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.

Guest Editor: G. Castaman GENE THERAPY FOR HEMOPHILIA: FACTS AND QUANDARIES IN THE 21ST CENTURY

“…Overall, 20–40% of candidates for intravascular AAV gene therapy are not eligible due to the presence of NAbs to the vector capsid resulting from cross-reactivity after natural exposure to WT-AAV. 125 To date, it has been challenging to compare the efficacy of a given serotype across distinct studies due to a lack of normalization of the assays as most are developed “in house.” Potential modifiers of the rates/titers of NAbs could include (1) reporter gene, (2) assay technique whether neutralizing assays, non-neutralizing antibody assays (ELISA) or cellular assays, (3) presence or absence of empty vector capsids, (4) age of the patient 65 , 126 and longitudinal studies in the same population over time, 63 (5) underlying disease, 62 , 63 , 65 , 126 and (6) vector manufacturing. Some candidates may also test positive for more than one serotype at a time.…”

Efficacy of AAV8-hUGT1A1 with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients

“… 13 , 14 Screening adult CNs type 1 patients showed that 30.6% were not eligible for AAV-mediated gene therapy because of preexisting NAbs toward AAV8. 15 …”

Anti-AAV Antibodies in AAV Gene Therapy: Current Challenges and Possible Solutions

“…Moreover, depending on the vector production method and purification scheme, the final vector preparations can also contain empty viral particles, particles with truncated vector genomes or particles with plasmid or genomic DNA. All of those particles are essentially inactive virus, and their presence in an NAb assay will artificially lower the NAb titer ( 17 ). It is also noteworthy to point out that so-called “neutralizing antibody assays” also incorporate inhibition by other factors in the serum [e.g., galectin-3 binding protein ( 18 )] that inhibit transduction.…”