Evidence for the in vivo safety of insulated foamy viral vectors.

Retroviral vector-mediated stem cell gene therapy is a promising approach for the treatment of hematopoietic disorders. However, genotoxic side effects from integrated vector proviruses are a significant concern for the use of retroviral vectors in the clinic. Insulated foamy viral (FV) vectors are potentially safer retroviral vectors for hematopoietic stem cell gene therapy. We evaluated two newly identified human insulators, A1 and A2, for use in FV vectors. These insulators had moderate insulating capacity and higher titers than previously developed insulated FV vectors. The A1-insulated FV vector was chosen for comparison with the previously described 650cHS4-insulated FV vector in human cord blood CD34+ repopulating cells in an immunodeficient mouse model. To maximize the effects of the insulators on the safety of FV vectors, FV vectors containing a highly genotoxic spleen focus forming virus promoter were used to elicit differences in genotoxicity. In vivo, the A1-insulated FV vector showed an approximate 50% reduction in clonal dominance compared with either the 650cHS4-insulated or control FV vectors, although the transduction efficiency of the A1-insulated vector was higher. This data suggests that the A1-insulated FV vector is promising for future preclinical and clinical studies.

Foamy viral vector integration sites in SCID-repopulating cells after MGMTP140K-mediated in vivo selection.

Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector-transduced human repopulating cells is needed. Human CD34(+) human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγ(null) mice, and selected in vivo for gene-modified cells. The retroviral insertion site profile of repopulating clones was examined using modified genomic sequencing PCR. We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy but also suggests additional safety features should be developed and evaluated.

Large animal models of hematopoietic stem cell gene therapy.

Large animal models have been instrumental in advancing hematopoietic stem cell (HSC) gene therapy. Here we review the advantages of large animal models, their contributions to the field of HSC gene therapy and recent progress in this field. Several properties of human HSCs including their purification, their cell-cycle characteristics, their response to cytokines and the proliferative demands placed on them after transplantation are more similar in large animal models than in mice. Progress in the development and use of retroviral vectors and ex vivo transduction protocols over the last decade has led to efficient gene transfer in both dogs and nonhuman primates. Importantly, the approaches developed in these models have translated well to the clinic. Large animals continue to be useful to evaluate the efficacy and safety of gene therapy, and dogs with hematopoietic diseases have now been cured by HSC gene therapy. Nonhuman primates allow evaluation of aspects of transplantation as well as disease-specific approaches such as AIDS (acquired immunodeficiency syndrome) gene therapy that can not be modeled well in the dog. Finally, large animal models have been used to evaluate the genotoxicity of viral vectors by comparing integration sites in hematopoietic repopulating cells and monitoring clonality after transplantation.

Foamy combinatorial anti-HIV vectors with MGMTP140K potently inhibit HIV-1 and SHIV replication and mediate selection in vivo.

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 x 10(7) transducing units ml(-1)) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34(+) cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Helper-free foamy virus vectors.

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.

Characterization of a rainbow trout Mx gene.

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.

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV.

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.

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.

Interferon-inducible Mx proteins in fish.

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.