Identification of Genetic Elements to Improve Liver-Specific Expression of a Novel Plasmid-Encoded Therapeutic Transgene

Abstract

As gene-encoded therapies advances to a stage where safety and efficacy translate to common standard of care for previously untreatable diseases, control of transgene expression is paramount. Gene control is exerted at a number of levels, however none more broadly applicable to the field of gene therapeutics then at the level of promoter design. By selecting the correct regulatory elements comprising a promoter one may precisely target a disease with tissue specificity at the afflicted cellular level, reducing the potential for off-target effects and limiting the liability of a therapeutic gene to inflict harm. Advances in bioinformatics have made the identification of these regulatory elements more precise and accessible. Chromatin immunoprecipitation (ChIP-Seq) coupled with Next Generation sequencing and in silico assembly have uncovered very exact regions of the genome responsible for tissue specific gene regulation. This study employed publicly available data from these modern tools to design, engineer, and test a novel liver specific promoter. After identifying CFHR2 as a gene highly enriched for expression in the liver further bioinformatical analysis of its genomic loci uncovered a region with strong indications of a gene regulatory element approximately 4kb upstream of the transcription start site. Epigenetic marks associated with gene regulation and evidence of active transcription were noted about this region as well as an abundance of transcription factor binding sites. A comparison of the CFHR2 core promoter alone to the inclusion of this 4kb region upstream of the CFHR2 core promoter however did not result in any increased expression of a transgene by luciferase reporter assay analysis. Ultimately these promoters demonstrated a 4-fold greater signal above background with minor off target expression in the kidney cell line NRK-52e. This off target expression was minor in comparison to a published liver specific promoter that demonstrated off target expression in several non-liver cell model systems.