Project Grant F31NS151915

ENGINEERING AN SIRNA-PEPTIDE CONJUGATE FOR ENHANCED BIODISTRIBUTION AND NEURON TARGETING IN THE CNS - PROJECT SUMMARY NEURODEGENERATIVE DISEASES AFFECT MILLIONS WORLDWIDE AND ARE CHARACTERIZED BY THE PROGRESSIVE LOSS OF NEURONS IN DEFINED BRAIN REGIONS. THESE DISORDERS ARE DRIVEN BY ABERRANT GENE EXPRESSION AND ACCUMULATION OF TOXIC PROTEINS, THEREFORE THERAPIES THAT CAN SELECTIVELY SILENCE DISEASE-ASSOCIATED TRANSCRIPTS HOLD PROMISE FOR SLOWING OR HALTING DISEASE PROGRESSION. SMALL INTERFERING RNAS (SIRNAS) ENABLE POTENT, SELECTIVE, AND DURABLE GENE SILENCING, BUT THEIR TRANSLATION FOR CENTRAL NERVOUS SYSTEM (CNS) DISEASES IS LIMITED BY POOR DELIVERY ACROSS CNS BARRIERS AND INEFFICIENT NEURONAL UPTAKE. CEREBROSPINAL FLUID (CSF) ADMINISTRATION, THE CURRENT CLINICAL ROUTE FOR OLIGONUCLEOTIDE DELIVERY, RESULTS IN RAPID CLEARANCE, LIMITED PERSISTENCE, AND MINIMAL CELLULAR UPTAKE, UNDERSCORING THE NEED FOR IMPROVED DELIVERY STRATEGIES THIS PROJECT AIMS TO DEVELOP A LIPID-SIRNA-PEPTIDE CONJUGATE PLATFORM FOR TARGETED NEURONAL GENE SILENCING. BUILDING ON OUR LABORATORY'S LIPID-SIRNA SYSTEM THAT EXPLOITS PERIVASCULAR FLOW PATHWAYS TO ACHIEVE BROAD CNS DISTRIBUTION AND SUSTAINED GENE KNOCKDOWN, THIS WORK SEEKS TO OVERCOME THE REMAINING CHALLENGE OF LIMITED NEURONAL UPTAKE. I WILL ENGINEER A LIPID-SIRNA-PEPTIDE CONSTRUCT DESIGNED TO ENABLE CARRIER-FREE, RECEPTOR- MEDIATED NEURONAL DELIVERY FOLLOWING CSF ADMINISTRATION. THE CONJUGATE WILL INTEGRATE BACKBONE CHEMICAL MODIFICATIONS FOR STABILITY, A LIPID MOIETY TO PROMOTE CNS TRANSPORT, AND A NEURON-TARGETING PEPTIDE TO FACILITATE UPTAKE BY HIPPOCAMPAL AND CORTICAL NEURONS, WHICH ARE KEY POPULATIONS AFFECTED IN NEURODEGENERATIVE DISEASE. THE PLATFORM WILL FIRST BE OPTIMIZED THROUGH IN VITRO SCREENING OF PEPTIDE CONJUGATES FOR STABILITY, UPTAKE, AND MRNA KNOCKDOWN EFFICIENCY. OPTIMIZED CANDIDATES WILL THEN ADVANCE TO IN VIVO STUDIES TO ASSESS BIODISTRIBUTION, CELL-SPECIFIC UPTAKE, AND GENE SILENCING EFFICACY IN THE BRAIN. TOGETHER, THESE STUDIES WILL DEFINE HOW INTEGRATING TRANSPORT-PROMOTING LIPIDS WITH RECEPTOR-TARGETING PEPTIDES CAN ACHIEVE BOTH WIDESPREAD DISTRIBUTION AND NEURON- SELECTIVE DELIVERY OF RNA THERAPEUTICS. BY ENABLING DURABLE, CELL-SPECIFIC SILENCING IN THE CNS, THIS WORK WILL DIRECTLY ADDRESS ONE OF THE KEY BARRIERS TO RNA-BASED THERAPIES FOR NEURODEGENERATIVE DISEASES. THE MODULAR DESIGN OF THE LIPID-SIRNA-PEPTIDE FRAMEWORK WILL ALLOW FACILE SUBSTITUTION OF TARGETING LIGANDS, ESTABLISHING A GENERALIZABLE PLATFORM FOR PRECISION NUCLEIC ACID DELIVERY ACROSS DIVERSE CNS CELL TYPES.