Use Of Retroviral Vectors Research Articles

Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein.

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein-histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.

Use of a retroviral vector for expression of a foreign gene.

Expression of foreign proteins in both cultured and intact mammalian cells has become essential to evaluating cellular biology and physiology including the proteins involved in the control of eicosanoid production. Many transient expression vectors are commercially available and are in widespread use. However, some cell types, such as the renal mesangial cell, have been difficult to transform with these vectors and require a different strategy to obtain stable expression of the exogenous gene. Viral vectors, including retroviruses, adenoviruses, and the Sinbis virus have been developed over the last decade for foreign gene expression in mammalian cells. This chapter will discuss the use of a replication-incompetent retroviral vector in cell culture to express an exogenous protein.

Development And Application Of A Minimal-Adenoviral Vector System For Gene Therapy Of Hemophilia A

To achieve efficient delivery and sustained expression of the human factor VIII cDNA in vivo, a minimal-adenoviral (mini-Ad) vector system was developed. The system is composed of a mini-Ad vector with essential cis-elements (less than 1 kb) of the viral genome, an E1-deleted ancillary Ad with packaging attenuation, and an E1-complementing production cell line. Based on this system, MiniAdFVIII was generated to deliver a 27 kb expression cassette consisting of a full-length human factor VIII cDNA flanked by human albumin promoter and genomic sequences. The MiniAdFVIII vector mediated expression of functional human factor VIII in HepG2 and 293 cells. A single-dose intravenous injection of 10(11) viral particles in hemophilic mice of MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml up to 369 days) which corrected the FVIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxic effects in mice and dogs after single intravessel doses of up to 3 x 10(11) and 6 x 10(12) viral particles, respectively. Studies for developing the MiniAdFVIII vector with a site-specific integration mechanism and progress in the development of a human factor VIII-tolerized mouse model for pre-clinical studies of MiniAdFVIII are reported. Further pre-clinical studies and product development of MiniAdFVIII for clinical trials are also discussed.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes.

Human immunodeficiency virus (HIV) infection ultimately leads to the destruction of the CD4+ lymphocyte subset and the onset of AIDS. In recent years, several gene therapy procedures making use of retroviral vectors that selectively target HIV susceptible cells have been proposed in order to interfere with HIV productive infection. However, the HIV glycoproteins' inability to be incorporated in other heterologous retroviruses considerably limits true HIV cell tropism of such vectors. We now report the use of murine leukemia virus (MuLV) viral particles harboring a truncated form of the HIV glycoprotein for specific gene delivery. Reporter lacZ gene transfer was determined to be appropriately specific to CD4+ cells when HeLaCD4 cells or peripheral blood lymphocytes (PBLs) were infected with these pseudotyped MuLV virus vectors. In contrast, MuLV viruses harboring amphotropic MuLV envelope glycoproteins displayed a broad and nonspecific infection of PBL subpopulations. This new approach, taking advantage of the ability of truncated HIV envelope glycoproteins to be incorporated into heterologous retroviral particles, may foreseeably be used in future interventions based on the coordinated delivery of therapeutic gene products specifically to cell types susceptible to HIV infection.

Cell cycle dependence of retroviral transduction: An issue of overlapping time scales.

Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility.

Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation.

The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by a cis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independent cis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy.

Cancer Vaccines

AbstractThis unit describes the use of retroviral vectors that can be successfully employed for gene transfer into both primary tumor cultures and established cell lines. The unit includes procedures for assaying the stability of the vaccine following gene transfer. Techniques for maintaining the retroviral producer lines and titering the retroviral vectors are also described. A protocol for frozen storage of the vaccine (transduced tumor cells) is provided. In addition, methods are described for characterizing the vaccine cells following gene transfer. Directions for testing the expression of the transferred gene in the transfected tumor line are also given. The final protocol provides suggestions for designing in vivo animal experiments and discusses what has to be observed in a clinical setting.

Evaluation of recommendations for replication-competent retrovirus testing associated with use of retroviral vectors.

With input from the gene therapy community, CBER is actively examining the recommendations for RCR testing during retroviral vector production, production of ex vivo-transduced cells, and in patients who receive such material. Our initial recommendations were made at a time when our experience with RCR detection assays and clinical use of retroviral vectors was limited. As the gene therapy field has matured, there is an increasing amount of data available on RCR detection assays and from monitoring of patients in clinical trials. The cumulative data give assurance that RCR detection assays in use are of sufficient sensitivity to provide a margin of safety to patients: no patients to date have evidence of RCR infection. However, CBER encourages members of the gene therapy community to continue to submit data to the FDA or to publish data that will enhance the cumulative data base on RCR testing assays, experience with different VPC, and patient monitoring. Based on the analysis of data accumulated to data, and ongoing discussions with members of the gene therapy community, CBER is proposing to discuss changes to the current RCR testing recommendations, as summarized below. RCR testing during production of retroviral vector and ex vivo-transduced cells. Development of characterized standards for RCR testing of supernatant and cells should allow comparison of assay sensitivity. One proposal under consideration is to apply statistical methods to determine how much material needs to be tested independent of the size of the production lot. Data and discussion are still needed to define a limit concentration and a value for probability of detection for RCR testing, while maintaining an appropriate margin of safety. These modifications of RCR testing strategies could lead to improvements in assay sensitivity. Additional discussion and data are also needed to evaluate the current recommendations of the testing for ex vivo-transduced cells: should both cells and supernatant be tested in all cases? RCR testing during patient follow-up. The time points required for RCR testing during patient follow-up need examination. One proposal under consideration is to sample and assay at three time points during the first year of treatment (e.g., 4-6 weeks, 3 months, and 1 year post-treatment). Further discussion is needed to define appropriate additional follow-up. Choice of assays to detect surrogate markers for RCR infection (i.e., serologic or PCR-based assays) should consider mode of vector administration and the patient population. Positive results with such assays should be pursued by direct culture assay to obtain and characterize the infectious viral isolate. These proposals will be the focal point for the discussion at the Retroviral Vector Breakout Session at the 1997 FDA/NIH Gene Therapy Conference. After the 1997 FDA/NIH Gene Therapy Conference, CBR plans to propose revised recommendations for RCR testing for public comment.

Hox genes and embryonic development

The hox genes specify regional differences along the anterior-posterior (A/P) axis of the vertebrate embryo. This function appears to reflect an ancestral role of the hox gene complex and is conserved across phyla. During the evolution of vertebrates, this gene complex has been recruited to perform other functions as well, many of which occur later in development. Although mutational analysis in the mouse is well-suited to the study of their early function, that same function limits the utility of mutational analysis in the investigation of later functions. The use of retroviral vectors to alter gene expression in the chick embryo has emerged as an effective way to address these later functions. This paper reviews that approach and its application to the study of the hox genes in the formation of the vertebrate limb.

Design of retroviral expression vectors.

Several characteristics of amphotropic murine retroviruses have made them useful as vectors for gene transfer with some distinct advantages over other methods of transduction First, the normal replication cycle of retroviruses includes integration of the viral genome into the host's chromosomal DNA, and these viruses have evolved a very efficient mechanism of stable gene transfer for this purpose Second, because the integration machinery uses the termini of the viral DNA as substrate, insertion of viral DNA takes place m a predictable manner with the long terminal repeat (LTR) sequences flanking the genes they carry. Third, retroviruses have a broad host range, which allows gene transfer and expression of foreign genes in many cell types, even including some cell types that are refractory to gene transfer by other means. Fourth, a cytopathic effect on infected cells is lacking, which is especially important for gene expression studies and the generation of stable cell lines Fifth, all retroviral proteins required for the assembly of infectious virions can be supplied in trans, thereby enabling the expression of exogenous genes up to approx 8 kbp. Finally, retroviral vectors can be conveniently manipulated in plasmid form and, as discussed below, are somewhat flexible in terms of vector design. There are, however, some limitations to the use of retroviral vectors. One limitation is that gene transfer only occurs when cells are actively dividing (1) A second limitation is that expression levels are only moderate compared to those achieved by other methods of gene transfer, owing m part to the low copy numbers of the integrated provirus generally achievable by these vectors. The first component of the retrovirus gene transfer system is the retroviral packaging cell line. Packaging cells provide all the retroviral proteins required for the encapsidation of the retroviral vector (in RNA form) into infectious

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders.

Chemoresistance genes have been identified as an impediment to anticancer drug treatment. In particular, P-glycoprotein, the product of the multidrug-resistance (MDR1) gene, plays a major role in clinical treatment failure. Conversely, expression of an MDR1 cDNA in bone marrow of transgenic animals renders hematopoietic cells chemoresistant. Efficient transfer of drug-resistance genes to normal hematopoietic progenitor cells has been achieved with the use of retroviral vectors. In this article we review approaches which use the multidrug-resistance gene to protect bone marrow from myelosuppression following chemotherapy and as a selectable markerin vivo to increase the expression of nonselectable genes which correct hereditary diseases of the hematopoietic system.

A novel murine retrovirus identified during testing for helper virus in human gene transfer trials.

An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses. To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients. This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses. The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors. All of the helper viruses were in a single interference group. We have now shown that treatment of the M. dunni marker rescue assay cells with 5-iodo-2'-deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M. dunni endogenous virus (MDEV). Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium. MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect.

Rapid and Efficient Selection of Human Hematopoietic Cells Expressing Murine Heat-Stable Antigen as an Indicator of Retroviral-Mediated Gene Transfer

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Use of Retroviral Vectors to Introduce and Express the β-Galactosidase Marker Gene in Cultured Chicken Primordial Germ Cells

Three methods of isolating primordial germ cells (PGCs) from gonads of 5-day-old chick embryos were compared. PGCs were then cultured in vitro in DMEM/F12 medium containing 10% fetal calf serum. BrdU incorporation showed that at least 10% of the PGC population were dividing, under our culture conditions, during the 2nd day of in vitro culture. During this culture period, PGCs were exposed to avian leukosis sarcoma virus-based retrovital vector pseudotyped with subgroup A envelope, carrying the LacZ reporter gene. X-Gal staining showed that PGCs were permissive to infection, with more than 50% of PGCs expressing the β-Gal protein. These data represent the first demonstration that PGCs, isolated from gonads of 5-day-old chick embryos, are able to divide in vitro and that it is possible to introduce and express exogenous DNA in chick PGCs maintained in vitro.

Integration and Germ-Line Transmission of a Pseudotyped Retroviral Vector in Zebrafish

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis.

Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis.

New approaches in brain tumor therapy using gene transfer and antisense oligonucleotides

Despite advances in neurosurgery, radiation, and chemotherapy, the prognosis of patients with malignant brain tumors remains grim. New approaches, particularly molecular strategies, have become the clinician's and scientist's hope for the future in brain tumor therapy. Several molecular approaches have been shown to have in vitro and in vivo activities. These approaches include the use of retroviral vectors, herpes simplex viruses, antisense vectors, and antisense oligonucleotides. Preclinical studies of the retroviral vector have already been extended to clinical trials, clearly demonstrating the clinical potential of these molecular therapies.