Methods for encapsulating a selected cargo in a lipid-based transfer-competent vesicle (TCV) for single-cell transfer, for preparation of compositions, kits, compositions, water-based solutions and uses.

Description

1 / 45 METHODS FOR ENCAPSULATING A SELECTED LOAD IN A TRANSFECTION-COMPETENT VESICLE (TCV) Lipid-based, for cell transfer, for preparation of compositions, kit, compositions, water-based solution and uses. Separated from BR112021006539-3, filed on October 9, 2019. CROSS-REFERENCE TO RELATED REQUESTS

[0001] This application claims the benefit of U.S. Provisional Patent Application 62 / 743,116, filed October 9, 2018, which application is incorporated herein by reference in its entirety. BACKGROUND

[0002] One of the important areas for scientific research and medical treatments is the desire to selectively and efficiently deliver RNA, DNA, other nucleic acid payloads, and / or proteins to target locations, such as specific target cells. This can be useful for a variety of reasons, including improved patient treatments, such as gene therapy, and for the treatment of cancer and other conditions. For example, gene therapy can be used in the brain and throughout the central nervous system to treat some of the horrific classic neurological disorders that humans can suffer from, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, spinal muscular atrophy, and many more.Current gene therapy approaches have several problems with their widespread application, especially to human patients, for example, due to the need for repeated dosing and the toxicity of packaging that transports therapeutic nucleic acids to the patient. The present compositions, methods, etc. in this document help to remedy one or more of these or other problems.

[0003] Returning to a more scientific discussion of the distribution of. Petition 870260070835, dated 07 / 16 / 2026, pp. 204 / 276 2 / 45 To deliver DNA and other nucleic acids to target locations, such as diseased cells in the brain, existing methods include lipid particles, sometimes called lipid nanoparticles (LNPs) or liposomes. The term lipid nanoparticles or LNPs is used to describe lipid-based particles around a neutral pH that typically contain nucleic acid and have an electron-dense core. Liposomes, also known as vesicles, are lipid-based structures with a single bilayer and an aqueous core. Typical established processes for LNP formation charge the vesicle with a specific charge at the time of initial vesicle formation.These processes still use specialized instrumentation, organic solvents and / or detergents, require large quantities of material and involve processing times on the order of days, which seriously impairs the usefulness, accessibility and therapeutic potential.

[0004] LNPs and other lipid particles typically comprise an ionizable cationic lipid, one or more phospholipids, cholesterol (Chol), and polyethylene glycol lipid (PEG lipid) (Maurer, Wong et al. 2001; Semple, Klimuk et al. 2001; Semple, Akinc et al. 2010; Belliveau, Huft et al. 2012; Leung, Hafez et al. 2012; Suhr, Coelho et al. (Several references are presented herein that discuss certain systems, devices, methods, and other information; all of them are incorporated herein by reference in their entirety and for all their teachings and descriptions, regardless of where the references may appear in this application. Citation of a reference in this document is not an admission that such reference constitutes the state of the art to the present application).An example of an LNP composition is the combination of ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipid in a ratio of 50 / 10 / 38.5 / 1.5 mol% (respectively). Petition 870260070835, dated 07 / 16 / 2026, pages 205 / 276 3 / 45 demonstrated that this composition exhibits potent gene silencing of hepatocytes (siRNA) or expression (mRNA) after intravenous administration (Semple, Akinc et al. 2010; Jayaraman, Ansell et al. 2012; Pardi, Tuyishime et al. 2015; Suhr, Coelho et al. 2015). Others have described LNP compositions composed of ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipid in a 50 / 10 / 38.5 / 1.5 mol% ratio as siRNA delivery from primary neuronal cells in culture and for delivery to the brain (Rungta, Choi et al. 2013).

[0005] LNP formulations can be generated by rapidly mixing lipid components dissolved in ethanol with an acidic aqueous phase consisting of the nucleic acid cargo (Jeffs, Palmer et al. 2005; Belliveau, Huft et al. 2012; Leung, Hafez et al. 2012). An established rapid mixing process for LNP fabrication includes microfluidic mixing via a staggered herringbone micromixer (SHM) (Belliveau, Huft et al. 2012; Rungta, Choi et al. 2013; Leung, Tam et al. 2015) or T-junction type mixing with specialized pumps (Jeffs, Palmer et al. 2005) or a more up-to-date approach of destabilized ethanol / detergent loading onto pre-formed vesicles (Wheeler, Palmer et al. 1999; Tam, Monck et al. 2000; Maurer, Wong et al. 2001; Semple, Klimuk et al. 2001).In all three methods, an ethanolic (or detergent) solution is required to provide sufficient membrane fluidity for lipid reorganization and retention to occur, and in the case of SHM and T-bonding techniques, particle formation also occurs after dilution of the ethanolic solution in the aqueous phase (Belliveau, Huft et al. 2012; Zhigaltsev, Belliveau et al. 2012; Zhigaltsev, Tam et al. 2016). However, the resulting suspension is not ready for use due to the organic solvent and acidic pH, and therefore requires downstream processing. Petition 870260070835, dated 07 / 16 / 2026, pages 206 / 276 4 / 45 substantial. In terms of material and time costs, these approaches have significant impediments to achieving a transfection-competent formulation on a laboratory scale for in vitro applications or for therapeutic levels for direct administration.

[0006] There remains a need for transfection reagents that effectively deliver a payload of nucleic acids and proteins into mammalian cells in a non-toxic manner, including cultured mammalian primary cells (generally, primary cells are untransformed, non-immortalized cells obtained directly from a target tissue). While the importance of using primary cells and their advantages over using cell lines is well understood, the difficulty encountered in transfecting such cells has almost entirely prevented their use in any type of discovery or validation studies requiring selective gene silencing (knockdown). Furthermore, a move towards personalized medicine is pushing for functional genomic screening and validation to be performed on primary patient cells, increasing the need for robust and non-toxic transfection methods for these difficult-to-transfect cell types.

[0007] The present systems and methods, etc., provide solutions to one or more of these difficulties and / or offer other advantages. SUMMARY

[0008] The systems, compositions, devices and methods, etc., in this document provide lipid-based vesicles, typically referred to herein as Transfection Competent Vesicles (TCVs), configured to safely and efficiently deliver proteins, ribonucleoproteins (RNPs), RNA, DNA and other nucleic acid payloads and other selected payloads to target cells. Safety and efficiency are each and both achieved in part by the elimination of agents Petition 870260070835, dated 07 / 16 / 2026, pages 207 / 276 5 / 45 Destabilizing agents, such as organic solvents like ethanol and detergents like sodium dodecyl sulfate, are removed from TCV loading processes (i.e., inserting a selected load into the TCV), TCV storage processes, and / or TCV distribution processes. Thus, the TCV is maintained in a destabilizing agent-free solution, for example, as a destabilizing agent-free suspension.

[0009] As used in this document, a TCV is a type of liposome or other vesicle that is lipid-based and is generated and / or stored without destabilizing agents and without a selected payload inside. An advantage of such TCVs is that they can be stored in solution or as a suspension without the presence of destabilizing agents, can retain selected payloads without the presence of destabilizing agents, and can deliver such selected payloads to target cells without the presence of destabilizing agents. Selected payload indicates RNPs, RNAs, DNAs, proteins, etc., that create a desired effect on a target cell and / or target patient that is transfected with the TCV containing the selected payload. Thus, unless otherwise evident from the context, the TCVs in this document ultimately do not have a selected payload and, in certain embodiments, are empty except as to ambient solution or similar.These TCVs are configured to safely and efficiently deliver nucleic acid and protein payloads, etc., to mammalian cells without the use of organic solvents or other destabilizing agents.

[0010] TCV delivery processes may involve transfection of mammalian cells, such as primary cells, with the selected cargo. Cargoes may also involve nucleic acids in complex with a protein, such as a ribonucleoprotein (RNP). The systems, compositions, Petition 870260070835, dated 07 / 16 / 2026, pages 208 / 276 6 / 45 devices and methods, etc. in this document, in some embodiments, may provide empty TCVs or loaded TCVs.

[0011] In some respects, the systems, compositions, devices, and methods, etc. in the present document provide transfection-competent vesicles (TCVs) without organic solvents and other destabilizing agents that were previously required to retain (or load) the cargo in lipid vesicles or liposomes and / or to store such vesicles. The compositions, methods, etc. in the present document can be used or obtained without the use of specialized instruments; for example, the pre-formed TCVs in the present document can be loaded by gently mixing the suspension containing empty TCVs with various types of selected cargo using a pipette's reciprocal action. The compositions, methods, etc. in the present document can be particularly useful for benchtop loading and can be used with small or large quantities of selected cargo material.Furthermore, a single batch of empty TCVs can be bench-loaded with several different selected loads in parallel.

[0012] In some embodiments, the systems, compositions, devices and methods, etc. in this document provide empty TCVs that are free of organic solvent and detergent. If ethanol or detergent or other destabilizing agent has been used to generate the TCVs, it is removed by dialysis or other suitable method to provide a TCV composition free of organic solvent and detergent. The TCVs can be loaded using a gentle mixing, such as repeated manual reciprocation of the TCV generating fluid in a pipette, SHM, T-junction type mixing or extrusion methods or other TCV mixing methods as desired.

[0013] In one aspect, lipid-based TCVs are composed of a mixture of an ionizable cationic lipid, phospholipid, Petition 870260070835, dated 07 / 16 / 2026, pp. 209 / 276 7 / 45 cholesterol and PEG-lipid and the composition containing TCV is free of organic solvent and / or detergent, which terminology is used in its normal usage to indicate that such organic solvent and detergent are essentially absent, so that no significant deleterious effect is caused by the organic solvent and / or detergent, although minor trace amounts may remain in the composition.

[0014] In some aspects, ionizable cationic lipid comprises between 20-50% of the lipid component of TCVs. In one aspect, empty lipid-based TCVs contain lipid components in a DODMA / DOPE / DSPC / Chol / PEG-lipid ratio of 20 / 30 / 10 / 39 / 1 mol. In another aspect, empty lipid-based TCVs contain lipid components in a DODMA / DOPE / DSPC / Chol ratio of 20 / 30 / 10 / 40 mol. In another aspect, empty lipid-based TCVs contain lipid components in a DODMA / DSPC / Chol / PEG-lipid ratio of 50 / 10 / 39 / 1 mol. In another aspect, empty lipid-based TCVs contain lipid components in a DODMA / DOPE / DSPC / Chol ratio of 50 / 10 / 39 / 1 mol%. Additional component ranges may also be used as desired. In certain aspects, the proportion of ionizable cationic lipid is reduced.For example, the proportion of ionizable cationic lipid can be approximately 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol%.

[0015] In one aspect, empty lipid-based TCVs are mixed with the selected nucleic acid payload for 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, or 2 minutes, as desired, for example, 10-30 seconds. The organic solvent- and detergent-free TCVs can then be stored in an organic solvent- and detergent-free environment and / or administered to target cells, such as mammalian cells, again in an organic solvent- and detergent-free environment. Petition 870260070835, dated 07 / 16 / 2026, pages 210 / 276 8 / 45

[0016] In some aspects, the selected nucleic acid payload may be double-stranded DNA, single-stranded DNA, RNA, small interfering RNA, short hairpin RNA, messenger RNA, complementary DNA, microRNA, plasmid DNA, or a combination thereof. In some aspects, the selected nucleic acid payload may comprise synthetic or chemically modified oligonucleotides, for example, to improve the stability of the selected payload. The selected payload may be a protein complexed with a nucleic acid (PNA). The selected protein payload may be proteins involved in gene editing or proteins that function as reporters for cell labeling (such as fluorescent markers and so forth). In some embodiments, the selected payload based on a protein that is complexed with a nucleic acid is a ribonucleoprotein.

[0017] In some respects, the present systems, devices and methods, etc., provide methods for encapsulating a selected payload in a transfection-competent lipid-based vesicle (TCV) comprising: - To provide a water-based solution comprising lipid-based TCV. The water-based solution may be free of destabilizing agents; and, - Mix the selected load in the solution under suitable conditions and for a sufficient time so that the selected load is encapsulated within the lipid-based TCV to provide a selected load encapsulated by lipid-based TCV. The mixing can be carried out without the presence of an organic solvent or detergent.

[0018] The destabilizing agent may be at least one organic solvent or a detergent. The organic solvent may be, for example, methanol, isopropyl alcohol, tetrahydrofuran (THF), sulfoxide Petition 870260070835, dated 07 / 16 / 2026, pages 211 / 276 9 / 45 of dimethyl (DMSO), dimethylformamide (DMF), or acetonitrile (ACN). The detergent can be, for example, sodium dodecyl sulfate (SDS). The destabilizing agent can be temperature. The water-based solution can be an acetate buffer at 25 mM to 100 mM.

[0019] Lipid-based TCV may be empty prior to encapsulation and the methods may still include: - Obtain the selected cargo encapsulated in lipid-based TCV in a water-based solution that is substantially free of solvents and detergents.

[0020] Lipid-based TCV may comprise a cationic lipid, such as an ionizable cationic lipid. Lipid-based TCV may comprise about 20 mol% to 50 mol% of cationic lipid. The ionizable cationic lipid may comprise 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA). Lipid-based TCV may comprise a mixture of 1,2-diooleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-diestearoyl-sn-glycero-3-phosphocholine (DSPC). The mixture may further comprise at least one polyethylene glycol (PEG) or cholesterol.

[0021] Lipid-based TCV may comprise a mixture of DODMA / DOPE / DSPC / Chol / PEG-lipid at about 20 / 30 / 10 / 39 / 1 mol%, may comprise a mixture of DODMA / DOPE / DSPC / Chol at about 20 / 30 / 10 / 40 mol%, a mixture of DODMA / DSPC / Chol at about 50 / 10 / 40 mol%, a mixture of DODMA / DSPC / Chol / PEG-lipid at about 50 / 10 / 39 / 1 mol% or a mixture of DODMA / DSPC / Chol / PEG at about 50 / 10 / 39 / 1 mol%.

[0022] The selected payload may be a nucleic acid, such as a modified nucleic acid. The modified nucleic acid may comprise, for example, at least one 2'-O-methylation (2'-O-ME), phosphorothioate or morpholino, a blocked nucleic acid. The acid Petition 870260070835, dated 07 / 16 / 2026, pp. 212 / 276 10 / 45 nucleic acid can be deoxyribonucleic acid (DNA). DNA can comprise double-stranded DNA, single-stranded DNA, complementary DNA (cDNA), or plasmid DNA. Nucleic acid can comprise ribonucleic acid (RNA). RNA can comprise small interfering RNA (siRNA), short loophole RNA, messenger RNA (mRNA), or microRNA (miRNA). The selected cargo can comprise a protein. The protein can be part of a ribonucleoprotein (RNP), which can be a functional ribonucleoprotein. The RNP can comprise at least one of a Cas9 protein or a guide RNA, a Cas9 protein and a guide RNA, or comprise a Cas9 protein and a guide RNA and single-stranded DNA (ssDNA).

[0023] The payload may comprise at least one of an enzyme, a nuclease, and an endonuclease or a primer. The payload may comprise at least one of zinc finger nuclease (ZFN), TALEN, Cas9, Cas10, Cas11, Cas12, or Cpf1. The payload may comprise at least one of an enzyme, a nuclease, and an endonuclease or a primer. The payload may comprise an mRNA encoding a nuclease or an antigen.

[0024] The methods may further comprise mixing the lipid-based TCV with the selected cargo. The selected cargo may be a nucleic acid that may be present in a ratio of about 0.022–0.058 mg of selected cargo per μmole of cationic lipid. The methods may further comprise mixing the lipid-based TCV with the selected cargo. The selected cargo may be a nucleic acid that may be present in a ratio of about 0.029–0.116 mg of selected cargo per μmole of cationic lipid. The lipid-based TCV and the selected cargo may be mixed in a mol ratio of lipid-based TCV:selected cargo of about 467. The selected cargo may be Petition 870260070835, dated 07 / 16 / 2026, pp. 213 / 276 11 / 45 a ribonucleoprotein (RNP). Lipid-based TCV and the selected filler can be mixed in a lipid-based TCV:selected filler ratio of about 400 to 1200 moles. Lipid-based TCV and the selected filler can be mixed in a lipid-based TCV:selected filler ratio of about 473 to 1173 moles. Lipid-based TCV and the selected filler can be mixed in a lipid-based TCV:selected filler ratio of up to about 3000 to 5000.

[0025] Lipid-based TCV and the selected load can be mixed at approximately room temperature for about 10 to 15 seconds or for about 10 to 30 seconds. Mixing can be performed using a stepped herringbone-type micromixer or a T-junction type mixer. Mixing can also be performed by reciprocity in a pipette.

[0026] In some aspects, the present systems, devices and methods, etc., provide compositions comprising a transfection-competent vesicle (TCV) based on lipids in a water-based solution. The compositions may be free of destabilizing agents, organic solvents and detergents. The composition and / or lipid-based TCV may further be configured as discussed in the Summary, Figures, Detailed Description or Claims. The present systems, devices and methods, etc., provide compositions comprising a selected payload encapsulated in a transfection-competent vesicle (TCV) based on lipids in a water-based solution substantially free of destabilizing agents such as organic solvents and detergents. The selected payload encapsulated in the lipid-based TCV is as discussed in this document.

[0027] The present systems, devices and methods, etc., also provide transfection methods, the methods comprising Petition 870260070835, dated 07 / 16 / 2026, pp. 214 / 276 12 / 45 transfect a target cell with a selected payload encapsulated in a lipid-based transfection-competent vesicle (TCV), as discussed in this document. The target cell can be a mammalian cell, such as a primary mammalian cell, a primary mammalian neuronal cell, a cultured mammalian cell, or a cell from a mammalian patient.

[0028] The methods in this document can be performed in a laboratory, for example, for benchtop loading. The methods can be performed in a factory to produce commercial quantities of transfected cells. The methods can be performed as part of an in vivo procedure, a medical procedure, a therapeutic procedure, or a gene therapy procedure. The methods can be performed as part of the treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, or spinal muscular atrophy. The methods may further comprise delivering the selected lipid-based TCV-encapsulated payload to the patient's brain.

[0029] In some additional aspects, the present systems, devices and methods, etc., provide kits comprising the compositions in this document. The compositions may be in a container and the kits may comprise instructions for use of the compositions. The instructions may direct the use of the compositions in accordance with any of the methods in this document. The container may be configured to administer at least one dose of the compositions to a mammal, the kit further comprising at least one label containing instructions for administration.

[0030] In some respects, the present systems, devices and methods, etc., provide isolated and purified compositions in this document for use in the manufacture of a medicament to inhibit, prevent or treat a disease or condition in a patient, which Petition 870260070835, dated 07 / 16 / 2026, pages 215 / 276 13 / 45 could be a mammal.

[0031] These and other aspects, features and embodiments are presented in this application, including in the Detailed Description below and attached drawings. Unless expressly indicated otherwise, all embodiments, aspects, features, etc., may be mixed and matched, merged and permuted in any manner desired. In addition, various references are presented in this document, including in the Cross-Reference to Related Applications, which discuss certain systems, devices, methods and other information; all of them are incorporated herein by reference in full and by all their teachings and descriptions, regardless of where the references may appear in this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A-1D. Transfection-competent vesicles (TCVs) produced according to the methods described herein exhibit knockdown in immortalized cells and primary neurons. (A) Relative luciferase expression in HEK-Luc cells after transfection with solvent- and detergent-free TCVs composed of 50% ionizable cationic lipid. (B) Cell viability of the same transfection set shown in (A) as % control wells (cells only). (C) Relative hdh mRNA expression in primary cortical neurons after solvent- and detergent-free TCV-mediated transfection of a selected siRNA load into neurons. The siRNA targeting hdh and TCV was produced using the same panel of ionizable cationic lipids shown in 1(A).(D) Cell viability measured by MTT reduction for an equivalent set of transfections in primary cortical neurons using solvent- and detergent-free TCVs to deliver an off-target siRNA (luciferase). Petition 870260070835, dated 07 / 16 / 2026, pages 216 / 276 14 / 45 N = 4 per condition for HEK-Luc cells, N = 3 cavities per condition for primary neurons. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 through Bonforroni post-hoc analysis after a one-sided ANOVA with each condition compared to the control condition (cells only).

[0033] Figures 2A-2D. The potency of solvent- and detergent-free TCVs does not depend on the mixing method used to generate the empty TCVs. (A) Relative luciferase expression in HEK-Luc cells after transfection with TCVs composed of 50% ionizable lipid and formed by T-joint mixing (DODMA50%) or extrusion (DODMA-50%-X). (B) Cell viability of the same transfection set shown in (A) as % control wells (cells only). (C) Relative hdh mRNA expression in primary cortical neurons after solvent- and detergent-free TCV-mediated transfection of an hdh-targeting siRNA using the same TCV panel shown in 2(A). (D) Cell viability measured by MTT reduction for an equivalent set of transfections in primary cortical neurons using solvent- and detergent-free TCVs to deliver an off-target siRNA (luciferase).N = 4 per condition for HEK-Luc cells, N = 3 cavities per condition for primary neurons. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 through Bonforroni post-hoc analysis after a one-sided ANOVA with each condition compared to the control condition (cells only).

[0034] Figures 3A-3D. Empty TCVs free of organic solvent and detergent containing a reduced amount of ionizable lipid facilitate the distribution of potent siRNA with reduced toxicity. (A) Relative expression of luciferase in HEK-Luc cells after transfection with TCVs composed of 20% or 50% of the ionizable lipid 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA). (B) Cell viability of Petition 870260070835, dated 07 / 16 / 2026, pages 217 / 276 15 / 45 same set of transfections presented in (A) as % of control wells (cells only). (C) Relative mRNA expression of hdh in primary cortical neurons after organic solvent- and detergent-free TCV-mediated transfection of an hdh-targeting siRNA using the same TCV panel presented in 3(A). (D) Cell viability measured by MTT reduction for an equivalent set of transfections in primary cortical neurons using organic solvent- and detergent-free TCVs to deliver an off-target siRNA (luciferase). N = 4 per condition for HEKLuc cells, N = 3 wells per condition for primary neurons. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 by Bonforroni post-hoc analysis after a one-sided ANOVA with each condition compared to the control condition (cells only).

[0035] Figures 4A, 4B. The incorporation of DOPE into an empty TCV free of organic solvent and detergent does not alter the TCV morphology. Cryo-TEM analysis was performed on TCVs of (A) DODMA / DOPE / DSPC / Chol / PEG-lipid at 20 / 30 / 10 / 39 / 1 mol% (DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC = 1,2-diestearoyl-sn-glycero-3-phosphocholine; PEG = polyethylene glycol) and (B) DODMA / DSPC / Chol / PEG-lipid (50 / 10 / 39 / 1 mol%). Scale bar = 100 nm.

[0036] Figure 5. Empty TCVs free of organic solvent and detergent can be used to deliver functional ribonucleoproteins (RNPs). HEK293 cells were transfected with Cas9 RNPs and a single-stranded oligodeoxynucleotide (ssODN) repair template, both delivered via solvent- and detergent-free TCVs produced according to the methods described in this document. The Cas9 protein was in complex with a guide targeting the GRN gene. DNA was extracted from the cells 48 hours after transfection. PCR was used to specifically detect the wild-type (WT) GRN allele or a mutant GRN allele, which is Petition 870260070835, dated 07 / 16 / 2026, pages 218 / 276 16 / 45 is present only when Homology-Directed Repair (HDR) incorporates the distributed ssODN into a double-strand DNA break produced by Cas9. The mutant GRN allele can be detected after TCV-mediated distribution of the Cas9 RMP, but is not present in control cells (Ctrl).

[0037] Figure 6. Photomicrographs of Cas9 located within a primary neuron. RNP distributed via solvent- and detergent-free TCV, as discussed in this document, can be detected using a fluorescent antibody against the Cas9 protein, shown in red. The untreated control does not have such red fluorescent signals. Blue signal = nucleic acid (DAPI staining), green signal = phalloidin (F-actin staining).

[0038] Figure 7. Graph demonstrating transcriptional knockdown by distributed RNP via benchtop loading of RNP into empty TCVs free of organic solvent and detergent in HEK cells. HEK293 cells were transfected with Cas9 RNPs targeting luciferase. Formula TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) were benchtop loaded. Relative luciferase mRNA levels in HEK cells show significant knockdown compared to the control (ctrl in graph) when measured by qPCR. N = 3 per group. Data represent mean ± SEM. p = 0.0018 using Student's t-test.

[0039] Figure 8. Graph demonstrating protein knockdown by RNP distributed via benchtop loading of RNP into an empty TCV free of organic solvent and detergent in HEK cells. HEK293 cells were transfected with Cas9 RNPs targeting luciferase. TCVs of formula (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) were benchtop loaded. The relative levels of luciferase protein in HEK cells show significant knockdown compared to the control (ctrl no Petition 870260070835, dated 07 / 16 / 2026, pp. 219 / 276 17 / 45 graph) when measured by ONE-Glo. N = 3 per group. Data represent mean ± SEM. p = 0.0003 using Student's t-test.

[0040] Figure 9. Graph demonstrating transcriptional knockdown by distributed RNP via bench loading of RNP into an empty, solvent- and detergent-free TCV in primary cortical neurons. Primary cortical neurons were transfected with Cas9 RNPs targeting Human Huntingtin (HTT). Formula TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) were bench loaded. Relative HTT mRNA levels in primary neurons show significant knockdown compared to control (gLuc in the graph) when measured by qPCR. N = 3 per group. Data represent mean ± SEM. p = 0.0039 using Student's t-test.

[0041] Figure 10. Graph showing mRNA expression for mRNA distributed via solvent- and detergent-free TCV in HEK cells. Luciferase mRNA was distributed via solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) in HEK cells at different ratios. Low = 0.029 mg mRNA / μmol lipid, Medium = 0.058 mg mRNA / μmol lipid, 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 measured by the ONE-Glo + Tox kit (Promega), N = 3 per condition.

[0042] Figure 11. Graph showing mRNA expression for mRNA distributed via solvent- and detergent-free TCV in primary cortical neurons. Luciferase mRNA was distributed via solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) in primary cortical neurons at different dosages. All dosages showed significant expression compared to the control. mRNA expression measured by the ONE kit. Petition 870260070835, dated 07 / 16 / 2026, pages 220 / 276 18 / 45 Glo + Tox (Promega), N = 3 per condition.

[0043] Figure 12. Graph demonstrating cell viability for RNPs in solvent- and detergent-free TCVs in HEK cells. RNPs were delivered via solvent- and detergent-free TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) or using RNAiMax (Rmax in the figure; ThermoFisher Scientific) in HEK cells for cytotoxicity assessment. The solvent- and detergent-free TCV was significantly less toxic compared to RNAiMax, p = 0.0002. Toxicity was assessed using the ONE-Glo + Tox kit (Promega), N = 3 per group.

[0044] Figure 13. Photomicrographs demonstrating cell viability for benchtop loading of siRNA in empty, solvent- and detergent-free TCVs into primary neurons. siRNA was delivered via solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol / PEG-lipid (20 / 30 / 10 / 39 / 1)) or using the commercially available Mirus TKO system (Mirus Bio) into primary neurons for cytotoxicity assessment. The siRNA delivered via benchtop loading in an empty, solvent- and detergent-free TCV was significantly less toxic compared to the Mirus TKO system, as shown by the photomicrographs.

[0045] Figure 14. Graph demonstrating the knockdown of organic solvent- and detergent-free TCVs in HEK cells. siRNAs were distributed across levels of 50% DODMA (D-50%) produced by T-joining, 50% (D-50% Ex, produced by extrusion), and the commercially available Mirus TKO system (Mirus Bio). The 50% DODMA formulations showed approximately 50% knockdown, however, the Mirus TKO system performed worse. DETAILED DESCRIPTION Petition 870260070835, dated 07 / 16 / 2026, pages 221 / 276 19 / 45

[0046] The systems, compositions, devices and methods, etc. in this document provide lipid-based vesicles, typically referred to herein as transfection-competent vesicles (TCVs), configured to safely and efficiently deliver DNA and other selected nucleic acid payloads to target cells. Safety and efficiency are both achieved in part by eliminating harmful agents, such as organic solvents like ethanol and detergents like sodium dodecyl sulfate, from the TCV loading and storage processes (i.e., inserting a selected payload into the TCV) and / or TCV delivery processes. TCV delivery processes may comprise transfecting mammalian cells, such as primary cells, with the selected payload. The selected payloads may also comprise nucleic acids in complex with a protein, such as a ribonucleoprotein (RNP).

[0047] In some embodiments, the systems, compositions, devices and methods, etc. in this document provide empty lipid-based TCVs that are free of organic solvent and detergent. Loaded TCVs can be generated using gentle mixing, such as repeated manual reciprocation of the TCV generating fluid in a pipette, SHM, T-junction type mixing or extrusion methods or other TCV mixing methods, as desired.

[0048] In one aspect, lipid-based TCVs consist of a mixture of an ionizable cationic lipid, phospholipid, cholesterol and PEG-lipid, and the TCV-containing composition is free of organic solvent and / or detergent.

[0049] Solvent- and detergent-free TCVs, as discussed in this document, can be used for the treatment of appropriate diseases and conditions, for example, through gene therapy. Solvent- and detergent-free TCVs, Petition 870260070835, dated 07 / 16 / 2026, pp. 222 / 276 20 / 45 as discussed in this document, improve the delivery of RNA, DNA, and RNP gene therapy products to human patients. Solvent- and detergent-free TCVs effectively deliver gene therapy products (including, but not limited to, mRNA, siRNA, and RNP) to brain cells or other target cells. The underlying cause of many human disorders is the loss of function of a necessary protein or the toxic gain of function of a mutant protein. These causes are treatable and even reversible using the solvent- and detergent-free TCVs of the present invention.

[0050] Some examples of such treatment include gene therapy in the central nervous system for the treatment of neurological disorders (some cases of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, spinal muscular atrophy, etc.).

[0051] Solvent- and detergent-free TCVs, as discussed in this document, can also cause gene silencing (knockdown) of mutant genes / gene products, for example, through targeted and safe delivery of siRNA or RNP, gene replacement therapy with mRNA, or correction of the causal mutation of native DNA through RNP-mediated gene editing. Two specific examples of human diseases that can be targeted in this way are Huntington's disease (HD) and frontotemporal dementia (FTD).

[0052] Huntington's disease is a progressive, incurable neurodegenerative disease with a dominant inheritance pattern. An expanded CAG nucleotide repeat sequence in the Huntingtin (HTT) gene is responsible for the disease. The Huntingtin (HTT) protein encoded by the mutant HTT gene contains an expanded polyglutamine repeat that confers a toxic gain-of-function to the gene product. Reduced brain levels of the mutant Huntingtin protein are Petition 870260070835, dated 07 / 16 / 2026, pp. 223 / 276 21 / 45 The main therapeutic strategy currently followed to slow or halt disease progression in HD can be achieved and improved using solvent- and detergent-free TCVs, as discussed in this document. TCVs loaded with siRNA targeting HTT expression or with selected RNP loads designed to decrease Huntingtin expression or toxicity will be effective therapies for HD. Frontotemporal dementia has many causes, but the loss of the progranulin protein (a potential brain survival factor) is a well-described cause. Solvent- and detergent-free TCVs, as discussed in this document, can deliver progranulin mRNA or RNPs designed to express progranulin or correct the underlying DNA mutation that causes progranulin loss (respectively) and increase brain progranulin levels and will be effective therapies for FTD.The increase in progranulin with TCVs expressing progranulin mRNA may also be a neuroprotective strategy for many common neurological diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. EXAMPLES OF MATERIALS AND METHODS Materials

[0053] 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA) was purchased from Cayman Chemical (Ann Arbor, MI). 1,2-Dioleyl-3-dimethylammoniumpropane (DODAP), 1,2-Dioleyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-diestearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was purchased from Sigma Aldrich (St. Louis, MO). PEG-DMG was synthesized as previously described (Akinc, Zumbuehl et al. 2008). All lipids were stored as stocks in ethanol. The siRNA targeting firefly luciferase (siLuc) (Basha, Ordobadi et al. 2016) was acquired from Petition 870260070835, dated 07 / 16 / 2026, pp. 224 / 276 22 / 45 from Integrated DNA Technologies (Coralville, IA). siRNA against murine hdh was purchased from Ambion (Silencer® Select Pre-design siRNA, Invitrogen, Carlsbad, CA). Preparation of Transfection-Competent Vesicles (TCVs)

[0054] Lipid components (ionizable cationic lipids, phospholipids, cholesterol, and PEG-lipid) were dissolved in ethanol in appropriate proportions to achieve a final total lipid concentration of 20-35 mM. An aqueous phase containing 25 mM sodium acetate buffer, pH 4, was prepared. The two solutions were combined using two established nanoparticle preparation techniques: rapid mixing and extrusion. Quick Mix:

[0055] The lipid-containing organic phase was mixed with the aqueous phase using a T-junction mixer manufactured to meet the specifications of the PEEK Low Pressure Tee Assembly (1 / 16, 0.02 inch through orifice, Part # P-712) at a final flow rate of 20 mL / min with an organic:aqueous ratio of 1:3 (v / v) (Jeffs, Palmer et al. 2005; Kulkarni, Tam et al. 2017; Kulkarni, Darjuan et al. 2018). The resulting suspension was dialyzed against a 1000-fold volume of 25 mM sodium acetate buffer, pH 4, to remove ethanol. Extrusion:

[0056] Lipids were dissolved in ethanol to a final concentration of 35 mM. Particles were generated by the rapid addition of 25 mM sodium acetate, pH 4, to the ethanol solution to achieve a final ethanol concentration of 30% (v / v) as described elsewhere (Maurer, Wong et al. 2001). The resulting nanoparticle suspension was extruded three times through 2x 80 nm polycarbonate membranes at room temperature. After extrusion, the particle buffer was changed to remove the ethanol. Analysis of Transfection-Competent Vesicles (TCVs) Petition 870260070835, dated 07 / 16 / 2026, pages 225 / 276 23 / 45

[0057] Lipid concentrations were determined by assay for cholesterol content using a Cholesterol T assay kit (Wako Chemicals, Mountain View, CA) and extrapolating the total lipid concentration as described elsewhere (Chen, Tam et al. 2014). Nucleic acid retention was determined using the RiboGreen assay as previously described (Chen, Tam et al. 2014; Leung, Tam et al. 2015). Cryogenic Transmission Electron Microscopy

[0058] Cryo-TEM was performed as previously described (Kulkarni, Darjuan et al. 2018). Briefly, TCVs were concentrated to a total lipid concentration of approximately 20 mg / mL using an Amicon centrifugal concentration unit (10 kDa NWCO). A small volume (3-5 μL) of material was applied to a light-discharge copper grid and immersion-frozen using a FEI Mark IV Vitrobot (Hillsboro, OR). The grids were stored under liquid nitrogen until imaging. All imaging was performed using a FEI Tecnai G2 instrument operating at 200 kV in low-dose mode. Images were captured using a FEI Eagle 4k CCD bottom-mount detector. All sample preparation and imaging were performed at the UBC BioImaging Facility (Vancouver, BC). Cell Culture and Reagents:

[0059] All basic cell culture media and neuronal supplement B27 were purchased from Gibco (Thermo Fisher, Waltham, MA). Hank's Balanced Saline (HBSS), penicillin-streptomycin, L-glutamine, and trypsin solutions were obtained from Hyclone (Logan, UT). HEK293 cells were seeded onto clear-bottomed, white-walled Corning plates (Corning, NY). Primary cortical cells were seeded onto tissue culture-treated plates (Fisher), coated with poly-D-lysine (Sigma, St. Louis, Petition 870260070835, dated 07 / 16 / 2026, pp. 226 / 276 24 / 45 (MO). Hygromycin B was obtained from Invitrogen (Carlsbad, CA). Recombinant ApoE4 was acquired from Peprotech (Rocky Hill, NJ).

[0060] To measure cell viability and luciferase levels in HEK293 cells, the ONE-Glo + Tox kit from Promega (Madison, WI) was used. Cell viability in primary neurons was measured using the Sigma MTT In Vitro Toxicology kit (St. Louis, MO). HEK293 Luciferase Reporter Cells:

[0061] The generation of the HEK293 cell line with a stably integrated luciferase reporter construct (HEK-Luc cells) has been previously described (De Souza, Islam et al. 2016). Cells were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2 in DMEM with high glucose concentration, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 125 μg / mL hygromycin B. Cells were placed in plates at a density of 12,000–20,000 cells / well in a 96-well white-walled plate. Primary Cell Culture:

[0062] Cortical cultures were prepared from C57BL / 6J and FVB.YAC128 mice on embryonic day E17.5. Briefly, the cortices were dissected in ice-cold HBSS and the tissue was digested using a 0.05% trypsin solution (Hyclone) for 10 minutes at 37 °C. The cortices were then ground using a 5 mL pipette 5 times and a further 5-7 times with the tip of a 200 μL pipette added. The cells were pelleted by centrifugation for 5 minutes at 800 rpm, washed with HBSS and then resuspended in warm neurobasal medium supplemented with B27, 2 mM L-glutamine (Hyclone) and 1% penicillin / streptomycin (Hyclone). Cortical neuron cultures were seeded in 24-well plates coated with poly-D-lysine at a density of 1.5 x 10⁵ cells / well. The cells were maintained at 37 °C in an atmosphere Petition 870260070835, dated 07 / 16 / 2026, pp. 227 / 276 25 / 45 humidified 95% air, 5% CO2. Transfection:

[0063] All reagents were mixed on the bench. Empty TCVs containing 50% cationic lipid were mixed with siRNA at a ratio of 0.058 mg siRNA per μmole of lipid. TCVs containing 20% ​​cationic lipid were mixed at 0.022 mg siRNA per μmole of lipid. The TCV suspension was rapidly mixed with siRNA using a pipette and incubated at room temperature for 10 minutes.

[0064] HEK293 cells were seeded 24 hours before transfection. Complete DMEM medium was added to the TCV:siRNA mixture to a final siRNA concentration of 3.3 μg / mL, and a complete medium exchange was performed at the time of transfection.

[0065] Primary neuronal cells were cultured in vitro for 7 days prior to transfection. Complete neurobasal medium with 2-6 μg / mL recombinant ApoE4 was added to the TCV:siRNA suspension and half of the medium was exchanged in each well. Luciferase assay:

[0066] Forty-eight to seventy-two hours post-transfection, HEK293 cells were tested for cell viability and luminescence using the ONE-Glo + Tox kit (Promega) according to the manufacturer's instructions. Briefly, live cell reagent was added to each well and the cells were incubated for 30 minutes at 37 °C. The plate was tested on a plate reader (POLARstar Omega plate reader, BMG LABTECH) at an excitation wavelength of 400 nm and read at an emission wavelength of 510 nm. The ONE-Glo reagent was then added and the plate incubated at room temperature for 3 minutes. Luminescence was measured by light emission through the lenses of the same plate reader. Values ​​are presented as % of control and represent N = 4 wells per Petition 870260070835, dated 07 / 16 / 2026, pages 228 / 276 26 / 45 condition. MTT assay:

[0067] Primary cortical neurons were tested for cell viability using the MTT assay 72 hours after transfection in a 24-well plate. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) was reconstituted in HBSS to a final concentration of 5 mg / mL and added to each well at 10% v / v. Cells were incubated at 37 °C for 4 hours. The medium was removed and 250 μL of solubilizing solution was added to each well. Absorbance was measured at 570 nm. Values ​​are presented as % of control and represent N = 3 wells per condition. Quantitative RT-PCR

[0068] Adherent primary cortical cells were washed once in sterile PBS before being scraped from the plate into 600 μL of lysis buffer containing 1% 2-mercaptoethanol and immediately frozen at -80 °C. Total RNA was subsequently extracted using the PureLink RNA mini kit (Invitrogen) according to the manufacturer's instructions. Reverse transcription of all samples was performed using the Superscript VILO kit (Invitrogen) according to the manufacturer's instructions, using 250 μg of total RNA as input for cDNA synthesis and 5 μg of diluted RNA for the quantitative PCR reaction. Quantification of hdh mRNA levels was performed using the standard curve method, with amplification of target mRNA and control genes in separate wells, performed on FastSybr (Applied Biosystems) and conducted on Step-One ABI System (Applied Biosystems). Each sample was passed in duplicate.The relative amount of mRNA in each well was calculated as the ratio between hdh mRNA and a control gene, Csnk2a2. Values ​​are shown as % of control and represent N = 3 wells per condition. Petition 870260070835, dated 07 / 16 / 2026, pp. 229 / 276 27 / 45 Ribonucleoprotein (RNP) Materials and Complex Formation

[0069] All materials for RNP formulation, including guide RNAs (gRNAs), tracrRNA, single-stranded oligodeoxynucleotides (ssODN), and recombinant Cas9 protein, were obtained from IDT (San Jose, CA). The gRNA sequence targeting luciferase was provided by IDT (San Jose, CA). The gRNA sequence targeting human progranulin (GRN) binds to exon 5 of the gene. The ssODN sequence used for homology-controlled repair (HDR) was designed to introduce a 4 bp deletion in exon 5 of GRN.

[0070] RNP assembly was performed according to the manufacturer's specifications. Briefly, the guide RNA (gRNA) complex was formed by incubating equiem mole ratios of crRNA:tracrRNA, such as 1 μM tracrRNA with 1 μM gRNA, at 95 °C for 5 minutes. The mixture was then allowed to cool to room temperature for 20–30 minutes. RNPs were subsequently formed by combining gRNA pairs with Cas9 protein in equiem mole ratios and allowing the mixture to stand at room temperature for 5 minutes before use. Transfection of Mammalian Cells with Nucleic Acid:

[0071] Empty TCVs free of organic solvent and detergent, as discussed in this document, as well as commercially available reagents for comparison, were mixed with the selected load on the bench. The TCVs were mixed with the selected nucleic acid load in a range of ratios from 0.01-0.2 mg of nucleic acid per μmole of lipid. The TCV suspension was rapidly mixed with siRNA using a pipette and incubated at room temperature for 10 minutes.

[0072] HEK293 cells were seeded 24 hours before transfection. Complete DMEM medium was added to the TCV:acid mixture. Petition 870260070835, dated 07 / 16 / 2026, pp. 230 / 276 28 / 45 nucleic acid to a final concentration of 0.33–3.3 μg / mL siRNA or 0.1–1 μg / mL mRNA for a complete medium exchange that was performed at the time of transfection. Primary neuronal cells were cultured in vitro for 7 days prior to transfection. Complete neurobasal medium with 2–6 μg / mL recombinant ApoE4 was added to the TCV:nucleic acid suspension, and half of the medium was exchanged in each well.

[0073] The cells were treated with Mirus TransIT-TKO according to the manufacturer's instructions. Briefly, Mirus TransIT-TKO was added to serum-free medium at a concentration of 5 μL Mirus / 100 μL serum-free medium. The siRNA was then added to the tube, gently pipetted to mix, and incubated at room temperature for 15–30 minutes. The solution was then transferred to the cells, and the final concentration of Mirus was 5 μL / 1 mL of complete medium. The final siRNA concentration was 25 nM. Transfection of Mammalian Cells with RNPs:

[0074] TCV at 0.5-20 mM and RNP at 0.5-20 μM were combined in a mole ratio of 467-5000 and incubated at room temperature for 10 minutes. Separately, ssODN solution at 1-10 μM was combined with TCV and this mixture was incubated at room temperature for 5-15 minutes. In some cases, an equimol amount of ssODN was added to the RNP complex solution before the addition of TCVs.

[0075] TCVs containing mixtures of RNPs and ssODN were combined and complete medium was added at a final concentration of 10200 nM of RNPs and ssODN each. A complete medium exchange was performed in HEK cells, which were seeded 24 hours prior. Primary neuronal cells were cultured in vitro for 5-7 days before transfection. Complete neurobasal medium with 2-6 μg / mL of recombinant ApoE4 was added to the TCV:RNP mixture and half of Petition 870260070835, dated 07 / 16 / 2026, pp. 231 / 276 29 / 45 was replaced in each cavity.

[0076] The cells were treated with Lipofectamine RNAiMAX reagent according to the manufacturer's instructions. Briefly, RNP complexes were prepared and added to a mixture of serum-free medium and RNAiMAX, incubated at room temperature for 5 minutes, and added to the cells in the plates. PCR for Homology-Controlled Repair Detection

[0077] Polymerase chain reaction (PCR) was used to amplify exon 5 of GRN from genomic DNA extracted from HEK293 cultures transfected using direct primers specific for wild-type (WT) or mutant GRN alleles and a common reverse primer. PCR was performed using MyTaq (Bioline, USA) according to the manufacturer's instructions. PCR products were separated by electrophoresis on a 1.5% agarose gel stained with SybrSafe and photographed under ultraviolet light. Immunocytochemistry

[0078] Cells were fixed for 15 minutes using a 3-4% paraformaldehyde solution. Cells were permeabilized for 15 minutes in PBS containing 0.1% Triton-X (PBST). Cells were incubated overnight at 4°C with PBST containing a 1:1000 mixture of anti-Cas9 antibody (Invitrogen). Cells were washed three times with PBS and incubated with a 1:1000 mixture of each of Alexa Fluor 594 fluorescent secondary antibodies (Invitrogen) and Phalloidin-iFluor 488 CytoPainter antibody (Abcam) for 1 hour at room temperature, washed again, and incubated for 5 minutes with a solution containing DAPI to visualize the nuclei. Statistic

[0079] All statistical comparisons were performed as Petition 870260070835, dated 07 / 16 / 2026, pp. 232 / 276 30 / 45 A one-sided analysis of variance (ANOVA) with Bonferroni post-hoc analysis was used to compare individual means for treated cells with controls and to correct for multiple comparisons (Prism 6, Graphpad Software Inc.). A Student's t-test was used to compare individual means in the case of only two groups. A p-value of less than 0.05 was considered significant. EXAMPLES RELATED TO RESULTS Example 1: Empty transfection-competent vesicles (TCVs) efficiently retain nucleic acid without organic solvents

[0080] Empty TCV formulations produced by T-bonding or SHM-type mixing exhibited retention efficiencies of the order of 85% or more. We first tested the ability of TCVs composed of ionizable cationic lipids spanning the range of in vivo gene silencing potencies (DODMA >> DLinDAP > DODAP) (DLinDAP is 1,2-dilinoyl-3-dimethylammonium-propane) to retain nucleic acid without the aid of organic solvents or detergents. Specifically, in the absence of either of the two, formulations composed of ionizable lipid / DSPC / Chol / PEG-lipid (50 / 10 / 39 / 1 mol %) achieve almost complete siRNA retention (> 85 %) when mixed at pH 4 in a ratio of 0.058 mg siRNA / μmol lipid, followed by neutralization with PBS (Table 1).The assay to determine retention is based on the exclusion of a nucleic acid RNA-binding dye by the lipid components. Thus, retention is considered the capture of RNA from the external medium in more than one transient form (i.e., stable retention). Despite the lack of organic solvents or detergents in the production processes, the TCV formulations obtained surprisingly exhibited retention efficiencies similar to those reported elsewhere for LNP-siRNA generated by rapid mixing techniques using organic solvents (Belliveau, Huft et al. 2012; Chen, Tam et al. 2014; Leung, Tam et al. 2015). Petition 870260070835, dated 07 / 16 / 2026, pp. 233 / 276 31 / 45 Chen, Tam et al. 2016). Table 1: Nucleic acid retention efficiencies for different TCV formulations Lipid Formulation Name | Formulation Process | Retention Efficiency, % (average ± SEM) | DODMA-50% | DODMA / DSPC / Chol / PEG (50 / 10 / 39 / 1) | T-link type mixture | 87.90 ± 3.94 | DLinDAP-50% | DLinDAP / DSPC / Chol / PEG (50 / 10 / 39 / 1) | T-link type mixture | 74.44 ± 7.18 | DODAP-50% | DODAP / DSPC / Chol / PEG (50 / 10 / 39 / 1) | T-link type mixture | 71.84 ± 5.76 Example 2: siRNA in empty TCVs free of organic solvent and detergent exhibits robust knockdown in immortalized cells and primary neurons.

[0081] The ability to retain nucleic acid and subsequently distribute it in a non-toxic manner presents two distinct hurdles. Having determined that the lipid-based TCVs free of organic solvents / detergents discussed above efficiently captured nucleic acids, their gene silencing ability and their effect on cell viability were tested in two scenarios, as shown in Figures 1A-1D. First, empty TCVs free of organic solvents and detergents were combined using benchtop loading with siLuc and used to treat HEK-Luc cells. Surprisingly, DODMA-based TCVs showed approximately 50% knockdown and performed better than DLinDAP and DODAP, which showed no indication of knockdown (Figure 1A). Neither formulation exhibited toxicity as measured by cell viability compared to untreated cells (Figure 1B).Secondly, the efficacy and toxicity of TCVs delivering siRNA were tested against the hdh gene in primary mouse cortical neurons. Again (Figure 1C), DODMAPetition 870260070835, 16 / 07 / 2026, p. 234 / 276. 32 / 45 TCVs exhibited a gene silencing (knockdown) efficacy of approximately 50%, while DLinDAP-TCVs exhibited less silencing (knockdown), and DODAP-TCVs showed no difference compared to untreated cells. It should be noted that primary neurons are quite susceptible to the toxic effects of harmful transfection reagents, such as those currently commercially available. Figure 13 highlights the differences in the effect on viability. Example 3: Multiple mixing processes can produce empty TCVs free of organic solvents and potent detergents.

[0082] In an effort to determine the role of the mixing aspect of the manufacturing process and the resulting particle size in achieving the potency shown in Figure 1, solvent- and detergent-free lipid-based empty TCVs containing ionizable lipids, such as DODMA, were produced via T-join mixing and extrusion. As shown in Figure 2A, after benchtop siRNA loading into empty TCVs, there was significant luciferase knockdown in HEK-Luc cells using both processes. There was no significant difference in cell viability between cells treated with particles produced via T-join mixing or extrusion (Figure 2B).Next, primary cortical neurons were treated with the same solvent- and detergent-free lipid-based TCV formulations, and it was determined that both processes result in particles with similar potency (Figure 2C) and similar cell viability (Figure 2D). In contrast, the toxicity of the present transfection methods (Figure 13) in primary cultures reduces the objective of a non-toxic but potent formulation. Example 4: Solvent- and detergent-free empty TCVs containing a reduced amount of ionizable lipid facilitate potent siRNA delivery with reduced toxicity. Petition 870260070835, dated 07 / 16 / 2026, pages 235 / 276 33 / 45

[0083] An established lipid composition currently used in clinical formulations (see Patisiran) includes a significant amount of ionizable cationic lipid (50 mol%) (Jayaraman, Ansell et al. 2012; Suhr, Coelho et al. 2015). While such high amounts allow for improvements in the effective dose to achieve 50% gene silencing (ED50) in vivo (Jayaraman, Ansell et al. 2012), the persistence of lipid metabolites after administration (Maier, Jayaraman et al. 2013; Sabnis, Kumarasinghe et al. 2018) and the toxicity associated with these molecules makes the formulation toxic in high-dose and repeated administration regimens (or for sensitive cell types). Present compositions, TCVs, etc., may be composed of materials that are biodegradable or facilitate disposal. In this document, the present compositions, TCVs, etc., reduce the amount of toxic ingredients while retaining transfection potency.

[0084] The transfection competence of a DODMA / DOPE / DSPC / Chol / PEG-lipid formulation (20 / 30 / 10 / 39 / 1 mol%, respectively) to silence luciferase in HEK-Luc cells was tested. A 40% knockdown of luciferase expression was observed (Figure 3A) without significant toxicity (Figure 3B). Next, we tested the ability of TCVs with 20 mol% ionizable lipid to deliver siRNA to primary neurons compared to DODMA-TCVs at 50 mol%. All formulations exhibited a ~60% knockdown of hdh (Figure 3C), while a DODMA formulation produced via T-junction mixing showed significantly enhanced cell viability (Figure 3C). Example 5: The incorporation of DOPE does not alter the morphology of empty TCVs free of organic solvent and detergent.

[0085] Improving the potential for Hii phase formation in a nucleic acid delivery vehicle may be an important factor. Petition 870260070835, dated 07 / 16 / 2026, pages 236 / 276 34 / 45 to facilitate membrane fusion in the endosome (Hafez, Maurer et al. 2001). In the present approach, two exemplary lipids capable of adopting Hii phases are DOPE (individually) and DODMA (when protonated and combined with anionic lipids). To determine if the incorporation of DOPE into solvent- and detergent-free TCVs resulted in the premature formation of the HII phase, cryo-TEM was performed on 20 mol% DODMA-TCVs and the equivalent formulation composed of 50 mol% DODMA. The resulting structures (Figures 4A, 4B) are visualized as two-layer structures with no indication of the HII phase.

[0086] Previous work by others suggested the presence of internal structures similar to Hii within the main LNP formulations, regardless of siRNA content (Leung, Hafez et al. 2012; Leung, Tam et al. 2015). Since then, it has been shown that siRNA LNPs do not contain such structures, but rather have siRNA immobilized between closely spaced layers of lipids (Kulkarni, Darjuan et al. 2018), giving the overall particle a multilamellar or onion-like morphology. In the absence of siRNA, LNP formulations adopt an electron-dense core containing an oil-phase lipid. Thus, the examples in this paper demonstrate that the TCV morphology is drastically different from LNP systems, but still has highly efficient transfection potential. Example 6: Empty TCVs free of organic solvent and detergent can be used to deliver functional ribonucleoproteins (RNPs)

[0087] As shown in Figure 5, empty TCVs free of organic solvent and detergent in this document were also tested for their ability to deliver a selected protein payload in nucleic acid complex in a mammalian cell. Briefly, a ribonucleoprotein complex consisting of recombinant Cas9 protein and a guide RNA targeting exon 5 of the gene of Petition 870260070835, dated 07 / 16 / 2026, pages 237 / 276 35 / 45 progranulin was assembled and combined with empty TCVs via benchtop loading (DODMA / DOPE / DSPC / Chol / (20 / 30 / 10 / 40 mol%, respectively)) at a mol ratio of 467:1 (TCV:RNP complex). Separately, a single-stranded oligodeoxynucleotide designed to introduce a 4 bp deletion in exon 5 of the progranulin gene was combined with TCVs at a mol ratio of 4275:1 (TCV:nucleic acid). The two TCV preparations containing their respective selected loads were combined to achieve a final concentration of 10 nM RNPs and 10 nM ssODN and then added to HEK cells. After 48 hours, PCR was used to determine if homology-controlled repair occurred in the progranulin target gene using direct primers specific for the wild-type allele. or mutant.As shown in Figure 5, cells exposed to the combination of TCVs containing each of the ribonucleoprotein and ssODN complexes resulted in the insertion of a 4 bp deletion via homology-controlled repair (second band of Figure 5, mutant labeled), while untreated control cells did not result in any genetic alteration at the exon 5 site of the progranulin gene (third and fourth bands of Figure 5). Example 7: Other examples of empty TCVs free of organic solvent and detergent used to deliver functional ribonucleoproteins (RNPs).

[0088] Empty TCVs free of organic solvent and detergent were used to distribute functional ribonucleoproteins (RNPs) using the methods as discussed above.

[0089] Figure 6 provides a photomicrograph of Cas9 located within a primary neuron. RNP distributed via TCV free of organic solvent and detergent, as discussed in this document, can be seen within a primary neuron as fluorescent signals; the untreated control does not exhibit such fluorescent signals. Petition 870260070835, dated 07 / 16 / 2026, pages 238 / 276 36 / 45 More specifically, immunocytochemistry of primary neuronal cells showed the localization of the Cas9 protein (red) within the nucleus (blue) of mouse-derived cortical neurons. The cells were also stained for phalloidin (green) to show cell morphology.

[0090] Figure 7 provides a graph demonstrating gene knockdown by RNP distributed via TCV without organic solvent and detergent in HEK cells. RNP distributed via TCV without organic solvent and detergent in HEK cells shows significant silencing (knockdown) of the luciferase transcript compared to the control (ctrl on graph). Figure 8 provides a graph demonstrating protein knockdown by RNP distributed via TCV without organic solvent and detergent in HEK cells. RNP distributed via TCV without organic solvent and detergent in HEK cells shows significant silencing (knockdown) of the luciferase protein compared to the control (ctrl on graph), p = 0.0003.

[0091] Figure 9 provides a graph demonstrating gene knockdown by RNP distributed via organic solvent- and detergent-free TCV in primary neurons. RNP distributed via organic solvent- and detergent-free TCV in primary cortical neurons shows significant mRNA knockdown by means of a quantitative real-time PCR assay (qRTPCR), p = 0.0039.

[0092] Returning to some further discussion of these Figures, Figures 7 and 8 show that the RNP distributed via bench loading of the empty TCV in this document shows robust knockdown of a reporter gene, luciferase, in HEK cells. This was demonstrated by measuring the output for luciferase (functional protein) as well as luciferase mRNA levels via qRT-PCR. Petition 870260070835, dated 07 / 16 / 2026, pp. 239 / 276 37 / 45 This same RNP distribution approach was used to disrupt Huntingtin gene expression in cortical neurons derived from FVB.YAC128 mice expressing the full-length human Huntingtin gene carrying a pathological mutation (Figure 9). Huntingtin mRNA levels were quantified from primary cortical neurons using qRT-PCR after 72 hours of incubation with the TCV:RNP mixture. Example 8: Examples of mRNA distributed via TCV free of organic solvent and detergent.

[0093] Benchtop loading of empty TCVs free of organic solvent and detergent used to distribute mRNA using the methods as discussed above.

[0094] Figure 10 provides a graph demonstrating the expression of mRNA distributed via solvent- and detergent-free TCV in HEK cells. mRNA was distributed via solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) in HEK cells at different ratios: Low = 0.029 mg mRNA / μmol lipid, Medium = 0.058 mg mRNA / μmol lipid, High = 0.116 mg mRNA / μmol lipid. All ratios showed significant expression compared to the control, with the lowest ratio showing the highest expression.

[0095] Figure 11 provides a graph demonstrating the expression of mRNA distributed via benchtop loading of organic solvent- and detergent-free empty TCV into primary cortical neurons. mRNA was distributed via organic solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) into primary cortical neurons at different dosages. All dosages demonstrated significant expression compared to the control.

[0096] Returning to some further discussion of these Figures, Figure 10 demonstrates that bench loading of empty TCVs Petition 870260070835, dated 07 / 16 / 2026, pages 240 / 276 Solvent- and detergent-free 38 / 45 saturates, as discussed in this document, can be used to deliver mRNA encoding a functional protein into cells. Briefly, solvent- and detergent-free TCVs were gently mixed using a pipette with firefly luciferase-encoding mRNA in a range of ratios from 0.029–0.116 mg nucleic acid:1 μmole lipid. This mixture was incubated at room temperature for 520 minutes before the addition of complete culture medium and then transferred to wells containing HEK cells (see Figure 10) or primary cortical neurons (Figure 11). Luciferase production was measured using the Promega ONE-Glo luciferase assay kit, according to the manufacturer's instructions. The results demonstrate the production of the luciferase protein within these cell types at a variety of selected loadings: concentrations and doses of TCV. Example 9: Comparison of solvent- and detergent-free TCVs with products available on the market.

[0097] Benchtop loading of empty organic solvent- and detergent-free TCVs was used to deliver siRNA or RNP to HEK cells or primary neurons using the methods as discussed above and then contrasted with equivalent transfections using commercially available systems. The organic solvent- and detergent-free TCVs in this paper outperformed the commercially available systems.

[0098] Figure 12 provides a graph demonstrating cell viability for solvent- and detergent-free TCVs in HEK cells. RNPs were delivered via solvent- and detergent-free TCVs (DODMA / DOPE / DSPC / Chol (20 / 30 / 10 / 40)) or using RNAiMax (Rmax in the figure; ThermoFisher Scientific) in HEK cells for cytotoxicity assessment. The solvent- and detergent-free TCV was significantly less toxic compared to the Petition 870260070835, dated 07 / 16 / 2026, pp. 241 / 276 39 / 45 RNAiMax, p = 0.0002.

[0099] Figure 13 provides photomicrographs demonstrating benchtop cellular viability for siRNA loading in empty, solvent- and detergent-free TCVs into primary neurons. siRNA was delivered via solvent- and detergent-free TCV (DODMA / DOPE / DSPC / Chol / PEG-lipid (20 / 30 / 10 / 39 / 1)) or using the commercially available Mirus TKO system (Mirus Bio) into primary neurons for cytotoxicity assessment. The solvent- and detergent-free TCV was significantly less toxic compared to the Mirus TKO system, as shown by the photomicrographs.

[00100] Figure 14 provides a graph demonstrating the knockdown of organic solvent- and detergent-free TCVs in HEK cells. siRNA was delivered via organic solvent- and detergent-free TCVs at DODMA levels of 50% (D-50%, via T-bonding type mixing), 50% (D-50% Ex, produced via extrusion), and via the commercially available Mirus TKO system (Mirus Bio). The present 50% DODMA formulations showed approximately 50% knockdown, while the Mirus TKO system performed significantly worse.

[00101] Returning to some further discussion of these Figures, Figure 12 demonstrates that benchtop loading of siRNA into empty TCVs free of organic solvent and detergent, as discussed in this document, has properties of low toxicity and efficient expression elimination in primary neuronal cells derived from mice and the HEK cell line. For the treatment of primary cortical neurons, TCVs were mixed with siRNA in a ratio of approximately 0.022–0.058 mg nucleic acid: 1 μmole lipid and transferred to cavities containing neuronal cells. Petition 870260070835, dated 07 / 16 / 2026, pp. 242 / 276 40 / 45 primary. The light micrographs in Figure 13, all taken at the same magnification, show that in the control and TCV-treated cavities, the cells remain healthy with intact processes and a very low number of dead cells. In the cavity treated with Mirus TransIT-TKO, the cells appear considerably less healthy, with disrupted cellular processes and an increased number of small condensed (dead) cells.

[00102] For Figure 12, HEK cells were treated with Lipofectamine RNAiMAX and TCVs containing the selected RNP load. HEK cells were treated with a final RNP concentration of 5-50 nM and TCV or RNAiMAX reagent. Cell viability was assessed using the Promega ONE-Glo + Tox kit and compared to untreated control cells. HEK cultures treated with RNAiMAX show a significant decrease in overall health compared to control and TCV-treated cavities.

[00103] Figure 14 demonstrates the effectiveness of bench-loaded empty TCVs free of organic solvent and detergent, as discussed in this document, in the distribution of the selected siRNA load compared to the commercially available product, Mirus LT-TKO. TCVs were incubated with siRNA targeting the luciferase gene for 5-10 minutes at room temperature at a ratio of approximately 0.022-0.058 mg nucleic acid:1 μmole lipid. HEK cells were treated with Mirus LT-TKO according to the manufacturer's instructions. At the time of treatment, the medium in the wells was completely replaced with fresh growth medium containing siRNA:TCV or siRNA:Mirus LT-TKO mixtures. After 72 hours of treatment, HEK cells were tested for cell viability and luciferase production using the Promega ONE-Glo + Tox kit, and all cells were compared to untreated control wells. Petição 870260070835, de 16 / 07 / 2026, pág. 243 / 276 41 / 45 REFERÊNCIAS Akinc, A., A. Zumbuehl, et al. (2008). A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 26(5): 561569. 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 Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA. Mol Ther Nucleic Acids 1: e37. Chen, S., Y. Y. Tam, et al. (2014). Development of lipid nanoparticle formulations 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 potency 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 manufactured by microfluidics and bulk self-assembly exhibit different transfection behavior. Biochem Biophys Res Commun 503(2): 508-512. Hafez, I. M., N. Maurer, et al. (2001). On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther 8(15): 1188-1196. Jayaraman, M., S. M. Ansell, et al. (2012). Maximizing the potency of siRNA lipid 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 for 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 Petição 870260070835, de 16 / 07 / 2026, pág. 244 / 276 42 / 45 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 Nanoparticles 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 for Encapsulating Macromolecules in Lipid Nanoparticle Systems. J Phys Chem B 119(28): 8698-8706. Lin, P. J., Y. Y. Tam, et al. (2013). Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations of siRNA. Nanomedicine 9(2): 233-246. Maier, M. A., M. Jayaraman, et al. (2013). Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol Ther 21(8): 1570-1578. Maurer, N., K. F. Wong, et al. (2001). Spontaneous entrapment of polynucleotides 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. T uyishime, et al. (2015). Expression kinetics of nucleosidemodified 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 cholesterol-containing lipoplexes. Biochim Biophys Acta 9(43): 22. Rungta, R. L., H. B. Choi, et al. (2013). Lipid Nanoparticle Delivery of Petição 870260070835, de 16 / 07 / 2026, pág. 245 / 276 43 / 45 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 mRNA 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 small 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 antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel 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 familial 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 systemic gene therapy. Gene Therapy 7: 1867. Wang, Y., L. Miao, et al. (2015). Delivery of oligonucleotides with lipid nanoparticles. Adv Drug Deliv Rev 87: 68-80. Wheeler, J. J., L. Palmer, et al. (1999). Stabilized plasmid-lipid particles: construction and characterization. Gene Ther 6(2): 271-281. Zhigaltsev, I. V., N. Belliveau, et al. (2012). Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing. Langmuir 28(7): 36333640. Zhigaltsev, I. V., Y. K. Tam, et al. (2016). Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery Petição 870260070835, de 16 / 07 / 2026, pág. 246 / 276 44 / 45 agents. J Liposome Res 26(2): 96-102.

[00104] All terms used in this document are used according to their common meanings, unless the context or definition clearly indicates otherwise. Furthermore, unless expressly stated otherwise in the descriptive report, the use of or includes and vice versa. Non-limiting terms should not be interpreted as limiting unless expressly stated or the context clearly indicates otherwise (for example, including, having and comprising normally indicate including, without limitation). Singular forms, including in claims, such as a, an and the include the plural reference, unless expressly stated or the context clearly indicates otherwise.

[00105] Unless otherwise indicated, adjectives in this document such as substantially and about that modify a condition or characteristic of a relationship of an aspect or aspects of a modality indicate that the condition or characteristic is defined within the tolerances that are acceptable for the operation of the modality for an application for which it is intended.

[00106] The scope of the present devices, systems and methods, etc., includes the concepts of means plus function and step plus function. However, the claims should not be interpreted as indicating a means plus function relationship unless the word means is specifically cited in a claim and should be interpreted as indicating a means plus function relationship where the word means is specifically cited in a claim. Similarly, the claims should not be interpreted as indicating a step plus function relationship unless the word step is specifically cited in a claim and should be interpreted as indicating a step plus function relationship where the word step is specifically cited in a claim. Petition 870260070835, dated 07 / 16 / 2026, pages 247 / 276 45 / 45

[00107] From the foregoing, it will be appreciated that, although specific embodiments have been discussed in this document for illustrative purposes, various modifications may be made without departing from the spirit and scope of the discussion in this document. Consequently, the systems and methods, etc., include such modifications, as well as all permutations and combinations of the subject matter presented in this document and are not limited, except as to the appended claims or other claims that find adequate support in the discussion and figures in this document. Petition 870260070835, dated 07 / 16 / 2026, pp. 248 / 276

Claims

1 / 7 CLAIMS 1. A method for preparing a water-based solution comprising an empty lipid-based transfection vesicle (TCV) free of a therapeutic and / or diagnostic load, the solution of which is free of organic solvents and detergents, the method characterized in that it comprises: (a) dissolving lipid components of the lipid-based TCV in an organic solvent to provide a lipid solution; (b) mixing the lipid solution with an aqueous solution to provide a suspension; and (c) removing the organic solvent from the suspension, wherein: (i) the lipid solution of (a) and the acidic aqueous solution of (b) do not comprise a therapeutic and / or diagnostic load; (ii) the lipid solution of (a) and the acidic aqueous solution of (b) do not comprise a detergent; (iii) the lipid components comprise (1) a cationic lipid consisting of one or more ionizable cationic lipids, (2) one or more phospholipids and (3) cholesterol (Chol);(iv) the lipid components comprise the cationic lipid in about 20 molar percent to about 50 molar percent; and (v) the lipid components do not comprise PEG-lipid.

2. Method according to claim 1, characterized in that the lipid components consist of: (1) one or more ionizable cationic lipids; (2) one or more phospholipids; and (3) Chol.

3. Method according to claim 1 or 2, characterized in that the lipid components comprise the cationic lipid: Petition 870260070835, dated 16 / 07 / 2026, page 249 / 276 2 / 7 (i) in about 20 mol% to about 30 mol%; or (ii) in about 20 mol%, about 30 mol%, about 40 mol% or about 50 mol%.

4. Method according to any one of claims 1 to 3, characterized in that one or more ionizable cationic lipids are or comprise 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) and / or 1,2-dilinoleyl-3-dimethylammoniumpropane (DLinDAP).

5. Method according to any one of claims 1 to 4, characterized in that one or more phospholipids are or comprise 1,2-diestearoyl-sn-glycero-3-phosphocholine (DSPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). 6.Method according to any one of claims 1 to 5, characterized in that: (1) one or more ionizable cationic lipids are or comprise DODMA and / or DLinDAP; and (2) one or more phospholipids are or comprise DSPC and / or DOPE.

7. Method according to any one of claims 1 to 6, characterized in that the lipid components consist of: (1) one or more ionizable cationic lipids, which are or comprise DODMA and / or DLinDAP; (2) one or more phospholipids, which are or comprise DSPC and / or DOPE; and (3) Chol.

8. Method, according to any one of claims 1 to 7, characterized in that the lipid components comprise a mixture of: (i) DODMA / DOPE / DSPC / Chol at 20 / 30 / 10 / 40% mol; or Petition 870260070835, dated 16 / 07 / 2026, p. 250 / 276 3 / 7 (ii) DODMA / DSPC / Chol at 50 / 10 / 40% molar. 9.A method according to any one of claims 1 to 8, characterized in that: (i) the acidic aqueous solution of (b) has or comprises a pH of about 4 and / or an acetate buffer of 25 mM to 100 mM; and / or (ii) the organic solvent of (a) is or comprises ethanol.

10. A method according to any one of claims 1 to 9, characterized in that the mixing of (b) is carried out by means of microfluidic mixing and / or extrusion.

11. A method according to any one of claims 1 to 10, characterized in that the removal of (c) is carried out by means of dialysis against an acidic buffer.

12. A method according to any one of claims 1 to 11, characterized in that the removal of (c) is carried out by means of dialysis against a buffer with a pH of about 4 and / or against an acetate buffer of 25 mM to 100 mM. 13.Water-based solution, characterized in that it comprises an empty lipid-based transfection competent vesicle (TCV) free of a therapeutic and / or diagnostic load, wherein: (i) the water-based solution is an acidic solution; (ii) the solution is free of organic solvents and detergents; (iii) the solution does not comprise a therapeutic and / or diagnostic load; (iv) the lipid-based TCV comprises lipid components that form the lipid-based TCV, wherein the lipid components comprise (1) a cationic lipid consisting of one or more ionizable cationic lipids, (2) one or more phospholipids and (3) cholesterol (Chol); (v) the lipid-based TCV comprises the cationic lipid Petition 870260070835, dated 07 / 16 / 2026, page. 251 / 276 4 / 7 in a proportion of about 20 molar percent to about 50 molar percent; and (vi) the lipid components do not comprise PEG-lipid. 14.Water-based solution according to claim 13, characterized in that the lipid components consist of: (i) one or more ionizable cationic lipids; (ii) one or more phospholipids; and (iii) Chol.

15. Water-based solution according to claim 13 or 14, characterized in that the lipid components comprise the cationic lipid: (i) in about 20 mol% to about 30 mol%; or (ii) in about 20 mol%, about 30 mol%, about 40 mol% or about 50 mol%.

16. Water-based solution according to any one of claims 13 to 15, characterized in that one or more ionizable cationic lipids are or comprise 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) and / or 1,2-dilinoyl-3-dimethylammoniumpropane (DLinDAP). 17.Water-based solution according to any one of claims 13 to 16, characterized in that one or more phospholipids are or comprise 1,2-diestearoyl-sn-glycero-3-phosphocholine (DSPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

18. Water-based solution according to any one of claims 13 to 17, characterized in that: (i) the ionizable cationic lipid is or comprises DODMA and / or DLinDAP; and (ii) one or more phospholipids are or comprise DSPC and / or DOPE. Petition 870260070835, dated 07 / 16 / 2026, p. 252 / 276 5 / 7 19. Water-based solution according to any one of claims 13 to 18, characterized in that the lipid components consist of: (i) one or more ionizable cationic lipids, which are or comprise DODMA and / or DLinDAP; (ii) one or more phospholipids, which are or comprise DSPC and / or DOPE; and (iii) Chol. 20.Water-based solution according to any one of claims 13 to 19, characterized in that the lipid components comprise a mixture of: (i) DODMA / DOPE / DSPC / Chol at 20 / 30 / 10 / 40 molar percent; or (ii) DODMA / DSPC / Chol at 50 / 10 / 40 molar percent.

21. Water-based solution according to any one of claims 13 to 20, characterized in that the water-based solution comprises a buffer with pH 4 and / or an acetate buffer of 25 mM to 100 mM.

22. Water-based solution, characterized in that the lipid component is made using a method as defined in any one of claims 1 to 12. 23.Method for encapsulating a therapeutic and / or diagnostic payload in a lipid-based transfection-competent vesicle (TCV), characterized in that it comprises: (a) providing the water-based solution as defined in any one of claims 13 to 22; and, (b) mixing the therapeutic and / or diagnostic payload in the water-based solution, wherein the mixing is carried out without the presence of an organic solvent or detergent, wherein the payload consists of a nucleic acid.

24. Method according to claim 23, Petition 870260070835, dated 16 / 07 / 2026, p. 253 / 276 6 / 7 characterized in that the nucleic acid comprises: (i) a deoxyribonucleic acid (DNA), optionally a double-stranded DNA, a single-stranded DNA, a plasmid DNA, a complementary DNA (cDNA) and / or a primer; and / or (ii) a ribonucleic acid (RNA), optionally a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA) and / or a microRNA (miRNA). 25.Composition, characterized in that it comprises a water-based solution comprising a lipid-based transfection-competent vesicle (TCV) encapsulating a therapeutic and / or diagnostic load, made using a method as defined in claim 23 or 24.

23. Use of the composition as defined in claim 25, characterized in that it is in the preparation of a medicament for the treatment of Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or spinal muscular atrophy.

27. Kit, characterized in that it comprises: (a) the composition as defined in claim 25 in a container; and (b) instructions for therapeutic or diagnostic use of the composition. 28.Use of the kit as defined in claim 27, characterized in that it is in the manufacture of a medicament to treat a disease or condition in a patient, optionally Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or spinal muscular atrophy.

29. Method for transfection, characterized in that it comprises transfecting an isolated target cell in vitro with the composition as defined in claim 25, optionally wherein Petition 870260070835, dated 07 / 16 / 2026, pp. 254 / 276 7 / 7 the target cell is a mammalian cell.

30. Use of transfected cells produced by the method as defined in claim 29, characterized in that it is in the manufacture of a medicament for the treatment of a disease or condition in a patient, optionally Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or spinal muscular atrophy.Petition 870260070835, dated 07 / 16 / 2026, pp. 255 / 276.

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