R15: Enhancement of Genome Editing in Hematopoietic Stem Cells by Nucleic Acids Nanoparticles Delivery of CRISPR/CAS System

Editing hematopoietic stem cells (HSCs) at the genetic level is a rapidly progressing field that has the potential to revolutionize the treatment of genetic blood disorders and related diseases. However, the effective implementation of HSCs therapy is hampered by challenges in the lack of efficient delivery of the CRISPR/Cas9 system into HSCs. Novel technologies that improve delivery efficiency and enable precise gene editing, hold great promise in hematopoietic stem cell transplant (HSCT)-based therapy. Here, the overall goal of this R15 proposal is to develop nucleic acids nanoparticle (NANP) technology for efficiently delivering the CRISPR/Cas9 system into HSCs, to guide genome editing. Precise control of the geometry, composition, structural switch, and specific ligand-receptor interactions of NANPs, will significantly enhance the delivery efficiency of CRISPR/Cas9, and improve the targeting precision of gene editing, while also decreasing off-target effects and minimizing potential toxicity. We will employ multidisciplinary approaches to assess the delivery efficiency of NANPs and the outcomes of genome editing in HSCs and relevant cell lines. We will also demonstrate its potential for gene knockout (KO) and knock-in (KI) to treat sickle cell disease. Innovation: Compared to traditional methods, NANPs offer several innovative features, including: (1) enhanced cellular uptake by controlling the shape and size of NANPs and spatial organization of ligand-receptor interactions; (2) enabling direct delivery of CRISPR/Cas into target cells, eliminating the necessity for mRNA delivery and translation; (3) facilitated cytosolic release and delivery of Cas9/sgRNA to nucleus by switchable NANP structures; (4) minimized toxicity by optimizing NANPs’ composition. These distinctive NANP characteristics should enhance the efficiency of gene delivery, improving targeting precision and efficacy of gene editing in HSCs. The proposed research will employ an interdisciplinary approach, incorporating nucleic acid nanotechnology, computation-guided design, CRISPR technology, and stem cell biology. The knowledge gained will not only progress gene delivery and genome editing, but also contribute to the broader field of nanomedicine. Significance and impact: The significance of this research lies in its potential to revolutionize treatment of various genetic disorders by enabling precise genome editing in HSCs. Successful in vitro delivery of CRISPR/Cas9 to HSCs could enhance HSCT-based therapy, offering the opportunity to correct disease-causing mutations, introduce therapeutic genes, and bolster the body's natural defense mechanisms against genetic disorders. Developing efficient NANP-based gene delivery to HSCs not only has significant clinical implications but could also contribute to our broader understanding of stem cell biology and gene therapy. This research's broad impact would enhance patient outcomes and provide hope for individuals and families affected by genetic disorders. For R15’s educational goals, it would improve Biomedical Sciences and STEM education at Rutgers- Camden and the Coriell Institute by involving graduate and undergraduate students in all research stages.