Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy

Abstract

Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…8 Although specific loci or general criteria identifying prospective GSHs have been proposed, 8,9 no GSH has yet been fully validated in a large animal model or a clinical setting. 10 Adeno-associated virus integration site 1 (AAVS1) locus in the first intron of PPP1R12C gene is one of most commonly used GSH sites in human cell studies. The AAVS1 locus has an open chromatin configuration in a variety of human cell types, including iPSCs, making this site readily accessible to site-specific endonucleases.…”

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

“…We developed an all-in-one expression vector for cis expression of dual nuclear localization signal Mad7 (nMad7n) and a pre-crRNA in vitro (Figure 4a). Using this expression vector and the workflow in Figure 4a, we targeted two therapeutically relevant "safe harbor" loci, AAVS1 and CCR5 (Papapetrou and Schambach 2016) ( Figure 4b, d). We additionally targeted the TRAC locus due to its reported value in generating Chimeric Antigen Receptor T cells ( Figure 4e) (Eyquem et al 2017).…”

Building with intent: Technologies and principles for engineering mammalian cell-based therapies to sense and respond

“…Transposon/transposase systems deliver larger payloads but are less efficient than lentiviral and retroviral vectors and integrate randomly, exhibiting a less biased pattern of integration [37]. Recombinase-based landing pads provide site-specific integration and more homogeneous gene expression, but thus far the required insertion of the initial landing pad precludes the use of primary cells [38], and it is not yet clear whether a given safe harbor site for targeting genomic integration is safe across all applications [39]. Targeted integration by Cas9-mediated DNA editing may confer site specificity, and sgRNAs are relatively straightforward to design, but integration efficiency remains a challenge [40].…”