Weekly CRISPR Digest

Cell Therapy: Editing in Immune Cells

CRISPR gene editing is powering a new generation of immuno-oncology cell therapies, allowing researchers to engineer immune cells with specific cancer-fighting properties. Specifically, CRISPR systems create targeted breaks in the immune cell genome, where engineered DNA templates can be inserted via the cell’s innate repair pathways. Armed with these new genetic inserts, immune cells can identify and target cancer cells with greatly improved effects.

While traditional immune cell therapies have relied on viral vector systems to deliver CRISPR components and DNA templates, safety concerns and manufacturing bottlenecks have shifted focus to non-viral approaches. Ex vivo non-viral CRISPR ribonucleoprotein (RNP) systems offer a safe, efficient, and inexpensive gene therapy alternative. In these systems, CRISPR guide RNA and Cas nuclease protein are complexed before being co-delivered via electroporation or lipofection into hematopoietic stem and progenitor cells (HSPCs), which are then reinjected into the patient. Ex vivo non-viral CRISPR ribonucleoprotein (RNP) systems offer a safe, efficient, and inexpensive immune cell therapy alternative. CRISPR guide RNA and Cas nuclease protein templates are complexed as RNPs before being co-delivered with DNA repair templates via electroporation or lipofection into T or NK immune cells, which are then reinjected into the patient.

Here we examine how researchers apply non-viral CRISPR RNP gene editing to develop novel immune cell therapies.

T Cells

CRISPR RNP gene knock-in techniques are currently being used to construct transgenic chimeric antigen receptor T (CAR-T) cells and T cell receptor T (TCR-T) cells for immunotherapy. Yet challenges remain to improve editing efficiency while reducing cytotoxicity.

Recently, significant efficiency improvements have been achieved using non-toxic GenExact single-stranded DNA (ssDNA) templates designed with a Cas9 Targeting Sequence (CTS) to bind the RNP for co-delivery via electroporation. This method achieved >90% knock-in efficiency with minimal cytotoxicity in multiple primary immune cell types.

NK Cells

NK cells are another attractive target for immune cell therapy. However, their highly sensitive and resistance to exogenous DNA limits the effectiveness of viral and plasmid-based approaches.

Non-viral CRISPR RNP systems are currently being optimized to engineer chimeric antigen receptor NK (CAR-NK) cells to improve cancer-killing activity and reduce tumor evasion. Non-viral approaches using Cas9 mRNA have also been applied to NK cell engineering to achieve >98% transfection efficiency with >90% cell viability.

B Cells

Antibody-producing B cells are another exciting target for immune cell therapy. While initial research focused on AAV-delivered antibody-expressing cassettes, B cells often produced insufficient antibody expression levels or duration.

Therefore, CRISPR RNP-based workflows are currently being optimized for applications in B cell therapy. Specifically, targeted DNA insertion at the B cell receptor loci allows researchers to reprogram antibody specificity.