Neuronal targeting of tau using a mRNA‐encoded tau‐specific antibody fragment

Abstract

AbstractBackgroundA promising target for the treatment of Alzheimer’s disease and related tauopathies is intraneuronal tau. We have generated conventional tau‐specific monoclonal antibodies, as well as engineered antibody fragments, and demonstrated their ability to reduce tau pathology in the brains of tau transgenic mice (Nisbet et al., Brain 2017, Bajracharya, Brici et al., ANC 2021, Bajracharya et al. JCR 2022). Therapeutic efficacy is limited, however, by the inability of these antibodies to readily cross the blood‐brain barrier (BBB) and neuronal membrane to target intraneuronal tau. The SARS‐CoV‐2 pandemic revealed the potential of mRNA technology as an accessible and affordable strategy for delivering exogenous proteins, such as antibodies, to cells. The aim of this study was to explore the potential of utilizing mRNA to improve the intraneuronal delivery of our lead‐candidate tau‐specific monoclonal antibody in a single chain variable fragment (scFv) format.MethodThe cDNA encoding the tau scFv was cloned into an expression plasmid with a T7 promotor. The plasmid DNA was linearized, and in vitro transcription was conducted. The resulting mRNA was capped at the 5’ end with anti‐reverse cap analogue (ARCA), and possessed a 3’ poly (A) tail. The mRNA was then loaded into synthetic and biological nanoparticles and cellular uptake of the encapsulated mRNA and expression of the scFv was determined using mouse primary hippocampal neurons and human neuroblastoma SH‐SY5Y cells. Finally, binding of the expressed scFv to cytoplasmic tau was determined using a GFP‐pulldown assay.ResultHighly‐pure synthetic mRNA encoding a tau scFv was successfully generated and encapsulated into synthetic and biological nanocarriers. Upon treatment of cells with the encapsulated mRNA in vitro, the tau scFv was expressed and capable of engaging intracellular tau.ConclusionCollectively, our results validate the use of mRNA encoding tau‐specific antibodies as an effective and economic alternative to conventional antibodies for specific targeting of intracellular antigens such as tau.