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News & Blogs » CRISPR News » Enabling Non-Viral T Cell Engineering with ssDNA HDR Templates

Homology-Directed Repair (HDR) Knock-in Templates

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Enabling Non-Viral T Cell Engineering with ssDNA HDR Templates

The success of cancer immunotherapies, such as CAR T cells for targeting tumor cells in hematological malignancies, has spurred an unprecedented push to develop more and improved living drugs. Conventionally, viral approaches have been used in manufacturing T cell therapies, where retroviral vectors deliver the necessary sequences encoding receptors for cancer antigens, essentially re-directing the T cell’s specificity.

FDA approved CAR T cell therapies. Retrieved from Pan et al. 2022 without modifications. https://creativecommons.org/licenses/by/4.0/

Nevertheless, recognizing specific safety concerns associated with some viral vectors, such as unwanted recombination events due to random genomic insertions, new efforts leverage CRISPR/Cas9-based editing strategies to achieve precise insertions of synthetic DNA payloads (i.e., single and double-stranded DNA or ssDNA and dsDNA). Investigators in the Marson lab at UCSF have found that targeted non-viral approaches not only offer increased safety but can also help improve the quality of T cell immunotherapies while reducing manufacturing time and cost (Shy et al. 2022).

Non-viral knock-in approaches offer increased safety and ease of workflow by circumventing the complexities of viral vector production. Nevertheless, one disadvantage has been their limited editing efficiency. New efforts undertaken by the Marson group have significantly helped optimize non-viral methods to achieve editing efficiencies suitable for cell immunotherapy manufacturing.

Non-viral precise genome editing strategies for improved cell therapies

To establish a non-viral editing approach, Marson’s team had already adopted the use of ribonucleoprotein (RNP) complexes, consisting of CRISPR/Cas9 protein reconstituted with synthetic guideRNA, which are directly electroporated into primary T cells. This approach provides some unique benefits, such as rapid genome editing and Cas9 turnover, ensuring limited Cas9 persistence within modified T cells and thus reducing off-target events and toxicities (Roth et al. 2018, Nguyen et al. 2019, Shy et al. 2021).

Next, Marson’s team stirred their attention to creating solutions to improve the integration efficiency of HDR templates. Having observed that large HDR templates supplied in their dsDNA form led to significant cytotoxicity, they evaluated the performance of ssDNA HDR templates, successfully demonstrating reduced cytotoxicity (Roth et al 2018). Nevertheless, optimization was still required to improve insertion efficiencies and ensure yields of edited cells fitting with those required for therapeutic use. Therefore, most recently, Marson and colleagues have developed a new approach that enables them to establish a GMP-compatible workflow for T cell engineering through fully non-viral methods (Shy et al. 2022).

Hybrid ssDNA HDR templates enable high-yield T cell editing

To leverage Cas9 as a payload shuttle, Marson’s team has introduced Cas9 target sequences (CTSs) onto the ends of long ssDNA HDR templates. The new hybrid structure, consisting of a therapeutic construct and homology arms flanked by short regions of double-stranded CTSs, enable the interaction between ssDNA HDR templates and RNPs and improve their knock-in efficiency. In addition, the generation of these hybrid constructs was simplified by the design of complementary oligonucleotides, which are easily annealed to the ends of the ssDNA HDR template.

Marson and colleagues found that such hybrid ssDNA HDR templates designed to deliver a 2.9kb BCMA CAR construct could achieve a high editing efficiency at the TRAC gene locus of 39% and modified T cells at high yield. Moreover, once optimized, hybrid ssDNA HDR templates could efficiently deliver a NY-ESO-1 recombinant TCR gene sequence and support higher (i.e., over 5 fold) knock-in and edited T cell yield than achieved by a hybrid dsDNA template.

To further improve T cell editing efficiency, Marson’s team evaluated a series of small molecule inhibitors. They found that combining the DNA-dependent protein kinase inhibitor (M3814), histone deacetylase class I/II inhibitor (Trichostatin A), and CDC7 inhibitor (XL413) could help achieve knock-in efficiencies above 80% for some long hybrid ssDNA templates.

Ultimately, under GMP-compatible conditions, including GMP-grade equipment and reagents, Marson and colleagues endeavored to develop a workflow for non-viral T cell engineering with the goal of testing the performance of their new approaches.

“We partnered with Genscript to develop a fully enzymatic GMP-compatible process for ssCTS template generation based on rolling circle amplification (RCA). Genscript templates encoding an anti-BCMA-CAR knock-in were able to be manufactured at large scale and consistently outperformed our internally generated HDRTs, showing lower levels of toxicity and higher knock-in efficiencies for both ssCTS and dsCTS constructs” Shy et al. 2022.

To this end, they edited a large number of primary T cells by targeting the TRAC locus for insertion of a BCMA-CAR construct. They found that under large-scale conditions, they could achieve high knock-in efficiency of ~46%, which could be further enhanced by using small-molecule inhibitors to over 60%.

Overall, by leveraging and optimizing the non-viral delivery of hybrid ssDNA HDR templates, the Marson group has developed a workflow for precise TRAC gene editing, producing T cell therapies safely, efficiently, and at a large scale.

Reference


    Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nature Biotechnology (2020) doi:10.1038/s41587-019-0325-6.

    Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature (2018) doi:10.1038/s41586-018-0326-5.

    Shy, B. R. et al. Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing (2021) bioRxiv 2021.09.02.458799; doi: https://doi.org/10.1101/2021.09.02.458799.

    Shy, B. R., et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nature (2022) https://doi.org/10.1038/s41587-022-01418-8.


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