Tailoring capsid directed evolution technology for improved AAV-mediated CAR-T generation.

in Molecular therapy : the journal of the American Society of Gene Therapy by Adrian Westhaus, Elena Barba-Sarasua, Yuyan Chen, Kenneth Hsu, Suzanne Scott, Maddison Knight, Florencia Haase, Santiago Mesa Mora, Benjamin C Houghton, Ramon Roca-Pinilla, Predrag Kalajdzic, Geraldine O'Neill, Adrian J Thrasher, Giorgia Santilli, Leszek Lisowski

TLDR

  • A new AAV capsid-directed evolution platform was developed to improve targeted CAR integration and reduce vector dose requirements in CAR-T therapies.

Abstract

Chimeric antigen receptor (CAR) T-cell (CAR-T) therapies present options for patients diagnosed with certain leukemias. Recent advances of the technology included a method to integrate the CAR into the T-cell receptor alpha constant (TRAC) locus to take advantage of the endogenous promoter and regulatory elements for CAR expression. This method used adeno-associated viral (AAV) vectors based on AAV6 to deliver the donor template encoding the CAR construct. Since the original publication, improvements have been made to this targeted CAR integration technique, however, none of those techniques focused on improving the AAV vector used to deliver the therapeutic cargo. The herein presented study developed a novel AAV capsid directed evolution platform that allows to specifically select for novel AAV capsid variants that enable more efficient targeted gene editing-mediated CAR construct integration into the TRAC locus in primary T-cells. Using this new platform, we selected several novel AAVs that enable more efficient editing in T-cells than AAV6. Two novel capsids, AAV-T1 and AAV-T2, were able to mediate five-fold improvement for on-target knock-in, which resulted in five-fold reduction of the vector dose to produce highly cytolytic T-cells against a brain tumor cell line.

Overview

  • The study aimed to improve previous targeted CAR integration techniques, specifically focusing on developing novel AAV vectors to deliver therapeutic cargo.
  • The research used an AAV capsid directed evolution platform to select novel AAV capsid variants for efficient targeted gene editing-mediated CAR construct integration into the TRAC locus in primary T-cells.
  • The primary objective was to improve targeted gene editing and reduce vector dose requirements to produce highly cytolytic T-cells against specific cancer cell lines.

Comparative Analysis & Findings

  • The novel AAV capsid-directed evolution platform selected several novel AAVs that enable more efficient editing in T-cells compared to AAV6.
  • Two novel capsids, AAV-T1 and AAV-T2, demonstrated a five-fold improvement in on-target knock-in, resulting in a five-fold reduction of the vector dose.
  • Compared to AAV6, the novel capsids showed improved efficiency in targeted gene editing-mediated CAR construct integration into the TRAC locus in primary T-cells.

Implications and Future Directions

  • The novel AAV capsid variants have the potential to improve the efficacy and efficiency of CAR-T therapies, reducing vector dose requirements and improving T-cell cytolytic activity.
  • Future studies can explore the optimal use of these novel capsids in different cancer types and patient populations, as well as investigate potential off-target effects.
  • The AAV capsid-directed evolution platform can be further developed and applied to select novel AAV capsids for targeted gene editing in various tissues and cell types.
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