Charcot-Marie-Tooth (CMT) disease is the most frequent inherited neuropathy affecting the peripheral nervous system and is characterized by a group of genetically and clinically heterogeneous disorders leading to progressive weakness and atrophy in distal muscles, sensory loss, hyporeflexia and skeletal deformity. CMT type 1A (CMT1A) is the most prevalent form, affecting 1 in 10,000 people, and is associated with a 1.4-Mbp duplication in the chromosome 17p11.2 region, which contains the peripheral myelin protein 22 (PMP22) gene. PMP22 is essential for the structure, development and maintenance of peripheral nerve myelin. PMP22 overexpression prompts cycles of demyelination-remyelination resulting in dysfunction in Schwann cells. Due to the association of PMP22 gene dosage with neuropathic phenotypes, therapeutic strategies are primarily focused on repressing PMP22 overexpression. RNA therapeutics, like antisense oligonucleotides (ASO) and siRNA, are attractive because they target messenger RNA and thus can modulate the expression of protein targets inaccessible to other therapeutic modalities. Challenges identifying safe and effective ways to deliver RNA therapeutics into cells outside the liver have limited the clinical deployment of this promising therapeutic class. For instance, a recent study used an ASO to decrease PMP22 mRNA in affected nerves, improving phenotypes in rat and mouse models of CMT1A. However, very high drug doses (multiple 100 mg/kg doses) were required to see a beneficial effect. At such high doses, the risk of toxicities related to ASO treatment, such as thrombocytopenia and renal dysfunction, preclude further development. DTx Pharma has identified a fatty acid motif that when covalently coupled to siRNA/ASO results in efficient delivery to multiple cells and tissues, including sciatic nerve (relevant for CMT), resulting in potent repression of target gene mRNA expression. Herein, we propose to explore whether DTx technology can be applied to PMP22-targeting siRNAs to correct its overexpression in Schwann cells in a mouse model of CMT1A, providing strong proof of concept for designing future therapeutic efficacy studies. We will explore this in 2 aims. Aim 1 will screen a library (~36 in addition to what weve screened to date) of siRNA candidates targeting PMP22 in vitro using both primary human Schwann cells and HEK293 cells engineered to express human PMP22 to identify potent and non-toxic siRNAs that will be conjugated to the DTx motif and further validated in vitro. In Aim 2, the 10 most active hits from aim 1 will be dosed in two parallel studies via intravenous or intrathecal administration in C3-PMP22 mice, a model of CMT1A, to assess dosing route, safety and target engagement in the sciatic nerve and Schwann cells. The more successful dosing route will be utilized in follow-on studies to explore dose range and duration of action for suppressing PMP22 expression to wildtype levels. Data from these studies will help us understand if DTx PMP22 siRNA is a viable approach for treatment of CMT1A and provide the foundation for a phase 2 SBIR grant focused on efficacy trials in rodents, non-GLP toxicity studies and validation in higher species.
Public Health Relevance Statement: NARRATIVE: There are currently no therapeutic treatments available for the more than 125,000 patients in the United States who suffer from Charcot-Marie-Tooth disease, the most prevalent hereditary demyelinating neuropathy leading to muscle atrophy, numbness and deformities in the arms and legs. The most common form, CMT1A, is caused by a duplication in the chromosome region containing peripheral myelin protein 22 (PMP22), leading to toxic overexpression of the protein. In this application, we propose to employ a novel drug delivery approach pioneered by DTx Pharma, based on the covalent conjugation of specific fatty acids to RNA therapeutics, to safely and effectively suppress the PMP22 gene in vivo in peripheral nerves in a mouse model of CMT1A as a critical step in developing a new therapeutic approach to this devastating, progressive disorder.
Project Terms: 17p11.2; Adult; Affect; afferent nerve; Anatomy; Animals; Antisense Oligonucleotides; arm; Atrophic; Automobile Driving; base; Biological; Biological Assay; Biological Markers; Cells; cellular engineering; Charcot-Marie-Tooth Disease; Chromosomes; Clinic; Clinical; Coupled; cytotoxicity; Data; Deformity; Demyelinations; design; Development; Disease; Disease model; Disease Outcome; Distal; Dose; Drug Delivery Systems; efficacy study; efficacy trial; Evaluation; experimental study; Fatty Acids; Foot Deformities; Foundations; Functional disorder; Future; Gene Dosage; Gene Targeting; Generations; Genes; Grant; hereditary neuropathy; Human; Hyporeflexia; improved; In Vitro; in vivo; in vivo evaluation; Inherited; Intrathecal Injections; Intravenous; intravenous injection; kidney dysfunction; knock-down; Leg; Libraries; Life; Liver; Maintenance; Measurement; Mediating; Messenger RNA; Modality; Modeling; Motor; mouse model; mRNA Expression; Mus; Muscle; Muscle Weakness; Muscular Atrophy; Myelin; Nerve; Neural Conduction; Neurons; Neuropathy; novel therapeutic intervention; novel therapeutics; Numbness; Occupational Therapy; Onset of illness; overexpression; Patients; Peripheral Nerves; Peripheral Nervous System; Peripheral Nervous System Diseases; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Physical therapy; PMP22 gene; Property; protein expression; Protein Overexpression; Proteins; Rattus; remyelination; Repression; Risk; Rodent; Route; Safety; Schwann Cells; sciatic nerve; screening; Sensory; siRNA delivery; skeletal; Small Business Innovation Research Grant; Small Interfering RNA; Structure; Technology; Testing; Therapeutic; therapeutic RNA; therapeutic siRNA; Thrombocytopenia; Tissues; Toxic effect; Transfection; Transgenes; Transgenic Mice; Treatment Efficacy; United States; uptake; Validation
