COMPARATIVE ASSESSMENT OF NANOPARTICLE DELIVERY VEHICLES
Provided herein are compositions and methods for identifying effective delivery vehicles (DVs) for mRNA delivery to and expression in specific cells or tissues in culture (in vitro) or in live animals (in vivo) from a pool of co-administered DVs.
1 . A method of assessing targeting efficacy of a delivery vehicle comprising:
(a) contacting each of at least two cell types with the delivery vehicle, wherein the delivery vehicle comprises a coding region for a Cas9 protein or a Base Editor protein and an sgRNA comprising a targeting sequence complementary to a portion of a genome of at least one of the at least two cell types;
(b) maintaining the at least two cell types under conditions suitable for:
(i) expressing the coding region for the Cas9 protein or the Base Editor protein;
(ii) forming a Cas9-sgRNA or a BE-sgRNA ribonucleoprotein; and
(iii) modifying the genome of the at least two cell types;
(c) measuring a level of genome modification in the at least two cell types; and
(d) identifying the delivery vehicle as effectively targeting the target cell type if the level of genome modification is greater in the target cell type than in at least one other cell type.
2 . The method of claim 1 , wherein the coding region encodes the Cas9 protein.
3 . The method of claim 1 , wherein the coding region encodes the Base Editor protein.
4 . The method of claim 1 , wherein the targeting sequence is complementary to the genome of each of the at least two cell types.
5 . The method of claim 4 , wherein the targeting sequence is perfectly complementary to the genome of each of the at least two cell types.
6 . The method of claim 1 , wherein the targeting sequence is complementary to a portion of a euchromatin of each of the at least two cell types.
7 . The method of claim 1 , wherein the targeting sequence is perfectly complementary to a portion of a euchromatin of each of the at least two cell types.
8 . The method of claim 1 , further comprising administering the delivery vehicle to a subject in need.
9 . A method of assessing target efficacy of a delivery vehicle comprising:
(a) contacting cells of a cell type with a plurality of delivery vehicles, wherein each delivery vehicle comprises a coding region for a Cas9 protein or a Base Editor protein and an sgRNA comprising a targeting sequence, wherein each delivery vehicle comprises a coding region for at least one gene-editing reagent and an sgRNA comprising a unique targeting sequence complementary to a portion of a genome of the cell type;
(b) maintaining the cells under conditions suitable for:
(i) expressing the coding region for the at least one gene-editing reagent;
(ii) forming a Cas9-sgRNA or a BE-sgRNA ribonucleoprotein; and
(iii) modifying the genome of the cells;
(c) measuring locations and levels of genome modifications in the cells; and
(d) identifying the delivery vehicle as effectively targeting a cell type if the level of genome modification is greater in the cell type compared to the level of genome modification resulting from use of at least one other delivery vehicle.
10 . The method of claim 9 , wherein the coding region encodes the Cas9 protein.
11 . The method of claim 9 , wherein the coding region encodes the Base Editor protein.
12 . The method of claim 9 , wherein the targeting sequence is complementary to the genome of each of the at least two cell types.
13 . A method of assessing target efficacy of a delivery vehicle comprising:
(a) contacting cells of a cell type with a plurality of delivery vehicles, wherein each delivery vehicle comprises a coding region for a Prime Editor protein and a pegRNA comprising a barcode sequence, wherein the barcode sequence in the pegRNA in each of the delivery vehicles is a unique barcode sequence;
(b) maintaining the cells under conditions suitable for:
(i) expressing the coding region for the Prime Editor protein;
(ii) forming a PE-pegRNA ribonucleoprotein; and
(iii) modifying a genome of the cells;
(c) measuring the levels of genome modifications in the cells; and
(d) identifying the delivery vehicle as effectively targeting a cell type if the level of genome modification resulting from use of the delivery vehicle is greater than the level of genome modification resulting from use of at least one other delivery vehicle.
14 . The method of claim 13 , wherein the barcode sequence is complementary to a genome of each of the at least two cell types.
15 . The method of claim 14 , wherein the barcode sequence is perfectly complementary to the genome of each of the at least two cell types.
16 . The method of claim 13 , wherein the barcode sequence is complementary to a portion of a euchromatin of each of the at least two cell types.
17 . The method of claim 13 , wherein the barcode sequence is perfectly complementary to a portion of a euchromatin of each of the at least two cell types.
18 . The method of claim 13 , further comprising administering the delivery vehicle to a subject in need.
19 . The method of claim 13 , wherein the unique barcode sequence is at least 2 nucleotides.
20 . The method of claim 13 , wherein the unique barcode sequence is at least 6 nucleotides.