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.
Refer: https://www.sciencedirect.com/science/article/pii/S2329050119300531#sec4
GFP-Based Anti-AAV5 NABs Assay
The assay entails incubation of the 1:50 dilution of test sera with an AAV5-based reporter vector that carries the GFP gene. This incubation allows any neutralizing antibodies, or other interfering factors present in the test serum, to bind to the reporter vector particles. These mixtures were subsequently transferred to wells seeded with HEK293 cells in a 96-well plate format, allowing non-neutralized reporter vector particles to transduce cells and express GFP. The cells were analyzed by flow cytometry for the percentage of GFP-expressing (and hence fluorescent) cells. Each analytical run included negative controls (control sample without AAV5-GFP reporter vector addition and pooled human serum control negative for anti-AAV5 NABs as determined during the assay development). Additional technical controls include a negative and positive assay control, consisting of monkey serum obtained pre- and post-immunization with AAV5-hFIX, respectively. The readout of the assay was the percent of inhibition of transduction, relative to normalized negative control serum. This percentage inhibition of transduction is then held against the pre-defined cut-point of 29%, which means that test sera that inhibit transduction by 29% or more are considered positive. Cut-point (cut-point = mean % inhibition + 2.33 × SD) was calculated at the 99% confidence level from the percent inhibition data obtained from the initial four test runs of 48 human sera samples screened in the development of GFP-based anti-AAV5 NABs assay.
Luciferase-Based Anti-AAV5 NABs Assay
The assay entails incubation of the test sera dilution series with an AAV5-based reporter vector that carries the luciferase gene. As in the GFP-based assay, this incubation allows neutralizing antibodies in the test serum to bind to the reporter vector particles. These mixtures are subsequently transferred to wells seeded with HEK293T cells in a 96-well plate format, where reporter vector particles can transduce cells and mediate expression of luciferase. After 2 h, the supernatants of each well are replaced by cell culture medium to maintain maximum cell viability. On the next day, all wells are analyzed for luciferase expression by luciferin substrate conversion-based chemiluminescent readout. The anti-AAV5 neutralizing antibody titer is determined with the use of LabKey software analysis that calculates the percent of neutralization for each serum dilution after subtraction of background activity and fits a curve to the neutralization profile. This curve is used to calculate neutralizing antibody titers, area under the curve (AUC), and error estimates. The four-parameter method is currently used to calculate curve fits. LabKey calculates IC50, the dilution at which the antibodies inhibit transduction by 50%. LabKey also calculates “point-based” titers. This is done by linearly interpolating between the two replicates on either side of the target neutralization percentage. Each analytical run includes positive controls (wells without sample sera but with AAV5-luciferase), negative controls (wells that have only medium, without sample sera and without AAV5-luciferase), and negative control sample serum (heat-inactivated fetal bovine serum [FBS]) to assess the specificity of AAV5-luciferase neutralization. MOI in the luciferase-based anti-AAV5 NABs assay was 378.4, whereas the target relative light units (RLUs) that were to be read in the luminometer after AAV5-luciferase transduction of HEK293T cells in the positive control wells were to be approximately 1000 RLUs, and negative control wells that consisted of only HEK293T cells would have reads of approximately 50 RLUs.
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