Editors' ChoiceGene Therapy

Neutralizing antibodies for AAV vectors: The strange case of AAV5

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Science Translational Medicine  19 Jun 2019:
Vol. 11, Issue 497, eaax9567
DOI: 10.1126/scitranslmed.aax9567


A clinical trial on hemophilia B demonstrated efficient liver targeting with AAV5 in the presence of neutralizing antibodies.

The recent approval by the U.S. Food and Drug Administration of gene therapies for eye and muscle disorders and the growing number of clinical trials with adeno-associated virus (AAV)–derived vectors clearly indicates the maturity of this method of gene replacement for the use in humans. The large clinical experience in liver gene transfer with AAV mostly derives from clinical trials for the treatment of two coagulation disorders, hemophilia A and B. Data from different trials strongly support the safety and the efficacy of the approach.

One major limitation of the systemic administration of AAV is the presence of preexisting neutralizing antibodies against the vector. Indeed, seropositivity for AAV is among the exclusion criteria in most of the AAV gene therapy trials. Early studies indicated that very low titers of neutralizing antibodies in circulation prevented vector entry and resulted in reduced liver transduction. So far, patients injected with AAV vectors had little to none anti-AAV neutralizing titers. This was true until the results of the clinical trial sponsored by uniQure for the treatment of hemophilia B with an AAV5 vector expressing human coagulation factor IX (hFIX) were public.

The assay used to measure preexisting neutralizing antibodies in the patients of the trial was based on green fluorescent protein (GFP) as a reporter and had a limited sensitivity. By using a more sensitive assay, based on luciferase as reporter, Majowicz et al. demonstrated that three of the patients included in the clinical trial were seropositive for AAV5 with titers that have been associated with in vivo neutralization of liver transduction in preclinical animal models. Importantly, in the patient who had the highest neutralizing titers, the expression of hFIX was similar, if not superior, to that of patients of the same dose cohort. This suggests that the serotype used in the trial, for some reason, was less sensitive to antibody neutralization. To support the clinical data, they performed an in vivo neutralization assay in nonhuman primates (NHPs). In this experiment, they tested four increasing doses of vector in a range that cover the doses of AAV normally administered in the clinic. The NHPs dosed were all seropositives for anti-AAV5 neutralizing antibodies with titers spanning from low (1/57) to relatively high (1/1030). Interestingly, they did not see the expected inverse correlation between the neutralizing titers and the transduction efficacy as measured by hFIX protein secreted in circulation.

One important caveat in the interpretation of the results of this study is the absence of standardized methods to measure vector and neutralizing antibody titers. This represents a major limitation in the field of gene therapy that hampers the comparison of data between the different laboratories and the exact reproduction of the data. Despite this, in 2018, Biomarin announced the dosing of the first hemophilia A patient having anti-AAV5 neutralizing titers. An eventual confirmation of the data obtained from Majowicz and colleagues in this second clinical trial would strongly suggest that AAV5 has a certain “resistance” to neutralization. This resistance, in principle, may allow for the inclusion of seropositive individuals, thus expanding the number of patients treatable by AAV gene therapy.

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