Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload-induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy-associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)-operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain-containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases.

Retroviruses depend on the virally encoded IN proteins to facilitate stable insertion of their reverse-transcribed genomes into host cell chromosomes. INs recognize the attachment (att) sites at the ends of long terminal repeats (LTRs) in viral DNA to carry out two sequential enzymatic reactions. In the first reaction, referred to as 3= processing, IN removes dinucleotides from the 3= ends of viral DNA to expose the 3= OH groups attached to the invariant CA dinucleotides. In the second reaction, DNA strand transfer, IN inserts the processed 3= termini into opposing strands of the host chromosomal DNA via a transesterification mechanism (1, 2). Host cell enzymes complete the process by repairing the single-stranded gaps on both sides of integrated viral DNA. Consequently, the resulting provirus is flanked by short duplications of the target DNA sequences. The duplication size appears to be retroviral genus specific, being 5 bp for human immunodeficiency virus type 1 (HIV-1) and 4 bp for murine leukemia virus (MLV) (3-5). The terminal cleavage and strand transfer steps can be observed in vitro with purified recombinant retroviral IN and DNA substrates, demonstrating that IN alone is sufficient to carry out these reactions (3, 6).Retroviral IN consists of three structural domains (reviewed in reference 7). The N-terminal domain (NTD) contains the zinc binding HHCC motif, and a highly conserved catalytic core domain (CCD) contains the essential active site Asp, Asp, and Glu (D, D-35-E motif) residues, which are directly involved in the catalytic activities of IN. The C-terminal domain (CTD) is least conserved (8-11). Mounting evidence suggests that IN functions as a tetramer (12-15). Recent crystal structures of the prototype foamy virus (PFV) IN bound to its viral and host DNA substrates revealed that all three IN domains participate in tetramerization and interactions with viral DNA (16,17).Retroviral integration into cellular DNA does not occur in a random manner with respect to various genomic features (reviewed in reference 18). HIV-1 and other lentiviruses show a remarkable preference for integration within active transcription units (19). In contrast, MLV, a gammaretrovirus, preferentially integrates near transcription start sites and CpG islands, features that are largely avoided by HIV-1 (20, 21). The remaining retroviral genera show other, albeit far less contrasting, integration patterns (22). Integration site selection of HIV-1 and other lentiviruses was shown to depend on the cellular protein lens epithelium-derived growth factor (LEDGF) (reviewed in reference 23). The IN binding domain (IBD) located within the C-terminal region of LEDGF mediates its interactions with HIV-1 and other lentiviral . LEDGF associates with chromatin via its N-terminal PWWP domain, which selectively binds to nucleosomes containing H3 trimethylated on Lys36 (27, 28), an epigenetic mark associated with bodies of transcription units (29). In cells depleted of LEDGF/p75, HIV-1 integration and replication were significantly affect...

No abstract

The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1alpha promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1alpha vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer-promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.

Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.