Subcutaneous delivery of hepatocyte targeted sub-50 nm nanoencapsulated siRNA mediates gene silencing
Authors: Kren BT , Korman VL , Tobolt DT , Unger Gretchen 
Published: October 2023

Gene augmentation for hepatocyte based metabolic disease has been a long-term goal of gene therapy. Subcutaneous (sq) administration of plasmid DNA is a desirable goal for patient-friendly genetic manipulation of target cells in chronic disease. However, two major delivery challenges that have impeded its development are avoiding sequstration at the injection site and in the target cells endosomes with subsequent degradation. A novel nonviral delivery technology developed by GeneSegues utilizes sub-50 nm crystalized capsules for intact delivery of nucleic acids (NA) from 19 bp to 15 kb. The capsule's crystalized shell formulated from any appropriate targeting protein, peptide or carbohydrate ligand specifies the uptake of the capsule by the specific cell-type in vivo via the receptor targeted by the capsule ligand shell. The nanocapsule's size, structure and charge mediates efficient in vivo penetration of tissues and cellular uptake via the non-endosomal lipid rafts pathway, delivering the NA cargo intact to the nucleus and cytoplasm of the target cell. We have previously demonstrated that asialoorasomucoid (ASOR) s50 capsules mediate hepatocyte specific delivery of transgenes in vivo following intravenous (iv) administration (J Clin Inv 119:2086, 2009). Here we show data comparing various sq regimens to iv administration for delivery of a secreted alkaline phosphatase (SEAP, pCpgSEAP) reporter packaged into ~ 10-15 nm ASOR s50 capsules. We found that RNA production at 48 hrs after final dose was approximately 15 – 25% of that produced by a maximum volume iv regimen. Target RNA production levels were similar between equivalent dose sq regimens with different schedules. Importantly for clinically relevant delivery, fluorescence microscopy showed siimilar uniform gene expression only in hepatocytes for both iv and sq liver sections. In contrast, higher expression levels were observed with a second reporter pcDNA3.1/LacZ in sq liver sections potentially reflecting differences in gene regulation between the plasmids. To study sq administration of s50 capsules independently of cargo transgene expression we used ASOR capsules carrying Dysprosium (Dy)-chelated dextran. Dy is a large nuclei, fluorescent lanthanide enabling sensitive isotopic measurements of tissues by neutron activation analysis of Dy content. A cohort of mice were injected sq over a 5 day period held in metabolism cages for the final 24 hrs. Over 5 days, ~ 4.3% of the injected dose accumulated in the liver (based on summation of 24-hr collection points for the 5 days) peaking at 3 days postinjection. Of note, no uptake was observed in spleen, kidney, lung, heart or testes. These data support the view that sq delivery of ASOR targeted s50 capsules to hepatocytes provides a depot effect, with prolonged availability of cargo and possible avoidance of duration-adverse effects such as saturation of cellular uptake, intracellular enzymatic degradation of cargo, etc. In conclusion, these studies provide a solid framework for further development of the nonviral ASOR s50-mediated sq nucleic acid based gene therapies for hepatocyte-based liver disease. Abstract 67