Genomic Safe Harbors in Human iPS Cells

Abstract SCI-47

Current hematopoietic stem cell (HSC) gene therapy relies on randomly integrated retroviral vectors and is hampered by the risk of insertional oncogenesis often leading to leukemia. This risk would be minimized if therapeutic transgenes could be inserted in selected sites of the genome that permit appropriate function without disruption or dysregulation of endogenous genes — referred to as “genomic safe harbors.” The advent of induced pluripotent stem (iPS) cell technology offered unprecedented opportunities for the genetic engineering of human cells. iPS cells, unlike HSC, can be extensively cultured in vitro, enabling the selection and study of unique sites of transgene integration for the first time in a relevant setting. We proposed a definition of safe harbor sites, based on their topology in the genome with relation to coding genes and other genomic landmarks, using five criteria: (i) distance of at least 50 kb from the 5’ end of any gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions of the human genome (1). To test them, we developed a strategy to select iPS cell clones harboring a single copy of a randomly integrating vector at sites that meet our safe harbor criteria. In a recent proof-of-principle study, using a model for genetic correction of β-thalassemia major, we demonstrated that erythroid progeny of patient-specific iPS cell clones harboring a lentivirally encoded β-globin transgene in a safe harbor site express therapeutic levels of β-globin without perturbing neighboring genes. This approach, entailing the prospective screening and selection of integration sites, based on combined bioinformatics and functional analyses, provides a robust and dependable strategy for the genetic engineering of human iPS cells. iPS cell-derived cell products used in regenerative medicine will need to be genetically engineered to correct a genetic disease or permit in vivo cell tracking or the elimination of residual undifferentiated cells or progeny gone astray. Our strategy should be broadly applicable to introducing reporter, suicide, or therapeutic genes in a clinically relevant manner. We are currently exploiting this strategy to express a conditional HSV-tk suicide gene for purging of iPS cell progeny from teratoma-initiating cells. With the emergence of improved technologies for homologous recombination into human cells, targeted gene addition may soon become a realistic option if predefined validated safe harbor sites in the human genome are identified. We are utilizing our selection strategy in iPS cells (using lentiviral vectors with reporter cassettes that can be exchanged using Cre recombinase-mediated cassette exchange) as a platform for the de novo discovery and characterization of putative universal safe harbor sites that can be broadly used for the genetic engineering of multiple human cell types.