Gene Writer Enables Stable In Vivo CAR T-Cell Engineering Through RNA Delivery

Compared with traditional lentivirus-based ex vivo therapies, a proposed platform of gene-editing tools derived from non–long terminal repeat (non-LTR) transposons enabled stable in vivo chimeric antigen receptor (CAR) T-cell engineering via RNA delivery. The proposed “toolbox” of gene-editing tools, referred to as Gene Writer, was discussed by Cecilia Cotta-Ramusino, PhD, senior vice president of technology development at Tessera Therapeutics, at the 2026 Joint American Society of Transplantation and Cellular Therapy + European Society for Blood and Marrow Transplantation Basic and Translational Scientific Meeting.¹

Researchers Explore Non-LTR Transposons for In Vivo CAR T-Cell Engineering

There is a need to develop a new class of gene-editing tools by leveraging natural biological processes, specifically non-LTR transposons, Cotta-Ramusino explained. In particular, she and her team were interested in these transposons because they naturally mobilize from a “donor to recipient” via an RNA intermediate, allowing insertion into a genome. Although many of these transposon elements had previously been identified computationally, there was a substantial lack of experimental validation confirming whether they remained active in cells. This gap prompted the team to undertake a large-scale computational effort to map and revive ancient transposon elements from approximately 700 genomes.^1-3

Another key objective of the research was to improve upon the natural “cis-activity” of these elements. In their native form, non-LTR transposons are often cis-preferential, Cotta-Ramusino said, meaning the proteins they encode typically recognize only their own messenger RNA. To address this limitation, the team sought to engineer a split system in which the driver protein, delivered as mRNA, is separated from the gene template carrying the therapeutic cargo. This approach was intended to create a safer therapeutic platform in which editing would occur only transiently and naturally stop once the driver protein degraded.¹

The study was also designed to shift the therapeutic paradigm away from traditional ex vivo editing toward a more scalable in vivo editing approach. Cotta-Ramusino and her team aimed to develop a system that would be more versatile and scalable than current lentivirus-based therapies while also addressing important safety concerns. Unlike traditional lentiviral vectors, which preferentially integrate into protein-coding regions of the genome and may increase the risk of harmful mutations, the researchers sought an alternative platform that integrates more randomly across the genome.

By synthesizing these ancient transposon elements and pairing them with specialized lipid nanoparticle (LNP) delivery systems, the investigators aimed to create a platform capable of delivering large gene cassettes—such as CAR constructs—directly into patients through a single infusion.¹

“We've been developing…a large ‘toolbox’ of gene editing tools that we collectively call gene drivers, and specifically have been focusing on proteins that encompass…all the enzymatic properties, as well as combine them with different template architectures that enable us to really reach this toolbox that now is capable of doing programmable small changes in the initial editing,” Cotta-Ramusino said. “….We are using and taking advantage of fusing these enzymes, as well as really enabling us to [find] a different form of [gene editing].”¹

In Vivo LNP-Based Delivery Generates Durable, Functional CAR T Cells Across Multiple Animal Models

In different animal models, including mice and nonhuman primates (NHPs), Gene Writer demonstrated strong in vivo efficacy. Specifically, in mouse studies, a single infusion of LNPs produced durable tumor killing and permanent B-cell clearance. Cotta-Ramusino also reported what she believed to be the first demonstration of permanent CAR T-cell generation in NHPs using LNP-mediated RNA delivery. In these studies, the treatment appeared to be about 10 times more potent than transient mRNA-based CAR T-cell approaches, achieving complete B-cell clearance after a single infusion. Average integration was limited to approximately 1 to 2 copies per cell, a range considered favorable for therapeutic safety and consistency. Additionally, edited CAR T cells were detected across multiple T-cell subsets, including naive and central memory T cells, which are essential for long-term persistence and sustained therapeutic efficacy.¹

Unlike lentiviral vectors, which often integrate into protein-coding regions of the genome, the gene driver platform exhibited random integration across the genome. Cotta-Ramusino suggested that this feature may help reduce concerns surrounding insertional mutagenesis and improve safety. Importantly, T cells engineered using this platform displayed durable, functional tumor-killing activity in laboratory assays, comparable to that of traditional lentiviral approaches.¹

Based on these findings, Cotta-Ramusino concluded that Gene Writer may offer greater scalability and versatility than traditional lentivirus-based ex vivo therapies. Because the system relies on RNA and LNP delivery rather than viral manufacturing, it avoids many logistical complexities and could support more flexible treatment strategies, including the simultaneous delivery of multiple CAR constructs.¹

“We believe that this platform has a higher scalability…and has a bigger versatility. We know that we can multiply 2 CARs, and the system is compatible for multiple different CARs, and also,…we believe we still have more flexibility for preserving the virus than really just the production of it,” Cotta-Ramusino concluded. “We really think this can be a new strong aspect in the T-cell space.”¹

REFERENCES

1. Cotta-Ramusino C. CAR T-cells and genome manipulation: targeted LNP delivery of an RNA Gene Writer in vivo enables generation of functional CAR-T cells. Presented at: Joint American Society of Transplantation and Cellular Therapy + European Society for Blood and Marrow Transplantation Basic and Translational Scientific Meeting; May 28-29, 2026; Philadelphia, PA.

2. Patel RP, Ghilardi G, Zhang Y, et al. CD5 deletion enhances the antitumor activity of adoptive T cell therapies. Sci Immunol. 2024;9(97):eadn6509. doi:10.1126/sciimmunol.adn6509

3. Angelos MG, Patel RP, Ruella M, Barta SK. Progress and pitfalls of chimeric antigen receptor T cell immunotherapy against T cell malignancies. Transplant Cell Ther. 2024;30(2):171-186. doi:10.1016/j.jtct.2023.10.013