid containing a Moloney murine leukemia virus (MLV) vector provirus with full length long terminal repeats (LTRs) was 144, 1230, and 4525 at 1, 2 and 4 days post nucleofection, respectively. Changing the orientation of the proviral DNA did not significantly change the results, consistent with an enhancer effect from the proviral LTRs. In comparison, the relative luciferase activation by a plasmid containing a self inactivating (SIN) MLV vector provirus with LTR deletions was only 59, 90 and 170, respectively, and was reduced by greater than 90% with the reverse orientation consistent with residual activation by read-through transcription. For plasmids containing the HIV SIN vector provirus, the relative luciferase activation was 48, 45 and 52, respectively at 1, 2 and 4 days post nucleofection, and decreased by >90% with the inverse orientation. The relative luciferase activation for plasmids containing the Foamy Virus (FV) vector provirus, also a “SIN” vector, was the least, only 3, 2 and 6, respectively, at 1, 2 and 4 days post nucleofection. Therefore, all three SIN vectors (MLV, HIV and FV) produced significantly less downstream gene activation than a conventional MLV vector, with the FV vectors being the lowest. Additional studies comparing various internal promoters and vectors will also be presented. These results demonstrate the usefulness of the pACT assay system to identify safer retroviral vectors.

A nuclear localization signal in the matrix of spleen necrosis virus (SNV) does not allow efficient gene transfer into quiescent cells with SNV-derived vectors

A major limitation in gene therapy for vectors derived from Moloney murine leukemia virus (MLV) is that they only deliver genes into dividing cells. In this study, a careful comparison of spleen necrosis virus (SNV)-derived vectors with MLV and human immunodeficiency virus (HIV)-1 retroviral vectors indicated that SNV vectors can deliver genes 4-fold more efficiently than MLV vectors into aphidicolin-arrested cells, although at a 25-fold lower efficiency than HIV-1-derived vectors. Furthermore, the addition of a NLS in the SNV matrix (MA) that mimics the one located in HIV-1 MA did not increase the ability of SNV vectors to transfer genes into arrested cells. Also, we found that the RD114 envelope was able to pseudotype SNV viral particles in a very efficient manner.

Redirecting Human T Lymphocytes Toward Renal Cell Carcinoma Specificity by Retroviral Transfer of T Cell Receptor Genes

Adoptive T cell therapy of renal cell carcinoma (RCC) is limited by the difficulty in generating sufficient numbers of RCC-reactive T cells in vitro. To circumvent this problem, we cloned T cell receptor (TCR) alpha and beta chains from a tumor-infiltrating lymphocyte clone specific for an RCC tumor antigen and transferred the TCR into human T cell lines and primary T lymphocytes. Efficient TCR expression in primary T lymphocytes was obtained only with a mouse myeloproliferative sarcoma virus (MPSV)-based retroviral vector, not with a Moloney murine leukemia virus (MLV)-based vector, although both viral supernatants were similar in titer, as shown by analysis of copy number integration in transduced T cells. Reverse transcription-polymerase chain reaction analysis revealed a higher amount of TCR-encoding transcripts when T cells were transduced with the MPSV vector in comparison with the MLV vector, indicating that high TCR expression levels can be achieved by appropriate cis-regulatory vector elements. The biological activity of the transferred TCR was shown by specific lysis of RCC cells ((51)Cr release assay) and by interferon gamma and tumor necrosis factor alpha release (enzyme-linked immunosorbent assay) in an antigen-specific and HLA-A*0201-restricted fashion. Comparison of the redirected T lymphocytes with the original tumor-infiltrating lymphocyte clone revealed similar killing and cytokine secretion capabilities. The functional activity of TCR-redirected T lymphocytes was stable over time. The results demonstrate that use of an optimized retroviral vector yielded a high TCR transduction efficiency and stable and high TCR expression in primary human T lymphocytes and redirected their specificity toward RCC cells.

Ubiquitin-dependent Degradation of Interferon Regulatory Factor-8 Mediated by Cbl Down-regulates Interleukin-12 Expression

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8 induced in peritoneal macrophages by interferon-gamma and lipopolysaccharide was degraded rapidly, and degradation of IRF-8 was blocked by MG132, the proteasome inhibitor, but inhibitors of calpain and lysosomal enzymes had no effect. The ubiquitination of IRF-8 was shown by co-immunoprecipitation from RAW264.7 macrophages retrovirally transduced with IRF-8 and hemagglutinin-ubiquitin. The dominant negative ubiquitin mutants K48R and K29R inhibited IRF-8 degradation in 293T cells, confirming the relationship between ubiquitination of IRF-8 and its degradation. IRF-8 carboxyl-terminal truncation mutants were not ubiquitinated and were consequently stable, indicating that the carboxyl-terminal domain of IRF-8 controls ubiquitination. The ubiquitin-protein isopeptide ligase (E3) that ubiquitinated IRF-8 was likely to be Cbl, which formed a complex with IRF-8, demonstrable by both immunoprecipitation and gel filtration. Furthermore, IRF-8 stability was increased by dominant negative Cbl, and IRF-8 ubiquitination was significantly attenuated in Cbl-/- cells. Reflecting increased stability and expression, the IRF-8 carboxyl-terminal deletion mutant induced interleukin (IL)-12 p40 promoter activity much more strongly than IRF-8 did. Furthermore, IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl-/- mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression.

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-β-cyclodextrin (MβC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Phenotype correction of fanconi anemia group a hematopoietic stem cells using lentiviral vector

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells.

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells.

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.

Retroviral Vectors for High-Level Transgene Expression in T Lymphocytes

Efficient expression of genes transferred by retroviral vectors is a prerequisite for gene therapy, especially when the biological effect depends on the amount of transgene product. High-level gene expression is desirable for several gene therapy approaches involving T lymphocytes. We evaluated standard retroviral vectors with cis-regulatory control elements of the Moloney murine leukemia virus (Mo-MLV) with or without the human T cell-specific CD2 enhancer. For comparison, vectors containing the long terminal repeat (LTR) of myeloproliferative sarcoma virus (MPSV) and an improved 5' untranslated region were used (MP71 vectors), with or without the woodchuck hepatitis virus posttranscriptional regulatory element (PRE). All vectors expressed the enhanced green fluorescent protein (GFP) to measure transgene expression. In mouse T cells MP71 vectors with and without the PRE yielded an up to 10-fold higher expression level compared with the Mo-MLV-based vectors currently used for gene transfer into T lymphocytes. A high multiplicity of infection (MOI) of standard Mo-MLV vectors could not reach expression levels obtained with a low MOI of MP71 vector. Ex vivo-transduced mouse T lymphocytes maintained the vector-dependent differences in level of transgene expression in Rag-1-deficient mice when adoptively transferred. In four human T cell lines and human primary T lymphocytes MP71 vectors yielded an up to 75-fold higher GFP expression level in comparison with the standard Mo-MLV vector. In contrast to mouse T cells, the integration of the PRE into MP71 vectors induced in human T cells a further significant increase in transgene expression level. Southern blot analysis of CEM T cells revealed that the superior performance of MP71 vectors was not due to a higher rate of viral integration. In summary, MP71 vectors are useful tools for stable, high-level gene expression in T lymphocytes, for example, in the expression of T cell receptor genes.

Generation of retroviral packaging and producer cell lines for large-scale vector production with improved safety and titer.

Retroviral vectors based on Moloney Murine Leukemia Virus (MLV), described more than 15 years ago (), first entered clinical trials in 1990 (). Since then, a greater understanding of the basic retrovirus biology and how it relates to the production of recombinant retroviral vectors has helped overcome some of the initial hurdles, including low titer, outbreak of replication-competent retrovirus (RCR), and complement inactivation of vector (, , , ). Subsequently, retroviral vectors have become a very safe and powerful tool for efficient in vitro, ex vivo, and in vivo gene transfer for proliferating cells. Several refinements in recombinant MLV vector technology were necessary, particularly for clinical trials using systemic administration of very high vector doses (>1010 transducing units), thus necessitating efficient large-scale manufacture of safe and high-titer retroviral vector preparations.

Lentiviral vector transduction of male germ line stem cells in mice

Transgenesis can be achieved in mice by retroviral transduction of male germ line stem cells (GSCs). However, the transduction efficiency by a Moloney murine leukemia virus (MMLV)-based vector is low, probably due to the characteristically slow cell cycle of stem cells. Since lentiviral vectors can transduce non-dividing cells, they have the potential to efficiently transduce GSCs. Here we report that male GSCs of mice can be transduced in vitro by a lentiviral vector and generate complete spermatogenesis when transplanted into infertile host testes. Transduction efficiencies were comparable to those for MMLV transduction using similar experimental conditions. The results suggest that both lentiviral and MMLV vectors could be effective in transducing GSCs of other species. In addition, these and previous studies suggest that transduction of immature donor stem cells transplanted into immature recipient testes will provide the most efficient system for male germ line modification.

Target Integration by a Chimeric Sp1 Zinc Finger Domain-Moloney Murine Leukemia Virus Integrase in vivo

A specificity protein 1 (Sp1) zinc finger domain containing two tandem zinc fingers was fused to the C terminus of the integrase (IN) protein of the Moloney murine leukemia virus (MuLV). The integrity of the MuLV IN was completely preserved, since the fusion was conducted at the last amino acid residue of the protein. The vector pMIN-Sp1, which carried the fused MuLV IN-Sp1 zinc finger domain gene, was cotransfected with a wild-type MuLV vector pMLV-K to NIH/3T3 cells. A nonradioactive reverse transcriptase assay was performed on culture supernatants collected from the cotransfected cells to confirm the production of recombinant viruses. The expression of the fusion protein and the integration of the MuLV genome by the fusion protein were confirmed by a Northern and then a Southern hybridization analysis on the total RNA or genomic DNA extracted from cells infected by viruses collected from the supernatants of the cotransfected cells. Regions of the host chromosome that were selected by the fusion protein as the integration targets were sequenced using the TOPO^TM cloning method on a series of PCR products generated with a nested set of primers. The percentage of positive clones screened that contained the DNA-binding sequence of the fused Sp1 zinc finger domain was around 13% (5 out of 39 clones). It was found that the Sp1 DNA-binding sequence was only present in regions that were proximal to one of the long terminal repeats of the integrated viral genome, suggesting that the fusion protein could select a target sequence for integration. The host flanking sequences determined for all the positive clones were also used as queries to perform a BLAST search on the GenBank mouse EST entries. Although matching scores for sequences of some of the clones computed were more significant than others, it was difficult to judge whether or not the integration in these clones had been targeted to some gene sequences. Most of the integration sites might exist in the introns, since we found that the probability of the gene sequences containing an Sp1 DNA-binding site was low.