ABSTRACT
Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Exons/genetics , Extracellular Vesicles/metabolism , Nanoparticles/chemistry , RNA, Guide, Kinetoplastida/metabolism , Base Sequence , Cell Survival , Dimerization , Gene Editing , Genetic Vectors/metabolism , HEK293 Cells , HIV Protease/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Ligands , Luciferases/metabolism , RNA Splicing/genetics , RNA, Catalytic/metabolism , Ribonucleoproteins/metabolism , Tissue Donors , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
PURPOSE OF REVIEW: To review the latest literature on otitis media diagnosis, guidelines, treatment, and pathophysiology. RECENT FINDINGS: Although otitis media remains one of the most common reasons for outpatient visits, antibiotic prescriptions, and surgery in the United States, little progress has been made in terms of developing novel treatments for the prevention and resolution of this condition, indicating the urgent need to continue investigations into the pathophysiology of this disorder. The recent past has seen the publication of new guidelines for the management of both acute otitis media, chronic otitis media and tympanostomy tube placement. Exciting technologies are being investigated into novel means to improve the diagnosis of otitis media, reviewed herein. Advancements in mucosal immunology and genetics have offered clues as to the underlying pathophysiology influencing otitis media propensity. Future research into modifying these pathophysiologic underpinnings, potentially through the usage of transtympanic drug delivery systems, should greatly influence the management of this condition. SUMMARY: Research into novel methods for otitis media pathophysiology, diagnosis and treatment has seen great strides in the recent past. Avenues towards markedly altering the evaluation and management of the condition are likely to be adopted into clinical practice over the coming years.
