Strategy to detect pre-existing immunity to AAV gene therapy

Abstract

Gene therapy may offer a new treatment option, particularly for patients with severe hemophilia, based on recent research. However, individuals with pre-existing immunity to adeno-associated viruses (AAVs) may be less likely to benefit from AAV vector-based therapies. To study pre-existing AAV5 immunity in humans, we validated two complementary, sensitive, and scalable in vitro assays to detect AAV5 total antibodies and transduction inhibition (TI). Using these two assays, we found that 53% of samples from 100 healthy male individuals were negative in both assays, 18% were positive in both assays, 5% were positive for total antibodies but negative for TI and, of interest, 24% were negative for total antibodies but positive for TI activity, suggesting the presence of non-antibody-based neutralizing factors in human plasma. Similar findings were obtained with 24 samples from individuals with hemophilia A. On the basis of these results, we describe the development of a dual-assay strategy to identify individuals without total AAV5 antibodies or neutralizing factors who may be more likely to respond to AAV5-directed gene therapy. These assays offer a universal, transferrable platform across laboratories to assess the global prevalence of AAV5 antibodies and neutralizing factors in large patient populations to help inform clinical development strategies.

Adenovirus‐associated antibodies in UK cohort of hemophilia patients: A seroprevalence study of the presence of adenovirus‐associated virus vector–serotypes AAV5 and AAV8 neutralizing activity and antibodies in patients with hemophilia A

“…Transduction inhibiton positive cut‐off titers were 44.9% for AAV5 and 38.4% for AAV8. These cut‐off values were determined by statistically analysing sufficient numbers of samples from healthy popluations, ensure distribution was normal by removing outliers and setting the desired rate of false positive results, generally 1%‐5% . No significant correlations were observed between the TAb and TI titers against either capsid, specifically AAV5 ( r = 0.68) or AAV8 ( r = 0.62) nor when comparing the inhibitor titer values between AAV5 vs AAV8 ( r = 0.56).…”

“…Despite progression in the current treatment of hemophilia A from plasma derived to recombinant products, natural exposure to AAV occurs at an early age, as shown in seroprevalence studies in both healthy and paediatric hemophilia populations . Therefore, screening may need to be considered for those most likely to respond to gene therapy with AAV vectors.…”

“…Total antibodies against AAV5 were measured in human plasma using a validated sequential bridging electrochemiluminescence (ECL) assay on the MSD platform as described previously . Sample results were expressed as an signal of noise (S/N) value, calculated by dividing sample ECL units by negative control ECL units.…”

“…Total antibodies against AAV5 were measured in human plasma using a validated sequential bridging electrochemiluminescence (ECL) assay on the MSD platform as described previously. 20…”

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

“…This poses a number of significant limitations to their standardization, as transduction efficiency is highly serotype‐dependent and, in general, the sensitivity of the assay decreases as the AAV dose increases, compromising the comparison of NAb titers between serotypes with distinct transduction efficiencies. As an example, the assay used by the authors to measure anti‐AAV5 NAbs requires an MOI of 25 000, supplemented with etoposide, an agent that promotes transduction, whereas the anti‐AAV8 NAb assay uses an MOI of 200 with no requirement for agents like etoposide . Other characteristics that impact NAb titers when evaluated using in vitro assays include the amount of serum used, the cell number on the plate and the reporter transgene .…”