Abstract
Efficient delivery of nucleic acids for the engineering of primary T cells is central to the study of the basic biology of these key immune effector cells and has clinical implications. To date, lentiviral vectors delivering guide RNAs for CRISPR-Cas9 editing are not optimal for use in primary cells. Herein, we describe the T cell optimized for packaging (TOP) vector for delivering guide RNAs and transgenes into primary T cells. The TOP vector produces high-titer virus compared to a routinely used guide RNA vector, resulting in a ~10-fold increase in transduction in T cells. Moreover, a TOP vector expressing a chimeric antigen receptor and a guide RNA targeting the T cell receptor showed an ~5- to 9-fold increased transduction efficiency with ~2- to 3-fold higher expression compared to the commonly used epHIV7 vector and was simultaneously able to mediate efficient knockout of the endogenous T cell receptor in >71% of transduced cells upon Cas9 electroporation. The increased packaging of the TOP vector genome into viral particles appears to contribute to its higher transduction efficiency. The TOP vector represents an optimal tool for tandem delivery of transgenes and guide RNAs to primary T cells for use in functional screens and immunotherapy applications.
Highlights
Gene engineering is central to both the transfer of specific genes or gene fragments and directed editing of specific gene targets for the purposes of modifying cell function
The simian virus 40 (SV40) polyadenylation signal was replaced with a bovine growth hormone (BGH) polyadenylation signal, and the promoter driving expression of the vector genomic transcript in producer cells was changed from the Rous sarcoma virus (RSV) enhancer/promoter to a cytomegalovirus (CMV) enhancer/promoter, which may serve to increase titer.[8]
The full-length human elongation factor 1ɑ (EF1-a) promoter was replaced with the mouse stem cell virus (MSCV) promoter, which is much shorter and has been shown to perform very well in primary T cells.[9]
Summary
Gene engineering is central to both the transfer of specific genes or gene fragments and directed editing of specific gene targets for the purposes of modifying cell function. Primary T cells represent a key potential target for gene engineering approaches because they can be modified and expanded ex vivo prior to re-introduction into a patient. This approach is fundamental to cutting edge immunotherapy approaches to promote T cell activity against specific immunological targets, including the introduction of combined antigen recognition and signaling molecules such as the chimeric antigen receptor (CAR). The use of CRISPRCas[9] technology for high-throughput gene knockout screens is well established for use in cell lines, but is much less widespread in primary T cells.[2,3] One major bottleneck in performing such CRISPR-Cas[9] screens in primary cells is the relative inefficiency of constructs delivering the guide RNAs to primary cells
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