Azalea Therapeutics, Inc. recently announced new preclinical data demonstrating robust in vivo generation of functional TRAC-CAR T cells using its proprietary Enveloped Delivery Vehicle (EDV) technology. The results will be presented during the Immune Cell session at the American Society of Gene and Cell Therapy’s (ASGCT’s) Breakthroughs in Targeted In Vivo Gene Editing.

Azalea’s dual-vector platform combines T cell-targeted EDVs delivering Cas9 RNP complexes with a human T cell-tropic AAV carrying a promoterless CAR template. This approach enables precise genomic insertion at the TRAC locus, bringing CAR expression under the control of the native T cell receptor promoter. In vivo TRAC insertion is designed to improve potency, persistence and safety by achieving regulated, physiologic CAR expression rather than continuous signaling from exogenous promoters.

In Vivo TRAC-CAR T Generation and Anti-tumor Activity in Mouse Models

Highlights from the presentation include new data showing that, across multiple hematologic and solid tumor humanized mouse models, Azalea’s in vivo engineered CAR T cells demonstrated:

• CAR detected on ~50% of all T cells following a single intravenous (IV) dose of EDV + AAV

• Physiologic CAR expression driven by the endogenous TRAC promoter

• Robust CAR T expansion in vivo, supporting persistence and anti-tumor effect

• Complete B cell aplasia as a pharmacodynamic marker of TRAC-CAR T activity

• Rapid and durable tumor clearance in hematologic models, including complete control of NALM6 acute lymphoblastic leukemia with >60-day survival

• Compelling anti-tumor activity in solid tumor settings, including demonstration of solid tumor rejection using an in vivo CAR T approach, with B7H3 CAR T cells eradicating sarcoma tumors

• No ex vivo cell manipulation or lymphodepletion required.

“These mouse studies show what becomes possible when T cells can be precisely engineered inside the body,” said Jenny Hamilton, Ph.D., co-founder, president and chief executive officer of Azalea Therapeutics. “A single off-the-shelf dose produced potent, persistent, physiologically regulated CAR T cells capable of clearing tumors without the need for lymphodepletion or any ex vivo manufacturing. These results reinforce that our fully programmable, modular system can reliably generate therapeutically active CAR T cells in vivo – an exciting validation of the platform and the potential for in vivo cell therapy.”

First Demonstration in Primates of In Vivo TRAC-CAR T Generation

In an ongoing nonhuman primate (NHP) study evaluating feasibility, pharmacodynamics and tolerability, a single IV dose of EDV + AAV achieved:

• Complete CD20+ B cell aplasia in peripheral blood by Day 10

• Expansion of TRAC-CAR T cells to ~35% of peripheral T cells, confirming in vivo engineering

• Complete CD20+ B cell aplasia in lymph nodes and bone marrow by Day 13

• Favorable tolerability profile, with no unexpected findings to date

“We believe this represents the first primate demonstration of in vivo TRAC-CAR T generation with both cell-selective delivery and genomic locus-specific integration,” said Jenny Hamilton, Ph.D. “The robust on-target editing, CAR T expansion and deep B cell depletion we’re seeing in primates is a pivotal step forward, supporting the notion that precise, programmable CAR insertion directly in patients may be within reach and could open the door to a new generation of off-the-shelf cell therapies.”

Azalea will present these data today at ASGCT’s Breakthroughs in Targeted In Vivo Gene Editing, taking place virtually and in San Diego.

Abstract Title: In vivo generation of TRAC CAR-T cells by leveraging enveloped delivery vehicles
Presenting Author: Jenny Hamilton, Ph.D., Azalea Therapeutics
Session 3: Immune Cell
Date/Time: Thursday, November 20, 2025 | 12:50 – 1:50 pm PST

Visit the ASGCT Breakthroughs in Targeted In Vivo Gene Editing website to view the published abstracts.

Azalea Therapeutics is a biotechnology company redefining precision genomic medicines in vivo. Its proprietary Enveloped Delivery Vehicles (EDV) platform is engineered to deliver transient CRISPR-Cas9 cargo to specific cells in the body for site-specific genome editing with curative intent. Azalea’s first programs leverage T cell-targeting EDVs and highly efficient T cell-tropic AAVs to enable programmable CAR gene insertion at a defined genomic locus, placing expression under control of the cell’s endogenous promoter for physiologic and sustained activity. This approach aims to generate potent, durable and safe therapies directly inside patients, avoiding the complexity of ex vivo manufacturing and unlocking new treatment modalities – including in vivo CAR T cell therapies – across cancer, autoimmune disease and genetic disorders. Azalea is headquartered in Berkeley, California. For more information, please visit azaleatx.com and follow us on LinkedIn.