An Open Letter to the Gene Therapy Community on CpG Content and AAV Vector Safety

To the Gene Therapy Community,

I write to you as a scientist, developer, and long-time participant in the evolution of adeno-associated virus (AAV) gene therapy, with deep respect for the extraordinary progress our field has achieved and with equal concern for risks that, in my view, remain insufficiently acknowledged. This letter is intended to encourage reflection, rigor, and precaution regarding the continued development and clinical testing of CpG-rich AAV vector genomes.

Over the past two decades, AAV gene therapy has transitioned from experimental promise to clinical reality, offering transformative benefit to patients with serious and often fatal diseases. Yet as the field has matured, so too has our understanding of innate immune sensing, therapeutic transgene persistence, and the molecular determinants that influence both safety and durability. Among these determinants, CpG dinucleotide content within the AAV vector genome has emerged as a factor of substantial biological consequence.

Unmethylated CpG motifs are recognized by the innate immune system, through Toll-like receptor 9, which can trigger inflammatory signaling, cytotoxic responses, and elimination of transduced cells. In preclinical and clinical settings, CpG-rich vector genomes have been associated with heightened immune activation, loss of expression, liver enzyme elevations, and, in some contexts, serious adverse events. The cumulative evidence indicates that CpG content is a controllable and mechanistically relevant variable in vector design.

Despite this knowledge, CpG-unmodified and even CpG-enhanced constructs continue to be advanced into clinical development. Abundant CpG motifs remain present not only within coding regions but also within regulatory elements such as promoters, introns, and polyadenylation signals, where they may contribute to immunogenicity. Given that CpG depletion strategies have repeatedly demonstrated improved safety and more durable transgene expression in preclinical and clinical trials, the continued use of CpG-rich genomes raises important scientific and ethical questions.

Clinical trials represent a covenant of trust between investigators and patients. Participants assume risk in the expectation that vector design reflects the best available understanding of safety. When modifiable genomic features known to influence immune activation are not optimized, we must ask whether we are meeting our obligation to minimize avoidable risk. The issue is whether knowingly advancing preventable immunostimulatory features is consistent with the standards of a mature therapeutic field.

Equally important is the role of patient awareness and empowerment. Individuals considering participation in a gene therapy clinical trial deserve clear, accessible information about the design of the therapy they may receive. Patients and families should feel empowered to ask informed questions about the CpG content within the AAV vector genome, including whether CpG motifs have been reduced, and how genomic design may influence immune response, durability of expression, and overall safety. Transparent dialogue between investigators and participants strengthens informed consent and reinforces trust in the clinical research process.

I urge the community to consider the following principles:

First, CpG content across the entire vector genome, including regulatory regions, should be treated as a critical quality attribute and reported transparently in preclinical and clinical disclosures.

Second, CpG depletion or reduction strategies should be incorporated into vector design as part of a broader, data-driven safety optimization framework.

Third, regulatory agencies, sponsors, and investigators should continue to work toward clearer guidance on genomic immunogenicity factors, including CpG content, to ensure consistent safety expectations across programs (many already are).

Finally, historical and ongoing clinical data should be systematically analyzed to better define correlations between CpG content, immune activation, and durability.

The gene therapy field stands at a pivotal moment. The choices we make now in vector design will shape not only clinical outcomes, but also public trust and the long-term credibility of genetic medicine. By confronting the risks associated with CpG-rich AAV vector genomes and committing to thoughtful, evidence-based optimization, we can better fulfill our shared mission: delivering safe, durable, and life-changing therapies to patients who depend on us.