Compositions and systems comprising transfection-competent vesicles free of organic-solvents and detergents and methods related thereto

Abstract

To provide a method of preparing a water-based solution comprising transfection competent vesicles (TCVs), configured to safely and efficiently deliver DNA, RNA, other nucleic acid and protein cargoes into target cells; and to provide the water-based solution.SOLUTION: There is provided a water-based solution that contains empty lipid-based transfection competent vesicles (TCVs) lacking a cargo, (i) wherein the water-based solution is an acidic solution; (ii) wherein the water-based solution does not contain an organic solvent or a detergent; (iii) wherein the water-based solution does not contain a cargo; (iv) wherein the lipid-based TCV comprises an ionizable cationic lipid, one or more phospholipids, and cholesterol (Chol); (v) wherein the lipid-based TCV comprises 20 mol%-50 mol% of the ionizable cationic lipid; and (vi) wherein the lipid-based TCV does not contain a PEG-lipid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of co-pending U.S. Provisional Patent Application No. 62 / 7 filed October 9, 2018. This application claims the benefit of US Pat. No. 4,333,116, which is incorporated herein by reference in its entirety. can be. [Background technology]

[0002] An important area of ​​scientific research and medical treatment is the synthesis of RNA, DNA, other nucleic acids and / or We want to selectively and efficiently deliver protein cargo to target sites, such as specific target cells. This is needed for improved patient treatments, such as gene therapy, and for the treatment of cancer and other conditions. This can be useful for a variety of reasons, including the treatment of Alzheimer's disease, Parkinson's disease, Human diseases include Chinton's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and spinal muscular atrophy. throughout the brain and central nervous system to treat some of the most classic and worst neurological disorders that can be suffered. Gene therapy may be used. Current gene therapy approaches include, for example, For example, the need for repeated administration of packaging that delivers therapeutic nucleic acids into a patient and its Toxicity presents some challenges to widespread use, especially in human patients. , methods, etc., help to ameliorate one or more of these or other such problems.

[0003] A more scientific discussion of the delivery of DNA and other nucleic acids to target sites, such as diseased cells in the brain. To address this, existing methods for such delivery include, in some cases, lipid nanoparticles (" Lipid particles called lipid nanoparticles or "LNPs" or liposomes. The term refers to a molecule that typically contains nucleic acids, has an electron-dense core, and is found at approximately neutral pH. Used to describe lipid-based particles. Liposomes, also known as vesicles, are LNPs are lipid-based structures with a single bilayer and an aqueous core. The established process loads specific cargo onto vesicles during initial vesicle formation. These processes further require the use of specialized equipment, organic solvents and / or surfactants, and require large amounts of materials and processing times measured in days, all of which contribute to availability, accessibility, and This significantly hampers the ease of treatment.

[0004] LNPs and other lipid particles typically contain ionizable cationic lipids, one or more Phospholipids, cholesterol (Chol), and polyethylene glycol lipids (PEG-lipids) quality) (Non-patent document 1; Non-patent document 2; Non-patent document 3; Non-patent document 4; Non-patent document 5; Non-patent document (Various references describing particular systems, devices, methods, and other information.) All such references are incorporated herein by reference in their entirety, as well as by their respective equivalents. for all teachings and disclosures thereof, regardless of where that reference appears in this application. , which are incorporated herein by reference. Citation to a reference herein is expressly incorporated by reference. No admission is made that any reference constitutes prior art to the present application.) Examples of LNP Compositions are ionizable cations in the ratios of 50 / 10 / 38.5 / 1.5 mol% (respectively) Lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipid combinations This composition induces potent hepatocyte gene silencing (SI) after intravenous administration. It has been shown that the expression of β-glucan is related to the expression of β-glucan in the nucleus of the thymus. ; Non-Patent Document 9; Non-Patent Document 10). Other references include SI in primary neuronal cultures. Ionizable cationic lipids, phospholipids for RNA delivery and for delivery to the brain , cholesterol and polyethylene glycol (PEG)-lipid 50 / 10 / 38.5 / 1.5 mol% (Non-Patent Document 11).

[0005] The LNP formulation rapidly dissociates the lipid components dissolved in ethanol into the acidic aqueous phase consisting of the nucleic acid cargo. can be produced by mixing 14) Established rapid mixing processes for LNP production include staggered herringbone Staggered Herringbone Micromixer Microfluidics using r:SHM (Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17) Mixing, or T-shaped mixing with special pumps (Non-Patent Document 18), or preformed vessels A more recent approach to ethanol / surfactant destabilization of the clonal antibody (Non-Patent Document 19) ; Non-Patent Document 20; Non-Patent Document 21; Non-Patent Document 22). In this case, the ethanol solution (or detergent) is sufficient for lipid reorganization and entrapment to occur. In the case of SHM and T-shaped methods, ethanol solution is necessary to provide adequate membrane fluidity. Dilution into an aqueous phase also results in particle formation (Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25 However, the resulting suspension is not "ready to use" due to the organic solvent and acidic pH. The resulting suspension therefore requires significant downstream processing. In terms of material cost and time, these approaches are difficult to implement in laboratory standards for in vitro applications. Transfection-competent formulations at therapeutic levels for clinical or direct administration significantly hinders its realization. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Maurer, Wong et al., 2001 [Non-patent document 2] Semple, Klimuk et al., 2001 [Non-patent document 3] Semple, Akinc et al., 2010 [Non-patent document 4] Belliveau, Huft et al., 2012 [Non-patent document 5] Leung, Hafez et al., 2012 [Non-patent document 6] Suhr, Coelho et al., 2015 [Non-Patent Document 7] Semple, Akinc et al., 2010 [Non-patent document 8] Jayaraman, Ansell et al., 2012 [Non-Patent Document 9] Pardi, Tuyishime et al., 2015 [Non-Patent Document 10] Suhr, Coelho et al., 2015 [Non-Patent Document 11] Rungta, Choi et al., 2013 [Non-Patent Document 12] Jeffs, Palmer et al., 2005 [Non-Patent Document 13] Belliveau, Huft et al., 2012 [Non-Patent Document 14] Leung, Hafez et al., 2012 [Non-Patent Document 15] Belliveau, Huft et al., 2012 [Non-Patent Document 16] Rungta, Choi et al., 2013 [Non-Patent Document 17] Leung, Tam et al., 2015 [Non-Patent Document 18] Jeffs, Palmer et al., 2005 [Non-Patent Document 19] Wheeler, Palmer et al., 1999 [Non-Patent Document 20] Tam, Monck et al., 2000 [Non-Patent Document 21] Maurer, Wong et al., 2001 [Non-Patent Document 22] Semple, Klimuk et al., 2001 [Non-Patent Document 23] Belliveau, Huft et al., 2012 [Non-Patent Document 24] Zhigaltsev, Belliveau et al., 2012 [Non-Patent Document 25] Zhigaltsev, Tam et al., 2016 Summary of the Invention [Problem to be solved by the invention]

[0007] Cultured mammalian primary cells (generally, primary cells are non-transgenic cells obtained directly from the target tissue) Nucleic acids and proteins can be expressed in a non-toxic manner in mammalian cells, including transformed, non-immortalized cells. There remains a need for transfection reagents that effectively deliver cargo. The importance of using progenitor cells and their advantages over the use of cell lines is well understood. However, the difficulty encountered in transfecting such cells is the lack of selective gene transcription. their use in any kind of discovery or validation research requiring breakdown Furthermore, with the shift to personalized medicine, functional differentiation in primary patient cells is becoming increasingly difficult. Genome screening and validation are required, and these transfectants There is a growing need for robust, non-toxic transfection methods for challenging cell types. do. [Means for solving the problem]

[0008] The present systems, methods, etc. provide a solution to one or more of these difficulties, and / or or provide other advantages.

[0009] Summary of the Invention The systems, compositions, devices, methods, etc. herein are directed to proteins, ribonucleoproteins, and the like. Delivering proteins (RNPs), RNA, DNA and other nucleic acid cargo and other selected cargo to target cells Typically referred to herein as transfectants, configured for complete and efficient delivery of Transfection competent vesicles The safety and efficacy of the lipid-based vesicle (TCV) are Each and both are involved in the TCV loading process (i.e., loading selected cargo into the TCV ethanol from the TCV storage process and / or the TCV delivery process. Remove any destabilizing agents, such as organic solvents and surfactants, such as sodium dodecyl sulfate. Thus, TCV can be used in part by, for example, destabilizer-free suspensions. It is maintained in a destabilizing agent-free solution as a suspension.

[0010] As used herein, a TCV is lipid-based, destabilizing agent-free, and A type of liposome or other vesicle that is produced and / or stored without encapsulating a selected cargo One advantage of such TCVs is that they can be used in solution or in the absence of destabilizing agents. can be stored as a suspension and can capture selected cargo in the absence of destabilizing agents; The ability to deliver such selected cargo to target cells in the absence of destabilizing agents. The selection cargo is transfected into target cells transfected with a TCV containing the selection cargo. / or RNP, RNA, DNA, protein, etc. that has a desired effect on the target patient. Therefore, unless otherwise clear from the context, TCV herein refers to the final selected cargo. In some embodiments, the T is empty except for the surrounding solution. CVs are designed to transport nucleic acid and protein cargo without the use of organic solvents or other destabilizing agents. It is designed for safe and efficient delivery to mammalian cells.

[0011] The TCV delivery process transfects mammalian cells, such as primary cells, with a cargo of choice. The cargo may also be complexed with proteins such as ribonucleoproteins (RNPs). In some embodiments, the systems, compositions, and devices described herein may include nucleic acids that are incorporated into the nucleic acid. The device and method may provide an empty TCV or a loaded TCV.

[0012] In some aspects, the systems, compositions, devices, methods, etc. herein are directed to lipid vesicles. for entrapment (or loading) of cargo into vesicles or liposomes and / or such vesicles A transesterification process that does not contain the organic solvents and other destabilizing agents previously required for storing The present invention also provides infection-competent vesicles (TCVs). etc. can be used or performed without the use of dedicated equipment, for example, The formed TCVs were then transferred to various types of selected samples by pipetting back and forth with the empty TCV-containing suspension. The compositions herein can be loaded by gently mixing with the selected cargo. Such methods may be particularly useful for "benchtop loading," allowing for the storage of small or large amounts of selected Additionally, a single batch of empty TCVs can be used with multiple different Different selected cargoes can be bench-top loaded in parallel.

[0013] In some embodiments, the systems, compositions, devices, methods, etc. herein may be used in organic solvents. Provide an empty TCV that is solvent-free and surfactant-free. If a destabilizing agent is used in TCV generation, it should be removed by dialysis or other suitable method. The TCV composition is organic solvent-free and surfactant-free. Repeated manual reciprocation of TCV producing fluid, SHM, T-mixing or extrusion methods, or as required If desired, gentle mixing such as other TCV mixing methods may be used to load the product.

[0014] In one embodiment, the lipid-based TCV comprises an ionizable cationic lipid, a phospholipid, a cholesterol-lowering agent ... The TCV-containing composition is composed of a mixture of sterols and PEG-lipids, and the TCV-containing composition is in the presence of an organic solvent and and / or surfactant-free, and the term does not, in its normal usage, refer to such organic solvents. The solvent and surfactant are essentially absent, and very small amounts may remain in the composition. However, no significant adverse effects are caused by organic solvents and / or surfactants. It is used to indicate that

[0015] In some embodiments, the ionizable cationic lipid is 20-50% of the lipid component of the TCV. In one embodiment, the empty lipid-based TCV comprises 20 / 30 / 10 / 39 / 1 The lipid components were mixed in the ratio of DODMA / DOPE / DSPC / Chol / PEG-lipid in mol%. In another embodiment, the empty lipid-based TCV contains 20 / 30 / 10 / 40 mol % of the lipid components in the ratio of DODMA / DOPE / DSPC / Chol. In the empty lipid-based TCV, 50 / 10 / 39 / 1 mol% DODMA / DSP In another embodiment, the lipid components are in the ratio of C / Chol / PEG-lipid. The TCV of the sachet was 50 / 10 / 39 / 1 mol% DODMA / DOPE / DSPC / Ch The lipid component is in the ratio of 1 / 2 to 1 / 2 of the original lipid component. If desired, a further range of ingredients may be used. In some embodiments, the ratio of ionizable cationic lipids is reduced. For example, The ratio of ionizable cationic lipids was approximately 10 mol%, 20 mol%, 30 mol%, It can be 40 mol %, 50 mol % or 60 mol %.

[0016] In one embodiment, empty lipid-based TCVs are added for 5 minutes, as needed, e.g., for 10-30 seconds. sec, 10 sec, 15 sec, 20 sec, 30 sec, 45 sec, 1 min or 2 min, nucleic acid selection The organic solvent-free and surfactant-free TCV is then mixed with the selected cargo. Store in an organic solvent-free and detergent-free environment and / or in an organic solvent-free and detergent-free environment. Once there, the compound can be administered again to target cells, such as mammalian cells.

[0017] In some embodiments, the nucleic acid selection cargo is double-stranded DNA, single-stranded DNA, RNA, low molecular weight nucleic acids, or the like. Interfering RNA, short hairpin RNA, messenger RNA, complementary DNA, microRNA A, plasmid DNA, or a combination thereof. The selection cargo may be a synthetic or chemically modified oligonucleotide, for example to improve the stability of the selection cargo. The cargo of choice may be a protein complexed with a nucleic acid (PNA). The protein selection cargo may be a protein involved in gene editing or a reporter of a cell marker. In some embodiments, the target protein may be a protein (such as a fluorescent marker) that functions as a tag. The protein-based selection cargo complexed with the nucleic acid is a ribonucleoprotein.

[0018] In some aspects, the present systems, devices, methods, etc., comprise transporting a cargo of choice to a lipid-based transporter. A method for encapsulating a transfection-competent vesicle (TCV), comprising: An aqueous solution comprising a lipid-based TCV is provided, wherein the aqueous solution is destabilizing agent-free. It may include The selected cargo is encapsulated in a lipid-based TCV to form a lipid-based TCV-encapsulated selected cargo. mixing the selected cargo in solution under suitable conditions and for a sufficient time to provide The mixing can be carried out in the absence of organic solvents or surfactants; The present invention provides a method comprising:

[0019] The destabilizing agent can be at least one of an organic solvent or a surfactant. Examples of the solvent include methanol, isopropyl alcohol, tetrahydrofuran (THF), dihydrofuran (DMF), and Methyl sulfoxide (DMSO), dimethylformamide (DMF) or acetonitrile (ACN). The surfactant may be, for example, sodium dodecyl sulfate (SDS). The destabilizing agent may be temperature. The aqueous solution may be 25 mM to 100 mM acetate buffer. It may be a liquid.

[0020] The lipid-based TCV may be empty prior to encapsulation, and the method may further comprise: Lipid-based TCV encapsulation selection carriers in aqueous solutions substantially free of solvents and surfactants Getting Go It may further include:

[0021] Lipid-based TCVs contain cationic lipids, e.g., ionizable cationic lipids. The lipid-based TCV may contain approximately 20 mol% to 50 mol% cationic The ionizable cationic lipid may include 1,2-dioleyloxy Lipid-based TCs can contain 3-dimethylamino-propane (DODMA). V is 1,2-dioleyloxy-3-dimethylamino-propane (DODMA), 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl yl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-distearate It can contain a mixture of aroyl-sn-glycero-3-phosphocholine (DSPC). The mixture contains at least one of polyethylene glycol (PEG) and cholesterol. It may further include one.

[0022] Lipid-based TCVs consist of DODMA / DOPE / DSPC / Chol / PEG-lipids. The mixture may contain approximately 20 / 30 / 10 / 39 / 1 mol% DODMA / DOPE The mixture of D / DSPC / Chol can contain about 20 / 30 / 10 / 40 mol %. It may contain a mixture of ODMA / DSPC / Chol at about 50 / 10 / 40 mol %; A mixture of DODMA / DSPC / Chol / PEG-lipids was prepared at approximately 50 / 10 / 39 / 1 mo. 1%, or a mixture of DODMA / DSPC / Chol / PEG at about 50 / 100%. It can contain 10 / 39 / 1 mol%.

[0023] The selection cargo can be a nucleic acid, such as a modified nucleic acid. 2'-O-ME, phosphorothioate or morpholino, locked nucleic acids The nucleic acid may comprise at least one of: It may contain double-stranded DNA, single-stranded DNA, complementary DNA (cDNA) or plasmid DNA. The nucleic acid can include ribonucleic acid (RNA). RNA can be used as a small interfering molecule. NA (siRNA), short hairpin RNA, messenger RNA (mRNA), micro The selected cargo may include a protein. The protein is part of a ribonucleoprotein (RNP) that can be a functional ribonucleoprotein. The RNP may comprise at least one of a Cas9 protein or a guide RNA, a Cas Both the Cas9 protein and guide RNA, or the Cas9 protein, guide RNA, and It may contain single-stranded DNA (ssDNA).

[0024] The cargo is at least one of an enzyme, a nuclease, and an endonuclease; or The cargo may comprise a zinc finger nuclease (ZFN), a T At least one of ALEN, Cas9, Cas10, Cas11, Cas12, or Cpf1 The cargo may comprise at least one of enzymes, nucleases and endonucleases. or a primer. The cargo may comprise a nuclease or can include mRNA encoding the antigen.

[0025] The method can further include mixing the lipid-based TCV with a selection cargo, The selected cargo was approximately 0.022-0.058 mg of selected cargo per μmole of cationic lipid. The method may involve mixing a lipid-based TCV with a selected cargo. and wherein the selection cargo is about 1000 mg of selection cargo per μmole of cationic lipid. The nucleic acid may be present in a ratio of 0.029 to 0.116 mg. Lipid-based TCV and The selected cargo can be mixed at a lipid-based TCV:selected cargo molar ratio of approximately 467. The selection cargo can be a ribonucleoprotein (RNP). The selected cargo may be mixed at a lipid-based TCV:selection cargo molar ratio of approximately 400-1200. The lipid-based TCV and selected cargo are mixed in a lipid-based TCV at a molar ratio of approximately 473 to 1173. The lipid-based TCV and the selected cargo can be mixed at approximately 3000 to 4000 kJ / ml. Lipid-based TCVs:selection cargoes may be mixed at molar ratios up to 5000.

[0026] The lipid-based TCV and selected cargo are then allowed to react for approximately 10-15 seconds, or approximately 10-30 seconds. The mixture can be mixed at room temperature. Mixing can be achieved by pipetting back and forth. do.

[0027] In some aspects, the present systems, devices, methods, etc., comprise lipid-based transfer media in aqueous solutions. A composition comprising a transfection-competent vesicle (TCV) is provided. The composition may be destabilizer-free, organic solvent-free and surfactant-free. or lipid-based TCVs as discussed in the Summary, Drawings, Detailed Description, or Claims. The present system, device, method, etc. can be further configured as follows. Lipid-based transfection in an aqueous solution that is substantially free of destabilizing agents such as surfactants. The present invention provides a composition comprising a transcription-competent vesicle (TCV)-encapsulated selection cargo. Lipid-based TCV-encapsulated select cargos are discussed in detail.

[0028] The systems, devices, methods, etc. also include transfection methods, as described herein. Lipid-based transfection-competent vesicles (T) as discussed in CV) Transfecting a target cell with an encapsulated selection cargo. The target cells may be, for example, primary mammalian cells, primary mammalian neural cells, cultured mammalian cells, or mammalian cells. The cells may be mammalian cells, such as cells from a mammalian patient.

[0029] The methods herein can be carried out in the laboratory, for example for benchtop loading. The method can be carried out in a factory to produce commercial quantities of transfected cells. The method can be used as part of an in vivo procedure, a medical procedure, a therapeutic procedure, or a gene therapy procedure. The method can be carried out by administering a therapeutic agent to treat Alzheimer's disease, Parkinson's disease, Huntington's disease, or other conditions. as part of the treatment for dementia, frontotemporal dementia, amyotrophic lateral sclerosis, or spinal muscular atrophy The method further includes delivering a lipid-based TCV-encapsulated cargo of choice to the brain of a patient. The method may include delivering the

[0030] In some further aspects, the systems, devices, methods, etc., comprise the compositions herein. The composition may be in a container, and the kit may include instructions for use of the composition. The instructions may direct use of the composition according to any of the methods herein. The container may be configured to administer at least one dose of the composition to a mammal. In some cases, the kit further comprises at least one label containing instructions for administration.

[0031] In some aspects, the systems, devices, methods, etc., are directed to treating a disease in a patient, which may be a mammal. a compound according to the present invention for use in the manufacture of a medicament for inhibiting, preventing, or treating a disease or condition. Isolated and purified compositions are provided.

[0032] These and other aspects, features and embodiments are described in detail in the following detailed description, including the accompanying drawings. Unless otherwise specified, all embodiments, aspects, features, etc. are intended to be illustrative, not restrictive. They may be mixed and matched, combined and substituted in any desired manner. Various references, including cross-references to related applications, describing systems, apparatus, methods, and other information are provided. All such references are incorporated herein by reference in their entirety, as well as for all of the teachings therein. and all of the disclosures herein are hereby incorporated by reference, regardless of where in this application the reference appears. and is incorporated herein by reference. [Brief explanation of the drawings]

[0033] [Figure 1]Figures 1A-1D show knockdown in immortalized cells and primary neurons using transfection-competent vesicles (TCVs) prepared according to the methods described herein. Figure 1A shows relative luciferase expression in HEK-Luc cells after transfection with organic- and detergent-free TCVs composed of 50% ionizable cationic lipids. Figure 1B shows cell viability from the same transfection set as in Figure 1A as a percentage of control (cell-only) wells. Figure 1C shows relative expression of hdh mRNA in primary cortical neurons after organic- and detergent-free TCV-mediated transfection of siRNA-selected cargoes into neurons. siRNA targeting hdh and TCVs were generated using the same panel of ionizable cationic lipids as in Figure 1A. Figure 1D shows cell viability, as measured by MTT reduction, of an equivalent transfection set in primary cortical neurons using organic- and detergent-free TCVs to deliver off-target (luciferase) siRNA. N=4 wells per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Bonforroni post-hoc analysis for each condition compared to the control (cells only) condition. [Figure 2]Figures 2A-2D show that the efficacy of organic- and detergent-free TCVs is independent of the mixing method used to generate the empty TCVs. Figure 2A shows relative luciferase expression in HEK-Luc cells after transfection with TCVs composed of 50% ionizable lipids and formed by either T-shaped mixing (DODMA-50%) or extrusion (DODMA-50%-X). Figure 2B shows cell viability from the same transfection set as in Figure 2A as a percentage of control (cell-only) wells. Figure 2C shows relative expression of hdh mRNA in primary cortical neurons after organic- and detergent-free TCV-mediated transfection of hdh-targeting siRNA using the same TCV panel as in Figure 2A. Figure 2D shows cell viability, as measured by MTT reduction, for an equivalent transfection set into primary cortical neurons using organic- and detergent-free TCVs to deliver off-target (luciferase) siRNA. N=4 wells per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Bonforroni post-hoc analysis for each condition compared to the control (cells only) condition. [Figure 3]Figures 3A-3D show that organic- and detergent-free empty TCVs with reduced ionizable lipid content promote potent siRNA delivery with low toxicity. Figure 3A shows relative luciferase expression in HEK-Luc cells after transfection with TCVs composed of 20% or 50% ionizable lipid 1,2-dioleyloxy-3-dimethylaminopropane (DODMA). Figure 3B shows cell viability from the same transfection set as in Figure 3A as a percentage of control (cell-only) wells. Figure 3C shows relative expression of hdh mRNA in primary cortical neurons after organic- and detergent-free TCV-mediated transfection of hdh-targeting siRNA using the same TCV panel as in Figure 3A. Figure 3D shows cell viability, as measured by MTT reduction, of an equivalent transfection set in primary cortical neurons using organic- and detergent-free TCVs to deliver off-target (luciferase) siRNA. N=4 wells per condition for HEK-Luc cells, N=3 wells per condition for primary neurons. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA followed by Bonforroni post-hoc analysis for each condition compared to the control (cells only) condition. [Figure 4] Figures 4A-4B show that the incorporation of DOPE into empty TCVs without organic solvent or surfactant does not alter the morphology of the TCVs. Cryo-TEM analysis was performed on (A) 20 / 30 / 10 / 39 / 1 mol% DODMA / DOPE / DSPC / Chol / PEG-lipid (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%) TCVs. Scale bar = 100 nm. [Figure 5]The organic solvent- and detergent-free empty TCVs described herein can be used to deliver functional ribonucleoproteins (RNPs). HEK293 cells were transfected with Cas9 RNP and a single-stranded oligodeoxynucleotide (ssODN) repair template, both of which were delivered by organic solvent- and detergent-free TCVs prepared according to the methods described herein. Cas9 protein was complexed to a guide targeting the GRN gene. DNA was extracted from cells 48 hours after transfection. PCR was used to specifically detect the wild-type (WT) GRN allele or a mutant GRN allele, which is present only when the delivered ssODN is integrated into the DNA double-strand break generated by Cas9 via homology-directed repair (HDR). The mutant GRN allele can be detected after TCV-mediated delivery of Cas9 RNP but is absent in control (Ctrl) cells. [Figure 6] Figure 1 shows a micrograph of Cas9 located within a primary neuron. RNPs delivered via the organic solvent-free and detergent-free TCV described herein can be detected using a fluorescent antibody against the Cas9 protein, shown in red. The untreated control has no such red fluorescent signal. Blue signal = nucleic acid (DAPI staining), green signal = phalloidin (F-actin staining). [Figure 7] Figure 1 shows transcriptional knockdown in HEK cells by RNP delivered by benchtop loading of RNP into empty, organic- and detergent-free TCVs. HEK293 cells were transfected with luciferase-targeting Cas9 RNP. TCVs containing DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40) were loaded on the bench. Relative levels of luciferase mRNA in HEK cells measured by qPCR show significant knockdown compared to the control ("ctrl" in the figure). N=3 per group. Data represent mean ± SEM. p=0.0018 by Student's t-test. [Figure 8]Figure 1 shows protein knockdown in HEK cells by RNP delivered by benchtop loading of RNP into empty, organic- and detergent-free TCVs. HEK293 cells were transfected with luciferase-targeting Cas9 RNP. TCVs containing DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40) were loaded on the bench. Relative luciferase protein levels in HEK cells measured by ONE-Glo demonstrate significant knockdown compared to the control ("ctrl" in the figure). N=3 per group. Data represent mean ± SEM. p=0.0003 by Student's t-test. [Figure 9] Figure 1 shows transcriptional knockdown in primary cortical neurons by RNP delivered by benchtop loading of RNP into empty, organic- and detergent-free TCVs. Primary cortical neurons were transfected with Cas9 RNP targeting human huntingtin (HTT). TCVs were loaded at the bench with a DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40) formulation. Relative levels of HTT mRNA in primary neurons measured by qPCR show significant knockdown compared to the control (labeled "gLuc" in the figure). N=3 per group. Data represent mean ± SEM. p=0.0039 by Student's t-test. [Figure 10] Graph showing mRNA expression in HEK cells for mRNA delivered via organic solvent-free and detergent-free TCVs. Luciferase mRNA was delivered to HEK cells via organic solvent-free and detergent-free TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) at various ratios: low = 0.029 mg mRNA / μmol lipid, medium = 0.058 mg mRNA / μmol lipid, and high = 0.116 mg mRNA / μmol lipid. All ratios showed significant expression compared to the control, with the lowest ratio showing the highest expression. mRNA expression was measured using the ONE-Glo+Tox kit (Promega) (n = 3 per condition). [Figure 11]Graph showing mRNA expression in primary cortical neurons for mRNA delivered via organic solvent-free and detergent-free TCV. Luciferase mRNA was delivered to primary cortical neurons at different doses via organic solvent-free and detergent-free TCV (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)). All doses showed significant expression compared to the control. mRNA expression was measured using the ONE-Glo+Tox kit (Promega) (N=3 per condition). [Figure 12] Figure 1 shows cell viability for RNPs in organic- and detergent-free TCVs in HEK cells. RNPs were delivered to HEK cells via organic- and detergent-free TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) or using RNAiMax ("Rmax" in the figure; ThermoFisher Scientific) for cytotoxicity assessment. Organic- and detergent-free TCVs were significantly less toxic than RNAiMax (p=0.0002). Toxicity was assessed using the ONE-Glo+Tox kit (Promega) with N=3 per group. [Figure 13] Figure 1 shows micrographs showing cell viability for benchtop loading of siRNA into empty, organic solvent-free, and detergent-free TCVs in primary neurons. siRNA was delivered to primary neurons for cytotoxicity assessment via organic solvent-free and detergent-free TCVs (DODMA / DOPE / DSPC / Chol / PEG-lipid (20 / 30 / 10 / 39 / 1)) or using the commercially available Mirus TKO system (Mirus Bio). As shown by the micrographs, siRNA delivered into empty, organic solvent-free and detergent-free TCVs by benchtop loading was significantly less toxic than that of the Mirus TKO system. [Figure 14]1 is a graph showing the knockdown of organic solvent-free and detergent-free TCV in HEK cells. siRNA was delivered using the commercially available Mirus TKO system (Mirus Bio) at organic solvent-free and detergent-free TCV DODMA levels of 50% ("D-50%"; produced by T-shape mixing) and 50% ("D-50% Ex"; produced by extrusion). The 50% DODMA formulation of the present invention showed approximately 50% knockdown, while the Mirus TKO system performed poorly. DETAILED DESCRIPTION OF THE INVENTION

[0034] The systems, compositions, devices, methods, etc. herein are directed to the detection of DNA and other nucleic acid selection groups. Exemplary embodiments of the present invention are configured to safely and efficiently deliver a polypeptide to target cells. Transfection-competent vesicles (TCVs) are lipid-based The safety and efficacy of the vesicles are evaluated individually and together with the TCV loading and and storage process (i.e., inserting the selected cargo into the TCV) and / or TCV delivery process. From the process, an organic solvent such as ethanol and a surfactant such as sodium dodecyl sulfate are added. This is achieved in part by removing disruptive agents such as steroids. This may involve transfection of mammalian cells, such as mammalian cells, with a selection cargo. The nucleic acid may also include a nucleic acid complexed with a protein such as a ribonucleoprotein (RNP). do.

[0035] In some embodiments, the systems, compositions, devices, methods, etc. herein may be used in organic solvents. Empty lipid-based TCVs are provided that are solvent-free and detergent-free. Loaded TCVs are , repeated manual reciprocation of TCV-generating fluid in a pipette, SHM, T-shaped mixing or extrusion method or other TCV mixing methods if desired. Cut.

[0036] In one embodiment, the lipid-based TCV comprises an ionizable cationic lipid, a phospholipid, a cholesterol-lowering agent ... The TCV-containing composition is composed of a mixture of sterols and PEG-lipids, and the TCV-containing composition is in the presence of an organic solvent and and / or surfactant-free.

[0037] The organic solvent-free and detergent-free TCVs discussed herein are useful for, for example, administering therapeutic agents via gene therapy. and can be used to treat appropriate diseases and conditions. The detergent-free TCV is suitable for the delivery of RNA, DNA, and RNP gene therapy products to human patients. The organic solvent-free and detergent-free TCV improves the delivery of gene therapy (mRNP). A, siRNA, and RNPs) to deliver products to brain cells or other target cells. The underlying cause of many human disorders is the loss of function of essential proteins. These causes are the loss of function or the gain of toxic function of mutant proteins. It is treatable and even reversible using detergent-free TCV.

[0038] Some examples of such treatments include: neurological disorders (Alzheimer's disease, Parkinson's disease, Several conditions, including Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and spinal muscular atrophy, are This includes gene therapy in the central nervous system to treat certain conditions (such as encephalopathy, encephalopathy, and encephalopathy).

[0039] The organic solvent-free and detergent-free TCVs discussed herein can also be used to prepare, e.g., siRNA. or targeted and safe delivery of RNP, gene replacement therapy with mRNA, or RNP Mutant genes through correction of the causative natural DNA mutation by mediated gene editing Gene products can also be genetically "knocked down." Two specific examples of human diseases that can be treated are Huntington's disease (HD) and frontotemporal dementia (FTD). FTD).

[0040] Huntington's disease is a progressive, incurable neurodegenerative disorder with a dominant inheritance pattern. An expanded CAG nucleotide repeat within the human thyroid tumor (HTT) gene causes the disease. The huntingtin protein (HTT) encoded by the mutant HTT gene is It contains an expanded polyglutamine repeat that confers a toxic gain-of-function on the gene product. Reducing brain levels of the protein may slow disease progression in HD. These are the major therapeutic strategies currently being pursued to prevent or halt HIV-1 infection and are discussed herein. To achieve and improve the performance of TCV by using organic solvent-free and surfactant-free TCV. siRNA targeting HTT expression or reducing huntingtin expression or toxicity can be used. TCVs loaded with RNP-selective cargos designed to induce vasoconstriction may be an effective treatment for HD. Frontotemporal dementia has many causes, but the protein progranulin (potentially a neurotransmitter) Loss of vital factors (vitamins) is one well-described cause. Can TCV containing and without surfactant deliver progranulin mRNA? or underlying factors that express progranulin or cause progranulin loss. RNPs (each) designed to correct a specific DNA mutation are progranulocyte-derived. This increases brain levels of progranulin, making it an effective treatment for FTD. Increasing progranulin in TCV also improves the risk of Alzheimer's disease, Parkinson's disease, and other conditions. This could be a neuroprotective strategy for many common neurological disorders, such as Son's disease and amyotrophic lateral sclerosis. do.

[0041] Examples of Materials and Methods

[0042] material 1,2-Dioleyloxy-3-dimethylamino-propane (DODMA) is a compound of the formula The compound was purchased from Man Chemical (Ann Arbor, Michigan). Dimethyl-3-ammonium-propane (DODAP), 1,2-dioleoyl-sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero- 3-Phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycerol Di-3-phosphocholine (DSPC) was purchased from Avanti Polar Lipids (Ara Cholesterol was purchased from Sigma Aldrich ( PEG-DMG was purchased from Bio-Rad (St. Louis, MO) as previously described. All lipids were synthesized using a method similar to that described in (Akinc, Zumbuehl et al., 2008). siRNA targeting firefly luciferase (siLuc) was maintained as a stock. (Basha, Ordobadi et al., 2016) The antibody was purchased from Technologies (Coralville, IA). The siRNAs used were purchased from Ambion (Silencer® Select). Pre-designed siRNA, Invitrogen, CA Carlsbad).

[0043] Preparation of transfection-competent vesicles (TCV) Lipid components (ionizable cationic lipids, phospholipids, cholesterol and PEG-lipids) The lipids were dissolved in ethanol in the appropriate ratio to achieve a final concentration of 20–35 mM total lipid. An aqueous phase containing 25 mM sodium acetate buffer at pH 4 was prepared. Nanoparticle Fabrication Technique: High speed mixing and extrusion were used to mix the two solutions.

[0044] Fast mixing: The lipid-containing organic phase was placed in a PEEK Low Pressure Tee Assembly Meets specifications for bly (1 / 16 inch, 0.02 inch through hole, part number P-712) A T-junction mixer fabricated as described above was used to mix the samples at a final flow rate of 20 mL / min with an organic:aqueous ratio of 1:3. (v / v) with the aqueous phase (Jeffs, Palmer et al., 2005; Kulkarn i, Tam et al., 2017; Kulkarni, Darjuan et al., 2018). The suspension was dialyzed against 1000 volumes of 25 mM sodium acetate pH 4 buffer to remove ethanol. was removed.

[0045] Extrusion: Lipids were dissolved in ethanol to a final concentration of 35 mM. Sea urchin (Maurer, Wong et al., 2001), 25 mM acetic acid in ethanol at pH 4 The final concentration of ethanol (v / v) was adjusted to 30% by rapid addition of sodium. The resulting nanoparticle suspension was heated at ambient temperature in a polycarbonate bath to produce 2 × 80 nm particles. After extrusion, the particles were buffer exchanged to remove the ethanol. Ta.

[0046] Analysis of transfection-competent vesicles (TCVs) T-Cholesterol Assay Kit (Wako Chemicals, Assayed for cholesterol content using ELISA kits (Mountain View, CA) However, as described elsewhere (Chen, Tam et al., 2014), the total lipid concentration was excluded. Lipid concentrations were determined by interpolation. Nucleic acid capture was performed as previously described (C Hen, Tam et al., 2014; Leung, Tam et al., 2015) RiboGreen was determined using the assay.

[0047] Cryogenic Transmission Electron Microscopy Cryo-TEM was performed as previously described (Kulkarni, Darjua Briefly, the centrifugation was performed using an Amicon centrifugal concentration unit (10 kDa N The TCV was concentrated to a total lipid concentration of approximately 20 mg / mL using a small volume (3–5 mL) of PBS. uL) of material was applied to a glow-discharge copper grid and used in an FEI Mark IV Vitro The grid was then frozen using a plunger freeze-thaw system (Hillsboro, Oregon). All imaging was performed at 200 kV in low-dose mode. The test was conducted using the FEI Tecnai G2 equipment manufactured by FEI Eagle 4k. Images were captured using a CCD bottom-mounted detector. and imaging were performed by the UBC BioImaging Facility (British Columbia University). The study was conducted in Vancouver, Columbia, Canada.

[0048] Cell culture and reagents: All base cell culture media and B27 neuronal supplements were purchased from Gibco (The Hanks' balanced salts were purchased from rmo Fisher, Waltham, MA. Hemoglobin-rich solution (HBSS), penicillin-streptomycin, L-glutamine and trypsin The solution was obtained from Hyclone (Logan, UT). HEK293 cells were cultured in Corning Primary cortical cells were seeded onto clear-bottom, white-walled plates manufactured by Corning, NY. , tissue coated with poly-D-lysine (Sigma, St. Louis, MO). The cells were seeded onto culture-treated plates (Fisher). Hygromycin B was added in vitro. Recombinant ApoE4 was obtained from Gen (Carlsbad, CA). protech (Rocky Hill, NJ).

[0049] To measure cell viability and luciferase levels in HEK293 cells, The ONE-Glo+Tox kit from romega (Madison, Wisconsin) was used. Cell viability in primary neurons was assessed using Sigma (St. Louis, MO). The toxicity was measured by MTT in vitro toxicity kit. Luciferase reporter HEK293 cells:

[0050] A HEK293 cell line (HEK293) carrying a stably integrated luciferase reporter construct was The generation of K-Luc cells has been described previously (De Souza, Islam et al., 2012). 016). Cells were cultured in 10% fetal bovine serum, 2 mM L-glutamine, and 125 μg / ml 95% air, 5% C in DMEM high glucose supplemented with L hygromycin B The cells were maintained at 37°C in a humidified atmosphere of O2. The cells were seeded at a density of 1000 μg / well into white-walled 96-well plates.

[0051] Primary cell culture: Cortical cultures were cultured from embryonic day E17.5 C57BL / 6J mice and FVB.YAC1 Briefly, cortices were dissected in ice-cold HBSS and diluted with 0.05% trypsin. The tissue was digested with Hyclone solution at 37°C for 10 minutes. Triturate five times with a 5 mL pipette and then add a 200 μL pipette tip. Triturate 5-7 times. Pellet the cells by centrifugation at 800 rpm for 5 minutes. The cells were then lysed in HBSS, washed, and then diluted with B27, 2 mM L-glutamine (Hyclone). and warm nerve base supplemented with 1% penicillin / streptomycin (Hyclone). The cortical neuron cultures were cultured at 1.5 x 10 cells / ml and resuspended in basal medium. 5 Cells / well density Cells were seeded onto lysine-coated 24-well plates and maintained in a 95% air, 5% CO2 atmosphere. The cells were maintained at 37°C in a humidified atmosphere.

[0052] Transfection: All reagents were mixed on the benchtop. Empty T containing 50% cationic lipid CV was mixed with siRNA at a ratio of 0.058 mg siRNA per μmole of lipid. TCV containing 0% cationic lipid was diluted with 0.022 mg of siRNA per μmole of lipid. The TCV suspension was briefly mixed with the siRNA by pipetting and then incubated at room temperature for 10 min. Incubated for minutes.

[0053] HEK293 cells were seeded 24 hours before transfection. TCV: siRN Mixture A with complete DMEM medium to give a final siRNA concentration of 3.3 μg / mL. The medium was completely changed at the time of transfection.

[0054] Primary neurons were grown in vitro for 7 days before transfection. Complete neurobasal medium containing 1 / mL recombinant ApoE4 was added to the TCV:siRNA suspension. , half of the medium was replaced from each well. Luciferase assay:

[0055] 48-72 hours after transfection, the ONE-Glo+Tox kit (Pro Cell viability and luminescence of HEK293 cells were measured using a 1000kJ / 2000kcal megachromatograph (1000kJ / 2000kcal) according to the manufacturer's instructions. Briefly, live cell reagent was added to each well and cells were incubated at 37°C for 30 minutes. The plate was then read by a plate reader (POLARstar Omega plate reader). Assay was performed using a 400 nm excitation and 510 nm emission spectrophotometer (Treida, BMG LABTECH). The plate was then read at 1000 kJ / s .... The ONE-Glo reagent was then added and the plate was incubated at room temperature for 3 minutes. Luminescence was measured by light output through the lens of the same plate reader. Values ​​are shown as % control and represent N=4 wells per condition.

[0056] MTT assay: Cell viability of primary cortical neurons after transfection in 24-well plates After 72 hours, the assay was carried out by MTT assay. (Methiazol-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. The cells were incubated at 37°C for 4 hours. The medium was removed and 250 μL of solubilization solution was added to each well. The absorbance was measured at 570 nm. Values ​​are shown as % of control and are Each figure represents N=3 wells.

[0057] Quantitative RT-PCR Adherent primary cortical cells were washed once in sterile PBS and then cultured in 1% 2-mercaptoethanol. Scrape the plate in 600 µL of lysis buffer containing 100 µL of lysate and immediately freeze at -80 °C. Subsequently, the PureLink RNA Mini Kit ( Total RNA was extracted using a PCR kit (Invitrogen). All samples were reverse transcribed using a PCR kit. Superscript VILO kit (Invitrogen) according to the manufacturer's instructions Therefore, use 250 μg of total RNA as input for cDNA synthesis and quantitative P 5 μg of diluted RNA was used for PCR reactions. Quantification of hdh mRNA levels The amplification was performed using a standard curve method in which target mRNA and a control gene were amplified in separate wells. Step-One ABI using ybr (Applied Biosystems) The experiment was carried out using the Applied Biosystems System. The relative amount of mRNA in each well was determined by comparing hdh mRNA with the control gene. The ratio was calculated to the gene Csnk2a2. Values ​​are shown as % of control, N=3 per condition. Represents a well.

[0058] Materials and production of ribonucleoprotein (RNP) complexes Guide RNA (gRNA), tracrRNA, single-stranded oligodeoxynucleotide ( All materials for the RNP formulation, including the ssODN, and recombinant Cas9 protein, were prepared by I The antibody was obtained from DT (San Jose, CA). The gRNA sequences used were provided by IDT (San Jose, CA). The gRNA sequence targeting toprogranulin (GRN) binds to exon 5 of the gene. The ssODN sequence used for homology-directed repair (HDR) is inserted into exon 5 of the GRN. It was engineered to introduce a 4 bp deletion.

[0059] RNP assembly was performed according to the manufacturer's specifications. Briefly, e.g., 1 μM tr Add 1 µM acrRNA and 1 µM gRNA to an equal molar ratio of crRNA:tracrRNA for 95 min. Form guide RNA (gRNA) complexes by incubating at 37°C for 5 min. The mixture was then cooled at room temperature for 20-30 minutes. Mix with Cas9 protein at a ratio of 0.01 to 0.1 and let the mixture stand at room temperature for 5 minutes before use. RNPs were formed.

[0060] Transfection of mammalian cells with nucleic acids: Empty organic solvent-free and surfactant-free TCV as discussed herein and The commercial reagent for comparison was mixed with the selected cargo on the benchtop. TCV was added to 1 μL of lipid. The nucleic acid was mixed with the selected cargo at ratios ranging from 0.01 to 0.2 mg of nucleic acid per mole. The suspension was briefly mixed with the siRNA by pipetting and incubated at room temperature for 10 minutes. .

[0061] HEK293 cells were seeded 24 hours before transfection. The final concentration of siRNA was 0.33-3.3 μg / mL, or mRNA was 0.1-1 μg / mL. Complete DMEM medium was added to the transfection medium at a concentration of 1 μg / mL. Primary neurons were grown in vitro for 7 days before transfection. Complete neurobasal medium containing 2–6 μg / mL recombinant ApoE4 was added to the TCV:nucleic acid suspension. and half of the medium was replaced from each well.

[0062] Cells were treated with Mirus TransIT-TKO according to the manufacturer's instructions. For cleanliness, Mirus TransIT-TKO was administered at 5 μL of Mirus / 100 μL of bloodless The siRNA was then added to the serum-free medium at the same concentration as in the supernatant. The solution was then mixed thoroughly with a pipette and incubated at room temperature for 15-30 minutes. The final concentration of Mirus was 5 μL / 1 mL of complete medium. The concentration was 25 nM.

[0063] Transfection of mammalian cells with RNPs: 0.5-20 mM TCV and 0.5-20 μM RNP were mixed at 467-5000 mol The mixture was mixed at the ratio of 1 to 10 μM and incubated at room temperature for 10 minutes. was mixed with TCV, and the mixture was incubated at room temperature for 5-15 minutes. added an equimolar amount of ssODN to the RNP complex solution before the addition of TCV.

[0064] The TCV containing RNP and the ssODN mixture were mixed, and complete medium was added to induce RNP and The final concentrations of ssODN and ssODN were set to 10–200 nM. A complete medium change was performed on K cells. Primary neurons were cultured for 5-7 days before transfection. The intact neuronal matrix was grown in vitro for 10 days and then cultured in a 2-6 μg / mL concentration of recombinant ApoE4. Bottom medium was added to the TCV:RNP mixture and half of the medium was replaced from each well.

[0065] Cells were transfected with Lipofectamine RNAiMAX reagent according to the manufacturer's instructions. Briefly, RNP complexes were prepared and mixed with serum-free medium and RNAiMAX. The mixture was added, incubated at room temperature for 5 minutes, and then added to the plated cells.

[0066] PCR for detection of homologous recombination repair Forward primers specific for either the wild-type (WT) or mutant GRN alleles and a common reverse primer were used to detect the transcripts. Amplify GRN exon 5 from genomic DNA extracted from infected HEK293 cultures PCR was performed using MyTaq (Bioline, USA) according to the manufacturer's instructions. PCR products were analyzed by gel electrophoresis on a 1.5% agarose gel stained with SybrSafe. They were separated by electrophoresis and imaged under UV light.

[0067] The cells were fixed with 3-4% paraformaldehyde solution for 15 minutes. The cells were permeabilized in PBS containing 0.1% Triton-X (PBST) for 15 minutes. Cells were cultured in PB containing a 1:1000 mixture of anti-Cas9 (Invitrogen) antibodies. The cells were incubated with ST overnight at 4°C. The cells were washed three times with PBS and then incubated with each Alexa Fluor 4000 (Alexa Fluor 4000) for 1 hour. Fluorescent 594 secondary antibody (Invitrogen) and phalloidin- iFluor 488 CytoPainter antibody (Abcam) 1:1000 mix Incubate with the solution for 1 hour at room temperature, wash again, and incubate for 5 minutes with a solution containing DAPI. The cells were incubated for 1 min to visualize the nuclei.

[0068] statistics All statistical comparisons were performed as one-way analyses of variance (ANOVAs) with Bonferroni post hoc. Analysis was performed to compare individual means with control-treated cells, correcting for multiple comparisons (Prism 6, Graphpad Software Inc.). Student's t-test was used. The mean individual values ​​for only two groups were compared using the ANOVA. A p value of less than 0.05 was considered significant. did.

[0069] Results Related Examples Example 1: Empty transfection-competent vesicles (TCVs) are soluble in organic solvents Efficiently captures nucleic acids

[0070] The empty TCV formulations produced by T-shaped or SHM mixing had a capture efficiency of approximately 85% or more. The inventors first demonstrated that TCVs composed of ionizable cationic lipids observed in vivo gene expression that captures nucleic acids without the aid of organic solvents or surfactants. Silencing efficacy (DODMA>>DLinDAP>DODAP) (DLinDAP " is 1,2-dilinyl-3-dimethylammonium-propane) Remarkably, in the absence of either of these, ionization Consists of lipid / DSPC / Chol / PEG-lipid (50 / 10 / 39 / 1 mol%) The resulting formulation was mixed at a ratio of 0.058 mg siRNA / μmol lipid at pH 4. Subsequent neutralization with PBS resulted in nearly complete (>85%) capture of siRNA (Table 1). The capture assay is based on the exclusion of an RNA-binding dye from nucleic acids by a lipid component. Therefore, the capture captures RNA from the external medium in more than a transient manner (i.e., a stable capture). Although there are no organic solvents or surfactants in the manufacturing process, The resulting TCV formulation was surprisingly produced by a rapid mixing technique using organic solvents. The LNP-siRNA produced by this method was compared with other literature (Belliveau, Huft et al., 2014). 12;Chen,Tam et al., 2014;Leung,Tam et al., 2015;Chen,T showed a capture efficiency similar to that reported in [Iam et al., 2016].

[0071] [Table 1]

[0072] Example 2: siRNA in organic solvent-free and detergent-free empty TCVs inhibits the expression of immortalized cells and demonstrates robust knockdown in primary neurons

[0073] The ability to capture nucleic acids and subsequently deliver them in a non-toxic manner presents two distinct challenges. The non-organic solvent / non-detergent lipid-based TCVs described above efficiently capture nucleic acids. If determined to be effective, their gene silencing capabilities and their effect on cell viability will be evaluated. The effect of this was examined in two scenarios, as shown in Figures 1A-1D. First, empty, organic solvent-free and detergent-free TCV was mixed with siLuc by benchtop loading, was used to treat HEK-Luc cells. Surprisingly, DODMA-based TCVs showed approximately DLinDAP and D showed 50% knockdown and no signs of knockdown. It performed better than ODAP (Figure 1A), as measured by cell viability compared to untreated cells. When tested, none of the formulations showed toxicity (Figure 1B). The efficacy and toxicity of TCV in hdh gene expression in primary mouse cortical neurons was tested. In this case as well (Fig. 1C), DODMA-TCV produced approximately 50% gene knockdown. DLinDAP-TCV showed lower knockdown, whereas DODAP- TCV showed no difference from untreated cells. Primary neurons are currently commercially available. It should be noted that these cells are highly susceptible to the toxic effects of harmful transfection reagents such as Figure 13 highlights the difference in effects on survival.

[0074] Example 3: Multiple mixing process produces strong organic solvent-free and surfactant-free empty TCVs can be generated

[0075] The role of the mixing aspect in the manufacturing process to achieve the efficacy shown in Figure 1 and the resulting To determine the particle size of the sample, an organic solvent containing an ionizable lipid such as DODMA was used. The lipid-based empty TCVs containing and surfactant-free were prepared by both T-mixing and extrusion. As shown in Figure 2A, siRNA was loaded into empty TCVs at the benchtop. Using both processes, luciferase was significantly increased in HEK-Luc cells. Viability of cells treated with T-shaped or extrusion-produced particles There was no significant difference in the results (Fig. 2B). and a lipid-based TCV formulation without the antimicrobial agent, and both processes showed similar efficacy (Figure 2C). It was found that the initial cleavage yielded particles with similar cell viability (Figure 2D). The toxicity of the transfection method of the present invention in subcultures (Figure 13) was non-toxic and strongly This undermines the goal of a potent formulation.

[0076] Example 4: Organic solvent-free and surfactant-free air with reduced content of ionizable lipids TCVs promote potent siRNA delivery with low toxicity

[0077] Established lipid compositions used in current clinical formulations (see Patisiran ) contains a significant amount of ionizable cationic lipids (50 mol%) (Jayar Aman, Ansell et al., 2012; Suhr, Coelho et al., 2015). Such a high concentration resulted in 50% gene silencing (ED) in vivo. 50 ) to achieve This allows for improved effective doses for the treatment of rheumatoid arthritis (Jayaraman, Ansell et al., 2012). , persistence of lipid metabolites after administration (Maier, Jayaraman et al., 2013; Sa bnis, Kumarasinghe et al., 2018) and the toxicity associated with these molecules. Therefore, high dose regimens and repeated administration (or to sensitive cell types) may be necessary. The compositions of the present invention, TCV, etc., are biodegradable or contain materials that facilitate excretion. The composition of the present invention, TCV, etc., can be composed of a transfectant. Reduces the amount of toxic ingredients to maintain efficacy.

[0078] DODMA / for silencing luciferase in HEK-Luc cells DOPE / DSPC / Chol / PEG-lipid (20 / 30 / 10 / 39 / 1 m respectively) The transfection ability of a formulation consisting of 100% of the luciferase gene was tested. A 40% knockdown of the expression was observed (Fig. 3A), without significant toxicity (Fig. 3B). B). Next, we compared 20 mol% DODMA-TCV with 50 mol% DODMA-TCV. The ability of TCVs with % ionizable lipids to deliver siRNA to primary neurons was investigated. All formulations showed 60% or less knockdown of hdh (Figure 3C), while DODMA formulations produced by T-mixing showed significantly improved cell viability. (Figure 3C).

[0079] Example 5: Incorporation of DOPE results in the formation of organic solvent-free and surfactant-free empty TCVs. Do not change

[0080] H in nucleic acid delivery vehicles II The improvement of the phase formation ability is related to membrane fusion in endosomes. This may be an important factor in promoting In the approach, H II Two exemplary lipids that can be employed are DOPE (alone) and ) and DODMA (when protonated and combined with anionic lipids). The incorporation of DOPE into organic solvent-free and surfactant-free TCV was rapid. II Phase formation To determine whether this effect was observed, 20 mol% DODMA-TCV and 50 mol% Cryo-TEM was performed on an equivalent formulation consisting of 1% DODMA. The resulting structure (Fig. 4A, Fig. 4B) is II visualized as a phase-free bilayer structure .

[0081] Previous studies by others (Leung, Hafez et al., 2012; Leung, Tam et al. , 2015) found that H was present in the core LNP formulation regardless of siRNA content. II The internal structure of This suggests that LNP-siRNA does not contain such a structure. rather, between closely adjacent lipid layers (Kulkarni, Darjuan et al., 2018). It has been shown that the particles have immobilized siRNA, giving the whole particle a multilayered or onion-like morphology. In the absence of siRNA, the LNP formulation is an electron-dense membrane containing oil-phase lipids. Thus, the examples herein demonstrate that the TCV morphology is dramatically different from the LNP system. Although these results are different in nature, they still have highly efficient transfection efficacies. .

[0082] Example 6: Organic Solvent-Free and Interfacially Active Inorganic Media for Delivery of Functional Ribonucleoproteins (RNPs) An empty TCV without activator can be used.

[0083] As shown in FIG. 5, the organic solvent-free and detergent-free empty TCVs of the present invention reacted with nucleic acids. The ability to deliver complexed protein-selected cargo into mammalian cells was also tested. Briefly, recombinant Cas9 protein and a gene targeting exon 5 of the progranulin gene were used. The ribonucleoprotein complex consisting of a guide RNA and a 467:1 (TCV:RN) Benchtop loading at a molar ratio of (DODMA / DOPE / DSPC / C) Combined with empty TCV by hol / (20 / 30 / 10 / 40mol%) A separate gene was designed to introduce a 4-bp deletion into exon 5 of the progranulin gene. The single-stranded oligodeoxynucleotide was added to the TCCV at a molar ratio of 4275:1 (TCV:nucleic acid). V. Two preparations of TCV containing each selected cargo were combined. The final concentrations were adjusted to 10 nM RNP and 10 nM ssODN, and then added to HEK cells. After 48 hours, forward primers specific for either the wild-type or mutant allele were detected. PCR using markers was performed to determine whether homologous recombination repair occurred at the progranulin gene target. As shown in Figure 5, the TCs containing each of the ribonucleoprotein complexes were Cells exposed to the combination of V and ssODN undergo homologous recombination repair (the second layer in Figure 5). The 4-bp deletion was introduced via the promoter (labeled mutant), whereas untreated control cells were This did not result in any genetic changes at exon 5 of the rhogranulin gene (Fig. 5 3rd and 4th lanes).

[0084] Example 7: Organic Solvent-Free Polymers Used to Deliver Functional Ribonucleoproteins (RNPs) Further examples of empty TCVs containing and without surfactant.

[0085] Using organic solvent-free and detergent-free empty TCVs, functional ribonucleic acid was synthesized by the above method. Protein (RNP) was delivered.

[0086] Figure 6 shows a photomicrograph of Cas9 located within a primary neuron. RNPs delivered via organic solvent-free and detergent-free TCVs exhibited a fluorescent signal. can be seen in primary neurons as fluorescent signals, whereas untreated controls show no such fluorescent signal More specifically, immunocytochemistry of primary neurons revealed that cortical neurons derived from mice The figure shows the localization of the Cas9 protein (red) within the nucleus (blue) of the rhodopsin. To do this, cells were stained with phalloidin (green).

[0087] Figure 7 shows the efficacy of TCV delivery via organic solvent-free and detergent-free TCV in HEK cells. 1 is a graph showing gene knockdown by RNPs in a solution containing no organic solvent and no surfactant. RNP delivered to HEK cells via empty TCV served as a control ("ctrl" in the figure). Figure 8 shows significant knockdown of luciferase transcripts compared to control. In cells, RNP delivered via organic solvent-free and detergent-free TCV Graph showing protein knockdown. Organic solvent-free and detergent-free TCV RNP delivered to HEK cells via ELISA showed significantly higher lucidation activity compared to the control ("Control" in the figure). This indicates significant knockdown of the enzyme protein, p=0.0003.

[0088] Figure 9 shows the effect of organic solvent-free and detergent-free TCV delivery on primary neurons. 1 is a graph showing gene knockdown achieved by RNP. RNPs delivered to primary cortical neurons via TCVs containing no active ingredient were quantitatively analyzed in real-time. demonstrated significant mRNA knockdown by quantitative real-time PCR (qRT-PCR) assay. p=0.0039.

[0089] Turning to further consideration of some of these figures, FIGS. 7 and 8 are RNPs delivered by benchtop loading of empty TCVs in HEK cells We demonstrate robust knockdown of the reporter gene, luciferase, in this This is the luciferase output (functional protein) as well as the mRNA of luciferase mRNA. This was demonstrated by measuring A levels by qRT-PCR. Using the same approach, we expressed the full-length human huntingtin gene carrying the disease mutation, F Huntingtin gene expression in cortical neurons derived from VB.YAC128 mice The mRNA levels of huntingtin were measured by infusion with the TCV:RNP mixture. After 72 hours of incubation, the expression of α-glucan was quantified from primary cortical neurons using qRT-PCR. Ta.

[0090] Example 8: Examples of mRNA delivered via organic solvent-free and detergent-free TCVs

[0091] The organic solvent-free and surfactant-free solutions used to deliver mRNA using the above methods are Benchtop loading of empty, agent-free TCVs.

[0092] Figure 10 shows the efficacy of organic solvent-free and detergent-free TCV-mediated delivery in HEK cells. 1 is a graph showing the mRNA expression achieved in a culture medium containing no organic solvent and no surfactant. Free TCV(DODMA / DOPE / DSPC / Chol(20 / 30 / 10 / 40) ) at various ratios, i.e., low = 0.029 mg mRNA / µmol lipid, medium = 0.029 mg mRNA / µmol lipid, = 0.058 mg mRNA / μmol lipid, High = 0.116 mg mRNA / μmol All ratios showed significant expression compared to the control, with the lowest The ratio of α- and β-actin-dependent markers showed the highest expression.

[0093] Figure 11 shows the effect of empty, organic solvent-free, and detergent-free TCs on primary cortical neurons. 10 is a graph showing expression of mRNA delivered by benchtop loading of V. mRNA was purified by organic solvent-free and detergent-free TCV (DODMA / DOPE / DSP C / Chol (20 / 30 / 10 / 40)) at various doses to induce primary cortical neurons All doses showed significant expression compared to controls.

[0094] Turning to further consideration of some of these figures, FIG. 10 is a diagram of the Use benchtop loading of an empty TCV that is free of organic solvents and surfactants, such as demonstrated that mRNA encoding a functional protein can be delivered to cells using Briefly, the TCV, which is free of organic solvents and surfactants, is 0.029 to 0. Firefly luciferase mRNA was encoded at a range of nucleic acid:lipid ratios of 116 mg to 1 μmole. The mixture was gently mixed with the mRNA to be injected using a pipette. The mixture was then incubated at room temperature for 5-20 minutes. After incubation, complete culture medium was added, followed by HEK cells (see Figure 10) or The cells were transferred to wells containing primary cortical neurons (Figure 11). Luciferase was detected using a luciferase assay kit according to the manufacturer's instructions. The results showed that the select cargo:TCV exerted its anti-inflammatory effect on these cells over a range of concentrations and doses. 1 shows the production of luciferase protein within the cytoplasm.

[0095] Example 9: Comparison of organic solvent-free and surfactant-free TCV with commercial products

[0096] Using benchtop loading of empty organic solvent-free and detergent-free TCVs siRNA or RNP is delivered to HEK cells or primary neurons by the above method, and then The results were compared with equivalent transfections using a commercially available system. The organic solvent-free and surfactant-free TCV outperformed the commercial system.

[0097] Figure 12 shows the cell proliferation of organic solvent-free and detergent-free TCV in HEK cells. 1 is a graph showing cell viability. RNP was incubated in organic solvent-free and detergent-free TCV (D ODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) or For cytotoxicity assessment, RNAiMax ("Rmax" in the figure; ThermoFisher The delivery was performed into HEK cells using a PEG-4000 (Primer Scientific) solution. The inhibitor-free TCV was significantly less toxic than RNAiMax (p=0.000 2).

[0098] Figure 13 shows the effect of empty, organic solvent-free, and detergent-free TCVs on primary neurons. 10 is a photomicrograph showing cell viability for benchtop loading of siRNA. siRNA was added to organic solvent-free and detergent-free TCV (DODMA / DOPE / DS PC / Chol / PEG-lipid (20 / 30 / 10 / 39 / 1)) or commercially available The Mirus TKO system (Mirus Bio) was used for the first time for cytotoxicity evaluation. The organic solvent-free and detergent-free TCVs were microscopically As shown, the toxicity was significantly lower than that of the Mirus TKO system.

[0099] Figure 14 shows the knockdown of organic solvent-free and detergent-free TCV in HEK cells. 1 is a graph showing the effect of 50% ("D-50%) siRNA produced by T-shaped mixing. ), 50% ("D-50% Ex", produced by extrusion) organic solvent-free and surfactant-free Drug-free TCV DODMA levels were measured using the commercially available Mirus TKO system (Mirus The 50% DODMA formulation of the present invention delivers approximately 50% knockdown. However, the Mirus TKO system was significantly inferior.

[0100] Turning to further discussion of some of these figures, FIG. 12 is a diagram of the Benchtop siRNA injection into empty, organic solvent-free, and detergent-free TCVs Loading has low toxicity characteristics and efficacy in primary neurons derived from mouse and HEK cell lines. For the treatment of primary cortical neurons, TCV was mixed with siRNA at a ratio of approximately 0.022–0.058 mg of nucleic acid:1 μmol of lipid. The cells were combined and transferred to wells containing primary neurons. All optical micrographs in Figure 13 are at the same magnification. In the control and TCV-treated wells, the cells were free of any process. This indicates that the wound remains healthy with very few dead cells. In transIT-TKO-treated wells, cells were small and shrunken, with disrupted cell processes. There is a large amount of (dead) cells and it appears to be in a very unhealthy state.

[0101] In Figure 12, HEK cells were transfected with Lipofectamine RNAiMAX and RNP-selected cargo. HEK cells were treated with TCV containing RNP at final concentrations of 5–50 nM. Treated with either drug or RNAiMAX reagent. Promega ONE-Glo+T Cell viability was assessed using the ox kit and compared with untreated control cells. Treated HEK cultures showed an overall increase in IL-1 expression compared to both control and TCV-treated wells. This indicates significantly lower health status.

[0102] Figure 14 shows the performance of the commercially available product Mirus LT-TK in delivering siRNA-selected cargo. The benefits of the empty, organic solvent-free, surfactant-free TCVs discussed herein compared to O This demonstrates the effectiveness of top-loading TCV targeting the luciferase gene. 1 μmol of siRNA and approximately 0.022 to 0.058 mg of nucleic acid at room temperature for 5 to 10 minutes. HEK cells were incubated with Mirus according to the manufacturer's instructions in a ratio of 1:1. At the time of treatment, the medium in the wells was replaced with siRNA:TCV or si Completely in fresh growth medium containing either the RNA:Mirus LT-TKO mixture After 72 hours of treatment, HEK cells were replated with Promega ONE-Glo+Tox Assay all cells for cell viability and luciferase production using a kit. was compared to untreated control wells.

[0103] All terms used herein are intended to be used interchangeably unless the context or definition clearly indicates otherwise. Unless otherwise specified, terms and conditions are used in accordance with their ordinary meaning. In this document, the use of "or" includes "and" and vice versa. Unless expressly stated or the context clearly indicates otherwise, should not be interpreted as a pun (for example, "including," "having," and "comprising" are typical examples). (The term "a," "an," and "the" typically refers to, but is not limited to, "the patent.") The singular forms "a," "an," and "the" are used in the claims unless expressly stated otherwise or the context clearly indicates otherwise. Includes plural references unless otherwise indicated.

[0104] References Akinc, A., A. Zumbuehl, et al. (2008). “A combinatorial library of lipid-like m aterials for delivery of RNAi therapeutics.” Nat Biotechnol 26(5): 561-569. Basha, G., M. Ordobadi, et al. (2016). "Lipid Nanoparticle Delivery of siRNA to Osteocytes Leads to Effective Silencing of SOST and Inhibition of Sclerostin In Vivo." Mol Ther Nucleic Acids 5(9): e363. Belliveau, N. M., J. Huft, et al. (2012). "Microfluidic Synthesis of Highly Pote nt Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA." Mol Ther Nucle ic Acids 1: e37. Chen, S., Y. Y. Tam, et al. (2014). "Development of lipid nanoparticle formulati ons of siRNA for hepatocyte gene silencing following subcutaneous administration ." J Control Release 196: 106-112. Chen, S., Y. Y. Tam, et al. (2016). "Influence of particle Size on the in vivo p otency of lipid nanoparticle formulations of siRNA." J Control Release. De Souza, R. A., S. A. Islam, et al. (2016). "DNA methylation profiling in human Huntington's disease brain." Hum Mol Genet 25(10): 2013-2030. Digiacomo, L., S. Palchetti, et al. (2018). "Cationic lipid / DNA complexes manufa ctured by microfluidics and bulk self-assembly exhibit different transfection be havior." Biochem Biophys Res Commun 503(2): 508-512. Hafez, I. M., N. Maurer, et al. (2001). "On the mechanism whereby cationic lipid s promote intracellular delivery of polynucleic acids." Gene Ther 8(15): 1188-11 96. Jayaraman, M., S. M. Ansell, et al. (2012). "Maximizing the potency of siRNA lip id nanoparticles for hepatic gene silencing in vivo." Angew Chem Int Ed Engl 51( 34): 8529-8533. Jeffs, L. B., L. R. Palmer, et al. (2005). "A Scalable, Extrusion-Free Method fo r Efficient Liposomal Encapsulation of Plasmid DNA." Pharm Res 22(3): 362-372. Kulkarni, J. A., M. M. Darjuan, et al. (2018). "On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA." ACS Nano 12(5): 4787-4795. Kulkarni, J. A., Y. Y. C. Tam, et al. (2017). "Rapid Synthesis of Lipid Nanopart icles Containing Hydrophobic Inorganic Nanoparticles." Nanoscale. Leung, A. K., I. M. Hafez, et al. (2012). "Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core ." J Phys Chem C Nanomater Interfaces 116(34): 18440-18450. Leung, A. K., Y. Y. Tam, et al. (2015). "Microfluidic Mixing: A General Method f or Encapsulating Macromolecules in Lipid Nanoparticle Systems." J Phys Chem B 11 9(28): 8698-8706. Lin, P. J., Y. Y. Tam, et al. (2013). "Influence of cationic lipid composition o n uptake and intracellular processing of lipid nanoparticle formulations of siRN A." Nanomedicine 9(2): 233-246. Maier, M. A., M. Jayaraman, et al. (2013). "Biodegradable lipids enabling rapidl y eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics." Mo l Ther 21(8): 1570-1578. Maurer, N., K. F. Wong, et al. (2001). "Spontaneous entrapment of polynucleotide s upon electrostatic interaction with ethanol-destabilized cationic liposomes." Biophys J 80(5): 2310-2326. Palchetti, S., D. Pozzi, et al. (2017). "Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells." Nanomedicine 13(2): 681-691. Pardi, N., S. Tuyishime, et al. (2015). "Expression kinetics of nucleoside-modif ied mRNA delivered in lipid nanoparticles to mice by various routes." J Control Release 217: 345-351. Pozzi, D., C. Marchini, et al. (2012). "Transfection efficiency boost of cholest erol-containing lipoplexes." Biochim Biophys Acta 9(43): 22. Rungta, R. L., H. B. Choi, et al. (2013). "Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain." Mol Ther Nucleic Acids 3(2): 65. Sabnis, S., E. S. Kumarasinghe, et al. (2018). "A Novel Amino Lipid Series for m RNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates." Mol Ther 26(6): 1509-1519. Scherphof, G. and H. Morselt (1984). "On the size-dependent disintegration of sm all unilamellar phosphatidylcholine vesicles in rat plasma. Evidence of complete loss of vesicle structure." Biochem J 221(2): 423-429. Semple, S. C., A. Akinc, et al. (2010). "Rational design of cationic lipids for siRNA delivery." Nat Biotechnol 28(2): 172-176. Semple, S. C., S. K. Klimuk, et al. (2001). "Efficient encapsulation of antisens e oligonucleotides in lipid vesicles using ionizable aminolipids: formation of n ovel small multilamellar vesicle structures." Biochimica et Biophysica Acta (BBA ) - Biomembranes 1510(1): 152-166. Suhr, O. B., T. Coelho, et al. (2015). "Efficacy and safety of patisiran for fam ilial amyloidotic polyneuropathy: a phase II multi-dose study." Orphanet Journal of Rare Diseases 10(1): 109. Tam, P., M. Monck, et al. (2000). "Stabilized plasmid-lipid particles for system ic gene therapy." Gene Therapy 7: 1867. Wang, Y., L. Miao, et al. (2015). "Delivery of oligonucleotides with lipid nanop articles." Adv Drug Deliv Rev 87: 68-80. Wheeler, JJ, L. Palmer, et al. (1999). "Stabilized plasmid-lipid particles: c struction and characterization." Gene Ther 6(2): 271-281. Zhigaltsev, IV, N. Belliveau, et al. (2012). "Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores usi ng millisecond microfluidic mixing." Langmuir 28(7): 3633-3640. Zhigaltsev, IV, YK Tam, et al. (2016). "Production of limit size nanolipos omal systems with potential utility as ultra-small drug delivery agents." J Lipo some Res 26(2): 96-102.

[0105] Unless otherwise specified, " " modifies the condition or relationship characteristics of one or more features of an embodiment. Adjectives used herein such as "substantially" and "about" mean that a condition or characteristic is within the range of its intended range. This indicates that the operating tolerances are defined within the acceptable ranges for operation of the embodiment for the intended use.

[0106] The scope of the present devices, systems, and methods includes means plus functions and steps. However, the claim does not include the word "means" and the concept of "function". Unless specifically recited in the claim, a "means-plus-function" relationship is not and should not be construed as indicating that the term "means" is specifically recited in the claim. In this case, it should be interpreted as indicating a "means plus function" relationship. A claim may be written as "step" unless the word "step" is specifically recited in the claim. should not be construed as indicating a "step-plus-function" relationship. If the claim specifically states that the step-plus-function relationship should be interpreted as

[0107] From the foregoing, while specific embodiments have been described herein for purposes of illustration, the spirit of the description herein is not to be construed as limiting the scope of the invention. It will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Accordingly, the systems, methods, etc., are intended to accommodate such modifications, as well as all of the subject matter described herein. and all permutations and combinations thereof as set forth in the appended claims or in the description and drawings of this specification. The present invention is not limited except as by other claims which are well supported in accordance with the present invention.

Claims

1. The cargo of choice is delivered to lipid-based transfection-competent vesicles (TCVs). A method of encapsulating in providing an aqueous solution comprising the lipid-based TCV, the aqueous solution being It does not contain stabilizers, encapsulating the selected cargo within the lipid-based TCV for lipid-based TCV encapsulation selection; The selected cargo is mixed into the solution under conditions suitable for a time sufficient to provide the cargo. wherein the mixing is carried out in the absence of organic solvents or surfactants; A method comprising:

2. 10. The method of claim 1, wherein the destabilizing agent is at least one of an organic solvent or a surfactant. The method described below.

3. The method of claim 1 , wherein the organic solvent is ethanol.

4. The method of claim 1 , wherein the organic solvent is methanol.

5. 10. The method of claim 1, wherein the organic solvent is isopropyl alcohol.

6. 2. The method of claim 1, wherein the organic solvent is tetrahydrofuran (THF).

7. 2. The method of claim 1, wherein the organic solvent is dimethyl sulfoxide (DMSO).

8. 2. The method of claim 1, wherein the organic solvent is dimethylformamide (DMF).

9. 2. The method of claim 1, wherein the organic solvent is acetonitrile (ACN).

10. 2. The method of claim 1, wherein the surfactant is sodium dodecyl sulfate (SDS).

11. Any of claims 1 to 10, wherein the aqueous solution is a 25 mM to 100 mM acetate buffer.

10. The method according to claim 1.

12. The method of claim 1 , wherein the destabilizing agent is temperature.

13. 13. The method of claim 1, wherein the lipid-based TCV is empty prior to encapsulation. The method described.

14. The method comprises: The lipid-based TCV encapsulation selection in an aqueous solution substantially free of solvents and surfactants. Getting cargo The method of any one of claims 1 to 13, further comprising:

15. 15. The method according to claim 1, wherein the lipid-based TCV comprises a cationic lipid. How to post.

16. 16. The method of claim 15, wherein the cationic lipid comprises an ionizable cationic lipid. 。

17. The lipid-based TCV comprises about 20 mol% to 50 mol% of cationic lipids.

17. The method according to any one of claims 1 to 16.

18. The ionizable cationic lipid is 1,2-dioleyloxy-3-dimethylamine.

17. The method of claim 16, comprising no-propane (DODMA).

19. The lipid-based TCV is 1,2-dioleyloxy-3-dimethylamino-propionate. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DODMA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOP C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE ), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) The method of any one of claims 1 to 17, comprising a mixture of

20. The mixture may contain at least one of polyethylene glycol (PEG) and cholesterol.

20. The method of claim 19, further comprising:

21. The lipid-based TCV is a DODMA / DOPE / DSPC / Chol / PEG-lipid 19. The method according to claim 1, wherein the mixture of the two substances is about 20 / 30 / 10 / 39 / 1 mol %. The method described in paragraph .

22. The lipid-based TCV contains a mixture of DODMA / DOPE / DSPC / Chol of about 20 / 30 / 10 / 40 mol % of the total amount of the hydroxybenzoates.

23. The lipid-based TCV contains a mixture of DODMA / DSPC / Chol in an amount of about 50 / 10. The method according to any one of claims 1 to 18, wherein the hydroxybenzoate comprises 40 mol% or more.

24. The lipid-based TCV is a mixture of DODMA / DSPC / Chol / PEG-lipid. The method of any one of claims 1 to 18, comprising about 50 / 10 / 39 / 1 mol% of

25. The lipid-based TCV contains a mixture of DODMA / DSPC / Chol / PEG in a volume of about 5 mL.

19. The method of any one of claims 1 to 18, comprising 0 / 10 / 39 / 1 mol%.

26. The method of any one of claims 1 to 25, wherein the selected cargo is a nucleic acid.

27. 27. The method of claim 26, wherein the nucleic acid is a modified nucleic acid.

28. The modified nucleic acid may be 2'-O-methylated (2'-O-ME), phosphorothioate, or monomethylated.

28. The method of claim 27, comprising at least one of:

29. 29. The method of claim 28, wherein the modified nucleic acid is a locked nucleic acid.

30. 27. The method of claim 26, wherein the nucleic acid is deoxyribonucleic acid (DNA).

31. 31. The method of claim 30, wherein the DNA comprises double-stranded DNA.

32. 31. The method of claim 30, wherein the DNA comprises single-stranded DNA.

33. 31. The method of claim 30, wherein the DNA comprises plasmid DNA.

34. 27. The method of claim 26, wherein the nucleic acid comprises ribonucleic acid (RNA).

35. 35. The method of claim 34, wherein the RNA comprises a small interfering RNA (siRNA).

36. 35. The method of claim 34, wherein the RNA comprises a short hairpin RNA.

37. 35. The method of claim 34, wherein the RNA comprises messenger RNA (mRNA).

38. 31. The method of claim 30, wherein the DNA comprises complementary DNA (cDNA).

39. 35. The method of claim 34, wherein the RNA comprises a microRNA (miRNA).

40. 40. The method of any one of claims 1 to 39, wherein the selected cargo comprises a protein.

41. 41. The method of claim 40, wherein the protein is part of a ribonucleoprotein (RNP). 。

42. 42. The method of claim 41, wherein the RNP is a functional ribonucleoprotein.

43. The RNP comprises at least one of a Cas9 protein or a guide RNA.

43. The method according to claim 41 or 42.

44. 43. The method of claim 41 or 42, wherein the RNP comprises a Cas9 protein and a guide RNA. How to do it.

45. The RNP contains the Cas9 protein and guide RNA and single-stranded DNA (ssDNA).

43. The method of claim 41 or 42, comprising:

46. The cargo is at least one of an enzyme, a nuclease, and an endonuclease.

46. ​​The method of claim 45, comprising:

47. The cargo may be a zinc finger nuclease (ZFN), a TALEN, a Cas9, a C as10, Cas11, Cas12, or Cpf1. Item 47. The method according to item 46.

48. 27. The method of claim 26, wherein the cargo comprises an mRNA encoding a nuclease or an antigen. How to do it.

49. further comprising mixing the lipid-based TCV with the selection cargo, The selected cargo is present at a ratio of about 0.022-0.058 mg of selected cargo per μmole of cationic lipid. The method according to any one of claims 1 to 48, wherein the nucleic acid is a nucleic acid present in the nucleic acid.

50. further comprising mixing the lipid-based TCV with the selection cargo, The cargo is present at a ratio of approximately 0.029-0.116 mg of selected cargo per μmole of cationic lipid. The method according to any one of claims 1 to 48, wherein the nucleic acid is a nucleic acid present in the nucleic acid.

51. The lipid-based TCV and the selected cargo are in a molar ratio of about 467.

49. The method of any one of claims 1 to 48, wherein the : is mixed with a cargo of choice.

52. 52. The method of claim 51, wherein the selected cargo is a ribonucleoprotein (RNP).

53. The lipid-based TCV and the selected cargo are in a lipid-based TCV molar ratio of about 400 to 1200.

53. The method of any one of claims 1 to 52, wherein the TCV is mixed with the selected cargo.

54. The lipid-based TCV and the selected cargo are in a lipid-based TCV molar ratio of about 473 to 1173.

53. The method of any one of claims 1 to 52, wherein the TCV is mixed with the selected cargo.

55. The lipid-based TCV and the selected cargo are in a molar ratio of about 3000-5000.

53. A lipid-based TCV according to any one of claims 1 to 52, admixed with a cargo of choice. How to do it.

56. 56. Any one of claims 1 to 55, wherein the selected cargo is a ribonucleoprotein (RNP). The method described below.

57. The lipid-based TCV and the selected cargo are mixed for about 10-15 seconds at about room temperature. The method of any one of claims 1 to 56,

58. The lipid-based TCV and the selected cargo are mixed for about 10-30 seconds at about room temperature. The method of any one of claims 1 to 56,

59. The mixing is carried out using a staggered herringbone micromixer or a T-type mixer.

59. The method of any one of claims 1 to 58.

60. 59. A method according to any one of claims 1 to 58, wherein the mixing is performed by reciprocating movements in a pipette. The method described.

61. Lipid-based transfection-competent vesicles (TCVs) in aqueous solution wherein the composition does not include a destabilizing agent.

62. 62. The composition of claim 61, wherein the destabilizing agent is an organic solvent and a surfactant.

63. The lipid-based TCV is an empty lipid-based TCV lacking a selected cargo.

63. The composition according to claim 61 or 62.

64. 64. The composition of claim 63, wherein the lipid-based TCV contains a selected cargo.

65. The composition comprises the lipid-based transfection competent protein in the aqueous solution. The composition according to any one of claims 61 to 64, consisting essentially of a tocopherol vesicle (TCV). Finished product.

66. 66. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises a cationic lipid. The composition described.

67. 67. The composition of claim 66, wherein the cationic lipid comprises an ionizable cationic lipid. thing.

68. The lipid-based TCV comprises about 20 mol% to 50 mol% of cationic lipids.

68. The composition of any one of claims 61 to 67.

69. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 10 mol% cationic lipid. The composition according to any one of the preceding claims.

70. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 20 mol % cationic lipid. The composition according to any one of the preceding claims.

71. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 30 mol% cationic lipid. The composition according to any one of the preceding claims.

72. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 40 mol % cationic lipid. The composition according to any one of the preceding claims.

73. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 50 mol % cationic lipid. The composition according to any one of the preceding claims.

74. 68. The lipid-based TCV of claim 61, wherein the lipid-based TCV comprises about 60 mol % cationic lipid. The composition according to any one of the preceding claims.

75. The ionizable cationic lipid is 1,2-dioleyloxy-3-dimethylamine.

75. The composition of any one of claims 67 to 74, comprising no-propane (DODMA).

76. The lipid-based TCV is 1,2-dioleyloxy-3-dimethylamino-propionate. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DODMA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOP C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE ), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) The composition of any one of claims 67 to 74, comprising a mixture of

77. The mixture may contain at least one of polyethylene glycol (PEG) and cholesterol.

77. The composition of any one of claims 67 to 76, further comprising one of:

78. The lipid-based TCV is a DODMA / DOPE / DSPC / Chol / PEG-lipid 78. The method of claim 61, wherein the mixture of the above-mentioned compounds contains about 20 / 30 / 10 / 39 / 1 mol % of the above-mentioned compounds. The composition described in any one of claims 1 to 4.

79. The lipid-based TCV is a mixture of DODMA / DOPE / DSPC / Chol / 78. The method of claim 61, comprising about 20 / 30 / 10 / 40 mol % of the hydroxybenzoates. The composition described above.

80. The lipid-based TCV contains a mixture of DODMA / DSPC / Chol in an amount of about 50 / 10.

78. The composition of any one of claims 61 to 75 or 77, comprising 40 mol% or more of:

81. The lipid-based TCV is a mixture of DODMA / DSPC / Chol / PEG-lipid.

78. The method of claim 61, comprising about 50 / 10 / 39 / 1 mol % of The composition described above.

82. The lipid-based TCV contains a mixture of DODMA / DSPC / Chol / PEG in a volume of about 5 mL.

78. The composition according to any one of claims 61 to 75 or 77, comprising 0 / 10 / 39 / 1 mol % Finished product.

83. The composition of any one of claims 61 to 82, wherein the organic solvent is ethanol.

84. 84. The composition of any one of claims 61 to 83, wherein the selected cargo is a nucleic acid.

85. 85. The composition of claim 84, wherein the nucleic acid is deoxyribonucleic acid (DNA).

86. 86. The composition of claim 85, wherein the DNA comprises double-stranded DNA.

87. 86. The composition of claim 85, wherein the DNA comprises single-stranded DNA.

88. 86. The composition of claim 85, wherein the DNA comprises plasmid DNA.

89. 85. The composition of claim 84, wherein the nucleic acid comprises ribonucleic acid (RNA).

90. 90. The composition of claim 89, wherein the RNA comprises a small interfering RNA (siRNA).

91. 90. The composition of claim 89, wherein the RNA comprises a short hairpin RNA.

92. 90. The composition of claim 89, wherein the RNA comprises messenger RNA (mRNA).

93. 86. The composition of claim 85, wherein the DNA comprises complementary DNA (cDNA).

94. 90. The composition of claim 89, wherein the RNA comprises a microRNA (miRNA).

95. 95. The composition of any one of claims 61 to 94, wherein the selected cargo comprises a protein. 。

96. 96. The composition of claim 95, wherein the protein is part of a ribonucleoprotein (RNP). thing.

97. 97. The composition of claim 96, wherein the RNP is a functional ribonucleoprotein.

98. The RNP comprises at least one of a Cas9 protein or a guide RNA.

98. The composition of claim 96 or 97.

99. 98. The method of claim 96 or 97, wherein the RNP comprises a Cas9 protein and a guide RNA. Composition of.

100. The RNP contains the Cas9 protein and guide RNA and single-stranded DNA (ssDNA).

98. The composition of claim 96 or 97, comprising:

101. Lipid-based transfection in an aqueous solution substantially free of organic solvents and detergents. A composition comprising a transcription-competent vesicle (TCV)-encapsulated selection cargo, Lipid-based TCV-encapsulated cargo of choice produced according to any one of claims 1 to 60. , composition.

102. A transfection method, comprising the steps of: Lipid-based transfection-competent vesicles (TCVs) encapsulating selection carriers transfecting a target cell with the vector.

103. The lipid-based transfection control according to any one of claims 61 to 101. Transfecting target cells with selected cargo encapsulated in transfectant vesicles (TCVs) A transfection method comprising:

104. 104. The method of claim 102 or 103, wherein the target cell is a mammalian cell.

105. 104. The method of claim 102 or 103, wherein the target cells are primary mammalian cells.

106. 104. The method of claim 102 or 103, wherein the target cells are mammalian primary neuronal cells.

107. 104. The method of claim 102 or 103, wherein the target cells are cultured mammalian cells.

108. 104. The method of claim 102 or 103, wherein the target cells are cells from a mammalian patient. 。

109. The method of any one of claims 102 to 108, wherein the method is carried out in a laboratory.

110. 102-103, which is carried out in a factory to produce commercial quantities of transfected cells.

08. A method according to any one of claims 08 to 08.

111. 109. The method of claim 102, wherein the method is performed as part of an in vivo procedure. method.

112. A method according to any one of claims 102 to 108, carried out as part of a medical procedure. 。

113. A method according to any one of claims 102 to 108, carried out as part of a therapeutic procedure. 。

114. 114. The method of claim 112 or 113, performed as part of a gene therapy procedure.

115. The method of claim 112 or 113, as part of a treatment for Alzheimer's disease. Law.

116. 114. The method of claim 112 or 113, which is carried out as part of a treatment for Parkinson's disease. 。

117. 114. The method of claim 112 or 113, carried out as part of a treatment for Huntington's disease. 。

118. The method of claim 112 or 113, as part of a treatment for frontotemporal dementia. Law.

119. 114. The method of claim 112 or 113, as part of a treatment for amyotrophic lateral sclerosis. method.

120. 114. The method of claim 112 or 113, which is carried out as part of a treatment for spinal muscular atrophy. 。

121. and further comprising delivering the lipid-based TCV-encapsulated cargo of choice to the brain of the patient.

121. The method of any one of claims 112 to 120.

122. A kit comprising the composition of any one of claims 61 to 101, in a container, said kit comprising instructions for use of said composition.

123. The instructions may be for preparing the composition according to the method of any one of claims 103 to 121.

123. The kit of claim 122, which includes instructions for use.

124. The container is configured to administer at least one dose of the composition to a mammal, The kit further comprises at least one label containing instructions for said administration. Item 124. The kit according to Item 122 or 123.

125. For use in the manufacture of a pharmaceutical product to inhibit, prevent, or treat a disease or condition in a patient 102. The isolated and purified composition of any one of claims 61 to 101.

126. 126. The composition of claim 125, wherein the patient is a mammal.

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