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.

Figure 1

The making of a GSH. Establishing one or few GSHs will require moving from a wish-list of desirable attributes, to well-defined criteria, an in silico or wet lab-based discovery phase and extensive validation in multiple cell types and stages of development, ideally including tests in both a human context and at the whole-organism level. GSH, genomic safe harbor.

Figure 2

A priori or prospective GSH determination. In the therapeutic example in the left, GSHs are prospectively selected after screening a number of clones with unique integration sites generated by random insertion. This approach is only feasible because hPSCs are endowed with extensive potential for in vitro self-renewal, subcloning, and expansion. Alternatively (right), a predetermined GSH can be targeted using a number of gene targeting tools (CRISPR/Cas9, TALENs, ZFNs, AAV, and others). If the cell type allows single cell subcloning, screening for verification of correctly targeted clones can be included as an optional step. AAV, adeno-associated virus; Cas9, CRISPR-associated nuclease 9; CRISPR, clustered regularly interspaced short palindromic repeats; GSH, genomic safe harbor; hPSCs, human pluripotent stem cells; TALENs, transcription activator-like effector nucleases; ZFNs, zinc finger nucleases.