Patent Application
20240336938
Non-viral genome editing for targeted insertion to reprogram T cells holds great therapeutic potentials. However, electroporation-mediated DNA delivery causes severe cellular toxicity, limiting its broad applications.
Non-viral reprogramming of primary human T cells provides a safe, fast and virus-free generation of CAR-T cells. Unlike lentivirus-mediated random integration or site-specific integration of CAR construct using AAV as templates, non-viral reprogramming of CAR-T cells circumvents the need of lentivirus or AAV preparation for donor DNA delivery and can be quickly adopted for manufactory process development. However, the biggest hurdle that holds back the non-viral delivery is the cellular toxicity after the electroporation of DNA donor and the relative low delivery efficiency into human T cells.
Improved methods for effective delivery of polynucleotides into cells, in particular into nuclei in the cells, are needed.
The present disclosure, in certain embodiments, provides compositions and methods for effective delivery of polynucleotides into cells, in particular into the nuclei of the cells. A new buffer system is developed that facilitates the delivery with greatly reduced cell toxicity. Also provided are methods of using the buffer system to effect the delivery. The polynucleotides may be DNA or RNA, whether single-stranded or double-stranded. The polynucleotides can also be provided in complexes such as RNA-protein complexes.
Accordingly, in one embodiment, the present disclosure provides an aqueous solution, comprising succinate, mannitol, a sugar selected from the group consisting of glucose, sucrose and inositol, glutamine or an analog thereof, and an antioxidant.
In some embodiments, the sugar is D-glucose. In some embodiments, the glutamine or analog thereof is selected from the group consisting of L-alanyl-L-glutamine, L-glutamine, and D-glutamine. In some embodiments, the succinate is sodium succinate, potassium succinate, or magnesium succinate. In some embodiments, the antioxidant is sodium pyruvate or acetylcysteine.
In some embodiments, the solution further comprises Na2HPO4 and/or NaH2PO4. In some embodiments, the solution further comprises serum.
In some embodiments, the solution includes no or limited NaCl, such as less than 80 mM NaCl, or preferably less than 70 mM, 60 mM, or 50 mM NaCl.
In a particular embodiment, the solution comprises 5 mM to 30 mM sodium succinate, 1 mM to 30 mM mannitol, 3 mM to 30 mM glucose, 50 mg/L to 800 mg/L L-alanyl-L-glutamine, 0.1 mM to 0.6 mM sodium pyruvate, and includes less than 60 mM NaCl.
Another embodiment of the present disclosure provides a method for introducing a polynucleotide into a cell, comprising applying a pulse of electricity to a sample comprising the polynucleotide and the cell in an aqueous solution provided herein.
Another embodiment of the present disclosure provides a method for introducing a polynucleotide into a cell, comprising applying a pulse of electricity to a sample comprising the polynucleotide and the cell in a culture medium that includes less than 80 mM NaCl, or preferably less than 70 mM, 60 mM, or 50 mM NaCl. In some embodiments, the culture medium comprises succinate, mannitol, a sugar, glutamine or an analog thereof, and an antioxidant.
In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the DNA is single-stranded DNA or double-stranded DNA. In some embodiments, the RNA is siRNA, sgRNA, messenger RNA or double-stranded RNA. In some embodiments, the RNA is provided in an RNA-protein complex.
In some embodiments, the cell is a mammalian cell. In some embodiments, the culture medium further comprises an agent that inhibits the cGAS-STING pathway. In some embodiments, the agent is an siRNA targeting the cGAS or STING protein.
In some embodiments, the method results in entry of the polynucleotide into the nucleus of the cell.
Also provided, in one embodiment, is a method for introducing a biomolecule into a cell, comprising applying a pulse of electricity to a sample comprising the biomolecule and the cell in an aqueous solution having an osmotic pressure greater than 310 mOsmol/kg, wherein the biomolecule is an RNA molecule, a protein, or the combination thereof.
In some embodiments, the aqueous solution has an osmotic pressure greater than 320 mOsmol/kg, 330 mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/kg. In some embodiments, the osmotic pressure is below 600 osmotic pressure.
In some embodiments, the aqueous solution is as described throughout the present disclosure. In some embodiments, the aqueous solution comprises a non-electrolyte agent for adjusting the osmotic pressure of the aqueous solution. In some embodiments, the non-electrolyte agent is selected from the group consisting of glucose, sucrose, fructose, Mannitol, Sorbitol, lactose, trehalose, Glycerol, PEG300, PEG400, PEG600, Glycine, Proline, Taurine, Betaine, and boric acid. In some embodiments, the non-electrolyte agent is present at concentration of 10 mM to 300 mM, 20 mM to 300 mM, 30 mM to 300 mM, 40 mM to 300 mM, 50 mM to 300 mM, 10 mM to 200 mM, 20 mM to 200 mM, 30 mM to 200 mM, 40 mM to 200 mM, 50 mM to 200 mM, 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, or 50 mM to 100 mM.
In some embodiments, the aqueous solution comprises an electrolyte agent for adjusting the osmotic pressure of the aqueous solution. In some embodiments, the electrolyte agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2), Ca(NO3)2, MgSO4, and NaHCO3. In some embodiments, the non-electrolyte agent is present at concentration of 3 mM to 90 mM, 4 mM to 90 mM, 5 mM to 90 mM, 6 mM to 90 mM, 7 mM to 90 mM, 8 mM to 90 mM, 9 mM to 90 mM, 10 mM to 90 mM, 15 mM to 90 mM, 20 mM to 90 mM, 3 mM to 60 mM, 4 mM to 60 mM, 5 mM to 60 mM, 6 mM to 60 mM, 7 mM to 60 mM, 8 mM to 60 mM, 9 mM to 60 mM, 10 mM to 60 mM, 15 mM to 60 mM, 20 mM to 60 mM, 3 mM to 40 mM, 4 mM to 40 mM, 5 mM to 40 mM, 6 mM to 40 mM, 7 mM to 40 mM, 8 mM to 40 mM, 9 mM to 40 mM, 10 mM to 40 mM, 15 mM to 40 mM, 20 mM to 40 mM, 3 mM to 20 mM, 4 mM to 20 mM, 5 mM to 20 mM, 6 mM to 20 mM, 7 mM to 20 mM, 8 mM to 20 mM, 9 mM to 20 mM, 10 mM to 20 mM, or 15 mM to 20 mM.
In some embodiments, the aqueous solution has a pH of 7 to 7.4. In some embodiments, the biomolecule is an siRNA, sgRNA, messenger RNA, double-stranded RNA, antisense RNA, tRNA or RNA aptamer. In some embodiments, the biomolecule is a protein or RNA-protein complex.
Also provided, in one embodiment, is a method for introducing a DNA molecule into a cell, comprising applying a pulse of electricity to a sample comprising the DNA molecule and the cell in an aqueous solution having an osmotic pressure between 270 and 330 mOsmol/kg. In some embodiments, the osmotic pressure inhibits cGAS-STING activation in the cell. In some embodiments, the DNA is double stranded DNA.
The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
Generally, introduction of foreign polynucleotides into a target cell, e.g., through electroporation, has low efficiency. When the target cell is a mammalian cell, another major challenge is cellular toxicity induced by the foreign polynucleotides. The mechanism of such cellular toxicity is not well-understood. It is contemplated that the presence of cytosolic double-stranded DNA per se triggers immune response. Accordingly, to reduce or avoid cellular toxicity, it is desirable to deliver the foreign polynucleotides directly into the cell nuclei. Compared to delivery to the cytoplasm, however, direct delivery to the nucleus has even lower efficiency.
The instant inventors explored the mechanism of cellular toxicity induced by DNA electroporation. When plasmid or double stranded DNA (dsDNA) was transfected into T cells, strong activation was observed for phosphorylated stimulator of interferon response cGAMP interactor 1 protein (p-STING), and its downstream signaling molecules, phosp-TBK1 (p-TBK1) and phosp-IRF3 (p-IRF3). Also observed was production of inflammatory cytokines and type I interferon (IFN) gene expression, downstream of cyclic GMP-AMP synthases (cGAS)-STING signaling.
To further confirm that the cellular toxicity is mediated by the cGAS-STING signaling pathway, the inventors knocked out cGAS and STING expression with the Cas-CRISPR technology. The results show that cGAS or STING knockout cells exhibited significantly increased cell viability, as compared to control cells, upon dsDNA electroporation. These results, therefore, demonstrate that cGAS-STING mediated immune activation is the major cause of electroporation-induced cell death, and inhibition of this signaling pathway can reduce the toxicity, thereby improving electroporation efficiency.
cGAS is a cytosolic DNA sensor and does not react with nucleus DNA. Accordingly, the instant inventor designed another strategy to reduce cellular toxicity, by promoting nuclear delivery of polynucleotides. Nuclear delivery would bypass the surveillance of cytosolic cGAS. To this end, the inventors performed high throughput screening of electroporation buffers, and identified buffer B. When combined with the commercial cell culture medium, Opti-MEM™ medium at different ratios, buffer B achieved the highest nuclear delivery of DNA in various types of mammalian cells. Notably, buffer B included succinate and mannitol, which were determined to be responsible for its exceptional ability to promote nuclear uptake of the foreign polynucleotides. The inventors further determined that, within Opti-MEM, the sugar (e.g., glucose) and glutamine analog (L-alanyl-L-glutamine) are responsible for the success. Also discovered, unexpectedly, is that high salt concentrations (e.g., 90 mM NaCl of buffer A) can be detrimental to the nuclear uptake of certain forms of polynucleotides.
Opti-MEM™ medium is a reduced serum medium commercially available from Fisher Scientific. It is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology. The main ingredients relevant to the present technology are provided in Table 1. In particular, the Opti-MEM™ medium includes i-Inositol, Ca(NO3)2·4H2O, sodium pyruvate, D-glucose and GlutaMAX (L-alanyl-L-glutamine).
In accordance with one embodiment of the present disclosure, provided is an aqueous solution that includes succinate, mannitol, a sugar, glutamine or an analog thereof, and/or an antioxidant. In some embodiments, the sugar is selected from glucose, sucrose and inositol.
In accordance with another embodiment of the present disclosure, provided is an aqueous solution that includes less than 80 mM NaCl, or preferably less than 70 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM or 20 mM NaCl, which is useful for electroporation. In some embodiments, the aqueous solution includes a sugar, glutamine or an analog thereof, and an antioxidant. In some embodiments, the aqueous solution further includes succinate and/or mannitol. In some embodiments, the sugar is selected from glucose, sucrose and inositol.
Succinate, such as sodium succinate, potassium succinate, and magnesium succinate, is contemplated to be able to increase cell viability. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM succinate (e.g., sodium succinate). In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM or 10 mM succinate (e.g., sodium succinate).
Mannitol is contemplated to promote homology directed DNA repair. The instant results show that mannitol improves transfer of foreign polynucleotides through the cytoplasm into the nuclei. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM mannitol. In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM or 10 mM mannitol.
The sugar included in the aqueous solution is contemplated to optimize the osmolality of the solution, for optimized electroporation and nuclear uptake. In some embodiments, the sugar is selected from glucose, sucrose and inositol. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM sugar. In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM sugar. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM D-glucose. In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM D-glucose. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM sucrose. In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM sucrose. In some embodiments, the aqueous solution includes at least 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM inositol. In some embodiments, the aqueous solution includes no more than 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM inositol.
Glutamine, or analogs, is contemplated to promote cell health and vitality. In some embodiments, the glutamine or analog thereof is selected from the group consisting of L-alanyl-L-glutamine, L-glutamine, and D-glutamine. In some embodiments, the aqueous solution includes at least 50 mg/L, 100 mg/L, 110 mg/L, 120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L, 170 mg/L, 180 mg/L, 190 mg/L, 200 mg/L, 210 mg/L, 220 mg/L, 230 mg/L, 240 mg/L, 250 mg/L, 260 mg/L, 270 mg/L, 280 mg/L, 290 mg/L, or 300 mg/L glutamine or an analog thereof. In some embodiments, the aqueous solution includes no more than 1000 mg/L, 900 mg/L, 850 mg/L, 800 mg/L, 750 mg/L, 700 mg/L, 650 mg/L, 600 mg/mL, 650 mg/L, 600 mg/L, 550 mg/L, 500 mg/L, 480 mg/L, 460 mg/L, 440 mg/L, 420 mg/L, 400 mg/L, 380 mg/L, 360 mg/L, 340 mg/L, 320 mg/L, 300 mg/L, 280 mg/L, 260 mg/L, 240 mg/L, 220 mg/L, 200 mg/L, 180 mg/L, 160 mg/L, 140 mg/L, 120 mg/L, or 100 mg/L glutamine or an analog thereof. In some embodiments, the aqueous solution includes at least 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, 300 mg/L, 350 mg/L, 400 mg/L, 450 mg/L, 500 mg/L, 550 mg/L, 600 mg/L, 650 mg/L, 700 mg/L, 750 mg/L, or 800 mg/L L-alanyl-L-glutamine. In some embodiments, the aqueous solution includes no more than 1000 mg/L, 900 mg/L, 850 mg/L, 800 mg/L, 750 mg/L, 700 mg/L, 650 mg/L, 600 mg/mL, 550 mg/L, 500 mg/L, 480 mg/L, 460 mg/L, 440 mg/L, 420 mg/L, 400 mg/L, 380 mg/L, 360 mg/L, 340 mg/L, 320 mg/L, 300 mg/L, 280 mg/L, 260 mg/L, 240 mg/L, 220 mg/L, 200, or 200 mg/L L-alanyl-L-glutamine.
An “antioxidant” refers to a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that destabilize the protein therapeutics and ultimately affect the product activity. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents, chelating agent and oxygen scavengers such as pyruvate, acetylcysteine, citrate, EDTA, DPTA, thiols, ascorbic acid or polyphenols. Non-limiting examples of antioxidants include ascorbic acid (AA, E300), thiosulfate, methionine, tocopherols (E306), propyl gallate (PG, E310), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA, E320) and butylated hydroxytoluene (BHT, E321).
In some embodiments, the antioxidant is pyruvate (e.g., sodium pyruvate). In some embodiments, the antioxidant is acetylcysteine. In some embodiments, the aqueous solution includes at least 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.4 mM or 0.5 mM sodium pyruvate. In some embodiments, the aqueous solution includes no more than 0.6 mM, 0.5 mM, 0.4 mM, 0.35 mM, 0.3 mM, 0.25 mM, 0.2 mM, or 0.15 mM sodium pyruvate. In some embodiments, the aqueous solution includes at least 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.4 mM or 0.5 mM acetylcysteine. In some embodiments, the aqueous solution includes no more than 0.6 mM, 0.5 mM, 0.4 mM, 0.35 mM, 0.3 mM, 0.25 mM, 0.2 mM, or 0.15 mM acetylcysteine.
In some embodiments, the aqueous solution further includes Na2HPO4 and/or NaH2PO4. Suitable concentrations for the Na2HPO4 and/or NaH2PO4 in the solution may be 20 mM to 200 mM, 30 mM to 180 mM, 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 130 mM, 70 mM to 110 mM, 80 mM to 95 mM, without limitation.
In some embodiments, the aqueous solution further includes KCl, MgCl2 or MgSO4, Ca(NO3)2, amino acids, phenol red, and/or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the aqueous solution has a pH of 6.8 to 7.6, 6.9 to 7.5, 7 to 7.4, 7.1 to 7.3, 7.15 to 7.25, or about 7.3, without limitation. In some embodiments, the aqueous solution further includes serum.
In some embodiments, the aqueous solution includes no or limited NaCl, such as less than 80 mM NaCl, or less than 70 mM, 60 mM, 55 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM or 25 mM NaCl.
In some embodiments, the aqueous solution includes 5 mM to 30 mM sodium succinate, 1 mM to 30 mM mannitol, 3 mM to 30 mM glucose, 50 mg/L to 900 mg/L L-Glutamine or L-alanyl-L-glutamine, 0.1 mM to 0.6 mM sodium pyruvate, and includes less than 60 mM NaCl.
In some embodiments, the aqueous solution includes 8 mM to 25 mM sodium succinate, 3 mM to 25 mM mannitol, 5 mM to 25 mM glucose, 100 mg/L to 700 mg/L L-Glutamine or L-alanyl-L-glutamine, 0.2 mM to 0.5 mM sodium pyruvate, and includes less than 50 mM NaCl.
In some embodiments, the aqueous solution includes 10 mM to 20 mM sodium succinate, 5 mM to 20 mM mannitol, 8 mM to 20 mM glucose, 200 mg/L to 500 mg/L L-Glutamine or L-alanyl-L-glutamine, 0.3 mM to 0.4 mM sodium pyruvate, and includes less than 40 mM NaCl.
In another surprising discovery, the instant inventors demonstrated that the osmotic pressure of the electroporation buffer has significant impact on the delivery of large molecules. For double stranded DNA (dsDNA), for instance, higher osmotic pressure tends to reduce the transfection efficiency (
Such an observation to dsDNA, however, did not hold for RNA. For both cultured Jurkat cells and primary human T cells, higher osmolality of the electroporation buffers (e.g., above 330 mOsmol/kg) was beneficial to both transfection efficiency and target cell viability (
In accordance with one embodiment of the present disclosure, therefore, provided is a method for introducing an RNA or protein into a cell, comprising applying a pulse of electricity to a sample that includes the RNA or protein and a target cell, in an aqueous solution having an osmotic pressure that is higher than 300 mOsmol/kg, or higher than 310 mOsmol/kg, 320 mOsmol/kg, 330 mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/kg.
In some embodiments, the solution has an osmotic pressure that is from 310 mOsmol/kg to 600 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 310 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, 400 mOsmol/kg, or 350 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 320 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, 400 mOsmol/kg, or 350 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 330 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, 400 mOsmol/kg, or 350 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 340 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, 400 mOsmol/kg, or 350 mOsmol/kg.
In some embodiments, the solution has an osmotic pressure that is from 350 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, or 400 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 360 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, or 400 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 370 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, or 400 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 380 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, or 400 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 390 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, 450 mOsmol/kg, or 400 mOsmol/kg. In some embodiments, the solution has an osmotic pressure that is from 400 mOsmol/kg to 600 mOsmol/kg, 550 mOsmol/kg, 500 mOsmol/kg, or 450 mOsmol/kg.
A suitable solution can be prepared by admixing a commonly used or presently disclosure buffer (e.g., BO14, BO11, B-ICSGG, and B-2xICSGG and their derivatives) with an electrolyte or a non-electrolyte, such as an organic molecule (e.g., sugar, amino acid, polymer).
An example buffer suitable for preparing an aqueous solution with the desired osmotic pressure includes succinate, mannitol, a sugar selected from the group consisting of glucose, sucrose and inositol, glutamine or an analog thereof, and an antioxidant.
In some embodiments, the sugar is D-glucose. In some embodiment, the glutamine or analog thereof is selected from the group consisting of L-alanyl-L-glutamine, L-glutamine, and D-glutamine. In some embodiments, the succinate is sodium succinate, potassium succinate, or magnesium succinate. In some embodiments, the antioxidant is sodium pyruvate or acetylcysteine. In some embodiments, the buffer further includes Na2HPO4 and NaH2PO4. In some embodiments, the buffer further includes serum. In some embodiments, the buffer includes less than 80 mM NaCl, or preferably less than 70 mM, 60 mM, or 50 mM NaCl.
Various additional examples of the buffer are described in the section above, under heading “Electroporation Buffers.” In a particular example, the buffer includes 5 mM to 30 mM sodium succinate, 1 mM to 30 mM mannitol, 3 mM to 30 mM glucose, 50 mg/L to 900 mg/L L-Glutamine or L-alanyl-L-glutamine, 0.1 mM to 0.6 mM sodium pyruvate, and includes less than 60 mM NaCl.
In some embodiments, the osmotic pressure of the buffer can be adjusted with a non-electrolyte agent, such as those listed in Table A below.
Example concentrations of these agents are provided in Table A. Generally, at such concentrations, the osmotic pressure of the buffer can be adjusted within the range of 300-600 mOsmol/kg. The actual concentration used can be determined based on the desired osmotic pressure, which can be measured experimentally.
In some embodiments, the concentration of the non-electrolyte agent is 10 mM to 200 mM or 300 mM. In some embodiments, the concentration of non-electrolyte agent is 20 mM to 200 mM or 300 mM, 30 mM to 200 mM or 300 mM, 40 mM to 200 mM or 300 mM, 50 mM to 200 mM or 300 mM, 60 mM to 200 mM or 300 mM, 70 mM to 200 mM or 300 mM, 80 mM to 200 mM or 300 mM, 90 mM to 200 mM or 300 mM, or 100 mM to 200 mM or 300 mM.
In some embodiments, the concentration of the non-electrolyte agent is 10 mM to 100 mM. In some embodiments, the concentration of non-electrolyte agent is 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 50 mM to 100 mM, 60 mM to 100 mM, 70 mM to 100 mM, 80 mM to 100 mM, or 90 mM to 100 mM.
In some embodiments, the osmotic pressure of the buffer can be adjusted with a non-electrolyte agent, such as those listed in Table B below.
Example concentrations of these agents are provided in Table B. Generally, at such concentrations, the osmotic pressure of the buffer can be adjusted within the range of 300-480 mOsmol/kg. The actual concentration used can be determined based on the desired osmotic pressure, which can be measured experimentally.
In some embodiments, the concentration of the electrolyte agent is 3 mM to 40 mM or 90 mM. In some embodiments, the concentration of the electrolyte agent is 4 mM to 40 mM or 90 mM, 5 mM to 40 mM or 90 mM, 6 mM to 40 mM or 90 mM, 7 mM to 40 mM or 90 mM, 8 mM to 40 mM or 90 mM, 9 mM to 40 mM or 90 mM, 10 mM to 40 mM or 90 mM, 12 mM to 40 mM or 90 mM, 14 mM to 40 mM or 90 mM, 15 mM to 40 mM or 90 mM, 17 mM to 40 mM or 90 mM, 19 mM to 40 mM or 90 mM, or 20 mM to 40 mM or 90 mM.
In some embodiments, the concentration of the electrolyte agent is 3 mM to 30 mM. In some embodiments, the concentration of the electrolyte agent is 4 mM to 30 mM, 5 mM to 30 mM, 6 mM to 30 mM, 7 mM to 30 mM, 8 mM to 30 mM, 9 mM to 30 mM, 10 mM to 30 mM, 12 mM to 30 mM, 14 mM to 30 mM, 15 mM to 30 mM, 17 mM to 30 mM, 19 mM to 30 mM, or 20 mM to 30 mM.
In some embodiments, the solution has a pH of 6 to 8, 6.1 to 8, 6.2 to 8, 6.3 to 8, 6.4 to 7.9, 6.5 to 7.8, 6.6 to 7.7, 6.8 to 7.6, 6.9 to 7.5, 7 to 7.4, 7.1 to 7.3, 7.15 to 7.25, or about 7.3, without limitation.
In some embodiment, the method is used to deliver an RNA molecule to a target cell, including prokaryotic cells and eukaryotic cells, which may be mammalian cells such as an animal cell including a human cell. In some embodiments, the target cell is an immune cell such as a T cell or an NK cell. In some embodiments, the RNA is an mRNA molecule, or an siRNA, shRNA, rRNA or tRNA. In some embodiments, the method is used to deliver a protein to a target cell. In some embodiments, the method is used to deliver a RNA-protein complex, without limitation.
Likewise, methods for introducing a DNA molecule to a target cell are also provided, wherein the osmotic pressure is provided as close to isotonic (e.g., between 270 and 330 mOsmol/kg, or between 280 and 320 mOsmol/kg, or between 290 and 310 mOsmol/kg, or between 295 and 305 mOsmol/kg). The isotonic osmotic pressure may help mitigate cGAS-STING signaling/activation in the cell that is activated by the DNA molecule. In some embodiments, the DNA is dsDNA. Compositions and methods useful for providing the desired osmotic pressure are described above.
Methods for introducing polynucleotides into target cells, e.g., through electroporation, are also provided. In one embodiment, a method is provided for introducing a polynucleotide into a cell. The method, in some embodiments, entails applying a pulse of electricity to a sample that includes the polynucleotide and the cell in an aqueous solution provided herein.
As demonstrated in the experimental examples, various types of polynucleotides can be effectively delivered to the nuclei of cells with the present technology. In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the DNA is single-stranded DNA or double-stranded DNA. In some embodiments, the RNA is siRNA, sgRNA, or single- or double-stranded RNA. In some embodiments, the RNA is provided in an RNA-protein complex.
In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immune cell, such as a natural killer (NK) cell, a T cell, a macrophage, or a monocyte, without limitation.
In some embodiments, the method entails applying a pulse of electricity to a sample that includes the polynucleotide and the cell which has been treated with an agent, or in the presence of an agent that inhibits the cGAS-STING pathway. In some embodiments, the method is carried out in an aqueous solution of the present disclosure.
In some embodiments, the agent is an siRNA or antibody targeting the cGAS or STING protein. Methods are well known for preparing inhibitory RNA and antibodies that can be effectively delivered to a cell to effect their inhibitory functions. In some embodiments, the agent is removed at some time following the electroporation, such as after 1 hour, 2 hours, 6 hours, 12 hours, a day, 2 days, 3 days, 7 days, 14 days, a month, or two months, without limitation.
Pulses of electricity can be generated with devices and systems that are commercially available, including those tested herein, such as 4D-nucleofector (Lonza). Each device is equipped with suitable electroporation programs, which can be further tuned for specific applications. The use and tuning are within the capability of skilled artisans.
In some embodiments, the method results in entry of the polynucleotide into the nucleus of the cell.
The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skills in the art that the techniques disclosed in the examples which follow represent techniques to function well in the practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skills in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
This example demonstrates that DNA delivery-induced toxicity in primary human T cell is mediated by cGAS-STING. To reduce DNA delivery-induced toxicity in T cells, this example systematically investigated electroporation conditions and identified a buffer composition which could significantly bypass the cytosolic cGAS-STING surveillance via enhanced DNA nucleus delivery. The identified BO14-buffer led to a five-fold increase of reprogrammed CAR-T cells compared with commercial standard after electroporation of DNA template. In addition, BO14-buffer generated CAR-T showed more potent tumor killing activity than CAR-T produced by AAV both in vitro and in vivo.
Primary Human T cell isolation and culture. Human T cells were isolated from healthy donors using fresh whole blood (SAILY BIO). In brief, peripheral blood mononuclear cells (PBMCs) were enriched by Ficoll (GE) density centrifugation using SepMate tube (STEMCELL). EasySep Human T Cell Isolation Kit (STEMCELL) was used to isolate CD3+ T cells from PBMC following the manufactory's instruction. Isolated T cells were activated by CD3/CD28 dynabeads (Thermo Fisher) at a bead to cell ratio of 1:3 for three days. CD3+ T cells were cultured in X-Vivo15 medium (Lonza) supplemented with 5% fetal bovine serum (FBS) (Gibco), 50 ng/ml human IL-2, 10 ng/ml IL-7 (Peprotech) and 1% Penicillin/Streptomycin. After electroporation, T cells were cultured in media with IL-2 at 100 ng/ml. Every 2-3 days after electroporation, additional media was added, along with fresh IL-2 to bring the final concentration to 100 ng/ml.
Double-stranded DNA (dsDNA). The homologous arms and inserts of the HDR template were amplified by PCR. All fragments of HDR template were ligated by Gibson assembly (TransGen Biotech) and sequence validated. 1 pg plasmid were used as PCR template per 50 μl reaction and PCR product were column purified to prepare dsDNA.
The 5′ end modified HDR template was prepared by PCR amplification with modified primers. Primers with 5′ phosphorothioate bonds and amine C6 were ordered from Sangon Biotech (Shanghai, China). Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588) and incubated with 10 μM primers with 5′ amine C6 group and 5′ phosphorothioate bonds at 1 mM concentration in ×1 borate buffer (LEAGENE, I1H0217) overnight at room temperature. Modified primers were then desalted by concentrator tube Amicon Ultra-0.5 (Millipore). The size and purity of the amplified HDR template were confirmed by 1% agarose gel electrophoresis. PCR products were purified using DNA Clean & Concentrator PCR purification kit (ZYMO, D4034). Concentrations of HDRTs were determined by nanodrop (Thermo Fisher).
Single-stranded DNA. ssDNA was produced using Guide-it Long ssDNA Production System (Takara Bio, 632644). In general, one regular primer and a 5′-phosphorylation labeled primer was used to amplify the target DNA. Phosphorylation labeled DNA strand were further digested and the resulting ssDNA were purified and checked on agarose gel.
Single-stranded oligonucleotides (ssODN) and double-stranded oligonucleotides (dsODN). ssODN were synthesized in Sangon Biotech (Shanghai, China). dsODN is prepared by hybridizing two complementary ssODN in hybridization buffer (10 mM Tris-HCl, pH 8.0, 20 mM NaCl) and further amplified by PCR to generate large quantity.
Electroporation (EP). Three days after T-cell stimulation, cells were electroporated using 4D-nucleofector (Lonza). In general, 0.6 to 3 million cells were resuspended in 20 μl indicated EP buffer and then mixed with RNP or siRNA or DNA repair template. Cell mixture were transferred to a 20 μl electroporation cuvette and electroporated under indicated EP program. Unless indicated, electroporation was performed under P3-EO115 condition as recommended by Lonza for human T cell. After electroporation, 80 μl of pre-warmed media was added to each cuvette, and cells were allowed to rest for 10 min at 37° C. before transferring to a 96-well plate. 100 ng chemically synthesized siRNA (GenScript) were used per EP. RNPs were produced by pre-complexing Cas9 protein with sgRNA at a 1:5 molar ratio. 3 μg chemical synthesized sgRNAs (GenScript) were incubated with 3 μg Cas9-NLS (Aldevron) at 25° C. for 10 min. In HDR experiment, 0.5-3 μg donor DNA were added to the RNP complex before mixing with cells.
Basic buffer A and Basic buffer B (Table 1) were prepared by admixing the respective ingredients. Basic buffer 0 is Opti-MEM medium adjusted to pH 7.2. The prepared basic buffer was filtered and sterilized with 0.2 m filter membrane. Basic buffer B or A was then mixed with opti-mem at different ratio. Five components were prepared at the following final concentration in buffer B: 0.13 mM i-inositol, 0.4 mM Ca(NO3)2, 0.25 mM Sodium Pyruvate, 10 mM D-glucose, 1% GlutaMAX. Mixed electroporation buffer can be stored at 4 degree for up to 6 months.
Flow cytometry. Flow cytometry analysis was performed on Agilent NovoCyte Flow Cytometer. The expression of GFP and absolute cell count were performed in a 96-well flat plate. For absolute cell count, 20-50 μl cell suspension was taken and diluted 2-4 times in FACS buffer. To detect CD19BBz-CAR expression, 5×105 cells were incubated with 0.5 μg of CD19-Fc (R&D Systems) in 40 μl FACS buffer (PBS+0.1% BSA+1% Penicillin/Streptomycin) for 30 min, and then stained with FITC-IgG-Fc, APC-TCRα/β antibodies (Biolegend) for 20 min on ice. The stained cells were measured on a NovoCyte flow cytometer, and analyzed with Novo Express Software. Intracellular staining was performed to detect the expression level of IFN-γ and TNF-α. AAV-CAR T and BO14-CAR T cells were co-cultured with FFluc-GFP NALM-6 at 1:1 ratio for 1 h. 2 μM Monensin (Biolegend) was added to the co-culture for another 5 hours before staining. CAR staining is performed first, followed by fixation and permeabilization. Antibodies against IFN-γ and TNF-α were incubated for 30 min and cells were analyzed by flow cytometer.
dsDNA labeling 20 μg of dsDNA (1350 bp) was incubated with 10 l Reagent of Cy5 Label-IT kit for 1 hour at 37° C. (Mirus Bio) prior to column purification (ZYMO RESEARCH). The purified DNA was quantified with NanoDrop and stored away from light at −20° C. 1×106 human primary T cells were transfected with 1 μg labeled dsDNA, and cells were collected 10 min post-EP for downstream analysis.
Staining and confocal analysis. Cells were fixed in 4% formaldehyde for 10 min at room temperature and were incubated with Phalloidin. Stained cells were resuspended to 50-100 μl with PBS and cytospinned on microscope slides for 10 min at 500 g. After the spin, 1 drop of DAPI-mounting medium (Sigma, F6057) was added on the slide and covered with a coverslip. Cy5-labeled dsDNA was localized and photographed using Zeiss LSM880 confocal microscope under a 60× oil objective. The field of vision was randomly selected and the focal length was adjusted based on Cy5 fluorescence. The DAPI was used to visualize the cell nuclei, phalloidin to estimate the cell borders and Cy5 is used to trace DNA distribution. Nuclear localization ratio was obtained by manual count. The mean fluorescence intensity of Cy5-labeled dsDNA within the nucleus and the entire cell were calculated using the image analysis tools of the Zeiss ZEN 2.3 Image Processing Software.
4 hours post-EP, 5×105 cells were collected for RNA extraction (Magen, #R4122-03) and cDNA preparation using HiScript II Q Select RT SuperMix (Vazyme, #R223-01). Real-time PCR reactions were performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme #Q711-02) on a Bio-Rad CFX Connect 96-well based on the manufacture instructions. The expression level of each indicated gene was normalized to GAPDH.
Nalm6 cells and THP1 cells (BNBIO, China) were cultured in RPMI1640 (Gibco) medium with 10% FBS (AusGeneX) and 1% Penicillin/Streptomycin (Gibco). HEK293T17 (ATCC) cells cultured in DMEM (Gibco) medium with 10% FBS and 1% Penicillin/Streptomycin.
Nalm6-luciferase-GFP cell line (FFluc-GFP Nalm6-CD19+) was generated by infecting Nalm6 with lentivirus carrying a GFP-P2A-luciferase construct. GFP expressing cells were sorted and expanded for further experiments. Nalm6-luciferase-GFP-CD19 knockout cell line (FFluc-GFP Nalm6-CD19−) were generated by knocking out CD19 via a lentivirus carrying sgRNA against CD19 and spCas9. CD19 negative cells were FACS sorted out to establish the control cell line. DNA analysis by TIDE were performed to confirm the genetic ablation of CD19.
5×105-1×106 cells were lysed by ice-cold RIPA buffer (Beyotime) containing protease inhibitors (Beyotime) and incubated on ice for 15 min. Protein supernatant was collected after centrifuging at 13,000 g 4° C. for 5 min. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime). Proteins were separated by 10% SDS-PAGE, followed by immunoblot analysis with the indicated antibodies.
AAV1 was produced by triple transfection of AAV2/CD19BBz, AAV1 serotype plasmid, and a helper plasmid via polyethyleneimine (PEI) in HEK293FT cells. Both cell supernatant and cell pellet were collected at 72 h for AAV purification. AAV is purified using iodixanol density gradient. In brief, cell pellet was lysed by repeated freeze-thaw cycles to release viruses, followed by benzonase (Sigma) treatment to remove gDNA and RNA. To collect AAV from supernatant, 0.0233 g/ml NaCl and 0.085 g/ml Polyethylene Glycol 8000 were added sequentially. The mixture was centrifuged at 6600 rpm at 4° C. for 15 min and the pellet was suspended in 1×GB buffer (0.5M NaCl, 0.1M MgCl2, 0.1M Tris pH 7.6). Resuspended pellets containing AAVs were added to a discontinuous iodixanol gradients and centrifuged at 48000 rpm for 2 h. AAVs located at 40-60% layer was extracted via a syringe and followed by concentration via Millipore Amicon filter unit (Millipore). Virus titer was determined by qPCR using ChamQ Universal SYBR qPCR Master Mix targeted to CAR. To generate AAV-CAR, stimulated T cells were first electroporated with RNP. 10 min post-EP, AAV1-CAR were added at a MOI=5×105. Cells were washed 1-2 h post AAV1 infection.
FFluc-GFP Nalm6-CD19+ was used as target antigen cells, and FFluc-GFP Nalm6-CD19− was used as control cells. The effector (AAV-CAR; BO14-CAR) and target cells or control cells were co-cultured in triplicates at indicated E/T ratios using 96 U-bottom well plate with 1×104 target cells or control cells in a total volume of 150 μl per well in Nalm-6 culture medium. 18 h later, luciferase assay intensity was measured by a plate reader (MD SpectraMax i3×) with Luciferase Assay System (Promega). The number of tumor cells was calculated according to the curvature.
6- to 8-week-old NOD/SCID/IL-2Rγ null mice (Gempharmatech, China) were inoculated with 2×105 FFluc-GFP Naml6 cells by tail vein injection. 4 days post-tumor cell injection, AAV-CAR or BO14-CAR T cells were injected at a dose of 5×105 per animal. Prior to imaging, 4.5 mg D-Luciferin potassium (Meilunbio, China) was intraperitoneal injected into each animal. Bioluminescence imaging were performed under the SI Imaging AmiX Imaging System (Spectral Instruments Imaging, USA) with Living Image software (Spectral Instruments Imaging) for acquisition of imaging datasets. All relevant animal-use guidelines and ethical regulations were followed.
Non-viral reprogramming of primary human T cells provides a safe, fast and virus-free generation of CAR-T cells. Unlike lentivirus-mediated random integration or site-specific integration of CAR construct using AAV as templates, non-viral reprogramming of CAR-T cells circumvents the need of lentivirus or AAV preparation for donor DNA delivery and can be quickly adopted for manufactory process development. However, the biggest hurdle that holds back the non-viral delivery is the cellular toxicity after the electroporation of DNA donor and the relative low delivery efficiency into human T cells.
To address this issue, we first explored the mechanism of DNA electroporation induced T cell toxicity. In healthy cells, DNA is restricted to the mitochondria and nucleus. Cyclic GMP-AMP synthases (cGAS) detects cytoplasmic DNA and signals through stimulator of interferon gene (STING) to initiate an innate immune response. We hypothesized that cGAS-STING was essential to trigger electroporation of DNA-induced cellular toxicity in human T cells.
To examine this hypothesis, we first determined which types of DNA was most detrimental to primary human T cells. Five types of DNA including a 3.5 kb plasmid, a 1.4 kb linear double-stranded DNA (dsDNA), a 1.4 kb single-stranded (ssDNA), a 99-mer double-stranded oligonucleotides (dsODN) and a 99-mer single-stranded oligonucleotide (ssODN) were electroporated into T cells and cell numbers were counted. dsDNA and plasmid showed ˜14-17 fold loss of cells when compared with MOCK electroporation (MOCK EP) (
To determine whether cell loss is a result from cGAS-STING activation, we examined STING phosphorylation (p-STING) and its downstream signaling pathways phosp-TBK1 (p-TBK1) and phosp-IRF3 (p-IRF3). Similar to MOCK EP control, dsODN, ssODN and ssDNA did not exhibit any activation, whereas plasmid and dsDNA transfected T cells showed strong activation of p-STING and p-TBK1 and p-IRF3 (
We next sought to ask if DNA induced T cell toxicity can be rescued by genetic ablation of cGAS-STING mediated immune response pathway. sgRNAs against cGAS or STING were screened and used to effectively knockout cGAS and STING expression prior to DNA electroporation (
To test whether this is the case in mouse, we isolated T cells from cgas or sting null mice and electroporated with 1 μg plasmid. Similar to human T cells, mouse cgas−/− or sting−/− T cells showed 17.5 fold more viable cells than wild-type, and significantly decreased cGAS-STING downstream gene expressions. To confirm that STING is the key player, we restored full-length or STING bearing a C88/91A or C88/91S mutation, a well-characterized palmitoylation site. T cells isolated from sting−/− mouse were infected with retrovirus and followed by plasmid challenge. Restoring full-length Sting induced significant cell death and upregulation of signature genes compared vector control. Interestingly, STING bearing C88/91A or C88/91S mutation expression still showed significant cell death, indicating that STING palmitoylation is not essential for T cell death. Taken together, these data suggested that cGAS-STING activation is the major cause of dsDNA electroporation-induced cell loss in primary human T cell.
cGAS-STING pathway is crucial for innate immune response and other biological functions. We next sought to transiently inhibit this pathway by small molecule inhibitor H151 or siRNA-mediated knockdown. Unfortunately, H151 treatment failed to rescue DNA-induced cell death and in align with this, IFNβ and ISG56 were upregulated in DNA transfected T cells (
cGAS is a cytosolic DNA sensor and does not react with nucleus DNA. Therefore, another strategy is to promote nuclear delivery of DNA, and thereby bypassing the surveillance of cytosolic cGAS sensor. To achieve this, we performed high throughput screening of different electroporation buffer and program combinations that enable nuclear delivery of DNA in primary human T cell (
Of three electroporation conditions, buffer BO14 paired with E0138 program had the highest number of GFP expressing cells, much higher than the commercial buffer (
In alignment with enhanced nuclear delivery, cells electroporated with dsDNA encoding an EF1α promoter followed by EGFP exhibited enhanced GFP cells number and expression level under BO14-mediated delivery compared with P3 commercial buffer (
Nucleus delivery of DNA donor is critical for homologous directed repair (HDR). We next tested whether the system could improve CRISPR/Cas9 mediated target insertion. We chose five different loci and tested three knock-in strategies including targeting exons or introns, or inserting an EF1a driven EGFP construct (
BO14 buffer is composed of basic buffer B mixed with opti-mem at 1:4 ratio. To determine the key components that are crucial in mediating nuclear delivery, we subtracted potential important components in opti-mem and added back to basic buffer B one by one. While each component showed a minor increase of knock-in rate compared with basic buffer alone, the mixture of all five components (Inositol, Calcium nitrate, Sodium pyruvate, Glucose, GlutaMAX, collectively “ICSGG”) showed a comparable knock-in rate and cell number to BO14 buffer (
Chimeric Antigen Receptor (CAR) reprogrammed T cells are a powerful immunotherapy option for several hematological malignancies. Recent study of CAR-T cell reprogramming has found out site-specific insertion of CARs into endogenous TRAC locus using CRISPR/Cas9 and an adeno-associated virus vector to deliver the donor template has improved functionality over the conventional lentivirus transduced counterparts.
To expand the usage of our system, we inserted the CD19-specific 19BBz CAR construct into TRAC locus so that the expression of CAR is under the endogenous promoter (
Next, to determine whether BO14-mediated target insertion of CAR-T is functional, we compared non-viral based methods with AAV-mediated donor delivery. In both cases, CAR sequence and RNP are the same to follow the same integration strategy. The only difference is the method of donor template delivery. In non-viral based methods, 5′ PS-PEG modified dsDNA were co-electroporated with RNP whereas in AAV-mediated delivery, RNP were electroporated first, followed by AAV addition. Equal amount CAR-T cells were co-cultured with tumor cells expressing CD19 antigen Nalm6-luciferase cells or Nalm6-CD19 KO cells. When CAR-T were incubated with tumor cells as 5:1 ratio, all tumor cells were lysed by non-viral delivered CAR or AAV-CAR; when incubated at 1:1 ratio, five-fold lower tumor cells were left in BO14 CAR-T, indicating that BO14 generated CAR-T has equal or higher tumor killing potency than AAV-CAR (
Likewise, the B+ICSGG buffer was tested for non-viral delivery, as compared to AAV- or lentivirus-mediated delivery. In lentivirus-based delivery, the same CAR sequence was used and purified lentivirus (MOI=10) was used to infect human T cells. Compared with lenti-CAR, AAV-CAR or P3-CAR, B+ICSGG-CAR exhibited the highest CAR expression intensity among all methods and significant enhanced CAR-T positive percentage than lentiviral or P3 based methods. Long term track of CAR-T production also showed that the absolute B+ICSGG-CAR T outnumbered that of lentivirus.
The higher lysis efficiency of non-viral delivered CAR likely resulted from the higher secretion of TNF-α, the anti-tumor cytokines (
In summary, this example described a simple and effective strategy for non-viral genome engineering by mitigating cGAS-STING activation through improved nuclear delivery of dsDNA. Evaluation of five different repair template DNA formats, we found plasmid and dsDNA induced the most profound loss of primary T cells and cGAS-STING activation compared with ssDNA, dsODN or ssODN (
To mitigate STING activation, we systematically optimized electroporation system and identified a new buffer BO14 that showed enhanced nuclear delivery of dsDNA with reduced cGAS-STING activation. The new methods exhibited 2-5 fold increase in targeted insertion cells across multiple loci in primary human T cells (
The present method has several advantages in combination (
This example tested the use of the developed buffer and a variant for nucleus-targeted DNA delivery in various types of cells.
The new electroporation (EP) protocols, e.g., BO buffers used with corresponding programs, were tested for plasmid and RNP delivery into 293T cells. As noted in Example 1, the BO14 buffer contained basic buffer B mixed with opti-mem at 1:4 ratio. Likewise, the BO11 buffer contained basic buffer B mixed with opti-mem at 1:1 ratio.
As shown in
Similarly, the new EP protocol with the BO11 buffer was used for gene delivery to U-2 OS, jurkat, and Raw264.7 cell lines. The results are presented in
In another experiment, the EP protocol (both BO11 and BO14) was used to deliver RNP into mouse primary T and macrophage cells. As shown in
In addition, a testing was conducted for delivery of DNA for mediating Cas9-based HDR in Jurkat cells. As shown
This example tested the use of the developed buffer for enhanced mRNA delivery into the human hematopoietic stem and progenitor cells (HSPC).
The buffer BO11 (1 part of buffer B and 1 part of Opti-mem) was used in this example. BO11 achieved three-fold increase in delivering mRNA to the nuclei of HSPC, when compared with the commercial standard (P3-Lonza).
The conditions for electroporation (EP) process for BO11, BO14, A041 and P3 were optimized using GFP mRNA in human CD34+ hematopoietic stem and progenitor cells (HSPCs) (
This example discovered that while an isotonic osmotic pressure (about 300 mOsmol/kg) of the electroporation buffers was optimal for dsDNA delivery efficiency, a minimum of 330 mOsmol/kg osmotic pressure was required for efficient delivery of RNA.
First, the impacts of osmotic pressure of the electroporation buffer on delivery of 0.5 μg of dsDNA (encoding GFP) to primary mouse T cells were tested. The buffers tested had osmotic pressures ranging from 190 to 512 mOsmol/kg. As shown in
Second, electroporation buffers of a range of osmotic pressures (158-500 mOsmol/kg) were tested to deliver 0.5 μg of GFP-encoding mRNA via electroporation to Jurkat cells. When the osmolality of electroporation buffer was above 330 mOsmol/kg, significantly enhanced MFI (
This experiment was repeated to deliver 0.5 μg GFP-encoding mRNA to primary human T cells. Similar results were observed. When the osmolality of the electroporation buffers was above 330 mOsmol/kg, significantly enhanced GFP positive cells (
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc., shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.
Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/107047 | Jul 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/106565 | 7/19/2022 | WO |
