Oligonucleotide therapeutics, like siRNA, are attractive because they target messenger RNA and thus, can modulate the expression of protein targets that are inaccessible to small molecules, antibodies and peptides. Challenges identifying safe and effective ways to deliver siRNA therapeutics into cells outside of the liver have greatly limited the expansion of oligonucleotides as a therapeutic class. DTx Pharma has identified a lipid motif that when covalently coupled to siRNA enables its activity in vivo in muscle, fat, heart, lung and liver following systemic administration. DTx-lipidated siRNA is also highly efficacious ex vivo in primary cells such as muscle, fat, stellate, endothelial and neuronal cells and at least an order of magnitude more potent at repressing mRNA expression relative to conjugates of other fatty acids that have been previously described. These preliminary data are highly encouraging and appear unique relative to other technologies reported to enable delivery of siRNA in vivo; however, we have generated very limited in vivo data with DTx-conjugated siRNAs and are yet to explore characteristics like mode of action, bioavailability, safety or relative activity compared to GalNAc and cholesterol, the gold-standard conjugation approaches for delivery of siRNA to and beyond the liver, respectively. Additionally, we are yet to apply learnings from the successful development of lipidated peptide therapeutics that are likely to enhance the activity of our original lead in tissues outside of the liver. In SA1, a novel lipid siRNA conjugate, DTx-EAB, with enhanced affinity for albumin but, similar activity in primary cells in the absence of albumin to the DTx-conjugated siRNA described above, will be evaluated in vivo in mice against the original conjugate for its ability to deliver siRNA beyond the liver. This novel conjugate was designed based on knowledge of half-life extension from peptide biologics and is expected to be far more potent at repressing mRNA expression in tissues outside of the liver because its enhanced albumin binding affinity will prolong residence time in circulation and increase exposure to these tissues. In SA2, we will advance our knowledge of the mode of action, bioavailability, safety and relative activity of DTx-EAB and the original DTx-conjugated siRNA compared with GalNAc and cholesterol. An analytical PK assay will be developed to understand whether any increased activity of DTx-EAB over cholesterol conjugates is likely due to half-life extension in the circulation. In the final aim, SA3, we will test whether DTx-EAB technology can be leveraged to improve outcomes in the mouse mdx model of muscular dystrophy. Therapeutics targeting myostatin, like cholesterol-conjugated myostatin siRNA, enhance muscle mass in animal models. DTx-EAB and cholesterol myostatin siRNA conjugates will be compared in the mdx model with the goal of demonstrating that DTx technology is far more potent at improving outcomes and thus, at delivering siRNA to muscle. Evaluation of the mode of action, the tissues to which DTx- conjugates deliver siRNA to, and the relative efficacy and safety, across these 3 aims will allow us to select indications and siRNA targets where this delivery approach is likely to have a meaningful benefit.
Public Health Relevance Statement: NARRATIVE: Despite enormous potential to access therapeutic targets that small molecules, antibodies and peptide biologics cannot reach, the success of oligonucleotide therapeutics as a class has been limited due to challenges delivering them into cells and tissues outside of the liver. DTx Pharma has developed a technology, based on the covalent conjugation of long-chain fatty acids (LCFAs) to oligonucleotides, that enables the delivery and activity of siRNA in vivo outside of the liver and ex vivo to multiple primary human cell types including T cells, muscle cells, endothelial cells, adipocytes, stellate cells and trabecular meshwork cells. It is anticipated that this technology will expand the reach of oligonucleotide therapeutics beyond the liver and unlock this class of therapeutics for treatment of cardiovascular, immune, muscular, neurodegenerative and ocular diseases, as well as other diseases where decreased expression of a gene target may be of benefit (e.g. genetic disease driven by dominant gain of function mutations).
NIH Spending Category: Biotechnology; Duchenne/ Becker Muscular Dystrophy; Gene Therapy; Genetics; Muscular Dystrophy; Orphan Drug; Pediatric; Pediatric Research Initiative; Rare Diseases
Project Terms: Adipocytes; Affinity; Albumins; Animal Model; Antibodies; base; Binding; Biological; Biological Assay; Biological Availability; Blood; Blood Circulation; Cardiovascular system; cell type; Cells; Characteristics; Cholesterol; commercialization; Coupled; Data; design; Development; Differentiation and Growth; Disease; Dose; Drug Kinetics; Duchenne muscular dystrophy; efficacy study; Endothelial Cells; Endothelium; Evaluation; Exposure to; Fatty acid glycerol esters; Fatty Acids; gain of function mutation; GDF8 gene; Gene Targeting; Generations; Genetic Diseases; Genetic Transcription; Goals; Gold; Half-Life; Head; Heart; Human; Immune; improved; improved outcome; in vivo; inhibitor/antagonist; Intravenous; Knowledge; Lead; Learning; Life Extension; Ligands; Lipids; Liver; long chain fatty acid; Lung; mdx mouse; Messenger RNA; Modeling; mouse model; Mus; Muscle; Muscle Cells; muscle form; Muscular Dystrophies; muscular dystrophy mouse model; Nerve Degeneration; Neurons; novel; Oligonucleotides; Organ; Pathology; peptide drug; Peptides; Pharmacology; Phenotype; protein expression; Publishing; receptor; Reporting; residence; response; Safety; Serum Albumin; Small Interfering RNA; small molecule; stellate cell; subcutaneous; success; T-Lymphocyte; targeted delivery; Technology; Testing; Therapeutic; therapeutic siRNA; therapeutic target; Time; Tissues; Trabecular meshwork structure; Treatment Efficacy; uptake; Validation
