NON-NUCLEASE BASED GENE EDITING FOR HUTCHINSON-GILFORD PROGERIA

Project Summary There is substantial interest in gene editing as a means to treat human genetic disorders such as Hutchinson- Gilford Progeria Syndrome (HGPS). Much effort has been focused on targeted nucleases such as CRISPR/Cas9, since site-directed DNA damage strongly promotes homologous recombination (HR). However,

clinical application of targeted nucleases is challenged by the risk of off-target cleavage in the genome, which can lead to carcinogenesis. As an alternative, we have shown that chemically modified triplex-forming peptide nucleic acids (TFPs) and donor DNAs (containing corrected base) delivered intravenously (IV) via poly(lactic-

co-glycolic) acid (PLGA) nanoparticles into a mouse model of human β-thalassemia produced almost complete amelioration of the disease, with clinically relevant β-globin gene correction frequencies in hematopoietic stem cells (HSCs) of up to 7%. TFPs can bind to duplex DNA in a sequence-specific manner and thereby stimulate

DNA repair and recombination. The mice showed alleviation of anemia, improvement in RBC morphologies, and reversal of splenomegaly and extramedullary hematopoiesis with extremely low off-target effects in the genome compared to nuclease-based approaches, a key advantage of this technology. The other key

advantage is that the components can be synthesized chemically and formulated into nanoparticles for simple IV administration. In the proposed work, we will test whether the same technology can be applied with the same efficiency for editing LMNA point mutation. Herein, our central hypothesis is to establish the feasibility of

a new minimally invasive and innovative therapeutic paradigm for HGPS disease: application of further advances in nucleic acid chemistry and nanoparticle technology for the site-directed editing of LMNA mutation in vivo by facile IV infusion with high efficiency and low toxicity. We will pursue two specific aims; Aim 1)

Development of new generation chemically modified PNAs to boost gene editing at the LMNA mutation site and in Aim 2) To test the gene editing efficiency at LMNA mutation site in vivo by simple IV infusion of PLGA NP. This work will lay the foundation for a novel gene editing therapy for HGPS that has a high efficiency and

much lower risk of off-target effects compared to existing nuclease based approaches.