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Rajeshwari D Koilkonda, William W Hauswirth, Vince Chiodo, Sanford L Boye, Phillip Gonzalez, John Guy; Evaluation of Pre-existing Neutralizing Antibodies Against Tyrosine-Mutant AAV2 in Leber Hereditary Optic Neuropathy (LHON) Patients . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):693.
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© ARVO (1962-2015); The Authors (2016-present)
Pre-existing neutralizing antibodies against adeno-associated virus (AAV), due to natural AAV infections could interfere with the recombinant AAV vector-mediated gene transfer, thus decreasing the efficiency and preventing effective vector administration. Here, we measured neutralizing antibody (NAb) titers against tyrosine-mutant AAV serotype 2 in Leber Hereditary Optic Neuropathy (LHON) patients.
Serum samples from LHON patients (n=53, age =33 + 1.9 yrs, average + se) were screened for the existence of NAbs to AAV2 using an invitro assay. For this, a self-complementary AAV2(Y444,500,703F)-smCBA-mCherry vector was pre-incubated with heat inactivated LHON patient serum samples at various dilutions, beginning with a 1:5 dilution and ending at 1:20,480. The serum-vector mixtures were used to infect RGC5 cells at multiplicity of infection of 5000 vector genomes/per cell. Two days post-infection, cell viability was confirmed by microscopy and transduction scored by flow cytometry. Expression of mCherry was quantified by multiplying the percentage of cells positive for mCherry by the mean fluorescence intensity. The neutralizing antibody (NAb) titer was reported as the highest serum dilution that inhibited self-complementary AAV2 (Y444,500,703F)-smCBA-mCherry transduction >50% compared to no serum control.
We found that the prevalence of NAbs to AAV2 (Y444,500,703F) among the LHON subjects was variable with majority, 49% (26/53) showing low NAb titers (< 1:80), 19% (10/53) showed moderate NAb titers (< 1:5120). However, 32% (17/53) showed a higher NAb titer <1:20,480. Prevalence of NAb’s was not associated to any specific age group (p>0.05).
Previous AAV2 exposure among LHON patients is low to high and these findings as they relate to the ongoing clinical human gene therapy trial (NCT02161380) will be discussed.
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