BackgroundIntravitreal delivery of adeno-associated virus (AAV) vectors offers a promising, minimally invasive strategy for retinal gene therapy, but remains limited by dose-dependent intraocular inflammation. Corticosteroid therapy, the current standard for managing gene therapy-associated uveitis (GTAU), can be ineffective or contraindicated, highlighting the need for alternative immunomodulatory approaches and deeper understanding of AAV-induced inflammation.MethodsUsing porcine and mice models, we characterized immune responses triggered by AAV2.7m8 vector.ResultsConsistent with previous data for this serotype, AAV-mediated transgene expression localized predominantly to retinal ganglion cells, although photoreceptor, bipolar, amacrine, horizontal, and glia cells were also transduced. Despite prophylactic methylprednisolone, AAV-treated animals developed GTAU, accompanied by increased MCP-1, IP-10, MIP-1α and IL-6 levels in ocular humors, along with microglial activation and peripheral leukocyte infiltration. Transcriptomic analysis revealed upregulation of antiviral interferon responses across all retinal cell populations, together with complement and DNA damage pathways. Accordingly, functional assays confirmed complement C3a accumulation in ocular humors and presence of γH2AX+ DNA damage foci in transduced retinas. Finally, a mechanistic study of intravitreal AAV delivery in mice using integrin-blocking antibodies revealed a role for peripheral leukocytes in mediating ocular inflammation in this species.ConclusionOur work identified new markers of ocular inflammation and potential targets to modulate GTAU.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

AAV gene transfer is a promising treatment for many patients with life-threatening genetic diseases. However, host immune response to the vector poses a significant challenge for the durability and safety of AAV-mediated gene therapy. Here, we characterize the innate immune response to AAV in human whole blood. We identified neutrophils, monocyte-related dendritic cells, and monocytes as the most prevalent cell subsets able to internalize AAV particles, while conventional dendritic cells were the most activated in terms of the CD86 co-stimulatory molecule upregulation. Although low titers (≤1:10) of AAV neutralizing antibodies (NAb) in blood did not have profound effects on the innate immune response to AAV, higher NAb titers (≥1:100) significantly increased pro-inflammatory cytokine/chemokine secretion, vector uptake by antigen presenting cells (APCs) and complement activation. Interestingly, both full and empty viral particles were equally potent in inducing complement activation and cytokine secretion. By using a compstatin-based C3 and C3b inhibitor, APL-9, we demonstrated that complement pathway inhibition lowered CD86 levels on APCs, AAV uptake, and cytokine/chemokine secretion in response to AAV. Together these results suggest that the pre-existing humoral immunity to AAV may contribute to trigger adverse immune responses observed in AAV-based gene therapy, and that blockade of complement pathway may warrant further investigation as a potential strategy for decreasing immunogenicity of AAV-based therapeutics.

Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.

Introduction Several studies have shown that the induction of antibodies by natural exposure to various AAV serotypes can compromise the subsequent use of AAV as a gene therapy vector, limiting patient eligibility for AAV-delivered therapeutics. The implications of pre-existing antibodies to AAV serotypes are very different: Levels of anti-AAV2 or anti-AAV8 neutralizing antibodies (NABs) as low as 5 have been related to a decrease or even total impairment of AAV liver transduction after systemic delivery in humans. However, successful gene transfer has been reported in patients with anti-AAV5 NABs titers up to 340 and in non-human primates with titers up to 1030. Extensive surveys on the prevalence of anti-AAV antibodies in humans have been published. Results from these studies indicate that prevalence varies dependent on serotype, and that a significant proportion of individuals develop humoral immunity against various AAV serotypes early in life, starting around 2 years of age. Furthermore, the prevalence of antibodies to different AAV serotypes has been reported to vary according to geographical location. Study Objective We performed a NABs seroprevalence study in South African hemophilia B patient population (n=44) using a panel of AAV serotypes suitable for liver targeted therapy, to determine the AAV serotypes likely to be of greatest clinical applicability for the South African hemophilia B population. Methods Forty-four hemophilia B patient serum samples were obtained from Hemophilia Comprehensive Care Center in Johannesburg (South Africa). All the patient serum samples were analyzed for the presence of NABs against AAV serotypes 1, 2, 5, 6 and 8 with the use of highly sensitive luciferase-based bioassays. The assays entail incubation of the test serum samples dilution series with an AAV1, 2, 5, 6 or 8-based reporter vector that carries the luciferase gene. This incubation allows neutralizing antibodies in the test serum to bind to the reporter vector particles. These mixtures are subsequently transferred onto Hek293T cells, where reporter vector particles can transduce cells and mediate expression of luciferase. Anti-AAV1, 2, 5, 6 or 8 NABs titers were determined by calculation of the percentage of neutralization for each sample dilution and fitting the neutralization curve with a four-parameter method. Anti-AAV1, 2, 5, 6 or 8 NABs titer (IC50) is the dilution at which antibodies inhibit Hek293T cell transduction with AAV1, 2, 5, 6 or 8-LUC by 50%. The lowest patient serum dilution used in every assay was 8 and samples were considered positive when calculated anti-AAV NAB titer was ≥8. All analytical runs included proper negative and positive controls. Results and Discussion The presence of NABs against the AAV serotypes 1, 2, 5, 6 and 8 was determined in the serum of the hemophilia B patients (Fig. 1). The highest prevalence of NABs was found to be against the AAV2 serotype, 95% (n=42/44) followed by the AAV6 serotype, 82% (n=36/44), and the AAV1 serotype 77% (n=34/44). The prevalence of NABs against AAV5 and AAV8 was lower with 66% (n=29/44) for AAV5 and 64% (n=28/44) for AAV8. The serum samples positive for anti-AAV2 NABs had a high occurrence of titers above 1030 (39%) in comparison to anti-AAV1 NABs (20%), anti-AAV5 NABs (5%) or anti-AAV8 NABs (7%).The occurrence of samples with low titers (ranging from titer of 8 to titer of 50) was the highest for anti-AAV8 NABs (32%) and for anti-AAV5 NABs (27%), followed by anti-AAV2 (18%) and anti-AAV1 (5%) (Fig.1). Currently, an anti-AAV NABs titer of 5 is used as an exclusion criteria in most of the systemic AAV-based gene therapies. When applying a similar cut-off of 8, 23% of the analyzed patients could be treated with AAV1, 5% with AAV2, 34% with AAV5, 18% with AAV6 and 36% with AAV8-based therapeutics (Fig.2). However, we have previously reported that AAV5-neutralizing antibodies do not impair the efficacy of in vivo transduction of AAV5-based vector up to a measured titer of 340 in humans and 1030 in non-human primates. Therefore, applying the cut-off of 340 or 1030, either 84% or 95% of the South African Hemophilia B patients could benefit from treatment with AAV5-based gene therapy (Fig.2). Disclosures Majowicz: uniQure N.V.: Employment. van Waes:uniQure N.V.: Employment. Timmer:uniQure N.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Mahlangu:Takeda: Consultancy, Honoraria, Speakers Bureau; LFB: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Baxalta: Consultancy, Research Funding, Speakers Bureau; Freeline Therapeutics: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; Spark: Consultancy, Speakers Bureau; Chugai: Consultancy; Biomarin: Research Funding; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Novartis: Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; uniQure: Research Funding; World Federation of Haemophilia: Speakers Bureau. Ferreira:uniQure N.V.: Employment.