HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 59 splice site upstream of the env open reading frame. To determine the role of this splice site in the 59-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 59 splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 59 splice site, SD4, and the free 59 end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position 11 of the 59 splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to 18 of the 59 splice site and all 11 nt constituting the single-stranded 59 end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.
The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.
The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.
IntroductionFanconi anemia (FA) is a complex recessive inherited disorder that is clinically characterized by variable congenital abnormalities, progressive bone marrow (BM) failure, and a high propensity to develop myeloid and epithelial malignancies. 1-5 On a cellular level, FA is characterized by a profound hypersensitivity upon exposure to DNA cross-linking agents such as mitomycin-C (MMC) or diepoxybutane (DEB). [6][7][8][9] Genetically, germ-line mutations in 13 genes (FANCA/B/C/D1/ D2/E/F/G/I/J/L/M/N) result in the clinical phenotype of FA. 2,8,[10][11][12][13][14] Spontaneous genetic correction of a germ-line mutation leading to repopulation of the entire hematopoietic system with normal progeny has been identified in a few FA patients. [15][16][17][18][19] These observations, in combination with the fact that the hematopoietic system can be functionally corrected in mice with targeted disruptions of FA genes by retroviral vectors expressing human analogues of the targeted mouse genes in stem cells, [20][21][22] have led to 3 clinical stem cell gene therapy phase 1 studies in FA-A and FA-C patients. So far, neither long-term marking/correction of cells nor clinical benefits for the patients were observed. 23,24 Due to the biologic characteristics of the gammaretroviral vectors used for transduction of stem cells, 25,26 optimal gene transfer protocols for delivery of genes to mammalian stem cells require a prestimulation period of 1 to 2 days with cytokines that promote the proliferation and survival of stem/progenitor cells. This is followed by a 2-to 3-day exposure of the target cells to vector containing supernatant on the recombinant fibronectin fragment 28 This gene transfer protocol was successful in transducing hematopoietic stem cells in humans, primates/monkeys, and mice. [29][30][31] However, in murine FA models, prolonged in vitro culture of Fancc Ϫ/Ϫ BM results in a length-of-culture-dependent reduction in myeloid progenitors and repopulating ability, 32,33 and the surviving untransduced Fancc Ϫ/Ϫ repopulating cells have an increased risk of developing cytogenetic abnormalities and myeloid malignancies. 22 Therefore, limiting the in vitro culture would be predicted to enhance both the efficacy and safety for genetic therapies of FA stem cells.Wild-type foamy viruses are the only retroviruses that are not associated with any disease in their natural hosts or in accidentally infected human beings. [34][35][36] It has been shown that vectors based on the prototype (formerly human) foamy virus (FV) can efficiently transduce hematopoietic stem cells from mice, 37 dogs, 38 and nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) repopulating human cells. [39][40][41] Further, FV vectors are at least equally efficient at transduction of CD34 ϩ umbilical cord blood cells engrafting in NOD/SCID mice as lentiviral vectors based on HIV-1. 40 In the present study, we demonstrated for the first time the ability of FV vectors encoding the human FANCC transgene to completely correct Fancc Ϫ/Ϫ myelo...
When transplanted into the rat heart by efficient intracoronary delivery, EGFP-expressing HUVECs cause the exclusive but transient labelling of cardiomyocytes. Our in vivo findings suggest that it is not cell fusion and/or transdifferentiation that occurs under these conditions but rather a horizontal gene transfer of the EGFP marker via apoptotic bodies from endothelial cells to cardiomyocytes.
The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.
The E2F1 transcription factor is an essential mediator of p53-dependent and p53-independent apoptosis as part of an anti-tumour safeguard mechanism. In this study, a functional so-called technical knockout (TKO) approach was applied to Saos-2ERE2F1 cells that conditionally activate E2F1 by the addition of 4-hydroxytamoxifen to search for p53-independent pro-apoptotic E2F1 targets. The approach was based on random inactivation of genes after retroviral transfer of an antisense cDNA library enriched of E2F1-induced genes, followed by the selection of Saos-2ERE2F1 cells that survive in the presence of the apoptotic stimulus. We identified 13 novel E2F1 target genes encoding proteins of known cellular function, including apoptosis and RNA binding. FACS analysis revealed that E2F1-induced apoptosis was significantly attenuated in cell clones containing the antisense cDNA fragments of these genes, demonstrating their participation in E2F1 death pathways. Moreover, inactivation of the target genes resulted in a clear increase of cell viability (>80%) in response to E2F1 activation compared with controls (∼30%). Four genes showed an increase in expression intensity in the presence of cycloheximide, suggesting a direct effect of E2F1 on gene transcription, whereas one gene was identified as an indirect target. Our data provide new insight in the regulation of E2F1-induced apoptosis.
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