The long-term goal of our laboratory is to develop efficient non-viral vectors for intracellular delivery of nucleic acids. Our focus is on three main research areas: 1. to understand cellular trafficking of nanoparticles in-vivo and develop approaches to overcome endosomal barriers; 2. to develop messenger (mRNA)-based gene therapy for the treatment of cystic fibrosis and eye disorders; 3. to exploit supramolecular assembly of peptide-based nanomaterials for cell type specific delivery.
Mechanisms of intracellular delivery
Intracellular delivery of messenger RNA (mRNA) boasts of rapid genetic transfer in recalcitrant cell populations and averts undesirable genomic integration. Lipid based nanoparticles (LNPs) are clinically approved delivery technology for delivery of nucleic acids. LNPs enter cells through highly dynamic endocytic pathways that are routed towards lysosomes for degradation. Endosomal sequestration of LNPs remains a formidable barrier for the delivery of nucleic acids to the cytosol. In studies with siRNA, even the most potent LNPs enable a mere <2% release of nucleic acid to its cytosolic target. The goal of this project is 1) Determine the gateways of cellular entry and endosomal escape of nanoparticles 2) Identify novel bioactive lipids that improve endosomal escape from productive endocytic compartments. We have shown that lysosome, once considered a dead-degradative organelle; controls spatiotemporal cell signaling that is essential for mRNA delivery. and identified bioactive lipids that breach endosomal barriers to boost intracellular delivery of modified mRNA. In another study, we found bioactive lipids that enhance LNP mediated gene transfection and have a polymorphic structure. We are understanding the overall structure of nanoparticles and their internal networks will enable new designs that lead to the improved subcellular release of genetic materials. This work is support through the NIBIB-1R15EB021581-01A (G.S).
Inhalable nanotherapeutics for gene delivery and treatment of cystic fibrosis
Cystic fibrosis (CF) is conferred by any of ≥1200 known mutations in the gene encoding the CFTR, an ion transporter protein, but in principle, a single gene therapy agent that delivers expression of functional CFTR could treat. Prior attempts to do so have used adeno-associated viral or liposomal delivery systems to deliver CFTR DNA but failed to produce consistent, impactful improvement of patient outcomes. LNPs are advantageous gene delivery agents because they are easy to generate, non-toxic, easy to scale and can deliver genes of any size. LNPs delivered CFTR mRNA, successfully restored up to 55% of normal CFTR-mediated chloride efflux in the nasal epithelium of CFTR-deficient mice). The long-term goal of this project is to overcome 1) the thick, sticky airway/lung epithelial mucus of CF that impedes gene carriers from reaching airway cells, and 2) inadequate cytosolic bioavailability of genetic material after uptake by cells, due to endosomal entrapment. There is urgent need to develop nanoparticles stable enough to cross CF mucus and reach airway cells, yet labile enough to facilitate endosomal escape of genetic material following cellular uptake. Based on our fundamental studies through modifications of LNPs’ core structure, we can enable disassembly and endosomal escape while maintaining a mucoinert surface. We will test LNPs ability to cross human CF sputum and deliver CFTR mRNA in CF state of the art mice models, and assess safety of sustained transmucosal transfection of CFTR mRNA via repeated aerosolization in CF rats, including toxicological analyses. These studies will significantly advance translational therapy, enabling long-term restoration of CFTR function to halt or reverse CF disease progression. This work is supported by NHLBI 1R01HL146736-01 (G.S) and Cystic Fibrosis Foundation (G.S) and is with collaboration with Dr. Kevin McDonald, a clinical CF specialist.
Nanomedicine based gene delivery to the eye
There are an estimated 100 million people around the world with advanced glaucoma, atrophic macular degeneration, advanced diabetic retinopathy and monogenic retinal degenerations. Each of these diseases results in the atrophy of retinal cells, which cannot regenerate. To treat a broad range of retinal disorders, non-viral vectors should 1) transduce degenerating retina using a less invasive injection technique that can be repeated throughout the patient’s life and 2) deliver genes of any size. We have shown that LNPs can deliver mRNA to the back of the eye after subretinal injections. We are engineering nanoparticles with various size and stability that can overcome barriers of the vitreous and traduce different regions of the retina in various animal models, including non-human primates. This work is being performed in collaboration with Dr. Mark Pennesi, a clinician with specialization in retinal degeneration and Drs. Trevor McGill and Martha Neuringer, experts in non-human primate eye biology. This work is supported through the Medical Research Foundation of Oregon (G.S).
Supramolecular assembly of peptide NanoDrills
Intracellular delivery of peptides can be used for selective targeting of tissues and organs. Through sequential ligation of peptide building blocks, we created cell penetrating self-assembling peptide nanomaterials that can form distinct shapes and morphology reminiscent of a “drill-bit”. These NanoDrills can deliver drugs in-vitro and in-vivo.