Tethosomes: a non-viral DNA platform for durable expression of therapeutic transgenes in specific target cells - Abstract Engage Bio is developing the Tethosome platform, a non-viral strategy for the delivery and potent expression of transgenes in target cells. Gene therapy offers the potential to treat many currently incurable heritable or acquired diseases. While non-viral gene delivery systems are of high interest due to their redosing capability and manufacturability, several challenges remain. mRNA-based approaches offer high peak expression but have limited tissue specificity and short duration. In contrast, non-viral DNA-based approaches achieve long expression duration, but are limited by low target cell delivery, low expression, and potentially dangerous or lethal innate immune activation. Thus, there remains a need for improved technologies able to achieve high peak expression levels for extended durations and with tissue or tumor selectivity. Engage Bio’s Tethosome platform addresses this need by increasing non-viral delivery of DNA to the nucleus, thus increasing the level of transgene expression. The system is comprised of 1) an mRNA encoding a proprietary DNA binding protein, and 2) a non- viral, recombinant therapeutic DNA expression vector containing a transgene. These are packaged in a lipid nanoparticle for co-formulation and delivery. Inside the cell, the DNA binding protein is translated and binds to the expression vector, shuttling it to the nucleus while evading detection by innate immune sensors. Once the Tethosome complex enters the nucleus, the DNA binding protein anchors the expression vector to the nucleus, supporting high expression levels for extended durations without significant DNA integration or replication. To demonstrate the potential of the Tethosome system, Engage Bio will evaluate the hepatic delivery of therapeutics for hemophilia and liver cancer. Hemophilia A is a prototypical gene therapy candidate due to its well-understood monogenic root: lack of FVIII. There are several adeno-associated viral vector-based therapies in various stages of clinical study, but these suffer from the cytotoxicity, immunogenicity, and lack of re-dosability. Moreover, traditional non-viral gene therapies for liver cancer suffer from low efficiency, short duration, and off- target effects. To advance Engage Bio’s system and validate the durable, tissue-specific expression of transgenes, the following Specific Aims are proposed: 1. The delivery of Tethosomes encoding a cocktail of clinically validated pro-inflammatory cytokines (IL-2, GM-CSF, IL-15su [sushi], IL-12sc [single chain]) will be evaluated in a humanized mouse model of hepatocellular carcinoma to demonstrate efficacy. Further, the safety and efficacy of Tethosomes encoding FVIII will be evaluated in a canine model of hemophilia; 2. The safety of the Tethosome platform will be demonstrated in the cynomolgus monkey; the biodistribution of Tethosome DNA and therapeutic protein expression will be determined in key organs including the liver, spleen, lung, and kidney by qPCR and in-situ hybridization; and 3. The GLP manufacture of Tethosome therapeutics will be initiated in preparation for future IND-enabling studies with the goal of producing 100-150 mg of GLP product for intravenous delivery.
