May 28, 2025

Overview of Current Allogeneic T Cell Therapy Trends

Traditional autologous CAR-T therapies, which rely on patient-derived T cells, face significant limitations, including lengthy manufacturing times, high costs, and variability in cell quality, particularly for patients with compromised immune systems. In contrast, allogeneic CAR-T therapies, derived from healthy donor cells, enable standardized, large-scale production—a single batch can treat hundreds of patients, drastically reducing costs and wait times while improving accessibility.

The emergence of allogeneic, or "off-the-shelf," CAR-T therapies like Sana Biotechnology’s SC291 and Allogene Therapeutics’ ALLO-329, both recently granted the Food and Drug Administration (FDA) Fast Track Designations (FTD) for autoimmune diseases, underscores the critical need for scalable and accessible treatments in this field.^1,2

SC291, a CD19-targeted hypoimmune (HIP)-modified CAR-T, leverages gene editing to evade immune rejection, enabling deep B-cell depletion to reset dysregulated immunity in conditions like systemic lupus erythematosus (SLE). Similarly, ALLO-329, a dual-targeted CD19/CD70 CAR-T, incorporates CRISPR-based engineering and Allogene’s Dagger® technology to eliminate dysfunctional cells and minimize lymphodepletion, addressing the major barrier in autoimmune patients.

The FDA’s FTD recognition for these allogeneic CAR-T therapies highlights the urgency to address unmet needs in refractory autoimmune diseases, where current therapies often fail to provide sustained remission. Allogeneic CAR-T platforms not only promise broader patient access but also pave the way for transformative, one-time treatments capable of achieving long-term immune tolerance, reshaping the therapeutic landscape for autoimmune diseases.

Safety concerns of multiple gene editing in allogeneic CAR-T development

Allogeneic cell therapies often require multiplex gene editing to eliminate immune rejection (e.g., disrupting TCR and MHC genes) and enhance therapeutic function. However, traditional CRISPR/Cas9-based methods, which induce double-strand breaks (DSBs), carry risks of chromosomal translocations and large deletions when multiple edits occur simultaneously.³ These genomic aberrations, caused by error-prone DNA repair mechanisms, raise safety concerns such as oncogenic transformation or loss of critical genes.

To mitigate these risks, base editing (BE) has emerged as a precise alternative. Unlike CRISPR/Cas9, base editors directly convert single DNA bases without creating DSBs, minimizing genomic instability. This approach enables efficient gene knockouts or functional corrections while preserving chromosomal integrity.

In cell therapies, base editing enhances safety for applications like hypoimmune CAR-T cells or correcting disease-causing mutations in hematopoietic stem cells.⁴ An early study demonstrates its potential to streamline allogeneic product development by reducing off-target effects and enabling multiplex editing with lower toxicity, positioning it as a transformative tool for next-generation genetic medicines.⁵

Evaluating multiplex gene editing efficiencies of base editors

To replace CRISPR/Cas9 with BE for multiplex gene disruption, we evaluated the editing efficiency of CBEmax mRNA and ABE protein targeting B2M, TRAC, PDCD1, CIITA in Jurkat cells. We electroporated BE and sgRNA in Jurkat cells and detected editing efficiency by Sanger sequencing and BEAT analysis 3 days after electroporation.

Both BE formats achieved efficient base conversion at splice donor sites of all target genes, enabling robust gene knockout. Notably, CBEmax and ABE demonstrated comparable editing rates (~90%), confirming the feasibility of BE for eliminating immune-related antigens and checkpoint molecules without double-strand breaks.

Combining Cas12 nuclease and nCas9-BE for multiplex genome editing in T cells

When combining nCas9-based BE for gene knockout with Cas9-mediated knock-in, unintended gRNA exchange between the two systems may compromise editing efficiency. To circumvent this difficulty, a Cas12-based knock-in system can be paired with BE-mediated knockout. This strategy leverages the orthogonal scaffold sequences required for the ribonucleoprotein (RNP) complex formation by Cas12 versus nCas9, effectively preventing cross-utilization of gRNAs.⁶ By utilizing distinct CRISPR-Cas enzymes (nCas9 for BE and Cas12 for knock-in), the systems operate independently, eliminating interference between base-editing and knock-in machinery.

To evaluate the multiplex editing efficacy of this combinatorial approach, we tested Cas12-mediated knock-in efficiency at the TRAC locus in primary T cells while simultaneously knocking out B2M and PDCD1 using ABE8e mRNA to establish an allogeneic CAR-T model.

We designed a single-stranded GFP donor template flanked by TRAC homology arms and incorporated Cas12-targeting sequences (CTS) at either the 5’ end (5’ only) or both ends (5’+ 3’). These CTS regions were annealed with complementary oligonucleotides to generate localized double-stranded structures, enhancing nuclear delivery and homology-directed repair (HDR) efficiency by facilitating Cas12 RNP binding.⁷

To achieve B2M/PDCD1/TRAC knockout and GFP knock-in at the TRAC locus, we electroporated T cells with ABE8e mRNA/sgRNA, Cas12/crRNA, and ssCTS template. We also set up a control group with GFP knock-in at the TRAC locus only.

Multiplex gene editing strategy in T cells

Cas12 RNP demonstrated robust knock-in efficiency at the TRAC locus, with multiple edited groups showing comparable performance to TRAC knock-in only controls. This finding confirms that BE-mediated B2M and PDCD1 knockout does not interfere with Cas12-driven integration.

This orthogonal approach enables efficient and precise multiplex genome editing in T cells without cross-system interference. Notably, ssCTS-5’+ 3’ designs significantly improved knock-in efficiency for most Cas12 variants (vs. ssCTS-5’-only), which is different from Cas9-mediated HDR, where the ssCTS-5’only design works better.

Validation of Cas12 and ABE8e multiplex editing efficiency by flow cytometry

Flow cytometry further validated the gene knockout efficiency, with ABE8e mRNA achieving>95% depletion of B2M and PD-1 proteins. Most Cas12 variants editing at TRAC eliminated TCR expression in 93% of edited cells. These results underscore the compatibility of combining BE and Cas12 systems to simultaneously disrupt MHC-I and immune checkpoints, deplete endogenous TCR, and integrate therapeutic transgenes via non-viral gene editing—a critical advancement for developing allogeneic CAR-T therapy with enhanced safety and efficacy.

Conclusion

Our data demonstrate the feasibility of combining base editing and Cas12-mediated knock-in to achieve efficient multiplex genome editing in primary T cells. This approach enables simultaneous disruption of B2M, PDCD1, and TRAC genes for allogeneic CAR-T engineering, which is ideal for developing “off-the-shelf” cell therapies. We will expand this platform by testing multiple editing strategies and optimizing donor templates. By systematically evaluating editing efficiency, off-target effects, and long-term functional persistence, we aim to refine multiplex editing workflows and accelerate the development of next-generation “off-the-shelf” allogeneic therapies with enhanced safety, potency, and manufacturability.

Reference

• (2024, December 2). Sana Biotechnology Announces Fast Track Designation for SC291 in Relapsed/Refractory Systemic Lupus Erythematosus. Https://Sana.com/. https://ir.sana.com/news-releases/news-release-details/sana-biotechnology-announces-fast-track-designation-sc291
• (2025, April 7). Allogene Granted Three U.S. FDA Fast Track Designations (FTD) for ALLO-329, a Next-Generation Dual-Targeted CD19/CD70 Allogeneic CAR T, for the Treatment of Lupus, Myositis and Scleroderma. Https://Allogene.com/. https://ir.allogene.com/news-releases/news-release-details/allogene-granted-three-us-fda-fast-track-designations-ftd-allo
• Tsuchida CA, Brandes N, Bueno R, et al. (2023). Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells. Cell. https://doi.org/10.1016/j.cell.2023.08.041
• Li C, Georgakopoulou A, Paschoudi K, et al. (2024). Introducing a hemoglobin G-Makassar variant in HSCs by in vivo base editing treats sickle cell disease in mice. Mol Ther. https://doi.org/10.1016/j.ymthe.2024.10.018
• Chiesa R, Georgiadis C, Syed F, et al. (2023). Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia. N Engl J Med. https://doi.org/10.1056/nejmoa2300709
• Glaser V, Flugel C, Kath J, et al. (2023). Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells. Genome Biol. https://doi.org/10.1186/s13059-023-02928-7
• Nitulescu AM, Du W, Glaser V, et al. Single-stranded HDR templates with truncated Cas12a binding sequences improve knock-in efficiencies in primary human T cells. bioRxiv. Published online September 11, 2024. https://doi.org/10.1101/2024.09.11.608426