Immunogenicity of Adeno-Associated Viral Vectors

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AAV is one of the most actively 
studied gene therapy vectors 
and it has been successfully 
developed as a commercial 
vector for gene therapies1–3

AAV vectors are less immunogenic when compared with other viral vectors; nevertheless, they can still trigger an immune response4

  • Example: Onasemnogene abeparvovec-xioi: an AAV9-delivered SMN gene therapy3

    • In clinical trials, evidence of prior exposure to AAV9 (i.e. anti-AAV9 antibodies) was uncommon3
    • Following treatment, anti-AAV9 antibody titers increased in all patients, with most exceeding 1:819,2003
    • Data on re-treatment in the presence of high anti-AAV9 antibody titers are not available3

Innate

An innate immune response can be triggered by:

Promoters

that are included in the AAV vector cassette, which can theoretically be recognized as foreign by the host cell5,6

AAV inverted terminal repeat (ITR) sequences.

Toll-like receptors (TLRs) are one of the most extensively studied and prominent components of the cellular innate immune response1. AAV ITRs are CpG-rich double-stranded DNA structures, which can act as antigens for TLR-95

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Adaptive

Humoral Immunity

Pre-existing binding or neutralizing antibodies* to naturally occurring AAV serotypes are common in the population, but are generally low in children7-9

  • Anti-AAV9 capsid antibodies are typically low in children8
  • In one study†, the presence of IgG binding anti-AAV9 antibodies in children aged 2 to 7 years was 6% (figure)8
  • In another study, the average prevalence of
    anti-AAV2 and anti-AAV8 neutralizing antibodies in infants (<1 year) was 15%‡9
Binding IgG Seropositivity Prevalence in 
Healthy Subjects4
PRAC-IOGT-Slide13 2

Assays have been developed to detect the presence of pre-existing anti-AAV antibodies10,11, and anti-AAV antibody screening is commonly employed in clinical trials of AAV-based gene therapies12,13

Figure created from data in Table 2 in Fu H, et al. Hum Gene Ther Clin Dev 2017;28(4):187–196. *Binding antibodies are defined as all isotypes capable of binding to the therapy, whereas neutralizing antibodies are a subpopulation of total binding antibodies that bind to epitopes crucial for facilitating successful transduction of the target cells14,15; †Carried out using serum samples taken from patients enrolled in a natural history study or an MPS III inflammation study at Nationwide Children’s Hospital, with control samples obtained from healthy subjects via BioServe or the MPS III inflammation study8; ‡Based on plasma samples from 175 anonymous infants from Children’s National Medical Center (Washington, DC) using a threshold titer of ≥1:205.
AAV, adeno-associated virus; IgG, immunoglobulin G; MPS, mucopolysaccharidosis.

Seropositivity for neutralizing factors* against AAV is prevalent in the general population; however, titers against certain serotypes may be low7

  • Among those who were seropositive, neutralizing factor* titers were lower for AAV5, AAV8, and AAV9 compared with other serotypes in the majority of patients7
Prevalence of Neutralizing Factors* 
in the Serum in Healthy Subjects

Based on an analysis of serum samples collected from 226 healthy volunteers

PRAC-IOGT-Slide14 2
Distribution of Neutralizing Factor* 
Titers in Healthy Subjects†
PRAC-IOGT-Slide14 3

Figures reproduced from Figure 3 in Boutin S, et al. Hum Gene Ther 2010;21(6):704–712. *Neutralizing factors are defined as neutralizing antibodies and unidentified factors present in the serum that may contribute to neutralization7; †The percentage of total neutralizing factor titers is shown for all the seropositive samples displayed in the graph on the right7. AAV, adeno-associated virus.

Cell-Mediated Immunity

AAV vectors can induce cell-mediated immunity via the presentation of capsid proteins to cytotoxic T cells16

  • CD8+ T cells can destroy cells that have been transduced by AAV vectors, hence leading to the loss of transgene expression16
PRAC-IOGT-Slide15-2c.svg

Figure reproduced from Figure 2 in Colella P, et al. Mol Ther Methods Clin Dev 2018;8:87–104. AAV, adeno-associated virus; CD, cluster of differentiation; ER, endoplasmic reticulum; MHC, major histocompatibility complex; sc, self-complementary; ss, single strand; TLR, toll-like receptor.

        1. Naso MF, et al. BioDrugs 2017;31(4):317–334;
        2. LuxturnaTM [package insert]. Philadelphia, PA; Spark Therapeutics, Inc.; 2017. Available at: http://sparktx.com/LUXTURNA_US_Prescribing_Information.pdf. Accessed July 24, 2019;
        3. ZolgensmaTM [package insert]. Bannockburn, IL: AveXis Inc.; 2019. Available at: https://www.avexis.com/content/pdf/prescribing_information.pdf. Accessed July 24, 2019;
        4. Sack BK, Herzog RW. Curr Opin Mol Ther 2009;11(5):493–503.
        5. Brown N, et al. Hum Gene Ther 2017;28(6):450–463;
        6. Megias J, et al. Stem Cells 2012;30(7):1486–1495.
        7. Boutin S, et al. Hum Gene Ther 2010;21(6):704–712;
        8. Fu H, et al. Hum Gene Ther Clin Dev 2017;28(4):187–196;
        9. Calcedo R, et al. Clin Vaccine Immunol. 2011;18(9):1586–1588;
        10. Vandamme C, et al. Hum Gene Ther 2017;28(11):1061–1074;
        11. Falese L, et al. Gene Ther 2017;24:768–778;
        12. Ferreira V, et al. Front Immunol 2014;5:82;
        13. FDA. FDA Briefing Document. Advisory Committee Meeting, October 12, 2017. Available at: https://www.fda.gov/media/108375/download. Accessed June 22, 2019;
        14. FDA. The immunogenicity of therapeutic proteins- what you don’t know can hurt you and the patient. Available at: https://www.fda.gov/media/89071/download. Accessed June 25, 2019;
        15. Fitzpatrick Z, et al. Mol Ther Meth Clin Dev 2018;9:119–129.
        16. Wang D, et al. Nat Rev Drug Discov 2019;18(5):358–378.
  1. Nayak S, Herzog RW. Gene Ther 2010;17(3):295–304.
  2. Bessis N, et al. Gene Ther 2004;11:S10–S17.