Hematopoietic stem cells are capable of forming the diverse components of an individual's blood throughout his/her lifetime. Inherited blood disorders such as ?-thalassemia and sickle cell anemia can potentially be treated or cured through genetic manipulation of hematopoietic stem and progenitor cells (HSPCs). It has been shown that triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 bp "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations. However, translation of PNA based therapies to the clinic is limited by challenges in intracellular delivery, especially in difficult-to-transfect HSPCs. Our preliminary data shows that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can deliver PNA and donor DNA for site-specific recombination at a ?-thalassemia site in human HSPCs. Our central hypothesis is that biodegradable nanoparticles can be engineered to deliver PNAs for site-specific editing of a ?-thalassemia locus, with high efficiency, low toxicity, and increased cell-specific targeting to human HSPCs.
SPECIFIC AIMS : We will test our overall hypothesis through three specific aims.
Our first aim will test the hypothesis that uptake of nanoparticles in HSPCs can be enhanced by adding specific ligands to the particle surface. We will accomplish this aim by surface-modifying fluorescent nanoparticles with cell-penetrating peptides, and analyzing their internalization in human HSPCs in vitro.
Our second aim will test the hypothesis that direct in vivo delivery of nanoparticles to HSPCs can be enhanced by adding cell-specific ligands to the particle surface. We will accomplish this aim by surface-modifying fluorescent nanoparticles with antibodies targeting HSPCs, and analyzing their uptake into HSPCs after systemic injection into a mouse model reconstituted with human hematopoietic cells. Ligands tested in Aim 1 will also be used if successful.
Our third aim will test the hypothesis that nanoparticle optimization will enhance editing at the ?-thalassemia locus both in vivo and in vitro.
This aim will be accomplished by optimizing PNA and DNA loading in nanoparticles, and using surface modifications explored in Aims 1 and 2. Optimized PNA-DNA nanoparticles will be assessed for in vitro gene modifying activity in human HSPCs, and for in vivo gene modifying activity after systemic injection in a mouse model reconstituted with human hematopoietic cells. Since our preliminary work has established that our nanoparticle system works well in HSPCs, and each aim tests a separate hypothesis that builds on our proven system, all three aims will be pursued concurrently.
This project will help translate gene therapies for ?-thalassemia to clinical practice through advances in drug delivery and develop highly versatile methods for delivery of therapeutics to hematopoietic stem cells for treatment of blood disorders.
|McNeer, N A; Schleifman, E B; Cuthbert, A et al. (2013) Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo. Gene Ther 20:658-69|