Comparative analysis of three genetic constructs for delivery and expression of a modified single-domain antibody gene in rAAV

Objectives. To obtain and compare the efficiency of three recombinant adeno-associated virus (rAAV) variants expressing the gene of the modified single-domain antibody B11-Fc specific to botulinum toxin type A (BoNT/A): rAAV-DJ-CMV-B11-Fc, rAAV-DJ-CASI-B11-Fc, and scAAV-DJ-CMV-B11-Fc. Methods. The AAV-DJ Packaging System ( Cell Biolabs , USA) was used to create target constructs and obtain rAAV. Expression of the B11-Fc antibody gene in the obtained rAAV was assessed in vitro (HEK293, CHO-S, and C2C12 cell lines) and in vivo (BALB/c mice) using biolayer interferometry. The protective properties of the drugs were investigated on the model of lethal intoxication of mice with BoNT/A. Results. The rAAV-DJ-CMV-B11-Fc drug demonstrated a high level of B11-Fc antibody production both in vitro and in vivo without a significant decrease in concentration for at least 6 months. Comparable levels of B11-Fc production were demonstrated by rAAV-DJ-CASI-B11-Fc and scAAV-DJ-CMV-B11-Fc drugs in both in vitro and in vivo studies, with the exception of C2C12 cells, where rAAV-DJ-CASI-B11-Fc demonstrated the highest efficacy. When investigating the protective activity of the drugs against a lethal dose of BoNT/A, it was found that rAAV-DJ-CASI-B11-Fc possessed more pronounced activity in the first two days following administration as compared to rAAV-DJ-CMV-B11-Fc. However, at later stages, starting from 3 months, the rAAV-DJ-CMV-B11-Fc drug product demonstrated the most pronounced protection against high doses of BoNT/A. Conclusions. The obtained data show that rAAV-DJ-CASI-B11-Fc should be used for the induction of protection against BoNT/A at early stages (24–48 h) after administration, whereas for protection against the highest doses of BoNT/A in the long term, rAAV-DJ-CMV-B11-Fc should be used. Studies into the specific activity of the drugs at later stages after administration are still ongoing.