Approximately a quarter of patients with hereditary spherocytosis, a common inherited anemia, suffer from alpha- spectrin linked recessive HS (rHS), the most severe form of the disease. rHS patients present in infancy with life-threatening hemolytic anemia, many are transfusion-dependent. In these patients, splenectomy is only palliative; the only cure is hematopoietic stem cell (HSC) transplant. Development of genome engineering has expanded the repertoire of potential strategies for treatment of inherited erythrocyte disorders. We have developed a non-nuclease based gene editing technique using biocompatible and biodegradable nanoparticles (NP) encapsulating chemically modified peptide nucleic acids (PNAs) and donor DNAs. Using this strategy, we have cured a murine model of beta-thalassemia using IV injections of NP-PNAs, achieving gene correction of 6% after a single treatment with unmodified PNA, establishing proof-of-principle and demonstrating feasibility of our approach. This technique avoids ex vivo manipulation and its associated challenges, as well as obviates most of the genotoxicity associated with nuclease-based methods. This project leverages a multi-PI collaborative effort to develop the NP-PNA approach for gene editing in sph mice, a spontaneous murine model of rHS due to a point mutation in the alpha-spectrin gene. It addresses the hypothesis that NP-PNAs and donor DNAs can be used to correct the alpha-spectrin mutation in an in vivo model of HS at clinically relevant frequencies sufficient to ameliorate the HS disease phenotype with minimal toxicity and extremely low genomic off-target effects. We will establish robust protocols for in vivo DNA modification in hematopoietic cells after simple IV administration of NP-PNAs, providing a facile, non-toxic strategy for treatment of HS without the need for complex transplantation procedures or ex vivo manipulation. The goal of aim one is to optimize triplex-forming PNAs for site-specific gene editing of the alpha-spectrin gene. Relevant studies include assays of gene editing, off target effects, and genotoxicity. The goal of aim two is to identify and develop nanoparticle formulations for systemic in vivo editing of the alpha-spectrin gene in hematopoietic stem and progenitor cells (HSPCs). Relevant studies include development and characterization of NPs with novel size and polymer composition to improve target delivery of PNAs after systemic administration to HSPCs as well as improving penetration of the bone marrow compartment. The goal of aim three is the establishment of robust gene editing protocols for in vivo modification of the alpha-spectrin gene in HSPCs in alpha-spectrin deficient sph/sph mice to ameliorate or cure the HS phenotype. Results will be monitored by detailed laboratory analyses of the HS phenotype, including functional analyses of the erythrocyte membrane. Gene editing efficiency in hematopoietic stem cells and genotoxicity will also be analyzed. These approaches are widely applicable not only to other inherited disorders of the erythrocyte, but also to many disorders arising in the hematopoietic stem cell. Many of the observations from this work will likely extend beyond the field of hematology.
The goal of this project is to develop and test tools for gene engineering to treat hereditary spherocytosis, a common inherited anemia. This new type of gene therapy could have uses in many different diseases, including many different types of inherited anemia such as sickle cell disease.