METHODS TO IMPROVE POTENCY OF ELECTROPORATION
Compositions and methods for reducing nucleotide oxidation during electroporation, specifically the use of free radical scavengers to reduce electroporation-induced oxidation, are described. Compositions and methods for enhancing transfection efficiency are also described.
1 . A method of reducing nucleotide oxidation during electroporation, the method comprising the steps of:
1) providing a composition comprising a) a polynucleotide, wherein the polynucleotide comprises a nucleotide alteration, b) a free radical scavenger, and c) a recipient entity; and
2) electroporating the composition, wherein the free radical scavenger reduces electroporation-induced oxidation of the nucleotide alteration.
2 . The method of claim 1 , wherein the polynucleotide comprises RNA.
3 . The method of claim 2 , wherein the RNA is selected from the group consisting of: siRNAs, miRNAs, antisense oligonucleotides, shRNAs, double-stranded RNAs, RNA oligonucleotides, mRNAs, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system RNA, and combinations thereof.
4 . The method of claim 2 , wherein the RNA is an siRNA.
5 . The method of claim 3 , wherein the CRISPR system RNA is selected from the group consisting of: a guide RNA (gRNA), a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and a single-guide crRNA and tracrRNA fusion (sgRNA), and combinations thereof.
6 . The method of claim 1 , wherein the polynucleotide comprises DNA.
7 . The method of claim 6 , wherein the DNA is selected from the group consisting of: circular plasmids, linear plasmids, vectors, single-stranded DNA, single-stranded oligonucleotides, double-stranded oligonucleotides, a CRISPR system expression vector, and combinations thereof.
8 . The method of claim 7 , wherein the CRISPR system expression vector encodes a CRISPR family enzyme, a gRNA, a crRNA, a tracrRNA, a sgRNA, and combinations thereof.
9 . The method of claim 1 , wherein the polynucleotide comprises a non-natural nucleic acid.
10 . The method of claim 9 , wherein the non-natural nucleic acid is a morpholino.
11 . The method of any of claims 1 - 10 , wherein the nucleotide alteration comprises a phosphorothioate internucleotide linkage.
12 . The method of any of claims 1 - 11 , wherein the free radical scavenger is a reducing agent.
13 . The method of claim 12 , wherein the reducing agent is selected from the group consisting of: L-Methionine, glutathione, L-cysteine, and ascorbic acid, and combinations thereof.
14 . The method of claim 13 , wherein the reducing agent is glutathione.
15 . The method of any of claims 1 - 14 , wherein the concentration of the free radical scavenger is between 0.1 mM to 100 mM.
16 . The method of any of claims 1 - 15 , wherein the recipient entity is a lipid-based entity.
17 . The method of claim 16 , wherein the lipid-based entity is selected from the group consisting of: a cell, a vesicle, a tissue, and a lipid-based nanoparticle.
18 . The method of claim 17 , wherein the lipid-based nanoparticle is selected from the group consisting of: a unilamellar liposome, a multilamellar liposome, a nanovesicle, and a lipid preparation.
19 . The method of claim 17 , wherein the vesicle is an extracellular vesicle.
20 . The method of claim 19 , wherein the extracellular vesicle is an exosome.
21 . The method of claim 17 , wherein the cell is selected from a eukaryotic cell or a prokaryotic cell.
22 . The method of claim 21 , wherein the eukaryotic cell is selected from the group consisting of: an animal cell, a fungal cell, and a plant cell.
23 . The method of claim 22 , wherein the animal cell is selected from a vertebrate cell or an invertebrate cell.
24 . The method of claim 23 , wherein the vertebrate cell is a mammalian cell.
25 . The method of claim 24 , wherein the mammalian cell is a human cell.
26 . The method of any of claims 23 - 25 , wherein the cell is selected from the group consisting of: a stem cell, an immune cell, an erythrocyte, a cancer cell, a cultured cell, an immortalized cell, and an isolated cell, and combinations thereof.
27 . The method of claim 26 , wherein the immune cell is selected from the group consisting of: a T cell, a B cell, a macrophage, and a dendritic cell.
28 . The method of claim 22 , wherein the fungal cell is a yeast cell.
29 . The method of claim 21 , wherein the prokaryotic cell is a bacterial cell.
30 . The method of any of claims 1 - 15 , wherein the recipient entity is a non-lipid entity.
31 . The method of claim 30 , wherein the non-lipid entity is a non-lipid nanostructure.
32 . The method of any of claims 1 - 31 , wherein the electroporating step is performed in vitro, in vivo, or ex vivo.
33 . The method of any of claims 1 - 32 , wherein the reduction in oxidation is determined through analyzing a molecular profile of the polynucleotide.
34 . The method of claim 33 , wherein the molecular profile is an anion exchange high-performance liquid chromatography (AEX-HPLC) chromatogram.
35 . The method of claim 33 , wherein the molecular profile is an ion-pairing reversed-phase chromatography (IPRP-HPLC) chromatogram.
36 . The method of claim 33 , wherein the molecular profile is a mass spectrometry spectrum.
37 . The method of any of claims 33 - 36 , wherein the molecular profile of the polynucleotide is shifted toward an unelectroporated polynucleotide relative to a polynucleotide electroporated in the absence of the free radical scavenger.
38 . The method of any of claims 1 - 37 , wherein the electroporating step comprises a voltage level higher than a viable electroporation voltage level in the absence of the free radical scavenger.
39 . The method of claim 38 , wherein the polynucleotide demonstrates a functional improvement at the voltage level.
40 . The method of claim 39 , wherein the functional improvement is an increased activity of the polynucleotide.
41 . The method of claim 40 , wherein the increased activity of the polynucleotide is an increase in RNA interference.
42 . The method of claim 40 , wherein the increased activity of the polynucleotide is an increase in CRISPR mediated gene editing.
43 . A method of enhancing transfection efficiency, comprising the steps of:
1) providing a composition comprising a) a polynucleotide, wherein the polynucleotide comprises a nucleotide alteration, b) a free radical scavenger, and c) a recipient entity; and
2) electroporating the composition, wherein the free radical scavenger reduces electroporation-induced oxidation of the electroporated polynucleotide.
44 . The method of claim 43 , wherein the polynucleotide comprises RNA.
45 . The method of claim 44 , wherein the RNA is selected from the group consisting of: siRNAs, miRNAs, antisense oligonucleotides, shRNAs, double-stranded RNAs, RNA oligonucleotides, mRNAs, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system RNA, and combinations thereof.
46 . The method of claim 44 , wherein the RNA is an siRNA.
47 . The method of claim 45 , wherein the CRISPR system RNA is selected from the group consisting of: a guide RNA (gRNA), a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and a single-guide crRNA and tracrRNA fusion (sgRNA), and combinations thereof.
48 . The method of claim 43 , wherein the polynucleotide comprises DNA.
49 . The method of claim 48 , wherein the DNA is selected from the group consisting of: circular plasmids, linear plasmids, vectors, single-stranded DNA, single-stranded oligonucleotides, double-stranded oligonucleotides, a CRISPR system expression vector, and combinations thereof.
50 . The method of claim 49 , wherein the CRISPR system expression vector encodes a CRISPR family enzyme, a gRNA, a crRNA, a tracrRNA, a sgRNA, and combinations thereof.
51 . The method of claim 43 , wherein the polynucleotide comprises a non-natural nucleic acid.
52 . The method of claim 51 , wherein the non-natural nucleic acid is a morpholino.
53 . The method of any of claims 43 - 52 , wherein the nucleotide alteration comprises a phosphorothioate internucleotide linkage.
54 . The method of any of claims 43 - 53 , wherein the free radical scavenger is a reducing agent.
55 . The method of claim 54 , wherein the reducing agent is selected from the group consisting of: L-Methionine, glutathione, L-cysteine, and ascorbic acid, and combinations thereof.
56 . The method of claim 55 , wherein the reducing agent is glutathione.
57 . The method any of claims 43 - 56 , wherein the concentration of the free radical scavenger is between 0.1 mM to 100 mM.
58 . The method of any of claims 43 - 57 , wherein the recipient entity is a lipid-based entity.
59 . The method of claim 58 , wherein the lipid-based entity is selected from the group consisting of: a cell, a vesicle, a tissue, and a lipid-based nanoparticle.
60 . The method of claim 59 , wherein the lipid-based nanoparticle is selected from the group consisting of: a unilamellar liposome, a multilamellar liposome, a nanovesicle, and a lipid preparation.
61 . The method of claim 59 , wherein the vesicle is an extracellular vesicle.
62 . The method of claim 61 , wherein the extracellular vesicle is an exosome.
63 . The method of claim 59 , wherein the cell is selected from a eukaryotic cell or a prokaryotic cell.
64 . The method of claim 63 , wherein the eukaryotic cell is selected from the group consisting of: an animal cell, a fungal cell, and a plant cell.
65 . The method of claim 64 , wherein the animal cell is selected from a vertebrate cell or an invertebrate cell.
66 . The method of claim 65 , wherein the vertebrate cell is a mammalian cell.
67 . The method of claim 66 , wherein the mammalian cell is a human cell.
68 . The method of any of claims 65 - 67 , wherein the cell is selected from the group consisting of: a stem cell, an immune cell, an erythrocyte, a cancer cell, a cultured cell, an immortalized cell, and an isolated cell, and combinations thereof.
69 . The method of claim 68 , wherein the immune cell is selected from the group consisting of: a T cell, a B cell, a macrophage, and a dendritic cell.
70 . The method of claim 64 , wherein the fungal cell is a yeast cell.
71 . The method of claim 63 , wherein the prokaryotic cell is a bacterial cell.
72 . The method of any of claims 43 - 57 , wherein the recipient entity is a non-lipid entity.
73 . The method of claim 72 , wherein the non-lipid entity is a non-lipid nanostructure.
74 . The method of any of claims 43 - 73 , wherein the electroporating step is performed in vitro, in vivo, or ex vivo.
75 . The method of any of claims 43 - 74 , wherein the reduction in oxidation is determined through analyzing a molecular profile of the polynucleotide.
76 . The method of claim 75 , wherein the molecular profile is an anion exchange high-performance liquid chromatography (AEX-HPLC) chromatogram.
77 . The method of claim 75 , wherein the molecular profile is an ion-pairing reversed-phase chromatography (IPRP-HPLC) chromatogram.
78 . The method of claim 75 , wherein the molecular profile is a mass spectrometry spectrum.
79 . The method of any of claims 75 - 78 , wherein the molecular profile of the polynucleotide is shifted toward an unelectroporated polynucleotide relative to a polynucleotide electroporated in the absence of the free radical scavenger.
80 . The method of any of claims 43 - 79 , wherein the electroporating step comprises a voltage level higher than a viable electroporation voltage level in the absence of the free radical scavenger.
81 . The method of claim 80 , wherein the polynucleotide demonstrates a functional improvement at the voltage level.
82 . The method of claim 81 , wherein the functional improvement is an increased activity of the polynucleotide.
83 . The method of claim 82 , wherein the increased activity of the polynucleotide is an increase in RNA interference.
84 . The method of claim 82 , wherein the increased activity of the polynucleotide is an increase in CRISPR mediated gene editing.
85 . A composition for reducing nucleotide oxidation during electroporation, the composition comprising a) a polynucleotide, wherein the polynucleotide comprises a nucleotide alteration, b) a free radical scavenger, and c) a recipient entity.
86 . The composition of claim 85 , wherein the polynucleotide comprises RNA.
87 . The composition of claim 86 , wherein the RNA is selected from the group consisting of: siRNAs, miRNAs, antisense oligonucleotides, shRNAs, double-stranded RNAs, RNA oligonucleotides, mRNAs, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system RNA, and combinations thereof.
88 . The composition of claim 86 , wherein the RNA is an siRNA.
89 . The composition of claim 87 , wherein the CRISPR system RNA is selected from the group consisting of: a guide RNA (gRNA), a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), and a single-guide crRNA and tracrRNA fusion (sgRNA), and combinations thereof.
90 . The composition of claim 85 , wherein the polynucleotide comprises DNA.
91 . The composition of claim 90 , wherein the DNA is selected from the group consisting of: circular plasmids, linear plasmids, vectors, single-stranded DNA, single-stranded oligonucleotides, double-stranded oligonucleotides, a CRISPR system expression vector, and combinations thereof.
92 . The composition of claim 91 , wherein the CRISPR system expression vector encodes a CRISPR family enzyme, a gRNA, a crRNA, a tracrRNA, a sgRNA, and combinations thereof.
93 . The composition of claim 85 , wherein the polynucleotide comprises a non-natural nucleic acid.
94 . The composition of claim 93 , wherein the non-natural nucleic acid is a morpholino.
95 . The method of any of claims 85 - 94 , wherein the nucleotide alteration comprises a phosphorothioate internucleotide linkage.
96 . The method of any of claims 85 - 95 , wherein the free radical scavenger is a reducing agent.
97 . The composition of claim 96 , wherein the reducing agent is selected from the group consisting of: L-Methionine, glutathione, L-cysteine, and ascorbic acid, and combinations thereof.
98 . The composition of claim 97 , wherein the reducing agent is glutathione.
99 . The method of any of claims 85 - 98 , wherein the concentration of the free radical scavenger is between 0.1 mM to 100 mM.
100 . The method of any of claims 85 - 99 , wherein the recipient entity is a lipid-based entity.
101 . The composition of claim 100 , wherein the lipid-based entity is selected from the group consisting of: a cell, a vesicle, a tissue, and a lipid-based nanoparticle.
102 . The composition of claim 101 , wherein the lipid-based nanoparticle is selected from the group consisting of: a unilamellar liposome, a multilamellar liposome, a nanovesicle, and a lipid preparation.
103 . The composition of claim 101 , wherein the vesicle is an extracellular vesicle.
104 . The composition of claim 103 , wherein the extracellular vesicle is an exosome.
105 . The composition of claim 101 , wherein the cell is selected from a eukaryotic cell or a prokaryotic cell.
106 . The composition of claim 105 , wherein the eukaryotic cell is selected from the group consisting of: an animal cell, a fungal cell, and a plant cell.
107 . The composition of claim 106 , wherein the animal cell is selected from a vertebrate cell or an invertebrate cell.
108 . The composition of claim 107 , wherein the vertebrate cell is a mammalian cell.
109 . The composition of claim 108 , wherein the mammalian cell is a human cell.
110 . The method of any of claims 107 - 109 , wherein the cell is selected from the group consisting of: a stem cell, an immune cell, an erythrocyte, a cancer cell, a cultured cell, an immortalized cell, and an isolated cell, and combinations thereof.
111 . The composition of claim 110 , wherein the immune cell is selected from the group consisting of: a T cell, a B cell, a macrophage, and a dendritic cell.
112 . The composition of claim 106 , wherein the fungal cell is a yeast cell.
113 . The composition of claim 105 , wherein the prokaryotic cell is a bacterial cell.
114 . The method of any of claims 85 - 99 , wherein the recipient entity is a non-lipid entity.
115 . The composition of claim 114 , wherein the non-lipid entity is a non-lipid nanostructure.
116 . A method of reducing nucleotide oxidation during electroporation, the method comprising the steps of:
1) providing a composition comprising the composition of any of claims 85 - 115 ; and
2) electroporating the composition, wherein the free radical scavenger reduces electroporation-induced oxidation of the nucleotide alteration.
117 . A method of enhancing transfection efficiency, the method comprising the steps of:
1) providing a composition comprising the composition of any of claims 85 - 115 ; and
2) electroporating the composition, wherein the free radical scavenger reduces electroporation-induced oxidation of the electroporated polynucleotide.