A Novel High-Throughput Lipid Nanoparticle Barcode Surveying Method for Developing Nanoparticles for Friedreich s Ataxia Genome Editing
Abstract
This proposal addresses the Peer Reviewed Medical Research Program (PRMRP) Topic Area Portfolio of Neuroscience, Topic Area Friedreich’s Ataxia, and the Strategic Goal of Treatment, focusing on developing novel treatment strategies for Friedreich’s ataxia. Friedreich’s ataxia is the most frequent hereditary ataxia caused by mutations in both copies of the frataxin gene. The disease affects multiple organs including the nervous system, the heart, and the metabolism system. Over 90% of the patients are caused by the expansion of the tri-nucleotide repeat in the non-coding region of the frataxin gene on chromosome 9. The gene products of frataxin are present in the heart, spinal cord, liver, skeletal muscle, and pancreas, and this explains why mutation of this gene affects multiple organs. The expanded tri-nucleotide repeat decreases the gene products of frataxin, leading to decreased enzymatic activities of mitochondria, the powerhouse of human cells. There is currently no cure or effective treatments for Friedreich ataxia. Providing a normal copy of the gene by viral vectors in disease model animals was observed to improve the phenotypes for a short time. However, too much frataxin protein expressed from the viral vectors was found to be toxic to mammalian cells. Using CRISPR/Cas9, the designer nuclease, to remove the expanded tri-nucleotide repeat, a process called genome editing, has been shown as a promising strategy. However, the lack of efficient strategy to send the nuclease into human cells hinders the clinical application of this strategy in Friedreich ataxia treatment. Nanoparticles are a promising tool for sending CRISPR/Cas9 nucleases into human cells. However, currently they are only efficient for sending nucleases to the liver but not to most of the other deep organs. To treat Friedreich ataxia effectively, the nuclease has to be sent to all organs affected by this disease, including the brain, the heart, and the pancreas. Current nanoparticles are inefficient in doing so, especially in sending to the brain due to the presence of the blood-brain barrier. This project aims to develop nanoparticles for sending CRISPR/Cas9 nuclease into multiple organs affected by Friedreich ataxia. By changing the compositions of the nanoparticles, the nanoparticles can be sent to one organ more efficiently than the other. This means that when screening enough types of different nanoparticles, one may find nanoparticles efficient in sending CRISPR/Cas9 nuclease to organs affected by Friedreich ataxia. However, current screening methods are not designed for this purpose. In this project, we will develop a novel screening method that can directly compare the gene-editing activities of hundreds types of nanoparticles in one experiment. Our method uses a unique barcode to label each type of nanoparticles. If the nanoparticles are efficient in sending the nuclease into the target organs, the barcode will be efficiently inserted into the genome of the cells. By identifying all barcodes inserted into the genome, which is easily achievable by next-generation sequencing, we can find the most efficient nanoparticles for a specific target organ. We will use this method to find the most efficient nanoparticles for sending the CRISPR/Cas9 nuclease into the organs affected by Friedreich ataxia and will test the application of these nanoparticles in removing the tri-nucleotide repeat in Friedreich ataxia model mice that show phenotypes similar to human Friedreich ataxia. This project is novel in that it will develop a new method to directly compare the genome-editing activities of hundreds types of nanoparticles in one experiment. This enables the development of nanoparticles that can efficiently send CRISPR/Cas9 nuclease into multiple organs affected by Friedreich ataxia. Our future plan is to use the nanoparticles found in this project to send CRISPR/Cas9 nuclease into Friedreich ataxia animal models and examine the improvement of
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310050
Entities
People
- Baisong Lu
Organizations
- United States Army
- Wake Forest University