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FDA Grand Rounds – Adeno-associated Virus-mediated Gene Therapy: Advances, Immune Challenges, and Research Innovations

Scientist in PPE operating automated lab instrument under teal lighting for AAV gene therapy immunogenicity analysis

Capabilities

Gene Therapy CDMO
CGT Bioanalysis
CGT Safety Assessment

Author: Karen Doucette, Senior Director of CGT Project Management at Pharmaron

Introduction

On February 19, 2026, the FDA Grand Rounds presented its monthly educational series which spotlighted AAV gene therapy immunogenicity and recent advances being made to overcome immunological challenges. The session was delivered virtually by Ronit Mazor, PhD, a principal investigator in FDA’s Center for Biologics Evaluation and Research (CBER). Dr. Mazor discussed both the substantial progress of gene therapy and the lingering immune challenges that shape the risk-benefit profile of AAV platforms today.

Promise and Challenges of  AAV Gene Therapy Immunogenicity

AAV vectors remain one of the most used delivery systems for human gene therapy because of their capacity for long-duration expression and favorable safety profile. There are currently eight (8) BLA approvals1 and many INDs ongoing for AAV gene therapies. Clinical programs have matured over the last few years across ocular, neuromuscular, hematologic, and metabolic indications, supported by advances in capsid design, promoter selection, and payload engineering. Despite these successes, the field continues to confront dose‑related toxicities, innate immune activation, adaptive immune response, and complex manufacturing requirements.

Immunogenicity can result in patient exclusion, increased risk of toxicity, and reduced efficacy. Factors contributing to immunogenicity come from various sources, including the patient (immune state, prior exposure, sex, age, etc.), the product (epitope content, impurities, PTMs, etc.), and the treatment (dose, ROA, frequency, etc.). Mazor reports that approximately 30-85% of the population have pre-existing AAV antibodies due to natural exposure, and females have significantly higher neutralizing and total antibodies than males.

Dr. Mazor presented on three main research areas:

  1. Reduction of T cell immunogenicity through capsid engineering
  2. CMC CQAs like PTMs that increase immunogenicity and impact T cell bioassays
  3. Immunogenic risk associated with emerging modalities like gene editing

Capsid Engineering

One of FDA’s focuses is for deimmunization, a strategy in which AAV capsids are engineered to specifically evade the immune system. By identifying and eliminating a specific T cell epitope in an AAV9 capsid, Dr. Mazor’s lab was successfully able to create a novel AAV vector that had similar in vitro and in vivo activity and biodistribution but elicited a reduced T-cell response. This type of technology should expand patient access and reduce toxicity. FDA has published this work to increase industry awareness and provide a roadmap for safer gene therapies2,3. The algorithm developed and validated by FDA to identify such opportunities for immune-engineering is known as Epitope Mutator and MHC Binding Predictor (EMPP). Access to EMPP is currently available to FDA users through their High-performance Integrated Virtual Environment (HIVE).

AAV Deamidation

Deamidation is a spontaneous  post translational modification (PTM) that AAV undergoes in storage. Since the effect of deamidation on immunogenicity had not been previously studied, Mazor tested the effect of WT and deamidated AAV vector on PBMCs from healthy donors. The results indicated changes in both binding stability to MHC II and cytokine secretion from CD4 T cells in vitro. This indicates that the chemical change that occurs during deamidation may increase the immunogenicity of AAV in individuals with certain HLA4. Because of this, Mazor recommends including deamidated peptides in the peptide mix used for ELISpot immune monitoring assays so as not to underpredict toxicity. FDA also encourages formulation studies designed to prevent deamidation.

Gene Editing

AAV’s use as a carrier for genome editing cargos introduces a new consideration for immune response. The most widely used nuclease, Cas9, is bacterial in origin and is in fact immunogenic. Pre‑existing antibodies and T‑cells have been detected against commonly used Cas9 transgenes, and standard short-term immunosuppression protocols are not sufficient in all clinical contexts5. Mazor’s team sought to further understand potential toxicity risks by modeling which Cas9 epitopes are most likely presented on MHC molecules and thereby recognizable by T cells. Similar to the work done for AAV capsid engineering, she used EMPP data modeling to identify a particular epitope on the bacterial Cas9 transgene that induces T cell toxicity in vitro and propose mutations to eliminate the epitope. In this case, deimmunization is aimed at the payload instead of the delivery vehicle.

Conclusions

The FDA’s Grand Rounds spotlight made one message unmistakable: the more we understand the immune system, the better our gene therapies will work. From optimizing capsids to controlling PTMs or deimmunizing payloads like Cas9, all of these steps bring us closer to treatments that actually last. It’s no longer about working around the immune system but rather designing with it in mind.  It’s encouraging to see the field leaning into this, and there’s a lot to look forward to.

Pharmaron Support

Pharmaron Lab Services plays a pivotal role in helping gene therapy developers navigate the complex immunogenicity landscape surrounding AAV products. With a comprehensive suite of analytical testing, our CMC team can identify product-related impurities, PTMs, and other CQAs to inform clinical risk assessment for AAV immunogenicity. Through an integrated suite of bioanalytical capabilities, Pharmaron provides the data needed to characterize immune risks from early discovery through clinical development. Services include neutralizing antibody assays, anti‑AAV and anti-transgene assays, and humoral and cellular immune‑response profiling. Our bioanalytical teams work closely with sponsors to design and validate assays aligned with FDA expectations for sensitivity, specificity, robustness, and clinical relevance. Pharmaron also applies a risk‑based bioanalytical framework, guiding clients in tailoring programs to vector serotype, route of administration, pre‑existing immunity, and transgene biology. By combining deep scientific expertise with regulatory awareness, Pharmaron helps developers anticipate immune‑mediated liabilities and build strong immunogenicity packages that support both regulatory review and patient safety.

References:

  1. Approved Cellular and Gene Therapy Products | FDA
  2. Bing et. al., Nat Biomed Eng 2024 Rational immunosilencing of a promiscuous T-cell epitope in the capsid of an adeno-associated virus
  3. Bing et.al., Nature Communications 2025 Can Sex-based Variations in the Immune Responses to AAV Gene Therapy Affect Safety and Efficacy? A Review of Current Understanding
  4. Bing et. a. 2022, Mol Ther Methods Clin Dev. Differential T cell immune responses to deamidated adeno-associated virus vector
  5. Stigzelius, Virpi et al., Molecular Therapy, Volume 33, Issue 10, 4714 – 4730. Peeling back the layers of immunogenicity in Cas9-based genomic medicine: Molecular Therapy