Editorial Article: The importance of investigating complement system in gene therapy

Immunology expert, Dr. Anna Majowicz, shares how Spark Therapeutics research is helping tackle immunogenicity challenges in AAV-mediated gene therapy

19 Sep 2023

Dr. Anna Majowicz, immunology expert for Spark Therapeutics research in gene therapy
Dr. Anna Majowicz, Preclinical Immunology Lead in the Research and Technology Department , Spark Therapeutics

Gene therapies offer potentially beneficial therapeutic options for patients affected by a variety of conditions. Adeno-associated virus (AAV) vectors are a leading non-pathogenic platform used for gene therapy delivery. While gene therapy – and delivery methodologies – have become more effective over the years, human immune responses to the vector remain a significant challenge for the safety, efficacy, predictability, and durability of AAV-mediated gene therapy today. 

The Immunology team at Spark Therapeutics, led by Dr. Klaudia Kuranda, Head of Immunology works to identify and characterize immune responses that might occur after infusion of AAV-based gene therapies. The group is studying targeted approaches to circumvent or treat such responses, with the objective of advancing the safe and efficacious use of gene therapies in patients. “Broadly, we explore potential immune responses in different therapeutic areas targeting hepatic, nervous, or ocular tissues,” explains Dr. Anna Majowicz, Preclinical Immunology Lead in the Research and Technology Department at Spark Therapeutics. “In the context of AAV gene therapy, we study immune responses in a preclinical setting in vivo, and in vitro using human blood, or samples from different animal species.”

A number of systemic AAV-based gene therapy clinical trials have reported asymptomatic elevation in serum levels of liver transaminases, which is an indication of liver inflammation and hepatotoxicity, mentions Dr. Majowicz. This increase in liver enzymes was correlated in some patients with an appearance of AAV capsid-specific T cells circulating in peripheral blood. It was also correlated with a concomitant decrease in transgene expression, which can limit or reduce the curative effect of the gene therapy. Fortunately, in many cases, prompt treatment with steroids successfully lowered liver transaminase levels, which prevented further loss of transgene expression.

Additionally, in some gene therapy trials in which high doses of AAV vectors were administered systemically, toxicities related to the activation of the complement system have been reported. “The complement system involves a complex cascade of proteins that enhance the body’s innate immune system”, shares Majowicz. “During a natural viral infection, complement activation leads to inflammation, opsonization, phagocytosis, and neutralization of the virus, and eventually, the activation of the adaptive immune response.”

AAV vector immunogenicity

There are three different pathways for activating the complement system, Majowicz tells us: the classical antibody-mediated pathway, the alternative pathway, and the lectin pathway. AAV vectors have been shown to activate complement via the classical pathway through binding of antibodies against AAV, and via the alternative pathway through direct binding of complement components. Additionally, complement proteins can potentially increase vector uptake into immune cells and promote proinflammatory cytokine release.

The reported cases of complement activation have occurred in a subset of AAV-based gene therapy clinical trials employing different AAV capsids, across multiple indications. “In some cases, this has resulted in severe adverse events, including atypical hemolytic uremic syndrome (aHUS), a type of thrombotic microangiopathy (TMA) which is a condition defined by the presence of hemolytic anemia, thrombocytopenia. Patients with severe complications like TMA have gone on to need urgent medical care post gene therapy infusion,” elaborates Majowicz.

Thus far in clinical studies, the overall incidence of complement activation has been low, considering the number of patients who have already been treated with gene therapy globally. Majowicz notes that a certain level of complement activation can occur without leading to observable symptoms like TMA. This could potentially contribute to the triggering of adaptive immune responses to AAV vectors.

Investigating complement activation in gene therapy

As identified in the recent Frontiers in Immunology article ,1 Majowicz suggests that monitoring complement activation in AAV-administered gene therapy in patients is potentially important for multiple reasons. “Monitoring of complement activation in patients over time can help us understand whether complement inhibition around AAV vector administration can mitigate immune responses against gene therapy vectors.”

To monitor complement activation in preclinical settings, one can measure different individual proteins that are part of the complement cascade. Because AAV can activate complement through the classical or the alternative pathway, it is of interest to understand the contribution of each of the pathways. “Using plasma samples, it is possible to measure a variety of proteins specific to the classical pathway such as C4a, as well as to the alternative pathway such as Ba and Bb,” says Majowicz. “Additionally, one can also look at protein levels of C3a and its downstream proteins C5a, and sC5b-9 (also known as TCC), which indicate general complement activation as C3 is the protein where the complement pathway converges. Monitoring proteins that regulate complement activation, such as factor H and factor I is possible as well.” Together, these results will allow scientists to obtain a comprehensive overview of the whole complement cascade during homeostasis and activation, providing potential insights for managing immune responses to AAV.

The future of AAV-based therapeutics

Given the limited number of patients in clinical trials in the AAV gene therapy field, Majowicz believes that increased collaboration among different companies, universities, and research institutions around strategies for immunomonitoring of immune response in clinical trials would be beneficial. Sharing immunology data will increase the understanding of immune responses to AAV, as well as the key biomarkers to be monitored. This could potentially provide valuable insight on how to modulate such responses with targeted immunosuppression protocols, ultimately contributing to the development of safer and more efficacious AAV gene therapies. In parallel, there is also room for the development of more potent and tissue-specific vectors, notes Majowicz. This can help reduce the viral vector infusion dose and lower potential immune responses.


  1. Smith CJ, Ross N, Kamal A, et al. Pre-existing humoral immunity and complement pathway contribute to immunogenicity of adeno-associated virus (AAV) vector in human blood. 2022. Front Immunol. Sep 16;13:999021. doi:10.3389/fimmu.2022.999021

Monitoring of complement activation - a critical focus in optimizing gene therapy

AAV vectors offer great promise in delivering life-saving gene therapy but come with the challenges of adverse activation of the complement system upon administration in high doses. Monitoring complement activation in AAV-administrated gene therapy patients is crucial to ensure the safety and durability of the gene therapy they are infused with.

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