128. Targeting a High-Expression FVIII Transgene to Exogenous Locations in the Genome without Disrupting Endogenous Gene Expression

Abstract

The technology that is now available for targeted genome editing consists of artificial nucleases that create double stranded breaks at specific genomic locations that can then be corrected by intrinsic cellular repair processes. Genome editing can be achieved by exploiting the repair mechanism of homologous recombination by providing a donor vector containing the gene(s) of interest flanked by sequences homologous to the site of the double strand break. To date, gene addition in this manner is targeted to defined inert safe harbor loci. We developed an innovative targeting strategy in which homologous recombination could be utilized to target a transcriptionally active genomic locus without disrupting expression of the endogenous gene. In this manner, essentially any location in the genome becomes a potential safe harbor for homologous recombination-mediated genome editing. One advantage to this strategy is that it allows for transgene expression to be under the precise control of endogenous regulatory elements, rather than using heterologous promoters. Therefore, the optimal locus with the most advantageous regulatory elements to drive transgene expression can be selected for gene addition. Pilot experiments confirmed the expression of exogenous reporter genes (such as the green fluorescent protein or the more therapeutically relevant biologically inert surface selectable marker delta NGFR) from either an exogenous ubiquitous promoter or from the exogenous promoter due to the addition of a T2A element incorporated downstream of the endogenous gene. Notably, expression of the endogenous gene was confirmed to be unaltered. These experiments were performed at three separate genomic locations. To demonstrate the feasibility of this strategy therapeutically however, a high-expression FVIII chimeric transgene (HPFVIII) was incorporated into two distinctly active genomic loci: the IL2R gamma chain locus and the adenosine deaminase locus. The donor vectors utilized contained the cDNA for the endogenous gene locus (either the IL2R gamma chain gene or the adenosine deaminase gene) followed by a ubiquitous promoter intended to drive the expression of HPFVIII, all of which was flanked by one kilobase arms of homology. Both the myeloid erythroid-leukemic K562 cell line and the acute T cell leukemic Jurkat cell line were modified upon nucleofection and confirmed to be targeted by sequencing. Single cell clones were produced and assessed for HPFVIII expression by a one-stage APTT based coagulation assay. Clones were found to express therapeutic levels of FVIII from both loci in both cell types (1 Unit / 2 million cells / 24 hrs). These experiments set the stage for targeting of HPFVIII into hematopoietic stem and progenitor CD34+ cells for the purpose of treating Hemophilia A.