Enhancing AAV gene therapy: Insights from Bayer’s immunogenicity testing study

Gene therapy is revolutionizing the treatment of genetic disorders, rare diseases, and chronic conditions. Among the most effective gene delivery vehicles are Adeno-Associated Viruses (AAVs), which efficiently transport genetic material to target cells. However, despite their therapeutic promise, AAV-based therapies face a critical obstacle: pre-existing immunity. Many individuals already carry antibodies against AAV, which can neutralize the viral vector before it delivers its intended therapy. Understanding and accurately detecting these antibodies is crucial for improving patient selection and optimizing clinical outcomes.

Pre-existing anti-AAV immunity: A barrier to treatment

AAVs are naturally occurring viruses that humans often encounter in the environment. Although they do not cause disease, they can still elicit immune responses. This pre-existing immunity is characterized by total anti-AAV binding antibodies (TAbs), which includes neutralizing antibodies (NAbs) and non-neutralizing antibodies. The NAbs can prevent AAV vectors from effectively transducing target cells, while non-NAbs don't directly block transduction but can potentially impact vector biodistribution¹.

Research has indicated that 30% to 60% of individuals possess pre-existing anti-AAV antibodies¹. The prevalence of these antibodies varies significantly according to geographic location, age, and AAV serotype. For instance, in the United States, the percentage of individuals with AAV1 antibodies ranges from 32% in Wisconsin to 67% in South Carolina. In Europe, NAb positivity for AAV1 is 48% in Sweden but rises to 79% in Poland and Hungary. Similarly, in Asia, the seroprevalence for AAV2 and AAV8 can reach up to 72%². These disparities suggest that tailored, population-specific approaches may be necessary when developing and administering AAV gene therapies.

How pre-existing immunity affects gene therapy success

The presence of anti-AAV antibodies can significantly impact gene therapy in various ways. Patients with high levels of NAbs may experience reduced efficacy of the therapy, as these antibodies bind to AAV vectors and impede their ability to deliver genetic material to target cells. Even in individuals with lower levels of antibodies, unpredictable immune responses can lead to inconsistent treatment outcomes.

Additionally, unlike many biologic therapies, AAV-based treatments are typically administered as a single-dose therapy. The immune response triggered by the initial administration can hinder successful repeat dosing, thereby limiting treatment options for patients who may require future interventions³. There are also safety concerns, as elevated pre-existing antibody levels may contribute to immune-mediated adverse events, including inflammation and liver toxicity, particularly at higher vector doses^4,5.

The need for robust pre-screening in AAV therapy

The variability in pre-existing immunity underscores the necessity of pre-screening patients before administering gene therapy. Accurate pre-screening ensures that only those patients most likely to benefit from AAV-based therapy receive treatment, thereby enhancing clinical success rates. Additionally, pre-screening can improve the reliability of clinical trial data by controlling for variability in immune responses across patient populations. This approach also supports personalized medicine, as patients with high antibody levels may require alternative treatment strategies, such as modified AAV serotypes or immune suppression protocols. Furthermore, pre-screening facilitates the efficient allocation of gene therapy resources, ensuring that costly treatments are directed towards patients with the best chance of success.

Bayer’s clinical screening study: A case for improved immunogenicity testing

A recent clinical study conducted by Bayer assessed the effectiveness of the Gyrolab^® Anti-AAVX Kit in comparison to traditional in-house ELISA methods for detecting anti-AAV antibodies in human and monkey serum samples. The study highlighted the significance of robust immunogenicity testing in optimizing patient selection and enhancing the predictability of AAV gene therapy outcomes

Key findings from the study

• Anti-AAV9 antibodies: The Gyrolab assay successfully identified positive and negative samples with good consistency across both human and monkey serum tests.
• Anti-AAV8 antibodies: The Gyrolab Anti-AAVX Kit accurately classified nine human serum samples as either positive or negative, demonstrating exceptional reliability.
• Cut-point calculations: The kit effectively differentiated positive samples from background noise, reinforcing its robustness for clinical screening applications.
• Assay validation: Sample preparation and dilution steps differ between ELISA and Gyrolab assays, requiring careful optimization when setting cut-point calculations. Additionally, centrifugation is a critical step in sample preparation to ensure reliable results in the Gyrolab system.

Detailed study results

• Human serum testing:
  • More than 300 human serum samples were tested for anti-AAV9 antibodies using bridging ELISA and indirect ELISA assays.
  • The Gyrolab Anti-AAVX Kit demonstrated a high correlation with ELISA results, confirming its ability to identify true positives and true negatives.
  • A confirmation assay using excess AAV capsid (% signal inhibition method) validated the kit’s ability to distinguish positive from negative samples.

• Monkey serum testing:
  • Ten monkey serum samples were analyzed for anti-AAV9 antibodies.
  • The Gyrolab assay accurately classified 7 out of 10 samples as either positive or negative.
  • Some samples displayed higher variability, likely due to interspecies immune response differences.

The impact of high-throughput testing on AAV research

Advancements in high-throughput testing, such as the Gyrolab Anti-AAVX Kit, are revolutionizing AAV immunogenicity assessments. While effective, traditional ELISA-based methods can be labour-intensive and prone to variability. High-throughput immunoassays offer several advantages:

• Increased efficiency: Automated high-throughput testing allows researchers to process large sample volumes quickly, streamlining preclinical and clinical studies.
• Greater accuracy and reproducibility: Minimizing manual handling reduces human error, leading to more reliable results.
• Improved scalability: As AAV gene therapies become more widely adopted, efficient screening is necessary to manage large-scale clinical trials and regulatory submissions. High-throughput testing, such as the Gyrolab Anti-AAVX Kit, plays a crucial role in this process. It ensures that the screening is not only efficient but also reliable, thereby providing reassurance about the robustness of the clinical trials.
• Optimised sample utilisation: Numerous patient populations, particularly those with rare diseases, face limited sample availability. High-throughput systems diminish the volume required per test, maximizing the utility of available samples.

Expanding the future of AAV-based gene therapies

With ongoing research, AAV gene therapy is poised to expand into new disease areas. One promising application is targeting the central nervous system (CNS), where AAV vectors with enhanced blood-brain barrier penetration are being developed for neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease. In oncology, AAV-based vectors are being investigated for cancer gene therapy, where they may be used to deliver immune-modulating genes to tumors. Additionally, advances in synthetic biology and vector engineering pave the way for personalized AAV gene therapies, where treatments can be tailored to individual patients' immune profiles and genetic conditions.

Conclusion

Bayer’s study underscores the importance of advanced immunoassay technologies like the Gyrolab® Anti-AAVX Kit in addressing the challenge of pre-existing anti-AAV immunity. As gene therapy continues to revolutionize medicine, precise, high-throughput screening tools will be essential for optimizing patient selection, improving clinical trial outcomes, and ensuring broader accessibility to these life-changing treatments.

The success of AAV-based gene therapy depends on overcoming barriers related to immunogenicity. By utilising state-of-the-art immunoassay technology and refining pre-screening protocols, researchers and clinicians can maximize treatment efficacy and unlock the full potential of gene therapy for a wider range of patients across the globe.

References

1. Schulz M, Levy DI, Petropoulos CJ, et al. Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy. Mol Ther. 2023;31(3):616-630. doi:10.1016/j.ymthe.2023.01.010
2. Weber T. Anti-AAV Antibodies in AAV Gene Therapy: Current Challenges and Possible Solutions. Front Immunol. 2021;12:658399. Published 2021 Mar 17. doi:10.3389/fimmu.2021.658399
3. Shi X, Bortolussi G, Collaud F, et al. Repeated dosing of AAV-mediated liver gene therapy in juvenile rat and mouse models of Crigler-Najjar syndrome type I. Mol Ther Methods Clin Dev. 2024;32(4):101363. Published 2024 Oct 28. doi:10.1016/j.omtm.2024.101363
4. Mendell JR, Connolly AM, Lehman KJ, et al. Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations. Mol Ther Methods Clin Dev. 2022;25:74-83. Published 2022 Feb 26. doi:10.1016/j.omtm.2022.02.011
5. Larrey D, Delire B, Meunier L, Zahhaf A, De Martin E, Horsmans Y. Drug-induced liver injury related to gene therapy: A new challenge to be managed. Liver Int. 2024;44(12):3121-3137. doi:10.1111/liv.16065