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Immunotherapy Solutions
Cancer continues to be a leading cause of death worldwide. In 2022, there were an estimated 20 million new cancer cases and 9.7 million deaths globally according to various institutes like the World Health Organization (WHO). This underscores the persistent and growing challenge that cancer poses to global health systems. In the United States, projections for 2024 estimate approximately 2,001,140 new cancer cases and 611,720 cancer-related deaths. In this 2024 report, the figures highlight the significant burden of cancer on the U.S. population and the critical need for continued research in cancer prevention, diagnosis, and immunotherapy development for treatment.
At the forefront of immunotherapy development, scientists are developing genetically engineered T cells using CRISPR and gRNA to create CAR-T cells that can be used as a therapeutic to treat cancer. The utilization of CRISPR gene-editing with CAR-T cell therapy can potentially lead to safer immunotherapies that offer hope for improved cancer treatments.
Chimeric antigen receptor T cells (CAR-T cells) are genetically modified T lymphocytes designed to recognize and eliminate tumor cells. These engineered T cells are derived from either the patient (autologous) or a healthy donor (allogeneic) and are modified to express chimeric antigen receptors, known as CARs, on the surface of the cell.
CARs are synthetic fusion proteins composed of:
The unique structure of CARs enables T cells to identify and bind to tumor-associated antigens (TAAs) on the surface of cancer cells in an MHC-independent manner, enhancing their ability to target a broad range of tumor types.
This MHC-independence distinguishes CAR-T cells from conventional T-cell receptors (TCRs), which require antigen presentation via HLA molecules. The design of CARs allows for robust and direct cytotoxic activity against antigen-expressing tumor cells, as demonstrated in hematologic malignancies like B cell acute lymphoblastic leukemia (B-ALL) and diffuse large B cell lymphoma (DLBCL) (June et al., 2018, Science; Maude et al., 2014, NEJM).
While early CAR-T therapies relied on viral vectors for gene delivery, these methods often resulted in random transgene integration, potentially leading to variable CAR expression or unintended genomic disruptions. This means the CAR-T cell therapy could become less effective, or worse, less safe. To overcome these limitations, researchers turned to CRISPR to make precise, targeted modifications of T cells resulting in safer CAR-T therapies.
The utilization of CRISPR in the development of CAR-T cell therapies has helped scientists design and optimize T cells to recognize and eliminate cancer cells. This can be done with CRISPR CAR-T guide RNA (gRNA) and nucleases to precisely edit the T-cell genome. By using their designed CRISPR CAR-T gRNA and nuclease, scientists can knock out genes, knock in chimeric antigen receptor constructs, or correct disease-relevant mutations on the T-cell receptors to improve its safety and efficacy as a CAR-T immunotherapy.
One of the most well-characterized targets for CRISPR CAR-T cell guide RNA is the insertion of the CAR transgene into the TRAC (T-cell receptor alpha constant) locus, replacing the endogenous T-cell receptor. This strategy was demonstrated by Eyquem et al. (2017, Nature), where the targeted integration of a CAR into TRAC led to uniform CAR expression, reduced tonic signaling, and enhanced anti-tumor activity. In this context, the CAR-T gRNA not only enables high-efficiency knock-in but also silences native TCR activity, minimizing the risk of graft-versus-host disease (GvHD). This represents a critical advancement in the design of allogeneic, off-the-shelf CAR-T therapies, where universal donor-derived cells are edited using CRISPR CAR-T cell gRNA to eliminate immunogenic components.
Base editing has emerged as a powerful addition to the CRISPR gene editing toolbox offering a safer and more precise approach to engineering NK- and CAR-T cell therapies. Unlike conventional CRISPR-Cas9 systems that rely on creating double-stranded DNA breaks, base editors such as cytosine base editors (CBEs) and adenine base editors (ABEs) enable targeted single-base conversions (C→T and A→G, respectively). These base conversions are achieved using a deaminase enzyme fused to a catalytically impaired Cas9 that helps facilitate the binding to the desired target site without generating a double-strand break. By avoiding double-strand breaks, base editing significantly reduces the risk of unintended gene editing that result in insertions, deletions, or chromosomal translocations, making it an attractive strategy for improving the safety and precision of cell-based immunotherapies.
In CAR-T cell therapy, base editing has enabled multiplexed gene modifications to improve therapeutic efficacy and safety. For instance, CBEs have been utilized to simultaneously disrupt multiple genes, including PD-1, TCR, and CD52, to create allogeneic CAR-T cells with reduced immunogenicity and enhanced persistence. Similarly, ABEs have been employed to generate CAR-T cells with precise edits that confer resistance to immunosuppressive tumor microenvironments, thereby enhancing anti-tumor activity.
Natural killer (NK) cells, another promising immuno- cell therapy, are also being engineered using base editors. Companies like Base Therapeutics are pioneering base editing approaches in NK cells with their engineered cytosine base editor, AccuBase™, enabling highly specific cytosine (C) to thymine (T) base conversions that improve NK cells persistence, cytotoxicity, and immune evasion.
These engineered base editors are helping unlock the full therapeutic potential of NK- and CAR-T cell therapies while maintaining a safer editing profile. Want to learn more about base editing? Explore our base editing guide.
To improve the function and persistence of CAR-T cells, researchers frequently design their CRISPR CAR-T cell guide RNA to target select genes that could influence T-cell efficacy. Common gene targets when developing CAR-T cell immunotherapies:
Scientists are not only limited to editing one gene for developing effective and safer CAR-T immunotherapies. Multiplexed editing with multiple CRISPR CAR-T guide RNAs allows for combinatorial genome engineering, such as CAR knock-in plus PD-1 and CTLA-4 knockout, in a single electroporation protocol. This approach has been explored for CAR-T cell clinical applications, exploring enhanced immune evasion and tumor cytotoxicity (Stadtmauer et al., 2020, Science).
That said, the efficiency and precision of using CRISPR to edit T cells depend heavily on the quality of the guide RNA used. Not all gRNA is synthesized equally across different vendors, which means finding the right supplier to synthesize your CAR-T gRNA is crucial for your immunotherapy development.
Once you design your CRISPR CAR-T guide RNA, selecting the right vendor that synthesizes best-in-class gRNA will help you achieve success when performing CRISPR gene editing on T cells. Synthego has proven to be a reliable supplier of quality CRISPR guide RNA for T-cell editing. A recent study using Synthego’s Research sgRNA demonstrated high cell survival of edited resting human CD4+ T cells. What made this CRISPR T-cell editing study remarkable is that compared to active CD4+ T cells, resting CD4+ T cells do not proliferate which can be challenging to generate knockout CD4+ T cells due to many reasons like having cell viability issues.
Not only did this study demonstrate successful CRISPR T-cell gene editing using Synthego’s Research sgRNA, the scientists were able to achieve efficient knockouts for both single and multiplexed gene knockout targets (Figure 1). After performing the CRISPR multiplexed T-cell gene editing targeting CD46, CXCR4, PSGL-1, CD4, TRIM5a, and CPSF6, cell viability remained relatively high following single and multiplexed knockouts. Upon activation, multiplexed edited T cells continued to express canonical activated T-cell markers CD25, CD39 CD69, and HLA-DR.
To further study Synthego’s Research sgRNA (freshly prepared or frozen and then thawed) against another major vendor’s synthetic sgRNAs, Synthego’s sgRNAs resulted in consistently greater (more than 2x) knockout efficiency than the other vendor’s (Figure 2).
This efficient gene editing in resting CD4+ T cells is important as these assays require the use of valuable donor cells from humans. Furthermore, using CRISPR gRNA that results in high editing efficiency in T cells is crucial for clinical applications like CAR-T cell therapies and other immunotherapies.
CAR-T cell therapies rely on gRNA and nucleases that exceed expectations for performance. Achieving high viability and on-target editing with minimized off-target edits is not just a goal, it is a necessity for making effective and safe CAR-T cells to treat diseases like cancer. Using Synthego’s best-in-class CRISPR guide RNAs, from research use only to cGMP quality, enables scientists to generate highly efficient knockouts for both single and multiplexed gene editing in T cells, which means they could be used as CRISPR CAR-T guide RNA for developing your CAR-T cell therapies.
We would love to learn about your CAR-T cell therapy and how we can help you take it to the clinic. Let’s start the discussions by contacting us here.
