Evaluation of anti-vector immune responses to adenovirus mediated lung gene therapy and modulation by aCD20

Although the last decade has seen tremendous progress in drugs that treat cystic fibrosis (CF) due to mutations that lead to protein misfolding, there are approximately 8-10% of subjects with mutations that result in no significant CFTR protein expression demonstrating the need for gene editing or gene replacement with mRNA or vector-based approaches. A limitation for vector-based approaches is the limitation of neutralizing humoral responses. Given that aCD20 has been used to manage post-transplant lymphoproliferative disease in CF subjects with lung transplants, we studied the ability of aCD20 to module both T and B cell responses in the lung to one of the most immunogenic vectors, E1-deleted adenovirus serotype 5. We found that aCD20 significantly blocked luminal antibody responses and efficiently permitted re-dosing. aCD20 had more limited impacts in the T-cell compartment, but reduced tissue resident memory T cell responses in bronchoalveolar lavage fluid. Taken together, these pre-clinical studies suggest that aCD20 could be re-purposed for lung gene therapy protocols to permit re-dosing. Overall design: For our scRNA-seq experiment, we compared naïve spleen cells from a publicly available database to interstitial T-cells isolated from mouse lung 4 weeks after lung-directed delivery of a human Adenovirus type 5 (HAdV) vector. To isolate mouse lung cells, mice were injected with anti-CD45.2-PE 2 minutes prior to euthanasia to label intravascular blood cells. Lungs were dissociated by mechanical disruption and digestion with collagenase I + DNAse. After filtration and RBC lysis, cells were subjected to magnetic negative selection using anti-PE microbeads and positive selection using anti-CD3 microbeads (Miltenyi). The resulting cells were analyzed by scRNA-seq, using a 5' VDJ and Gene Expression Profiling assay.