Enhanced nanoparticle delivery systems

Abstract

A method for enhancing nucleic acid delivery systems is disclosed.The method may use a compound and / or RNAi molecule treatment in combination with nucleic acid to improve nucleic acid uptake into cells.In particular, the disclosed method may be useful for improving gene therapy technology.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 123,519, entitled "Enhanced Nanoparticle Delivery Systems," filed December 10, 2020, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under EB023800 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] The introduction of nucleic acids, including double-stranded and single-stranded DNA and RNA, into eukaryotic cells is the most essential step in any gene transfer, repair, or editing technology. Nucleic acid introduction can be achieved using many types of delivery vehicles, including cationic lipids, viral vectors, and nucleic acid nanoparticles condensed with cationic polymers such as polylysine or polyethyleneimine. However, the significant costs associated with the preparation of these materials significantly limit their use as both research tools and transfer applications such as gene therapy. Furthermore, the effectiveness of nucleic acid introduction, with or without vector modification, remains an area requiring improvement. The present disclosure seeks to address one or more of the aforementioned needs in the art.

[0004] A brief overview A method for enhancing a nucleic acid delivery system is disclosed. The method may involve the use of a compound and / or a nucleic acid molecule, such as one or more molecules selected from RNAi, miRNA, shRNA, tRNA, siRNA, single-stranded and double-stranded DNA, in combination with the administration of a nucleic acid, for example, before or at the same time. In particular, the disclosed method may be useful for improved gene therapy techniques, in which the disclosed RNAi and / or the disclosed compound can be administered before or at the same time as a gene therapy delivery vehicle containing a nucleic acid. [Brief explanation of the drawings]

[0005] The application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0006] Those skilled in the art will understand that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Figure 1] This is a schematic diagram of the partial NNP (DNP and RNP) interactome, including nucleolin, APC, and SPTAN1, identified by MS analysis of two gel bands from DNP and RNP pulldowns that were not present in the beads-only control. Lighter circles indicate interactions that enhance NNP-mediated gene transfer, while darker circles indicate inhibitory interactions. A (+) or (-) along the arrow indicates an effect on the interaction with DNP. A (+) or (-) by a pharmacological agent reflects an effect on GR, CDK1, or CKII activation. For example, cortisone increases membrane nucleolin via GR (10), while spermine increases it via stimulation of CKII-mediated phosphorylation of nucleolin. Pulldowns were initially performed in primary hepatocytes and repeated three times in wd-AECs for two non-CF subjects and three CF subjects. This DNP interactome was observed in all hepatocytes and wd-AEC studies, both CF and non-CF. [Figure 2]1 shows immunoprecipitation of protein interactors of DNA nanoparticles in HeLa cells. [Figure 3] 1 shows the enhancement of DNA nanoparticle transfection by siRNA expression. [Figure 4] Transfection of human primary airway epithelia is shown after 48 hours of pretreatment with either scrambled shRNA, APC-specific shRNA, or SPTAN1-specific shRNA. Luciferase expression was measured 2 days after transfection. *Denotes difference from saline pretreatment (triplicate of three experiments, p<0.01). [Figure 5] Primary airway epithelial cell cultures were transfected with DNP containing a 5.4 kb plasmid encoding luciferase driven by the ubiquitin B promoter. Infection with shRNA lentivirus was performed 48 h prior to transfection, but spermine (a CK11 inducer), roscovitine (a CDK1 inhibitor), resveratrol (a CDK1 agonist), or cortisone (a GR agonist) was added 4 h prior to transfection. Treatments included saline (S), APC shRNA (-APC), SPTAN1 shRNA (-SPTAN1), hydrocortisone (c), spermine (Sper), roscovitine (Ros), or resveratrol (RES). Luciferase expression was measured 2 days after transfection. * indicates a difference from saline (p<0.01). [Figure 6] Schematic diagram showing the DNP gene transfer process and barriers to gene transfer. Barriers to gene transfer prevent DNP from completing these processes. Protein-DNP interactions can affect how DNP passes through these barriers. [Figure 7] FIG. 1 is a schematic diagram showing protein-vector interactions to circumvent intracellular barriers. [Figure 8] 1 is a schematic diagram showing protein-vector interactions to circumvent intracellular barriers.Cortisone concentration versus gene expression as a percentage of vehicle treatment. [Figure 9]Interactome analysis to identify DNP / protein interactions [Figure 10]

[0033] Figure 1 shows a graph demonstrating that luciferase gene transfer is enhanced by pharmacological manipulation in vitro. HeLa cells were transfected with luciferase-DNP either 4 hours after treatment with drugs, simultaneously with treatment with drugs, or 4 hours before treatment with drugs: DNP alone, RX001 (roscovitine), RX011 (spermine), and RX008 (ruxolitinib). [Figure 11]

[0033] Figure 1 shows a graph demonstrating that luciferase gene transfer is enhanced by pharmacological manipulation in vitro. HeLa cells were transfected with luciferase DNP either 4 hours after treatment with drug, simultaneously with drug treatment, or 4 hours before treatment with drug, DNP alone, RX012 (doxorubicin), RX013 (acetohexamide), and RX014 (sildenafil citrate). [Figure 12] Luciferase gene transfer is enhanced by pharmacological manipulation in vitro. HeLa cells were treated with luciferase-DNP for 4 hours before administration. Drugs were obtained from the blind plate. Labels on the graph indicate the drug position on the blind plate. *: p<0.05, **: p<0.01, ***: p<0.001, ***: p<0.0001 (compared to the DNP alone group). [Figure 13]This shows that the enhancement of in vivo DNP transfection by pharmaceuticals is maintained over time. Mice were administered roscovitine (10 mg / kg, a CDK1 inhibitor), spermine (20 mg / kg, a CKII activator), and ruxolitinib (1 mg / kg, a JAK inhibitor) 2 hours before intratracheal administration of 100 μg of luciferase-induced DNP. A. BLI images taken 2, 3, 7, and 14 days after DNP administration of a single mouse from a group of mice receiving DNP only (D), roscovitine (Rs), spermine (S), and ruxolitinib (Rx). Total photon measurements were taken from the chest of each mouse, with ROIs outlined in red. Background luminescence was subtracted from the saline control mice (not shown). B. Total photons collected over a 10-minute BLI exposure from the ROI on each day. (n>10 for each group, mean and SD shown, p<0.05, p<0.01, p<0.001 compared to the DNP alone group). Note: On day 14, spermine was significantly different (P<0.5) from the DNP alone group as well as roscovitine (p<0.01). Means graphed with SD, significance for each time point is in the order ruxo, sper, rosc (top to bottom). *: p<0.05, **: p<0.01, ***: p<0.001, ***: p<0.0001 compared to the DNP alone group on the same day after treatment. [Figure 14] Pharmacological manipulation of interactome proteins enhances luciferase gene transfer in vivo. Bioluminescence imaging (BLI) analysis, RX001, RX011, RX008, days 2, 3, 7, and 14. *: p<0.05, **: p<0.01, ***: p<0.001, ***: p<0.0001 (compared to the DNP alone group on the same day after treatment). [Figure 15] We show that pharmacological manipulation of interactome proteins enhances luciferase gene transfer in vivo BLI image analysis and luciferase activity assays for DNP alone, RX001 (roscovitine), RX011 (spermine), and RX008 (ruxolitinib). [Figure 16]hCFTR gene transfer is enhanced by pharmacological manipulation of interactome proteins: DNP alone, RX001 (roscovitine), and RX008 (ruxolitinib), 2 days after DNP administration, 4 days after DNP administration, and 7 days after DNP administration. [Figure 17] Pharmacological manipulations were performed before, after, and during DNP transfection. HeLa cells were treated with roscovitine (1 μM, a CDK1 inhibitor), spermine (1 μM, a CKII activator), or ruxolitinib (0.1 μM, a JAK inhibitor) at various times during luciferase-DNP transfection. A. HeLa cells were treated with the drug for 4 hours, washed, and then treated with luciferase-DNP for 24 hours. B. HeLa cells were treated with the drug and luciferase-DNP simultaneously for 24 hours. C. HeLa cells were treated with luciferase-DNP for 4 hours, washed, and then treated with the drug for 24 hours. All cells were then lysed and analyzed for luciferase activity using a light-based assay. (n = 8 per group, mean p < 0.05, mean p < 0.001). [Figure 18] This demonstrates pharmaceutical enhancement of DNP gene delivery efficacy in vivo. Mice were administered 2 hours prior to intratracheal administration of 100 μg of luciferase-inducing DNP with roscovitine (10 mg / kg, a CDK1 inhibitor), spermine (20 mg / kg, a CKII activator), and ruxolitinib (1 mg / kg, a JAK inhibitor). All data were collected 14 days after DNP administration. Representative BLI images of mice receiving A-I) DNP only, B-II) roscovitine, C-III) spermine, and D-IV) ruxolitinib. ROIs outlined in red were consistently drawn on each mouse and used to quantify total photons in each mouse. B. Total photons collected from the ROI over a 10-minute BLI exposure. Mice receiving saline instead of DNP (not shown) were used to subtract background from experimental mice. C-B: Lungs from mice used in C were harvested immediately after BLI imaging and assayed for luciferase activity. (Each group n>10, p<0.05, p<0.01, p<0.001) [Figure 19]Pharmacological manipulations before, after, and during DNP transfection are shown. HeLa cells were treated with roscovitine (1 μM, a CDK1 inhibitor), spermine (1 μM, a CKII activator), or ruxolitinib (0.1 μM, a JAK inhibitor) at various times during luciferase-DNP transfection. A. HeLa cells were treated with the drug for 4 hours, washed, and then treated with luciferase-DNP for 24 hours. B. HeLa cells were treated with the drug and luciferase-DNP simultaneously for 24 hours. C. HeLa cells were treated with luciferase-DNP for 4 hours, washed, and then treated with the drug for 24 hours. All cells were then lysed and analyzed for luciferase activity using a light-based assay. (n = 8 per group, mean and SEM shown, p < 0.05, mean p < 0.001). [Figure 20] This demonstrates pharmaceutical enhancement of DNP gene delivery efficacy in vivo. Mice were administered 2 hours prior to intratracheal administration of 100 μg of luciferase-inducing DNP with roscovitine (10 mg / kg, a CDK1 inhibitor), spermine (20 mg / kg, a CKII activator), and ruxolitinib (1 mg / kg, a JAK inhibitor). All data were collected 14 days after DNP administration. Representative BLI images of mice receiving A-I) DNP only, B-II) roscovitine, C-III) spermine, and D-IV) ruxolitinib. ROIs outlined in red were consistently drawn on each mouse and used to quantify total photons in each mouse. B. Total photons collected from the ROI over a 10-minute BLI exposure. Mice receiving saline instead of DNP (not shown) were used to subtract background from experimental mice. C-B: Lungs from mice used in C were harvested immediately after BLI imaging and assayed for luciferase activity. (n>10 for each group, mean and SEM shown, p<0.05, p<0.01, p<0.001) [Figure 21]This shows that the enhancement of in vivo DNP transfection by pharmaceuticals is maintained over time. Mice were administered roscovitine (10 mg / kg, a CDK1 inhibitor), spermine (20 mg / kg, a CKII activator), and ruxolitinib (1 mg / kg, a JAK inhibitor) 2 hours before intratracheal administration of 100 μg of luciferase-induced DNP. A. BLI images taken 2, 3, 7, and 14 days after DNP administration of a single mouse from a group of mice receiving DNP only (D), roscovitine (Rs), spermine (S), and ruxolitinib (Rx). Total photon measurements were taken from the chest of each mouse, with ROIs outlined in red. Background luminescence was subtracted from the saline control mice (not shown). B. Total photons collected over a 10-minute BLI exposure from the ROI on each day. (n>10 for each group, mean and SEM shown, p<0.05, p<0.01, p<0.001 compared to the DNP alone group). Note: On day 14, spermine was significantly different (P<0.5) from the DNP alone group as well as roscovitine (p<0.01). Means graphed with SEM, significance for each time point is in the order ruxo, sper, rosc (top to bottom). *: p<0.05, **: p<0.01, ***: p<0.001, ***: p<0.0001 compared to the DNP alone group on the same day after treatment. [Figure 22] Comparison of CFTR expression between NHBE cells (normal human bronchial / tracheal epithelial cells), untreated mice, and DNP-treated mice on day 2, as well as comparison of CFTR expression in mice treated with vehicle (DNP alone) or drug on day 4. Day 4 data represents vector-subtracted values ​​for PCR of CFTR. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0008] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0009] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" may mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0010] As used herein, the term "effective amount" refers to an amount of one or more active ingredients sufficient to produce the desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.

[0011] The terms "individual," "host," "subject," and "patient" are used interchangeably to refer to an animal that is the object of treatment, observation, and / or experiment. Generally, the term refers to a human patient, although the methods and compositions may be equally applicable to non-human subjects, such as other mammals. In some embodiments, the term refers to a human. In further embodiments, the term may refer to a child.

[0012] As used herein, "a pharmaceutically acceptable form thereof" includes any pharmaceutically acceptable salt, prodrug, tautomer, isomer, and / or isotopically labeled derivative of the compounds provided herein, as defined below and herein.

[0013] As used herein, the term "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and does not neutralize the biological activity and properties of the compounds described herein. As used herein, the disclosed compounds also include their pharmaceutically acceptable salts.

[0014] As used herein, the term "prodrug" refers to a derivative of a parent compound that requires conversion in the body to release the parent compound. A prodrug can be inactive when administered to a subject, but is converted to an active compound in vivo, for example, by hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug has improved physical and / or delivery properties compared to the parent compound. A prodrug is typically designed to enhance pharmaceutical and / or pharmacokinetic properties associated with the parent compound. The advantage of a prodrug can be its physical properties, for example, enhanced water solubility for parenteral administration at physiological pH compared to the parent compound, or it can enhance absorption from the gastrointestinal tract, or it can enhance drug stability for long-term storage. (See, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entireties.

[0015] The term "prodrug" is also meant to include any covalently bonded carrier that releases the active compound in vivo when such prodrug is administered to a subject. Prodrugs of the active compounds described herein can be prepared by modifying functional groups present in the active compound such that the modifications are cleaved to the parent active compound, either by routine manipulation or in vivo. Prodrugs include compounds in which a hydroxy group, an amino group, or a mercapto group is bonded to any group that cleaves to form a free hydroxy group, a free amino group, or a free mercapto group, respectively, when the prodrug of the active compound is administered to a subject. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or acetamides in the active compound, formamide and benzamide derivatives of amine functional groups, and the like. Other examples of prodrugs include compounds containing -NO, -NO2, -ONO, or -ONO2 moieties. Prodrugs can be prepared using well-known methods, such as those typically described in Burger's Medicinal Chemistry and Drug Discovery, 172-178, 949-982 (Manfred E. Wolff ed., 5th ed., 1995), and Design of Prodrugs (H. Bundgaard ed., Elselvier, NY, 1985).

[0016] For example, if a disclosed compound or a pharmaceutically acceptable form of the compound contains a carboxylic acid functional group, a prodrug can be prepared by replacing the hydrogen atom of the acid group with a (C1-C8) alkyl, (C2-C 12Alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 1-(alkoxycarbonyl)aminomethyl having 4 to 10 carbon atoms, These may include pharmaceutically acceptable esters formed by substituting with groups such as N-(alkoxycarbonyl)amino)ethyl, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolactone-4-yl, di-N,N-(C1-C2 alkylamino(C2-C3)alkyl (e.g., (3-dimethylaminoethyl), carbamoyl-(C1-C2 alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2 alkyl and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl).

[0017] Similarly, when a disclosed compound or a pharmaceutically acceptable form of a compound contains an alcohol functional group, a prodrug can be formed by replacing the hydrogen atom of the alcohol group with a group such as (1-C6 alkanoyloxymethyl, 1-((C1-C6)alkanoyloxyethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl (C1-C6 alkoxycarbonyloxymethyl, N—(C1-C6 alkoxycarbonylaminomethyl, succinoyl, (C1-C6 alkanoyl, α-amino (C1-C4 alkanoyl, arylacyl and α-aminoacyl, or α-aminoacyl-α-aminoacyl (each α-aminoacyl group is independently selected from naturally occurring L-amino acids, P(O)(OH)), P(O)(OH), —P(O)(O(C1-C6)alkyl) or glycosyl (the radical resulting from removal of the hydroxyl group of a hemiacetal form of a carbohydrate).

[0018] When a disclosed compound or a pharmaceutically acceptable form of the compound incorporates an amine functional group, a prodrug can be formed by replacing a hydrogen atom in the amine group with a group such as R-carbonyl, RO-carbonyl, NRR′-carbonyl, and the like, where R and R′ are each independently (C1-C 10 ) alkyl, (C3-C7 cycloalkyl, benzyl, natural α-aminoacyl or natural α-aminoacyl-natural α-aminoacyl, -C(OH)C(O)OY 1 (In the formula, Y 1 is H, (C1-C6) alkyl or benzyl), —C(OY 2 )Y 3 (In the formula, Y 2 is (C1-C4) alkyl, and Y 3 is (C1-C6) alkyl, carboxy(C1-C6) alkyl, amino(C1-C4 alkyl or mono-N- or di-N,N-(C1-C6 alkylaminoalkyl), C(Y 4 )Y 5 (wherein Y 4 is H or methyl, and Y 5 is mono-N- or di-N,N-(C1-C6)alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl).

[0019] Active agents may form salts, which are also within the scope of preferred embodiments. Reference to an active agent compound herein is understood to include reference to its salts, unless otherwise indicated. As used herein, the term "salt" refers to acid and / or base salts formed with inorganic and / or organic acids and bases. Furthermore, when an active agent contains both a basic moiety (e.g., but not limited to, an amine or a pyridine or imidazole ring) and an acidic moiety (e.g., but not limited to, a carboxylic acid), zwitterions ("internal salts") may be formed, which are included within the term "salt" as used herein. Although pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, other salts are also useful, for example, in isolation or purification steps that may be used during preparation. Salts of active agent compounds can be formed, for example, by reacting the active agent compound with an amount (e.g., equivalent amount) of an acid or base in a medium (e.g., a medium in which the salt precipitates or an aqueous medium) followed by lyophilization. When compounds are in the form of salts, they may include pharmaceutically acceptable salts.Such salts may include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium salts and alkylated ammonium salts.Acid addition salts include salts of inorganic acids and organic acids.Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, bismethylenesalicylic acid, ethanedisulfonic acid, gluconic acid, citraconic acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, boric acid, acetic acid, benzoic acid, hydroxynaphthoic acid, glycerophosphoric acid, ketoglutaric acid, and the like.Examples of metal salts include lithium, sodium, potassium, magnesium salts, etc. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts, etc. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline, etc.

[0020] As used herein, "sequence identity" refers to a nucleic acid sequence that has the same nucleic acid sequence as a reference sequence or a specified percentage of nucleotides that are the same at corresponding positions within the reference sequence when the two sequences are optimally aligned. For example, a nucleic acid sequence may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference nucleic acid sequence. The length of the comparison sequence is generally at least 5 contiguous nucleotides, preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides, and most preferably the full-length nucleotide sequence. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) with default settings. Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0021] The term "NNP" refers to nucleic acid nanoparticles. Non-limiting examples include complexes of DNA or RNA with lysine polymers (e.g., 15-45 lysines in length).

[0022] The term "DNP" refers to DNA nanoparticles.

[0023] The term "RNP" refers to RNA nanoparticles.

[0024] The term "interactome" refers to the entire set of molecular interactions in a particular cell. The term specifically refers to the physical interactions between molecules (such as between proteins, also known as protein-protein interactions), but can also describe the set of indirect interactions between genes (genetic interactions).

[0025] The term "APC" refers to adenomatous polyposis coli protein.

[0026] The term "wd-AEC" refers to fully differentiated airway epithelial cells.

[0027] The term "SPTAN1" refers to αII-spectrin, also known as spectrin α chain, a protein encoded by the SPTAN1 gene in humans. αII-spectrin is expressed in various tissues and is highly expressed in cardiac muscle in Z-disc structures, costameres, and sarcosal membranes.

[0028] The term "GR" refers to the glucocorticoid receptor.

[0029] The term "CDK1" refers to cyclin-dependent kinase 1.

[0030] The term "CKII" refers to casein kinase II.

[0031] The term "spermine" refers to a polyamine found in all eukaryotic cells and involved in cellular metabolism.

[0032] The term "shRNA" refers to small hairpin RNA or short hairpin RNA (shRNA), an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi).

[0033] Disclosed herein are methods for enhancing nucleic acid delivery systems by combined treatment with one or more compounds disclosed herein and / or one or more RNAi molecules disclosed herein. For example, the disclosed methods may be used in conjunction with the delivery of nucleic acids such as genes, gene fragments, or fragments containing active portions of proteins encoded by genes. Further examples of nucleic acids that can be delivered include nucleic acid components of CRISPR / CAS9, or short nucleic acids such as microRNAs or DNA or RNA oligonucleotides. The disclosed RNAi molecules and / or compounds may be administered to an individual in need of nucleic acid administration prior to or simultaneously with administration of the nucleic acid delivery system.

[0034] In one aspect, the method may be a method for introducing genes into eukaryotic cells, and may include compacted nucleic acid nanoparticles; and compounds such as sonolicib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), afuresertib (substituted benzene, phenethylamine, amphetamine), cationic vemurafenib (aryl-phenylketone), suramin (benzene derivative, benzanilide), uprosertib (benzene derivative, phenethylamine, amphetamine), flutamide (benzene derivative, trifluoromethyl ketone), phenethylamine, amphetamine, ... The method may include administering to the eukaryotic cell one or more agents selected from: benzophenone-3 (benzotriazole derivative, N-alkyl indole), enzastaurin (indole derivative, N-alkyl indole), fasudil (isoquinoline derivative), ruboxistaurin (macrolactam), pentamidine (phenol ether), uprosertib (benzene derivative, phenethylamine, amphetamine), lestaurtinib (indole derivative, indolocarbazole), adenine (imidazole pyrimidine), pimozide (diphenylbutylpiperidine), chlorpromazine (phenothiazine), afrestib (benzene derivative, phenethylamine, amphetamine), and combinations thereof.

[0035] In one embodiment, the method may include administering an inhibitor of a protein that inhibits nanoparticle delivery uptake. In this embodiment, the inhibitor may be selected from one or more of RNAi, miRNA, shRNA, tRNA, siRNA, single-stranded DNA, double-stranded DNA, and combinations thereof. In this embodiment, the nucleic acid may inhibit the synthesis of one or more proteins that inhibit nucleic acid delivery vehicle uptake. Exemplary proteins may include one or more proteins selected from the proteins in Table 1.

[0036] In one aspect, the method can include administering an active agent that promotes cellular uptake of the compacted nucleic acid nanoparticles. The active agent can inhibit the synthesis of one or more proteins that inhibit nucleic acid delivery vehicle uptake.

[0037] In one embodiment, the RNAi molecule may inhibit expression of a gene encoding a protein selected from Table 1.

[0038] In one embodiment, the method may further comprise administering a second active agent selected from the agents listed in Table 2 or Table 3.

[0039] In one aspect, the active agent may be selected from one or more of roscovitine, geldanamycin, acetohexamide, and ruxolitinib, or a combination thereof.

[0040] In one aspect, the nucleic acid delivery vehicle may be a nanoparticle containing one or more of the aforementioned genes.

[0041] In one aspect, the compacted nucleic acid nanoparticles may comprise a nucleic acid plasmid and a polymer, and the nanoparticles may be compacted in the presence of counterions selected from trifluoroacetate (TFA), bromide, bicarbonate, glutamate, hydroxyl ions, or combinations thereof.

[0042] In one embodiment, the nucleic acid may be single-stranded or double-stranded DNA, or a combination thereof.

[0043] In one embodiment, the polymer may be a polycation. In one embodiment, the polycation may be a lipid. In a further embodiment, the polycation may be a cysteine ​​(C)-containing polymer of lysine (K), such as CK30, a cysteine ​​(C)-containing polymer of arginine (R), such as CR30, or a combination thereof. In a further embodiment, the polycation is selected from a cysteine ​​(C)-containing polymer of lysine (K) and arginine (R), e.g., C(K5R)5 or C(R5K)5 (e.g., CK15-90), a polymer of arginine (e.g., CR15-90), or a polymer of lysine mixed with arginine (e.g., C(KR5KR5KR5KR5) or C(K5RK5RK5RK5RK5R)), conjugated to PEG, and complexed with a nucleic acid. In a further embodiment, the polymer may be a lysine polymer, e.g., a polyethylene glycol (PEG)-substituted lysine polymer or polyethyleneimine.

[0044] In one aspect, the compacted nucleic acid nanoparticles may have a shape selected from rod-shaped, ellipsoidal, spheroidal, or toroidal, and may have a diameter of about 25 to about 400 nm in length as measured by electron microscopy.

[0045] The method, in certain aspects, comprises: The method may include contacting the cell with an RNAi molecule or active agent, which may be in an amount sufficient to inhibit synthesis of one or more proteins that inhibit nucleic acid delivery vehicle uptake; The eukaryotic cell is contacted with the nucleic acid delivery vehicle.

[0046] The cell may be, for example, a eukaryotic cell of human origin.

[0047] In one aspect, the disclosed methods may be used to treat an individual in need of such treatment. The individual may be one in which administration of a therapeutically effective amount of a protein would be beneficial for reversing, preventing, or ameliorating a disease state. Protein delivery may be achieved via administration of a gene, or a portion of a gene encoding an active portion of the protein, which may then be expressed in the individual to provide a therapeutically effective amount of functional protein or functional protein fragment. In this aspect, the method includes administering to a subject a compound selected from Tables 2 or 3, such as, but not limited to, sonolicib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), afuresertib (substituted benzene, phenethylamine, amphetamine), cationic vemurafenib (aryl-phenylketone), suramin (benzene derivative, benzanilide), uprosertib (benzene derivative, phenethylamine, amphetamine), or the like. The method may include administering an agent selected from the group consisting of flutamide (benzene derivative, trifluoromethylbenzene), enzastaurin (indole derivative, N-alkylindole), fasudil (isoquinoline derivative), ruboxistaurin (macrolactam), pentamidine (phenol ether), uprosertib (benzene derivative, phenethylamine, amphetamine), lestaurtinib (indole derivative, indolocarbazole), adenine (imidazole pyrimidine), pimozide (diphenylbutylpiperidine), chlorpromazine (phenothiazine), afrestib (benzene derivative, phenethylamine, amphetamine), and combinations thereof. The RNAi or agent may be administered simultaneously with, before, or after administration of a drug delivery vehicle containing a nucleic acid encoding the gene of interest (or in some cases, an active portion of the gene).

[0048] The amount of compound and / or RNAi required to practice the methods of the present disclosure can be determined by one of ordinary skill in the art. The dose administered to a subject, particularly a human, can be sufficient to produce a desired response in the subject over a reasonable period of time. The dose may be determined by the strength of the particular compound used, the condition of the subject, and the body weight of the subject being treated. The existence, nature, and extent of any adverse side effects that may accompany the administration of a particular compound also determine the dose size and particular route of administration used for a particular patient. For example, a compound may be therapeutically effective at low doses. Exemplary dose ranges may be from about 0.001 mM or less to about 100 mM or more, or from about 0.01, 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, or 0.9 mM to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM. Thus, compounds may generally be administered at low doses.

[0049] In one embodiment, the gene is the CF gene and the individual in need of treatment is an individual with cystic fibrosis.

[0050] In one embodiment, the RNAi molecule may inhibit the expression of a gene encoding a protein selected from the proteins in Table 1.

[0051] [Table 1-1]

[0052] [Table 1-2]

[0053] [Table 1-3]

[0054] [Table 1-4]

[0055] Table 1-5

[0056] Table 1-6

[0057] Table 1-7

[0058] Table 1-8

[0059] Table 1-9

[0060] Table 1-10

[0061] Table 1-11

[0062] Table 1-12

[0063] Table 1-13

[0064] Table 1-14

[0065] [Table 1-15]

[0066] [Table 1-16]

[0067] In one aspect, the active agent may be selected from one or more compounds listed in Table 2.

[0068] [Table 2-1]

[0069] [Table 2-2]

[0070] [Table 2-3]

[0071] Drug descriptions in Table 2. Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90 and activates the heat shock response in mammalian cells. Entasoburin is the first anticancer drug in development that involves two mechanisms of action: tubulin and topoisomerase II inhibition. Entasoburin exhibits distinct modes of action, including proapoptotic and antiangiogenic properties. Dihydrotestosterone (DHT) (INN: androstanolone) is the biologically active metabolite of the hormone testosterone and is formed primarily in the prostate, testes, hair follicles, and adrenal glands by reduction of the alpha-4,5 double bond by the enzyme 5-alpha-reductase. Dihydrotestosterone belongs to a class of compounds called androgens and is commonly referred to as the androgen hormone or testosterone. DHT is thought to be approximately 30 times more potent than testosterone due to its higher affinity for the androgen receptor. Spermine is a polyamine found in all eukaryotic cells and is involved in cellular metabolism. The synthetic precursor of spermine is the amino acid ornithine. It is found in a wide range of organisms and tissues and is an essential growth factor in some bacteria. It is found as a polycation at physiological pH. Spermine is thought to associate with nucleic acids and stabilize helical structures, particularly in viruses. Cortisone is a corticosteroid. Its mechanism of action is as a corticosteroid hormone receptor agonist. Quercetin is a flavonoid, more specifically a flavonol, accounting for 60% of total dietary flavonol intake. The term flavonoid includes thousands of plant-derived compounds that share a common phenyl-chroman skeleton. This basic structure allows for numerous substitution patterns, resulting in several flavonoid subclasses, including flavonols, flavones, flavanones, catechins, anthocyanidins, isoflavones, dihydroflavonols, and chalcones. First-generation sulfonylureas include acetohexamide, chlorpropamide, tolazamide, and tolbutamide, oral hypoglycemic agents used to treat type 2 diabetes. Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin.It is a stilbenoid, a derivative of stilbene, and is produced in plants with the aid of the enzyme stilbene synthase. It exists as two structural isomers: cis- (Z) and trans- (E), with the trans isomer shown in the image above. The trans isomer can isomerize to the cis isomer upon exposure to heat or ultraviolet radiation. In a 2004 issue of Science, Dr. Sinclair of Harvard University stated that resveratrol is not an easy molecule to protect from oxidation. It has been claimed to be easily degraded by exposure to light, heat, and oxygen. However, studies have found that trans-resveratrol undergoes negligible oxidation at room temperature and in normal atmospheres. Doxorubicin is a drug used in cancer chemotherapy. It is an anthracycline antibiotic, closely related to the natural product daunomycin, and, like all anthracyclines, intercalates DNA. It is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinomas, and soft tissue sarcomas. The drug is administered intravenously in the form of its hydrochloride salt. It may be sold under the trade names Adriamycin PFS, Adriamycin RDF, or Rubex. It is photosensitive and is often packaged in an aluminum bag to protect it from light. Ruxolitinib (INCB018424) is a selective oral JAK1 / JAK2 inhibitor. This drug has the potential to modulate two key kinases that may be involved in myeloproliferative neoplasms, including primary myelofibrosis. Roscovitine is a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2, and cdk5. Sildenafil is a selective PDE5 inhibitor used to treat erectile dysfunction and pulmonary arterial hypertension. Teniposide / Vumon is a semisynthetic derivative of podophyllotoxin with antitumor activity. Teniposide forms a ternary complex with the enzyme topoisomerase II and DNA, resulting in dose-dependent single- and double-strand breaks in DNA, DNA:protein crosslinks, inhibition of DNA strand religation, and cytotoxicity. The drug acts at the late S or early G phase of the cell cycle.

[0072] [Table 3]

[0073] In one embodiment, one or more compounds or derivatives thereof may be used to facilitate the introduction of nanoparticles. These include one or more of the following: doxorubicin, sildenafil androstanolone, acetohexamide, and teniposide (which may be administered to the patient approximately 30-60 minutes prior to administering DNP), roscovitine (an imidazopyrimidine), spermine (a dialkylamine), geldanamycin (a macrolactam), ruxolitinib (a pyrrolopyrimidine), teniposide (a podophyllotoxin), sildenafil, androstanolone. (benzenesulfonamide), androstanolone (anabolic steroid), acetohexamide (alkyl-phenyl ketone), doxorubicin (anthracycline), sonolisib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), afrese ultib (substituted benzene, phenethylamine, amphetamine), cationic vemurafenib (aryl-phenyl ketone), suramin (benzene derivative, benzanilide), uprosertib (benzene derivative, phenethylamine, amphetamine), flutamide (benzene derivative, trifluoromethylbenzene), enzastaurin (indole derivative, N-alkylindole), fasudil (isoquinoline derivative), ruboxistaurin (macrolactam), pentamidine (phenol ether), uprosertib (benzene derivative, phenethylamine, amphetamine), lestaurtinib (indole derivative, indolocarbazole), adenine (imidazole pyrimidine), pimozide (diphenylbutylpiperidine), chlorpromazine (phenothiazine), and afrestib (benzene derivative, phenethylamine, amphetamine).

[0074] These individual compounds, alone or in combination, may be combined with a nanoparticle formulation or administered before or after intranasal (IN) administration (eg, 5 μg (with respect to DNA) in a 25 μl solution).

[0075] Gene transfer can occur in connection with administration to cells in humans, i.e., administration of a vector containing a nucleic acid to a mammal, particularly a human. For example, a compound and / or RNAi disclosed herein can be administered to an individual prior to administration of a nucleic acid delivery system known in the art (exemplary nucleic acid delivery systems are known in the art and disclosed in References 11-16). The nucleic acid can be single-stranded or double-stranded, or in certain instances, multiple delivery vehicles can be utilized, which can use one or the other or both.

[0076] The nanoparticle delivery vehicle may take a variety of forms. For example, in one embodiment, the nucleic acid delivery vehicle may be a nanoparticle containing the gene of interest. In one embodiment, the nucleic acid delivery vehicle may be a nanoparticle containing a lysine polymer conjugated to PEG and complexed with a nucleic acid containing the gene.

[0077] In one aspect, the protein that inhibits nucleic acid delivery vehicle uptake may be selected from keratin 13, APC protein, protocadherin 17, spectrin alpha (non-erythroid type 1), or a combination thereof.

[0078] In one embodiment, there is a period of time between step a and step b. In embodiments in which the nucleic acid delivery vehicle is administered after delivery of the RNAi and / or compounds disclosed herein, the nucleic acid delivery vehicle may be administered to the individual in need thereof, for example, 30 minutes, 60 minutes, 90 minutes, or 120 minutes after administration of the compounds and / or RNAi disclosed herein. In the case of RNAi, in some embodiments, the RNAi may be administered about 12 hours before the nucleic acid delivery vehicle, about 20 hours before the nucleic acid delivery vehicle, about 24 hours before the nucleic acid delivery vehicle, or about 30 hours before administration of the delivery vehicle.

[0079] For example, for RNAi applications, patient stem cells or patient-derived iPSCs are harvested, cultured, and treated with RNAi directed against a gene in Table 1 for 24 hours. NNPs formulated to contain an expression cassette for a therapeutic gene are then added to the cells for 72 hours. The reagents and delivery vectors are replaced daily. As an example of the time involved in this method of active agent application, the patient is treated with one or more of the compounds listed in Tables 2 and 3 about 30 to about 60 minutes prior to administration of the gene delivery vector. The agent treatment may be administered one or more times prior to gene therapy. NNPs containing an expression cassette for the therapeutic gene may then be administered to the patient's airways, for example, by nebulization.

[0080] In one embodiment, the method may include providing a transfection-facilitating reagent. In one embodiment, the agent may be a cationic lipid transfection reagent (e.g., lipofectamine or GL67), which may be mixed with the nucleic acid in a given formulation to produce a nucleic acid / lipid complex. With regard to lipid (or protein) nucleic acid complexes, any formulation that produces lipid / nucleic acid or protein / nucleic acid complexes (there are thousands) can be combined with the methods herein. This may similarly apply to protein polymers such as PEGylated poly-L-lysine or PEI. With regard to viral vectors, the vectors can be produced in cell lines, purified, and used for therapy according to the disclosed methods.

[0081] Compositions comprising RNAi and / or compounds of Tables 2 and / or 3 may be administered intranasally. In such embodiments, the compositions may further comprise other agents suitable for improving delivery across the nasal mucosa. For example, in certain embodiments, agents such as penetration enhancers, polymers capable of increasing mucoadhesion of the composition, or combinations thereof may be included in the composition. In one embodiment, the disclosed compositions may comprise, consist of, or consist essentially of any of the foregoing features in any combination.

[0082] It will be understood by those skilled in the art that the particular method of administration will depend on a variety of factors, all of which are routinely considered when administering therapeutic agents, particularly in the context of gene transfer. However, it will also be understood that the specific dose level for any given patient will depend on a variety of factors, including the activity of the particular compound used, the patient's age, the patient's weight, the patient's general health, the patient's sex, the patient's diet, the time of administration, the route of administration, the rate of excretion, the drug combination, and the severity of the condition being treated. Furthermore, those skilled in the art will understand that the optimal treatment course, i.e., the treatment mode and the number of daily doses given for a specified number of days, can be ascertained by those skilled in the art using conventional treatment trials.

[0083] Nanoparticle counterions Nanoparticles containing nucleic acids, such as DNA or RNA, which may be double-stranded or single-stranded, and may be protein-coding or antisense-coding or non-coding, are disclosed. The nucleic acids may include RNA and / or DNA analogs (including, for example, miRNA, shRNA, tRNA, siRNA, single-stranded and double-stranded DNA) modified to enhance degradation in vivo.

[0084] Methods for producing nanoparticles according to the present invention are known in the art. See, for example, U.S. Patent No. 8,017,577, entitled "Lyophilizable and enhanced compacted nucleic acids," and / or "Chapter 33: Real-Time Imaging of Gene Delivery and Expression with DNA Nanoparticle Technologies" (Sun and Ziady, filed herewith), both of which are incorporated herein by reference in their entireties. Disclosed herein are alternative counterions to those disclosed in the art that can be used to produce nucleic acid nanoparticles. The counterion of the polycation used to compact the nucleic acid is known to affect the shape of the particles formed. Shape is related to nuclease resistance and colloidal stability. Furthermore, shape affects the suitability and efficacy of the compacted nucleic acid complex for transfecting cells via various routes into mammalian cells.

[0085] Counterions that may be used in creating compacted nucleic acid complexes may also affect the stability of the complexes against lyophilization. Disclosed herein are nanoparticles that are compacted using one or more counterions selected from trifluoroacetate (TFA), bromide, bicarbonate, glutamate, aspartate, hydroxyl ions, or combinations thereof, which may be used prior to compaction of the nucleic acid.

[0086] Polycations may include polyamino acids, such as polylysine and derivatives of polylysine. Polycations may contain 15 to 60 lysine residues, preferably in the range of 15 to 30, 30 to 45, or 45 to 60 residues. Exemplary derivatives of polylysine are CK15, CK30, and CK45, which have additional cysteine ​​residues attached to polylysine polymers of 15, 30, and 45 residues in length, respectively. Other amino acids can be readily attached to polylysine. Other polycationic amino acid polymers (e.g., polyarginine or copolymers of arginine and lysine) can be used. Polymers of non-protein amino acids, such as ornithine or citrulline, can also be used. Any pharmaceutically approved or suitable polycation can be used, including, but not limited to, protamine, histones, polycationic lipids, putrescine, spermidine, spermine, peptides, and polypeptides. Polycations may also contain targeting moieties, which are typically ligands that bind to receptors on specific types of cells. The targeting ligand may be a polyamino acid or other chemical moiety. The specificity of the ligand-receptor interaction is important for targeting purposes. In one embodiment, polycations may be reacted with bifunctional PEG (e.g., PEG-maleimide (PEG-Mal) or ortho-pyridyl disulfide (OPSS) (PEG-OPSS)) to allow for the attachment of targeting moieties.

[0087] In one aspect, a) the compacted nucleic acid nanoparticles described above; and b) a nucleic acid nanoparticle selected from the group consisting of sonolicib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), afuresertib (substituted benzene, phenethylamine, amphetamine), cationic vemurafenib (aryl-phenylketone), suramin (benzene derivative, benzanilide), uprosertib (benzene derivative, phenethylamine, amphetamine), flutamide (benzene derivative, trifluoromethylbenzene), enzastaurin (indole derivative), and optionally, one or more agents selected from Table 1 or Table 2. Disclosed are compositions comprising one or more agents selected from: one or more agents selected from: benzodiazepine (benzoyl benzoate), benzoyl benzoate (benzoyl benzoate, ...

[0088] Kits are also provided. In one embodiment, the kit may comprise or consist essentially of an agent or composition described herein. The kit may be a package housing a container that may contain one or more compounds or solutions containing the RNAi disclosed herein, and also housing instructions for administering the agent or composition to a subject. In one embodiment, a pharmaceutical pack or kit is provided that includes one or more containers filled with one or more compositions disclosed herein. Such container(s) may be accompanied by various documents, such as instructions for use or notices in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for manufacture, use, or sale for human administration.

[0089] Because it may be advantageous to mix the components of the compositions described herein and the pharmaceutically acceptable carrier, excipient, or vehicle near the time of use, kits are disclosed in which the components of the compositions are packaged separately. For example, the kit can contain the active ingredient in powder or other dry form, e.g., in a sterile vial or ampoule, and the carrier, excipient, or vehicle, or the components of the carrier, excipient, or vehicle (liquid or dry form) in a separate container within the kit. In one aspect, the kit can contain the components in dry form, typically as a powder, often in lyophilized form, e.g., in a sterile vial or ampoule, and in a separate container within the kit. Alternatively, the kit may contain the components in the form of a concentrated solution to be diluted before administration. Any of the components described herein, any of the carriers, excipients, or vehicles described herein, and any combination of components with carriers, excipients, or vehicles can be included in the kit.

[0090] Optionally, the kit may also contain instructions for preparation or use (e.g., instructions printed on a leaflet disposed on or within the outer container), as well as one or more devices to aid in the preparation of the solution and / or its administration to a patient (e.g., one or more syringes, needles, filters, tape, tubing (e.g., tubing to facilitate intravenous administration), alcohol swabs, and / or Band-Aid® applicators). Compositions can be prepared that are more concentrated than those administered to a subject. Thus, such compositions can optionally be included in the kit along with appropriate materials for dilution (e.g., water, saline, or other physiologically acceptable solution). The instructions included in the kit can include instructions for dilution, if appropriate.

[0091] In other embodiments, the kit may include a premixed composition and instructions for solubilizing any precipitates that may form during transport or storage. Kits containing solutions of one or more of the aforementioned active agents or pharmaceutically acceptable salts thereof and one or more carriers, excipients, or vehicles may also contain any of the materials described above (e.g., any devices for assisting in the preparation of the composition for administration or the administration itself). The instructions in these kits may describe appropriate indications (e.g., a description of patients suitable for treatment) and instructions for administering the solution to patients. [Example]

[0092] Methods for enhancing nucleic acid transfer Applicants have discovered methods to enhance the efficiency of gene transfer through the use of interfering RNA (RNAi) technology or pharmacological agents that modulate the interactome of nucleic acid nanoparticles composed of polymers of lysine conjugated to PEG and complexed with nucleic acids (Figure 1). Both of these approaches have been scaled to implement and achieve significantly higher levels of gene transfer in the context of condensed DNA nanoparticle vectors, resulting in as much as 50-fold higher gene transfer efficiencies. These techniques represent significant enhancements over gene transfer technologies.

[0093] Using a novel immunocapture procedure (Figure 2), we identified protein interactors of polyethylene glycol-conjugated DNA nanoparticles. This investigation revealed 474 unique proteins that interact with the nanoparticles, as listed in Table 3. While many of these proteins represent the nanoparticle-specific transfection interactome, some proteins, such as prohibitin 1 and 2, are also involved in viral and liposomal gene delivery. Some of these protein interactors may inhibit cellular uptake of DNA nanoparticles and other vectors for nucleic acid delivery. The interactome was segregated to the intracellular site to which the nucleic acid particles are delivered (Table 4). In this method, we used RNAi and / or pharmacological agents to modulate the particle interactome and enhance nucleic acid delivery to the nucleus (DNA) or ribosomes (RNA).

[0094] [Table 4]

[0095] For RNAi applications, RNAi molecules can be delivered to cells prior to the desired nucleic acid delivery vehicle, or to an individual in the case of delivery to an individual. The RNAi molecule is administered in an amount sufficient to target and knock down specific cellular proteins that negatively affect the uptake of the nucleic acid delivery vehicle. RNAi reduces the cellular levels of these proteins, reducing their detrimental effects on downstream delivery of nucleic acids. RNAi-mediated knockdown of four of these proteins was tested by the present applicant, resulting in significant enhancement of gene transfer in three-quarters of the constructs tested. RNAi targeted to disrupt the synthesis of four proteins—keratin 13 (GI:81891678), APC protein (GI:97535708), protocadherin 17 (GI:94538350), and spectrin α (non-erythroid 1, GI:119608216)—which are detrimental to gene transfer via DNA nanoparticles—improved gene delivery (Figure 3).

[0096] In addition to, or in addition to, pharmacological agents that modulate the DNP interactome, nucleic acid delivery to the nucleus or ribosomes (in the case of RNA delivery) can be enhanced. Applicant has identified 13 compounds and their derivatives (see Table 2) that modulate 71 interactors and can be administered to patients approximately 30 to 60 minutes prior to DNP administration. These are categorized by their cellular site of action. For example, doxorubicin and sildenafil act to inhibit or promote interactions in the cytosol. Androstanolone modulates interactions in the ribosome. Acetohexamide promotes non-nucleolin-mediated interactions at the cell membrane. Ruxolitinib and teniposide may also be used to modulate nuclear interactions. Applicant's data also point to the importance of interactions with nucleolin and how modulation of this interaction at the plasma membrane significantly impacts DNP-mediated gene delivery (Figures 3-5). Modulation of these cellular interactions is expected to have different effects on RNPs versus DNPs because the cellular compartment targeting of these formulations of NNPs changes (ribosomes versus the nucleus, respectively). For example, a drug that promotes cell entry may benefit both DNPs and RNPs. However, a drug that reduces interactions at the ribosome is expected to benefit only DNPs. Conversely, a drug that reduces nuclear interactions should benefit RNPs.

[0097] The compounds summarized in Tables 2 and 3 may be used to modulate nucleolin-associated nucleic acid nanoparticle (NNP) uptake. Nucleolin translocation to the cell surface can be enhanced by IP injection of animals with roscovitine (inhibiting CDK1 at 10 mg / kg), spermine (inducing CKII at 50 mg / kg), geldanamycin (inhibiting HSP90 interaction with nucleolin at 15 mg / kg), or hydrocortisone (increasing GR shuttling of nucleolin to the cell surface at 7.5 mg / kg) 60 min prior to intranasal (IN) administration of 25 μl of 5 μg (relative to DNA) NNPs containing the CFTR gene, as previously reported (1). Control groups injected with either DMSO or transgene-containing NNPs without drug may also be used. As previously reported (1), CF mice may undergo NPD measurements 1 week before treatment (background / baseline measurement), followed by NPD measurements 4, 7, and 14 days after transfection with nasally applied CFTR-containing NNPs. Two weeks after transfection, mice are sacrificed, lungs are harvested, paraffin-embedded, and sectioned for immunohistochemistry. Sections may be probed with CF3 or 24:1 anti-CFTR Abs, which do not cross-react with mouse CFTr, as previously reported (1). Studies can be replicated in F508del and S489X homozygous mice.

[0098] Uses in Research: RNAi and / or pharmacological approaches to enhance gene transfer may be developed as additives to current gene transfer and transfection vectors. For example, they may be used as auxiliary additives to the cationic lipid transfection reagent Lipofectamine, allowing for either greater gene transfer or reduced amounts of transfection reagent used, resulting in either reduced costs or increased efficiency. Alternatively, pharmacological and RNAi treatments may be used prior to or simultaneously with viral vector delivery in in vitro or ex vivo gene transfer applications. This may allow for greater cellular gene modification and higher expression of therapeutic transgenes in these cells, or reduced viral titers required to provide curative levels of cellular modification. This may increase the efficacy of these genes or reduce the associated costs associated with producing sufficient amounts of virus, which is currently a significant obstacle in gene therapy protocols.

[0099] Use in Human Therapy. CF is the most common inherited recessive disorder in Caucasians, and advances in small molecule therapy have not provided significant benefit to the majority of patients. Gene therapy (repair or replacement) offers the possibility of corrective treatment of the disease, regardless of mutation type. The disclosed methods may be useful for enhancing corrective DNA and / or RNA delivery by synthetic vectors to airway epithelial cells, the cells most affected in CF. This example concerns the biology of NNP, a vector shown to have partial efficacy in correcting CFTR in CF patients (2). Applicants have identified 71 specific protein interactors (e.g., some of the interactors and associated regulation are shown in Figure 2, and others are listed in Table 1) that help define the particle's biology in cells and can be targeted with 13 FDA-approved drugs (Table 2). Other compounds are listed in Table 3.

[0100] We have demonstrated that by modulating the NNP interactome, gene transfer can be enhanced 10-50-fold, the highest level of enhancement achieved to date in 20 years of engineering and testing DNP-based vectors (see Figures 3 and 4). Based on these results, and considering the fact that DNP achieved partial clinical correction in a Phase 1 trial in CF patients (2), the disclosed method has the potential to provide pharmacological agents that can enhance gene transfer to fully therapeutic levels in humans. While airway epithelial cells are the primary site of disease and the most important gene therapy target in CF, a better understanding of the determinants of successful gene transfer into these cells would significantly benefit gene delivery for many other diseases, including chronic obstructive pulmonary disease (COPD; approximately 12,000,000 patients in the United States) and epithelial lung cancer (approximately 200,000 patients in the United States). This disclosure provides a novel approach to implementing NNP biology. The findings in the airway epithelium are likely relevant to other cellular targets where NNPs have been successful, including cells in the brain (3-6) and retina (7-10), and may be relevant to the cellular uptake of other non-viral polyplex-based vectors as well as viral and liposomal vectors.

[0101] In therapeutic settings, siRNA can be applied to human cells ex vivo just before or during gene delivery to optimize gene transfer obtained with DNA / RNA nanoparticles, or pharmacological agents can be applied to humans, as is also possible with liposomal and viral vectors.

[0102] References 1.Ziady AG,Kelley TJ,Milliken E,Ferkol T,Davis PB.Functional evidence of CFTR gene transfer in nasal epithelium of cystic fibrosis mice in vivo following luminal application of DNA complexes targeted to the serpin-enzyme complex receptor.Mol.Ther.2002 Apr;5(4):413-9 2.Konstan MW,Davis PB,Wagener JS,Hilliard KA,Stern RC,Milgram LJ,Kowalczyk TH,Hyatt SL,Fink TL,Gedeon CR,et al.Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution.Hum.Gene Ther.2004 Dec;15(12):1255-69 3.Yurek DM,Fletcher AM,Smith GM,Seroogy KB,Ziady AG,Molter J,Kowalczyk TH,Padegimas L,Cooper MJ.Long-term transgene expression in the central nervous system using DNA nanoparticles.Mol.Ther.2009 Apr;17(4):641-50 4.Yurek DM,Flectcher AM,Kowalczyk TH,Padegimas L,Cooper MJ.Compacted DNA nanoparticle gene transfer of GDNF to the rat striatum enhances the survival of grafted fetal dopamine neurons.Cell Transplant.2009;18(10):1183-96.PMCID:PMC3031110 5.Yurek DM,Fletcher AM,McShane M,Kowalczyk TH,Padegimas L,Weatherspoon MR,Kaytor MD,Cooper MJ,Ziady AG.DNA Nanoparticles:Detection of Long-term Transgene Activity In Brain Using Bioluminescence Imaging.Mol.Imaging 2011 Apr 26; 6.Fletcher AM,Kowalczyk TH,Padegimas L,Cooper MJ,Yurek DM.Transgene expression in the striatum following intracerebral injections of DNA nanoparticles encoding for human glial cell line-derived neurotrophic factor.Neuroscience 2011 Oct 27;194:220-6.PMCID:PMC3408714 7.Farjo R,Skaggs J,Quiambao AB,Cooper MJ,Naash MI.Efficient non-viral ocular gene transfer with compacted DNA nanoparticles.PLoS.One.2006;1:e38.PMCID:PMC1762345 8.Ding XQ,Quiambao AB,Fitzgerald JB,Cooper MJ,Conley SM,Naash MI.Ocular delivery of compacted DNA-nanoparticles does not elicit toxicity in the mouse retina.PLoS.One.2009;4(10):e7410.PMCID:PMC2756629 9.Cai X,Conley SM,Nash Z,Fliesler SJ,Cooper MJ,Naash MI.Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.FASEB J.2010 Apr;24(4):1178-91.PMCID:PMC2845431 10.Koirala A,Makkia RS,Conley SM,Cooper MJ,Naash MI.S / MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA.Hum.Mol.Genet.2013 Apr 15;22(8):1632-42.PMCID:PMC3605833 11.Ziady AG,Davis PB,Konstan MW.Non-viral gene transfer therapy for cystic fibrosis.Expert.Opin.Biol.Ther.2003 Jun;3(3):449-58 12.Ziady AG,Davis PB.Current prospects for gene therapy of cystic fibrosis.Curr.Opin.Pharmacol.2006 Oct;6(5):515-21 13.Ahmed H,Shubina-Oleinik O,Holt JR.Emerging Gene Therapies for Genetic Hearing Loss.J.Assoc.Res.Otolaryngol.2017 Aug 16; 14.Naso MF,Tomkowicz B,Perry WL,III,Strohl WR.Adeno-Associated Virus(AAV) as a Vector for Gene Therapy.BioDrugs.2017 Jul 1.PMCID:PMC5548848 15.Huang J,Wang Y,Zhao J.CRISPR Editing in Biological and Biomedical Investigation.J.Cell Physiol 2017 Aug 8; 16. Zhang X, Wang L, Liu M, Li D. CRISPR / Cas9 system: a powerful technology for in vivo and ex vivo gene therapy.Sci.China Life Sci.2017 May;60(5):468-75 All percentages and ratios are calculated by weight unless otherwise indicated.

[0103] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0104] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0105] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "20 mm" is intended to mean "approximately 20 mm."

[0106] All documents cited herein, including any cross-references or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. All registered information (e.g., identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession number) and publications are incorporated by reference in their entirety into this disclosure to more fully describe the state of the art as known to those of skill in the art as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with any other reference(s), teaches, suggests, or discloses any such invention. Furthermore, if any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control.

[0107] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. A pharmaceutical composition for gene transfer into eukaryotic cells, comprising a compacted nucleic acid nanoparticle; and a pharmaceutical composition for gene transfer into eukaryotic cells, comprising Sonolisib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), Trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), Afuresertib (substituted benzene, phenethylamine, amphetamine), cationic Vemurafenib (aryl-phenylketone), Suramin (benzene derivative, benzanilide), Uprosertib (benzene derivative, phenethylamine, amphetamine), Flutamide (benzene derivative, trifluoromethacrylate), 16. A pharmaceutical composition comprising one or more agents selected from: benzoylbenzene), enzastaurin (indole derivative, N-alkyl indole), fasudil (isoquinoline derivative), ruboxistaurin (macrolactam), pentamidine (phenol ether), uprosertib (benzene derivative, phenethylamine, amphetamine), lestaurtinib (indole derivative, indolocarbazole), adenine (imidazole pyrimidine), pimozide (diphenylbutylpiperidine), chlorpromazine (phenothiazine), afrestib (benzene derivative, phenethylamine, amphetamine), and combinations thereof.

2. The pharmaceutical composition of claim 1, further comprising an inhibitor of a protein that inhibits nanoparticle delivery uptake, said inhibitor being selected from one or more of RNAi, miRNA, shRNA, tRNA, siRNA, single-stranded DNA, double-stranded DNA, and combinations thereof, said nucleic acid inhibiting synthesis of one or more proteins that inhibit nucleic acid delivery vehicle uptake, said one or more proteins being: Apolipoprotein B-100 (Apo B-100), Leiomojin-1, Leiomojin, SM-Lmod, elongation factor 1-alpha1, Pot. HLA-DP-alpha 1, Utrophin, Myosin light chain 3, Cytochrome b-c1 complex subunit 2, mitochondria, ubiquitin-conjugating enzyme E2 S, coagulation factor VIII, Heterogeneous nuclear ribonucleoprotein M, Myosin light chain polypeptide 6, Adenomatous Polyposis Coli Protein, tyrosine-protein kinase JAK1, Myosin-6, 40S ribosomal protein S3a, Golgin subfamily A member 2, Peripherin, Heterogeneous nuclear ribonucleoprotein A / B, Spectrin beta chain, red blood cells, Dual specificity protein kinase TTK, 78 kDa glucose-regulated protein, CACCC box binding protein, 60S ribosomal protein L11, eukaryotic initiation factor 4A-III, Interleukin enhancer binding factor 2, nucleolar and coiled body phosphoprotein 1, Neblin, Microtubule-associated proteins, DNA replication licensing factor MCM4, Centromere protein F, cytoskeleton-associated protein 5, Chromodomain-helicase-DNA binding protein 4, Solute carrier family 12 member 3, phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, Periodic tryptophan protein 2 homologue, DNA mismatch repair protein Msh3, ubiquitin-protein ligase E3A, SWI / SNF complex subunit SMARCC1, Histone H2B type 1-C / E / F / G / I, histone-lysine N-methyltransferase EZH1, endonuclease III-like protein 1, General transcription factor 3C polypeptide 1, histone-lysine N-methyltransferase 2D, uracil nucleotide / cysteinyl leukotriene receptor, Catenin delta-2, Heme oxygenase 2, Periplakin, Ryanodine receptor 3, Karin-3, Small subunit processome component 20 homologue, T13D8.9 protein, RNA-binding motif protein, X chromosome, Protein CBFA2T3, Peripheral benzodiazepine receptor-related protein 1, Actin, gamma-intestinal smooth muscle, Regulators of chromosome condensation, CHC1 protein, elongation factor 2, Histone H3.1t, prohibitin, isoform CRA a, prohibitin, prohibitin variant, 60S ribosomal protein L24, epididymal secretory protein Li 310, Ribosomal protein, large, P2, isoform CRA_a; 60S acidic ribosomal protein P2, 40S ribosomal protein S10, 40S ribosomal protein S11, 40S ribosomal protein S13, 40S ribosomal protein S17, Ribosomal protein S23, isoform CRA_a, 40S ribosomal protein S23, 40S ribosomal protein S25, 40S ribosomal protein S4, 40S ribosomal protein S4,X isoform, 40S ribosomal protein S8, Ski oncogene, sperm-binding protein 1a, cDNA FLJ38670 fis, clone HSYRA2000190, voltage-dependent anion-selective channel protein 1, transcriptional repressor protein YY1, A-kinase anchor protein 9, Histone H1.3, Zinc finger C2HC domain-containing protein 1A, Prelamin-A / C, Lamin-B1, Alcohol dehydrogenase, epididymal secretory protein Li6, ATP5H protein, ATP synthase subunit d, mitochondrion, myosin regulatory light chain 12A, Uncharacterized proteins, Gag-Pro-Pol-Env protein, High mobility group proteins HMG-I / HMG-Y, Cytochrome c oxidase subunit 4 isoform 1, mitochondrial, tyrosine-protein kinase BAZ1B, Septin-9, 40S ribosomal protein S18, MCG23000, isoform CRA_b, meiosis required protein 1 homologue, Heterogeneous nuclear ribonucleoprotein H3, Low density lipoprotein receptor-related protein 1B, MICOS complex subunit MIC60, MICOS complex subunits, MICOS complex subunit MIC19, Rhomboid domain-containing protein 2, armadillo repeat-containing X-linked protein 5, ATP-dependent RNA helicase DDX39A, leucine-rich repeat-containing protein 31, coiled-coil domain-containing protein 170, ABC50 protein, ATP-binding cassette subfamily F member 1, cell division cycle 5-like protein, Myosin-10, 60S ribosomal protein L14, Sperm flagellar protein 2, Keratin, type II cytoskeleton 7, As with ribosomal protein L23, gamma-tubulin complex component 6, Ribosomal protein L15, 60S ribosomal protein L15, KLHL20 protein, high mobility group nucleosome binding domain-containing protein 5, Nucleolar protein 7, 27 kDa, isoform CRA_a, nucleolar protein 7, 60S ribosomal protein L10a, DNA-dependent protein kinase catalytic subunit, cDNA FLJ51732, peroxisomal NADH pyrophosphatase NUDT12, 60S ribosomal protein L28, Heterogeneous nuclear ribonucleoprotein A2 / B1, serine / threonine protein kinase SMG1, 40S ribosomal protein S9 (ribosomal protein S9, isoform CRA_a), 40S ribosomal protein S9 (ribosomal small subunit protein uS4), Remodeling and spacing factor 1, Actin, cytoplasmic 1 (beta actin), BRCA1-A complex subunit RAP80, galactose-3-0-sulfotransferase 3, 40S ribosomal protein S24, Bromodomain and WD repeat-containing protein 1, Mating plakoglobin, isoform CRA_a, Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1; 60S ribosomal protein L13, Phosphorylase b kinase regulatory subunit alpha, hepatic isoform Heterogeneous nuclear ribonucleoprotein Q, Arsine (Amyotrophic lateral sclerosis chromosome 2 candidate gene 6), Synaptotagmin-like protein 1, Ribosomal protein L34, isoform CRA_a, 60S ribosomal protein L34, Cingulin, Centromere protein T, Cingulin-like protein 1, Coiled-coil domain-containing protein 42, Heterogeneous nuclear ribonucleoprotein F, mitochondrial import receptor subunit TOM40 homologue, FBL protein, ESF1 homologue (ABT 1 related protein), calcium / calmodulin-dependent protein kinase type II subunit delta, 60S ribosomal protein L18, Protein Shroom3, dynein heavy chain 5, axoneme; PC4 and SFRS1 interacting proteins, epididymal tissue sperm-binding protein Li 14m, Trifunctional enzyme subunit alpha, mitochondrial, Bcl-2-like protein 12, Core histone macro-H2A.1, MTRF1 protein, Alström syndrome protein 1, UHRF1-binding protein 1-like, U5 small nuclear ribonucleoprotein 200 kDa helicase, SWI / SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4; the transcriptional activator BRG1, eukaryotic elongation factor 2 kinase, EVI 5-like protein (ecotropic virus integration site 5-like protein), Protein ECT2 (epithelial cell transforming sequence 2 oncogene), CSC1-like protein 2, cDNA FLJ35251 fis, clone PROST2003635, Nesprin-1, Chromodomain-helicase-DNA binding protein 5, Midasin, Nesprin-2, voltage-dependent anion-selective channel protein 3, ATPase family AAA domain-containing protein 5, N-α-acetyltransferase 15, NatA auxiliary subunit, Histone H2A type 2-B, Rootletin (ciliary rootlet coiled-coil protein), Histone H2A, Histone H2A.J, PDZ domain-containing protein 8, Maintenance of the structure of chromosomal protein 1A, SMC1A protein, Vacuolar protein sorting-related protein 13B; Centriolin, Pericentrin, Testis secretory protein Li 14, Glutamate dehydrogenase 2, mitochondrial, Myomesin-3, Acidic ribosomal phosphoprotein P1, nucleolar protein 56, nucleolar protein 58, Uncharacterized proteins, FLJ00279 protein, cDNA FLJ16045 fis, clone CTONG2000042, Putative C-mannosyltransferase DPY19L4, Coiled-coil domain-containing protein 18 (sarcoma antigen NY-SAR-24), FYVE, RhoGEF and PH domain-containing protein 6 (zinc finger FYVE domain-containing protein 24); ATP synthase subunit alpha, mitochondrial, Super killer antiviral activity 2-like 2, Phosphate carrier protein, mitochondria, 40S ribosomal protein S6, EEF1D protein (fragment), sterile alpha motif domain-containing protein 9; antigen MLAA-44, coiled-coil and C2 domain-containing protein 1B, elongation factor 1-gamma, Heterogeneous nuclear ribonucleoprotein U, Malignant fibrous histiocytoma amplified sequence 1, arachidonate 15-lipoxygenase, GC-rich sequence DNA binding factor 2, 60S ribosomal protein L4, Regulator of G protein signaling 20, Harmonin, Plectin, kinesin-like protein KIF1B, Neuropilin-2, Neuropilin, Vacuolar protein sorting-associated protein 13D; Lymphoid-specific helicase, Histone H2B type 1-A, ankyrin repeat domain-containing protein 11, Histone H2B, Keratin, type II cytoskeleton 6A, Nip-B-like protein, ATPase family AAA domain-containing protein 1, mitochondrial dynamin-like 120 kDa protein, Striated muscle preferentially expressed protein kinase, disc large-associated protein 1, Cytokinesis protein 11, Myogaline, putative uncharacterized protein DKFZp666F1010; putative uncharacterized protein DKFZp666M0710; putative uncharacterized protein DKFZp686P17171, Activation-dependent raft-mobilizing ADAP-like phosphoprotein, unconventional myosin-XVIIIa, Nucleolin, isoform CRA_b, Nucleolin, isoform CRA_c, Nucleolin (protein C23), Keratin, type I cytoskeleton 9, Cilia and flagella-associated proteins 58; terminal uridylyltransferase 4, WD repeat and SOCS box-containing protein 1, Centromere protein J, FRAS1-related extracellular matrix protein 1, Machoilin, NARG1 protein, calpastatin isoform a variant, Ribosomal protein L5 variants, Insulin-like growth factor binding protein 2 variant, Rho-associated protein kinase 2, 60S acidic ribosomal protein P0, elongation factor 1-alpha, Glial fibrillary acidic protein, beta-actin-like protein 2, Fanconi anemia, complementation group D2, isoform CRA_b, Fanconi anemia group D2 protein, IQ and AAA domain-containing protein 1, Protein FAM92A, Teneurin-3, Coiled-coil and C2 domain-containing protein 2A, the putative uncharacterized protein C3orf49, Heterogeneous nuclear ribonucleoprotein A1, Dynein heavy chain 2, axoneme, Ribosomal proteins, RSL1D1 protein, ATP-dependent DNA helicase PIF1, Keratin, type II cytoskeleton 73, ATP synthase subunit beta, cDNA FLJ39263 fis, clone OCBBF2009571, ATP-dependent RNA helicase A, 290 kDa centrosomal protein, CEP290 protein, Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1, La-related protein 7, pumilio homolog 3, Ovochymase-1, INO80 complex subunits, RWD domain-containing protein 3, Dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A, histone-lysine N-methyltransferase ASH1L, ATP-binding cassette subfamily C member 8, cilia and flagella-associated proteins 157; M-phase phosphoprotein 1, isoform CRA_a, 60S ribosomal protein L3, A kinase (PRKA) anchor protein 6, isoform CRA_b; Lamin B2, isoform CRA_b, Nuclear mitotic apparatus protein 1, isoform CRA_a; Coiled-coil domain-containing protein 40, TBK1 binding protein 1, isoform CRA_a, Membrane-associated polypeptide 2, isoform beta / gamma, KIAA1212, isoform CRA_a, Histone H2B, Septin 11, isoform CRA_b, Heterogeneous nuclear ribonucleoprotein A3, isoform CRA_a, MORC family CW-type zinc finger protein 4, cytoplasmic dynein heavy chain 1, unconventional myosin VIIb, Protein unc-13 homolog C, HSPA1L, Heat shock 70 kDa protein 1-like, Laminin subunit beta-4, leucine-rich repeat-containing protein 43, Protocadherin Fat 3, Death-associated protein kinase, ADP / ATP translocase 2, SLC25A5 protein, ADP / ATP translocase 3, SLC25A6 protein, HNRPR protein, Poly[ADP-ribose]polymerase (PARP), Poly[ADP-ribose]polymerase 1, Protein AHNAK2, Myb-binding protein 1A, leucine-rich repeat and IQ domain-containing protein 3, cDNA FLJ76880, cDNA FLJ76071, Maintenance of chromosomal protein structure, coiled-coil domain-containing protein 180, dynein heavy chain 17, axoneme; Histone H2A, cDNA, FLJ95007, Protein phantom, Mpv17-like protein, Protein CASP, cDNA FLJ44920 fis, clone BRAME 3011501, ATPase family AAA domain-containing protein 3A, cDNA FLJ35376 fis, clone SKMUS2004044, SNF2LT, dynein heavy chain 6, axoneme; Nuclear export mediator factor NEMF, cDNA FLJ51361, cDNA FLJ57651, cDNA FLJ50007, cDNA FLJ52940, Protein RUFY3, leucine-rich repeat and coiled-coil domain-containing protein 1, cDNA FLJ60374, G protein-coupled receptor 176, cDNA FLJ61500, Small nuclear ribonucleoprotein Sm D3, cDNA FLJ54081, cDNA FLJ58017, Piwi-like protein 2, Serine / threonine-protein kinase N2, Dysferlin, putative uncharacterized protein DKFZp469P0721, Short transient receptor potential channel 4, GTP-binding nuclear protein Ran, HNRNPL protein, cDNA FLJ52713, cDNA FLJ50492, TBC1 domain family member 1, Non-POU domain-containing octamer binding proteins, 60S ribosomal protein L31, cDNA FLJ51382, Protein Jumonji, von Willebrand factor A domain-containing protein 5B2, VWA5B2 protein, 128 kDa centrosomal protein, proliferation marker protein KI-67; Ribosomal protein L19, RPL27 / NME2 fusion protein, TPR and ankyrin repeat-containing protein 1, cDNA FLJ90678 fis, clone PLACE1005736, pleckstrin homology domain-containing family A member 1, SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5; Golgin subfamily B member 1, Heterogeneous nuclear ribonucleoprotein D-like, and Heterogeneous nuclear ribonucleoprotein D-like A pharmaceutical composition selected from the group consisting of

3. A pharmaceutical composition as described in claim 1 or claim 2, further comprising an active agent that promotes uptake of the compressed nucleic acid nanoparticles into cells, wherein the active agent inhibits synthesis of one or more proteins that inhibit nucleic acid delivery vehicle uptake.

4. The pharmaceutical composition according to claim 2, wherein the RNAi is Apolipoprotein B-100 (Apo B-100), Leiomojin-1, Leiomojin, SM-Lmod, elongation factor 1-alpha1, Pot. HLA-DP-alpha 1, Utrophin, Myosin light chain 3, Cytochrome b-c1 complex subunit 2, mitochondria, ubiquitin-conjugating enzyme E2 S, coagulation factor VIII, Heterogeneous nuclear ribonucleoprotein M, Myosin light chain polypeptide 6, Adenomatous Polyposis Coli Protein, tyrosine-protein kinase JAK1, Myosin-6, 40S ribosomal protein S3a, Golgin subfamily A member 2, Peripherin, Heterogeneous nuclear ribonucleoprotein A / B, Spectrin beta chain, red blood cells, Dual specificity protein kinase TTK, 78 kDa glucose-regulated protein, CACCC box binding protein, 60S ribosomal protein L11, eukaryotic initiation factor 4A-III, Interleukin enhancer binding factor 2, nucleolar and coiled body phosphoprotein 1, Neblin, Microtubule-associated proteins, DNA replication licensing factor MCM4, Centromere protein F, cytoskeleton-associated protein 5, Chromodomain-helicase-DNA binding protein 4, Solute carrier family 12 member 3, phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1, Periodic tryptophan protein 2 homologue, DNA mismatch repair protein Msh3, ubiquitin-protein ligase E3A, SWI / SNF complex subunit SMARCC1, Histone H2B type 1-C / E / F / G / I, histone-lysine N-methyltransferase EZH1, endonuclease III-like protein 1, General transcription factor 3C polypeptide 1, histone-lysine N-methyltransferase 2D, uracil nucleotide / cysteinyl leukotriene receptor, Catenin delta-2, Heme oxygenase 2, Periplakin, Ryanodine receptor 3, Karin-3, Small subunit processome component 20 homologue, T13D8.9 protein, RNA-binding motif protein, X chromosome, Protein CBFA2T3, Peripheral benzodiazepine receptor-related protein 1, Actin, gamma-intestinal smooth muscle, Regulators of chromosome condensation, CHC1 protein, elongation factor 2, Histone H3.1t, prohibitin, isoform CRA a, prohibitin, prohibitin variant, 60S ribosomal protein L24, epididymal secretory protein Li 310, Ribosomal protein, large, P2, isoform CRA_a; 60S acidic ribosomal protein P2, 40S ribosomal protein S10, 40S ribosomal protein S11, 40S ribosomal protein S13, 40S ribosomal protein S17, Ribosomal protein S23, isoform CRA_a, 40S ribosomal protein S23, 40S ribosomal protein S25, 40S ribosomal protein S4, 40S ribosomal protein S4,X isoform, 40S ribosomal protein S8, Ski oncogene, sperm-binding protein 1a, cDNA FLJ38670 fis, clone HSYRA2000190, voltage-dependent anion-selective channel protein 1, transcriptional repressor protein YY1, A-kinase anchor protein 9, Histone H1.3, Zinc finger C2HC domain-containing protein 1A, Prelamin-A / C, Lamin-B1, Alcohol dehydrogenase, epididymal secretory protein Li6, ATP5H protein, ATP synthase subunit d, mitochondrion, myosin regulatory light chain 12A, Uncharacterized proteins, Gag-Pro-Pol-Env protein, High mobility group proteins HMG-I / HMG-Y, Cytochrome c oxidase subunit 4 isoform 1, mitochondrial, tyrosine-protein kinase BAZ1B, Septin-9, 40S ribosomal protein S18, MCG23000, isoform CRA_b, meiosis required protein 1 homologue, Heterogeneous nuclear ribonucleoprotein H3, Low density lipoprotein receptor-related protein 1B, MICOS complex subunit MIC60, MICOS complex subunits, MICOS complex subunit MIC19, Rhomboid domain-containing protein 2, armadillo repeat-containing X-linked protein 5, ATP-dependent RNA helicase DDX39A, leucine-rich repeat-containing protein 31, coiled-coil domain-containing protein 170, ABC50 protein, ATP-binding cassette subfamily F member 1, cell division cycle 5-like protein, Myosin-10, 60S ribosomal protein L14, Sperm flagellar protein 2, Keratin, type II cytoskeleton 7, As with ribosomal protein L23, gamma-tubulin complex component 6, Ribosomal protein L15, 60S ribosomal protein L15, KLHL20 protein, high mobility group nucleosome binding domain-containing protein 5, Nucleolar protein 7, 27 kDa, isoform CRA_a, nucleolar protein 7, 60S ribosomal protein L10a, DNA-dependent protein kinase catalytic subunit, cDNA FLJ51732, peroxisomal NADH pyrophosphatase NUDT12, 60S ribosomal protein L28, Heterogeneous nuclear ribonucleoprotein A2 / B1, serine / threonine protein kinase SMG1, 40S ribosomal protein S9 (ribosomal protein S9, isoform CRA_a), 40S ribosomal protein S9 (ribosomal small subunit protein uS4), Remodeling and spacing factor 1, Actin, cytoplasmic 1 (beta actin), BRCA1-A complex subunit RAP80, galactose-3-0-sulfotransferase 3, 40S ribosomal protein S24, Bromodomain and WD repeat-containing protein 1, Mating plakoglobin, isoform CRA_a, Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1; 60S ribosomal protein L13, Phosphorylase b kinase regulatory subunit alpha, hepatic isoform Heterogeneous nuclear ribonucleoprotein Q, Arsine (Amyotrophic lateral sclerosis chromosome 2 candidate gene 6), Synaptotagmin-like protein 1, Ribosomal protein L34, isoform CRA_a, 60S ribosomal protein L34, Cingulin, Centromere protein T, Cingulin-like protein 1, Coiled-coil domain-containing protein 42, Heterogeneous nuclear ribonucleoprotein F, mitochondrial import receptor subunit TOM40 homologue, FBL protein, ESF1 homologue (ABT 1 related protein), calcium / calmodulin-dependent protein kinase type II subunit delta, 60S ribosomal protein L18, Protein Shroom3, dynein heavy chain 5, axoneme; PC4 and SFRS1 interacting proteins, epididymal tissue sperm-binding protein Li 14m, Trifunctional enzyme subunit alpha, mitochondrial, Bcl-2-like protein 12, Core histone macro-H2A.1, MTRF1 protein, Alström syndrome protein 1, UHRF1-binding protein 1-like, U5 small nuclear ribonucleoprotein 200 kDa helicase, SWI / SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4; the transcriptional activator BRG1, eukaryotic elongation factor 2 kinase, EVI 5-like protein (ecotropic virus integration site 5-like protein), Protein ECT2 (epithelial cell transforming sequence 2 oncogene), CSC1-like protein 2, cDNA FLJ35251 fis, clone PROST2003635, Nesprin-1, Chromodomain-helicase-DNA binding protein 5, Midasin, Nesprin-2, voltage-dependent anion-selective channel protein 3, ATPase family AAA domain-containing protein 5, N-α-acetyltransferase 15, NatA auxiliary subunit, Histone H2A type 2-B, Rootletin (ciliary rootlet coiled-coil protein), Histone H2A, Histone H2A.J, PDZ domain-containing protein 8, Maintenance of the structure of chromosomal protein 1A, SMC1A protein, Vacuolar protein sorting-related protein 13B; Centriolin, Pericentrin, Testis secretory protein Li 14, Glutamate dehydrogenase 2, mitochondrial, Myomesin-3, Acidic ribosomal phosphoprotein P1, nucleolar protein 56, nucleolar protein 58, Uncharacterized proteins, FLJ00279 protein, cDNA FLJ16045 fis, clone CTONG2000042, Putative C-mannosyltransferase DPY19L4, Coiled-coil domain-containing protein 18 (sarcoma antigen NY-SAR-24), FYVE, RhoGEF and PH domain-containing protein 6 (zinc finger FYVE domain-containing protein 24); ATP synthase subunit alpha, mitochondrial, Super killer antiviral activity 2-like 2, Phosphate carrier protein, mitochondria, 40S ribosomal protein S6, EEF1D protein (fragment), sterile alpha motif domain-containing protein 9; antigen MLAA-44, coiled-coil and C2 domain-containing protein 1B, elongation factor 1-gamma, Heterogeneous nuclear ribonucleoprotein U, Malignant fibrous histiocytoma amplified sequence 1, arachidonate 15-lipoxygenase, GC-rich sequence DNA binding factor 2, 60S ribosomal protein L4, Regulator of G protein signaling 20, Harmonin, Plectin, kinesin-like protein KIF1B, Neuropilin-2, Neuropilin, Vacuolar protein sorting-associated protein 13D; Lymphoid-specific helicase, Histone H2B type 1-A, ankyrin repeat domain-containing protein 11, Histone H2B, Keratin, type II cytoskeleton 6A, Nip-B-like protein, ATPase family AAA domain-containing protein 1, mitochondrial dynamin-like 120 kDa protein, Striated muscle preferentially expressed protein kinase, disc large-associated protein 1, Cytokinesis protein 11, Myogaline, putative uncharacterized protein DKFZp666F1010; putative uncharacterized protein DKFZp666M0710; putative uncharacterized protein DKFZp686P17171, Activation-dependent raft-mobilizing ADAP-like phosphoprotein, unconventional myosin-XVIIIa, Nucleolin, isoform CRA_b, Nucleolin, isoform CRA_c, Nucleolin (protein C23), Keratin, type I cytoskeleton 9, Cilia and flagella-associated proteins 58; terminal uridylyltransferase 4, WD repeat and SOCS box-containing protein 1, Centromere protein J, FRAS1-related extracellular matrix protein 1, Machoilin, NARG1 protein, calpastatin isoform a variant, Ribosomal protein L5 variants, Insulin-like growth factor binding protein 2 variant, Rho-associated protein kinase 2, 60S acidic ribosomal protein P0, elongation factor 1-alpha, Glial fibrillary acidic protein, beta-actin-like protein 2, Fanconi anemia, complementation group D2, isoform CRA_b, Fanconi anemia group D2 protein, IQ and AAA domain-containing protein 1, Protein FAM92A, Teneurin-3, Coiled-coil and C2 domain-containing protein 2A, the putative uncharacterized protein C3orf49, Heterogeneous nuclear ribonucleoprotein A1, Dynein heavy chain 2, axoneme, Ribosomal proteins, RSL1D1 protein, ATP-dependent DNA helicase PIF1, Keratin, type II cytoskeleton 73, ATP synthase subunit beta, cDNA FLJ39263 fis, clone OCBBF2009571, ATP-dependent RNA helicase A, 290 kDa centrosomal protein, CEP290 protein, Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1, La-related protein 7, pumilio homolog 3, Ovochymase-1, INO80 complex subunits, RWD domain-containing protein 3, Dual 3',5'-cyclic-AMP and -GMP phosphodiesterase 11A, histone-lysine N-methyltransferase ASH1L, ATP-binding cassette subfamily C member 8, cilia and flagella-associated proteins 157; M-phase phosphoprotein 1, isoform CRA_a, 60S ribosomal protein L3, A kinase (PRKA) anchor protein 6, isoform CRA_b; Lamin B2, isoform CRA_b, Nuclear mitotic apparatus protein 1, isoform CRA_a; Coiled-coil domain-containing protein 40, TBK1 binding protein 1, isoform CRA_a, Membrane-associated polypeptide 2, isoform beta / gamma, KIAA1212, isoform CRA_a, Histone H2B, Septin 11, isoform CRA_b, Heterogeneous nuclear ribonucleoprotein A3, isoform CRA_a, MORC family CW-type zinc finger protein 4, cytoplasmic dynein heavy chain 1, unconventional myosin VIIb, Protein unc-13 homolog C, HSPA1L, Heat shock 70 kDa protein 1-like, Laminin subunit beta-4, leucine-rich repeat-containing protein 43, Protocadherin Fat 3, Death-associated protein kinase, ADP / ATP translocase 2, SLC25A5 protein, ADP / ATP translocase 3, SLC25A6 protein, HNRPR protein, Poly[ADP-ribose]polymerase (PARP), Poly[ADP-ribose]polymerase 1, Protein AHNAK2, Myb-binding protein 1A, leucine-rich repeat and IQ domain-containing protein 3, cDNA FLJ76880, cDNA FLJ76071, Maintenance of chromosomal protein structure, coiled-coil domain-containing protein 180, dynein heavy chain 17, axoneme; Histone H2A, cDNA, FLJ95007, Protein phantom, Mpv17-like protein, Protein CASP, cDNA FLJ44920 fis, clone BRAME 3011501, ATPase family AAA domain-containing protein 3A, cDNA FLJ35376 fis, clone SKMUS2004044, SNF2LT, dynein heavy chain 6, axoneme; Nuclear export mediator factor NEMF, cDNA FLJ51361, cDNA FLJ57651, cDNA FLJ50007, cDNA FLJ52940, Protein RUFY3, leucine-rich repeat and coiled-coil domain-containing protein 1, cDNA FLJ60374, G protein-coupled receptor 176, cDNA FLJ61500, Small nuclear ribonucleoprotein Sm D3, cDNA FLJ54081, cDNA FLJ58017, Piwi-like protein 2, Serine / threonine-protein kinase N2, Dysferlin, putative uncharacterized protein DKFZp469P0721, Short transient receptor potential channel 4, GTP-binding nuclear protein Ran, HNRNPL protein, cDNA FLJ52713, cDNA FLJ50492, TBC1 domain family member 1, Non-POU domain-containing octamer binding proteins, 60S ribosomal protein L31, cDNA FLJ51382, Protein Jumonji, von Willebrand factor A domain-containing protein 5B2, VWA5B2 protein, 128 kDa centrosomal protein, proliferation marker protein KI-67; Ribosomal protein L19, RPL27 / NME2 fusion protein, TPR and ankyrin repeat-containing protein 1, cDNA FLJ90678 fis, clone PLACE1005736, pleckstrin homology domain-containing family A member 1, SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5; Golgin subfamily B member 1, Heterogeneous nuclear ribonucleoprotein D-like, and Heterogeneous nuclear ribonucleoprotein D-like A pharmaceutical composition for inhibiting the expression of a gene encoding a protein selected from the group consisting of:

5. The pharmaceutical composition of claim 1, further comprising a second active agent selected from geldanamycin, alvespimycin, entasobrine, androstanolone / dihydrotestosterone, spermine, cortisone, quercetin, acetohexamide, resveratrol, doxorubicin, ruxolitinib, roscovitine / seliciclib, sildenafil, teniposide / Vumon, memantine, etoposide, INO 1001, diazoxide, tedisamil, glimepiride, epirubicin, anamycin, As(2)O(3), repaglinide, TOP53, elsamitrucin, ketamine, NK109, thifenazoxide, olaparib, and intopricin.

6. The pharmaceutical composition described in any one of claims 1 to 5, wherein the active agent is selected from roscovitine, geldanamycin, acetohexamide, and ruxolitinib, or a combination thereof.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein the nucleic acid delivery vehicle is a nanoparticle containing the gene.

8. The pharmaceutical composition of claim 1, wherein the compressed nucleic acid nanoparticles comprise a nucleic acid plasmid and a polymer, and the nanoparticles are compressed in the presence of a counterion selected from trifluoroacetate (TFA), bromide, bicarbonate, glutamate, hydroxyl ion, or a combination thereof.

9. 9. The pharmaceutical composition of claim 8, wherein the nucleic acid is single-stranded or double-stranded DNA.

10. 10. The pharmaceutical composition according to claim 8 or 9, wherein the polymer is a polycation.

11. The pharmaceutical composition according to any one of claims 8 to 10, wherein the polycation is a lipid.

12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the polycation is a cysteine ​​(C)-containing polymer of lysine (K) such as CK30, a cysteine ​​(C)-containing polymer of arginine (R) such as CR30, or a combination thereof.

13. The pharmaceutical composition according to any one of claims 8 to 12, wherein the polycation is selected from a cysteine ​​(C)-containing polymer of lysine (K) and arginine (R), or a polymer of lysine mixed with arginine, conjugated to PEG, and complexed with a nucleic acid.

14. The pharmaceutical composition according to any one of claims 8 to 13, wherein the polymer is a lysine polymer, preferably a polyethylene glycol (PEG)-substituted lysine polymer or a polyethylenemine.

15. The pharmaceutical composition of claim 1, wherein the compressed nucleic acid nanoparticles have a shape selected from rod-shaped, elliptical, spheroidal, or toroidal.

16. The pharmaceutical composition of claim 1, wherein the compressed nucleic acid nanoparticles have a diameter of 25 to 400 nm in length as measured by electron microscopy.

17. 1. A composition comprising: a) a compacted nucleic acid nanoparticle according to any one of claims 8 to 16; and b) Sonolisib (steroid lactone), LY294002 (benzopyran), TG100115 (pteridine), trifluoperazine (benzothiazine, phenothiazine), CEP5214 (indole derivative, pyrrolocarbazole), afuresertib (substituted benzene, phenethylamine, amphetamine), cationic vemurafenib (aryl-phenylketone), suramin (benzene derivative, benzanilide), uprosertib (benzene derivative, phenethylamine, amphetamine), flutamide (benzene derivative, trifluoromethylbenzene), enzastaurin (indole derivatives, N-alkyl indoles), fasudil (isoquinoline derivatives), ruboxistaurin (macrolactams), pentamidine (phenol ethers), uprosertib (benzene derivatives, phenethylamines, amphetamines), lestaurtinib (indole derivatives, indolocarbazoles), adenine (imidazole pyrimidines), pimozide (diphenylbutylpiperidines), chlorpromazine (phenothiazines), afrestib (benzene derivatives, phenethylamines, amphetamines), and combinations thereof; and Optionally, the composition comprises one or more agents selected from doxorubicin, ruxolitinib, roscovitine / seliciclib, sildenafil, teniposide / Vumon, memantine, etoposide, INO 1001, diazoxide, tedisamil, glimepiride, epirubicin, anamycin, As(2)O(3), repaglinide, TOP53, elsamitrucin, ketamine, NK109, thifenazoxide, olaparib, and intopricin.

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