Identification of genetic insulator elements to increase the safety of viral gene therapy vectors

Haematopoeitic system diseases, both acquired and inherited, can be currently cured by allogeneic haematopoietic stem cell transplantation. This treatment provides highly successful immune function recovery for patients receiving grafts of HLA-compatible donors but has still a great risk of complications and even failure if no suitable donor is available. Among the alternative therapeutic options, ex vivo retrovirus-mediated gene transfer into haematopoietic progenitor cells has been shown to be an efficient strategy for a substantial number of severe combined immunodeficiency-suffering patients. A recent gene therapy trial has been remarkably effective for the immunological reconstitution of patients suffering from X-linked severe combined immunodeficiency. This treatment was able to provide full correction of disease phenotype and thus, clinical benefit. However, the appearance of leukemia in several patients has put in question the safety of the procedure. This severe adverse event has been attributed to the integration of the therapeutic transgene-carrying viral vector into a known T-cell oncogene, LMO2, thereby contributing to the development of T-cell leukemia by causing aberrant expression of LMO2. Further studies mentioned the possible retroviral-mediated cis-activation of the LMO2 promoter underlying the potential ability of retroviral regulatory elements to influence neighboring gene transcription. This project aimed at decreasing the risk associated with the use of viral vectors for gene therapy through the identification of genetic insulator elements capable of isolating the vector regulatory elements in order to prevent the activation of chromosomal genes by the viral enhancers. We have established a standardized screening procedure whereby the potency of insulators can be assessed quantitatively on relevant vector elements. This assay system consists of a series of plasmids containing two reporter genes: one mimicking a therapeutic gene under the control of strong viral long terminal repeat (LTR) enhancer, and the other one standing for an endogenous gene close to the chromosomal vector integration site. The assay allowed the quantification of the enhancer blocking activity of the well-characterized chicken beta-globin 5'HS4 insulator (cHS4) in cultured cells. We assessed the insulator activity of novel synthetic elements, constructed from optimized binding sites for the insulator protein CTCF. In addition, we demonstrated the enhancer blocking activity of the nuclear factor 1 protein family (CTF/NF1) and showed that it also displays barrier properties, protecting transgene expression from silencing. We showed that both CTCF and CTF/NF1 binding sites act as insulators that mediate potent enhancer-blocker activity, resulting in a commensurate reduction of genotoxicity when implemented in viral gene therapy vectors. Finally, we used the same approach to analyze the enhancer blocking activity of well-known chromatin domain delimiters, matrix attachment regions (MAR), mainly characterized by their barrier properties. We could demonstrate the enhancer blocking ability of the 1-68 MAR that is mainly harbored by an A-T rich core sub-region

Ini1/hSNF5 is dispensable for retrovirus-induced cytoplasmic accumulation of PML and does not interfere with integration.

International audienceRetroviral infection triggers the cytoplasmic translocation of two Crm1-dependent shuttle factors, namely the Ini1 (integrase interactor 1, hSNF5) and the promyelocytic leukemia (PML) protein. Blocking nuclear export of shuttle factors by leptomycin B increases the efficiency of retroviral integration, suggesting that some may mediate antiviral activity. While PML was shown to counteract proviral establishment, it remained unclear whether Ini1, a protein implicated in various processes during human immunodeficiency virus replication, has the same potential. Employing RNA interference-mediated knock-down of Ini1, we show here that the simultaneous accumulation of both proteins in the cytoplasm likely reflects two non-interdependent phenomena. Furthermore, Ini1 does not interfere with retroviral integration, as cells lacking Ini1 show no increased infection susceptibility

Nuclear factor I revealed as family of promoter binding transcription activators.

ABSTRACT: BACKGROUND: Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge. RESULTS: As a study model, we chose the NFI/CTF family of mammalian transcription factors and we compared the results obtained from a genome-wide study of its binding sites with chromatin structure assays, gene expression microarray data, and in silico binding site predictions. We found that NFI/CTF family members preferentially bind their DNA target sites when they are located around transcription start sites when compared to control datasets generated from the random subsampling of the complete set of NFI binding sites. NFI proteins preferably associate with the upstream regions of genes that are highly expressed and that are enriched in active chromatin modifications such as H3K4me3 and H3K36me3. We postulate that this is a causal association and that NFI proteins mainly act as activators of transcription. This was documented for one member of the family (NFI-C), which revealed as a more potent gene activator than repressor in global gene expression analysis. Interestingly, we also discovered the association of NFI with the tri-methylation of lysine 9 of histone H3, a chromatin marker previously associated with the protection against silencing of telomeric genes by NFI. CONCLUSION: Taken together, we illustrate approaches that can be taken to analyze large genomic data, and provide evidence that NFI family members may act in conjunction with specific chromatin modifications to activate gene expressio