In vivo site-specific engineering to reprogram T cells

Area of Science:

• Immunology
• Gene Therapy
• Cancer Research

Background:

• Engineered T cells (CAR-T, TCR-T) show promise for cancer, autoimmune, and infectious diseases.
• Genome editing enhances T cell function but ex vivo manufacturing is costly and time-consuming.
• In vivo generation of CAR-T cells is desirable but faces challenges with transient expression or random integration.

Purpose of the Study:

• To develop a method for stable, cell-specific transgene expression via in vivo site-specific integration of large DNA payloads.
• To overcome the limitations of ex vivo T cell manufacturing and current in vivo approaches.

Main Methods:

• A two-vector system was developed using CRISPR-Cas9 ribonucleoproteins and a DNA donor template.
• Enveloped delivery vehicles and adeno-associated viruses were optimized for T cell-specific delivery.
• CAR transgene was integrated into a T cell-specific locus in humanized mouse models.

Main Results:

• Stable and cell-specific transgene expression was achieved in vivo.
• Therapeutic levels of CAR T cells were generated in vivo in models of B cell aplasia, hematological, and solid malignancies.
• The method demonstrated efficient T cell-specific delivery and gene-targeting.

Conclusions:

• In vivo site-specific integration enables precise and stable CAR T cell generation.
• This approach offers a more efficient, precise, and accessible alternative to ex vivo manufacturing.
• The findings pave the way for broader application of T cell-based therapies.