There is substantial interest in gene editing as a means to treat human genetic disorders such as sickle cell disease (SCD). 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 ?-thalassemia produced almost complete amelioration of the disease, with clinically relevant ?-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 codon 6 of the ?-globin gene, the site of the sickle cell disease mutation. Herein, our central hypothesis is to establish the feasibility of a new minimally invasive and innovative therapeutic paradigm for sickle cell disease: application of further advances in nucleic acid chemistry and nanoparticle technology for the site-directed editing of SCD mutation in the ?-globin gene in vivo by facile IV infusion with high efficiency and low toxicity. This project will eventually help translate gene therapies for SCD to clinical practice through advances in nucleic acid chemistry and drug delivery. We will pursue Aim 1) Development of new generation chemically modified PNAs to boost gene editing at the SCD mutation site. The efficacy of the approach will be evaluated in a sickle cell disease mouse model. We will also explore the mechanism of PNA based gene editing.
In Aim 2) Identify novel nanotherapeutics based strategies to deliver reagent to HSCs by enabling penetrance into the bone marrow following simple IV infusion of NP. This work will lay the foundation for a novel gene editing therapy for SCD that has a high efficiency and much lower risk of off-target effects compared to existing nuclease based approaches.

Public Health Relevance

PNAs are synthetic pieces of DNA that can bind to the chromosomes of cells in a targeted manner and stimulate recombination of the target site with single-stranded donor DNAs to produce gene editing, with extremely low off target effects compared to other approaches. We will test the novel PNAs for correction of a sickle cell-associated mutation in the ?-globin gene in blood stem cells, using nanoparticle-mediated delivery via IV administration in mice. We expect this work to yield potent PNAs and nanoparticle based approach to achieve curative gene editing for sickle cell disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular and Cellular Hematology Study Section (MCH)
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Qasba, Pankaj
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University of Connecticut
Schools of Pharmacy
United States
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