Illustration of RNA aptamer-mediated base editing for gene therapyIntroduction

Researchers from Revvity have developed and validated a novel RNA aptamer-mediated base editing platform (Pin-point™ system) for simultaneous gene knock-in and multiple gene knockout in human primary T cells. This platform aims to advance the generation of allogeneic CAR-T cells by overcoming the limitations of traditional CRISPR-Cas9 technology such as off-target effects and genomic instability. The researchers demonstrated that this technology could efficiently and safely introduce multiple gene edits and transgene integrations, thereby facilitating the creation of advanced cell therapies with improved safety and functionality.

Base Editing and Comparison with CRISPR Technology

Base Editing

Base editing is a precision genome editing technology that enables the direct conversion of one DNA base pair into another without inducing double-strand breaks (DSBs). There are two primary types of base editors:

  • Cytosine Base Editors (CBEs): Convert cytosine (C) to thymine (T).
  • Adenine Base Editors (ABEs): Convert adenine (A) to guanine (G).

Comparison with CRISPR Technology

  • CRISPR-Cas9: Introduces DSBs at specific genomic locations, which can be repaired by the cell’s natural repair mechanisms, often leading to insertions or deletions (indels) that disrupt the target gene.
  • Base Editing: Achieves precise base conversions without creating DSBs, thus reducing the risk of unintended genomic alterations and genotoxicity. It provides a safer alternative for therapeutic applications, especially when multiple edits are required.

Advantages of RNA Aptamer-Mediated Base Editing

High Efficiency and Specificity of Base Editing

The RNA Aptamer-Mediated Base Editing (Pin-point™ platform) demonstrated high efficiency in converting cytosine (C) to thymine (T) at target loci, achieving conversion rates of 76-90% across four genes (B2M, CD52, TRAC, and PDCD1) when delivered to human T cells using synthetic RNA reagents. Minimal off-target effects were observed, with only 4 out of 456 analyzed sites showing low-level base editing, indicating high specificity. The Pin-point™ platform’s ability to perform gene editing without generating DSBs significantly reduces the risk of chromosomal aberrations and genomic instability, which are major concerns with conventional CRISPR-Cas9 systems. This enhances the safety profile of the edited cells, making them more suitable for clinical applications.

Simultaneous Multi-Gene Knockout

The platform successfully achieved high levels of protein knockout for B2M, CD52, TRAC, and PDCD1 in both non-activated and activated T cells, with approximately 80% reduction in protein expression. Flow cytometry analysis confirmed the efficient knockout of multiple target proteins simultaneously, with a high degree of simultaneous target knockout observed in individual cells.

Reduced Chromosomal Translocations

The Pin-point™ platform significantly reduced the incidence of chromosomal translocations compared to SpCas9. Translocations were undetectable in cells edited with the Pin-point™ platform, whereas SpCas9 editing resulted in translocations in 1 in 17-25 diploid cells.

Improved Proliferative Capacity

T cells edited with the Pin-point™ platform exhibited better proliferative capacity compared to those edited with SpCas9, suggesting reduced activation of DNA-damage responses and better cell fitness.

Simultaneous Knock-In and Knockout

The study demonstrated the ability to perform simultaneous site-specific knock-in of a CD19-CAR transgene at the TRAC locus and multi-gene knockout using the Pin-point™ platform. This was achieved without detectable AAV integration at other sgRNA-targeted sites. The engineered CAR-T cells retained their functionality, showing comparable ability to kill antigen-positive target cells and produce effector cytokines (TNFα and IFNγ) as SpCas9-engineered controls.

Implications of the Findings in CAR-T Cell and Other Cell Therapies

The RNA aptamer-mediated base editing (Pin-point™ system) offers significant advantages for CAR-T cell therapies and other allogeneic cell therapies due to its capabilities in multiplex gene editing, simultaneous knock-in and knockout, and enhanced safety profile.

Improved Manufacturing of CAR-T Cells

The high efficiency and specificity of the RNA aptamer-mediated base editing platform, combined with its ability to perform simultaneous knock-in and knockout, streamline the manufacturing process of CAR-T cells. This capability can potentially reduce production costs and improve the scalability of CAR-T cell therapies.

Broader Applications in Cell Therapy

The modularity and flexibility of the RNA aptamer-mediated base editing platform allow for its application across various cell types and therapeutic contexts. It can be used to engineer complex genetic modifications, making it a valuable tool for developing advanced cell therapies beyond CAR-T cells, including other allogeneic cell therapies.

Potential for Precision Medicine

The ability to introduce precise genetic modifications with minimal off-target effects and reduced genomic instability aligns with the goals of precision medicine. The RNA aptamer-mediated base editing platform can be leveraged to develop personalized therapies tailored to individual patients’ genetic profiles.

Conclusion

The RNA aptamer-mediated base editing method (Pin-point™ system) represents a significant advancement in the field of gene editing. Its high efficiency, specificity, and safety profile make it a promising tool for generating engineered cell therapies, particularly allogeneic CAR-T cells. The ability to perform simultaneous knock-in and multi-gene knockout in a single intervention without inducing DSBs addresses key limitations of existing gene editing technologies and opens new avenues for the development of complex engineered cell therapies.

Reference

Porreca P. et al. An aptamer-mediated base editing platform for simultaneous knock-in and multiple gene knockout for allogeneic CAR-T cells generation. Molecular Therapy. Prep-proof Published online. https://doi.org/10.1016/j.ymthe.2024.06.033

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