Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy

Area of Science:

• Biotechnology
• Gene Therapy
• Immunotherapy

Background:

• On-target toxicity to normal cells is a significant safety challenge in targeted immune and gene therapies.
• CD33 is a surface protein targeted by certain therapeutics, and its expression on normal cells can lead to toxicity.

Purpose of the Study:

• To develop a base editing (BE) strategy to mitigate on-target toxicity by modifying CD33 expression.
• To assess the efficacy and safety of CD33 base editing in hematopoietic stem and progenitor cells (HSPCs) for improved immunotherapies.
• To demonstrate multiplexed base editing for broader gene therapy applications, including HbF reactivation.

Main Methods:

• Utilized a base editing (BE) approach targeting a CD33 single nucleotide polymorphism to reduce surface expression.
• Performed CD33 editing in human and nonhuman primate (NHP) HSPCs.
• Demonstrated multiplexed adenine base editing of CD33 and gamma globin genes.
• Evaluated long-term persistence and HbF reactivation in NHPs.
• Assessed *in vitro* enrichment of dual gene-edited cells using gemtuzumab ozogamicin (GO).

Main Results:

• CD33 editing in HSPCs protected cells from CD33-targeted therapeutics without affecting hematopoiesis *in vivo*.
• Highly efficient (>70%) multiplexed adenine base editing of CD33 and gamma globin genes was achieved.
• Dual gene-edited cells showed long-term persistence and HbF reactivation in NHPs.
• *In vitro* studies demonstrated enrichment of dual gene-edited cells with GO treatment.

Conclusions:

• Adenine base editing offers a promising strategy to reduce off-leukemia toxicity in targeted immunotherapies.
• Multiplexed base editing can enable dual gene modifications for enhanced gene therapies, such as inducing fetal hemoglobin production.
• This approach holds potential for developing safer and more effective immune and gene therapies.