Developing a Platform for Multiplexed Large Payload Delivery

Abstract BackgroundNew approaches to therapeutic development using cellular engineering are rapidly emerging and evolvingApplications such asbut not limited tochimeric antigen receptorCART cellsregenerative medicineand drug delivery using liposomes and nanoparticles are steadily transitioning from idea to realityIn factsuch cellular therapeutics are increasingly becoming a viable option for treatment of patients with cancer and genetic diseaseswith the first two CAR T therapies receiving FDA approval inHoweverit is becoming increasingly clear with CAR T that multiple editing steps will be needed to confer the desired phenotypeas well as improved safety and toxicity profilesCurrent approaches for gene editing and intracellular delivery include chemical methodse glipofectaimeelectroporationand viral vectorsChemical methods are widely used in research labs because they are affordable and easy to usebut they are not very effective at transfecting primary cellsElectroporation is another widely used method that is capable of treating primary cellsbut it is only effective at delivering highly charged moleculesi enucleic acidsViral vectors were the earliest gene delivery technologythey are quite efficient at delivering DNAbut are also limited to nucleic acid delivery and have safety concerns related to viral integration in the genomeWith the growing interest in cellular engineeringa need for more efficient and flexible intracellular delivery technologies has emergedOpenCell TechnologiesOCThas developed a proprietary technologyPOROSto deliver macromolecules such as DNARNAproteinliposomes and nanoparticles to a wide variety of cell typesPOROS uses acoustic waves to drive cells through an array of nozzles one cell at a timethus creating a mechanoshear force to porate cells in a uniform mannerThe acoustic shear porationASPis coupled with an electrophoresisEPstep that uses a low strength electric field to actively drive molecules into the cells through the pores already createdIn this SBIR projectOCT will expand the capabilities of its POROS platform by developing the POROS multipayload deliveryMPDplatformApproachIn this Phase I feasibility studyOCT will demonstrate that its delivery technologyPOROScan be combined with unique reagents and protocols to enable efficient delivery of multiple large molecules to cellsIn this projectwe will first optimize protocols for nucleic acidproteinliposome and nanoparticle delivery using three model systems that use reporter gene readoutssuch as GFPFollowing protocol development and optimizationthis technology will be tested for two relevant applicationslentivirus packaging for viral gene delivery and CAR T cell production using CRISPR CasBoth of these applications require delivery of two or more large molecular payloads and will serve as the proof of concept that the POROS MPD platform improves delivery Developing a platform for multiplexed intracellular delivery Project Narrative Cellular therapeutics are rapidly emerging as an exciting new area in both cancer treatment and regenerative medicineTo develop such therapeuticscellular engineering is used to edit the genes of interestNext generation cellular therapieshoweverwill require multiple editing steps to ensure safety and efficacyExisting methods for gene editing are struggling to keep up with these demands because they generally only insert single molecules during a treatment and the types of molecules they can deliver are limitedOpenCell Technologies is developing a unique platform that will allow the simultaneous insertion of a large range of heterogenous molecules into cellsThis will enable the end user to perform multiple editing events in one fell swoopwithout harming the cellsThe result will be decreased time to produce cellular therapeutic products with enhanced efficacy and reduced toxicity