SBIR-STTR Award

Gene correction therapy is one of the most important application directions in regenerative medicine. Emerging technologies such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9), Zinc Finger Nuclease (ZFN), and Transcription Activator-Like Effector Nuclease (TALEN) have enabled efficient and precise gene editing in a wide spectrum of species, and hold promises for eventually achieving gene correction/therapy in therapeutic settings. However, several major challenges remain to be addressed, including low knock-in efficiency, off-targeting effect and lack of an efficient delivery system in vivo. The present proposal focuses on the challenge of low knock-in efficiency. Recently we reported that RS-1, a homology directed repair (HDR) enhancer improves the efficiency of Cas9 or TALEN mediated knock-in in rabbit embryos. Microinjecting human RAD51 (hRAD51) mRNA to the embryos mimicked the beneficial effects of RS-1 treatment. In the present project, we propose experiments to further improve nuclease mediated HR rates. In Aim 1, we will first develop a RAD51 augmentation method to improve Cas9 mediated HR. On RAD51, Threonine 13 (T13) and Serine 14 (S14) are the two best known sites that are phosphorylated/activated in DNA repair processes. So we hypothesize that the replacement of T13 and S14 with their phosphomimetics (T13E and S14D) and the use of such mutant RAD51 mRNAs will lead to consecutively active RAD51 which leads to enhanced Cas9-mediated HR rate. BRCA2 is a key player in HR. It is recruited to processed double strand breaks (DSBs), and facilitates the assembly of RAD51. In Aim 2, we will develop a TALE and BRCA2 exon27 fusion protein (TALE-BE27) to help recruiting RAD51 at the DSB to further improve the HR rate. In Aim 3, we will validate these HR improving methods in rabbit embryos. The proposal aims to address a bottleneck problem in regenerative medicine (i.e. low knock-in efficiency). Its success will have significant impacts on the entire field, as a majority of stem cell based therapy will require targeted gene modifications.

Public Health Relevance Statement:
Project Narrative Emerging technologies such as CRISPR (clustered regularly interspaced short palindromic repeats) associated protein 9 (Cas9) have enabled high efficient gene knockout (KO) in human cells and model animals. However, the knock-in efficiency remains to be further improved. We propose novel methods to improve the Cas9 mediated knock-in efficiency.

Project Terms:
Address; Animal Model; arm; ATPase Domain; base; Binding; Biomedical Research; Biotechnology; BRCA2 gene; Cell Line; Cell model; Cell Therapy; Cells; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; design; direct application; DNA; DNA Damage; DNA Repair; Elements; Embryo; Emerging Technologies; Enhancers; Exons; experimental study; Filament; gene correction; Gene Targeting; Gene-Modified; Genes; Genome; homologous recombination; Human; human pluripotent stem cell; improved; in vivo; inhibitor/antagonist; Knock-in; knockout gene; Mediating; Messenger RNA; Methods; Michigan; Modeling; Mus; mutant; Mutation; Nature; Nonhomologous DNA End Joining; novel; nuclease; nuclease I; Nucleoproteins; Organism; Oryctolagus cuniculus; Phase; plasmid DNA; Play; Process; Proteins; Recruitment Activity; Regenerative Medicine; repaired; Reporter; Reporting; response; Role; Serine; Site; Stem cells; success; Supplementation; Support System; System; Testing; Therapeutic; Threonine; tool; transcription activator-like effector nucleases; treatment group; Universities; Work; zinc finger nuclease

The 2020 Nobel Prize in Chemistry was awarded to Drs. Emmanuelle Charpentier and Jennifer Doudna fortheir development of a revolutionary gene-editing tool, CRISPR/Cas9. It allows precise edits to the genomeand has swept through the life science field. It has countless applications. Scientists hope to use it to developtherapeutic strategies for treating human genetic diseases. However, there are still several hurdles that need tobe overcome before achieving clinical applications. One of the major concerns is the undesirable insertion ordeletion (indel) events at off-target sites, as well as at the on-target site where the goal is to introductionprecise correction or mutation. Another aspect that remains to be further improved is the low efficiency ofknockin (KI) when a large size donor fragment is used, which is often below 1%. In Phase I of this STTRproject, we engineered the spCas9 protein by fusing a 36 amino acid long peptide encoded by BRCA2 Exon27 (Brex27), which has been reported to bind RAD51 to enhance homology-directed repair (HDR). We namedthis new variant the meticulous integration spCas9 (mi-spCas9), which possesses a unique combination ofdesirable features, including improving knock-in rates, reducing undesirable off-target events, and reducingundesirable on-target insertion or deletion (indel) events, providing a "one small stone for three birds" tool ingene editing. In this Phase II project, we propose studies to further engineer Brex27, to develop an AdenoAssociated Virus (AAV) friendly mi-saCas9 and demonstrate its clinically relevant applications. Specifically, i)in Aim 1, we will develop next-generation mi-Cas9s (mi-spCas9-v2) towards near-complete abolishment ofundesirable on-target and off-target indels; ii) in Aim 2, we will develop and optimize an AAV-friendly mi-saCas9 for in vivo gene editing; iii) in Aim 3, we will demonstrate the advantages of mi-Cas9s in clinicallyrelevant applications. We expect that mi-spCas9-v2 and mi-saCas9 lead to a multi-fold increase in gene knock-in rates and close to zero on-target and off-target indel rates. Completion of the proposed studies will enhancethe safety and efficacy of genome editing, propelling novel mi-Cas9 tools closer to an emerging multi-billion-dollar market of basic research and therapeutic.

Public Health Relevance Statement:
Project Narrative CRISPR/Cas9 has become a major tool in biomedical research and offers great promise for gene editing based therapies for many human diseases. Through Phase I, we have developed miCas9 which dramatically improves the efficacy and safety in gene editing. In Phase II, we propose to develop next generation mi-Cas9s, and to demonstrate the advantages of miCas9s in clinically relevant applications.

Project Terms: