Efficient and scalable microfluidic gene delivery to hematopoietic stem cells to treat blood diseases

Project Summary Ex vivo genetic modification of autologous CDHematopoietic stem progenitor cellsHSPCis a proven route to durable correction of blood diseases such as sickle cell diseasehemophiliaand HIVAt the core of these therapeutic approaches is delivery of exogenous genes or gene modifying elements to HSPCCurrent delivery methodsviral delivery or electroporationare a major limitation to the widespread adoption of HSPC cell therapy due to their high costvariable or limited effectivenessand difficulty to scale up during developmentThus the pharmaceutical industry has a clear and urgent need for new delivery systemsThese systems must be compact and enclosedscalable from research to patient scaleand must preserve the viability and engraftment potential of the modified HSPCFurthermoresuch a system may be readily integrated into a point of carePOCor even bedside manufacturing systemWe have recently demonstrated a novel non viral approach for efficient delivery of gene products through microfluidic mechanoporation that meets these design requirementsIn this processcells undergo a series of rapid and large intensity compressions to transiently open membrane pores and to induce active transport of target molecules into the cell interiorThe advantage of this innovative approach for microfluidic intracellular delivery is large macromoleculesandgtMDaare transported to the cells with high efficiencyhigh cell viabilityfast processing speedM cells hrand overall processing simplicityfew skilled stepsThese outcomes have been met in proof of concept studies with cancer cell linesThe goal of the proposed work is to reproduce these studies in HSPCand thus demonstrate feasibility of our device as a route to HSPC cell therapy manufacturingThe results of this study will define and validate microfluidic mechanotransfection as the first truly scalablemillion cells tobillion cells using same process conditionsand POC cell therapy manufacturing technology We propose to develop high throughput and high efficiency microfluidic platform for intracellular delivery of gene transfection reagents to CDhematopoietic stem progenitor cellsHSPCby adapting our previously developed proof of concept device to these therapeutically relevant cellsThis platform will be optimized for the efficient delivery of mRNADNA plasmidsand CRISPR Casgene editing reagentswhile maintaining high cell viabilityengraftment potentialand global gene expression pattern