Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for ß-Thalassemia.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for ß-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of ß-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for ß-thalassemia.

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 ß-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 ß-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.