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...