• Lipid nanoparticles (LNPs) are the leading carriers for mRNA therapies as to the approved SARS-CoV-2 vaccines. We present our approaches and experiences focusing on key steps of CMC for the clinical development of LNP–mRNA formulations including formulation design and scalable manufacturing process.
• LNPs typically contain four　lipids: an ionizable cationic lipid, a phospholipid, a cholesterol, and a PEG-lipid. Ionizable lipids are defined typically by a tertiary amine that is deprotonated under neutral conditions and is positively charged in pH conditions below the pKa of the lipid. We designed proprietary ionizable lipids suitable for LNP-based mRNA delivery with improved efficiencies of mRNA delivery and protein expression. Results suggesting potential application to cancer immunotherapy will be introduced as well.
• LNPs are usually prepared by an ethanol injection method in which an aqueous solution of mRNA at acidic condition is rapidly mixed with　an ethanol solution of lipids. Appropriate selection of microfluidics is key for seamless scale-up of LNP formation process.　Furthermore, and of importance is to appropriately design downstream equipment and Fill & Finish processes considering the desired scale.

Speaker:

Shigetomo Tsujihata
Senior scientist, Bio Science & Engineering Laboratory
FUJIFILM Corporation

Inefficient knock-in of transgene cargos limits the potential for cell-based medicines. We hypothesized that targeting a site within an exon of an essential gene with CRISPR could drive efficient knock-in through a selection mechanism. An HDR template was designed so that correct integration would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive indels would die. We termed this method SLEEK (SeLection by Essential-gene Exon Knock-in) and achieved HDR >90% in clinically relevant cells.

Speaker:

John Zuris, Ph.D.
Associate Director, Editing Technologies
Editas Medicine