New Echota Biotechnology proposes to wed two highly promising technologies, CRISPR/Cas genome editing and cell-penetrating peptides (CPPs) to enhance genome editing in a wide array of cell lines, tissues and organisms. CPPs are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargos to the cell interior. CPP delivery of cargos to the interior compartments of cells is a potentially transformative platform technology for the development of therapeutics, diagnostics and research tools. This SBIR Phase 1 application proposes to develop an enhanced CRISPR/Cas9 product based on our novel CPP-adaptor technology. Our innovative approach to cargo delivery is the use of high affinity but reversible noncovalent binding to attach cargo to CPPs. We have designed CPP-adaptor fusion proteins consisting of the cell penetrating moiety from HIV transactivator of transcription or other CPP fused to calmodulin (CaM) or other EF hand proteins. Our prototypes bind CaM binding-site (CBS)-containing cargos with nanomolar affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell. The largest technical hurdle to development of CPP therapeutics is failure to escape from endosomes our technology solves this problem. We will deliver active Cas9/sgRNA complexes to the nuclei of mammalian cells using our proprietary CPP- adaptor technology, and using an established CRISPR assay, optimize delivery for efficiency of easily assayable editing. We will then use the optimized conditions to perform proof-of-concept experiments, knocking out F9, which encodes blood coagulation factor IX (FIX), defects in which cause hemophilia B. We will also design and execute a small mouse study to demonstrate efficacy of our approach in animals, modeling from a previous studies in which editing was effected via injection of CRISPR plasmids in tail veins. New Echota Biotechnologys (NEB) system has speed, safety and efficiency advantages over competing methods and ease of use and dosing advantages over other CPP methods. The significance of this application is that success will enable utilization of CPP-adaptors to enhance CRISPR technology in a way that overcomes currently intractable limitations.
Public Health Relevance Statement: PROJECT NARRATIVE This project seeks to develop its core technology to enhance the delivery of CRISPR/Cas gene editing components to living cells using cell penetrating peptides attached to adaptor molecules that bind payloads with high affinity and release them in the cell interior. CRISPR/Cas is currently limited by the need to transfect or transduce components into cells which is often problematic. Success will broaden the application of gene editing technology and further validate that our technology is an adaptable tool for delivery of a wide array of macromolecules, potentially improving the delivery of cancer therapeutics, antivirals, and proteins to ameliorate a variety of disorders as well as creating versatile research and diagnostic tools.
Project Terms: Address; Affinity; Animal Model; Animals; Antiviral Agents; Architecture; base; Binding; Binding Sites; Biological Assay; Biotechnology; Calcium; Calmodulin; Cell Compartmentation; Cell Culture Techniques; Cell Line; Cell membrane; Cell Nucleus; cell type; Cells; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Coagulation Process; Complex; CRISPR/Cas technology; Defect; design; Development; Diagnostic; Diagnostics Research; Disease; Dose; EF Hand Motifs; Endosomes; enzyme activity; experimental study; F9 gene; Factor IX; Failure; Funding; gene therapy; Genes; Genetic Diseases; Genetic Models; Genetic Transcription; Genome; genome editing; Hemophilia A; Hemophilia B; Hepatocyte; HIV; improved; in vivo; Injections; innovation; instrumentation; Investments; Isomerism; Knock-out; Licensing; Liver; macromolecule; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Modeling; Modernization; Modification; Mus; Mutate; Mutation; nanomolar; novel; Nuclear; Organ; Organism; Patients; Penetration; peptide drug; Peptides; Phase; Plasmids; Positioning Attribute; Problem Solving; Proteins; prototype; Reagent; Research; Research Contracts; research facility; Research Personnel; Rest; Risk; RNA; Safety; Small Business Innovation Research Grant; Speed; success; System; Tail; Technology; technology development; Therapeutic; therapeutic development; Tissues; tool; Toxic effect; Trans-Activators; Transfection; Veins
