Innate Immune Response to AAV-Based Gene Therapy Vectors: Dissecting Mechanisms and Approaches for Safer Next-Generation Therapies

 

The Promise and Peril of AAV Gene Therapy

Recombinant adeno-associated viruses (rAAVs) have rapidly established themselves as the leading vectors for in vivo gene therapy, a position reinforced by the regulatory approvals of Zolgensma for spinal muscular atrophy, Roctavian for hemophilia A, and Elevidys for Duchenne muscular dystrophy. Their broad tissue tropism, ability to transduce non-dividing cells, and favorable safety profile compared to other viral vectors underscore their therapeutic promise. However, immune responses to rAAV remain a critical barrier to clinical success, limiting transduction efficiency, jeopardizing transgene durability, and in severe cases, triggering adverse events, particularly at high systemic doses. While adaptive immune responses such as neutralizing antibodies and T cell-mediated clearance have been well characterized, increasing attention has turned to the innate immune system, which acts as the first responder and initiator of downstream adaptive immunity. A recent article published in Molecular Therapy-Methods and Clinical Development by a research team from the Roche Innovation Center Basel explores how rAAV2 and rAAV8 vectors engage with the innate immune system (1) . The study specifically examines complement activation, the release of cytokines and chemokines, and the influence of pre-existing antibodies.

Setting the Stage for Innate Immunity in AAV Therapy

The understanding of innate immune responses to rAAV has evolved significantly. Early studies primarily focused on adaptive immunity. The landmark hemophilia B trial (2) first linked loss of transgene expression to capsid-specific CD8+ T cell responses, detected via IFN-γ ELISpot. However, the inability of animal models to fully recapitulate this highlighted species-specific differences in the Roche Innovation Center Basel study.

The innate arm began to attract attention when studies in mice and non-human primates (NHPs) revealed rapid (within hours) and transient increases in hepatic transcripts encoding cytokines and chemokines (e.g., IL-6, TNF-α, MCP-1) following intravenous (i.v.) rAAV injection (3,4). Crucially, innate immunity was shown to prime adaptive responses. Previous study has demonstrated that anti-AAV antibody production, limiting re-dosing potential, is dependent on IL-6 and IL-1β in vitro and in vivo (5) .

Key innate sensors were identified:

  1. Toll-like receptor 9 (TLR9): Plasmacytoid dendritic cells (pDCs) sense unmethylated CpG motifs in the rAAV genome via TLR9, leading to robust type I interferon (IFN-α/β) production (3). CpG-depleted AAV vectors demonstrated the ability to maintain long-term transgene expression, evade immune surveillance, and limit effector cell infiltration (6) .
  2. Toll-like receptor 2 (TLR2): Recognizes the AAV capsid itself, contributing to inflammatory cytokine production (7).
  3. Complement: Clinical trials administering high systemic rAAV doses reported sepsis associated encephalopathies (SAEs), notably thrombotic microangiopathy (TMA), linked to complement activation within days to weeks post-infusion (8). Complement inhibitors (eculizumab – anti-C5, APL-9 – anti-C3) showed efficacy in mitigating these toxicities (9) . In vitro and in vivo studies confirmed that complement components (e.g., C3) bind directly to the capsid, promoting activation, cytokine induction, and enhanced vector uptake by phagocytes (10).
  4. cyclic GMP-AMP synthase (cGAS)-activating Stimulator of Interferon Genes (STING) (cGAS-STING) and Interferon gamma inducible Protein 16 (IFI16) : Beyond TLR9, cytosolic sensors such as cGAS-STING and IFI16 may detect episomal AAV DNA. These pathways converge on type I interferon production, creating an antiviral state that diminishes transgene expression (11) .
  1. Capsid recognition and cellular stress responses: The AAV capsid itself can also contribute to innate activation. Uptake of capsids through clathrin-mediated endocytosis and subsequent trafficking to endosomal compartments expose them to TLRs. For instance. Additionally, proteasome processing of capsids generates peptides for MHC class I presentation, bridging innate and adaptive immunity (12, 13) .

 
The Roche Innovation Center Basel study build upon this foundation by systematically dissecting the mechanisms of complement activation and cytokine release in a human whole blood assay (WBA), incorporating the critical variable of pre-existing immunity and investigating the specific role of the viral genome.

Mechanisms of Complement Activation: Antibody-Dependence and Alternative Pathway Involvement

The Roche Innovation Center Basel study provides compelling evidence that rAAV capsids (AAV2 and AAV8) activate the complement cascade in human blood, primarily through the classical pathway dependent on pre-existing anti-AAV antibodies (Abs), but also revealing a significant contribution from the alternative pathway.

  1. Antibody-Dependent Classical Pathway: Using a whole blood assay (WBA) measuring C3a-des-Arg and C5a-des-Arg (stable cleavage products), the researchers demonstrated dose-dependent complement activation by both AAV2 and AAV8 empty capsids. This activation was significantly more pronounced in donors seropositive for anti-AAV IgG/IgM. Crucially, adding intravenous immunoglobulin (IVIg) containing anti-AAV2 IgGs to seronegative donor blood recapitulated this activation, proving the sufficiency of anti-capsid Abs in driving the classical pathway. Pathway-specific ELISAs confirmed C3 deposition on AAV2-coated plates under conditions favoring classical/lectin pathway activation, correlating strongly with anti-AAV IgG titers, particularly the IgG1 and IgG3 subclasses known for potent complement activation.
  2. Antibody-Independent Alternative Pathway: A critical finding was the observation of complement activation (elevated C3a-des-Arg) in some seronegative donors lacking detectable anti-AAV IgG/IgM. The pathway-specific ELISAs provided direct evidence: sera from all tested donors, including seronegatives, induced C3 deposition on AAV2-coated plates under conditions selective for the alternative pathway. This demonstrates that AAV capsids themselves can directly trigger the alternative pathway, likely through spontaneous hydrolysis of C3 binding to the capsid surface. This finding resonates with clinical observations of TMA in patients receiving Zolgensma who lacked high-titer pre-existing NAbs, suggesting alternative pathway involvement (8) .
  3. Capsid vs. Genome: Importantly, the study confirmed that complement activation is driven solely by the capsid, as empty capsids were as effective as genome-containing vectors. This implies that empty capsids, common impurities in rAAV preparations, contribute significantly to complement-mediated toxicity risks.

Cellular Players: Unmasking Responders via Transcriptomics

To identify the immune cell types responsible for cytokine production, researchers employed single-cell RNA sequencing (scRNA-seq) on PBMCs from whole blood incubated with rAAV8. This revealed:

  • Myeloid Cells (Monocytes): Were major contributors, upregulating transcripts for key chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CXCL8/IL-8, CXCL10/IP-10) and cytokines (IL-1β, TNF-α). They also showed strong induction of IFN-stimulated genes (ISGs: MX1, ISG15, HERC5, OAS1/2, IFITs), indicating response to type I IFN.
  • NK Cells: Upregulated transcripts for CCL3, CCL4, IFN-γ, TNF-α, and ISGs.
  • T Cells (CD4+ and CD8+): Upregulated CCL3, CCL4, IFN-γ, TNF-α, and ISGs. Crucially, donors with high anti-AAV8 IgG titers showed pronounced transcriptomic changes in T cells, particularly central memory T cells, and strong differential expression of genes involved in NF-κB signaling (early, 4h) and IFN-I responses (later, 24h). This suggests reactivation of pre-existing capsid-specific memory T cells by the vector, amplified by antibodies, aligning with the original hemophilia B trial findings.
  • B Cells: Also showed induction of ISGs.

 
Notably, while ISG induction was strong, IFN-α transcripts were not detected, likely due to the scarcity of pDCs in PBMCs and their potential loss during isolation. This highlights the importance of pDCs as the primary source of IFN-α, sensed via. The transcriptomic data confirms that multiple innate (monocytes, NK cells) and adaptive (T cells, B cells) immune cell types are engaged by rAAV, with pre-existing antibodies significantly amplifying the response, particularly in the T cell compartment.

In Vivo Innate Immune Activation

To extend these findings beyond in vitro assays, the researchers evaluated whether cytokines, chemokines, and interferons detected in human whole blood following rAAV exposure were also released in vivo. Although the in vitro data suggested that AAV8 was less inflammatory than AAV2, AAV8 was selected for the animal study due to its widespread use in systemic gene therapy applications. Mice were administered a high intravenous dose of rAAV8 (4.04 × 1014 vg/kg), and serum samples were collected at baseline and at 2 hours, 24 hours, and 14 days post-injection. Analysis confirmed the induction of multiple cytokines, chemokines, and interferons, providing direct evidence that systemic administration of high-dose rAAV8 elicits measurable innate immune activation in vivo.

Clinical Implications and Future Directions

The findings of the Roche Innovation Center study have profound implications for the development and clinical application of rAAV gene therapies:

  1. Complement activation as a major safety risk: The study solidifies complement activation, via both classical (Ab-dependent) and alternative (Ab-independent) pathways, as a key mechanism underlying SAEs like TMA observed in high-dose systemic trials. This necessitates:
    • Patient screening: Beyond just NAbs, assessing complement activity (e.g., baseline complement levels, genetic variants in complement regulators) might identify high-risk patients.
    • Prophylaxis/mitigation: Complement inhibitors (eculizumab, APL-9, others in development) should be considered for high-risk patients or high-dose regimens. Monitoring complement activation markers (C3a, C5a, sC5b-9) post-infusion is crucial.
    • Vector engineering: Developing capsids with reduced complement activation potential (e.g., mutated surface residues) is a promising strategy.
  2. Pre-existing immunity beyond neutralization: The study demonstrates that pre-existing anti-AAV Abs, even at sub-neutralizing levels, significantly amplify both complement activation and cytokine release. This “sub-neutralizing immunity” can contribute to inflammation and potentially prime adaptive responses, impacting safety and efficacy. Strategies to temporarily modulate humoral immunity (e.g., B cell depletion, plasmapheresis, IgG-cleaving endopeptidases) need refinement to more completely remove Ab effects.
  3. CpG reduction is paramount: The direct correlation between CpG content and IFN-α release underscores the critical importance of genome engineering to minimize CpG motifs, especially within the transgene ORF. This is a well-established strategy to reduce TLR9 activation and subsequent inflammation and adaptive T cell responses. Codon optimization and CpG depletion should be standard practice.
  4. Serotype selection matters: The differential innate response to AAV2 vs. AAV8 highlights that serotype choice impacts immunogenicity. While AAV8 is often preferred for systemic delivery due to lower liver sequestration, its potentially lower innate activation (as seen in vitro) might be advantageous, though this needs careful consideration alongside tropism and efficacy. Engineering capsids to combine desirable tropism with reduced innate immune recognition is key.
  5. Need for predictive assays: The human WBA combined with scRNA-seq emerges as a valuable ex vivo tool to assess the innate immunogenicity potential of rAAV vectors, including the impact of pre-existing immunity and CpG content. Such assays could aid in vector selection and candidate ranking during preclinical development.

Conclusion

The Roche Innovation Center study provides a comprehensive and mechanistically detailed dissection of the innate immune response to rAAV vectors in a human context. It unequivocally demonstrates that complement activation occurs via both antibody-dependent classical and antibody-independent alternative pathways, driven solely by the capsid. It further reveals that pre-existing anti-AAV antibodies act as powerful amplifiers of both complement activation and the release of a broad array of pro-inflammatory cytokines and chemokines from diverse immune cells (monocytes, NK cells, T cells, B cells). Critically, the viral genome, specifically its CpG content, is identified as the key determinant of TLR9-mediated IFN-α release by pDCs. These findings illuminate the complex interplay between vector components (capsid, genome) and host factors (pre-existing immunity, complement genetics) that underlies the innate immune barriers to safe and effective rAAV gene therapy. They provide a strong scientific foundation for developing improved strategies – including complement inhibition, modulation of pre-existing immunity, CpG-depleted genome design, and engineered capsids – to mitigate innate immune activation and unlock the full therapeutic potential of rAAV vectors for a wider range of diseases and patients. Overcoming the innate immune hurdle is essential for the next generation of safer and more effective gene therapies.

References

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Our Recent Publication/Meeting Presentation on Gene Therapy

1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

2. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1.

4. American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

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