Research Projects

Background: An estimated 415 million people worldwide suffer from diabetic kidney disease (DKD), which continues to be the primary cause of end-stage renal disease (ESRD). The prevalence of ESRD in DKD patients is still rising, and the remission of albuminuria has not surpassed 45% despite strict blood pressure or glucose control. In order to improve albuminuria and podocyte damage in DKD patients, it is imperative to investigate alternative therapy approaches. Hypothesis: We hypothesize that a nano-enabled targeted delivery of CRISPR-Cas9 plasmid that simultaneously knockout three of the lncRNAs, significantly upregulated in DKD, may be a viable therapeutic strategy against DKD. Objectives: In our 1st objective, we will design, synthesize and physicochemically characterize the targeted non-viral system for CRISPR-Cas9 delivery. The system will be made up of new ionizable lipids having polyunsaturated aliphatic backbones. The in vitro efficacy of the system in successfully delivering the CRISPR plasmid inside the damaged podocytes will then be monitored in our 2nd objective. Finally, in vivo efficacy of the system will be determined in DKD mouse models. We will further undertake a “See-Treat-See” approach in our final objective as we will additionally incorporate an exogeneous contrast probe inside the nanoparticle assembly for photoacoustic imaging. This system will therefore be capable of non-invasive monitoring of DKD remediation with the treatment progress. Study Design: To increase the safe and efficient uses of gene editing, CRISPR-Cas9 delivery using non-viral synthetic nanoparticles is of prime necessity. Herein, we attempt to develop a new class of ionizable lipids that are uniquely suitable for delivery of three lncRNA targeted and three respective guide RNA embedded CRISPR-Cas9 plasmid. The polyunsaturated backbone will presumably improve biocompatibility of the lipid nanoparticles (LNPs) in kidney. Arginine-Leucine-Aspartate (RLD) peptide tagged LNPs will specifically bind integrin αvβ3 overexpressed on the basal surface of damaged podocytes and will facilitate cellular entry. The LNPs will then ensure membrane destabilization and induce endosomal escape leading to efficient genome editing. The context-responsive nano-enabled delivery system will then be evaluated for their in vitro and in vivo efficacy in model DKD systems in downregulating the expression of three lncRNAs, i.e., plasmacytoma variant translocation-1 (PVT1), antisense RNA to INK4 locus (ANRIL) and ENST00000436340, simultaneously. Impact: Overall, we will attempt to knockout the expression of three lncRNAs by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system having adequate photoacoustic contrast. We believe that the successful execution of this project will revolutionize the treatment regimen for DKD and harbinger a more effective, less toxic strategy with improved survival rates.