(G-261) Kidney-Targeted Gene Delivery with Polymeric Mesoscale Nanoparticles

Introduction:: Kidney diseases, both acute and chronic, have few approved pharmacological interventions while affecting up to 20% of the population each year. With disease progression, acute kidney injury (AKI) can advanced to chronic kidney disease (CKD), which can advance to end-stage renal disease and/or renal carcinoma. Local inflammation, interstitial fibrosis, and oxidative stress are substantial contributors to both AKI and CKD. In our prior studies, we designed a polymeric mesoscale nanoparticle (MNP) system, approximately 350 nm in diameter, which targets the kidneys up to 26-fold greater than any other organ. We used that system to inhibit oxidative stress in chemotherapy-induced AKI through delivery of a small molecule reactive oxygen species scavenger. In these studies, we sought to adapt the MNP system to encapsulate gene therapies for longer-acting gene silencing and replacement therapy at the site of AKI/CKD.


Materials and Methods:: Using model gene therapies, we evaluated a two-step mixture-nanoprecipitation formulation to design MNPs that retain the appropriate size and chemistry necessary for kidney targeting while optimizing cargo loading. We investigated various excipients and cargo ratios for several classes of cargoes, including mRNA, siRNA, and CRISPR gRNA/Cas9 loaded within poly-lactic-co-glycolic polyethylene glycol di-block (PLGA-PEG) polymeric MNPs. We performed dynamic light scattering and electron microscopy to characterize nanoparticle size, polydispersity, and charge, as well as fluorescence intercalator assays to determine RNA loading. We then determined functionality in vitro using fluorescence reporter assays via fluorescence microscopy with renal epithelial cell lines. In ongoing work, we are using targeted gene therapies that reduce inflammation and oxidative stress in renal disease in order to halt disease progression in rodent models, including salt-sensitive hypertensive renal disease.


Results, Conclusions, and Discussions:: We found that an optimized formulation of nucleic acids with polymer exists for each cargo class while maintaining the 350 – 400 nm diameter of the PLGA-PEG MNPs required for renal targeting. Our in vitro studies revealed the ability to knockdown gene expression in renal cell lines with MNP-loaded siRNA and to delete gene expression with CRISPR gRNA/Cas9-loaded MNPs. We additionally demonstrated mRNA expression in vitro within one hour of incubation. In vivo, we have observed successful knockdown of target genes only in the kidneys with no off-target effects. We anticipate ongoing therapeutic efficacy studies will exhibit a reduction in blood pressure and increase in renal filtration associated with reduced CKD progression. We are hopeful that long-term work will lead to clinical trials in patients with hypertensive CKD and a reduction in the number of patients that progress to renal cancer or end stage renal disease.


Acknowledgements (Optional): : We acknowledge support from the CCNY-MSK Partnership for Cancer Research, NCI-funded under grants U54 CA132378 (CCNY) / U54 CA137788 (MSK).

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