SBIR-STTR Award

An estimated $1.8 billion dollars is spent for each new successful drug developed, primarily in failed clinical trials due to lack of efficacy and safety. New approaches for rapid identification and early preclinical validation of novel therapeutic targets are crucial to make important 'go/no-go' decisions and curb the cost of developing new cancer treatments. For decades, genetically engineered mouse models have provided a powerful platform to study disease initiation and maintenance, the tumor microenvironment and the responsiveness of cancers to known or novel therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models with various cancer predisposing gene combinations have limited their practical utility in the drug discovery process. RNA interference (RNAi), a mechanism that controls gene expression, can be exploited experimentally to silence nearly any gene target. By expressing synthetic short hairpin RNAs (shRNAs) in mice, RNAi serves as a fast alterative to gene deletion. In theory, RNAi- GEMMs are a powerful platform to validate candidate cancer genes and drug targets, however, using traditional approaches, up to 2 years of extensive intercrossing would be required to produce experimental cohorts, thus preventing their routine use. Hypothesis: We hypothesize that RNAi-GEMMs of cancer can be developed rapidly and cost-effectively using our embryonic stem cell (ESC) rederivation method coupled with new genome editing technologies (TALENs/CRISPRs) to introduce additional sensitizing lesions and recombinase-mediated cassette exchange (RMCE) for precise integration of tetracycline inducible shRNAs to silence specific gene targets. Preliminary data: We have previously used this technique to generate entire experimental cohorts of functional mouse models of cancer without any breeding. Specific Aims: This project entails preclinical testing of RNAi-mediated Ptgs2 (Cox-2) inhibition in a new mouse model of Familial Adenomatous Polyposis (FAP) and determining its ability to model therapeutic administration of the Cox-2 inhibitor, Celecoxib, which reduces disease in mice and is the FDA approved treatment for FAP. In Specific Aim #1, we will use the CRISPR/Cas9 system to create a truncating mutation in the Apc tumor suppressor gene in newly derived ESCs, monitor the mice generated by blood sampling and macroscopically examine their intestines for polyp formation to confirm their ability to phenocopy the APCMin mouse model and mirror the human condition. In Specific Aim #2, we will introduce an shRNA targeting Cox-2 and compare its effects to the FDA approved therapy, Celecoxib, to further assess the therapeutic potential of our ESC-derived RNAi- GEMMs. Together, these studies will define a new paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAi-GEMMs that will serve as innovative research tools, guiding the development of novel and effective therapeutics.

Thesaurus Terms:
Adenomatous Polyposis Coli;Alleles;Antineoplastic Agents;Blastocyst;Blood Specimen;Breeding;Cancer Therapy;Cancer-Predisposing Gene;Celecoxib;Clinical Trials;Clustered Regularly Interspaced Short Palindromic Repeats;Cohort;Complex;Cost;Cost Effective;Coupled;Cox Models;Data;Derivation Procedure;Development;Disease;Disease Model;Drug Development;Drug Discovery;Drug Targeting;Early Identification;Embryonic Stem Cell;Es Cell Line;Evaluation;Fda Approved;Flexibility;Gene Combinations;Gene Deletion;Gene Expression;Gene Silencing;Gene Targeting;Generations;Genetic Engineering;Genetically Engineered Mouse;Genome;Goals;Human;In Vivo;Industry;Infancy;Inhibitor/Antagonist;Injection Of Therapeutic Agent;Innovation;Intestinal Polyps;Lead;Lesion;Maintenance;Malignant Neoplasms;Marketing;Measures;Mediating;Medicine;Methods;Modeling;Monitor;Mouse Model;Mus;Mutant Mouse Model;Mutant Strains Mice;Mutation;New Therapeutic Target;Novel;Novel Strategies;Novel Therapeutics;Oncogenes;Patients;Pharmaceutical Preparations;Phase;Phenocopy;Polyps;Pre-Clinical;Preclinical Testing;Predictive Value;Prevent;Process;Production;Public Health Relevance;Recombinase;Research;Research Personnel;Rna Interference;Safety;Side;Small Business Innovation Research Grant;Small Hairpin Rna;System;Techniques;Technology;Tetracyclines;Theories;Therapeutic;Time;Tool;Toxic Effect;Tumor;Tumor Microenvironment;Tumor Suppressor Genes;Validation;Xenograft Model;

New approaches for rapid identification and early preclinical validation of novel therapeutic targets are crucial to make important “go/no-go” decisions and curb the cost of developing new cancer treatments. Genetically engineered mouse models (GEMMs) are a powerful platform to study disease initiation and maintenance, the tumor microenvironment and the responsiveness of cancers to known or novel therapeutics; however, the long lead times and high costs required to develop, intercross and maintain models with various cancer predisposing gene combinations have limited their practical utility in the drug discovery process. Recently, we have shown RNA interference (RNAi) in mice can serve as a fast alterative to gene deletion and be exploited experimentally to silence nearly any gene target, by the expression of synthetic short hairpin RNAs (shRNAs). Importantly, because it is reversible, gene silencing by RNAi better mimics the dynamics of small molecule inhibition than permanent genetic knockouts. Furthermore, with the advent of new genome editing techniques, such as CRISPR/Cas9 technology, we are able to introduce additional sensitizing lesions to induce disease pathogenesis. In synergy with RNAi technology, complex multi-allelic ESC based GEMMs can be generated without extensive intercrossing. Using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in mice, giving us unprecedented capabilities to perform preclinical studies in vivo. Hypothesis: We hypothesize that CRISPR/Cas9-RNAi-GEMMs of cancer can be developed rapidly using new genome editing technologies (CRISPRs) to introduce additional sensitizing lesions and recombinase-mediated cassette exchange (RMCE) for precise integration of tetracycline inducible shRNAs to silence specific gene targets. Preliminary data: We have previously used CRISRP/Cas9 and RMCE to generate RNAi-GEMMs without any breeding. Specific Aims: As a proof-of-concept, we will develop a model of lung adenocarcinoma by using the CRISPR/Cas9 system to introduce a conditional KrasG12D allele into the endogenous locus and in situ delivery of sgRNAs targeting Trp53 which will be activated by a conditionally expressed Cas9 allele. We will further modulate mutant Kras or Mek1/2 activity by introducing tetracycline inducible shRNAs to model therapeutic inhibition. Finally, we will expand our flexible platform by producing validated, ‘off-the-shelf’ viral vectors carrying combination sgRNAs targeting commonly altered genes in NSCLC. Together, these studies will define a new paradigm and accelerate drug discovery research by creating a flexible platform for the generation of RNAi- GEMMs that will serve as innovative research tools, guiding the development of novel and effective therapeutics.

Public Health Relevance Statement:
Narrative The goal of this project is to revolutionize drug discovery research by developing a pipeline for the rapid production of genetically engineered CRISPR/Cas9-RNAi mouse models of cancer – powerful tools with combined features for both conditional gene-specific mutagenesis to drive development of genetically-defined cancers and inducible shRNA therapy to evaluate candidate targets for efficacy and safety, all within the same animal. This transformative platform technology will enable rapid and cost-effective creation of better model systems to identify and validate new targets, predict potential toxicities and ultimately lead to better therapeutic success in our fight against cancer.

Project Terms:
Adult; Alleles; Animals; Biological Models; blastocyst; Breathing; Breeding; Cancer Model; cancer therapy; Cancer-Predisposing Gene; cohort; Complex; cost; cost effective; CRISPR/Cas technology; Data; Development; Disease; Disease model; Doxycycline; drug discovery; Drug Targeting; embryonic stem cell; Engineering; ES Cell Line; Evaluation; fight against; flexibility; Gene Combinations; Gene Deletion; Gene Silencing; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genetically Engineered Mouse; genome editing; Goals; Guide RNA; In Situ; in vivo; Injection of therapeutic agent; innovation; Knock-out; Lead; Lesion; Lung Adenocarcinoma; Lung Adenoma; Maintenance; Malignant Neoplasms; Measures; Mediating; Modeling; mouse model; Mus; Mutagenesis; mutant; Mutation; new therapeutic target; next generation; Non-Small-Cell Lung Carcinoma; novel; novel strategies; novel therapeutics; Oncogenes; Oncogenic; Pathogenesis; Pathology; Pharmacotherapy; Phase; pre-clinical; preclinical study; Process; Production; Publishing; recombinase-mediated cassette exchange; repaired; Research; RNA; RNA Interference; Safety; Side; Small Business Innovation Research Grant; small hairpin RNA; small molecule; success; synergism; System; Techniques; Technology; Tetracyclines; Therapeutic; Time; tool; Toxic effect; TP53 gene; Treatment Efficacy; tumor microenvironment; Validation; vector; Viral Vector