Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity

Abstract

Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

Fig. 1

Schematic diagrams for summarizing the different methods available to identify proviral insertion sites. (A)Methods requiring restriction enzyme digestion adjacent to insertion sites, including inverse PCR, LM-PCR, LAM-PCR and multi-arm optimized LAMPCR. (B) Methods without restriction enzyme digestion, including FLEA-PCR, nrLAM-PCR and transposase MuA based-PCR. (Timeline shows the year when each method was first described.)