Editing Lipid for protein delivery (section)

== Lipid Nanoparticle Formulation. ==

Lipid was formulated into nanoparticles&nbsp;for all delivery applications. The as-synthesized lipid was&nbsp;mixed with cholesterol, DOPE, and C16-PEG2000-Ceramide&nbsp;(Avanti Polar Lipids) at a wt/wt ratio of 16:4:1:1. Briefly, lipid,&nbsp;cholesterol, and DOPE were mixed in a chloroform solution in a&nbsp;0.5-dr vial. The organic solvent was evaporated under vacuum&nbsp;and further dried over night to form a thin layer film on the vial.&nbsp;The lipid film was hydrated using a mixed solution of ethanol/&nbsp;sodium acetate buffer (200 mM, pH = 5.2, vol:vol = 9:1). The&nbsp;above solution was added dropwise to an aqueous solution of&nbsp;C16-PEG2000-ceramide, and the resulted solution was incubated&nbsp;at 37 °C for 30 min before subjecting to dialysis against water or&nbsp;PBS in 3500 molecular weight cut-off (MWCO) cassettes (Pierce/&nbsp;Thermo Scientific).

Delivery of proteins complexed with cationic lipids in cell culture

Cultured cells were plated in 48-well format (250 µL volume) in Dulbecco’s Modified Eagle’s Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ~70% confluence the next day. Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery. Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 µM protein (based on a 275 µL final volume) with 0.2–2.5 µL commercially available cationic lipids in 25 µL OPTIMEM media (Life Technologies) according to the manufacturer’s protocol for normal plasmid transfection, including incubation time. 25 µL of the OPTIMEM mixture containing cationic lipids and protein was then added to cells in 250 µL of full serum media. For Cas9 delivery in vitro, purified sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent in 25 µL OPTIMEM and treating cells in 250 µL of full serum media. All complexing steps were performed at room temperature. For all protein deliveries that lasted longer than 12–16 hours, media containing lipid complexes was replaced with fresh full serum media without antibiotics after 12–16 hours unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for optimal gene activation either 4 hours (TALE) or 12–16 hours (dCas9-VP64) after delivery, and for gene modification (Cas9) 48 hours after delivery. Delivery of proteins complexed with cationic lipids in cell culture

Cultured cells were plated in 48-well format (250 µL volume) in Dulbecco’s Modified Eagle’s Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ~70% confluence the next day. Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery. Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 µM protein (based on a 275 µL final volume) with 0.2–2.5 µL commercially available cationic lipids in 25 µL OPTIMEM media (Life Technologies) according to the manufacturer’s protocol for normal plasmid transfection, including incubation time. 25 µL of the OPTIMEM mixture containing cationic lipids and protein was then added to cells in 250 µL of full serum media. For Cas9 delivery in vitro, purified sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent in 25 µL OPTIMEM and treating cells in 250 µL of full serum media. All complexing steps were performed at room temperature. For all protein deliveries that lasted longer than 12–16 hours, media containing lipid complexes was replaced with fresh full serum media without antibiotics after 12–16 hours unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for optimal gene activation either 4 hours (TALE) or 12–16 hours (dCas9-VP64) after delivery, and for gene modification (Cas9) 48 hours after delivery.

https://patents.google.com/patent/WO2014134445A1

Compositions 31 and 32 were prepared using a thin film hydration method described below. Compound 1-016B or 1-018B, cholesterol, and DOPE were mixed at a weight ratio of 16:4: 1 in chloroform, which was then evaporated under vacuum, leaving a thin film. Re-hydrating the thin film in PBS yielded a solution of 1-016B or 1-018B at a concentration of 1 mg/mL. Saporin was added (1-016B or 1-018B solution : saporin = 15: 1 by weight). The mixture was incubated for 15 minutes at room temperature followed by addition of mPEG2000-ceramide C16/DSPE-PEG2000-Biotin (purchased from Avanti Polar Lipids, weight/weight = 8: 1, PEG 10% by weight of l-Ol 6B, this component not shown in the table above). The mixture was again incubated for 15 minutes to yield a nanocomplex composition, Composition 3 lor 32. See the table above for the weight ratios between Compound 1-016B or 1-018B, cholesterol, DOPE, and saporin. These two compositions thus prepared were subjected to an in vivo assay described in Example 47 below.[[Category:Transfection]] [[Category:GenomeEngineering]]