Adeno-associated virus (AAV) vectors have emerged as versatile and promising tools in gene therapy due to their favorable safety profile, broad tissue tropism, and long-term gene expression. However, pre-existing immunity, especially in the form of neutralizing antibodies (NAbs) remains a significant barrier, reducing vector efficacy and restricting patient eligibility. This review provides a comprehensive overview of the immunological landscape affecting AAV gene therapy, including global seroprevalence, environmental influences, and antibody cross-reactivity stemming from natural parvovirus exposure or vaccination of animal research models.We detail the mechanisms underlying immune detection and vector clearance, covering innate pattern recognition receptors, complement activation, and adaptive immune effector functions such as antibody-dependent complement deposition, cytotoxicity, and phagocytosis.We further analyze how species, age, serotype, administration route, and target tissue contribute to immune susceptibility and variable transduction outcomes. To overcome these challenges, we propose a three-pronged classification of mitigation strategies: (1) immune-focused strategies, such as plasmapheresis, immunoadsorption, enzymatic antibody cleavage, corticosteroids, and B cell depletion; (2) delivery-focused strategies, which include targeting immune-privileged sites, localized or intrathecal delivery, and timing of vector administration; and (3) capsid-focused strategies, comprising rational capsid engineering and the use of decoy particles or empty capsids.We also discuss promising advances such as AAV-specific regulatory T cells and re-dosable AAV platforms. This strategic framework offers a roadmap for tailoring gene therapy approaches to individual immune profiles and improving the safety, efficacy, and accessibility of AAVbased therapeutics.
OsunkwoI, AndemariamB, MinnitiCP, et al.Impact of sickle cell disease on patients’ daily lives, symptoms reported, and disease management strategies: Results from the international Sickle Cell World Assessment Survey (SWAY). Am J Hematol, 2021; 96(4):404–417.
2.
CooperDN, ChenJ-M, BallEV, et al.Genes, mutations, and human inherited disease at the dawn of the age of personalized genomics. Hum Mutat, 2010; 31(6):631–655.
3.
DunbarCE, HighKA, JoungJK, et al.Gene therapy comes of age. Science, 2018; 359(6372):eaan4672.
4.
EmeryDW. Gene therapy for genetic diseases: On the horizon. Clin Appl Immunol Rev, 2004; 4(6):411–422.
5.
RamamoorthM, NarvekarA. Non viral vectors in gene therapy-an overview. J Clin Diagn Res, 2015; 9(1):GE01–GE06.
6.
DingB, LiT, ZhangJ, et al.Advances in liver-directed gene therapy for hepatocellular carcinoma by non-viral delivery systems. Curr Gene Ther, 2012; 12(2):92–102.
7.
TomaninR, ScarpaM. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curr Gene Ther, 2004; 4(4):357–372.
8.
LukashevAN, ZamyatninAA. Viral vectors for gene therapy: Current state and clinical perspectives. Biochemistry (Mosc), 2016; 81(7):700–708.
9.
ChiraS, JacksonCS, OpreaI, et al.Progresses towards safe and efficient gene therapy vectors. Oncotarget, 2015; 6(31):30675–30703.
10.
CarterBJ. Adeno-associated virus and the development of adeno-associated virus vectors: A historical perspective. Mol Ther, 2004; 10(6):981–989.
11.
BullerRM, RoseJA. Characterization of adenovirus-associated virus-induced polypeptides in KB cells. J Virol, 1978; 25(1):331–338.
12.
DongB, NakaiH, XiaoW. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther, 2010; 18(1):87–92.
13.
YanZ, ZakR, LuxtonGWG, et al.Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. J Virol, 2002; 76(5):2043–2053.
14.
Costa VerderaH, KurandaK, MingozziF. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther, 2020; 28(3):723–746.
15.
MartinoAT, MarkusicDM. Immune response mechanisms against AAV vectors in animal models. Mol Ther Methods Clin Dev, 2020; 17:198–208.
16.
CalcedoR, VandenbergheLH, GaoG, et al.Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis, 2009; 199(3):381–390.
17.
GordonJC, AngrickEJ. Canine parvovirus: Environmental effects on infectivity. Am J Vet Res, 1986; 47(7):1464–1467.
18.
WangF, ZhanQ, YuS-P, et al.Environmental monitoring of parvovirus B19 in the kidney transplantation ward of a chinese teaching hospital. Infect Drug Resist, 2022; 15:1903–1910.
19.
GuoL, YangZ, ZhangL, et al.Systematic review of the effects of environmental factors on virus inactivation: Implications for coronavirus disease 2019. Int J Environ Sci Technol (Tehran), 2021; 18(9):2865–2878.
20.
KlamrothR, HayesG, AndreevaT, 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.
21.
ZhaoQ, YuS, FuD, et al.Pre-existing Anti-AAV9 antibodies in the Chinese healthy and rare disease populations: Implications for gene therapy. Virus Res, 2025; 354:199549.
22.
CaoL, LedeboerA, PanY, et al.Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials. Gene Ther, 2023; 30(1–2):150–159.
EmmanuelSN, MietzschM, TsengYS, et al.Parvovirus capsid-antibody complex structures reveal conservation of antigenic epitopes across the family. Viral Immunol, 2021; 34(1):3–17.
25.
GurdaBL, DiMattiaMA, MillerEB, et al.Capsid antibodies to different adeno-associated virus serotypes bind common regions. J Virol, 2013; 87(16):9111–9124.
26.
LiH, LinS-W, Giles-DavisW, et al.A preclinical animal model to assess the effect of pre-existing immunity on AAV-mediated gene transfer. Mol Ther, 2009; 17(7):1215–1224.
YangT-Y, BraunM, LembkeW, et al.Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper. Mol Ther Methods Clin Dev, 2022; 26:471–494.
29.
MuhuriM, MaedaY, MaH, et al.Overcoming innate immune barriers that impede AAV gene therapy vectors. J Clin Invest, 2021; 131(1):e143780.
30.
NidetzNF, McGeeMC, TseLV, et al.Adeno-associated viral vector-mediated immune responses: Understanding barriers to gene delivery. Pharmacol Ther, 2020; 207:107453.
31.
RogersGL, SuzukiM, ZolotukhinI, et al.Unique roles of TLR9- and MyD88-dependent and -independent pathways in adaptive immune responses to aav-mediated gene transfer. J Innate Immun, 2015; 7(3):302–314.
32.
ZhuJ, HuangX, YangY. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J Clin Invest, 2009; 119(8):2388–2398.
33.
WestAP, Khoury-HanoldW, StaronM, et al.Mitochondrial DNA stress primes the antiviral innate immune response. Nature, 2015; 520(7548):553–557.
34.
Costa-VerderaH, MeneghiniV, FitzpatrickZ, et al.AAV vectors trigger DNA damage responses and STING-dependent inflammation in human CNS cells [Internet]. Research Square; 2024 [cited 2025 Apr 5] Available from: https://www.researchsquare.com/article/rs-4171795/v1
35.
UnterholznerL, KeatingSE, BaranM, et al.IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol, 2010; 11(11):997–1004.
36.
HornungV, AblasserA, Charrel-DennisM, et al.AIM2 recognizes cytosolic dsDNA and forms a caspase-1 activating inflammasome with ASC. Nature, 2009; 458(7237):514–518.
37.
SutterSO, ToblerK, SeyffertM, et al.Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way. J Virol, 2024; 98(7):e00110-24.
ThoresenD, WangW, GallsD, et al.The molecular mechanism of RIG‐I activation and signaling. Immunol Rev, 2021; 304(1):154–168.
40.
KatoH, TakeuchiO, SatoS, et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature, 2006 May;441(7089):101–105.
41.
ShaoW, EarleyLF, ChaiZ, et al.Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction. JCI Insight, 2018; 3(12):e120474.
42.
KropfE, MarkusicDM, MajowiczA, et al.Complement System Response to Adeno-Associated Virus Vector Gene Therapy. Hum Gene Ther, 2024; 35(13–14):425–438.
43.
ZaissAK, CotterMJ, WhiteLR, et al.Complement is an essential component of the immune response to adeno-associated virus vectors. J Virol, 2008; 82(6):2727–2740.
44.
GuillouJ, de PellegarsA, PorcheretF, et al.Fatal thrombotic microangiopathy case following adeno-associated viral SMN gene therapy. Blood Adv, 2022; 6(14):4266–4270.
45.
SalabarriaSM, CortiM, ColemanKE, et al.Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies. J Clin Invest, 2024; 134(1):e173510.
46.
ChandlerTL, YangA, OteroCE, et al.Protective mechanisms of nonneutralizing antiviral antibodies. PLoS Pathog, 2023; 19(10):e1011670.
47.
GreenbergB, ButlerJ, FelkerGM, et al.Prevalence of AAV1 neutralizing antibodies and consequences for a clinical trial of gene transfer for advanced heart failure. Gene Ther, 2016; 23(3):313–319.
48.
MannoCS, PierceGF, ArrudaVR, et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 2006; 12(3):342–347.
49.
Basner-TschakarjanE, MingozziF. Cell-Mediated immunity to AAV vectors, evolving concepts and potential solutions. Front Immunol, 2014; 5:350.
50.
HsiJ, MietzschM, ChipmanP, et al.Structural and antigenic characterization of the avian adeno-associated virus capsid. ParrishCR, editor. J Virol. 2023; 97(10):e00780-23.
51.
WestC, FederspielJD, RogersK, et al.Complement Activation by Adeno-Associated Virus-Neutralizing Antibody Complexes. Hum Gene Ther, 2023June1; 34(11–12):554–566.
52.
SmithCJ, RossN, KamalA, 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.
53.
BenhniaMR-E-I, McCauslandMM, MoyronJ, et al.Vaccinia Virus Extracellular Enveloped Virion Neutralization In Vitro and Protection In Vivo Depend on Complement. J Virol, 2009; 83(3):1201–1215.
54.
ChandDH, ZaidmanC, AryaK, et al.Thrombotic Microangiopathy Following Onasemnogene Abeparvovec for Spinal Muscular Atrophy: A Case Series. J Pediatr, 2021; 231:265–268.
55.
KeshwaraR, HagenKR, Abreu-MotaT, et al.A recombinant rabies virus expressing the marburg virus glycoprotein is dependent upon antibody-mediated cellular cytotoxicity for protection against marburg virus disease in a murine Model. García-Sastre A, editor. J Virol, 2019; 93(6):e01865-18.
56.
TerminiJM, Martinez-NavioJM, GaoG, et al.Glycoengineering of AAV-delivered monoclonal antibodies yields increased ADCC activity. Mol Ther Methods Clin Dev, 2021; 20:204–217.
57.
FuchsSP, Martinez-NavioJM, PiatakM, et al.AAV-Delivered antibody mediates significant protective effects against SIVmac239 challenge in the absence of neutralizing activity. PLoS Pathog, 2015; 11(8):e1005090. PLOS Pathogens.
58.
BarouchDH, StephensonKE, BorducchiEN, et al.Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys. Cell, 2013; 155(3):531–539.
59.
GaoGP, AlviraMR, WangL, et al.Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A, 2002; 99(18):11854–11859.
60.
PabingerI, Ayash-RashkovskyM, EscobarM, et al.Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia. Gene Ther, 2024; 31(5–6):273–284.
61.
MajowiczA, NijmeijerB, LampenMH, 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.
62.
PerocheauDP, CunninghamS, LeeJ, et al.age-related seroprevalence of antibodies against AAV-LK03 in a UK population cohort. Hum Gene Ther, 2019Jan; 30(1):79–87.
63.
ArjomandnejadM, DasguptaI, FlotteTR, et al.Immunogenicity of recombinant adeno-associated virus (AAV) vectors for gene transfer. BioDrugs, 2023 May;37(3):311–329.
CarsonMJ, DooseJM, MelchiorB, et al.CNS immune privilege: Hiding in plain sight. Immunol Rev, 2006; 213:48–65.
66.
VandammeC, AdjaliO, MingozziF. Unraveling the complex story of immune responses to AAV vectors trial after trial. Hum Gene Ther, 2017; 28(11):1061–1074.
67.
MonteilhetV, SahebS, BoutinS, et al.A 10 patient case report on the impact of plasmapheresis upon neutralizing factors against adeno-associated virus (AAV) types 1, 2, 6, and 8. Mol Ther, 2011; 19(11):2084–2091.
68.
ChicoineLG, MontgomeryCL, BremerWG, et al.Plasmapheresis eliminates the negative impact of AAV antibodies on microdystrophin gene expression following vascular delivery. Mol Ther, 2014; 22(2):338–347.
69.
MingozziF, ChenY, MurphySL, et al.Pharmacological modulation of humoral immunity in a nonhuman primate model of aav gene transfer for hemophilia B. Mol Ther, 2012; 20(7):1410–1416.
70.
BertinB, VeronP, LeborgneC, et al.Capsid-specific removal of circulating antibodies to adeno-associated virus vectors. Sci Rep, 2020; 10(1):864.
71.
PotterRA, PetersonEL, GriffinD, 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.
72.
KaplanA. Complications of Apheresis. Semin Dial, 2012; 25(2):152–158.
73.
FitzpatrickZ, LeborgneC, BarbonE, 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.
74.
FuchsK, RummlerS, RiesW, et al.Performance, clinical effectiveness, and safety of immunoadsorption in a wide range of indications. Ther Apher Dial, 2022; 26(1):229–241.
75.
SalasD, KwikkersKL, ZabaletaN, et al.Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates. Blood Adv, 2019; 3(17):2632–2641.
76.
JordanSC, LorantT, ChoiJ, et al.IgG endopeptidase in highly sensitized patients undergoing transplantation. N Engl J Med, 2017; 377(5):442–453.
77.
LeborgneC, BarbonE, AlexanderJM, 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.
78.
Bou-JaoudehM, DelignatS, DaventureV, et al.The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice. Haematologica, 2023; 108(5):1322–1334.
79.
LonzeBE, TatapudiVS, WeldonEP, et al.IdeS (Imlifidase): A novel agent that cleaves human IgG and permits successful kidney transplantation across high-strength donor-specific antibody. Ann Surg, 2018; 268(3):488–496.
80.
CaoM, KatialR, LiuY, et al.Safety, efficacy, and immunogenicity of a novel IgG degrading enzyme (KJ103): results from two randomised, blinded, phase 1 clinical trials. Gene Ther, 2025; 32(3):223–236.
81.
SharpTH, BoyleAL, DiebolderCA, et al.Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b. Proc Natl Acad Sci U S A, 2019; 116(24):11900–11905.
82.
SmithTJ, ElmoreZC, FuscoRM, 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.
83.
SalabarriaSM, CortiM, ColemanKE, et al.Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies. J Clin Invest, 2024; 134(1):e173510.
84.
HandysideB, ZhangL, YatesB, et al.Prophylactic prednisolone promotes aav5 hepatocyte transduction through the novel mechanism of aav5 coreceptor platelet-derived growth factor receptor alpha upregulation and innate immune suppression. Hum Gene Ther, 2024; 35(1–2):36–47.
85.
VrellakuB, Sethw HassanI, HowittR, et al.A systematic review of immunosuppressive protocols used in AAV gene therapy for monogenic disorders. Mol Ther, 2024; 32(10):3220–3259.
86.
BiswasM, PalaschakB, KumarSRP, et al.B Cell depletion eliminates FVIII memory B cells and enhances AAV8-coF8 immune tolerance induction when combined with rapamycin. Front Immunol, 2020; 11:1293.
87.
NeubertK, MeisterS, MoserK, et al.The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat Med, 2008; 14(7):748–755.
88.
ChoiSJ, YiJS, LimJ-A, et al.Successful AAV8 readministration: Suppression of capsid‐specific neutralizing antibodies by a combination treatment of bortezomib and CD20 mAb in a mouse model of Pompe disease. J Gene Med, 2023; 25(8):e3509.
89.
KarmanJ, GumlawNK, ZhangJ, et al.Proteasome inhibition is partially effective in attenuating pre-existing immunity against recombinant adeno-associated viral vectors. PLoS One, 2012; 7(4):e34684.
90.
ChaanineAH, NonnenmacherM, KohlbrennerE, et al.Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure. Gene Ther, 2014; 21(4):379–386.
91.
MonahanPE, LothropCD, SunJ, et al.Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: A strategy for broad clinical application. Mol Ther, 2010; 18(11):1907–1916.
92.
DoshiB, GeorgeL, FraiettaJ, et al.Compositions and methods for b cell directed immunotherapies for anti-aav neutralizing alloantibodies [Internet]. WO2024098054A1, 2024 [cited 2025 June 8]. Available from: https://patents.google.com/patent/WO2024098054A1/en/
MingozziF, ChenY, EdmonsonSC, et al.Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue. Gene Ther, 2013; 20(4):417–424.
95.
KaplittMG, FeiginA, TangC, et al.Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: An open label, phase I trial. Lancet, 2007; 369(9579):2097–2105.
96.
MaguireAM, SimonelliF, PierceEA, et al.Safety and efficacy of gene transfer for leber’s congenital amaurosis. N Engl J Med, 2008; 358(21):2240–2248.
97.
LeWittPA, RezaiAR, LeeheyMA, et al.AAV2-GAD gene therapy for advanced Parkinson’s disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol, 2011; 10(4):309–319.
98.
QiaoC, LiJ, ZhengH, et al.Hydrodynamic limb vein injection of adeno-associated virus serotype 8 vector carrying canine myostatin propeptide gene into normal dogs enhances muscle growth. Hum Gene Ther, 2009; 20(1):1–10.
99.
SamaranchL, SebastianWS, KellsAP, et al.AAV9-mediated expression of a non-self protein in nonhuman primate central nervous system triggers widespread neuroinflammation driven by antigen-presenting cell transduction. Mol Ther, 2014Feb; 22(2):329–337.
100.
BoboRH, LaskeDW, AkbasakA, et al.Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci U S A, 1994; 91(6):2076–2080.
101.
BankiewiczKS, EberlingJL, KohutnickaM, et al.Convection-Enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach. Exp Neurol, 2000 July;164(1):2–14.
102.
MingozziF, AnguelaXM, PavaniG, et al.Overcoming preexisting humoral immunity to aav using capsid decoys. Sci Transl Med, 2013; 5(194):194ra92.
103.
GaoK, LiM, ZhongL, et al.Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Molecular Therapy - Methods & Clinical Development, 2014; 1:9.
104.
WrightJF. AAV empty capsids: For better or for worse? Mol Ther, 2014; 22(1):1–2.
105.
GaoK, LiM, ZhongL, et al.Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side-effects. Mol Ther Methods Clin Dev, 2014; 1(9):20139.
106.
WangC, MulagapatiSHR, ChenZ, et al.Developing an anion exchange chromatography assay for determining empty and full capsid contents in AAV6.2. Mol Ther Methods Clin Dev, 2019; 15:257–263.
