Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector.
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ABSTRACT:
SUBMITTER: Sweeney NP
PROVIDER: S-EPMC4982542 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Viral vectors are effective tools in gene therapy, but their limited packaging capacity can be restrictive. Larger clinically-relevant vectors are needed. Foamy viruses have the largest genomes among mammalian retroviruses and their vectors have shown potential for gene therapy in preclinical studies. However, the effect of vector genome size on titre has not been determined. We inserted increasing lengths of the dystrophin open reading frame in a foamy virus vector and quantified packaged vector RNA and integrated DNA. For both measures, a semi-logarithmic reduction in titre was observed as genome size increased. Concentrated titres were reduced 100-fold to approximately 106 transducing units per ml when vector genomes harboured a 12 kb insert, approximately twice that reported for lentivirus vectors in a comparable study. This potential was applied by optimising foamy virus vectors carrying the full-length dystrophin open-reading frame for transduction of human muscle derived cells. Full-length dystrophin protein was expressed and transduced cells remained able to form myotubes in vitro. Foamy virus vectors are well-suited for stable delivery of large transgene cassettes and warrant further investigation for development as a therapy for Duchenne or Becker muscular dystrophy.
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Project description:Foamy viruses (FVs) or spumaviruses are retroviruses that have been developed as vectors, but their integration patterns have not been described. We have performed a large-scale analysis of FV integration sites in unselected human fibroblasts (n = 1,008) and human CD34(+) hematopoietic cells (n = 1,821) by using a bacterial shuttle vector and a comparable analysis of lentiviral vector integration sites in CD34(+) cells (n = 1,331). FV vectors had a distinct integration profile relative to other types of retroviruses. They did not integrate preferentially within genes, despite a modest preference for integration near transcription start sites and a significant preference for CpG islands. The genomewide distribution of FV vector proviruses was nonrandom, with both clusters and gaps. Transcriptional profiling showed that gene expression had little influence on integration site selection. Our findings suggest that FV vectors may have desirable integration properties for gene therapy applications.
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Project description:BackgroundAfter injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo; however, protocols for transduction of kidney mesenchymal cells have not been established.MethodsWe evaluated the transduction profiles of various pseudotyped AAV vectors expressing either GFP or Cre recombinase reporters in mouse kidney and human kidney organoids.ResultsOf the six AAVs tested, a synthetic AAV called Anc80 showed specific and high-efficiency transduction of kidney stroma and mesangial cells. We characterized the cell specificity, dose dependence, and expression kinetics and showed the efficacy of this approach by knocking out Gli2 from kidney mesenchymal cells by injection of Anc80-Cre virus into either homozygous or heterozygous Gli2-floxed mice. After unilateral ureteral obstruction, the homozygous Gli2-floxed mice had less fibrosis than the Gli2 heterozygotes had. We observed the same antifibrotic effect in β-catenin-floxed mice injected with Anc80-Cre virus before obstructive injury, strongly supporting a central role for canonical Wnt signaling in kidney myofibroblast activation. Finally, we showed that the Anc80 synthetic virus can transduce the mesenchymal lineage in human kidney organoids.ConclusionsThese studies establish a novel method for inducible knockout of floxed genes in mouse mesangium, pericytes, and perivascular fibroblasts and are the foundation for future gene therapy approaches to treat kidney fibrosis.
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Project description:Vector systems based on different retroviruses are widely used to achieve stable integration and expression of transgenes. More recently, transient genetic manipulation systems were developed that are based on integration- or reverse transcription-deficient retroviruses. Lack of viral genome integration is desirable not only for reducing tumorigenic potential but also for applications requiring transient transgene expression such as reprogramming or genome editing. However, all existing transient retroviral vector systems rely on virus-encoded encapsidation sequences for the transfer of heterologous genetic material. We discovered that the transient transgene expression observed in target cells transduced by reverse transcriptase-deficient foamy virus (FV) vectors is the consequence of subgenomic RNA encapsidation into FV particles. Based on this initial observation, we describe here the establishment of FV vectors that enable the efficient transient expression of various transgenes by packaging, transfer, and de novo translation of nonviral RNAs both in vitro and in vivo. Transient transgene expression levels were comparable to integrase-deficient vectors but, unlike the latter, declined to background levels within a few days. Our results show that this new FV vector system provides a useful, novel tool for efficient transient genetic manipulation of target tissues by transfer of nonviral RNAs.
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Project description:Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes the retroviral and lentiviral vectors commonly used for deriving induced pluripotent stem cells (iPSCs). We previously used integrating foamy virus (FV) vectors expressing OCT4, SOX2, MYC and KLF4 to reprogram osteogenesis imperfecta mesenchymal stem cells (MSCs). Here, we have studied the effects of 10 FV vector proviruses on neighboring gene expression in four iPSC lines and their corresponding iPSC-derived MSC (iMSCs). Gene expression profiles in these iPSC lines showed that none of the 38 genes within 300?kb up- or downstream of integrated proviruses had a significant difference in mRNA levels, including five genes with proviruses in their transcription units. In the iMSCs derived from these iPSCs, the same type of analysis showed a single dysregulated transcript out of 46 genes found near proviruses. This frequency of dysregulation was similar to that of genes lacking nearby proviruses, so it may have been due to interclonal variation and/or measurement inaccuracies. While the number of integration sites examined in this paper is limited, our results suggest that integrated FV proviruses do not impact the expression of chromosomal genes in pluripotent human stem cells or their differentiated derivatives. This interpretation is consistent with previous reports that FV vectors have minimal genotoxicity, even when integrating near or within genes.
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