A Review of the Challenge of Pre-Existing Humoral Immunity in Adeno-Associated Virus Gene Therapy and Potential Solutions

Abstract

Adeno-associated virus (AAV) vectors are promising platforms used in a growing number of approved gene therapy (GT) products across diverse therapeutic areas. Due to the potential safety and efficacy concerns associated with AAV-based immune responses, patients with pre-existing anti-AAV antibodies (Abs) are routinely excluded from GT trials to prevent treatment of patients who are hypothesized to have potentially higher risk and/or little or no benefit. However, the exclusion of seropositive patients without prior GT exposure is not based on data-driven Ab titer cut-offs, and diagnostics to identify levels of pre-existing immunity have not been standardized, precluding data generation that would substantiate or reject this hypothesis. There are also significant gaps in clinical data comparing the impact of pre-existing immunity with treatment-induced immune responses for a variety of disease states. This review aims to address these gaps by examining the impact of pre-existing anti-AAV Abs on the safety and efficacy of approved and failed GT products and ongoing clinical trials. Together, these data suggest that pre-existing immunity may not be the principal determinant of GT success. Therefore, to expand the number of patients eligible for treatment, novel AAV GTs should be optimized to mitigate against the effects of anti-AAV Abs and avoid the need for exclusionary screening; in turn, including both seronegative and seropositive patients in clinical trials will enable characterization of the clinical relevance of anti-AAV Abs. Strategies to prevent an undesirable immune response to GT, including immunosuppressive regimens and modifications to GT manufacturing, design and delivery, are presented for consideration in future GT trials.

Keywords

pre-existing immunity neutralizing antibodies adeno-associated virus gene therapy

INTRODUCTION

Gene therapy (GT) involves the manipulation of gene expression within target cells using a variety of nonviral or viral vectors, offering the potential to achieve therapeutic disease modification.¹ Over the past decade, adeno-associated virus (AAV) vectors have emerged as the leading platform for GT interventions, particularly for monogenic disorders including hemophilia, various neurological diseases, distinct forms of muscular dystrophy, and inherited retinal degenerations.² Advances in these therapy areas have catalyzed a paradigm shift in the field, prompting the application of AAV-based vectors toward more prevalent and genetically complex conditions. These expanded indications now encompass refractory neurodegenerative disorders such as Parkinson’s disease, genetic forms of cardiomyopathy, and various malignancies, reflecting the growing versatility and therapeutic potential of AAV vectors in addressing both monogenic and multifactorial diseases.³ However, challenges remain in AAV GT development, notably risk of adverse events or lack of efficacy due to an undesirable immune response. An important open question is the impact of naturally occurring, pre-existing immunity to the AAV capsid on the immune response to GT at first administration.

At least 12 wildtype AAV (wtAAV) serotypes of human and non-human primate (NHP) origin have documented tissue tropism in humans (AAV1–9, rh8 rh10, rh74),¹ and over 1000 wtAAV variants have been identified from Adenovirus preparations, human/NHP tissues and other mammalian/nonmammalian species.¹ Pre-existing immunity to the AAV capsid can arise due to previous exposure to natural AAV infection, which stimulates host anti-viral immune responses to produce anti-AAV antibodies (Abs).^4,5 Due to exposure to wtAAV, a relatively large proportion of humans carry circulating Abs directed against the AAV capsid.⁶ Globally, seroprevalence rates are highest for anti-AAV2 Abs, ranging from 30% to 60% of the population depending on geographic location, age, and ethnicity.^1,7 In preclinical monkey studies, the lowest seroprevalence was for AAV5, and highest was against AAV8 and AAV9, depending on origin.⁸ Moreover, there is significant cross-reactivity of serotype-specific Abs due to a high degree of AAV capsid sequence homology.⁹ For example, studies in NHPs have also shown infection with AAV induces broadly cross-reactive Ab response to multiple AAV serotypes, and a Phase 1/2 study in males with hemophilia A reported detectable cross-reactivity to other AAV serotypes after treatment with an AAV5-based GT, but this response was not associated with any safety signals.¹⁰

Abs against the AAV capsid can be classified as neutralizing (NAbs) and non-neutralizing (nNAbs) depending on their ability to block virus infectivity.^4,6 Pre-existing anti-AAV NAbs generally bind to the capsid and inhibit vector transduction by blocking AAV attachment to target cell receptors or intracellular trafficking.^4,9 While non-neutralizing Abs do not directly block vector transduction, they can indirectly reduce GT efficacy through vector clearance via opsonization or complement activation, processes that are not exclusive to non-neutralizing Abs, as neutralizing Abs can also mediate Fc-dependent clearance mechanisms.^4,9 In laboratory settings, current assay approaches to detect anti-AAV Abs are limited to identifying anti-AAV NAb or total antibody (TAb) titers,¹¹ therefore these Ab categories will be the focus of discussion in the rest of this review.

The interplay between pre-existing anti-AAV Abs and immune pathways may compromise the therapeutic benefit of AAV-mediated GT and increase the risk of immune-related adverse events. These deleterious effects are mediated via diverse immunological mechanisms, including immune complex formation, complement activation, and enhanced APC-mediated antigen presentation.⁹ For example, binding of anti-AAV Abs to AAV vectors facilitates their sequestration in secondary lymphoid organs and promotes uptake by antigen-presenting cells, a process mediated through AAV-specific B-cell receptors. This interaction alters AAV biodistribution and accelerates vector clearance from the bloodstream. As a consequence, the efficiency of vector delivery to target tissues is diminished, resulting in suboptimal transduction of intended cell populations and potentially provoking localized or systemic inflammatory responses.^9,12

Furthermore, regardless of previous AAV exposure, GT vector administration can trigger nonspecific, early innate immune responses and the development of treatment-induced anti-AAV Abs within days or weeks following GT administration, with potentially serious implications for safety and efficacy.¹³ Treatment-induced anti-AAV Abs can be triggered by the AAV GT capsid itself or the transgenic protein, which may be recognized as a novel antigen in patients lacking native gene expression.^4,5,14,15 Although lack of immune tolerance is more likely if the therapeutic gene is absent, an undesirable immune response could also potentially occur due to transgene modifications or overexpression.¹⁵ Production of treatment-induced anti-AAV Abs following vector dosing can limit the ability of vector readministration, which may be required if treatment efficacy begins to diminish.⁴

The extent to which AAV GT safety and efficacy is driven by pre-existing immunity versus the post-treatment immune response is not fully understood, and no consistent relationship between pre-existing anti-AAV NAb titers and vector transduction levels has been established to date.⁵ Despite this, patients are routinely screened for pre-existing anti-AAV Abs ahead of administration of approved AAV GTs.² As a result, a significant proportion of patients are unable to access GT due to seropositivity;¹³ thus, strategies are needed to circumvent pre-existing anti-AAV immunity to potentially expand the GT-eligible patient population.⁵ New perspectives, technical innovations and novel approaches are reinforcing the hope that historic setbacks to AAV GT development and delivery will be overcome, allowing evasion of GT-targeted immune responses to successfully treat as many patients as possible. In this review, we examine the major pitfalls and failures in AAV GT attributed to undesirable immune responses, highlight the most promising novel AAV GT candidates, discuss strategies to overcome pre-existing anti-AAV immunity, and characterize their uncharted clinical relevance.

Search methodology

We conducted a structured literature search within PubMed for the following terms ([Neutralizing OR NAb OR NAbs] AND [Safety OR Serious Adverse Event (SAE) OR Adverse] AND [AAV]) to identify relevant papers regarding safety issues in subjects with various Ab levels when receiving GT products. A total of 187 publications were returned in the search dated up to February 2026. These results were systematically reviewed by the authors using predefined inclusion and exclusion criteria focused on clinical relevance to develop the data tables and comparative analyses. Inclusion criteria were study quality, clinical data (all phases), treatment with an AAV GT, and availability of efficacy or safety outcomes relating to pre-existing humoral immunity. Exclusion criteria were observational studies or GT techniques, manufacturing, design, and review articles. To capture information for Figure 1, a separate search was performed for reported cases of thrombotic microangiopathy (TMA), including press releases and congress materials. We also incorporated selected supplementary references beyond the primary search results to provide necessary background context including relevant preclinical findings.

Figure 1.

Summary of clinical experience with TMA. AAV, adeno-associated virus; aHUS, atypical hemolytic uremic syndrome; DMD, Duchenne muscular dystrophy; GLA, galactosidase alpha; LAMP2B, lysosome-associated membrane protein 2B; MMA, methylmalonic acidemia; MMUT, methylmalonyl coenzyme A mutase; pts, patients; SMA, spinal muscular atrophy; rAAV, recombinant adeno-associated virus; SMN, survival motor neuron; TMA, thrombotic microangiopathy; vg, vector genomes; yrs, years.¹ (Chand et al 2021a, Chand et al 2021b, Guillou et al 2022, Novartis Gene Therapies Inc 2024, Silver et al 2024)^{22,28,48,64,102}^,2 (Greenberg et al 2021, Rocket Pharmaceuticals 2021, Silver et al 2024)^101,102,128^,3 (4D Molecular Therapeutics 2023a, 4D Molecular Therapeutics 2023b, Silver et al 2024)^102,129,130^,4 (NCT03362502)^46,131^,5 (Mendell et al 2021, Silver et al 2024)^3,102^,6 (Solid Biosciences press release February 18, 2025)¹⁰⁴^,⁷ (Global Genes 2022).¹⁰³

SECTION 1: OVERVIEW OF INNATE AND ADAPTIVE IMMUNE RESPONSE TO GT

Collectively, the adaptive and innate immune systems elicit a rapid and robust immune response to the AAV capsid.^4,10

Early-stage response

Activation of the innate immune system can occur via toll-like receptor (TLR)-mediated recognition of pathogen-associated molecular patterns on/in the AAV vector. Activation occurs either via TLR2 binding of capsid glycoproteins on AAV cell surface or TLR9 binding of viral material exposed by degradation in endosomes. TLR binding recruits MyD88, activating NF-kB signaling and induction of pro-inflammatory cytokines and interferons (IFNs).¹⁶ It includes activation of the complement system, as well as macrophage and neutrophil activity. Zaiss et al demonstrated that AAVs interacted with macrophages, stimulating cellular activation and the expression of genes related to inflammatory pathways.¹⁷

Later-stage response

The innate immune system has no immunological memory component, and is not primed to produce rapid Ab responses.⁴ In contrast, long-lasting immunological memory is mediated by persisting T cells (CD4+ and CD8+) and memory B (CD19+) cells and plasma cells, priming of which can elicit a faster and stronger response resulting in efficient clearance of antigens with subsequent exposures (e.g., with AAV GT).⁴ T-cell responses directed toward AAV capsids are dose-dependent, with vector-specific T-cell responses increasing with vector dose¹⁸ and higher vector doses associated with faster generation of detectable immune responses.¹⁹ Similar dose-dependent responses have been observed for AAV-specific Ab generation following AAV administration.²⁰

Complement activation serves as a bridge between both pre-existing and treatment-induced innate and adaptive immune responses to AAV GT.²¹ Anti-AAV Abs can form immune complexes with circulating vectors, which can activate the complement system via the classical pathway. AAV capsid-triggered activation of the alternative pathway has also been observed in the absence of anti-AAV Abs, with some studies suggesting that either pathway can be activated within a few days post-AAV administration.^9,21–23 Both pathways converge at C3 convertases leading to the opsonization and the potential removal of AAV vectors by phagocytosis.¹⁶ Mice lacking C3 convertases, have delayed anti-AAV Ab development and significantly lowered NAb titers, highlighting the important role of the complement system in mediating the immune response to AAV.¹⁷

GT and adverse immune responses

Activation of both the classical and alternative complement pathways following high-dose AAV GT has been implicated in hepatotoxicity, neurotoxicity, and cell-mediated adverse immune responses, as well as severe and life-threatening inflammatory responses including complement-related thrombocytopenia, TMA, and atypical hemolytic uremic syndrome (HUS) leading to organ failure, even in the absence of pre-existing immunity.^15,21,22 However, a key advantage of AAV vectors is that they display lower immunogenicity compared with other viral vectors (e.g., adenovirus); due to their smaller size and simplicity, they present limited viral epitopes available for immune recognition.^16,24 Consequently, AAVs may activate complement pathways to a lesser extent compared with other viral vectors (e.g., adenovirus).

Antibody screening in AAV GT

Historically, where pre-screening is indicated in AAV GT clinical trials, either TAb or NAb assays have been implemented,²⁵ to exclude subjects with titers above a predefined threshold. One meta-analysis reported that 45% of AAV clinical trials excluded patients with pre-existing anti-AAV NAbs, although exclusion rates varied significantly by therapeutic area.² See Table 1 for a list of approved products and related anti-AAV Ab testing requirements. A variety of sponsor-specific assays have been used for detecting pre-existing anti-AAV Abs across different clinical trials. Not all anti-AAV assays used in clinical trials are validated for clinical use nor are these always required.^{32,34,36,38,40} Diverse approaches to cut-off determination have been adopted, with thresholds generally established based on technical or statistical assays, rather than by clinical relevance.¹³ Although researchers have questioned whether TAb or NAb assays are more informative, TAb assays may be more suitable for patient prescreening and selection due to greater ease of use, and lower cost of validation and deployment.²⁵

Table 1.

Characteristics of Approved AAV GTs

Product	Vector	Primary receptor for cellular attachment/Tissue tropism¹	Disease	Dose; route of admin	Key clinical observations	SPC approach to anti-AAV NAbs		Immune suppression protocol
Product	Vector	Primary receptor for cellular attachment/Tissue tropism¹	Disease	Dose; route of admin	Key clinical observations	Screening required?	Assay	Required?
Alipogene tiparvovec (GLYBERA) (UniQure)²⁶ EMA approval only; UniQure did not apply for market reauthorization due to lack of demand, authorization withdrawn in 2017	AAV1	N-linked sialic acid Skeletal muscle, CNS, airway, retina, and pancreas	Familial lipoprotein lipase deficiency	1E12 gc/kg; IM injection	Clinical improvements in plasma triglyceride levels and reduction in incidence of pancreatitis²⁷	✗	Not required	✓	Required prior to injection Initiate three days prior to injection, continue for 12 weeks; recommend ciclosporin (3 mg/kg/day) and mycophenolate mofetil (2 × 1 g/day) Administer IV bolus of methylprednisolone (1 mg/kg) 30 min prior to injection
Voretigene neparvovec (LUXTURNA) (Spark)²⁸	AAV2	Heparan sulfate proteoglycan Retina and CNS	Retinal dystrophy	1.5E11 vg/eye; subretinal injection	Restoration of RPE65 enzymatic activity and clinically meaningful improvements in ability to navigate independently in low-to-moderate light conditions²⁹	✗	Not required	✓	Required prior to injection Initiate three days prior to injection, continue for 7 days before tapering; recommend oral prednisolone (1 mg/kg/day) or equivalent
Eladocagene exuparvovec-tneq (KEBILIDI) (PTC Therapeutics)³⁰	AAV2	Heparan sulfate proteoglycan Retina and CNS	Aromatic L-amino acid decarboxylase deficiency	1.8E11 vg; intraputaminal infusion	Improvement in motor and cognitive function associated with increased levels of dopamine in the brain at 5-years³¹	✗	Not required	✗	Not required
Valoctocogene roxaparvovec (ROCTAVIAN) (BioMarin)³²	AAV5	N-linked sialic acid Retina, CNS, kidney, pancreas, and liver	Hemophilia A	6E13 vg/kg; IV infusion	Significant increase in factor VIII activity and reduced bleeding rate over a period of up to 2 years³³	✓	Anti-AAV5 Ab negative; FDA-approved companion diagnostic	✗	Consider post-infusion if indicated Consider corticosteroid treatment for ALT elevation
Etranacogene dezaparvovec (HEMGENIX) (CSL Behring)³⁴	AAV5	N-linked sialic acid Retina, CNS, kidney, pancreas, and liver	Hemophilia B	2E13 gc/kg; IV infusion	Favorable safety and efficacy profile, with sustained increase in factor IX activity and durable hemostatic improvements regardless of pre-existing AAV5 NAb status up to titer of 700³⁵	✗	Not required	✗	Consider post-infusion if indicated Consider corticosteroid treatment for transaminase elevation
Onasemnogene abeparvovec (ZOLGENSMA) (Novartis)³⁶	AAV9	Galactose Heart and CNS	Spinal muscular atrophy	1.1E14 vg/kg; IV infusion	Extended survival, improved motor function and reduced need for permanent ventilation³⁷	✓	Anti-AAV9 Ab titers ≤1:50; enzyme-linked immunosorbent assay (ELISA)	✓	Required prior to infusion Initiate one day prior to infusion, continue for 30 days; recommend oral prednisolone (1 mg/kg/day) or equivalent
Fidanacogene elaparvovec (BEQVEZ) (Pfizer)³⁸	AAVrh74	Unknown Skeletal muscle	Hemophilia B	5E11 vg/kg; IV infusion	Stable factor IX expression and reduced annualized bleeding rate compared with factor IX prophylaxis therapy³⁹	✓	Anti-AAVRh74var Ab negative; FDA-approved companion diagnostic	✗	Consider post-infusion if indicated Consider corticosteroid treatment for transaminase elevation or a decline in factor IX activity
Delandistrogene moxeparvovec (ELEVIDYS) (Sarepta)⁴⁰	AAVrh74	Unknown Skeletal muscle	Duchenne muscular dystrophy	1.33E14 vg/kg (<70 kg), 9.31E15 vk/kg (>=70 kg); IV infusion	Robust dystrophin expression and improvement and durable, sustained stabilization of motor function over 2 years⁴¹	✓	Anti-AAVrh74 total binding Ab titers <1:400; total binding Ab enzyme-linked immunosorbent assay (ELISA)	✓	Required prior to infusion Initiate glucocorticoid (prednisone equivalent) one day prior to infusion, continue for a minimum of 60 days

Green = Anti-AAV NAb screening or immune suppression protocol not required; Yellow = Immune suppression protocols may be considered under specific circumstances; Red = Anti-AAV NAb screening or immune suppression protocol required.

AAV, adeno-associated virus; Ab, antibody; ALT, alanine transaminase; CNS, central nervous system; ELISA, enzyme-linked immunosorbent assay; EMA, European Medicines Agency, FDA, Food and Drug Administration; IM, intramuscular; IV, intravenous; NAbs, neutralizing antibodies; SPC, statistical process control.

Between 30% and 90% of the population have pre-existing immunity to naturally occurring AAVs.^9,42 Consequently, requiring a negative anti-AAV Ab result for trial enrollment may exclude a substantial number of patients from AAV GT trials. However, interpretation of such screening is complicated by the lack of assay standardization, variability in thresholds for positivity, and known technical limitations of NAb/TAb assays.^4,5 These issues can lead to both false positives (inappropriately excluding patients) and false negatives (identifying AAV-exposed patients as seronegative).

Beyond humoral immunity, pre-existing cellular responses to AAV may also affect GT outcomes. Following natural AAV infection, memory B and T cell responses may be reactivated upon subsequent AAV vector exposure. Indeed, Veron et al. found that 36% of healthy donors with pre-existing AAV1-specific cellular responses—identified via IFN-γ enzyme-linked immunospot (ELISPOT) or intracellular staining—were seronegative for anti-AAV1 Abs, highlighting the need to consider cellular immunity alongside antibody screening.⁴³ While cellular immunity is an important component of the immune response, it represents a broad and complex research area. A detailed evaluation of how cellular responses to AAV may affect GT outcomes is beyond the scope of this review, which focuses on pre-existing humoral immunity.

Beyond the above-mentioned issues related to inconsistent and false measurement, these diagnostic tests are a costly addition to AAV GT treatment¹³ and require early consideration for codevelopment. Given that it is unknown whether and to what degree pre-existing anti-AAV Abs impact GT success, it is also not clear whether the assays would need to be considered as companion diagnostics (required for GT) or as complementary diagnostics (as informative). Moreover, patients may still derive benefit despite Ab positivity, raising critical questions about the appropriateness of exclusionary screening for pre-existing immunity.

SECTION 2: CURRENT LANDSCAPE OF AAV GTS

As of 2025, eight in vivo AAVs have been approved by the U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA); however, EMA approval for alipogene tiparvovec (Glybera) was not renewed and Glybera was withdrawn from the market in 2017 (Table 1).⁴⁴ These targeted GTs leverage diverse AAV serotypes with distinct tissue tropism. The difference between serotypes is primarily within the amino acid sequence of their capsid proteins and this difference, coupled with the presence of receptors or co-receptors on the host cell, determines cell/tissue tropism and drives vector selection.⁵ The clinically approved AAV vectors can be grouped into three broad categories based on receptor usage: heparan sulfate proteoglycan (HSP) for AAV2, sialic acid for AAV1 and AAV5, and galactose for AAV9.⁴⁵ The wide range of tissue tropisms facilitates the application of these AAV vectors to a wide variety of therapeutic indications, including blood disorders, muscular dystrophy, inherited forms of blindness and metabolic diseases (Table 1).¹³ The most commonly used and best characterized AAV is AAV2, with documented tropism for renal tissue, hepatocytes, retina, non-mitotic cells of the central nervous system (CNS), and skeletal muscles; beyond being the most studied AAV, the innovation of mosaicism and cross-packaging has enabled broader tissue tropism of AAV2 with a wider transduction spectrum.⁴⁶

Currently, voretigene neparvovec (Luxturna) for inherited retinal dystrophies, eladocagene exuparvovec-tneq (Kebilidi) for inherited movement disorder, etranacogene dezaparvovec (Hemgenix) for hemophilia B, and alipogene tiparvovec (Glybera) for familial lipoprotein lipase deficiency (EMA approval withdrawn in 2017) do not require anti-AAV NAb screening prior to treatment initiation based on local administration to immuneprivileged sites (by subretinal injection or intraputaminal infusion) or broad recruitment into Phase 3 trials, respectively.^28,30,35,44 All other approved AAV GTs—which are administered systemically by intravenous (IV) infusion—currently require patients to be negative for serotype-specific anti-AAV Abs or have titers below an indicated threshold (Table 1).

In a Phase 3 open-label trial for the hemophilia B AAV GT fidanacogene elaparvovec (Beqvez), approximately 60% of patients were considered ineligible for enrollment due to the presence of anti-AAV NAbs,³⁹ emphasizing the high exclusion rate for patients based on this parameter. Conversely, Hemgenix, administered via IV infusion, demonstrated favorable safety and efficacy despite pre-existing anti-AAV5 NAbs.^35,47 Together, these data raise questions about the appropriateness of exclusionary screening for pre-existing immunity. Equivalent studies have not been undertaken for AAV GT products for the treatment of hemophilia A, and patients with pre-existing AAV Abs continue to be ineligible for these AAV GT studies.⁴⁸

To date, all approved AAV GTs, except for Kebilidi, require use of an immunosuppression protocol, either prior to vector administration or in response to a clinical manifestation that may be linked to an immune response (e.g., transaminitis) (Table 1). Some immunosuppressive drugs target naïve adaptive immune system responses, while others have greater impacts on immunological memory,¹⁵ therefore understanding the implications of pre- versus post-treatment immune responses is in AAV GT safety and efficacy is important for establishing appropriate immunosuppressive protocols.

SECTION 3: ANTI-AAV ABS AND SAFETY IN APPROVED AND FAILED AAV GT PRODUCTS

The magnitude of the immune response to an AAV GT product is unpredictable due to individual patient characteristics, with AAV serotype, delivery method and dose playing an important role. Patient age can also impact the likelihood of adverse immune reactions following AAV GT; younger patients have less serotype exposure and time to develop pre-existing immunity, while elderly patients may be less reactive to AAV vectors due to age-related decline in their innate and adaptive immune systems.¹⁵ In addition to age, both preclinical and clinical studies have shown that sex can impact immunological response to AAV vectors.⁴⁹ Pre-clinically, female mice demonstrated increased NAb response to AAV8 GT, resulting in reduced treatment efficacy compared with their male counterparts, which may be attributable to the interplay between sex specific hormone-mediated signaling pathways and immune-modulatory receptor expression.⁵⁰ In a U.S.-based analysis, females were reported to have higher pre-existing anti-AAV Ab levels, including NAb activities against a selection of AAV serotypes, and AAV dose-dependent differences in inflammatory response between males and females suggest that enhanced immune response in females could lead to neutralization and faster clearance of AAV vectors with potential to impact the efficacy of GT.⁵¹ These initial observations underscore the necessity for prospective clinical investigations to systematically evaluate the influence of clinical and demographic variables, including sex, on pre-existing or treatment-emergent anti-AAV Ab responses to help inform patient selection criteria in future studies.

Complement-related thrombocytopenia, TMA and atypical HUS reactions can be triggered by AAV GT and carry a high risk for fatality and kidney failure if not detected and treated early; therefore, the occurrence and management of TMA/HUS is an important issue for AAV GT safety (Fig. 1).^4,21 As such, TMA has been added to the safety label of some GTs, including onasemnogene abeparvovec (Zolgensma), where immunosuppression, additional monitoring, and avoidance of immune activators concurrent with treatment are advised.³⁶ The majority of TMA/HUS have occurred in pediatric patients; this is not unexpected given that the diseases targeted by these GTs predominately impact pediatric patients. We estimate TMA has occurred in ∼19 patients after various AAV GT. Extensive searches of AE reporting underpin our estimate; other sources have reported 13 cases.²³

Studies have suggested that treatment-induced anti-AAV Abs may play an important role in triggering these complement-mediated AEs. For example, it has been shown that anti-AAV Abs rapidly increased following AAV9 infusion, coinciding with activation of the classical and alternative complement pathways, in addition to clinically significant thrombocytopenia.^52,53 However, confirmation of low or no pre-existing immunity using companion diagnostic tests may not translate to clinical efficacy and safety in AAV GT clinical trials¹³ (Table 2).

Table 2.

Impact of Pre-existing Immunity to the AAV Capsid on Safety and Efficacy in Clinical Studies

AAV GT	Dose	ROA	Pre-existing anti-AAV NAb or TAb Titers	Safety observations in subjects with pre-existing anti-AAV Abs	Efficacy observations in subjects with pre-existing anti-AAV Abs	Immune response potentially triggering the adverse effects
AAV1-AAT for Alpha-1 antitrypsin (AAT) deﬁciency (n = 9, including 4 previously treated with AAV2)⁵⁴	6.9E12, 2.2E13, 6E13 vg/kg	Intramuscular	NAb titers <20 to 640	No clinically important changes in hematology, serum chemistry, or urinalysis parameters	—	NA; pre-existing Abs not associated with any adverse observations
AAV2-REP1 for choroideremia⁵⁵	1E11 vg	Subretinal injection	NAb titers up to 225	Potential association with higher % of TEAEs related to ocular inflammation or reduced visual acuity	Three patients with pre-existing NAbs showed a decrease in visual acuity over 12 months; no reasons were identified for this	NA; pre-existing Abs not associated with the occurrence of retinal inflammation TEAEs
AAV2-hFIX (n = 8) for hemophilia B⁵⁶	2E11–1.8E12 vg/kg	Intramuscular	NAb titers up to 1000	No significant systemic or local toxicities	No impact on gene transfer or transgene expression 2–10 months post vector administration	NA; pre-existing Abs not associated with any adverse observations
AVV2-hFIX (n = 7) for hemophilia B⁵⁶	8E10, 4E11, 2E12 vg/kg	Hepatic artery infusion	NAb titers of 2 & 17	NAb titer of 17: transaminitis	NAb titer of 17: reduced FIX expression	Cytotoxic T cell activity on transduced cells Immune complex formation with AAV capsid
AAV5-wthFIX (AMT-060) (n = 10) for hemophilia B^57–59	5E12, 2E13 gc/kg	Intravenous	NAb titers up to 340 in 3/10 patients TAb IgG titers of 87 & 256 in 2/10 patients	No ALT elevation or capsid-specific T cell activation	No loss of transgene activity; NAbs titer of 340 associated with highest FIX expression after low dose administration	NA; pre-existing Abs and transient T cell response to AAV capsid not associated with any adverse observations
AAV5-hFIXco-Padua (AMT-061, HEMGENIX^®) for hemophilia B^34,59	2E13 vg/kg	Intravenous infusion	NAb titers up to 678; NAb titer of 3212	Asymptomatic mild elevation of ALT/AST	Nab titers up to 678: hemostatic protection but lower mean FIX expression NAb titer of 3212: no hemostatic protection, no FIX expression	Injury of transduced hepatocytes Immune complex formation with AAV capsid
AAV9-SMN1 (ZOLGENSMA^®) for spinal muscular atrophy^22,36,60	1.1E14 vg/kg	Intravenous infusion	TAb titer ≤50	Transient (and mostly mild) elevation of ALT, AST, and/or bilirubin concentrations; TMA	—	Increase of treatment-induced anti-AAV9 TAbs coincidental with liver enzyme elevation TMA associated with complement activation (alternate pathway)
sc-AAV2/8-LP1-hFIXco for severe hemophilia B⁶¹	2E11, 6E11, 2E12 vg/kg	Intravenous infusion	TAb titers 1–37 relative units	Not reported	Participants with low pre-existing anti-AAV8 Abs still achieved durable factor IX expression over 13 years	Pre-existing Abs not associated with any adverse observations
sc-rAAV2.5IL-1Ra for knee osteoarthristis⁶²	1E11, 1E12, 1E13 vg	Intra-articular injection into index knee	NAb titers <1:8, one patient 1:512	No serious adverse events related to therapy	Participant with 1:512 NAb titer; vector genomes were not detected and no efficacy was observed, whereas patients with low titers showed sustained IL-1Ra expression.	NAb titers rose after injection in most patients, but no T-cell responses were detected

Green = Positive safety or efficacy outcomes/NAbs not associated with adverse observations; Yellow = Impact of safety or efficacy outcome undetermined; Red = Negative safety or efficacy outcomes/NAbs associated with adverse observations. AAT, alpha-1 antitrypsin; AAV, adeno-associated viral vector; ALT, alanine transaminase; AST, aspartate aminotransferase; FIX, factor IX; gc, genome copies; GT, gene therapy; hFIX, human factor IX; IgG, immunoglobulin G; NA, not applicable; NAb, neutralizing antibody; REP1, Rab escort protein 1; ROA, route of administration; SMN1, survival motor neuron 1; TAb, total antibody; TEAE, treatment emergent adverse events; TI, transduction inhibition; TMA, thrombotic microangiopathy; vg, viral genomes; wthFIX, wild-type human factor IX.

Safety concerns, including SAEs and death, have been associated with immune responses after AAV GT administration in subjects who had minimal or no pre-existing anti-AAV Abs (i.e., in trials where patients with non-negligible anti-AAV Ab titers were excluded).⁶³ Nine cases of TMA, including one case of fatality, have been reported in 1,400 patients treated with Zolgensma, and TMA with complement activation has been reported in other AAV9-based gene therapies.⁶⁴ Additionally, dose-limiting toxicities reported in AAV GT-based clinical trials and the postmarketing setting include: hepatoxicity, complement activation resulting in cytopenias and renal toxicity and fatalities due to progression of pre-existing hepatobiliary disease.^3,65 Furthermore, an increase in treatment-induced anti-AAV9 TAbs with Zolgensma was coincidental with elevated liver enzymes;²² we interpret this to suggest that the risk of observing SAEs is more likely associated with treatment-induced anti-AAV Abs and complement-mediated inflammatory reactions than with pre-existing anti-AAV Abs.^66,67 In further support of this conclusion, in a DMD AAV GT clinical study that excluded patients with NAb at screening, participants with complement-mediated TMA had rapid rises in NAbs within 2 weeks post-AAV administration.⁵³ While these findings indicate that TMAs and hepatic adverse effects can occur in individuals without pre-existing AAV Abs (as the trials excluded participants with high Ab titers), it could not be assessed whether the frequency, kinetics, and severity of these AEs may have been altered in patients with versus without pre-existing immunity.

There are, however, some clinical data exploring whether the presence of pre-existing anti-AAV Abs impacts safety outcomes. Clinical trials for AMT-060, the precursor to Hemgenix, demonstrated that vector administration in patients with pre-existing anti-AAV5 NAbs did not result in capsid-specific T cell activation or alanine aminotransferase elevation.⁵⁷ In a Phase 3 trial of Hemgenix, AEs and efficacy were comparable in participants, irrespective of whether they had pre-existing AAV5 NAbs.³⁵ Similarly, intramuscular injection of an AAV vector encoding human factor IX (FIX) did not induce significant systemic or local toxicities, including in patients with high-titer pretreatment NAbs.⁵⁶ Moreover, baseline NAb positivity was unclear in a Phase 2 trial of timrepigene emparvovec in patients with genetically confirmed choroideremia; however, as AAV2 GT was administered via subretinal injection,⁵⁵ the immune-privileged status of this tissue may limit access of pre-existing and treatment-induced AAV Abs in the blood, limiting conclusions on the impact of NAbs.⁶⁸

Preclinical evidence further supports the hypothesis that pre-existing anti-AAV Ab titers do not impact the magnitude of the treatment-induced anti-AAV Ab response. In a preclinical study characterizing anti-AAV2 NAb levels in sheep before and after intravitreal AAV exposure, while the degree of serum neutralization in the pretreatment seronegative group increased significantly postintravitreal injection, there was no significant difference in NAb titers between pretreatment seronegative and seropositive groups 12-weeks after administration. Furthermore, patterns of postoperative serum neutralization were comparable between preoperatively seronegative sheep and those with pre-existing anti-AAV2 Abs. However, only sheep with pre-existing anti-AAV2 Abs presented with signs of postoperative inflammation, suggesting activation of other branches of the immune system, such as the innate immune response.⁶⁹ Further preclinical data from NHPs showed similar kinetics for the onset of anti-AAV5 Ab response following IV administration of valoctocogene roxaparvovec (Roctavian) regardless of pre-existing Ab status. All animals developed anti-AAV5 immune responses following AAV vector administration, as demonstrated by TAb assay, which remained until at least the end of the study period at day 56 without any effect of pre-existing immunity.⁷⁰ Further clinical data are required to solidify the relationship between pre- and post-treatment Ab titers in patients and to clarify whether the magnitude and kinetics of Ab responses may differ following local administration (e.g., intravitreal) compared with IV administration.

Comparing the management of TMA cases across multiple AAV GT trials, certain themes are observed, as summarized in Figure 1. The timing suggests a potential hypothesis that the anti-AAV Abs produced after AAV dosing are the main trigger of complement activation, as TMA/HUS after GTs has been observed between 3 days and 2 months after dosing. This variance in the timing of the development of an immune reaction suggests that the response is not primarily dependent on pre-existing Abs, since a more consistently rapid response may be expected in this case. In keeping with this hypothesis, anti-AAV Abs have been reported to be generated within days after administration of AAV GT and remain elevated for months–years¹⁰ and it has been demonstrated that intact AAV5 capsids can be detected in plasma for at least 1 or 2 weeks after dosing (at 4E13 and 6E13 vg/kg).⁶⁶ The capsids may persist even longer due to potential underestimation of capsid levels. Thus, treatment-induced anti-AAV Abs can arise in a time window in which AAV capsids may still be present within the circulation, particularly if administered systemically at high doses.

The large immune complexes hypothesized to trigger SAEs, including TMA, require a specific antigen to Ab ratio;⁷¹ the probability that this ratio is reached with pre-existing Ab titers is low and it is more likely achieved following the induction of treatment-induced anti-AAV Abs. However, an unanswered question is whether the treatment-induced anti-AAV Ab response has faster kinetics or a greater magnitude among individuals with versus without pre-existing anti-AAV Abs, and what the clinical impact of any difference in response magnitude would be. Hepatotoxicity is another adverse effect that can occur after AAV GT, with one potential mechanism for this being T-cell-mediated cytotoxicity in response to the AAV capsid.⁷² Elevations in liver transaminases have been shown to peak at 8 weeks following AAV administration,³³ and sometimes occur up to 4 months after administration.³⁴ The timing⁷³ of these immune events is indicative of delayed onset of immune reactions more likely associated with treatment-induced Ab responses.

Differences in safety monitoring, AE definitions, and reporting practices across studies weaken cross-trial comparisons and limit a definitive conclusion on the impact of pre-existing immunity on patient safety.

SECTION 4: ANTI-AAV ABS AND EFFECTIVENESS OF GENE TRANSFER IN APPROVED AAV GT PRODUCTS

Reduced efficacy in the presence of anti-AAV NAbs has historically been reported in AAV GT trials using the IV route of administration, thus, pre-existing anti-AAV NAb titers ≤20 and anti-AAV TAb titers <400 are often used as the cut-off for treatment selection, unless the AAV GT was administered to an immune-privileged organ such as the eye and brain.¹³ For example, treatment guidelines for Zolgensma recommend performing baseline testing (and retesting as needed) for the presence of anti-AAV9 TAbs, as the safety and efficacy has not been tested in patients with titers above 1:50.^36,74 Despite this, there are some exceptions where a limited number of trials administering IV or intramuscular doses did not select based on the pre-existing anti-AAV Ab titers (Table 2),^{22,34,36,54–60} setting a precedent for assessing the potential benefit of AAV GT treatment against the potential risks associated with pre-existing anti-AAV Abs.

Initial trials for hemophilia B screened patients for anti-AAV5 NAbs, but retrospective analysis using more sensitive assays revealed that pre-existing anti-AAV5 NAbs titers <340 had no impact on treatment safety/efficacy.⁵⁷ As a result, seropositive patients were included in subsequent trials, demonstrating no correlation between anti-AAV5 NAb titers up to 678 with FIX activity and hemostatic protection at 18 months after treatment. Sufficient and sustained FIX activity to achieve hemostasis was observed in patients with and without anti-AAV NAbs treated with Hemgenix in the Phase 3 HOPE-B trial over 5 years.⁴⁷ Only one patient with an anti-AAV5 NAb titer of 1:3212 showed no FIX expression at all.^34,35,75 Accordingly, Hemgenix is currently approved in the U.S., UK, and European Union for all patients with no requirement for anti-AAV NAb testing during patient selection.³⁴ However, patients are encouraged to enroll in a postapproval monitoring study. Pre-existing anti-AAV5 Abs differ in both prevalence, titer and binding avidity compared with AAV2 or AAV8,^57,76 their impact on efficacy may vary by serotype, titer or antibody activity. Pre-existing circulating anti-AAV Abs are hypothesized to have limited impact on locally administered AAV gene therapies, for example, retinal AAV GT Luxturna does not mandate pre-existing anti-AAV2 Ab testing,²⁸ due to local subretinal administration and observation of improvements in light sensitivity even in patients with pre-existing Abs to the vector and/or transgene.⁷⁷

The GEMINI (NCT03507686) open-label, Phase II study investigating the administration of the retinal AAV GT timrepigene emparvovec in patients with choroideremia did not exclude patients on the basis of pre-existing anti-AAV NAbs; however, data from this study suggested that the occurrence of ocular-inflammation-related treatment-emergent adverse events and reduced visual acuity after treatment may be associated with pre-existing NAb positivity.⁵⁵ This complex interaction needs further investigation to assess or determine causality and may have wider implications for GT trial design and use.

In most cardiac AAV GT trials (e.g., the CUPID trials [NCT00454818; NCT01643330]) patients with pre-existing anti-AAV NAbs have been excluded. Despite this, multiple products have failed to progress through clinical trials; the most common reason for failure is lack of efficacy. However, the Phase 2 GenePHIT clinical trial of AB-1002 GT for the treatment of congestive heart failure (NCT05598333) is proceeding without screening for pre-existing anti-AAV NAbs.^78,79 AB-1002 is currently in development with FDA fast-track designation status; it is delivered locally through a single intracoronary infusion. In the Phase 1 study of AB-1002, patients with anti-AAV NAb titers >1:5 within 6 months prior to administration were excluded; and excellent myocardial transduction was reported alongside clinically meaningful improvements across a range of efficacy parameters alongside a favorable safety profile.⁸⁰

AB-1002 study design features could maintain effectiveness of the treatment without increasing the risks for patients with pre-existing anti-AAV antibodies. For example, AB-1002 has an optimized tropism for myocardium and muscle with a 30-fold higher transduction efficiency in humans when compared with pigs, reducing off-target effects like liver enzyme elevation.⁸⁰ Furthermore, local administration of AB-1002 into the coronary arteries should limit the interaction with pre-existing anti-AAV NAbs, potentially reducing their impact on efficacy. The local administration should also limit the interaction and uptake of vectors by circulating immune cells and reduce immune cell response to vector proteins.⁸¹ This is supported by preclinical data in swine indicating that efficacy with AB-1002 occurred in the presence of pre-existing AAV2i8 NAbs ≤1:16 after intracoronary administration of 1E13 vg; or 1E14 vg per pig.⁸² Similarly, a recent study assessing AAV6 vector delivery in a swine ischemic heart failure model demonstrated improved cardiac gene expression, correlating with virus uptake with no increase in extra-cardiac gene expression, in pigs with relatively-high pre-existing NAb titers.⁸³ Therefore, to optimize treatment access and firmly establish or refute the clinical relevance of Abs, detectable Abs will not preclude study enrollment and their impact on treatment safety and efficacy will be assessed during the GenePHIT study.

SECTION 5: STRATEGIES TO MANAGE THE IMPACT OF ANTI-AAV IMMUNOGENICITY

The implementation of immunosuppression regimens to reduce treatment-induced anti-AAV immunogenicity (alongside certain AAV GT product design features) may enhance safety and efficacy of AAV GTs, irrespective of pre-existing anti-AAV Ab status, and provide an avenue to expand the pool of eligible patients.

Immunosuppression

It is estimated that around 38% of AAV GT studies suggest potential use of immunosuppressants.² A combination of a variety of immunosuppressive drugs is used, including steroids, TLR inhibitors, and cytokine, B-cell and T-cell inhibitors. A systematic review of immunosuppressive protocols used in AAV GT identified corticosteroids (e.g., methylprednisolone and prednisone) as the most used immunosuppressive drug, administered in 95% of studies analyzed.⁸⁴ Corticosteroids work to downregulate TLR expression, suppress proinflammatory cytokines and upregulate anti-inflammatory cytokines, having a broad inhibitory effect on innate and adaptive immune cells.⁸⁵ Prednisolone has been shown to successfully reduce elevated serum aminotransferase levels in patients following administration of Zolgensma, an AAV GT for spinal muscular atrophy, for which it is recommended to be given prophylactically prior to vector administration.³⁷ Despite this, other studies have shown that corticosteroid treatment initiated in response to elevated aminotransferase levels was unable to prevent loss of transgene activity.⁸⁶

TLRs recognize pathogen-associated molecular patterns on viral capsids or viral DNA, triggering a signaling cascade leading to activation of antigen-presenting cells (APCs) and initiation of the innate immune response to AAV vectors.^15,16 Pharmacological inhibition of this innate signaling pathway can be achieved using the antimalarial drug hydroxychloroquine, which acts as a TLR-9 inhibitor. Subretinal injection of hydroxychloroquine with AAV vector has been shown to increase transgene expression, sustained for up to 8 weeks across different AAV vector serotypes.⁸⁷

Some cell-mediated immune responses occurring in AAV GT trials are hypothesized to be caused by complement activation via the alternative pathway triggered by the AAV capsid.²² Complement can opsonize AAV antigens for phagocytosis and co-stimulate B-cell activation and Ab production. Complement-mediated immune responses can be overcome using complement inhibitors such as the PEGylated synthetic cyclic peptide, APL-9, or the monoclonal Ab, eculizumab.⁸⁸ APL-9 has been shown to lower AAV-induced complement activation, and it lowered vector uptake and activation of APCs in blood with high anti-AAV NAb titers.¹² Furthermore, eculizumab was used to treat patients who developed complement TMA following Zolgensma GT administration for spinal muscular atrophy.²²

Several pharmacological options, including rituximab, rapamycin and tacrolimus, are also available to inhibit B- and T-cell activation and/or proliferation to suppress immunological memory responses by the adaptive immune system. A combination of rituximab, tacrolimus and methylprednisolone resulted in no vector-induced adverse events in a recent clinical trial for CNS-directed AAV delivery.⁸⁹ Similarly, rapamycin has been used successfully in combination with rituximab and corticosteroids in clinical trials to prevent treatment-induced anti-AAV Ab formation.⁹⁰ In addition to targeting treatment-induced anti-AAV immunity, there is evidence to suggest effective depletion of pre-existing anti-AAV Abs can also be achieved with broad immune system targeting. For example, a combination of rapamycin and prednisolone was also shown to be effective in reducing anti-AAV9 Abs by up to 93% following 8-weeks of treatment in a mouse model of pre-existing immunity.⁹¹

A key factor influencing the efficacy of immunosuppression is timing of treatment initiation. Early AAV GT trials used a reactive approach responding to evidence of adverse immune responses; however, subsequent trials began to incorporate prophylactic immunosuppression regimens administered before or at the time of AAV dosing and continued after vector administration.⁸⁵ A systematic review of immunosuppressive protocols used in AAV GT identified 74% of clinical trials used prophylactic immunosuppression prior to initiation of therapy; in the remaining 26% of cases, immunosuppressive treatments were used in response to a range of AEs and SAEs associated with the GT product.⁸⁴ The timing of immunosuppression initiation has been shown to modulate the likelihood of developing treatment-induced anti-AAV Abs.⁹² For example, NHPs receiving concomitant administration of immunosuppressive treatment with AAV vector developed immune responses directed toward the transgene protein, whereas animals receiving delayed immunosuppression produced no such immune reaction.⁹² These results support a critical period surrounding vector administration when T-cell-dependent processes occur that can initiate peripheral immune tolerance; early intensive immunosuppression can disrupt these processes and result in failure to develop immune tolerance.⁹² The optimal timing of immunosuppressive regimens remains to be determined and requires further investigation; several factors can influence optimal strategy including immunogenicity of AAV GT product, as well as site of administration (i.e., systemic vs. localized delivery).

The development of adverse effects associated with immunosuppressant use is a major limitation of all immunosuppressive strategies discussed, calling to question the benefit-to-risk ratio of immunosuppression.⁹³ This is highlighted by a study showing that 62% of patients treated with steroids experienced steroid-associated adverse events including hypertension, diabetes and increased infection rates.⁹⁴ Furthermore, it remains to be determined whether one or a combination of some of the above-mentioned strategies will be most beneficial to mitigate the undesirable immune response to AAV GT.⁵ Currently, no consensus or specific guidelines are available for determining the most appropriate combination and timings for immunosuppressive treatment regimens and no studies directly comparing available regimens have been conducted.

Immunomodulation

More recently, alternative immunomodulatory strategies were explored to enable efficient systemic gene transfer in the presence of low-to-moderate pre-existing anti-AAV Abs. Plasmapheresis and immunoadsorption are extracorporeal blood purification techniques that are commonly used to remove immunoglobulins from patient plasma across a variety of diseases.^95,96 Immunoadsorption, which selectively removes immunoglobulins from plasma, has been shown to lower pre-existing anti-AAV Abs in humans.⁹⁵ Plasmapheresis, which nonselectively removes large molecular weight plasma proteins,⁹⁵ lowered pre-existing AAVrh74 Ab levels in NHPs, permitting sustained gene transfer in NHPs with pre-existing anti-AAV Abs.⁹⁶ However, these procedures are costly, and the benefits may be reduced in patients with high pre-existing NAb titers. Similarly, pre-existing anti-AAV Abs, including NAbs, are also being targeted with bacterial-derived immunoglobulin cleavage proteases. Imlifidase (IdeS), a cysteine proteinase derived from Streptococcus pyogenes, successfully degraded anti-AAV8 NAbs and rescued transgene expression and liver transduction in a mouse model of hemophilia B passively immunized with human IV IgG following administration of an AAV8 vector expressing hFIX.⁹⁷ The ability of IdeS to prevent the inhibitory activity of NAbs on AAV-mediated transduction in mice was subsequently confirmed,⁹⁸ and these strategies have now advanced to clinical testing and have demonstrated safety and preliminary efficacy.^99–102 An important advantage of this approach is that it is not capsid-specific, so it can be applied to any AAV serotype. In addition to cleaving free IgG, IdeS treatment can also cleave membrane-associated IgG-type B cell receptors on the surface of CD19⁺ memory B-cells, thus also potentially targeting B-cell-mediated immune responses to the transgene protein or vector.¹⁰³ Advancements to this technology are being made to further target IgM in addition to IgG, using IgM-cleaving enzymes (IceMG), which can also cleave B-cell surface IgM to block complement activation; however, whether this will translate to clinical benefits remains to be studied.¹⁰⁴ FcRN inhibitors have also been shown to be effective in removing pre-existing AAV Abs is the use of in pre-clinical studies and may represent a novel strategy to overcome the presence of pre-existing anti-AAV NAbs in AAV GT approaches.¹⁰⁵ These alternative steroid-sparing treatment options may also be beneficial from a patient acceptability perspective as they avoid some of the toxicity/tolerability concerns associated with steroid use.⁹⁴

Effective strategies for overcoming pre-existing anti-AAV NAbs may likely include a combination of approaches, broadening cell populations targeted; however, the increased likelihood of adverse effects should be considered when using combination strategies. Additionally, a rational approach should be taken on a case-by-case basis as such measures will not always be required. In the future, well-designed AAV GT clinical trials could monitor complement activation to mitigate the risks of SAEs. This approach is being taken in the Phase 2 trial, GenePHIT, for AB-1002, which will only administer immunosuppressants if early signs of complement activation are observed.^78,79 It has also been suggested that testing for activated complement prior to AAV administration could mitigate the risks associated with anti-AAV Abs and inform treatment decisions for seropositive patients.⁷³

Manufacturing, design, dose, and delivery considerations

Product strength can impact AAV immunogenicity,^15,106 with SAEs tending to be correlated with vector dose, increasing in both prevalence and severity with higher doses.¹⁰⁶

The SAEs outlined in Figure 1 were exclusively observed in patients receiving systemic administration of high-dose GT (i.e., at viral loads ≥ 1E13 vg/kg).^60,107–109 Immune-mediated SAEs occurred across nearly all of the agents and dose levels studied. In SGT-001, four patients experienced TMA, despite treating patients in both a low-dose and high-dose arm; patients were nonambulatory adolescents with Duchenne muscular dystrophy (IGNITE DMD clinical trial; NCT03368742).^3,108 The underlying mechanism may reflect the inability of low to moderate doses to induce detectable T cell responses (likely primary) associated with inflammatory/immune-mediated adverse events, although they may stimulate AAV capsid-specific Ab responses,¹⁵ suggesting that these may not be related to AEs). The next-generation SGT-003 (AAV-SLB101) recently reported no instances of TMA or aHUS among the first six patients treated in the INSPIRE DUCHENNE trial (NCT06138639).¹¹⁰ This is a key product design modification that may be responsible for the differences in immune-mediated SAEs observed between SGT-001 and SGT-003. For example, SGT-003 employs a novel AAV-SLB101 capsid with enhanced skeletal and cardiac muscle biodistribution and decreased liver biodistribution compared with the SGT-001 AAV9 vector.^{22,64,110–112}

Conversely, quantitative evidence from NHPs has shown that high levels of pre-existing immunity can influence AAV biodistribution and uptake in target tissues, indicating that higher vector doses may be required to reach therapeutic effect in the presence of high-titer anti-AAV NAbs. However, administration of higher vector doses could, in turn, increase host immune activation.¹¹³

Sandza et al. (2022) demonstrated that AAV capsid levels in patient plasma increase with increased doses and patients receiving high vector doses had intact AAV capsids present in their plasma for two weeks or longer following AAV5-GT administration. These capsids can form large immune complexes with treatment-induced Abs, which rise quickly (within 4 days) following dose administration.⁶⁶ In support of these findings, AAV capsids have also been detected in hepatocytes 21 days post vector administration.¹¹⁴

Dose can be fixed or calculated based on viral vg per kilogram of body weight; however, the actual number of administered AAV particles is higher due to empty viral particles, which lack the vector genome. These have no therapeutic effect but can potentially increase the adverse immunogenic reaction. Studies report high variability in percentage of empty capsids (a range of 10%–98%), making product purity an important and modifiable variable.¹¹⁵ Products with higher full-to-empty/partial ratios may be safer and more effective at a lower dose compared with products with lower ratios.¹¹⁶ Doses containing excess empty capsids have shown dose-dependent toxicity.^89,93 For example, administration of AAV vectors at high doses caused neurotoxicity in the CNS and triggered activation of genes associated with antiviral responses and recruitment of immune cells. Higher doses also led to localized disruption of vascular endothelial tight junctions permeabilizing the blood–brain barrier to Abs in mice.¹¹⁷ The IGNITE clinical trial investigating an AAV GT for DMD (SGT-001) was placed on clinical hold pending changes to the manufacturing process, including better tropism and product purity.¹¹⁸

On the other hand, decoy/empty capsids can potentially be used as a strategy to saturate NAbs and allow effective treatment of seropositive patients, since more full vectors can successfully reach target cells, but this approach carries a higher risk of immunogenicity and increased manufacturing costs.⁹³ Mingozzi et al. (2013) demonstrated that empty AAV8 capsid adsorbed NAbs from passively immunized mice resulting in a dose-dependent drop in anti-AAV8 NAb titer and increased transgene expression from the AAV8-F.IX vector.¹¹⁹ While saturation of NAbs may enhance transduction, empty capsids could also reduce overall transduction of full capsids, unless increased vector doses are administered. One limitation in optimizing dose is in accurately quantifying the full-to-empty capsid ratio. Currently, this approach is not being actively pursued, but technological advances in analytical tools may help to achieve this cost-effectively, quickly and at scale to optimize manufacturing and enhance product purity.¹¹⁵

Utilizing an alternative AAV serotype with low immunogenicity may also help to overcome pre-existing immunity. It has been hypothesized that AAV5 may be less likely to elicit immune response as it is the most structurally divergent wtAAV serotype.¹²⁰ Low levels of natural immunity to AAVs can be further tailored with immune evasion engineering strategies to modify the capsid. Distinct binding sites of the viral capsid are thought to determine its tropism; therefore, efforts are being made around these sites to engineer AAV variants with enhanced transduction capabilities for specific cells or tissues.¹ Enhanced transduction to target tissue will reduce off-target effects through targeted transgene optimization. A study in mice observed that polyploid AAV vectors engineered from co-transfection of different serotypes enhanced transduction efficacy, increased transgene expression and escaped anti-AAV NAb neutralization compared with the parental AAV vector.¹²¹ It remains to be seen whether engineered capsids could contribute to reduced pre-existing immunity.

Adjusting the route of vector administration can also help reduce the impact of NAbs on vector transduction. Direct delivery to target organs through local administration (e.g., intracerebral or intracoronary) should limit the interaction of AAV vectors with circulating immune cells and systemic anti-AAV Abs including NAbs, increasing vector uptake and reducing immune cell response to capsid proteins compared with systemic administration. A caveat to this approach is that localized administration may only help reduce the impact of NAbs on vector transduction if all binding and subsequent transduction of AAV occurs at the first pass through local circulation and encounter with the tissue. Another consideration is that local administration of AAV vectors allows for lower doses (<7.5E5 vg) compared with systemic administration (<1.5E17 vg), which requires higher doses to compensate for systemic dilution; as a result, local administration is less likely to induce anti-AAV immune responses. Undesirable immune reactions can still occur with localized, targeted delivery, but are less likely than with high systemic doses²

Furthermore, AAV vectors administered to immune-privileged sites such as the brain, eye or immunosuppressive microenvironments such as the liver are also less likely to trigger strong responses than vectors given systemically or to the muscle.¹⁵ These factors are dependent on the local anti-AAV Ab concentration and transduction efficiency. It should be noted that the eye and CNS are immune-privileged, but are not immune-free, and administration to these target tissues may not eliminate immune responses.^122,123

While these approaches may help to overcome the challenges associated with pre-existing anti-AAV Abs, vector re-administration may still be prevented by the development of treatment-induced immune responses. This presents further issues for patients who experience initial treatment failure or progressive loss of treatment efficacy over time if safe redosing is not possible.¹²⁴ However, the development of immunomodulation strategies and techniques such as plasmapheresis and B-cell depletion techniques, such as CAR-T cell therapy, offer promising tools that may allow for successful vector redosing in the future. A recent study has shown that CD19 CAR-T cell therapy can markedly reduce high-titer anti-AAV NAbs allowing for successful transgene expression following AAV readministration.¹²⁵

Assay harmonization

Historically, there has been a mismatch between assay prediction and outcomes, and this has led to both over- and underestimation of pre-existing immunity impact. Early studies used assays no longer considered fit for purpose (i.e., not meeting current regulatory standards for accuracy, specificity, and reproducibility). No consistent correlation has been shown between AAV Ab levels and adverse outcomes, and regulatory guidance on assay use differs across approved therapies (Table 1).¹³ On one hand, the neutralizing effect of even low titers has been reported; conversely, the accidental inclusion of patients with pre-existing immunity led to a positive outcome for patients, but this was only uncovered post-trial with an improved assay.^76,126 There is an increasing focus on optimizing assays, but while guidelines exist for assay validation (see https://clsi.org/shop/standards/ila21/ and https://www.fda.gov/media/119788/download), numerous parameters still require industry standardization. The essential parameters for a well-validated and standardized antibody assay include the type, assay cut point, analytical sensitivity, specificity and selectivity, inter- and intra-assay precision, accuracy, susceptibility to endogenous interferents such as hemoglobin and lipids, and exogenous interferents such as medications, stability of samples and reagents used, and robustness of the assay. Guidelines should differentiate between systemic and localized dosing, product design, and delivery and take into account population background immunity levels to the selected capsid.¹³

SECTION 6: ETHICAL AND SAFETY CONSIDERATIONS

Enrollment of seropositive patients in AAV GT trials raises important ethical and safety considerations. Pre-existing immunity can impact trial design by excluding patients with an unmet need or, conversely, necessitating costly and burdensome testing or antibody-clearing strategies.¹³ In addition, there are long-term concerns for treatment durability and redosing. This review acknowledges these issues, but challenges the assumption that seropositive status will always impact efficacy or safety.

Strategies proposed to mitigate immunogenicity, including immunosuppression, immunomodulation, and dose and trial design highlight important safety concerns.^7,15 AEs have been shown to increase in both prevalence and severity with higher doses.¹⁵ While higher vector doses may be required to overcome immune barriers in the presence of neutralizing antibodies, increasing dosing may exacerbate immune activation, creating a trade-off between achieving therapeutic success and increasing immune-mediated toxicity.^7,15 Although, as discussed, it should be recognized that immune-related SAEs have also been reported in individuals with minimal or no detectable pre-existing anti-AAV antibodies, highlighting that immune risk is not confined to seropositive patients.^35,56,57

The durability of treatment success remains uncertain; it is not yet established whether treatment-induced anti-AAV antibody responses differ in kinetics or magnitude for seropositive individuals, or how such differences may influence long-term clinical impact. If therapeutic durability was significantly reduced in seropositive populations (particularly in relation to magnitude of adverse events), the benefit–risk balance of AAV GT would need to be considered regarding the immune and treatment-related risks presented.

Additionally, the safety data presented here are limited by cross-trial comparison where differences in safety monitoring, AE definitions, and reporting practices prevent any strong conclusions on the impact of pre-existing immunity on safety. Theories relating to the tragic deaths occurring during the ASPIRO program (Astellas/Audentes, AT132, AAV8) suggested a combined role of pre-existing immunity with concurrent liver disease contributing to hepatotoxicity cases, particularly as a result of the systemic high doses.¹²⁷ GT trial failures are usually multifactorial, and in the literature, few have been considered a program failure solely based on pre-existing immunity. We suggest there should be more guidance on how to assess the potential impact of pre-existing immunity. Communication of the uncertainty and level of risk to HCPs and patients is essential to ensure ethical access and informed decision-making in GT treatment.^128,129

CONCLUSION

The impact of pre-existing immunity on AAV GT safety and efficacy is not clearly understood. Currently, due to many GTs excluding seropositive patients through anti-AAV Ab prescreening, patients that could potentially benefit from AAV GT do not receive treatment, while patients given treatment could still be at risk of an immune response despite having no measurable anti-AAV Abs. Treatment-induced immune responses are a considerable barrier to AAV GT re-administration. Future studies should focus on confirming the relationship between anti-AAV NAb or TAb titers, vector transduction, and AAV GT safety, while characterizing the impact of pre-existing versus treatment-induced immune responses. There needs to be a particular focus on long-term outcomes such as response durability. To that end, treatment regardless of serostatus will generate the evidence base necessary to determine the clinical relevance of anti-AAV Abs and could justify exclusionary screening. In turn, screening may be better approached on a case-by-case basis, weighing individual factors pertaining to the disease area and product features, rather than based on an overarching strategy to guide all treatment decision-making. A clear differentiation should be made between different risk levels associated with high-dose systemic or low-dose localized delivery of gene therapies. Such a pragmatic approach could be supported by clinical mitigation strategies including continued monitoring, proactive management through immunosuppression protocols and improved design, manufacturing and optimized delivery. Ultimately, a combination of alternative AAV variants, alternate routes of administration with minimal immune exposure, and techniques to reduce pre-existing anti-AAV Ab levels by physical methods or pharmacological modulation of the humoral immune response may be needed to overcome any impact of pre-existing anti-AAV Abs in patients who would otherwise not be eligible for AAV-mediated GT.

AUTHORS’ CONTRIBUTIONS

All authors were involved in conceptualization and writing—review and editing. Additional contributions include: R.H.: Writing, overall design and strategy. L.Roberts: Overall design and strategy. L.Roessig: Overall design and strategy. M.A.: Investigation, data curation, and project administration. D.B.: Investigation, data curation and project administration. S.S.: Project administration. M.B.: Writing—review and editing. S.G.: Data curation. S.L.: Methodology and supervision. P.P.: Investigation, data curation, and project administration.

Footnotes

ACKNOWLEDGMENTS

Medical writing support was provided by Avalere Health.

AUTHOR DISCLOSURE

M.A., S.S., S.G., and S.L. are employees of AskBio Inc. L.Roberts and L.Roessig are employees of AskBio and Bayer AG. D.B. acted as a consultant to AskBio. M.B. and P.P. are employees of Bayer AG. S.L. acted as a consultant to Trogenix.

FUNDING INFORMATION

This article was funded by AskBio.

References

1. Wang

J-H

, Gessler

, Zhan

, et al. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther 2024;9(1):78; doi: 10.1038/s41392-024-01780-w

2. Au

HKE

, Isalan

, Mielcarek

. Gene therapy advances: A meta-analysis of AAV usage in clinical settings. Front Med (Lausanne) 2021;8:809118; doi: 10.3389/fmed.2021.809118

3. Mendell

, Al-Zaidy

, Rodino-Klapac

, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol Ther 2021;29(2):464–488; doi: 10.1016/j.ymthe.2020.12.007

4. Mendell

, Connolly

, Lehman

, 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; doi: 10.1016/j.omtm.2022.02.011

5. Dhungel

, Winburn

, Pereira

CDF

, et al. Understanding AAV vector immunogenicity: From particle to patient. Theranostics 2024;14(3):1260–1288; doi: 10.7150/thno.89380

6. Fitzpatrick

, Leborgne

, Barbon

, et al. Influence of pre-existing anti-capsid neutralizing and binding antibodies on AAV vector transduction. Mol Ther Methods Clin Dev 2018;9:119–129; doi: 10.1016/j.omtm.2018.02.003

7. Costa Verdera

, Kuranda

, Mingozzi

. AAV vector immunogenicity in humans: A long journey to successful gene transfer. Mol Ther 2020;28(3):723–746; doi: 10.1016/j.ymthe.2019.12.010

8. Pajaziti

, Michgehl

, Blazevic

, et al. Preclinical assessment of antibody responses to adeno-associated virus (AAV) vector-based capsids of AAV2, AAV5, AAV8, or AAV9 in laboratory cynomolgus macaques (Macaca fascicularis) of Asian or Mauritian origin. Hum Gene Ther 2026;37(3–4):114–129; doi: 10.1177/10430342251376043

9. Schulz

, Levy

, Petropoulos

, 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

10.

10. Long

, Veron

, Kuranda

, et al. Early phase clinical immunogenicity of valoctocogene roxaparvovec, an AAV5-mediated gene therapy for hemophilia A. Mol Ther 2021;29(2):597–610; doi: 10.1016/j.ymthe.2020.12.008

11.

11. Haar

, Blazevic

, Strobel

, et al. MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies. Mol Ther Methods Clin Dev 2022;25:360–369; doi: 10.1016/j.omtm.2022.04.008

12.

12. Smith

, Ross

, Kamal

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

13.

13. Braun

, Lange

, Schatz

, et al. Preexisting antibody assays for gene therapy: Considerations on patient selection cutoffs and companion diagnostic requirements. Mol Ther Methods Clin Dev 2024;32(1):101217; doi: 10.1016/j.omtm.2024.101217

14.

14. Gorovits

, Azadeh

, Fiscella

, et al. Assessment of immune responses against AAV encoded transgene products. Aaps J 2026;28(1):43; doi: 10.1208/s12248-025-01192-w

15.

15. Ertl

HCJ

. Immunogenicity and toxicity of AAV gene therapy. Front Immunol 2022;13:975803; doi: 10.3389/fimmu.2022.975803

16.

16. Muhuri

, Maeda

, Ma

, et al. Overcoming innate immune barriers that impede AAV gene therapy vectors. J Clin Invest 2021;131(1):e143780; doi: 10.1172/jci143780

17.

17. Zaiss

, Cotter

, White

, et al. Complement is an essential component of the immune response to adeno-associated virus vectors. J Virol 2008;82(6):2727–2740; doi: 10.1128/jvi.01990-07

18.

18. Nathwani

, Tuddenham

EGD

, Rangarajan

, et al. Adenovirus-associated virus vector–Mediated gene transfer in hemophilia B. N Engl J Med 2011;365(25):2357–2365; doi: 10.1056/NEJMoa1108046

19.

19. Mingozzi

, Meulenberg

, Hui

, et al. AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood 2009;114(10):2077–2086; doi: 10.1182/blood-2008-07-167510

20.

20. Casazza

, Cale

, Narpala

, VRC 603 Study Team. et al.; Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: A phase 1, dose-escalation trial. Nat Med 2022;28(5):1022–1030; doi: 10.1038/s41591-022-01762-x

21.

21. Kropf

, Markusic

, Majowicz

, et al. Complement system response to adeno-associated virus vector gene therapy. Hum Gene Ther 2024;35(13–14):425–438; doi: 10.1089/hum.2023.194

22.

22. Chand

, Zaidman

, Arya

, et al. Thrombotic microangiopathy following onasemnogene abeparvovec for spinal muscular atrophy: A case series. J Pediatr 2021;231:265–268; doi: 10.1016/j.jpeds.2020.11.054

23.

23. Schwotzer

, El Sissy

, Desguerre

, et al. Thrombotic microangiopathy as an emerging complication of viral vector-based gene therapy. Kidney Int Rep 2024;9(7):1995–2005; doi: 10.1016/j.ekir.2024.04.024

24.

24. Ferreira

, Petry

, Salmon

. Immune responses to AAV-vectors, the glybera example from bench to bedside. Front Immunol 2014;5:82; doi: 10.3389/fimmu.2014.00082

25.

25. Pan

, Rohde

, Zeitler

, et al. A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies. Mol Ther Methods Clin Dev 2023;31:101126; doi: 10.1016/j.omtm.2023.101126

26.

26.UniQure biopharma. Glybera. European Medicines Agency website; 2017. Available from: https://www.ema.europa.eu/en/documents/product-information/glybera-epar-product-information_en.pdf [Last accessed: January 23, 2025].

27.

27. Gaudet

, Methot

, Dery

, et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: An open-label trial. Gene Ther 2013;20(4):361–369; doi: 10.1038/gt.2012.43

28.

28.Spark Therapeutics Inc. Luxturna. U.S. Food and Drug Administration website; 2022. Available from: https://www.fda.gov/media/109906/download?attachment [Last accessed: January 23, 2025].

29.

29. Russell

, Bennett

, Wellman

, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: A randomised, controlled, open-label, phase 3 trial. Lancet 2017;390(10097):849–860; doi: 10.1016/S0140-6736(17)31868-8

30.

30.PTC Therapeutics International Limited. Keblidi. U.S. Food and Drug Administration website; 2024. Available from: https://www.fda.gov/media/183530/download?attachment [Last accessed: January 27, 2025].

31.

31. Tai

, Lee

, Chien

, et al. Long-term efficacy and safety of eladocagene exuparvovec in patients with AADC deficiency. Mol Ther 2022;30(2):509–518; doi: 10.1016/j.ymthe.2021.11.005

32.

32.BioMarin Pharmaceutical Inc. Roctavian. U.S. Food and Drug Administration website; 2023. Available from: https://www.fda.gov/media/169937/download?attachment [Last accessed: January 23, 2025].

33.

33. Ozelo

, Mahlangu

, Pasi

, GENEr8-1 Trial Group. et al.; Valoctocogene roxaparvovec gene therapy for hemophilia A. N Engl J Med 2022;386(11):1013–1025; doi: 10.1056/NEJMoa2113708

34.

34.CSL Behring LLC. Hemgenix. U.S. Food and Drug Administration website; 2022. Available from: https://www.fda.gov/media/163467/download [Last accessed: January 23, 2025].

35.

35. Pipe

, Leebeek

FWG

, Recht

, et al. Gene therapy with etranacogene dezaparvovec for hemophilia B. N Engl J Med 2023;388(8):706–718; doi: 10.1056/NEJMoa2211644

36.

36.Novartis Gene Therapies Inc. Zolgensma. U.S. Food and Drug Administration website; 2024. Available from: https://www.fda.gov/media/126109/download?attachment [Last accessed: January 23, 2025].

37.

37. Mendell

, Al-Zaidy

, Shell

, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med 2017;377(18):1713–1722; doi: 10.1056/NEJMoa1706198

38.

38.Pfizer Inc. Beqvez. U.S. Food and Drug Administration website; 2024. Available from: https://www.fda.gov/media/178140/download?attachment [Last accessed: January 23, 2025].

39.

39. Cuker

, Kavakli

, Frenzel

, BENEGENE-2 Trial Investigators. et al.; Gene therapy with fidanacogene elaparvovec in adults with hemophilia B. N Engl J Med 2024;391(12):1108–1118; doi: 10.1056/NEJMoa2302982

40.

40.Sarepta Therapeutics Inc. Elevidys [highlights of prescribing information]. U.S. Food and Drug Administration website; 2024. Available from: https://www.fda.gov/media/169679/download [Last accessed: January 23, 2025].

41.

41. Mendell

, Shieh

, McDonald

, et al. Expression of SRP-9001 dystrophin and stabilization of motor function up to 2 years post-treatment with delandistrogene moxeparvovec gene therapy in individuals with Duchenne muscular dystrophy. Front Cell Dev Biol 2023;11:1167762; doi: 10.3389/fcell.2023.1167762

42.

42. Klamroth

, Hayes

, Andreeva

, et al. Global seroprevalence of pre-existing immunity against AAV5 and other AAV serotypes in people with hemophilia A. Hum Gene Ther 2022;33(7–8):432–441; doi: 10.1089/hum.2021.287

43.

43. Veron

, Leborgne

, Monteilhet

, et al. Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors. J Immunol 2012;188(12):6418–6424; doi: 10.4049/jimmunol.1200620

44.

44.Chiesi. Glybera▼ (alipogene tiparvovec) in Europe. 2017. Available from: https://www.chiesi.com/en/glybera-alipogene-tiparvovec-in-europe/ [Last accessed: February 12, 2026].

45.

45. Pupo

, Fernández

, Low

, et al. AAV vectors: The Rubik’s cube of human gene therapy. Mol Ther 2022;30(12):3515–3541; doi: 10.1016/j.ymthe.2022.09.015

46.

46. Issa

, Shaimardanova

, Solovyeva

, et al. Various AAV serotypes and their applications in gene therapy: An overview. Cells 2023;12(5):785.

47.

47. von Drygalski

, Gomez

, Giermasz

, et al. Completion of phase 2b trial of etranacogene dezaparvovec gene therapy in patients with hemophilia B over 5 years. Blood Adv 2025;9(14):3543–3552; doi: 10.1182/bloodadvances.2024015291

48.

48. Bala

, Thornburg

. Gene therapy in hemophilia A: Achievements, challenges, and perspectives. Semin Thromb Hemost 2025;51(1):28–40; doi: 10.1055/s-0044-1785483

49.

49. Abdelhamid

, Mazor

. Can sex-based variations in the immune responses to AAV gene therapy affect safety and efficacy? A review of current understanding. Aaps J 2025;27(6):141; doi: 10.1208/s12248-025-01127-5

50.

50. Piechnik

, Amendum

, Sawamoto

, et al. Sex difference leads to differential gene expression patterns and therapeutic efficacy in mucopolysaccharidosis IVA murine model receiving AAV8 gene therapy. Int J Mol Sci 2022;23(20):12693.

51.

51. Warrington

, Hoang

, Seirup

, et al. Unveiling the sex bias: Higher preexisting and neutralizing titers against AAV in females and implications for gene therapy. Gene Ther 2025;32(4):339–348; doi: 10.1038/s41434-025-00528-7

52.

52. Salabarria

, Corti

, Coleman

, et al. Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies. J Clin Invest 2024;134(1):e173510; doi: 10.1172/JCI173510

53.

53. Byrne

, Butterfield

, Shieh

, et al. Complement activation in a phase Ib study of fordadistrogene movaparvovec for Duchenne muscular dystrophy. Mol Ther 2025;33(9):4226–4238; doi: 10.1016/j.ymthe.2025.06.032

54.

54. Brantly

, Chulay

, Wang

, et al. Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy. Proc Natl Acad Sci U S A 2009;106(38):16363–16368; doi: 10.1073/pnas.0904514106

55.

55. MacLaren

, Audo

, Fischer

, et al. An open-label phase II study assessing the safety of bilateral, sequential administration of retinal gene therapy in participants with choroideremia: The GEMINI study. Hum Gene Ther 2024;35(15–16):564–575; doi: 10.1089/hum.2024.017

56.

56. Manno

, Chew

, Hutchison

, et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 2003;101(8):2963–2972; doi: 10.1182/blood-2002-10-3296

57.

57. Majowicz

, Nijmeijer

, Lampen

, et al. Therapeutic hFIX activity achieved after single AAV5-hFIX treatment in hemophilia B patients and NHPs with pre-existing anti-AAV5 NABs. Mol Ther Methods Clin Dev 2019;14:27–36; doi: 10.1016/j.omtm.2019.05.009

58.

58. Miesbach

, Meijer

, Coppens

, et al. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B. Blood 2018;131(9):1022–1031; doi: 10.1182/blood-2017-09-804419

59.

59. Thornburg

. Etranacogene dezaparvovec for hemophilia B gene therapy. Ther Adv Rare Dis 2021;2:26330040211058896; doi: 10.1177/26330040211058896

60.

60. Chand

, Mohr

, McMillan

, et al. Hepatotoxicity following administration of onasemnogene abeparvovec (AVXS-101) for the treatment of spinal muscular atrophy. J Hepatol 2021;74(3):560–566; doi: 10.1016/j.jhep.2020.11.001

61.

61. Reiss

, Davidoff

, Tuddenham

EGD

, et al. Sustained clinical benefit of AAV gene therapy in severe hemophilia B. N Engl J Med 2025;392(22):2226–2234; doi: 10.1056/NEJMoa2414783

62.

62. De la Vega

, Sellon

, Smith

, et al. A phase 1 clinical trial shows safe, sustained, AAV-mediated expression of IL-1Ra in the human osteoarthritic knee joint. Sci Transl Med 2025;17(801):eadu9804; doi: 10.1126/scitranslmed.adu9804

63.

63. Duan

. Lethal immunotoxicity in high-dose systemic AAV therapy. Mol Ther 2023;31(11):3123–3126; doi: 10.1016/j.ymthe.2023.10.015

64.

64. Guillou

, de Pellegars

, Porcheret

, et al. Fatal thrombotic microangiopathy case following adeno-associated viral SMN gene therapy. Blood Adv 2022;6(14):4266–4270; doi: 10.1182/bloodadvances.2021006419

65.

65.Zolgensma Prescribing Information. Novartis gene therapies. 2019. Available from: https://www.fda.gov/media/126109/download [Last accessed: February 16, 2025].

66.

66. Sandza

, Clark

, Koziol

, et al. Ultra-sensitive AAV capsid detection by immunocapture-based qPCR following factor VIII gene transfer. Gene Ther 2022;29(1–2):94–105; doi: 10.1038/s41434-021-00287-1

67.

67. Day

, Finkel

, Mercuri

, et al. Adeno-associated virus serotype 9 antibodies in patients screened for treatment with onasemnogene abeparvovec. Mol Ther Methods Clin Dev 2021;21:76–82; doi: 10.1016/j.omtm.2021.02.014

68.

68. Willett

, Bennett

. Immunology of AAV-mediated gene transfer in the eye. Front Immunol 2013;4:261; doi: 10.3389/fimmu.2013.00261

69.

69. Ross

, Sade

, Obolensky

, et al. Characterization of anti-AAV2 neutralizing antibody levels in sheep prior to and following intravitreal AAV2.7m8 injection. Gene Ther 2024;31(11–12):580–586; doi: 10.1038/s41434-024-00495-5

70.

70. Long

, Sandza

, Holcomb

, et al. The impact of pre-existing immunity on the non-clinical pharmacodynamics of AAV5-based gene therapy. Mol Ther Methods Clin Dev 2019;13:440–452; doi: 10.1016/j.omtm.2019.03.006

71.

71. Kroenke

, Barger

, Hu

, et al. Immune complex formation is associated with loss of tolerance and an antibody response to both drug and target. Front Immunol 2021;12:782788; doi: 10.3389/fimmu.2021.782788

72.

72. Roy

, Bansal

, Kulkarni

, et al. Hepatic manifestations following gene therapy. Gastro Hep Adv 2025;4(8):100681; doi: 10.1016/j.gastha.2025.100681

73.

73. Notarte

, Catahay

, Macasaet

, et al. Infusion reactions to adeno-associated virus (AAV)-based gene therapy: Mechanisms, diagnostics, treatment and review of the literature. J Med Virol 2023;95(12):e29305; doi: 10.1002/jmv.29305

74.

74. Proud

, Kichula

, Matesanz

, et al. Onasemnogene abeparvovec gene therapy for treatment of patients with spinal muscular atrophy: Updated real-world practical considerations. J Neuromuscul Dis 2025;0(0):22143602251391258; doi: 10.1177/22143602251391258

75.

75. Von Drygalski

, Giermasz

, Castaman

, et al. Etranacogene dezaparvovec (AMT-061 phase 2b): Normal/near normal FIX activity and bleed cessation in hemophilia B. Blood Adv 2019;3(21):3241–3247; doi: 10.1182/bloodadvances.2019000811

76.

76. Majowicz

, Ncete

, van Waes

, et al. Seroprevalence of pre-existing nabs against AAV1, 2, 5, 6 and 8 in South African hemophilia B patient population. Blood 2019;134(Supplement_1):3353; doi: 10.1182/blood-2019-128217

77.

77. Bennett

, Wellman

, Marshall

, et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: A follow-on phase 1 trial. Lancet 2016;388(10045):661–672; doi: 10.1016/s0140-6736(16)30371-3

78.

78. Henry

, Chung

, Alvisi

, et al. GenePHIT phase 2 study design: A double blind, placebo-controlled trial to assess efficacy, safety, and tolerability of AB-1002 gene therapy in adults with heart failure. Eur Heart J 2024;45(Supplement_1); doi: 10.1093/eurheartj/ehae666.3642

79.

79.ClinicalTrials.gov. Phosphatase inhibition by intracoronary gene therapy in subjects with non-ischemic NYHA class III heart failure (GenePHIT). 2025. Available from: https://clinicaltrials.gov/study/NCT05598333 [Last accessed: November 27, 2025].

80.

80. Henry

, Chung

, Alvisi

, et al. Cardiotropic AAV gene therapy for heart failure: A phase 1 trial. Nat Med 2025;31(11):3845–3852; doi: 10.1038/s41591-025-04011-z

81.

81. Ishikawa

, Weber

, Hajjar

. Human cardiac gene therapy. Circ Res 2018;123(5):601–613; doi: 10.1161/circresaha.118.311587

82.

82. Watanabe

, Ishikawa

, Fish

, et al. Protein phosphatase inhibitor-1 gene therapy in a swine model of nonischemic heart failure. J Am Coll Cardiol 2017;70(14):1744–1756; doi: 10.1016/j.jacc.2017.08.013

83.

83. Mazurek

, Tharakan

, Mavropoulos

, et al. AAV delivery strategy with mechanical support for safe and efficacious cardiac gene transfer in swine. Nat Commun 2024;15(1):10450; doi: 10.1038/s41467-024-54635-x

84.

84. Vrellaku

, Sethw Hassan

, Howitt

, et al. A systematic review of immunosuppressive protocols used in AAV gene therapy for monogenic disorders. Mol Ther 2024;32(10):3220–3259; doi: 10.1016/j.ymthe.2024.07.016

85.

85. Prasad

, Dimmock

, Greenberg

, et al. Immune responses and immunosuppressive strategies for adeno-associated virus-based gene therapy for treatment of central nervous system disorders: Current knowledge and approaches. Hum Gene Ther 2022;33(23–24):1228–1245; doi: 10.1089/hum.2022.138

86.

86. Konkle

, Walsh

, Escobar

, et al. BAX 335 hemophilia B gene therapy clinical trial results: Potential impact of CpG sequences on gene expression. Blood 2021;137(6):763–774; doi: 10.1182/blood.2019004625

87.

87. Chandler

, Barnard

, Caddy

, et al. Enhancement of adeno-associated virus-mediated gene therapy using hydroxychloroquine in murine and human tissues. Mol Ther Methods Clin Dev 2019;14:77–89; doi: 10.1016/j.omtm.2019.05.012

88.

88. Arjomandnejad

, Dasgupta

, Flotte

, et al. Immunogenicity of recombinant adeno-associated virus (AAV) vectors for gene transfer. BioDrugs 2023;37(3):311–329; doi: 10.1007/s40259-023-00585-7

89.

89. Flotte

, Cataltepe

, Puri

, et al. AAV gene therapy for Tay-Sachs disease. Nat Med 2022;28(2):251–259; doi: 10.1038/s41591-021-01664-4

90.

90. Corti

, Liberati

, Smith

, et al. Safety of intradiaphragmatic delivery of adeno-associated virus-mediated alpha-glucosidase (rAAV1-CMV-hGAA) gene therapy in children affected by pompe disease. Hum Gene Ther Clin Dev 2017;28(4):208–218; doi: 10.1089/humc.2017.146

91.

91. Velazquez

, Meadows

, Pineda

, et al. Effective depletion of pre-existing anti-AAV antibodies requires broad immune targeting. Mol Ther Methods Clin Dev 2017;4:159–168; doi: 10.1016/j.omtm.2017.01.003

92.

92. Samelson-Jones

, Finn

, Favaro

, et al. Timing of intensive immunosuppression impacts risk of transgene antibodies after AAV gene therapy in nonhuman primates. Mol Ther Methods Clin Dev 2020;17:1129–1138; doi: 10.1016/j.omtm.2020.05.001

93.

93. Earley

, Piletska

, Ronzitti

, et al. Evading and overcoming AAV neutralization in gene therapy. Trends Biotechnol 2023;41(6):836–845; doi: 10.1016/j.tibtech.2022.11.006

94.

94. Oh

, Waldo

, Paez-Cruz

, et al. Steroid-associated side effects in patients with primary proteinuric kidney disease. Kidney Int Rep 2019;4(11):1608–1616; doi: 10.1016/j.ekir.2019.08.019

95.

95. Boedecker-Lips

, Judel

, Holtz

, et al. Efficient removal of antibodies to adeno-associated viruses by immunoadsorption. J Clin Apher 2023;38(5):590–601; doi: 10.1002/jca.22069

96.

96. Potter

, Peterson

, Griffin

, et al. Use of plasmapheresis to lower anti-AAV antibodies in nonhuman primates with pre-existing immunity to AAVrh74. Mol Ther Methods Clin Dev 2024;32(1):101195; doi: 10.1016/j.omtm.2024.101195

97.

97. Leborgne

, Barbon

, Alexander

, et al. IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nat Med 2020;26(7):1096–1101; doi: 10.1038/s41591-020-0911-7

98.

98. Ros-Ganan

, Hommel

, Trigueros-Motos

, et al. Optimising the IgG-degrading enzyme treatment regimen for enhanced adeno-associated virus transduction in the presence of neutralising antibodies. Clin & Trans Imm 2022;11(2):e1375; doi: 10.1002/cti2.1375

99.

99.ClinicalTrials.gov. Efficacy and safety of GNT0003 following imlifidase pre-treatment in severe crigler-NajjarSyndrome. 2025. Available from: https://clinicaltrials.gov/study/NCT06518005 [Last accessed: October 10, 2025].

100.

100.ClinicalTrials.gov. A gene transfer therapy study to evaluate the safety and efficacy of delandistrogene moxeparvovec (SRP-9001) following imlifidase infusion in participants with Duchenne muscular dystrophy (DMD) determined to have pre-existing antibodies to recombinant adeno-associated virus serotype (rAAVrh74). 2025. Available from: https://clinicaltrials.gov/study/NCT06241950 [Last accessed: October 10, 2025].

101.

101.Genethon. Data from GNT-018-IDES trial supports feasibility of imlifidase as pretreatment in gene therapy treatment for patients with Crigler-Najjar syndrome who are immune to AAV. 2025. Available from: https://www.genethon.com/data-from-gnt-018-ides-trial-supports-feasibility-of-imlifidase-as-pretreatment-in-gene-therapy-treatment-for-patients-with-crigler-najjar-syndrome-who-are-immune-to-aav/ [Last accessed: October 28, 2025].

102.

102.Hansa Biopharma. Hansa Biopharma announces supportive data from treatment with imlifidase prior to the administration of gene therapy for Duchenne muscular dystrophy. 2025. Available from: https://www.hansabiopharma.com/media/press-releases/2025/hansa-biopharma-announces-supportive-data-from-treatment-with-imlifidase-prior-to-the-administration-of-gene-therapy-for-duchenne-muscular-dystrophy/ [Last accessed: October 28, 2025].

103.

103. Lorant

, Bengtsson

, Eich

, et al. Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients. Am J Transplant 2018;18(11):2752–2762; doi: 10.1111/ajt.14733

104.

104. Smith

, Elmore

, Fusco

, et al. Engineered IgM and IgG cleaving enzymes for mitigating antibody neutralization and complement activation in AAV gene transfer. Mol Ther 2024;32(7):2080–2093; doi: 10.1016/j.ymthe.2024.05.004

105.

105. Horiuchi

, Hinderer

, Shankle

, et al. Neonatal Fc receptor inhibition enables adeno-associated virus gene therapy despite pre-existing humoral immunity. Hum Gene Ther 2023;34(19–20):1022–1032; doi: 10.1089/hum.2022.216

106.

106. Kishimoto

, Samulski

. Addressing high dose AAV toxicity - ‘one and done’ or ‘slower and lower’? Expert Opin Biol Ther 2022;22(9):1067–1071; doi: 10.1080/14712598.2022.2060737

107.

107. Greenberg

, Eshraghian

, Battiprolu

, et al. Abstract 10727: Results from first-in-human clinical trial of RP-A501 (AAV9:LAMP2B) gene therapy treatment for danon disease. Circulation 2021;144(Suppl_1):A10727; doi: 10.1161/circ.144.suppl_1.10727

108.

108. Silver

, Argiro

, Hong

, et al. Gene therapy vector-related myocarditis. Int J Cardiol 2024;398:131617; doi: 10.1016/j.ijcard.2023.131617

109.

109.Global Genes. FDA places hold on LogicBio’s trial of LB-001 for the treatment of pediatric patients with MMA. Global genes website; 2022. Available from: https://globalgenes.org/raredaily/fda-places-hold-on-logicbios-trial-of-lb-001-for-the-treatment-of-pediatric-patients-with-mma/ [Last accessed: March 7, 2025].

110.

110.Solid Biosciences. Solid biosciences reports positive initial clinical data from next-generation Duchenne gene therapy candidate SGT-003. 2025. Available from: https://investors.solidbio.com/news-releases/news-release-details/solid-biosciences-reports-positive-initial-clinical-data-next [Last accessed: November 27, 2025].

111.

111. Flanigan

, Shieh

, Gonorazky

, et al. 22OUpdate on INSPIRE DUCHENNE: A phase 1/2 study of SGT-003, a next-generation microdystrophin gene therapy for Duchenne muscular dystrophy. Neuromuscul Disord 2025;53:106188; doi: 10.1016/j.nmd.2025.106188

112.

112. Day

, Mendell

, Mercuri

, et al. Clinical trial and postmarketing safety of onasemnogene abeparvovec therapy. Drug Saf 2021;44(10):1109–1119; doi: 10.1007/s40264-021-01107-6

113.

113. Ballon

, Rosenberg

, Fung

, et al. Quantitative whole-body imaging of I-124-labeled adeno-associated viral vector biodistribution in nonhuman primates. Hum Gene Ther 2020;31(23–24):1237–1259; doi: 10.1089/hum.2020.116

114.

114. Wang

, Bell

, Somanathan

, et al. Comparative study of liver gene transfer with AAV vectors based on natural and engineered AAV capsids. Mol Ther 2015;23(12):1877–1887; doi: 10.1038/mt.2015.179

115.

115. Nam

, Ju

, Lee

, et al. Distinguishing between DNA-loaded full and empty capsids of adeno-associated virus with atomic force microscopy imaging. Langmuir 2023;39(19):6740–6747; doi: 10.1021/acs.langmuir.3c00241

116.

116. McColl-Carboni

, Dollive

, Laughlin

, et al. Analytical characterization of full, intermediate, and empty AAV capsids. Gene Ther 2024;31(5–6):285–294; doi: 10.1038/s41434-024-00444-2

117.

117. Stone

, Aubert

, Jerome

. Breaching the blood-brain barrier: AAV triggers dose-dependent toxicity in the brain. Mol Ther Methods Clin Dev 2023;31:101105; doi: 10.1016/j.omtm.2023.09.001

118.

118.Solid Biosciences. Solid biosciences provides update regarding SGT-001 phase I/II clinical hold on IGNITE DMD. 2020. Available from: https://investors.solidbio.com/news-releases/news-release-details/solid-biosciences-provides-update-regarding-sgt-001-phase-iii [Last accessed: November 26, 2025].

119.

119. Mingozzi

, Anguela

, Pavani

, et al. Overcoming preexisting humoral immunity to AAV using capsid decoys. Sci Transl Med 2013;5(194):194ra92; doi: 10.1126/scitranslmed.3005795

120.

120. Martino

, Markusic

. Immune response mechanisms against AAV vectors in animal models. Mol Ther Methods Clin Dev 2020;17:198–208; doi: 10.1016/j.omtm.2019.12.008

121.

121. Chai

, Sun

, Rigsbee

, et al. Application of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody evasion. J Control Release 2017;262:348–356; doi: 10.1016/j.jconrel.2017.08.005

122.

122. Ma

, Shen

. Immune responses in retinal gene therapy: Challenges, mechanisms, and future strategies. Front Immunol 2025;16:1664968; doi: 10.3389/fimmu.2025.1664968

123.

123. Wu

, Xue

, Li

, et al. A comprehensive analysis of scRNA-Seq and RNA-Seq unveils B cell immune suppression in the AAV-loaded brain. Immunol Res 2025;73(1):57; doi: 10.1007/s12026-025-09609-6

124.

124. Gardin

, Ronzitti

. Current limitations of gene therapy for rare pediatric diseases: Lessons learned from clinical experience with AAV vectors. Arch Pediatr 2023;30(8S1):8S46–8S52; doi: 10.1016/S0929-693X(23)00227-0

125.

125. Doshi

, Markmann

, Novak

, et al. Use of CD19-targeted immune modulation to eradicate AAV-neutralizing antibodies. Mol Ther 2025;33(7):3073–3085; doi: 10.1016/j.ymthe.2025.03.003

126.

126. Scallan

, Jiang

, Liu

, et al. Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood 2006;107(5):1810–1817; doi: 10.1182/blood-2005-08-3229

127.

127. Shieh

, Kuntz

, Dowling

, et al. Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): A multinational, open-label, dose-escalation trial. Lancet Neurol 2023;22(12):1125–1139; doi: 10.1016/s1474-4422(23)00313-7

128.

128.Rocket Pharmaceuticals. Rocket pharmaceuticals announces positive updates from phase 1 clinical trial of RP-A501 in Danon disease. 2021. Available from: https://www.businesswire.com/news/home/20211115005497/en/Rocket-Pharmaceuticals-Announces-Positive-Updates-from-Phase-1-Clinical-Trial-of-RP-A501-in-Danon-Disease

129.

129.4D Molecular Therapeutics. 4D molecular therapeutics presents interim data from 4D-310 INGLAXA phase 1/2 clinical trials & development plans for fabry disease cardiomyopathy at WORLDSymposium™. 4D Molecular Therapeutics website; 2023. Available from: https://ir.4dmoleculartherapeutics.com/news-releases/news-release-details/4d-molecular-therapeutics-presents-interim-data-4d-310-inglaxa/ [Last accessed: March 7, 2025].

130.

130.4D Molecular Therapeutics. Update from INGLAXA phase 1/2 clinical trials & development plans for 4D-310 genetic medicine for Fabry disease cardiomyopathy. 2023.

131.

131.ClinicalTrials.gov. A study to evaluate the safety and tolerability of PF-06939926 gene therapy in Duchenne muscular dystrophy. 2025. Available from: https://clinicaltrials.gov/study/NCT03362502 [Last accessed: October 8, 2025].