SBIR-STTR Award

Direct germline editing in donor spermatogonial stem cells will streamline production of genetically modified model organisms and provide unprecedented quality control to accurately predict germline transmission rates for individually selected, or multiple, co-selected targeted alleles. GenomeDesigns' direct germline editing technologies will transform the custom rat model market by providing the broadest range of targeted genomic DNA insertion sizes available in the broadest range of tractable rat genetic backgrounds. Here, we propose three specific aims to produce frozen spermatogonial stocks that can be used as germline vectors to build custom designed, genetically modified rat models. In Specific Aim 1, spermatogonial stocks derived from up to 12 different popular outbred and inbred rat genetic backgrounds will be generated to seed GenomeDesigns with the most comprehensive catalogue of custom designable rat models. In Specific Aim 2, precise spermatogonial gene targeting efficiencies will be measured by droplet digital PCR (ddPCR) to guide animal production protocols, benefiting individual customers and GenomeDesigns. We will apply ddPCR to project real production cost and time savings received by co-transmitting, co-selected gene targeting events from an individual germline, or by polyclonal rat production where distinct spermatogonial lines are pooled and transplanted into a single recipient rat. In Specific Aim 3, developing recipient males compatible with diverse donor spermatogonial strains will build an unmatched portfolio of biomedically relevant rat strains amenable to genetic engineering. By completing Phase I Specific Aims, GenomeDesigns will provide proof-of-concept for building a genetically diverse catalogue of spermatogonial stocks that uniquely enable strong genetic selection for precise, sexually transmittable, genomic modifications directly in the rat germline. Benchmarks for this Phase I SBIR project are: 1) Significantly expand spermatogonial gene targeting services into new rat genetic backgrounds 2) Establish ddPCR as prognostic for germline transmission of co-selected gene targeting events 3) Produce recipient rat models for diverse donor rat spermatogonial genetic backgrounds

Public Health Relevance Statement:
Narrative This project will facilitate the production of clinically relevant rat models to study biological processes important for human health. The project will positively impact biomedical discovery in numerous, diverse biomedical fields allowing researchers to study relationships between gene function and human health using the rat as a genetic model.

Project Terms:
Adult; Alleles; Allogenic; Animal Model; Animals; Benchmarking; Biological Assay; Biological Process; Biomedical Research; Businesses; Catalogs; Cell Culture Techniques; clinically relevant; cost; Custom; design; digital; Event; Fertility; Freezing; gene function; Gene Targeting; Genes; Genetic; Genetic Engineering; Genetic Models; genetic selection; Genomic DNA; Genomics; germline stem cells; Health; Human; Inbreeding; Individual; male; Measures; Methods; Modeling; Modification; mutant; novel; Organism; Phase; Production; prognostic; Protocols documentation; Quality Control; Rat Strains; Rattus; Research Personnel; Savings; Scientist; Seeds; Services; Small Business Innovation Research Grant; sperm cell; Stem cells; Technology; Testis; Time; transmission process; Transplantation; vector

Rat research models are established to investigate a broad spectrum of human health issues significant to most NIH Institutes and are reported in >1.5 million publications listed on NCBI PubMed Central. Direct germline editing products and services generated by this SBIR project will streamline production of the most sophisticated, genetically designed rat models used to study gene, cell and organ function. Robust gene targeting by homology directed DNA repair in potent rat spermatogonial stem cell lines embodies technology GenomeDesigns Laboratory, LLC. (GDL) uses to introduce custom designed genomic modifications directly into the rat’s germline. Genetically modified rat germlines are transplanted into rat testes to produce fertilization-competent spermatozoa. Genetically engineered donor germline-derived spermatozoa then sexually transmit targeted alleles by breeding to generate new rat strains that harbor heritable genomic modifications needed to investigate leading biomedical questions. Phase I experiments have demonstrated feasibility to: 1) derive functionally robust, genetically tractable rat spermatogonial lines from diverse rat genetic backgrounds, 2) predict donor germline transmission rates prior to animal production, and 3) sexually transmit donor germline alleles on diverse rat genetic backgrounds from male-sterile recipients. Phase II will deliver Carrier Sperm Technology, Gene Sculptor Sperm Technology and Tissue Tracker Germlines into the model organism market. Carrier Sperm and Gene Sculptor Sperm technologies will establish spermiogenetic services exclusively offered by GDL for generating rat models that sexually transmit relatively large, allele-specific, targeted germline genomic insertions. Tissue Tracker Germlines will provide novel services for producing rat models with inducible gene regulation in target cell types of interest. Thus, Phase II deliverables will empower research requiring highly controlled, tissue-dependent and/or inducible transgene regulation using a wide assortment of inbred and outbred rat strains. Benchmarks for this Phase II SBIR project are: Specific Aim 1: Standardize Carrier Sperm Technology for transgenesis in popular rat strains Specific Aim 2: Launch Gene Sculptor Sperm services for in germline selectable marker auto-excision Specific Aim 3: Produce Tissue Tracker Germlines for inducible transgene regulation in rats

Public Health Relevance Statement:
Narrative This project will facilitate the production of clinically relevant rat models to study biological processes important for human health. The project will positively impact biomedical discovery in numerous, diverse biomedical fields allowing researchers to study relationships between gene function and human health using the rat as a genetic model.

Project Terms:
Alleles; Animal Model; Animals; Benchmarking; Biological Process; Breeding; Catalogs; Cell Line; Cell physiology; cell type; Cells; clinically relevant; Custom; design; Development; Devices; DNA; DNA Markers; DNA Modification Process; DNA Repair; Engineering; Excision; experimental study; Fertilization; Gene Activation; Gene Expression Regulation; gene function; Gene Silencing; Gene Targeting; Gene Transfer; Gene Transfer Techniques; Genes; Genetic; Genetic Engineering; Genetic Models; genetic selection; Genomics; Haplotypes; Health; Heritability; Human; Inbreeding; Institutes; interest; Laboratories; Male Sterility; model design; Modeling; Modification; novel; Organ; Partner in relationship; Performance; Phase; Procedures; Production; Publications; PubMed; Rat Strains; Rattus; recombinase; Regulation; Reporting; Research; Research Personnel; Scientist; Services; Sexual Transmission; Small Business Innovation Research Grant; sperm cell; Spermatids; Spermatogonia; Standardization; Stem cells; success; System; Techniques; Technology; Technology Transfer; Testis; Tissues; Transgenes; transmission process; Transplantation; United States National Institutes of Health