. Hemoglobinopathies such as ?-thalassemia and sickle cell disease are genetic disorders caused by mutations in the HBB gene that codes for the ?-globin component of hemoglobin. Currently, the only gene therapy available for these prevalent hereditary diseases is based on transplantation of genetically corrected hematopoietic stem cells (HSPCs) from fully matched donors. However, the efficacy of this approach is limited by multiple factors. Gene editing is a promising alternative approach for curing hemoglobinopathies. Using this approach, synthetic mRNA-based drugs encoding nucleases that target the HBB gene can be utilized to permanently correct the patient?s DNA. Combining nanoparticle-based drug delivery with zinc- finger nucleases (ZFNs) has the potential to facilitate targeted gene-editing in HSPCs. However, the reliance on in vitro screening of nanoparticles impedes the discovery of safe and efficient in vivo delivery vehicles. Furthermore, current ZFN-mRNA based drugs targeting the HBB gene in HSPCs exhibit immunogenicity and are expressed in off-target cells. The PIs have recently been shown that DNA barcoded nanoparticles can ?evolve? nanoparticles to target endothelial cells more efficiently than hepatocytes directly in vivo. The team has also demonstrated that it is possible to (i) design low immune stimulating mRNA via nucleotide modification and HPLC purification, and that (ii) mRNAs can be designed to completely preclude translation in hepatocytes using rationally designed ?on? and ?off? switches. Based on these supporting data, it is posited that nanoparticles can be evolved to specifically target HSPCs while avoiding hepatocytes, and that ZFN- mRNA based drugs can be rationally optimized to generate safe gene editing therapeutics targeting HSPCs. Thus, the team proposes to create an mRNA-based drug that safely and specifically edits HSPCs in non-human primes in two phases. The development (UG3) phase will address 2 aims: (1) to iteratively evolve nanoparticles that target HSPCs and avoid hepatocytes in vivo, and (2) to reduce mRNA immunogenicity and improve cell type specific delivery to HSPCs. The demonstration (UH3) phase will address the aim (3) to analyze functional gene editing in non-human primates (Rhesus macaques). These will be achieved using a cutting edge multidisciplinary approaches recently developed. Specifically, the team will combine a DNA barcoded nanoparticle technology to screen 4,500 nanoparticles in vivo, synthesize mRNA-based drugs with low immunogenicity and cell type-specific expression, and utilize customized bioinformatics pipeline that facilitates ?big data? experiments with a statistical power new to nanomedicine. By creating an mRNA-based drug that safely edits HSPCs, the project is poised to advance gene editing as a viable therapeutic approach for curing genetic blood disorders and pave the way for clinical trials.
The only gene therapy currently available to patients with prevalent genetic blood disorders such as ?- thalassemia and sickle cell disease is based on transplantation of genetically corrected hematopoietic stem cells (HSPCs) from fully matched donors. However, the effectiveness of this approach is limited by factors that range from limited stem cell dose, quality, and gene transfer efficiency ex vivo to the toxicity of the conditioning regimens used to prepare patients for transplantation. The proposed studies will combine novel (i) in vivo nanoparticle barcoding screens and (ii) mRNA rationally designed for expression in HSPCs to create a mRNA-based drug that safely and specifically edits HSPCs in non-human primes, thereby advancing gene editing as a viable therapeutic approach for curing genetic blood disorders and paving the way for clinical trials.