Replication-defective Retroviral Vectors Research Articles

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Split genome-based retroviral replicating vectors achieve efficient gene delivery and therapeutic effect in a human glioblastoma xenograft model

The murine leukemia virus-based semi-retroviral replicating vectors (MuLV-based sRRV) had been developed to improve safety and transgene capacity for cancer gene therapy. However, despite the apparent advantages of the sRRV, improvements in the in vivo transduction efficiency are still required to deliver therapeutic genes efficiently for clinical use. In this study, we established a gibbon ape leukemia virus (GaLV) envelopepseudotyped semi-replication-competent retrovirus vector system (spRRV) which is composed of two transcomplementing replication-defective retroviral vectors termed MuLV-Gag-Pol and GaLV-Env. We found that the spRRV shows considerable improvement in efficiencies of gene transfer and spreading in both human glioblastoma cells and pre-established human glioblastoma mouse model compared with an sRRV system. When treated with ganciclovir after intratumoral injection of each vector system into pre-established U-87 MG glioblastomas, the group of mice injected with spRRV expressing the herpes simplex virus type 1-thymidine kinase (HSV1-tk) gene showed a survival rate of 100% for more than 150 days, but all control groups of mice (HSV1-tk/PBS-treated and GFP/GCV-treated groups) died within 45 days after tumor injection. In conclusion, these findings sug-gest that intratumoral delivery of the HSV1-tk gene by the spRRV system is worthy of development in clinical trials for the treatment of malignant solid tumors. [BMB Reports 2022; 55(12): 615-620].

General steps in the production and establishment of transgenic chicken lines carrying a bacterial lacZ gene expressing β-galactosidase

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. In this study, performance of the SNTZ vector carrying the Eschrichia coli lacZ gene to express a nuclear-localized β-galactosidase was injected into the subgerminal cavity of embryos collected from newly laid eggs was reviewed.

Abstract 5261: Transfection of nerve growth factor cDNA into SK-N-BE(2)C neuroblastoma modulates TrkA and p75 receptor expression and activation

Abstract The diverse biological role of nerve growth factor (NGF) as a necessary modulator of neuroectodermal cell survival, proliferation and differentiation has been previously well described in both normal and neoplastic cell lines. However, recent studies demonstrate that neuroectodermal tumors, such as neuroblastoma, contain genetic alterations that result in limited secretion of active NGF, as well as expression abnormalities of its receptors TrkA and p75. To better define the differential effects of exogenous and endogenously synthesized NGF, the in vitro response of wild-type and genetically engineered neuroblastoma cells was investigated. Replication-defective NGF-expressing retroviral vectors were prepared using the calcium phosphate co-precipitation method and used to transfect SK-N-BE(2)C cells. Presence of the NGF cDNA was confirmed by RT-PCR, and secretion of NGF into the conditioned media by western immunoblotting. NGF transfection resulted in decreased cell proliferation as determined by nonradioactive cell proliferation assay. Wild type and NGF transfected (BE(2)C-NGF) cells were then incubated with exogenous NGF at 200ng/mL for varying time periods up to 24 hours. The expression of TrkA and p75, as well as proteins of their associated survival and apoptosis cascades were determined by Western immunoblotting of whole cell lysates. p75 expression in the NGF transfected cell line was equivalent to the parental wild type cell, but TrkA expression was significantly lower in BE(2)C-NGF. At baseline, BE(2)C-NGF cells demonstrate significant down-regulation of phospho-cRAF and its downstream effector phospho-p42/44 in comparison to the wild type cell line. Additionally, NGF stimulation further down-regulated phospho-c-RAF expression in the NGF transfected cell line, with maximal down-regulation occurring at 24 hours. BE(2)C-NGF cells also demonstrate significant baseline activation of phospho-p38 in comparison to the wild type cell line, with expression further up-regulated with NGF stimulation at 5 minutes and 1 hour. In conclusion, nerve growth factor cDNA gene transfer into wild type SK-N-BE(2)C neuroblastoma results in decreased activation of the p-42/44 survival pathway and concomitant up-regulation of the p-38 apoptotic pathway, leading to a overall decreased rate of proliferation. This effect is likely the result of a decreased TrkA to p75 receptor ratio, and further understanding of these pathways may facilitate the discovery of novel therapeutic targets for neuroblastoma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5261.

Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector

Replication-defective retroviral or lentiviral vectors have been used for the production of transgenic animals. Chicken primordial germ cells (PGCs) are the precursors for ova and spermatozoa. Here, we describe the production of transgenic chickens via a germline transmission system using PGCs infected with a replication-defective lentiviral vector. PGCs were sorted with a fluorescence-activated cell sorter based on the expression of stage-specific embryonic antigen-1 from 2.5- and 5.5-day embryos. PGCs from both stages of embryo were infected with a lentiviral vector at a similar efficiency in vitro. PGCs were then transferred into the bloodstream of 2.5-day recipient embryos. The efficiency with which the PGCs were delivered and settled in the gonads was lower for PGCs from 5.5-day embryos than those from 2.5-day embryos when a limited number of PGCs was transferred, while the difference was not obvious upon the transfer of increased number of cells. Using a high number of 5.5-day PGCs infected with a lentiviral vector, transgenic chimeras (G(0)) with an acceptable efficiency for germline transmission were obtained. G(0) female chickens produced transgenic progeny (G(1)) with higher efficiency compared to G(0) male chickens. In G(1) transgenic chickens obtained by this method, enhanced green fluorescent protein was effectively expressed under the control of the actin promoter.

Late Origin of Glia-Restricted Progenitors in the Developing Mouse Cerebral Cortex

In order to unravel the molecular determinants of cell fate, it is important to understand when fate restriction occurs during brain development. Lineage analysis suggested that bi- or multipotent progenitors persist into late developmental stages in some central nervous system regions, whereas most progenitor cells in the cerebral cortex appeared to be restrained to generate only a single cell type already at early stages. Here we discuss this previous work and present new data demonstrating that cortical progenitors generating exclusively glial cells appear late in development. In utero transduction of cortical progenitors at early and mid-neurogenesis using a combination of replication-defective retroviral vectors encoding different fluorescent proteins indicated that the early developing cortex is devoid of glia-restricted progenitors, although these are frequent during mid- and late neurogenesis. Clonal analyses in vitro using retroviral vectors and live cell tracking by video time-lapse microscopy confirmed these findings, revealing that the early developing cortex harbors 2 main progenitor types: neuron-restricted and bipotent (neuron-glial) progenitors. The latter are responsible for the generation of glial-restricted progenitors at mid- and late neurogenesis.

FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation

Critical steps in coronary vascular formation include the epithelial–mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.

Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16

Insertional activation of cellular proto-oncogenes by replication-defective retroviral vectors can trigger clonal dominance and leukemogenesis in animal models and clinical trials. Here, we addressed the leukemogenic potential of vectors expressing interleukin-2 receptor common gamma-chain (IL2RG), the coding sequence required for correction of X-linked severe combined immunodeficiency. Similar to conventional gamma-retroviral vectors, self-inactivating (SIN) vectors with strong internal enhancers also triggered profound clonal imbalance, yet with a characteristic insertion preference for a window located downstream of the transcriptional start site. Controls including lentivirally transduced cells revealed that ectopic IL2RG expression was not sufficient to trigger leukemia. After serial bone marrow transplantation involving 106 C57Bl6/J mice monitored for up to 18 months, we observed leukemic progression of six distinct clones harboring gamma-retroviral long terminal repeat (LTR) or SIN vector insertions in Evi1 or Prdm16, two functionally related genes. Three leukemic clones had single vector integrations, and identical clones manifested with a remarkably similar latency and phenotype in independent recipients. We conclude that upregulation of Evi1 or Prdm16 was sufficient to initiate a leukemogenic cascade with consistent intrinsic dynamics. Our study also shows that insertional mutagenesis is required for leukemia induction by IL2RG vectors, a risk to be addressed by improved vector design.

Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements that Modulate Splicing

Retroviruses are well known for their ability to incorporate envelope (Env) proteins from other retroviral strains and genera, and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous Env proteins. We investigated the possibility of “genetically pseudotyping” replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. Earlier, we developed replication-competent versions of MLV that stably transmit and express transgenes inserted into the 3′ untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high-titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, extended cultivation of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site, and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution as an approach to efficiently generating and screening for replicating mutants of replication-impaired recombinant viruses.

An Alternative Method for the Rapid Generation of Stable, High-Expressing Mammalian Cell Lines

Biopharmaceutical companies are constantly evaluating new methods for mammalian cell line development that offer any of the following benefits: shorter time lines, improved consistency, higher production, better genetic stability, and increased flexibility. Each of these advantages extends a large cost benefit to companies as their recombinant protein products are moved from development into the clinic and finally to commercial launch. A versatile system has been developed that is capable of transferring genes of interest into a wide variety of mammalian host cells and offers a number of the above advantages over other methods. The system, which is referred to as GPEx™ (an acronym for “gene product expression”), utilizes replication-defective retroviral vectors, derived from Moloney murine leukemia virus (MLV) and pseudotyped with vesicular stomatitis virus G protein (VSV-G), to stably insert single copies of genes into dividing cells. Retrovectors deliver genes coded as RNA that, after entering the cell, are reverse transcribed to DNA and integrated stably into the genome of the host cell. Two enzymes, reverse transcriptase and integrase, provided transiently in the vector particle, perform this function. These integrated genes are maintained through subsequent cell divisions as if they were endogenous

VSV-G pseudotyped, MuLV-based, semi-replication-competent retrovirus for cancer treatment

Low levels of gene delivery in vivo using replication-defective retroviral vectors have severely limited their application for clinical protocols. To overcome this problem, we describe here a semi-replication-competent retrovirus (s-RCR) in which the gag-pol and envelope (VSV-G, vesicular stomatitis virus G protein) genes were split into two vectors. This system offers potential advantages over both replication-defective vectors, in terms of efficiency of in vivo spread through a tumor, and all-in-one replication-competent vectors in terms of the payload of therapeutic genes that can be carried. We achieved a viral titer of s-RCR viruses approximately 70-fold higher than VSV-G pseudotyped, replication-defective vectors. In addition, s-RCR vectors induced tumor killing by the cytotoxicity of VSV-G during viral spread. Inclusion of the herpes simplex virus thymidine kinase (HSVtk30) gene into vectors significantly improved tumor killing activity followed by ganciclovir (GCV) treatment in vitro under conditions of low-level viral replication. However, at high levels of viral spread, VSV-G-mediated cytotoxicity predominated. Xenografts of human fibrosarcoma HT1080 cells, preinfected by semi-replicative green fluorescent protein vectors (semi-GFP), were completely non-tumorigenic in nude mice. Implantation of cells preinfected by semi-replicative TK30 vectors (semi-TK30) mixed with parental HT1080 cells at a ratio of 1:1 efficiently prevented tumor growth in mice treated by GCV. Direct intratumoral injection of HT1080 tumors growing in nude mice, or B16 murine melanoma in immunocompetent mice, with semi-TK30 viruses significantly prolonged survival. Injection of autologous cells (B16) producing semi-TK30 vector into B16 tumors prolonged survival only in mice treated with GCV but not with phosphate-buffered saline (PBS). In contrast, when xenogeneic cells (293T) producing semi-TK30 vectors were injected into B16 tumors, an optimal survival advantage was obtained in mice treated with PBS rather than GCV. These data indicate that complex interactions exist between direct cytotoxicity of VSV-G and HSVtk expression when placed in the context of additional immune parameters, which combine to determine the efficacy of the therapy. Taken together, our data suggest that s-RCR vectors have some potential advantages for development to deliver genes into tumors for cancer treatment but that a combination of factors will impact on the decision as to whether the s-RCR strategy is worth developing to full clinical trials.

Clonal Dominance of Hematopoietic Stem Cells Triggered by Retroviral Gene Marking

Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover.

Characterization of a semi‐replicative gene delivery system allowing propagation of complementary defective retroviral vectors

Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.

PH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN.

The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.

742. Generation and Evaluation of a New Replication-Competent Retrovirus (RCR) Vector Coding for the SuperCD Suicide Gene

Limited efficiency of in vivo gene transfer constitutes the main problem in cancer gene therapy applications employing replication-defective retroviral (RDR) vectors based on Moloney murine leukaemia virus (MoMLV). Therefore, replication-competent retroviral (RCR) vectors have been developed being able to transmit inserted transgenes very efficiently not only in cell culture, but also throughout entire tumor tissues. For efficient elimination of transduced tumor cells RCRs encoding well-known suicide genes like herpes simplex virus thymidine kinase (HSV TK) or yeast cytosine deaminase (YCD) have been made. Here, we generated a new RCR carrying the highly effective SuperCD gene which is composed of a bifunctional fusion suicide gene of YCD and yeast uracil phosphoribosyltransferase (YUPRT). We then compared the in vitro killing potential of this viral vector with the parental one (coding for YCD only). Viral vectors were produced by transient transfection of 293T producer cells with plasmids containing the proviral DNA. Viral supernatant was harvested and titered using real-time-PCR. Human hepatoma cells were transduced with each vector with equal multiplicity of infections; subsequently, incubation with the prodrug 5-FC was carried out. We observed enhanced cell cytotoxicity for the new RCR-SuperCD vector compared to parental RCR-YCD vector. Our results demonstrate that RCR-SuperCD vectors are more effective in toxifying the 5-FC prodrug.

Production of anti-CD2 chimeric antibody by recombinant animal cells

Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H-and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5′Cap- and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5′Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein.

Production of Anti-CD2 Chimeric Antibody by Recombinant Animal Cells

Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H-and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5′Cap- and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5′Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein.

The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery

Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitatively, if not quantitatively, predictable. In addition, it is clear that the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence.

Suppression of tumor growth by viral vector-mediated gene transfer of N-terminal truncated platelet factor 4.

Platelet factor four (PF4), an inhibitor of endothelial cell proliferation in vitro, inhibits angiogenesis and tumor growth in vivo in experimental animals. The present study was designed to determine whether gene therapy-mediated expression of a form of PF4 lacking 16 amino acids of N-terminus from tumor cells could inhibit angiogenesis and tumor growth in vivo. Two replication-defective recombinant retroviral vectors were constructed. One encodes human PF4 (rRV-PF4) and the other encodes the N-truncated peptide (rRVp17-70). These vectors were then used to transduce KB cells, a human head and neck squamous carcinoma cell line. Expression of PF4 and p17-70 transgenes was confirmed by Western blot analysis. In vitro, both rRV-PF4 and rRVp17-70 were able to inhibit selectively the proliferation of human umbilical vascular endothelial cells (HUVEC) but not KB cells. In vivo activity was assessed by injecting 10(7) KB cells subcutaneously into nude mice and by monitoring subsequent tumor growth, xenograft vascular histochemistry, and animal survival. Viral vector-mediated cDNA transfer of PF4 and p17-70 resulted in inhibiting solid tumors through an anti-angiogenic action in vivo. Our data indicate that targeting tumor angiogenesis using viral-mediated gene transfer of full-length and N-terminal truncated PF4 represents a promising strategy for cancer gene therapy.

Development of transgenic chickens expressing bacterial beta-galactosidase.

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. From one of the eight lacZ-positive G(0) males, two lacZ-positive male chickens were produced from a total of 224 G(1) progeny for a germline transmission rate of 0.89%. Both G(1) male chickens carrying the lacZ gene were mated with wild-type female chickens and 46.5% of the G(2) progeny contained the lacZ gene, which is consistent with the expected Mendelian 50% ratio for a heterozygous dominant allele. The product of the lacZ gene, nuclear localized beta-galactosidase, was expressed in primary myoblast cultures derived from G(2) chickens, and it was also expressed in whole G(2) chicken embryos.