Abstract
Purpose :
The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%-2% of cases with autosomal dominant retinitis pigmentosa (adRP) (PMID: 17564971). The mutant G56R allele in the first zinc finger of NR2E3 is hypothesized to have a reduced DNA-binding potential, while maintaining its interaction profile, thereby exerting a dominant-negative effect on the wild-type allele. Several attempts have been done to knockout or silence the mutant G56R allele using CRISPR/Cas or antisense oligonucleotide (AON)-mediated knockdown respectively. Here, we aimed to investigate single domain antibodies, also known as nanobodies, as macromolecular ligand analogues to modulate NR2E3 activity, in the absence of (a) known endogenous ligand(s) of NR2E3.
Methods :
The DNA-binding domain of NR2E3 G56R was expressed in E. coli and purified through tandem-affinity purification. Subsequently, the recombinant protein was used to immunize a llama for the generation of single domain antibodies. From the acquired nanobody bank, at least one nanobody from each family was selected for further validation. Furthermore, through recombinant co-immunoprecipitation of each nanobody with wild-type and mutant NR2E3, the presence of allele-specific nanobodies was assessed.
Results :
Thirty-three nanobodies targeting the DNA-binding domain of NR2E3 G56R were acquired from llama immunization and categorized into five families, from which ten nanobodies were selected. Through co-immunoprecipitation, we identified two nanobodies exhibiting an increased affinity for one of the variants. Therefore, our collection encompasses two allele-specific nanobodies capable of discriminating between wild-type and mutant NR2E3. In addition, these nanobodies have shown to stabilize their antigen upon co-immunoprecipitation, which is promising for upcoming experiments.
Conclusions :
As no endogenous ligands of NR2E3 are known, we hypothesized that single domain antibodies could be exploited as a modulator of G56R in the context of NR2E3-adRP. Our nanobody bank yielded two allele-specific nanobodies capable of discriminating the wild-type from the G56R NR2E3 allele. Taken together, this is the first step towards the use of single domain antibodies as an alternative to small molecules in treating NR2E3-associated adRP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.