Nonviral Modification of T Cell Gene Expression
There is provided a lipid mix composition comprising ionizable lipid, a structural lipid such as DSPC, a sterol, and a surfactant such as polysorbate 80, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, or D-α-Tocopherol polyethylene glycol 1000 succinate. The lipid mix compositions find particular use in transfecting difficult to transfect cells and maintaining the viability of those cells. The lipid mix compositions are particularly well suited to T cell transfection ex vivo.
1 . A lipid mix composition for forming lipid particles in association with a nucleic acid, for use in transfecting nucleic acid into T cells, the composition comprising about 40-50 Mol % ionizable lipid, about 10-20 Mol % DSPC, about 35 to 40 Mol % sterol, and about 0.1-3 Mol % stabilizer.
2 . The lipid mix composition of claim 1 , wherein said transfecting takes place ex vivo or in vitro.
3 . The lipid mix composition of claim 1 , wherein said stabilizer is polyoxyethylene (10) stearyl ether.
4 . The lipid mix composition of claim 1 , wherein said stabilizer is polysorbate 80.
5 . The lipid mix composition of claim 1 , wherein said stabilizer is polyoxyethylene (20) stearyl ether.
6 . The lipid mix composition of claim 1 , wherein said stabilizer is D-α-Tocopherol polyethylene glycol 1000 succinate.
7 . The lipid mix composition of claim 1 wherein the ionizable lipid is an aminolipid.
8 . The lipid mix composition of claim 7 wherein the aminolipid is selected from the group consisting of BOCHD-C3-DMA, Dlin-MC3-DMA, DODMA, and DLin-KC2-DMA.
9 . The lipid mix composition of claim 1 wherein the ionizable lipid is C12-200.
10 . The lipid mix composition of claim 1 , wherein the ionizable lipid is 40 Mol %, the structural lipid is 20 Mol % DSPC, the sterol is from 37-40 Mol %, and the stabilizer is from 0.5 Mol % to 2.5 Mol %, and the stabilizer is selected from 2.5 Mol % polyoxyethylene (10) stearyl ether, 1.5 Mol % polysorbate 80, 0.5 Mol % TPGS 1000, 2.5 Mol % TPGS, and 2.5 Mol % polyoxyethylene (10) stearyl ether.
11 . The lipid mix composition of claim 1 , wherein the ionizable lipid is 50 Mol %, the structural lipid is 10 Mol % DSPC, the sterol is from 37-40 Mol %, and the stabilizer is about 0.5 Mol % to 2.5 Mol %, and the stabilizer is selected from 2.5 Mol % polyoxyethylene (10) stearyl ether, 1.5 Mol % polysorbate 80, 0.5 Mol % TPGS 1000, 2.5 Mol % TPGS, and 2.5 Mol % polyoxyethylene (10) stearyl ether.
12 . The lipid mix composition of claim 1 wherein an N/P ratio is from 4-12.
13 . The lipid mix composition of claim 12 wherein the N/P ratio is from 8-10.
14 . A method of treating T cells in vitro comprising isolating T cells from a bodily fluid, and contacting said cells with a nucleic acid therapeutic encapsulated in the lipid mix composition of claim 1 .
15 . The method of claim 14 wherein the T cells are in the log phase of growth initiated by T cell activation when contact is made.
16 . The method of claim 14 wherein the T cells are just beginning the log phase of growth after activation.
17 . The method of claim 14 wherein the T cells are at the end of the log phase of growth after activation.
18 . The method of claim 14 wherein contact is made from day 3 to day 7 after activation.
19 . The method of claim 14 wherein contact is made on day 4 after activation.
20 . The method of claim 14 wherein the T cells have previously been cryopreserved.
21 . The method of claim 14 wherein the contact is made when CD25 positive population is greater than 70%.
22 . A method of treating T cells obtained via differentiation of other mammalian cells and contacting said cells with a nucleic acid therapeutic encapsulated in a lipid mix composition of claim 1 .