Prevalence and Relevance of Pre-Existing Anti-Adeno-Associated Virus Immunity in the Context of Gene Therapy for Crigler-Najjar Syndrome

Abstract

Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler-Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants.

Keywords: AAV gene therapy; Crigler–Najjar syndrome; UGT1A1; anti-AAV neutralizing antibodies; pre-existing immunity; unconjugated hyperbilirubinemia.

Figure 1.

Management of CN in the selected population. Results are reported as percentage of total (n = 49). CN, Crigler–Najjar syndrome.

Figure 2.

Subjects with pre-existing anti-AAV8 NAbs were older than seronegative subjects. The age in years at time of NAb determination of subjects with NAb titer >1:1 (positive, n = 15) versus NAb titer ≤1:1 (negative, n = 34). Two-tailed t-test was used to compare groups (*p < 0.05). NAbs, neutralizing antibodies.

Figure 3.

Correlation between anti-AAV8 NAb titers and total IgG and IgM to AAV serotypes 8, 5, and 2 in subjects affected by CN. (A) Anti-AAV8 NAb titer versus total IgG binding antibodies to AAV8 measured in seropositive subjects (n = 15). (B) IgM binding antibodies to AAV8 (n = 49). (C) Anti-AAV5 NAb titer versus total IgG binding antibodies to AAV5. (D) IgM binding antibodies to AAV5. (E) Anti-AAV2 NAb titer versus total IgG binding antibodies to AAV2. (F) IgM binding antibodies to AAV2. NAb titers are reported as reciprocal dilutions (1:x). The dotted line represents the limit of quantification for IgM.

Figure 4.

VGCNs in liver of passively immunized mice after receiving AAV vector preparations obtained with different manufacturing methods. (A) Experimental design, indicating passive immunization regimens with IVIg at day 0 followed by the administration of two different AAV vector preparations at day 1; a total of six experimental groups with n = 6 per group were included in the study and all animals were sacrificed at day 22 postvector infusion. (B) Anti-AAV8 NAb titer (reciprocal dilution, 1:x) measured by an in vitro reporter assay, 24 h after the three immunization regimens. (C, D) VGCN assessed by qPCR in liver tissue at the time of sacrifice (d22) in animals receiving vectors made by triple transfection of adherent HEK293 cells (AAV8 ADH) or HEK293 cells cultured in suspension (AAV8 SUSP). Results are expressed as fold relative to PBS-treated animals. Statistical significance was determined with one-way ANOVA with Bonferroni post-test comparison of all treatment groups (**p < 0.01). Data are represented as mean ± standard deviation. IVIg, intravenous immunoglobulin; PBS, phosphate-buffered saline; qPCR, quantitative PCR; VGCN, vector genome copy number.