COMPOSITIONS AND SYSTEMS COMPRISING TRANSFECTION-COMPETENT VESICLES FREE OF ORGANIC-SOLVENTS AND DETERGENTS AND METHODS RELATED THERETO

Abstract

Lipid-based vesicles, typically herein called transfection competent vesicles (TCVs), configured to safely and efficiently deliver DNA, RNA, other nucleic acid and protein cargoes into target cells. The safety and efficiency are each, and both, achieved in part by eliminating organic solvents such as ethanol and detergents such as sodium dodecyl sulfate from the TCV loading processes (i.e., inserting a cargo into the TCV), TCV storage processes, and/or TCV delivery processes. The cargoes can also comprise nucleic acids complexed with a protein, such as a ribonucleoprotein (RNP). The systems, compositions, devices and methods, etc., herein, in some embodiments, can provide empty TCVs that can if desired be loaded at the bench without use of specialized equipment.

Claims

1-153. (canceled)

154. A method of encapsulating a ribonucleoprotein (RNP) into an ionizable cationic lipid-based transfection competent vesicle (TCV) comprising: providing a water-based solution comprising the ionizable cationic lipid-based, wherein the water-based solution is free of organic solvents and detergents and wherein the ionizable cationic lipid-based TCV comprises about 20 mol% to about 30 mol% ionizable cationic lipid; and, mixing the RNP into the water-based solution under conditions suitable and for a time sufficient for the RNP to encapsulate within the ionizable cationic lipid-based TCV to provide an ionizable cationic lipid-based TCV-encapsulated RNP, wherein the mixing is performed without the presence of an organic solvent or detergent.

155. The method of claim 154, wherein the ionizable cationic lipid of the ionizable cationic lipid-based TCV comprises 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) and/or 1,2—Bis(linoleoyloxy)—3—(dimethylamino)propane (DLinDAP).

156. The method of claim 154, wherein the ionizable cationic lipid-based TCV comprises a mixture of: (a) the ionizable cationic lipid, (b) one or more phospholipids, optionally 1,2—distearoyl—sn—glycero—3—phosphocholine (DSPC) and/or 1,2—dioleoyl—sn—glycero—3—phosphoethanolamine (DOPE), (c) cholesterol and (d) PEG-lipid, optionally at about 1 mol%.

157. The method of claim 154, wherein the ionizable cationic lipid-based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol optionally at about 20/30/10/40 mol% or the ionizable cationic lipid- based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol/PEG—lipid optionally at about 20/30/10/39/1 mol%.

158. The method of claim 154, wherein the water-based solution (i) comprises an acetate buffer, optionally a 25 mM to 100 mM acetate buffer, and/or (ii) is pH4.

159. The method of claim 154, wherein: (a) the ionizable cationic lipid-based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol optionally at about 20/30/10/40 mol% or comprises a mixture of DODMA/DOPE/DSPC/Chol/PEG-lipid optionally at about 20/30/10/39/1 mol%; and (b) the RNP comprises a Cas9 protein and a guide RNA.

160. The method of claim 154, wherein the mixing is performed using staggered herringbone micromixing, T-junction mixing or via reciprocation in a pipette.

161. The method of claim 154, wherein the mixing is at about room temperature for about 10 to 30 seconds, preferably about 10 to 15 seconds.

162. The method of claim 154, wherein the ionizable cationic lipid- based TCV is empty prior to the encapsulation.

163. A composition comprising an ionizable cationic lipid-based transfection competent vesicle (TCV)-encapsulated RNP in a water-based solution substantially free of organic solvents and detergents, wherein the ionizable cationic lipid-based TCV-encapsulated RNP was produced according to the method of claim 154, optionally wherein the ionizable cationic lipid of the ionizable cationic lipid-based TCV comprises 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) and/or 1,2—Bis(linoleoyloxy)—3—(dimethylamino)propane (DLinDAP).

164. A composition comprising an ionizable cationic lipid-based transfection competent vesicle (TCV)-encapsulated RNP in a water-based solution substantially free of organic solvents and detergents, wherein the ionizable cationic lipid-based TCV-encapsulated RNP was produced according to the method of claim 160.

165. A composition comprising an ionizable cationic lipid-based transfection competent vesicle (TCV) in a water-based solution, wherein the composition is free of organic solvents and detergents and wherein the ionizable cationic lipid-based TCV comprises about 20 mol% to about 30 mol% ionizable cationic lipid.

166. The composition of claim 165, wherein the ionizable cationic lipid of the ionizable cationic lipid-based TCV comprises 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) and/or 1,2—Bis(linoleoyloxy)—3—(dimethylamino)propane (DLinDAP).

167. The composition of claim 165, wherein the ionizable cationic lipid-based TCV comprises about 20 mol% ionizable cationic lipid or comprises about 30 mol% ionizable cationic lipid, optionally wherein the ionizable cationic lipid-based TCV comprises 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) and/or 1,2—Bis(linoleoyloxy)—3—(dimethylamino)propane (DLinDAP).

168. The composition of claim 165, wherein the ionizable cationic lipid-based TCV comprises (i) a mixture of the ionizable cationic lipid, phospholipid, cholesterol and PEG-lipid; or (ii) a mixture of 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) /1,2—dioleoyl—sn—glycero—3—phosphoethanolamine (DOPE)/ 1,2—distearoyl—sn—glycero—3—phosphocholine (DSPC)/cholesterol (Chol); or (iii) 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA)/ 1,2—dioleoyl—sn—glycero—3—phosphoethanolamine (DOPE)/ 1,2—distearoyl—sn—glycero—3—phosphocholine (DSPC)/ cholesterol (Chol)/PEG-lipid.

169. The composition of claim 165, wherein the ionizable cationic lipid- based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol, optionally respectively at about 20/30/10/40 mol% or the ionizable cationic lipid- based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol/PEG-lipid, optionally respectively at about 20/30/10/39/1 mol%.

170. The composition of claim 165, wherein the water-based solution (i) comprises an acetate buffer, optionally a 25 mM to 100 mM acetate buffer, and/or (ii) is pH4.

171. The composition of claim 165, wherein: (a) the ionizable cationic lipid-based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol, respectively optionally at about 20/30/10/40 mol%; and (b) the water-based solution comprises 25 mM to 100 mM acetate buffer and/or is pH4.

172. The composition of claim 165, wherein the composition consists essentially of the ionizable cationic lipid-based TCV in the water-based solution, optionally wherein the ionizable cationic lipid-based TCV comprises 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA).

173. The composition of claim 165, wherein the ionizable cationic lipid- based TCV is an empty lipid-based TCV.

174. The composition of claim 165, wherein a therapeutic cargo is encapsulated in the ionizable cationic lipid-based TCV, optionally wherein said therapeutic cargo comprises a nucleic acid, ribonucleoprotein or nucleic acid protein complex, or a protein.

175. The composition of claim 174, wherein said nucleic acid or nucleic acid complex comprises a RNA, DNA, RNA protein complex, or DNA protein complex, or a protein.

176. The composition of claim 165, wherein a RNP is encapsulated in the ionizable cationic lipid-based TCV.

177. The composition of claim 176, wherein the RNP comprises a Cas9 protein and a guide RNA.

178. The composition of claim 165, wherein: (a) the water-based solution pH4 buffer is comprises a25 mM to 100 mM acetate buffer and/or is pH4; (b) the ionizable cationic lipid-based TCV comprises a mixture of DODMA/DOPE/DSPC/Chol, optionally respectively at about 20/30/10/40 mol%, or comprises a mixture of DODMA/DOPE/DSPC/Chol/PEG-lipid, optionally respectively at about 20/30/10/39/1 mol%; and (c) a RNP is encapsulated in the ionizable cationic lipid-based TCV, wherein the RNP comprises a Cas9 protein and a guide RNA.

179. A method for use in inhibiting, preventing, or treating a disease or condition in a patient, comprising administering to the patient a therapeutically effective amount of a composition according to claim 164, optionally wherein the patient comprises a human.

180. A method for use in inhibiting, preventing, or treating a disease or condition in a patient, comprising administering to the patient a therapeutically effective amount of a composition according to claim 174, optionally wherein the patient comprises a human.

181. A method of transfection, the method comprising transfecting a target cell with an ionizable cationic lipid-based TCV-encapsulated RNP produced according to a method of claim 154, wherein the target cell is a mammalian cell, optionally a human cell.

182. A method of transfection, the method comprising transfecting a target cell with a composition according to claim 174, which composition comprises an ionizable cationic lipid-based TCV comprising an encapsulated nucleic acid or nucleic acid protein complex cargo, wherein the transfected target cell comprises a mammalian cell, optionally a human cell.

183. A method of transfection, the method comprising transfecting a target cell with an composition according to claim 176, which composition comprises an ionizable cationic lipid-based TCV comprising an encapsulated RNP, wherein the transfected target cell comprises a mammalian cell, optionally a human cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIGS. 1A-1D. Transfection-competent vesicles (TCVs) produced according to the methods herein display knockdown in immortalized cells and primary neurons. (A) Relative luciferase expression in HEK-Luc cells after transfection with organic solvent-free, detergent-free TCVs composed of 50% ionizable cationic lipid. (B) Cell viability from the same set of transfections presented in (A) as % of control (cells only) wells. (C) Relative expression of hdh mRNA in primary cortical neurons after organic solvent-free, detergent-free TCV-mediated transfection of an siRNA selected cargo into the neurons. The siRNA targeted hdh, and the TCV were produced using the same panel of ionizable cationic lipids presented in 1(A). (D) Cell viability as measured by MTT reduction for an equivalent set of transfections in primary cortical neurons using organic solvent-free, detergent-free TCVs to deliver an off-target (luciferase) siRNA. N=4 per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by Bonforroni post-hoc analysis after a one-way ANOVA with each condition compared to the control (cells only) condition.

[0036] FIGS. 2A-2D. The potency of organic solvent-free, detergent-free TCVs does not depend on the method of mixing used to generate the empty TCVs. (A) Relative luciferase expression in HEK-Luc cells after transfection with TCVs composed of 50% ionizable lipid and formed by either T-junction mixing (DODMA-50%) or extrusion (DODMA-50%-X). (B) Cell viability from the same set of transfections presented in (A) as % of control (cells only) wells. (C) Relative expression of hdh mRNA in primary cortical neurons after organic solvent-free, detergent-free TCV-mediated transfection of an siRNA targeting hdh using the same panel of TCVs presented in 2(A). (D) Cell viability as measured by MTT reduction for an equivalent set of transfections into primary cortical neurons using organic solvent-free, detergent-free TCVs to deliver an off-target (luciferase) siRNA. N=4 per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent the mean ±SEM. *p<0.05, **p<0.01, ***p<0.001 by Bonforroni post-hoc analysis after a one-way ANOVA with each condition compared to the control (cells only) condition.

[0037] FIGS. 3A-3D. Organic solvent-free, detergent-free empty TCVs containing a reduced amount of ionizable lipid facilitate potent siRNA delivery with decreased toxicity. (A) Relative luciferase expression in HEK-Luc cells after transfection with TCVs composed of either 20% or 50% of the ionizable lipid 1,2-Dioleyloxy-3-dimethylamino-propane (DODMA). (B) Cell viability from the same set of transfections presented in (A) as % of control (cells only) wells. (C) Relative expression of hdh mRNA in primary cortical neurons after organic solvent-free, detergent-free TCV-mediated transfection of an siRNA targeting hdh using the same panel of TCVs presented in 3(A). (D) Cell viability as measured by MTT reduction for an equivalent set of transfections in primary cortical neurons using organic solvent-free, detergent-free TCVs to deliver an off-target (luciferase) siRNA. N=4 per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by Bonforroni post-hoc analysis after a one-way ANOVA with each condition compared to the control (cells only) condition.

[0038] FIGS. 4A, 4B. Incorporation of DOPE in an organic solvent-free, detergent-free empty TCV does not change TCV morphology. Cryo-TEM analysis was performed on TCVs of (A) DODMA/DOPE/DSPC/Chol/PEG-lipid at 20/30 /10/39/1 mol% (DOPE = 1,2—dioleoyl—sn—glycero—3—phosphoethanolamine; DSPC = 1,2—distearoyl—sn—glycero—3—phosphocholine; PEG = polyethylene glycol), and (B) DODMA/DSPC/Chol/PEG-lipid (50/10 /39/1 mol%). Scale bar = 100 nm.

[0039] FIG. 5. Organic solvent-free, detergent-free empty TCVs herein can be used to deliver functional ribonucleoproteins (RNPs). HEK293 cells were transfected with Cas9 RNPs and a single-stranded oligodeoxynucleotide (ssODN) repair template, both delivered by organic solvent-free, detergent-free TCVs produced according to the methods herein. The Cas9 protein was complexed to a guide targeting the GRN gene. DNA was extracted from the cells 48 hrs post-transfection. PCR was used to specifically detect the wild-type (WT) GRN allele or a mutant GRN allele, which is only present when homology-directed repair (HDR) incorporates the delivered ssODN into a DNA double-stranded break produced by Cas9. The mutant GRN allele can be detected after TCV-mediated delivery of the Cas9 RMP but is not present in control (Ctrl) cells.

[0040] FIG. 6. Photomicrographs of Cas9 located within a primary neuron. RNP delivered via organic solvent-free, detergent-free TCV as discussed herein can be detected using a fluorescent antibody against the Cas9 protein, displayed in red. The untreated control has no such red fluorescent signals. Blue signal = nucleic acid (DAPI stain), green signal = phalloidin (F-actin stain).

[0041] FIG. 7. Graph demonstrating transcriptional knockdown by RNP delivered via bench-top loading of RNP into empty organic solvent-free, detergent-free TCV in HEK cells. HEK293 cells were transfected with Cas9 RNPs targeting luciferase. TCVs of formula (DODMA/DOPE/DSPC/Chol (20/30/10/40)) were loaded at the bench. Relative levels of luciferase mRNA in HEK cells show significant knockdown compared to control (“ctrl” in the graph) when measured by qPCR. N=3 per group. Data represent the mean ± SEM. p=0.0018 by Student’s t-test.

[0042] FIG. 8. Graph demonstrating protein knockdown by RNP delivered via bench-top loading of RNP into empty organic solvent-free, detergent-free TCV in HEK cells. HEK293 cells were transfected with Cas9 RNPs targeting luciferase. TCVs of formula (DODMA/DOPE/DSPC/Chol (20/30/10/40)) were loaded at the bench. Relative levels of luciferase protein in HEK cells show significant knockdown compared to control (“ctrl” in the graph) when measured by ONE-Glo. N=3 per group. Data represent the mean ± SEM. p=0.0003 by Student’s t-test.

[0043] FIG. 9. Graph demonstrating transcriptional knockdown by RNP delivered via bench-top loading of RNP into empty organic solvent-free, detergent-free TCV in primary cortical neurons. Primary cortical neurons were transfected with Cas9 RNPs targeting Human Huntingtin (HTT). TCVs of formula (DODMA/DOPE/DSPC/Chol (20/30/10/40)) were loaded at the bench. Relative levels of HTT mRNA in primary neurons show significant knockdown compared to control (“gLuc” in the graph) when measured by qPCR. N=3 per group. Data represent the mean ± SEM. p=0.0039 by Student’s t-test.

[0044] FIG. 10. Graph demonstrating mRNA expression for mRNA delivered via organic solvent-free, detergent-free TCV in HEK cells. Luciferase mRNA was delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) into HEK cells at different ratios Low=0.029 mg mRNA/.Math.mol lipid, mid=0.058 mg mRNA/.Math.mol lipid, high=0.116 mg mRNA/.Math.mol lipid. All ratios demonstrated significant expression compared to the control, with the lowest ratio demonstrating the highest expression. mRNA expression measured by ONE-Glo+Tox kit (Promega), N=3 per condition.

[0045] FIG. 11. Graph demonstrating mRNA expression for mRNA delivered via organic solvent-free, detergent-free TCV in primary cortex neurons. Luciferase mRNA was delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) into primary cortical neurons at different dosages. All dosages demonstrated significant expression compared to the control. mRNA expression measured by ONE-Glo+Tox kit (Promega), N=3 per condition.

[0046] FIG. 12. Graph demonstrating cell viability for RNP in organic solvent-free, detergent-free TCVs in HEK cells. RNP were delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) or using RNAiMax (“Rmax” in the figure; ThermoFisher Scientific) into HEK cells for cytotoxicity assessment. The organic solvent-free, detergent-free TCV were significantly less toxic compared to RNAiMax, p = 0.0002. Toxicity was assed using ONE-Glo + Tox kit (Promega), N=3 per group.

[0047] FIG. 13. Photomicrographs demonstrating cell viability for bench-top loading of siRNA into empty organic solvent-free, detergent-free TCVs in primary neurons. siRNA were delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol/PEG-lipid (20/30/10/39/1)) or using the commercially available Mirus TKO system (Mirus Bio) into primary neurons for cytotoxicity assessment. The siRNA delivered by bench-top loading into empty organic solvent-free, detergent-free TCV was significantly less toxic compared to the Mirus TKO system, as shown by photomicrographs.

[0048] FIG. 14. Graph demonstrating knockdown for organic solvent-free, detergent-free TCVs in HEK cells. siRNA were delivered via organic solvent-free, detergent-free TCV DODMA levels at 50% (“D-50%”), made by T junction mixing), 50% (“D-50% Ex”, made by extrusion) and via the commercially available Mirus TKO system (Mirus Bio). The current 50% DODMA formulations showed about 50% knockdown, however, the Mirus TKO system performed worse.

DETAILED DESCRIPTION

[0049] The systems, compositions, devices and methods, etc., herein provide lipid-based vesicles, typically herein called transfection competent vesicles (TCVs), configured to safely and efficiently deliver DNA and other nucleic acid selected cargoes into target cells. The safety and efficiency are each, and both, achieved in part by eliminating disrupting agents such as organic solvents such as ethanol and detergents such as sodium dodecyl sulfate from the TCV loading and storage processes (i.e., inserting a selected cargo into the TCV), and/or TCV delivery processes. TCV delivery processes can comprise transfection of mammalian cells such as primary cells with the selected cargo. The selected cargoes can also comprise nucleic acids complexed with a protein, such as a ribonucleoprotein (RNP).

[0050] In some embodiments, the systems, compositions, devices and methods, etc., herein provide empty lipid-based TCVs that are organic solvent-free and detergent-free. The loaded TCVs may be generated using gentle mixing such as repeated manual reciprocation of the TCV-generating fluid in a pipette, SHM, T-junction mixing or extrusion methods, or other TCV-mixing methods as desired.

[0051] In one aspect, the lipid-based TCVs are comprised of a mixture of an ionizable cationic lipid, phospholipid, cholesterol and PEG-lipid, and the TCV-containing composition is organic-solvent and/or detergent free.

[0052] The organic solvent-free, detergent-free TCVs as discussed herein can be used for treatment of appropriate diseases and conditions, for example via gene therapy. The organic solvent-free, detergent-free TCVs as discussed herein improve the delivery of RNA, DNA, and RNP gene therapy products to human patients. The organic solvent-free, detergent-free TCVs effectively deliver gene therapy (including but not limited to mRNA, siRNA, and RNP) products to brain cells or other target cells. The underlying cause of many human disorders is the loss-of-function of a required protein or the toxic gain-of-function of a mutant protein. These causes are treatable, and even reversible, using the organic solvent-free, detergent-free TCVs herein.

[0053] Some examples of such treatment include gene therapy in the central nervous system for treating neurological disorders (some cases of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, spinal muscular atrophy, etc.).

[0054] The organic solvent-free, detergent-free TCVs as discussed herein can also genetically “knockdown” the mutant genes/gene products, for example via targeted, safe delivery of siRNA or RNP, gene replacement therapy with mRNA, or correction of the causal, native DNA mutation with RNP-mediated gene editing. Two specific examples of human diseases that can be targeted in this manner are Huntington’s disease (HD) and frontotemporal dementia (FTD).

[0055] Huntington’s disease is a progressive, incurable, neurodegenerative disease with a dominant pattern of inheritance. An expanded CAG nucleotide repeat sequence in the huntingtin (HTT) gene is responsible for the disease. The huntingtin protein (HTT) encoded by the mutant HTT gene contains an expanded polyglutamine repeat that confers a toxic gain-of-function to the gene product. Lowering the brain levels of mutant huntingtin protein is the principal therapeutic strategy currently being pursued to slow or stop disease progression in HD, and can be effected and improved using the organic solvent-free, detergent-free TCVs as discussed herein. TCVs loaded with siRNA targeting HTT expression or with RNP selected cargos designed to decrease huntingtin expression or toxicity will be effective therapies for HD. Frontotemporal dementia has many causes, but loss of the protein progranulin (a potential brain survival factor) is one well-described cause. The organic solvent-free, detergent-free TCVs as discussed herein can deliver progranulin mRNA or RNPs designed to either express progranulin or correct the underlying DNA mutation causing progranulin loss (respectively) will increase brain levels of progranulin and will be effective therapies for FTD. Increasing progranulin with TCVs expressing progranulin mRNA can also be a neuroprotective strategy for many common neurological diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis.

EXAMPLES OF MATERIALS AND METHODS

Materials

[0056] 1,2—Dioleyloxy—3—dimethylamino—propane (DODMA) was purchased from Cayman Chemical (Ann Arbor, MI). 1,2—dioleoyl—3—dimethylammonium—propane (DODAP), 1,2—dioleoyl—sn—glycero—3—phosphocholine (DOPC), 1,2—dioleoyl—sn—glycero—3—phosphoethanolamine (DOPE), and 1,2—distearoyl—sn—glycero—3—phosphocholine (DSPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Sigma Aldrich (St. Louis, MO). PEG-DMG was synthesized previously described (Akinc, Zumbuehl et al. 2008). All lipids were maintained as ethanol stocks. siRNA targeting firefly luciferase (siLuc) (Basha, Ordobadi et al. 2016) was purchased from Integrated DNA Technologies (Coralville, IA). siRNA against murine hdh was purchased from Ambion (Silencer® Select Pre-designed siRNA, Invitrogen, Carlsbad, CA).

Preparation of Transfection Competent Vesicles (TCVs)

[0057] Lipid components (ionizable cationic lipids, phospholipids, cholesterol and PEG-lipid) were dissolved in ethanol at appropriate ratios to achieve a final concentration of 20-35 mM total lipid. An aqueous phase was prepared containing 25 mM sodium acetate pH 4 buffer. The two solutions were combined using two established nanoparticle preparation techniques: rapid-mixing and extrusion.

Rapid-Mixing

[0058] The organic phase containing lipids was mixed with the aqueous phase through a T-junction mixer fabricated to meet the specifications of the PEEK Low Pressure Tee Assembly (1/16", 0.02 in thru hole, Part # P-712) at a final flow rate of 20 mL/min with a 1:3 organic:aqueous (v/v) ratio (Jeffs, Palmer et al. 2005; Kulkami, Tam et al. 2017; Kulkami, Darjuan et al. 2018). The resulting suspension was dialyzed against 1000-fold volume of 25 mM sodium acetate pH 4 buffer to remove ethanol.

Extrusion

[0059] Lipids were dissolved in ethanol to a final concentration of 35 mM. Particles were generated by rapidly adding 25 mM sodium acetate pH 4 was to the ethanolic solution to achieve a final concentration of 30% ethanol (v/v) as described elsewhere (Maurer, Wong et al. 2001). The resulting nanoparticle suspension was extruded three times through 2x 80 nm polycarbonate membranes at ambient temperature. Following extrusion, the particles were buffer-exchanged to remove ethanol.

Analysis of Transfection Competent Vesicles (TCVs)

[0060] Lipid concentrations were determined by assaying for the cholesterol content using a T-Cholesterol Assay Kit (Wako Chemicals, Mountain View, CA) and extrapolating total lipid concentration as described elsewhere (Chen, Tam et al. 2014). Nucleic acid entrapment was determined using the RiboGreen Assay as previously described (Chen, Tam et al. 2014; Leung, Tam et al. 2015).

Cryogenic Transmission Electron Microscopy

[0061] Cryo-TEM was performed as described previously (Kulkarni, Darjuan et al. 2018). Briefly, TCVs were concentrated to a total lipid concentration of approximately 20 mg/mL using an Amicon centrifugal concentration unit (10 kDa NWCO). A small volume (3-5 uL) of material was applied to a glow-discharged copper grid and plunge-frozen using an FEI Mark IV Vitrobot (Hillsboro, OR). The grids were stored under liquid nitrogen until imaged. All imaging was performed using an FEI Tecnai G2 instrument operating at 200 kV in low-dose mode. Images were captured using an FEI Eagle 4k CCD bottom-mount detector. All sample preparation and imaging was performed at the UBC BioImaging Facility (Vancouver, BC).

Cell Culture and Reagents

[0062] All base cell culture media and B27 neuronal supplement were purchased from Gibco (Thermo Fisher, Waltham, MA). Hank’s balanced salt solution (HBSS), penicillin-streptomycin, L-glutamine, and trypsin solutions were obtained from Hyclone (Logan, UT). HEK293 cells were plated on clear-bottom, white-walled plates from Corning (Corning, NY). Primary cortical cells were plated onto tissue culture-treated plates (Fisher), coated with poly-D-lysine (Sigma, St. Louis, MO). Hygromycin B was obtained from Invitrogen (Carlsbad, CA). Recombinant ApoE4 was acquired from Peprotech (Rocky Hill, NJ).

[0063] To measure cell viability and luciferase levels in HEK293 cells, the ONE-Glo + Tox kit from Promega (Madison, WI) was used. Cell viability in primary neurons was measured via MTT in vitro toxicology kit from Sigma (St. Louis, MO).

Luciferase Reporter HEK293 Cells

[0064] Generation of the HEK293 cell line with a stably-integrated luciferase reporter construct (HEK-Luc cells) has been described previously (De Souza, Islam et al. 2016). Cells were maintained at 37° C. in a humidified atmosphere of 95% air, 5% CO.sub.2 in DMEM high glucose, supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 125 .Math.g/mL hygromycin B. Cells were plated at a density of 12,000-20,000 cells/well in a white-walled 96-well plate.

Primary Cell Culture

[0065] Cortical cultures were prepared from embryonic day E17.5 C57BL/6J and FVB.YAC128 mice. Briefly, cortices were dissected in ice cold HBSS, and the tissue was digested using a 0.05% trypsin (Hyclone) solution for 10 minutes at 37° C. The cortices were then triturated through a 5 mL pipette 5 times, and an additional 5-7 times with a 200 .Math.L pipette tip added. Cells were pelleted by centrifugation for 5 minutes at 800 rpm, washed with HBSS, and then resuspended in warm neurobasal media supplemented with B27, 2 mM L-glutamine (Hyclone) and 1% penicillin/streptomycin (Hyclone). Cortical neuronal cultures were plated onto poly-D lysine-coated 24-well plates at a density of 1.5 x 10.sup.5 cells/well. Cells were maintained at 37° C. in a humidified atmosphere of 95% air, 5% CO.sub.2.

Transfection

[0066] All reagents were mixed on the bench-top. Empty TCVs containing 50% cationic lipid were mixed with siRNA at a ratio of 0.058 mg siRNA per .Math.mole lipid. TCVs containing 20% cationic lipid were mixed at 0.022 mg siRNA per .Math.mole lipid. TCV suspension was mixed with siRNA briefly by pipette and incubated at room temperature for 10 minutes.

[0067] HEK293 cells were plated 24 hours before transfection. Complete DMEM media was added to the TCV:siRNA mixture for a final concentration of 3.3 .Math.g/mL siRNA, and a complete change of media was performed at the time of transfection.

[0068] Primary neuronal cells were grown in vitro for 7 days before transfection. Complete neurobasal media with 2-6 .Math.g/mL of recombinant ApoE4 was added to the TCV:siRNA suspension, and half the media was replaced from each well.

Luciferase Assay

[0069] Forty-eight to seventy-two hours post-transfection, HEK293 cells were assayed for cell viability and luminescence using the ONE-Glo + Tox kit (Promega) according to the manufacturer’s instructions. Briefly, live cell reagent was added to each well, and cells were incubated for 30 minutes at 37° C. The plate was assayed on a plate reader (POLARstar Omega plate reader, BMG LABTECH) at an excitation of 400 nm, and read at an emission wavelength of 510 nm. ONE-Glo reagent was then added, and the plate incubated at room temperature for 3 minutes. Luminescence was measured via light output through the lens of the same plate reader. Values are presented as % control and represent N=4 wells per condition.

MTT Assay

[0070] Primary cortical neurons were assayed for cell viability via MTT assay 72 hours after transfection in a 24-well plate. MTT reagent (3—(4,5—dimethylthiazol—2—yl)—2,5—diphenyltetrazolium bromide, or MTT) was reconstituted in HBSS to a final concentration of 5 mg/mL, and added to each well at 10% v/v. Cells were incubated at 37 ° for 4 hours. The media was removed, and 250 .Math.L solubilization solution was added to each well. Absorbance was measured at 570 nm. Values are presented as % control and represent N=3 wells per condition.

Quantitative RT-PCR

[0071] Adherent primary cortical cells were washed once in sterile PBS prior to being scraped off the plate in 600 .Math.L lysis buffer containing 1% 2—mercaptoethanol and immediately frozen at –80° C. Total RNA was subsequently extracted using the PureLink RNA mini kit (Invitrogen) performed according to the manufacturer’s instructions. Reverse transcription of all samples was carried out using the Superscript VILO kit (Invitrogen) according to the manufacturer’s instructions, using 250 ηg of total RNA as input for cDNA synthesis and 5 ηg diluted RNA for the quantitative PCR reaction. Quantification of hdh mRNA levels was accomplished using the standard curve method, with amplification of target mRNA and control genes in separate wells, performed using FastSybr (Applied Biosystems) and conducted on a Step-One ABI System (Applied Biosystems). Each sample was run in duplicate. The relative amount of mRNA in each well was calculated as the ratio between hdh mRNA and a control gene, Csnk2α2. Values are presented as % control and represent N=3 wells per condition.

Ribonucleoprotein (RNP) Complex Materials and Formation

[0072] All materials for RNP formulation, including guide RNAs (gRNAs), tracrRNA, single-stranded oligodeoxynucleotides (ssODN), and recombinant Cas9 protein, were obtained from IDT (San Jose, CA). The gRNA sequence used to target luciferase was provided by IDT (San Jose, CA). The gRNA sequence targeting human progranulin (GRN) binds to exon 5 of the gene. The ssODN sequence used for homology-directed repair (HDR) was engineered to introduce a 4bp deletion into exon 5 of GRN.

[0073] RNP assembly was performed according to the manufacturer’s specifications. Briefly, the guide RNA (gRNA) complex was formed by incubating equimolar ratios of crRNA:tracrRNA, such as 1 .Math.M tracrRNA with 1 .Math.M gRNA, at 95° C. for 5 minutes. The mixture was then allowed to cool at room temperature for 20-30 minutes. RNPs were subsequently formed by combining gRNA duplexes with Cas9 protein at equimolar ratios and allowing the mixture to stand at room temperature for 5 minutes prior to use.

Transfection of Mammalian Cells with Nucleic Acid

[0074] Empty organic solvent-free, detergent-free TCVs as discussed herein, as well as commercially available reagents for comparison, were mixed with selected cargo on the bench-top. TCVs were mixed with nucleic acid selected cargo at a range of ratios of 0.01-0.2 mg nucleic acid per .Math.mole lipid. TCV suspension was mixed with siRNA briefly by pipette and incubated at room temperature for 10 minutes.

[0075] HEK293 cells were plated 24 hours before transfection. Complete DMEM media was added to the TCV:nucleic acid mixture for a final concentration of 0.33 - 3.3 .Math.g/mL siRNA, or 0.1 - 1 .Math.g/mL mRNA a complete change of media was performed at the time of transfection. Primary neuronal cells were grown in vitro for 7 days before transfection. Complete neurobasal media with 2-6 .Math.g/mL of recombinant ApoE4 was added to the TCV:nucleic acid suspension, and half the media was replaced from each well.

[0076] Cells were treated with Mirus TransIT-TKO per manufacturer’s instructions. Briefly, Mirus TransIT-TKO was added to serum-free media at a concentration of 5 .Math.L Mirus/100 .Math.L serum-free media. siRNA was then added to the tube, pipetted gently to mix, and incubated at room temperature for 15-30 minutes. The solution was then transferred onto cells, and the final concentration of Mirus was 5 .Math.L/1 mL of complete media. The final concentration of siRNA was 25 nM.

Transfection of Mammalian Cells with RNPs

[0077] 0.5-20 mM TCV and 0.5-20 .Math.M RNP were combined at a 467-5000 molar ratio and allowed to incubate at room temperature for 10 minutes. Separately, 1-10 .Math.M solution of ssODN was combined with TCV and this mixture was incubated at room temperature for 5-15 minutes. In some instances, an equimolar amount of ssODN was added to the RNP complex solution prior to the addition of TCVs.

[0078] TCVs containing RNPs and ssODN mixtures were combined, and complete media was added to a final concentration of 10-200 nM of RNPs and ssODN each. A full media change was performed on HEK cells, which were plated 24 hours prior. Primary neuronal cells were grown in vitro for 5-7 days before transfection. Complete neurobasal media with 2-6 .Math.g/mL of recombinant ApoE4 was added to the TCV:RNP mixture, and half the media was replaced from each well.

[0079] Cells were treated with Lipofectamine RNAiMAX reagent per manufacturer’s instructions. Briefly, RNP complexes were prepared and added to a mixture of serum-free media and RNAiMAX, incubated at room temperature for 5 minutes, and added onto plated cells.

PCR for Detection of Homology-Directed Repair

[0080] Polymerase chain reaction (PCR) was used to amplify GRN exon 5 from genomic DNA extracted from transfected HEK293 cultures using forward primers specific for either the wild-type (WT) or mutant GRN alleles and a common reverse primer. PCR was performed using MyTaq (Bioline, USA) according to the manufacturer’s instructions. PCR products were separated by gel electrophoresis on a 1.5% agarose gel stained with SybrSafe and imaged under UV light.

Immunocytochemistry

[0081] Cells were fixed for 15 minutes using a solution of 3-4% paraformaldehyde. The cells were permeabilized for 15 minutes in PBS containing 0.1% Triton-X (PBST). Cells were incubated overnight at 4° C. with PBST containing a 1:1000 mixture of anti-Cas9 (Invitrogen) antibody. Cells were washed thrice with PBS and incubated with 1:1000 mixture of each Alexa Fluor 594 fluorescent secondary antibody (Invitrogen) and Phalloidin-iFluor 488 CytoPainter antibody (Abcam) for 1 hour at room temperature, washed again and incubated for 5 minutes with a solution containing DAPI to visualize nuclei.

Statistics

[0082] All statistical comparisons were performed as a one-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis to compare individual means to control-treated cells and correct for multiple comparisons (Prism 6, Graphpad Software Inc.). A Student’s t-test was used to compare individual means in the case of only two groups. Ap-value less than 0.05 was considered significant.

RESULTS-RELATED EXAMPLES

Example 1: Empty Transfection-Competent Vesicles (TCVs) Entrap Nucleic Acid Efficiently without Organic Solvents

[0083] Empty TCVs formulation produced by T-junction or SHM mixing exhibited entrapment efficiencies on the order of 85% or greater. We first tested the ability of TCVs composed of ionizable cationic lipids that span the range of observed in vivo gene silencing potencies (DODMA >> DLinDAP > DODAP) (“DLinDAP” is 1,2—dilineoyl—3—dimethylammonium—propane) to entrap nucleic acid without the aid of organic solvents or detergent. Remarkably, in the absence of either, formulations composed of ionizable lipid/DSPC/Chol/PEG—lipid (50/10/39/1 mol%) achieve near complete entrapment of siRNA (> 85%) when mixed at pH 4 at a ratio of 0.058 mg siRNA/.Math.mol lipid followed by neutralisation with PBS (Table 1). The assay to determine entrapment is based on the exclusion of an RNA-binding dye from the nucleic acid by the lipid components. Thus, entrapment is considered the sequestration of RNA from the external medium in more than a transient manner (i.e., stable entrapment). Despite the lack of organic solvents or detergents in the production processes, the obtained TCV formulations surprisingly displayed entrapment efficiencies similar to those reported elsewhere for LNP-siRNA generated by rapid-mixing techniques using organic solvents (Belliveau, Huft et al. 2012; Chen, Tam et al. 2014; Leung, Tam et al. 2015; Chen, Tam et al. 2016).

TABLE-US-00001 Entrapment efficiencies of nucleic acids for different formulations of TCVs Name Lipid formulation Formulation process Entrapment efficiency, % (mean ± SEM) DODMA-50% DODMA/DSPC/Chol/PEG (50/10/39/1) T-junction mixing 87.90 ± 3.94 DLinDAP-50% DLinDAP/DSPC/Chol/PEG (50/10/39/1) T-junction mixing 74.44 ±7.18 DODAP-50% DODAP/DSPC/Chol/PEG (50/10/39/1) T-junction mixing 71.84 ±5.76

Example 2: siRNA in Organic Solvent-Free, Detergent-Free Empty TCVs Displays Robust Knockdown in Immortalized Cells and Primary Neurons

[0084] The ability to both entrap nucleic acid and subsequently deliver it in a non-toxic manner represent two separate hurdles. Upon determining that the non-organic solvent/non-detergent lipid-based TCVs discussed above efficiently entrapped nucleic acids, their ability to silence genes and their effect on cell viability was tested in two scenarios, as shown in FIGS. 1A-1D. First, the empty organic solvent-free, detergent-free TCVs were combined using bench-top loading with siLuc and used to treat HEK-Luc cells. Surprisingly, DODMA-based TCVs shows about 50% knockdown and performed better than DLinDAP and DODAP, which showed no indication of knockdown (FIG. 1A). None of the formulations displayed toxicity as measured by cell viability compared to untreated cells (FIG. 1B). Second, the efficacy and toxicity of the TCVs delivering siRNA was tested against the hdh gene in primary mouse cortical neurons. Once again (FIG. 1C), DODMA-TCVs displayed an about 50% gene knockdown efficacy, while DLinDAP-TCVs displayed less knockdown, and DODAP-TCVs displayed no difference from untreated cells. It should be noted that primary neurons are quite susceptible to the toxic effects of harmful transfection reagents such as those currently available commercially. FIG. 13 highlights the differences in the effect on viability.

Example 3: Multiple Mixing Processes Can Produce Potent Organic Solvent-Free, Detergent-Free Empty TCVs

[0085] In an effort to determine the role of the mixing aspect of the manufacturing process and the resulting particle size to achieve the potency shown in FIG. 1, organic solvent-free, detergent-free lipid-based empty TCVs containing ionizable lipids such as DODMA were produced through both T-junction mixing and extrusion. As shown in FIG. 2A, following bench-top loading of siRNA into empty TCVs there was significant knockdown of luciferase in HEK-Luc cells using both processes. There was no significant difference in the viability of cells treated with particles produced by T-junction or extrusion (FIG. 2B). Next, primary cortical neurons were treated with the same solvent-free, detergent-free lipid-based TCV formulations and it was determined that both processes result in particles with similar potency (FIG. 2C) and similar cell viability (FIG. 2D). In contrast, the toxicity of current transfection methods (FIG. 13) in primary cultures detracts from the goal of a non-toxic yet potent formulation.

Example 4: Organic Solvent-Free, Detergent-Free Empty TCVs Containing a Reduced Amount of Ionizable Lipid Facilitate Potent siRNA Delivery with Decreased Toxicity

[0086] An established lipid composition, currently used in clinical formulations (see Patisiran), includes a significant amount of ionizable cationic lipid (50 mol%) (Jayaraman, Ansell et al. 2012; Suhr, Coelho et al. 2015). While such high amounts allow for improvements in the effective dose to achieve 50% gene silencing (ED.sub.50) in vivo (Jayaraman, Ansell et al. 2012), the persistence of lipid metabolites following administration (Maier, Jayaraman et al. 2013; Sabnis, Kumarasinghe et al. 2018) and the toxicity associated with those molecules renders the formulation toxic in high-dose regimens and repeat administration (or to sensitive cell types). The current compositions, TCVs, etc., can be comprised of materials that are biodegradable or facilitate elimination. Here, the current compositions, TCVs, etc., decrease the amount of toxic ingredients maintaining transfection potency.

[0087] The transfection competency of a formulation composed of DODMA/DOPE/DSPC/Chol/PEG-lipid (20/30/10/39/1 mol% respectively) to silence luciferase in HEK-Luc cells was tested. A 40% knockdown of luciferase expression was observed (FIG. 3A) with no significant toxicity (FIG. 3B). Next, we tested the ability of TCVs with 20mol% of ionizable lipid to deliver siRNA to primary neurons as compared to DODMA-TCVs at 50 mol%. All formulations displayed -60% knockdown of hdh (FIG. 3C), while a DODMA formulation produced via T junction mixing showed significantly improved cell viability (FIG. 3C).

Example 5: Incorporation of DOPE Does Not Change Organic Solvent-Free, Detergent-Free Empty TCV Morphology

[0088] Improving the potential for H.sub.II phase formation in a nucleic acid delivery vehicle can be an important factor to facilitate membrane fusion in the endosome (Hafez, Maurer et al. 2001). In the present approach, two exemplary lipids able to adopt H.sub.II phases are DOPE (in isolation) and DODMA (when protonated and combined with anionic lipids). To determine if the incorporation of DOPE in organic solvent-free, detergent-free TCVs resulted in premature H.sub.II phase formation, cryo-TEM was performed on DODMA-TCVs at 20 mol% and the equivalent formulation composed of 50 mol% DODMA. The resulting structures (FIGS. 4A, 4B) are visualized as bilayer structures with no indication of H.sub.II phase.

[0089] Previous work by others has suggested the presence of H.sub.II-like internal structures within the core LNP formulations regardless of siRNA content (Leung, Hafez et al. 2012; Leung, Tam et al. 2015). It has since been shown that LNP-siRNA do not contain such structures, but rather have siRNA immobilised between closely apposed layers of lipid (Kulkarni, Darjuan et al. 2018) giving the overall particle a multi-lamellar or onion-like morphology. In the absence of siRNA, LNP formulations adopt an electron-dense core containing an oil-phase lipid. Thus, the examples herein demonstrate that TCV morphology is drastically different from LNP systems but still have highly efficient transfection potency.

Example 6: Organic Solvent-Free, Detergent-Free Empty TCVs Can Be Used to Deliver Functional Ribonucleoproteins (RNPs)

[0090] As shown in FIG. 5, organic solvent-free, detergent-free empty TCVs herein were also tested for their ability to deliver a protein selected cargo complexed with nucleic acid into a mammalian cell. Briefly, a ribonucleoprotein complex consisting of recombinant Cas9 protein and a guide RNA targeting exon 5 of the progranulin gene was assembled and combined with empty TCVs via bench-top loading (DODMA/DOPE/DSPC/Chol/(20/30/10/40 mol% respectively) at a molar ratio of 467:1 (TCV:RNP complex). Separately, a single stranded oligodeoxynucleotide designed to introduce a 4bp deletion into exon 5 of the progranulin gene was combined with TCVs at a molar ratio of 4275:1 (TCV:nucleic acid). The two preparations of TCVs containing their respective selected cargoes were combined to achieve a final concentration of 10 nM RNPs and 10 nM ssODN, and then added to HEK cells. After 48 hours, PCR was employed to determine whether homology-directed repair had occurred at the progranulin gene target using forward primers specific for either the wild type or mutant allele. As shown in FIG. 5, cells exposed to the combination of TCVs containing each of ribonucleoprotein complexes and the ssODN resulted in the insertion of the 4bp deletion via homology-directed repair (second lane of FIG. 5, labelled mutant), whereas untreated control cells did not result in any genetic alternation at the exon 5 site of the progranulin gene (third and fourth lanes of FIG. 5).

Example 7: Further Examples of Organic Solvent-Free, Detergent-Free Empty TCVs Used to Deliver Functional Ribonucleoproteins (RNPs)

[0091] Organic solvent-free, detergent-free empty TCVs were used to deliver functional ribonucleoproteins (RNPs) using methods as discussed above.

[0092] FIG. 6 provides a photomicrograph of Cas9 located within a primary neuron. RNP delivered via organic solvent-free, detergent-free TCV as discussed herein can be seen within a primary neuron as fluorescent signals; the untreated control has no such fluorescent signals. More specifically, immunocytochemistry of primary neuronal cells showed localization of Cas9 protein (red) within the nucleus (blue) of cortical neurons derived from mice. Cells were also stained for phalloidin (green) to show cellular morphology.

[0093] FIG. 7 provides a graph demonstrating gene knockdown by RNP delivered via organic solvent-free, detergent-free TCV in HEK cells. RNP delivered via organic solvent-free, detergent-free TCV into HEK cells show significant knockdown of luciferase transcript compared to control (“ctrl” in the graph). FIG. 8 provides a graph demonstrating protein knockdown by RNP delivered via organic solvent-free, detergent-free TCV in HEK cells. RNP delivered via organic solvent-free, detergent-free TCV into HEK cells show significant knockdown of luciferase protein compared to control (“control” in the graph), p = 0.0003.

[0094] FIG. 9 provides a graph demonstrating gene knockdown by RNP delivered via organic solvent-free, detergent-free TCV in primary neurons. RNP delivered via organic solvent-free, detergent-free TCV into primary cortical neurons show significant mRNA knockdown by quantitative real-time PCR (qRT-PCR) assay, p = 0.0039.

[0095] Turning to some further discussion of these Figures, FIGS. 7 and 8 show that RNP delivered via bench-top loading of the empty TCV herein show robust knockdown of a reporter gene, luciferase, in HEK cells. This was demonstrated by measuring the luciferase output (functional protein) as well as mRNA levels of the luciferase mRNA via qRT-PCR. This same approach of RNP delivery was used to disrupt expression of the huntingtin gene in cortical neurons derived from FVB.YAC128 mice expressing the full-length human huntingtin gene carrying a disease mutation (FIG. 9). mRNA levels of huntingtin were quantified from primary cortical neurons using qRT-PCR after 72 hours of incubation with TCV:RNP mixture.

Example 8: Examples of mRNA Delivered via Organic Solvent-Free, Detergent-Free TCV

[0096] Bench-top loading of organic solvent-free, detergent-free empty TCVs used to deliver mRNA using methods as discussed above.

[0097] FIG. 10 provides a graph demonstrating expression of mRNA delivered via organic solvent-free, detergent-free TCV in HEK cells. mRNA was delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) into HEK cells at different ratios, Low=0.029 mg mRNA/.Math.mol lipid, mid=0.058 mg mRNA/.Math.mol lipid, high=0.116 mg mRNA/.Math.mol lipid. All ratios demonstrated significant expression compared to the control, with the lowest ratio demonstrating the highest expression.

[0098] FIG. 11 provides a graph demonstrating expression of mRNA delivered via bench-top loading of empty organic solvent-free, detergent-free TCV in primary cortical neurons. mRNA was delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) into primary cortical neurons at different dosages. All dosages demonstrated significant expression compared to the control.

[0099] Turning to some further discussion of these Figures, FIG. 10 demonstrates that bench-top loading of organic solvent-free, detergent-free empty TCVs as discussed herein can be used to deliver mRNA encoding for a functional protein into cells. Briefly, organic solvent-free, detergent-free TCVs were mixed gently using a pipette with mRNA encoding the firefly luciferase mRNA, in a range of ratios from 0.029-0.116 mg nucleic acid:1 .Math.mole lipid. This mixture was incubated at room temperature for 5-20 minutes, before addition of complete culture media and then transferred into wells containing HEK cells (see FIG. 10) or primary cortical neurons (FIG. 11). Luciferase output was measured using the ONE-Glo luciferase assay kit from Promega, per manufacturer’s instructions. The results demonstrate the production of luciferase protein within these cells types across a range of selected cargo:TCV concentrations and doses.

Example 9: Comparison of Organic Solvent-Free, Detergent-Free TCVs to Market-Available Products

[0100] Bench-top loading of empty organic solvent-free, detergent-free TCVs was used to deliver siRNA or RNP to HEK cells or primary neurons using methods as discussed above, and then were contrasted to equivalent transfections using commercially available systems. The organic solvent-free, detergent-free TCVs herein outperformed the commercially available systems.

[0101] FIG. 12 provides a graph demonstrating cell viability for organic solvent-free, detergent-free TCVs in HEK cells. RNP were delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol (20/30/10/40)) or using RNAiMax (“Rmax” in thefigure; ThermoFisher Scientific) into HEK cells for cytotoxicity assessment. The organic solvent-free, detergent-free TCV were significantly less toxic compared to RNAiMax, p = 0.0002.

[0102] FIG. 13 provides photomicrographs demonstrating cell viability for bench-top loading of siRNA into empty organic solvent-free, detergent-free TCVs in primary neurons. siRNA were delivered via organic solvent-free, detergent-free TCV (DODMA/DOPE/DSPC/Chol/PEG—lipid (20/30/10/39/1)) or using the commercially available Mirus TKO system (Mirus Bio into primary neurons for cytotoxicity assessment. The organic solvent-free, detergent-free TCV were significantly less toxic compared to the Mirus TKO system, as shown by photomicrographs.

[0103] FIG. 14 provides a graph demonstrating knockdown for organic solvent-free, detergent-free TCVs in HEK cells. siRNA were delivered via organic solvent-free, detergent-free TCV DODMA levels at 50% (“D-50%”, made by T junction mixing), 50% (“D-50% Ex”, made by extrusion), and via the commercially available Mirus TKO system (Mirus Bio). The current 50% DODMA formulations showed about 50% knockdown, while the Mirus TKO system performed significantly worse.

[0104] Turning to some further discussion of these Figures, FIG. 12 demonstrates that the bench-top loading of siRNA into empty organic solvent-free, detergent-free TCVs as discussed herein have low-toxicity properties and efficient expression knockdown in primary neuronal cells derived from mice and HEK cell line. For treatment of primary cortical neurons, TCVs were mixed with siRNA at a ratio of about 0.022-0.058 mg nucleic acid: 1 .Math.mole lipid and transferred into wells containing primary neuronal cells. The light micrographs in FIG. 13, all taken at the same magnification, show that in the control and TCV-treated wells, the cells remain healthy with processes intact and very low numbers of dead cells. In the Mirus TransIT-TKO-treated well, the cells appear considerably less healthy, with broken cellular processes and an increased amount of small, condensed (dead) cells.

[0105] For FIG. 12, HEK cells were treated with Lipofectamine RNAiMAX and TCVs containing RNP selected cargo. HEK cells were treated with a final concentration of 5-50 nM RNP and either TCV or RNAiMAX reagent. Cell viability was assessed using Promega ONE-Glo + Tox kit, and compared with untreated control cells. HEK cultures treated with RNAiMAX show a significant decrease in overall health compared to both control and TCV-treated wells.

[0106] FIG. 14 demonstrates the effectiveness of bench-top loading of empty organic solvent-free, detergent-free TCVs as discussed herein in delivering siRNA selected cargo compared to the commercially-available product, Mirus LT-TKO. The TCVs were incubated with siRNA targeting the luciferase gene for 5-10 minutes at room temperature at a ratio of about 0.022-0.058 mg nucleic acid: 1 .Math.mole lipid. HEK cells were treated with Mirus LT-TKO according to manufacturer’s instructions. At the time of treatment, media in the wells was completely replaced with fresh growth media containing either siRNA:TCV or siRNA:Mirus LT-TKO mixtures. After 72 hours of treatment, HEK cells were assayed for cell viability and luciferase output using the Promega ONE-Glo + Tox kit, and all cells were compared to untreated control wells.

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[0138] All terms used herein are used in accordance with their ordinary meanings unless the context or definition clearly indicates otherwise. Also unless expressly indicated otherwise, in the specification the use of “or” includes “and” and vice-versa. Non-limiting terms are not to be construed as limiting unless expressly stated, or the context clearly indicates, otherwise (for example, “including,” “having,” and “comprising” typically indicate “including without limitation”). Singular forms, including in the claims, such as “a,” “an,” and “the” include the plural reference unless expressly stated, or the context clearly indicates, otherwise.

[0139] Unless otherwise stated, adjectives herein such as “substantially” and “about” that modify a condition or relationship characteristic of a feature or features of an embodiment, indicate that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended.

[0140] The scope of the present devices, systems and methods, etc., includes both means plus function and step plus function concepts. However, the claims are not to be interpreted as indicating a “means plus function” relationship unless the word “means” is specifically recited in a claim, and are to be interpreted as indicating a “means plus function” relationship where the word “means” is specifically recited in a claim. Similarly, the claims are not to be interpreted as indicating a “step plus function” relationship unless the word “step” is specifically recited in a claim, and are to be interpreted as indicating a “step plus function” relationship where the word “step” is specifically recited in a claim.

[0141] From the foregoing, it will be appreciated that, although specific embodiments have been discussed herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the discussion herein. Accordingly, the systems and methods, etc., include such modifications as well as all permutations and combinations of the subject matter set forth herein and are not limited except as by the appended claims or other claim having adequate support in the discussion and figures herein.