Genome-Wide Analysis of Alpharetroviral Integration in Human Hematopoietic Stem/Progenitor Cells

Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN) alpha-retroviral vectors in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and compared it to previously reported gamma-retroviral and lentiviral vectors integration profiles obtained in the same experimental setting. Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications). Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications

Mechanisms of retroviral integration and mutagenesis

Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therap

Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors.

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34(+) human hematopoietic stem/progenitor cells (HSPCs). We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant) and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile

Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts

Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human β-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5ι long terminal repeat and gag gene as well as in the β-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity

Distribution of SIN-MLV and MLV integrations in the genome of human HSPCs.

<p>Percentage of intergenic, TSS-proximal or intragenic integration sites in the SIN-MLV, MLV and random control site datasets, together with the percentage of sites at a distance of ±1,000 bp from at least one CpG island or conserved non coding region (CNC).</p

Genomic distribution and association with histone modifications of MLV and SIN-MLV integrations in human HSPCs.

<p>Distribution of the distance of SIN-MLV (green bars) and MLV (red bars) integration sites from the TSS of targeted genes at 2,500 bp (<b>a</b>) or 50 bp (<b>b</b>) resolution. The percentage of genes targeted at each position is plotted on the y axis. The number of plotted sites is higher than the actual number of mapped integrations sites, since each site may relate to more than one gene. The black line indicates the distribution of random control sites. (<b>c</b>) The distribution of H3K4me1 (top panels) and H3K4me3 (lower panels) epigenetic marks in a 10-kb window around vector integration sites (IS) is shown for MLV integrations (left panels) or SIN-MLV integrations (right panels). The mean density of tags (tag/50 bp) of the reference dataset (i.e. each chromatin feature) is plotted on the y axis. The scale of the graph is shown at the top left of each panel.</p

MLV and SIN-MLV integration sites and clusters in CD34⁺ HSPC-specific loci.

<p>Distribution of MLV (red) and SIN-MLV (green) integration clusters (horizontal solid bars) and integrations (vertical marks) in the LMO2, RUNX1, EVI2A/B, and ZNF217-BCAS1 loci as displayed by the UCSC Genome Browser. The base position feature at the top (scale bar and chromosome number) identifies the genomic coordinates of the displayed region.</p