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Principal Investigator: Gaurav Sahay
Organization: OREGON STATE UNIVERSITY
Fiscal Year: 2019
Award: $675,953
Funding agency: National Heart Lung and Blood Institute
ABSTRACT:
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. Although some of these reached Phase I-IIB clinical trials, they failed to produce consistent,
impactful improvement of patient outcomes. We developed a novel lipid nanoparticle (LNP)-based system to
deliver mRNA, and found it successfully restored up to 55% of normal CFTR-mediated chloride efflux in the
nasal epithelium of CFTR-deficient mice. To exploit and apply these novel discoveries, the long-term goal of this
project is to overcome two key remaining biological barriers that limit entry of all gene therapy vectors into the
lung: 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. Overcoming these two obstacles will require opposing particle characteristics: mucosal penetration
is achievable primarily by stabilizing particles with a muco-inert polymer, whereas intracellular bioavailability of
genetic payloads relies on particle destabilization. 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. We will meet these criteria, by altering the stability of the LNP core, while maintaining
a muco-inert LNP surface. We found that replacing cholesterol with its naturally-occurring analogues improves
intracellular gene delivery by 200-fold, vs. that seen with a clinically successful cholesterol-containing LNP. We
posit that this major improvement in gene transfer occur via modifications in the enhanced LNP (eLNPs) core
structure and trafficking. Modifications in eLNPs' core structure as we propose will enable disassembly,
endosomal escape and cytosolic delivery of mRNA, while maintaining their muco-inert surface properties. Our
translational project's goal is to analyze LNP structure and intracellular trafficking within the CF lung. Our
objectives are thus 1) elucidate and optimize the structural features of nanocarriers that drive endosomal
trafficking of eLNPs and enhanced gene delivery, 2) test and optimize the ability of altered core structures to
enhance mucopenetration across human CF sputum and state of art mice models, and 3) 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 model CF disease progression. Our novel approach alters internal
particle stability via small structural modifications of cholesterol, rather modifying LNP surface coating or cationic
lipid as in prior studies. Our discoveries will propel toward translation new non-viral vector carriers that can
traverse the thick sticky mucus and endosomal barriers to deliver genes for efficacious CF treatment.
Terms: <Aerosols><Apical><Atomic Force Microscopy><Bioavailability><Biologic Availability><Biological><Biological Availability><Body Tissues><Bronchoalveolar Lavage Fluid><CF mucus><CF patients><CF sputum><CFTR><CFTR Protein><Carrier Proteins><Cations><Cell Body><Cells><Characteristics><Chemicals><Chloride Channels><Chloride Ion Channels><Chlorides><Cholesterol><Clinical><Clinical Trials><Common Rat Strains><Complex><Confocal Microscopy><Cystic Fibrosis><Cystic Fibrosis Transmembrane Conductance Regulator><Cystic Fibrosis sputum><DNA><DNA Therapy><Dehydration><Deoxyribonucleic Acid><Disease><Disease Progression><Disorder><Dose><Drugs><ENaC><Electrons><Engineering><Epithelial><Exposure to><Force Microscopy><Gene Delivery><Gene Expression><Gene Therapy Agent><Gene Therapy Vectors><Gene Transduction Agent><Gene Transduction Vectors><Gene Transfer><Gene Transfer Clinical><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic Materials><Genetic defect><Goals><Half-Life><Histology><Human><In Vitro><Inflammatory><Inhalation><Inhaling><Investigation><Ions><KO mice><Knock-out Mice><Knockout Mice><Label><Lipids><Liquid substance><Location><Lung><Lung Respiratory System><Lung diseases><Measures><Mediating><Medication><Messenger RNA><Mice><Mice Mammals><Modeling><Modern Man><Modification><Morphology><Movement><Mucosa><Mucosal Tissue><Mucous Membrane><Mucous body substance><Mucoviscidosis><Mucus><Murine><Mus><Mutation><Nasal Epithelium><Nebulizer><Negative Beta Particle><Negatrons><Non-Polyadenylated RNA><Non-Viral Vector><Nucleic Acids><Null Mouse><Obstruction><Particle Size><Patient outcome><Patient-Centered Outcomes><Patient-Focused Outcomes><Patients><Penetration><Pharmaceutic Preparations><Pharmaceutical Preparations><Phase><Physiologic Availability><Polymers><Process><Production><Property><Pulmonary Diseases><Pulmonary Disorder><Pulmonary Fibrosis><Pulmonary Pathology><RNA><RNA Gene Products><Rat><Rats Mammals><Rattus><Recovery><Respiratory Disease><Respiratory System Disease><Respiratory System Disorder><Ribonucleic Acid><Safety><Scanning Force Microscopy><Sputum><Structure><Surface><Surface Properties><System><Testing><Therapeutic Effect><Thick><Thickness><Tissues><Toxic effect><Toxicities><Toxicology><Transfection><Translations><Transport Protein Gene><Transport Proteins><Transporter Protein><Variant><Variation><Viral><X ray diffraction><X ray diffraction analysis><aerosolized><analog><base><body movement><body water dehydration><cholesterol analog><cystic fibrosis mucus><cystic fibrosis patients><cystic fibrosis transmembrane regulator><cytokine><disease of the lung><disease phenotype><disorder of the lung><drug/agent><epithelial Na+ channel><epithelial sodium channel><fluid><gene correction><gene replacement><gene therapy><gene-based therapy><gene-corrected><genetic payload><genetic therapy><genome mutation><genomic therapy><genotyped patients><improved><in vivo><individuals with CF><individuals with cystic fibrosis><lipid nanoparticle><liposomal delivery><liposome delivery><liquid><loss of function><lung disorder><lung fibrosis><lung pathology><mRNA><mouse model><mucous><murine model><nano carrier><nano particle><nano-sized particle><nanocarrier><nanoparticle><nanosized particle><new approaches><next generation><nonviral vector><novel><novel approaches><novel strategies><novel strategy><overexpress><overexpression><particle><patients with CF><patients with cystic fibrosis><prevent><preventing><protein expression><protein function><pulmonary><restoration><success><surface coating><trafficking><uptake>