Search

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)-expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/microL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical.

Progress in retroviral gene transfer to large animal hematopoietic stem cells (HSCs) has led to efficient, reproducible long-term marking in both canine and nonhuman primate models. Successes for HSC gene therapy have occurred in the severe combined immunodeficiency setting, in which transduced cells have a selective advantage. However, for most diseases, the therapeutic transgene does not confer a sufficient survival advantage, and increasing the percentage of gene-marked cells in vivo will be necessary to observe a therapeutic effect. In vivo amplification should expand the potential of HSC gene therapy, and progress in this area has benefited greatly from the use of large animal models where efficacy and toxicity have often not correlated with results in murine models. To date, the best results have been observed with O(6)-methylguanine-DNA methyltransferase (MGMT) selection, with which increases in gene-marked repopulating cells have been maintained long-term, likely because of the toxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea and temozolomide to quiescent HSCs. Using MGMT selection, long-term marking levels exceeding 50% can now be routinely attained with minimal toxicity. There is cause to be optimistic that HSC gene therapy with in vivo amplification will soon allow the treatment of several genetic and infectious diseases.

Adeno-associated virus (AAV) vectors can be used to introduce site-specific mutations into homologous chromosomal sequences. There are many potential applications of this technique, but the process of AAV-mediated gene targeting and factors that influence targeting efficiency are not completely understood. We investigated the dependence of AAV-mediated gene targeting on the host cell-cycle status. The frequency of gene targeting by AAV vectors was compared in dividing and serum-arrested normal human fibroblast cultures. Gene targeting occurred in arrested fibroblast cultures at 0.15 to 1.1% the frequency of dividing cultures, and only took place in cells that had undergone DNA synthesis. Gene targeting was also reduced when DNA synthesis was inhibited by hydroxyurea.

Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.

Foamy virus (FV) vectors are a promising gene delivery system for use in hematopoietic stem cell gene therapy. Previous FV vector marking studies in the NOD/SCID xenotransplantation model used umbilical cord blood (UCB)-derived SCID repopulating cells (SRCs) that were assayed 5-10 weeks posttransplantation. We now report efficient FV vector transduction (>65%) of UCB-derived primitive, long-term SRCs engrafted for 18 weeks. In addition, we evaluated gene transfer into mobilized peripheral blood (MPB)-derived SRCs by improved, deleted FV vectors containing minimal cis-acting sequences and packaged by split helper constructs that would be appropriate for use in clinical trials. When used at a multiplicity of infection of 1 in a 10-hr transduction protocol, these improved vectors transduced 34% of engrafted MPB-derived SRCs.

Foamy virus (FV) vectors show promise for gene therapy applications. However, existing FV vectors either retain a significant portion of the wild-type virus genome or are produced at low titers. We describe a transient cotransfection system that produces high-titer FV vectors with minimal cis-acting regions. These vector genomes have deletions in the gag, pol, env, and bel1-3 accessory genes, as well as the LTR U3 region, but retain an essential 2.5-kb cis-acting region. In addition, stop codons were introduced into the remaining gag sequences to prevent expression of viral peptides and to eliminate dominant-negative effects of a Gag-Pol fusion protein. Although these deleted foamy (deltaphi) vectors were produced at relatively low titers with our prior packaging construct, we designed separate helper plasmids for Gag, Pol, and Env expression that allowed us to routinely produce helper-free, unconcentrated vector stocks with titers of over 10(5) transducing units/ml by four-plasmid transient transfection. The deltaphi vector stocks were then concentrated by ultracentrifugation to titers over 10(7) transducing units/ml. A deltaphi vector containing a 9.2-kb transgene cassette was produced at unconcentrated titers of over 10(5) transducing units/ml, demonstrating the utility of these deleted vectors for large therapeutic genes.

Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34(+) cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34(+) cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy. (Blood. 2001;98:604-609)

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

Mx proteins are members of a family of interferon-inducible genes expressed when cells are treated with double-stranded RNA or virus infection. These proteins are important components of the antiviral response and form the first line of the body's defense against virus infections. The exact mechanism of action for these proteins has not been discovered, but mice missing the Mx genes are extremely sensitive to influenza virus infection. Mammals have between two and three Mx genes whose functions may vary with regard to the inhibition of a specific virus, cellular localization, and activity. The cDNA of three rainbow trout Mx proteins has been cloned and a comparison of their sequences with that of avian and mammalian species reveals striking conservation of domains. They all maintain the tripartite ATP/GTP binding domain and the dynamin family signature in the amino terminal half of the protein. In the carboxyl terminal half of the Mx proteins are the localization signals and the leucine zipper motifs which account for the trimerization of Mx in the cell. Like the rat and human Mx proteins, the different trout Mx proteins exhibit distinctly different immunohistochemical staining patterns in cells transfected with plasmids expressing RBTMx1, RBTMx2, or RBTMx3. To date, the antiviral function of the trout Mx proteins has not been satisfactorily established.

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.

Current efforts to develop vaccines, particularly for aquacultured species, have turned largely to biotechnology because it provides the means to inexpensively produce sufficient quantities of the immunoprotective antigen. These efforts have resulted in several prototype vaccines for fish and the publication of a large number of articles on the subject. However, there are only a few recombinant DNA-based vaccines for aquaculture in the licensing pipeline. Continued funding of research on recombinant DNA vaccines comes from the recognition by industry and government funding agencies that this research can lead to an increased understanding of the mechanisms in protective immunity. This is especially important for fish and shellfish species since our knowledge of the immune mechanisms in these animals is pitifully meagre. This presentation discusses the relative merits of the different recombinant DNA technologies that have been used to produce viral vaccines for fish and the promising approaches that are under consideration to increase the efficacy of these vaccines. There are many approaches to antigen production by recombinant DNA techniques including: (i) the preparation of purified antigenic proteins produced from the cloned viral genes in a variety of vector/host expression systems, (ii) chemical synthesis or the use of fusion vectors to produce peptides corresponding to known epitopes, (iii) defined attenuations, i.e. specific genetic alterations, of live virus vaccines, (iv) the use of live bacterial or viral vectors to deliver resistance genes or viral antigens, (v) anti-idiotype antibodies, and (vi) DNA vaccines where purified plasmid DNA expressing the pathogen gene under a eucaryotic promoter is injected. All of these technologies have been used more or less successfully in the development of vaccines for aquacultured species. However, the requirements for safety, effectiveness, ease of application and low cost/dose restrict their commercial development for aquaculture. The ideal viral vaccine for aquaculture must be effective in preventing death, be inexpensive to produce and license, provide immunity of long duration, and be easily administered. In addition, these vaccines must not only provide protection against the lethal effects of virus infection but prevent the formation of virus persistence. This is especially true for infectious haematopoietic necrosis virus (IHNV) which has been shown to persist in survivors in the presence of high antibody levels. Since resolution of virus persistence is thought to be correlate with cell-mediated immunity, vaccines designed to augment the cell-mediated immunity must be developed for fish. Approaches that are being considered include the use of cytokines in combination with subunit vaccines and the use of specific MHC-I inducer adjuvants with the vaccine. The "tailoring" of vaccine immunogenicity using different combinations of antigen and adjuvant will be presented.

A full-length cDNA clone of a rainbow trout (Oncorhynchus mykiss) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from poly (I).(C)-induced rainbow trout gonad cells (RTG-2). Mx was previously identified in rainbow trout by Staeheli et al. by hybridization with a partial perch genomic Mx probe to induced rainbow trout mRNA. The 2.5 kb rainbow trout cDNA clone contains an open reading frame of 1863 nt (nucleotides) encoding a 621 amino acid protein. The deduced rainbow trout Mx protein is 70.6 kD and contains the characteristic tripartite GTP binding motif common to all Mx protein. Southern blot analysis with the rainbow trout Mx probe demonstrated the presence of Mx homologous genes in four other salmonid fish species, including chinook, coho, and kokanee salmon and brook trout. Poly (I).(C) treatment of both RTG-2 and chinook salmon cells (CHSE-214) induced two transcripts whose appearance was observed first at 24 h and as long as 72 h after treatment. Infection of rainbow trout with the salmonid rhabdovirus, IHNV (infectious hematopoietic necrosis virus), also induced the synthesis of Mx mRNA. A comparison of the rainbow trout Mx protein with other reported Mx proteins indicates that the piscine Mx is highly homologous to the mammalian Mx proteins.