High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k “Helper” Proteins

Abstract

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

“…Expanding on previous work, we explored several methods for increasing the efficiency of HDR. For example, the use of adenoviral helper mRNAs 35 increased HDR with lower efficiency donor templates in our experiments with the anti-BCMA CAR and BFP donor templates at the TRAC locus (Figures 5, S6E, and S6F). Additionally, we confirmed the importance of removing the nuclease target site within the AAV donor (Figure S5) and found that reduction of viral packaging size by reducing homology arm length to 300 bp within candidate AAV donor constructs did not significantly decrease HDR (Figure S6).…”

“…We performed HDR editing of CD3 + PBMCs using the TRAC megaTAL nuclease, with AAV TRAC -anti-BCMA CAR as a donor template, to generate TRAC [BCMACAR] T cells. Of note, we have recently reported that delivery of mRNA encoding wild-type (WT) adenoviral helper proteins, Ad5 E4orf6/E1b55k WT, can enhance AAV transduction 35 . Further, co-delivery of mRNA encoding specific mutant helper proteins in association with nuclease mRNA and AAV donor can improve HDR rates in cases where donor template delivery may be rate limiting 35 (Y.H., unpublished data).…”

“…Of note, we have recently reported that delivery of mRNA encoding wild-type (WT) adenoviral helper proteins, Ad5 E4orf6/E1b55k WT, can enhance AAV transduction 35 . Further, co-delivery of mRNA encoding specific mutant helper proteins in association with nuclease mRNA and AAV donor can improve HDR rates in cases where donor template delivery may be rate limiting 35 (Y.H., unpublished data). Therefore, we used this combined approach to promote HDR within the TRAC locus.…”

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

“…(Lentz and Samulski, 2015; Gwiazda et al , 2016) To evaluate the ability of these proteins to augment targeted gene integration at CD40LG in HSC, adenoviral serotype 5 helper proteins (E4Orf6 and E1B55K H354 mutant) were co-introduced as mRNA during electroporation with TALENs or CRISPRs. Figure 3 (D & E) shows that PBSC samples treated with helper protein achieved a 2-fold enhancement in gene modification (p<0.0001) regardless of nuclease platform.…”

Application of Genome Editing Techniques in Immunology

“…Indeed, numerous of such attempts have been made (Chi et al 2016; Chu et al 2016; Gundry et al 2016; Gwiazda et al 2016; Hendel et al 2015; Li et al 2015; Liu et al 2017; Mandal et al 2014; Schumann et al 2015, Su et al 2016, 2017), as exemplified in Table 3. Various clinically important genes were disrupted in human primary CD4 + T cells and/or CD34 + hematopoietic and progenitor cells with high on-target efficacy and low incidence of off-target mutagenesis using CRISPR/Cas9 approaches e.g.…”