High-Throughput Screening for Lipid Nanoparticles Efficient for Systemic Delivery to Tissues Affected by DMD
Abstract
Majority of Duchenne muscular dystrophy (DMD) patients carry deletions and duplications in the DMD gene. These mutations abolish dystrophin expression. Loss of dystrophin expression is the fundamental cause of DMD. Recent studies from several laboratories suggest that DMD can be treated using the CRISPR technology in mice and dogs. All these studies have used adeno-associated virus (AAV) as the vector to deliver the CRISPR editing machinery to muscle. AAV is currently being used in systemic microdystrophin trials because AAV vector can (a) reach all muscles in the body and (b) lead to persistent expression of the gene it carries (e.g., the microdystrophin gene). While these are advantageous for dystrophin gene replacement therapy, persistent expression of Cas9 will cause problems to patients in the context of CRISPR therapy. CRISPR therapy relies on a protein called Cas9 to bypass the mutation in order to restore dystrophin expression. Cas9 is a bacteria protein. Persistent Cas9 protein expression leads to immune responses and untoward cutting at wrong places. A delivery vehicle that can reach all body muscles and yet only express Cas9 transiently will be ideal for DMD CRISPR therapy. Lipid nanoparticles are emerging RNA delivery vesicles, exemplified by their successful application in the delivery of mRNA vaccines for COVID-19. DMD CRISPR therapy machineries can be delivered in the form of Cas9 mRNA and guide RNA, the former expresses Cas9 protein which forms a complex with guide RNA to be fully functional. Cas9 mRNA and guide RNA cannot enter cells by themselves, and lipid nanoparticles would be ideal to help them get into human cells. A few studies successfully delivered CRISPR/Cas9 machineries to muscle tissues by local delivery, suggesting the potential application of lipid nanoparticles as a delivery tool for DMD CRISPR therapy. However, local delivery is not ideal for treating DMD which affects many muscles. Current lipid nanoparticles do not efficiently reach muscle tissues affected by DMD when delivered systemically, necessitating the development of new ones. It is found that tuning the components of lipid nanoparticles changes tissue preference, thus it is possible to find lipid nanoparticles with high delivery efficiency for DMD-affected organs. Unfortunately, lipid nanoparticle’s in vitro performance cannot predict in vivo performance due to the completely different environments. Although barcode-based high throughput screening methods are developed for in vivo screening of many lipid nanoparticles in one experiment, they cannot assay gene editing activity or provide cell type information. Most importantly, they cannot distinguish the 95% nonfunctional lipid nanoparticles trapped in endosomes from the 5% functional ones successfully escaping from the endosome and entering the cytoplasm, thus suffer from high false-positive results. Here we developed a novel barcode integration nanoparticle screen (BINS) method, which can assay gene editing activity, efficiently eliminate nonfunctional lipid nanoparticles from the screening, and provide cell type information. We will use BINS to screen for lipid nanoparticles that can deficiently deliver Cas9 mRNA and guide RNA into all muscle tissues affected by DMD. Aim 1: Will screen for lipid nanoparticles efficient for dystrophin-expressing cells, which will restore dystrophin expression for immediate therapeutic effects. Aim 2: Will screen for lipid nanoparticles efficient for Pax7-expressing muscle stem cells for sustained therapeutic effects. Restoring the DMD gene in muscle stem cells provide the possibly for the edited stem cells to regenerate the whole muscle tissue. Aim 3: Will use the best LNPs from preliminary study, Aims 1 and 2 to deliver CRISPR/Cas9 RNA to all muscles of humanized DMD model mice. We will examine how efficient the LNPs can deliver CRISPR/Cas9 RNA to edit DMD gene and restore dystrophin, in differentiated my
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310685
Entities
People
- Baisong Lu
Organizations
- United States Army
- Wake Forest University