Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

Abstract

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8+ T cells and gene targeting of a GFP transgene cassette in >40% of CD8+ and CD4+ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.

Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome

“…2,43 Therefore, it is important to extend this work to autologous hematopoietic stem cells, where targeting efficiencies using codelivery of nuclease and AAV have now begun to reach clinically relevant levels. 31,39 Thus, our combined observations support the potential of this methodology for treatment of X-HIGM syndrome and, likely, other primary immunodeficiency diseases including those where regulated expression of a transgene is required or highly advantageous. For personal use only.…”

“…Notably, although this potential advantage has been largely theoretical to date, recent work has led to important advances in gene-editing efficiencies in primary human T cells. 31,38,39 Here, we demonstrate the direct application of these advances in correction of X-HIGM syndrome through CD40LG gene editing, providing a promising pathway for near-term clinical application. Our strategy replaces and restores normal regulated CD40L protein expression in primary human T cells, restoring helper T-cell function at efficiency rates amenable to clinical application.…”

Going non-viral: theSleeping Beautytransposon system breaks on through to the clinical side

“…The common advantage of these technologies is that they enable site-specific engineering of the genome, thereby allowing introduction of facile and highly specific changes in the genetic code. These technologies are making their way toward therapeutic applications (Yu et al, 2016); for example, site-specific gene addition at reasonable efficiencies has recently been demonstrated by mRNA encoding ZFN and a homology template delivered as AAV in both HSCs (Wang et al, 2015) and T cells (Wang et al, 2016a).…”

Clinical Applications of Genome Editing to HIV Cure

“…Using this method, HR efficiencies of up to 40-60% have been achieved in primary T cells and HSPCs at the CCR5, IL2RgG, HBB, and AAVS1 loci. 37,45,56,57 Nevertheless, HR efficiencies vary widely by loci and nature of template, with the highest efficiencies resulting from editing of AAVS1, the natural AAV integration locus. Further, editing levels tend to be lower in the primitive stem cell population within a heterogeneous bulk HSPC mixture, resulting in lower editing levels in cells that persist in immunodeficient mice or nonhuman primates in vivo.…”