Process for producing DNA formulations using lipopolymer delivery systems

Abstract

Certain embodiments of the present invention relate to methods of making a concentrated nucleic acid composition, the methods comprising: (a) combining (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium, wherein the nucleic acid complexes with the cationic lipopolymer, thereby forming a nucleic acid mixture having a concentration of at least 0.15 mg / mL; and (b) concentrating the nucleic acid mixture of (a) by tangential flow filtration to form a concentrated nucleic acid composition having a concentration of at least 0.75 mg / mL, wherein the concentrated nucleic acid composition is suitable for pharmaceutical use, storage at or below -20°C, 4°C, and / or lyophilization. The present invention further relates to pharmaceutical compositions prepared using the disclosed methods and methods employing same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 482,441, filed January 31, 2023, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the fields of molecular biology and biochemistry. For example, the present disclosure relates to methods and processes for preparing formulations comprising nucleic acids and lipopolymers. [Background technology]

[0003] GEN-1 is a gene-based cancer therapy containing a human IL-12 gene expression plasmid and a synthetic lipopolymer delivery system (Thaker, Premal H. et al., Future Oncol. (2019) 15(4), 421-438). GEN-1 can be delivered intraperitoneally (ip) to produce localized and sustained levels of IL-12 at the tumor site in patients with advanced ovarian cancer. GEN-1 can be administered alone or in combination with chemotherapy. GEN-1 contains a plasmid vector encoding the p35 and p40 subunits of the human IL-12 gene, each under the control of the cytomegalovirus (CMV) promoter, and a synthetic lipopolymer delivery system, polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC). GEN-1 is currently formulated as a lyophilized powder that can be reconstituted at the bedside to doses up to approximately 0.5 mg / mL.

[0004] Pharmaceuticals containing DNA that self-assemble into nanoparticles often exhibit poor stability, especially when the formulation is an aqueous suspension.In these formulations, DNA with synthetic vectors typically aggregates over time, especially at the concentrations required for optimal administration in clinical settings.These formulations are often difficult to prepare at DNA concentrations above 0.3 mg / mL, which limits their commercial use, especially for local delivery, where volume constraints limit flexible administration.DNA aggregation can reduce or eliminate the activity of DNA, making the composition unsuitable for therapeutic use.

[0005] Freeze-drying is a useful method for improving the long-term stability of several pharmaceuticals. However, this process presents challenges for drying DNA complexes with synthetic vectors, as they tend to alter their physicochemical properties and result in aggregation and loss of transfection upon reconstitution.

[0006] Approaches have been attempted to prevent formulation aggregation and damage during lyophilization. In some cases, lyophilization of DNA complexes in the presence of cryoprotectants such as low-molecular-weight sugars, dextran, and polyethylene glycol can provide better product stability, but this approach is not thought to improve administration flexibility. The addition of sugars is often the most commonly used approach for this purpose. Many tested sugars have been found to prevent formulation damage and particle aggregation to some extent, but the quality of this effect varies depending on the type of sugar and delivery vector used.

[0007] While lyophilization offers some improvement in formulation shelf life, the conditions required to produce lyophilized DNA products allow for only limited pharmaceutical applications. Even the most effective cryoprotectant sugars require very high sugar / DNA molar ratios (typically greater than 1000:1) for stability. As a result, lyophilized products often must be diluted by very large factors to obtain isotonic formulations, resulting in a reduction in the final DNA concentration to that of the DNA before lyophilization. For many cationic carriers, the final DNA concentration can typically be approximately 0.1–0.2 mg / mL, often less than 0.1 mg / mL.

[0008] A method for achieving compositions with higher DNA concentrations (e.g., 0.15 mg / mL to 0.5 mg / mL) is disclosed in U.S. Patent No. 9,144,546. This process can yield 6 mg of lyophilized product / vial reconstituted to 0.5 mg / mL in 12 mL of sterile water for injection (WFI). For example, approximately 100 mg / mL 2 The clinical dose was 1.8m on average in subjects. 2 For a surface area of ​​1000 μg, approximately 180 mg of DNA is required. A 180 mg DNA dose would require 30 vials (50 mL vials) to reconstitute, with each vial carrying 6 mg of DNA. This is a commercially difficult preparation.

[0009] While low-concentration formulations are sufficient for in vitro studies, their clinical application may be limited by the large volumes required for optimal administration. Improved methods for preparing DNA therapeutics, such as for concentrated, temperature-stable, biologically active DNA formulations, are needed. Summary of the Invention

[0010] Certain aspects of the present disclosure relate to methods of making a concentrated nucleic acid composition, the methods comprising: (a) combining (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a filler excipient in an aqueous medium to complex the nucleic acid with the cationic lipopolymer, thereby forming a nucleic acid mixture having a concentration of at least 0.10-0.2 mg / mL (e.g., at least 0.15 mg / mL, e.g., 0.15-0.2 mg / mL); and (b) concentrating the nucleic acid mixture by tangential flow filtration to form a concentrated nucleic acid composition having a concentration of at least 0.7 mg / mL-0.8 mg / mL (e.g., at least 0.75 mg / mL, e.g., 0.75-1 mg / mL), wherein the concentrated nucleic acid composition is suitable for pharmaceutical use, storage at about 4°C or below, storage at about -20°C or below, and / or lyophilization.

[0011] In some embodiments, the recovery of nucleic acids complexed with the cationic lipopolymer after tangential flow filtration is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%.

[0012] In some embodiments, the method further comprises storing the concentrated nucleic acid composition at about 4° C. or below for at least 24 hours, at least 1 week, at least 1 month, at least 1 year, or at least 30 months.

[0013] In some embodiments, the method further comprises storing the concentrated nucleic acid composition at about -20°C or below for at least 24 hours, at least 1 week, at least 1 month, or at least 1 year.

[0014] In some embodiments, the concentrated nucleic acids are storage stable, ie, one or more of particle size, DNA concentration, PPC concentration, PPC / DNA ratio, osmolality, and / or pH are stable for at least 30 months at 4° C. or below.

[0015] In some embodiments, the nucleic acid mixture has a concentration of at least 0.15 mg / mL, hi some embodiments, the nucleic acid mixture has a concentration of between about 0.15 mg / mL and 0.2 mg / mL.

[0016] In some embodiments, the concentrated nucleic acid composition has a concentration of at least 0.75 mg / mL, hi some embodiments, the concentrated nucleic acid composition has a concentration of between about 0.75 mg / mL and 1 mg / mL.

[0017] In some embodiments, the method further includes (c) lyophilizing the concentrated nucleic acid composition having a concentration of at least 0.7 mg / mL to 0.8 mg / mL (e.g., at least 0.75 mg / mL) in a volume of 20 to 40 mL, thereby forming a lyophilized formulation containing at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, or at least 30 mg of DNA plasmid (e.g., about 10 to 50 mg).

[0018] In some embodiments, the lyophilized formulation contains 10 to 50 mg (eg, about 30 mg) of DNA plasmid.

[0019] In some embodiments, the method further comprises (d) reconstituting the lyophilized formulation in a diluent (e.g., water, 5% dextrose, or saline), thereby forming a reconstituted composition.

[0020] In some embodiments, the nucleic acid mixture comprises GEN-1 nanoparticles.

[0021] In some embodiments, the ratio of amine nitrogens in the cationic polymer backbone to phosphate in the nucleic acid is from about 10:1 to about 100:1.

[0022] In some embodiments, the filler comprises a sugar, a sugar alcohol, a starch, a cellulose, or a combination thereof. In some embodiments, the filler comprises one or more of lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, Ficoll, sodium chloride, calcium phosphate, calcium carbonate, and / or polyethylene glycol. In some embodiments, the filler comprises lactose.

[0023] Certain embodiments of the present disclosure provide methods that can process low-concentration nucleic acid compositions (e.g., 0.15 mg / mL) using a method that includes a tangential flow filtration (TFF) step to prepare concentrated (e.g., 0.30 mg / mL to 0.75 mg / mL or greater) nucleic acid compositions without affecting the physicochemical or biological properties of the nucleic acid or nucleic acid composition. In some embodiments, the nucleic acid composition includes a DNA plasmid containing a polynucleotide encoding a therapeutic protein (e.g., a hIL-12-encoding nucleic acid) and a lipopolymer (e.g., PPC). In some embodiments, the nucleic acid composition includes GEN-1. In some embodiments, GEN-1 can be concentrated to a solution containing 0.30 mg / mL to 0.75 mg / mL according to the methods of the present disclosure.

[0024] In some embodiments, the (pre-lyophilized) concentrated nucleic acid composition at any concentration (e.g., 0.30 mg / mL to 0.75 mg / mL or higher) can be used directly as a dosing regimen for administration to a subject. In some embodiments, the concentrated nucleic acid composition can be stored at -20°C.

[0025] In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing 10 mg to 100 mg (e.g., 10 mg to 100 mg, 10 mg to 90 mg, 10 mg to 80 mg, 10 mg to 70 mg, 10 mg to 60 mg, 10 mg to 50 mg, 10 mg to 40 mg, or 10 mg to 30 mg) of DNA. In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing 10 mg to 50 mg (e.g., 10 mg to 40 mg, 15 mg to 30 mg, 20 mg to 40 mg, 25 mg to 40 mg, 25 mg to 35 mg, or about 30 mg) of DNA. In some embodiments, concentrated lyophilized nucleic acid compositions can be stably stored (e.g., 1 year, at least 2 years, at least 3 years).

[0026] In some embodiments, the concentrated lyophilized nucleic acid composition can be reconstituted (e.g., in water, 5% dextrose, or saline). In some embodiments, the concentrated nucleic acid composition allows for a wide range of dosing regimens in vivo after reconstitution of the lyophilized composition.

[0027] In some embodiments, the concentrated lyophilized nucleic acid composition can be reconstituted at 0.5 mg / mL, or 1 mg / mL, or 1.5 mg / mL, 2 mg / mL, or 2.5 mg / mL, or 3 mg / mL, or 3.5 mg / mL, or 4 mg / mL, or 4.5 mg / mL, or 5 mg / mL, or 5.5 mg / mL, or 6 mg / mL, or 6.5 mg / mL, or 7 mg / mL, or 7.5 mg / mL, or 8 mg / mL, or 8.5 mg / mL, or 9 mg / mL, or 9.5 mg / mL, or 10 mg / mL.

[0028] Certain aspects of the present disclosure relate to concentrated nucleic acid compositions (pre-lyophilized and post-lyophilized) prepared according to the methods disclosed herein.

[0029] Certain aspects of the present disclosure relate to pharmaceutical compositions comprising concentrated nucleic acid compositions (pre-lyophilized and post-lyophilized) prepared according to the methods disclosed herein. In some aspects, the pharmaceutical compositions comprise concentrated nucleic acid compositions or reconstituted compositions prepared according to the methods comprising TFF disclosed herein.

[0030] In some embodiments, the pharmaceutical composition comprises more than 6 mg (e.g., 10-100 mg, 10-90 mg, 10-80 mg, 10-70 mg, 10-60 mg, 10-50 mg, 10-40 mg, 20-50 mg, 20-45 mg, 20-40 mg, 20-25 mg, or 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of less than 70 mL (e.g., 20-70 mL, 20-60 mL, 20-50 mL, or 20-45 mL) of diluent. In some embodiments, the pharmaceutical composition comprises more than 6 mg (e.g., 10-50 mg, 10-40 mg, 20-50 mg, 20-25 mg, 20-40 mg, or 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of less than 50 mL (e.g., 20-45 mL) of diluent. In some embodiments, the pharmaceutical composition comprises about 30 mg of DNA plasmid (complexed with PPC) in a volume of 20 to 45 mL (e.g., about 40 mL) of diluent (e.g., water, 5% dextrose, or saline). In some embodiments, the pharmaceutical composition comprises about 50 mg of DNA plasmid (complexed with PPC) in a volume of 20 to 45 mL (e.g., about 20 mL) of diluent (e.g., water, 5% dextrose, or saline).

[0031] In some embodiments, the pharmaceutical composition is in a container capable of holding up to 100 mL, 150 mL, 200 mL, 250 mL, or 500 mL (e.g., a 250 mL vial) containing 10-50 mg, 10-40 mg, 20-50 mg, 20-45 mg, 20-40 mg, 20-25 mg, or 25-35 mg of DNA in solution (e.g., 200 mL), where the concentration of DNA is at least 0.7 mg / mL to 0.8 mg / mL (e.g., at least 0.75 mg / mL).

[0032] In some embodiments, the pharmaceutical composition is used to prepare a dose of 100-200 mg (e.g., 180 mg) of plasmid DNA (complexed with PPC) in less than 300 mL (e.g., 200-300 mL) of diluent (e.g., water, 5% dextrose, or saline).

[0033] Certain embodiments of the present disclosure relate to kits that include a first vial (e.g., a 250 mL vial) containing a nucleic acid mixture comprising (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide, (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC), and (iii) a bulking agent in an aqueous medium, wherein the nucleic acid is complexed with the cationic lipopolymer, and the nucleic acid mixture comprises at least 25 mg to 50 mg (e.g., about 30 mg) of the DNA plasmid, and optionally further includes a second vial containing a diluent (e.g., about 20 to 45 mL).

[0034] Certain embodiments of the present disclosure relate to kits comprising a vial containing a nucleic acid mixture comprising: (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium, wherein the nucleic acid is complexed with the cationic lipopolymer and the nucleic acid mixture is in aqueous solution at a concentration of at least 0.7 mg / mL to 0.8 mg / L (e.g., at least 0.75 mg / mL).

[0035] In some embodiments, the vial (e.g., the first vial and / or the second vial) can hold up to 100 mL, 150 mL, 200 mL, 250 mL, or 500 mL (e.g., a 250 mL vial). In some embodiments, the vial contains a volume of diluent (e.g., water, 5% dextrose, or saline) of 200-300 mL (e.g., about 200 mL). In some embodiments, the vial contains 10-50 mg, 10-40 mg, 20-50 mg, 20-25 mg, 20-40 mg, or 25-35 mg (e.g., about 30 mg) of DNA plasmid (complexed with PPC). In some embodiments, the first vial and / or the second vial can hold up to about 100 mL, about 150 mL, about 200 mL, about 250 mL, or about 500 mL (e.g., a 250 mL vial). In some embodiments, the second vial contains about 200-300 mL (e.g., about 200 mL) of diluent (e.g., water, 5% dextrose, or saline) in a volume of about 10-50 mg, about 10-40 mg, about 20-50 mg, about 20-25 mg, about 20-45 mg, about 20-40 mg, or about 25-35 mg (e.g., about 30 mg) of DNA plasmid (complexed with PPC).

[0036] In some embodiments, the kit can be stored at -20°C or below.

[0037] Certain embodiments of the present disclosure relate to methods comprising a mixture of a cationic lipopolymer and at least about 10 mg / mL of nucleic acid, the mixture being suspended in an isotonic solution. The cationic lipopolymer comprises a cationic polymer backbone having cholesterol and polyethylene glycol groups independently covalently attached thereto. The molar ratio of cholesterol to cationic polymer backbone is within the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to cationic polymer backbone is within the range of about 0.1 to about 10.

[0038] The method may further include a bulking agent. In certain embodiments, the mixture of nucleic acid and lipopolymer forms a complex.

[0039] In certain aspects, the method comprises condensing nucleic acids by tangential flow filtration.

[0040] In another aspect, the present disclosure provides a method for lyophilizing nucleic acids and lipopolymers. The lyophilization method of the present disclosure includes a mixture of a bulking agent, a condensed nucleic acid, and a cationic lipopolymer. As described above, the cationic lipopolymer comprises a cationic polymer backbone having cholesterol and polyethylene glycol covalently attached thereto, wherein the molar ratio of cholesterol to the cationic polymer backbone is within the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to the cationic polymer backbone is within the range of about 0.1 to about 10. In certain aspects, the lyophilization method produces at least about 30 mg of nucleic acid and lipopolymer.

[0041] In another embodiment, the present disclosure provides a method comprising a mixture of a cationic lipopolymer and at least about 0.75 mg / mL of nucleic acid, wherein the mixture is suspended in an isotonic solution. The cationic lipopolymer comprises a cationic polymer backbone having cholesterol and polyethylene glycol groups independently covalently attached thereto. The molar ratio of cholesterol to the cationic polymer backbone is within the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to the cationic polymer backbone is within the range of about 0.1 to about 10. The method may further comprise a bulking agent. In certain embodiments, the mixture of nucleic acid and lipopolymer forms a complex. In certain embodiments, the method comprises condensing the nucleic acid by tangential flow filtration. In certain embodiments, the condensed nucleic acid is stored at a temperature of about 4°C or below or at least about -20°C (e.g., about 4°C to about -20°C).

[0042] The present disclosure further provides a method for using the compositions described herein in the treatment of diseases and / or disorders, for example, by transfecting various cells and tissues.A particular embodiment of the present disclosure relates to a method for treating cancer in a subject, comprising administering to a subject in need of cancer treatment a pharmaceutical composition prepared by the method disclosed herein.In some embodiments, the pharmaceutical composition is formulated for intratumoral, intraperitoneal, intravesicular, intravenous, intraarterial, intratracheal, intrahepatic portal vein, intracranial, intramuscular, or intraarticular administration.In some embodiments, the cancer is ovarian cancer. [Brief explanation of the drawings]

[0043] [Figure 1A]

[0023] Figure 1 shows a schematic diagram of the process for preparing a lyophilized formulation of plasmid IL-12 DNA and PEI-PEG-cholesterol polymer (PPC). The process is shown using a static mixer. [Figure 1B] Figure 1B shows a schematic of the process for preparing a lyophilized formulation of plasmid IL-12 DNA and PEI-PEG-cholesterol polymer (PPC). The process further includes a tangential flow filtration (TFF) step and optional prior lyophilization storage, resulting in a 5-fold higher yield of DNA / vial compared to the process in Figure 1A.

[0044] [Figure 2A] 1 shows a graph of particle size and osmolality for nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x. Particle size and osmolality for 1x and 5x concentrations are shown. [Figure 2B] Graphs of particle size and osmolality of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x are shown. Particle size and polydispersity of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 10 L formulation scale are shown. [Figure 2C]Graphs of particle size and osmolality of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x are shown. Osmolality and pH of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 10 L formulation scale are shown. [Figure 2D] Graphs of particle size and osmolality of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x are shown. Osmolality of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 20 L formulation scale is shown. [Figure 2E] Graphs of particle size and osmolality of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x are shown. Diameters of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 20 L formulation scale are shown.

[0045] [Figure 3A] Figure 1 shows DNA recovery after a tangential flow filtration (TFF) process for PEI-PEG-cholesterol polymer (PPC) and nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x. The PPC concentration and DNA ratios for 1x and 5x concentrations are shown. [Figure 3B] Figure 1 shows DNA recovery after tangential flow filtration (TFF) of PEI-PEG-cholesterol polymer (PPC) and 1x, 2x, 3x, 4x, and 5x concentrated nucleic acid formulations. Figure 2 shows PPC and DNA concentrations of 1x, 2x, 3x, 4x, and 5x concentrated nucleic acid formulations from a 10 L formulation scale. [Figure 3C] Figure 1 shows DNA recovery after tangential flow filtration (TFF) process for PEI-PEG-cholesterol polymer (PPC) and nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x. Figure 2 shows PPC / DNA recovery and PPC / DNA ratios for nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 10 L formulation scale. [Figure 3D]Figure 1 shows DNA recovery after tangential flow filtration (TFF) of PEI-PEG-cholesterol polymer (PPC) and 1x, 2x, 3x, 4x, and 5x concentrated nucleic acid formulations. Figure 2 shows PPC and DNA concentrations of 1x, 2x, 3x, 4x, and 5x concentrated nucleic acid formulations from a 20 L formulation scale.

[0046] [Figure 4A] 1 shows gel electrophoresis of nucleic acid formulations concentrated 1x, 2x, 3x, 4x and 5x. 1 shows gel electrophoresis of nucleic acid formulations concentrated 1x and 5x. [Figure 4B] Gel electrophoresis of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 10 L formulation scale with and without dextran sulfate is shown. [Figure 4C] Gel electrophoresis of nucleic acid formulations concentrated 1x, 2x, 3x, 4x, and 5x from a 20 L formulation scale with and without dextran sulfate is shown.

[0047] [Figure 5A] 1 shows the in vitro expression levels of IL-12 in COS-1 cells from nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x. 1 shows the in vitro expression levels of IL-12 in COS-1 cells from nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x. 1 shows the expression levels of IL-12 at different doses from 1x and 5x concentrations. [Figure 5B] 1 shows the in vitro expression levels of IL-12 in COS-1 cells from nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x concentrations. 1 shows the expression levels of IL-12 at different doses from 1x, 2x, 3x, 4x, and 5x concentrations at an 8.5 L formulation scale. [Figure 5C] 1 shows the in vitro expression levels of IL-12 in COS-1 cells from nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x concentrations. 10 L formulation scale IL-12 expression levels at different doses from 1x, 2x, 3x, 4x, and 5x concentrations are shown. [Figure 5D]1 shows the in vitro expression levels of IL-12 in COS-1 cells from nucleic acid formulations concentrated at 1x, 2x, 3x, 4x, and 5x concentrations. 1 shows the expression levels of IL-12 at different doses from 1x, 2x, 3x, 4x, and 5x concentrations at a 20 L formulation scale.

[0048] [Figure 6A] Graph of hIL-12 expression levels (total pg) in tumor-bearing mice following treatment with 1x, 2x, 3x, 4x and 5x concentrated nucleic acid formulations is shown. [Figure 6B] Graph of hIL-12 expression levels (pg / mL) in tumor-bearing mice after treatment with 1x, 2x, 3x, 4x, and 5x concentrated nucleic acid formulations is shown.

[0049] [Figure 7A] 1 shows a graph of hIL-12 expression levels (total pg) in tumor-bearing mice after treatment with nucleic acid formulations from different lots. [Figure 7B] 1 shows a graph of hIL-12 expression levels (pg / mL) in tumor-bearing mice after treatment with nucleic acid formulations from different lots.

[0050] [Figure 8A] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage for up to 30 months at 4° C. and −20° C. Particle size of the reference standard stored at −80° C. is shown, and stored at 5x concentration at 4° C. and −20° C. for up to 30 months. [Figure 8B] Graphs of nucleic acid / cationic lipopolymer compositions are shown after storage at 4° C. and −20° C. for up to 30 months. DNA concentrations of reference standards stored at −80° C. are shown, and at 5x concentrations stored at 4° C. and −20° C. for up to 30 months. [Figure 8C] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage at 4° C. and −20° C. for up to 30 months. The PPC concentrations of the reference standard stored at −80° C. are shown, and at 5x concentrations stored at 4° C. and −20° C. for up to 30 months. [Figure 8D]1 shows a graph of nucleic acid / cationic lipopolymer composition after storage for up to 30 months at 4° C. and −20° C. The PPC / DNA ratio of the reference standard stored at −80° C. is shown, and stored at 5x concentration at 4° C. and −20° C. for up to 30 months. [Figure 8E] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage for up to 30 months at 4° C. and −20° C. The osmolality of the reference standard stored at −80° C. is shown, and at 5x concentration stored at 4° C. and −20° C. for up to 30 months. [Figure 8F] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage for up to 30 months at 4° C. and −20° C. The pH of the reference standard stored at −80° C. is shown, and stored at 5x concentration at 4° C. and −20° C. for up to 30 months. [Figure 8G] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage at 4° C. and −20° C. for up to 30 months. IL-12 expression levels at different doses from a reference standard stored at −80° C., a 1× concentration stored at −20° C. for up to 30 months, and a 5× concentration stored at 4° C. and −20° C. for up to 30 months are shown. [Figure 8H] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage at 4° C. and −20° C. for up to 30 months. It shows the relative potency of 5x concentrations stored at 4° C. and −20° C. for up to 30 months compared to a reference standard stored at −80° C. [Figure 8I] 1 shows a graph of nucleic acid / cationic lipopolymer compositions after storage for up to 30 months at 4° C. and −20° C. 2 shows IL-12 expression levels at different doses from 1× and 5× concentrations stored at 4° C. and −20° C. for a period of 30 months. DETAILED DESCRIPTION OF THE INVENTION

[0051] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0052] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0053] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an"), and the terms "one or more" and "at least one" may be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single." In other embodiments, the term "a" or "an" includes "two or more" or "plurality."

[0054] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10% above or below (even higher or lower).

[0055] Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. The numerical ranges described are inclusive of the numbers defining the range and include each integer within the defined range.

[0056] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is understood that each intervening integer and each fractional integer between the stated upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can be independently included in or excluded from the range, and each range in which either, neither, or both limits are included is also encompassed within the present disclosure. Thus, ranges described herein are understood to be shorthand for all values ​​within the range, including the stated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0057] Where a value is explicitly recited, it is understood that values ​​of approximately the same quantity or amount as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded alone or in any combination with other alternatives are also hereby disclosed. Multiple elements of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0058] The term "and / or" used herein should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0059] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0060] As used herein, the term "nucleic acid" refers to DNA and RNA, as well as their synthetic homologs. In addition, nucleic acids can vary in size, ranging from oligonucleotides to chromosomes. Nucleic acids can be of human, animal, plant, bacterial, viral, or synthetic origin. They can be obtained by any technique known to those skilled in the art.

[0061] A "coding sequence," or a sequence "encoding" a particular molecule (e.g., a therapeutic molecule), is a nucleic acid that is transcribed (in the case of DNA) or translated into a polypeptide (in the case of mRNA) in vitro or in vivo when operably linked to appropriate regulatory sequences, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid but is considered to be part of the coding region; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. A transcription termination sequence is typically located 3' to the coding sequence.

[0062] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence operably linked to the promoter / regulatory sequence, which is necessary for expressing a gene product. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. The term "inducible" promoter means that, when operably linked to a polynucleotide encoding a particular gene product, an inducer corresponding to the promoter essentially results in the production of the gene in a cell only when the nucleotide sequence of the product is present in the cell.

[0063] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any expression of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. It includes, but is not limited to, the transcription of a gene into messenger RNA (mRNA) and the translation of such mRNA into a polypeptide. Expression of a gene produces a "gene product."

[0064] As used herein, a gene product can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcription product. Gene products as described herein further include nucleic acids that have post-transcriptional modifications, e.g., polyadenylation, or polypeptides that have post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.

[0065] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide. In some embodiments, the expression vector is a DNA plasmid.

[0066] As used herein, the term "operably linked" or "transcriptional control" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the latter. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when they are placed in a functional relationship. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Operable linked DNA sequences can be adjacent to each other, for example, when two protein coding regions need to be linked, the DNA sequences are in the same reading frame.

[0067] As used herein, the term "transfer vector" refers to a composition containing an isolated nucleic acid and a substance that can be used to deliver the isolated nucleic acid into the interior of a cell.Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.The term transfer vector should also be interpreted as including non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc.

[0068] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In certain aspects, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur in subsequent generations, or to integration of the nucleic acid molecule into the host cell genome.

[0069] As used herein, the terms "condensed nucleic acid" and "partially condensed nucleic acid" refer to nucleic acids contacted with a cationic lipopolymer of the present disclosure. In certain embodiments, the condensed nucleic acid remains in contact with the cationic lipopolymer. Condensed nucleic acids typically occupy a significantly smaller volume than non-condensed nucleic acids. However, it is recognized that the amount of condensed nucleic acid may vary depending on the local environment (e.g., a lipid environment as opposed to an aqueous environment). In various embodiments of the present disclosure, the condensed nucleic acid is in a nucleic acid or cationic lipopolymer nanoparticle having a size of about 50 nm to about 300 nm, more preferably about 50 to 200 nm, and even more preferably about 50 to 150 nm. "Partially condensed nucleic acid" refers to a nucleic acid contacted with a cationic lipopolymer of the present disclosure that is not fully condensed but still occupies a significantly smaller volume than non-condensed nucleic acid.

[0070] As used herein, the term "complex" refers to a nucleic acid associated with a lipopolymer, e.g., a cationic lipopolymer. The complex comprising the condensed nucleic acid and the cationic lipopolymer typically exists as a particle, e.g., a nanoparticle.

[0071] As used herein, the term "concentrated" refers to a composition that is reduced in dilution. In some embodiments, a concentrated composition comprises, for example, condensed DNA in an isotonic solution.

[0072] As used herein, the term "polymer backbone" refers to a collection of polymer backbone molecules having a weight-average molecular weight within a specified range. Therefore, when a molecule such as cholesterol is described as being covalently attached within any range of molar ratios, such ratio should be understood to represent the average number of cholesterol molecules attached to the collection of polymer backbone molecules. For example, when cholesterol is described as being covalently attached to the polymer backbone at a molar ratio of 0.5, on average, half of the polymer backbone molecules are attached with cholesterol. As another example, when cholesterol is described as being covalently attached to the polymer backbone at a molar ratio of 1.0, on average, one cholesterol molecule is attached to each polymer backbone molecule. However, in reality, in this case, some polymer backbone molecules may not have any cholesterol molecules attached, while other polymer backbone molecules may have multiple cholesterol molecules attached, and it should be understood that this ratio is derived from the average number of attached cholesterol molecules. The same reasoning applies to the molar ratio of polyethylene glycol to polymer backbone.

[0073] As used herein, the term "peptide" can refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another amino acid. Peptides of the present disclosure are not limited by length, and thus "peptide" can include polypeptides and proteins.

[0074] As used herein, the terms "covalent bond" and "covalently" refer to a chemical bond in which electrons are shared between a pair of atoms.

[0075] As used herein, a "composition" refers to a mixture of two or more compounds, elements, or molecules. In some embodiments, the term "composition" can be used to refer to a mixture of a nucleic acid and a delivery system (e.g., a cationic lipopolymer).

[0076] As used herein, "aqueous medium" or "aqueous solution" refers to a solution or mixture in which water is the carrier or solvent.

[0077] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, formulations and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0078] The term "excipient" refers to any substance that is not itself a therapeutic agent and that may be used in a composition for delivering an active therapeutic agent to a subject, or that may be combined with an active therapeutic agent to improve its handling or storage characteristics, or to enable or facilitate the formation of a dosage unit of the composition (e.g., to create a pharmaceutical composition). Excipients include, but are not limited to, solvents, penetration enhancers, humectants, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances used to form hydrogels. Any such excipients can be used in any dosage form according to the present disclosure. The aforementioned classes of excipients are not meant to be exhaustive but merely exemplary, as one of ordinary skill in the art will recognize that additional types and combinations of excipients can be used to achieve the desired goal of drug delivery. Excipients can be inert, inactive, and / or pharmaceutically non-active substances. Excipients can serve a variety of purposes.

[0079] Those skilled in the art can select one or more excipients for specific desired properties without undue burden through routine experimentation.The amount of each excipient used can vary within the range conventional in the art.The techniques and excipients that can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0080] As used herein, "N:P ratio" refers to the molar ratio of amine nitrogens in the functionalized cationic lipopolymer to phosphate groups in the nucleic acid.

[0081] As used herein, "physicochemical properties" refers to various properties such as, but not limited to, particle size and surface charge of the nucleic acid complex with the cationic polymer, pH and osmolality of the particle solution.

[0082] As used herein, the terms "transfect" and "transfection" refer to the transport of nucleic acids from the external environment of a cell to the internal environment of a cell, such as the cytoplasm and / or nucleus. It should be understood that in some embodiments, nucleic acids can be delivered to a cell after being encapsulated within, attached to, or incorporated into one or more cationic polymer / nucleic acid complexes. A specific transfection example delivers nucleic acids to the nucleus of a cell. Without being bound by any particular theory, it should be understood that nucleic acids can be delivered to a cell after being encapsulated within, attached to, or incorporated into one or more cationic polymer / nucleic acid complexes. A specific transfection example delivers nucleic acids to the nucleus of a cell. Nucleic acids include DNA and RNA and their synthetic analogs. Such nucleic acids include missense, antisense, nonsense, and proteinogenic nucleotides, as well as on-off and rate-regulating nucleotides that control the production of proteins, peptides, and nucleic acids. In particular, but not limited to, they may be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences, or synthetic or semi-synthetic sequences, and may be of natural or artificial origin. In addition, nucleic acids may vary in size, ranging from oligonucleotides to chromosomes. These nucleic acids may be of human, animal, plant, bacterial, viral, or synthetic origin. They may be obtained by any technique known to those skilled in the art.

[0083] As used herein, a "vial" or "container" refers to a vessel capable of containing or storing a substance. In some embodiments, a vial can be shaped like a tube or bottle and have a flat or rounded bottom. In some embodiments, a vial can be used to contain or store a liquid. In some embodiments, a vial can be made of glass or plastic. In some embodiments, a vial can be sealed with a lid or can have no lid. In some embodiments, a vial can hold a volume of at least 50 mL to 500 mL.

[0084] As used herein, "subject" refers to a mammal, particularly a mammal that can benefit from the administration of the compositions of the present disclosure. Examples of subjects include humans, and may also include other mammals such as mice, horses, pigs, cows, dogs, cats, rabbits, and aquatic mammals.

[0085] As used herein, the terms "administration," "administering," and "delivering" refer to the manner in which a composition is presented to a subject or cell. Administration to a subject can be achieved by various routes known in the art, such as oral, parenteral, transdermal, inhalation, implantation, etc. In some embodiments, oral administration can be achieved by swallowing, chewing, or inhaling an oral dosage form containing the composition. In some embodiments, parenteral administration can be achieved by injecting the composition intravenously, intraarterially, intramuscularly, intraarticularly, intrathecally, intraperitoneally, subcutaneously, intratumorally, etc. Injectables for such use can be prepared in conventional forms, as liquid solutions or suspensions, or in solid forms suitable for preparation as solutions or suspensions in liquid prior to injection, or as emulsions. Additionally, transdermal administration can be achieved by applying, sticking, rolling, attaching, injecting, pressing, rubbing, etc., a transdermal composition to the skin surface. These and additional administration methods are well known in the art. In one aspect, administration can include delivering the composition to a subject so that the composition circulates systemically and binds to target cells where it is taken up by endocytosis.

[0086] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0087] As used herein, the term "primary tumor" refers to the original or first tumor that formed in a subject's body.

[0088] As used herein, the terms "metastasis," "metastatic," "secondary tumor," or "metastatic tumor" refer to a cancer (e.g., tumor) formed by cancer cells derived from a primary cancer (e.g., tumor) that spreads to an additional location or area of ​​the body.

[0089] As used herein, the term "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells (e.g., malignant cells) in the body. Unregulated cell division and growth lead to the formation of malignant tumors that can infiltrate adjacent tissues through local spread and metastasize to distant parts of the body via the lymphatic system or bloodstream. In some embodiments, the methods of the present disclosure can be used to reduce the size of or treat primary or metastatic tumors. Conditions that can be treated or prevented by the methods of the present disclosure include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like. The methods of the present disclosure can achieve inhibition and / or reversal of the unwanted hyperproliferative cell growth involved in such conditions. In some embodiments, the cancer can be ovarian cancer.

[0090] As used herein, "ovarian cancer" refers to cancer that originates in or involves the ovaries, for example, originates in or involves the ovarian epithelium. As used herein, the term "cancer" or "tumor" refers to the uncontrolled growth of cells that interferes with the normal function of bodily organs and systems. A subject with cancer or tumor is one that has objectively measurable cancer cells present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer that migrates from its original location and disseminates to vital organs can ultimately lead to the death of the subject through functional deterioration of the affected organ. Ovarian cancer is typically treated by cytoreductive surgery (also referred to herein as "debulking") followed by chemotherapy.

[0091] As used herein, the term "combination therapy" refers to a therapy that includes more than one treatment (e.g., active agents or procedures). In some embodiments, the compositions of the combination therapy are formulated together in a single composition or as separate compositions.

[0092] As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to a wide variety of chemotherapeutic agents that may be used in accordance with the present disclosure. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" may connote a compound or composition administered in the treatment of cancer.

[0093] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in common lists for convenience. However, these lists should be construed as though each member of the list were individually identified as a separate and unique member. Accordingly, the individual members of such lists should not be construed as de facto equivalents of any other members of the same list solely based on their presentation in a common grouping in the absence of any contradictory designation.

[0094] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range format may be used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "about 1 to about 5" should be interpreted not only to include the explicitly recited values ​​of about 1 to about 5, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 2, 3, and 4, as well as subranges such as 1 to 3, 2 to 4, and 3 to 5, and 1, 2, 3, 4, and 5 individually. This same principle also applies to ranges reciting only a single numerical value as the minimum or maximum value. Furthermore, such interpretation should apply regardless of the breadth or character of the range described.

[0095] II. Methods for Preparing Nucleic Acid Compositions Certain aspects of the present disclosure relate to methods of making a concentrated nucleic acid composition, the method comprising: (a) combining (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium, wherein the nucleic acid complexes with the cationic lipopolymer, thereby forming a nucleic acid mixture having a concentration of at least 0.15 mg / mL; and (b) concentrating the nucleic acid mixture of (a) by tangential flow filtration to form a concentrated nucleic acid composition having a concentration of at least 0.75 mg / mL, wherein the recovery of the nucleic acid complexed with the cationic lipopolymer after tangential flow filtration is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%, forming a concentrated nucleic acid composition that is suitable for pharmaceutical uses, storage at -20°C or below, storage at 4°C or below, and / or lyophilization.

[0096] Certain embodiments of the present disclosure relate to methods of making a concentrated nucleic acid composition, the method comprising: (a) combining (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium to complex the nucleic acid with the cationic lipopolymer, thereby forming a nucleic acid mixture having a concentration of at least about 0.15 mg / mL; and (b) concentrating the nucleic acid mixture of (a) by tangential flow filtration to form a concentrated nucleic acid composition having a concentration of at least about 0.75 mg / mL, wherein the recovery of the nucleic acid complexed with the cationic lipopolymer after tangential flow filtration is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, and wherein the concentrated nucleic acid composition is suitable for pharmaceutical uses, storage at about −20° C. or below, storage at about 4° C. or below, and / or lyophilization.

[0097] In some embodiments, the method further comprises storing the concentrated nucleic acid composition at or below about −20° C. for at least 24 hours, at least 1 week, at least 1 month, or at least 1 year. In some embodiments, the method further comprises storing the concentrated nucleic acid composition at or below about −20° C. for at least about 24 hours, at least about 1 week, at least about 1 month, or at least about 1 year.

[0098] In some embodiments, the method further comprises storing the concentrated nucleic acid composition at or below about 4° C. for at least 24 hours, at least 1 week, at least 1 month, at least 1 year, or at least 30 months. In some embodiments, the method further comprises storing the concentrated nucleic acid composition at or below about 4° C. for at least about 24 hours, at least about 1 week, at least about 1 month, at least about 1 year, or at least about 30 months.

[0099] In some embodiments, the concentrated nucleic acids are storage stable, in which one or more of particle size, DNA concentration, PPC concentration, PPC / DNA ratio, osmolality, and / or pH are stable for at least 30 months at or below about 4° C.

[0100] In some embodiments, the methods provide concentrated nucleic acids with improved relative potency in 5x formulations compared to 1x formulations after 30 months of storage at 4°C.

[0101] In some embodiments, the nucleic acid mixture has a concentration of at least 0.15 mg / mL, hi some embodiments, the nucleic acid mixture has a concentration of between about 0.15 mg / mL and 0.2 mg / mL.

[0102] In some embodiments, the concentrated nucleic acid composition has a concentration of at least 0.75 mg / mL. In some embodiments, the concentrated nucleic acid composition has a concentration of about 0.75 mg / mL to 1 mg / mL. In some embodiments, the method further comprises (c) lyophilizing the concentrated nucleic acid composition having a concentration of at least 0.75 mg / mL to a volume of 20 to 40 mL, thereby forming a lyophilized formulation containing at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, or at least 30 mg of DNA plasmid.

[0103] In some embodiments, the method further comprises (c) lyophilizing the concentrated nucleic acid composition having a concentration of at least about 0.75 mg / mL in a volume of about 20-40 mL, thereby forming a lyophilized formulation containing at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, or at least about 30 mg of DNA plasmid.

[0104] In some embodiments, the lyophilized formulation contains 10 to 50 mg (eg, about 30 mg) of DNA plasmid.

[0105] In some embodiments, the method further comprises (d) reconstituting the lyophilized formulation in a diluent (e.g., water, 5% dextrose, or saline), thereby forming a reconstituted composition.

[0106] In some embodiments, the nucleic acid mixture comprises GEN-1 nanoparticles.

[0107] In some embodiments, the ratio of amine nitrogens in the cationic polymer backbone to phosphate in the nucleic acid is from about 10:1 to about 100:1.

[0108] In some embodiments, the filler comprises a sugar, a sugar alcohol, a starch, a cellulose, or a combination thereof. In some embodiments, the filler comprises one or more of lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, Ficoll, sodium chloride, calcium phosphate, calcium carbonate, and / or polyethylene glycol. In some embodiments, the filler comprises lactose.

[0109] Certain embodiments of the present disclosure provide methods that can process low-concentration nucleic acid compositions (e.g., 0.15 mg / mL) using a method that includes a tangential flow filtration (TFF) step to prepare concentrated (e.g., 0.30 mg / mL to 0.75 mg / mL or greater) nucleic acid compositions without affecting the physicochemical or biological properties of the nucleic acid or nucleic acid composition. In some embodiments, the nucleic acid composition includes a DNA plasmid containing a polynucleotide encoding a therapeutic protein (e.g., an hIL-12-encoding nucleic acid) and a lipopolymer (e.g., PPC). In some embodiments, the nucleic acid composition includes GEN-1. In some embodiments, GEN-1 can be concentrated to a solution containing 0.30 mg / mL to 0.75 mg / mL according to the methods of the present disclosure. In some embodiments, GEN-1 can be concentrated to a solution containing about 0.30 mg / mL to about 0.75 mg / mL according to the methods of the present disclosure.

[0110] In some embodiments, the (pre-lyophilized) concentrated nucleic acid composition at any concentration (e.g., 0.30 mg / mL to 0.75 mg / mL or higher) can be used directly as a dosing regimen for administration to a subject. In some embodiments, the concentrated nucleic acid composition can be stored at -20°C. In some embodiments, the (pre-lyophilized) concentrated nucleic acid composition at any concentration (e.g., about 0.30 mg / mL to about 0.75 mg / mL or higher) can be used directly as a dosing regimen for administration to a subject.

[0111] In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing 10 mg to 100 mg (e.g., 10 mg to 100 mg, 10 mg to 90 mg, 10 mg to 80 mg, 10 mg to 70 mg, 10 mg to 60 mg, 10 mg to 50 mg, 10 mg to 40 mg, or 10 mg to 30 mg) of DNA. In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing 10 mg to 50 mg (e.g., 10 mg to 40 mg, 15 mg to 40 mg, 20 mg to 40 mg, 25 mg to 40 mg, 25 mg to 35 mg, or about 30 mg) of DNA. In some embodiments, concentrated lyophilized nucleic acid compositions can be stably stored (e.g., 1 year, at least 2 years, at least 3 years).

[0112] In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing about 10 mg to about 100 mg of DNA (e.g., about 10 mg to about 100 mg, about 10 mg to about 90 mg, about 10 mg to about 80 mg, about 10 mg to about 70 mg, about 10 mg to about 60 mg, about 10 mg to about 50 mg, about 10 mg to about 40 mg, or about 10 mg to about 30 mg). In some embodiments, nucleic acid compositions can be lyophilized according to the present disclosure to provide concentrated lyophilized nucleic acid compositions containing about 10 mg to about 50 mg of DNA (e.g., about 10 mg to about 40 mg, about 15 mg to about 40 mg, about 20 mg to about 40 mg, about 25 mg to about 40 mg, about 25 mg to about 35 mg, or about 30 mg). In some embodiments, the concentrated lyophilized nucleic acid composition is capable of stable storage (eg, at least about 1 year, at least about 2 years, at least about 3 years).

[0113] In some embodiments, the concentrated lyophilized nucleic acid composition can be reconstituted (e.g., in water, 5% dextrose, or saline). In some embodiments, the concentrated nucleic acid composition allows for a wide range of dosing regimens in vivo after reconstitution of the lyophilized composition.

[0114] In some embodiments, the concentrated lyophilized nucleic acid composition can be reconstituted at 0.5 mg / mL, or 1 mg / mL, or 1.5 mg / mL, 2 mg / mL, or 2.5 mg / mL, or 3 mg / mL, or 3.5 mg / mL, or 4 mg / mL, or 4.5 mg / mL, or 5 mg / mL, or 5.5 mg / mL, or 6 mg / mL, or 6.5 mg / mL, or 7 mg / mL, or 7.5 mg / mL, or 8 mg / mL, or 8.5 mg / mL, or 9 mg / mL, or 9.5 mg / mL, or 10 mg / mL.

[0115] In some embodiments, the concentrated lyophilized nucleic acid composition can be reconstituted at about 0.5 mg / mL, or about 1 mg / mL, or about 1.5 mg / mL, or about 2 mg / mL, or about 2.5 mg / mL, or about 3 mg / mL, or about 3.5 mg / mL, or about 4 mg / mL, or about 4.5 mg / mL, or about 5 mg / mL, or about 5.5 mg / mL, or about 6 mg / mL, or about 6.5 mg / mL, or about 7 mg / mL, or about 7.5 mg / mL, or about 8 mg / mL, or about 8.5 mg / mL, or about 9 mg / mL, or about 9.5 mg / mL, or about 10 mg / mL.

[0116] Certain aspects of the present disclosure relate to concentrated nucleic acid compositions (pre-lyophilized and post-lyophilized) prepared according to the methods disclosed herein.

[0117] Certain aspects of the present disclosure relate to pharmaceutical compositions comprising concentrated nucleic acid compositions (pre-lyophilized and post-lyophilized) prepared according to the methods disclosed herein. In some aspects, the pharmaceutical compositions comprise concentrated nucleic acid compositions or reconstituted compositions prepared according to the methods comprising TFF disclosed herein.

[0118] In some embodiments, the pharmaceutical composition contains more than 6 mg (e.g., 10-100 mg, 10-90 mg, 10-80 mg, 10-70 mg, 10-60 mg, 10-50 mg, 10-40 mg, 20-50 mg, 20-25 mg, 20-40 mg, or 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of less than 70 mL (e.g., 20-70 mL, 20-60 mL, 20-50 mL, or 20-45 mL) of diluent. In some embodiments, the pharmaceutical composition contains more than 6 mg (e.g., 10-50 mg, 10-40 mg, 20-50 mg, 20-25 mg, 20-40 mg, or 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of less than 50 mL (e.g., 20-45 mL) of diluent. In some embodiments, the pharmaceutical composition comprises about 30 mg of DNA plasmid (complexed with PPC) in a volume of 20-45 mL (e.g., about 40 mL) of diluent (e.g., water, 5% dextrose, or saline). In some embodiments, the pharmaceutical composition comprises about 150 mg of DNA plasmid (complexed with PPC) in a volume of 200-300 mL (e.g., about 200 mL) of diluent (e.g., water, 5% dextrose, or saline).

[0119] In some embodiments, the pharmaceutical composition comprises more than about 6 mg (e.g., about 10-100 mg, about 10-90 mg, about 10-80 mg, about 10-70 mg, about 10-60 mg, about 10-50 mg, about 10-40 mg, about 20-50 mg, about 20-25 mg, about 20-45 mg, about 20-40 mg, or about 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of diluent of less than about 70 mL (e.g., about 20-70 mL, about 20-60 mL, about 20-50 mL, or about 20-45 mL). In some embodiments, the pharmaceutical composition comprises more than about 6 mg (e.g., about 10-50 mg, about 10-40 mg, about 20-50 mg, about 20-25 mg, about 20-45 mg, about 20-40 mg, or about 25-35 mg) of DNA plasmid (complexed with PPC) in a volume of diluent less than about 50 mL (e.g., about 20-45 mL). In some embodiments, the pharmaceutical composition comprises about 30 mg of DNA plasmid (complexed with PPC) in a volume of diluent (e.g., water, 5% dextrose, or saline) of about 20-45 mL (e.g., about 40 mL). In some embodiments, the pharmaceutical composition comprises about 150 mg of DNA plasmid (complexed with PPC) in a volume of diluent (e.g., water, 5% dextrose, or saline) of about 200-300 mL (e.g., about 200 mL).

[0120] In some embodiments, the pharmaceutical composition is used to prepare a dose of 100-200 mg (e.g., 180 mg) of plasmid DNA (complexed with PPC) in less than 300 mL (e.g., 200-300 mL) of diluent (e.g., water, 5% dextrose, or saline). In some embodiments, the pharmaceutical composition is used to prepare a dose of about 100-200 mg (e.g., about 180 mg) of plasmid DNA (complexed with PPC) in less than about 300 mL (e.g., about 200-300 mL) of diluent (e.g., water, 5% dextrose, or saline).

[0121] In some embodiments, the pharmaceutical composition is in a vial capable of holding up to 100 mL, 150 mL, 200 mL, 250 mL, or 500 mL (e.g., a 250 mL vial). In some embodiments, the vial contains a diluent (e.g., water, 5% dextrose, or saline) in a volume of 200-300 mL (e.g., about 200 mL). In some embodiments, the vial contains 10-50 mg, 10-40 mg, 20-50 mg, 20-25 mg, 20-40 mg, or 25-35 mg (e.g., about 30 mg) of DNA plasmid (complexed with PPC). In some embodiments, the pharmaceutical composition is in a vial capable of holding up to about 100 mL, about 150 mL, about 200 mL, about 250 mL, or about 500 mL (e.g., a 250 mL vial). In some embodiments, the vial contains about 200-300 mL (e.g., about 200 mL) of diluent (e.g., water, 5% dextrose, or saline) in volume. In some embodiments, the vial contains about 10-50 mg, about 10-40 mg, about 20-50 mg, about 20-25 mg, about 20-45 mg, about 20-40 mg, or about 25-35 mg (e.g., about 30 mg) of DNA plasmid (complexed with PPC).

[0122] In some embodiments, the present disclosure provides methods for preparing concentrated, stable pharmaceutical compositions. In some embodiments, the present disclosure relates to pharmaceutical compositions produced by the methods of the present disclosure, comprising at least about 10 mg / mL of nucleic acid, wherein the nucleic acid is complexed with a cationic lipopolymer, and the complex is suspended in an isotonic solution. The complex suspended in the isotonic solution comprises partially or fully condensed nucleic acid molecules. The cationic lipopolymer comprises cholesterol and a cationic polymer backbone having polyethylene glycol groups (i.e., molecules) covalently attached thereto. The molar ratio of cholesterol molecules to the cationic polymer backbone is within the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol molecules to the cationic polymer backbone is within the range of about 0.1 to about 10. In another embodiment, the molar ratio of polyethylene glycol molecules to the cationic polymer backbone in the cationic lipopolymer is within the range of about 1 to about 10. In yet another embodiment, the molar ratio of polyethylene glycol molecules to the cationic polymer backbone in the cationic lipopolymer is within the range of about 1 to about 5. In a further embodiment, the molar ratio of cholesterol molecules to cationic polymer backbone in the cationic lipopolymer is within the range of about 0.3 to about 5. In a further embodiment, the molar ratio of cholesterol molecules to cationic polymer backbone in the cationic lipopolymer is within the range of about 0.4 to about 1.5.

[0123] In some embodiments, the composition can further comprise a filler. In some embodiments, the filler can comprise a sugar, a sugar alcohol, a starch, a cellulose, or a combination thereof. In some embodiments, the filler is selected from one or more of lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, Ficoll, sodium chloride, calcium phosphate, calcium carbonate, and / or polyethylene glycol. In some embodiments, the filler can comprise lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, or any combination thereof. In some embodiments, the filler comprises or is sucrose. In some embodiments, the filler comprises or is lactose.

[0124] The resulting compositions are suitable for delivery of nucleic acids to target cells to induce, inhibit, or modify a biological response depending on the function of the nucleic acid.

[0125] In one embodiment, the cholesterol and polyethylene glycol molecules may be independently directly and covalently bound to the cationic polymer backbone. In another embodiment, the cholesterol and polyethylene glycol molecules are each indirectly and covalently bound to the cationic polymer backbone. For example, the cholesterol molecule may be directly or indirectly coupled to the polyethylene glycol molecule via a linker or spacer, and the polyethylene glycol molecule is covalently bound to the cationic polymer backbone. Alternatively, the cholesterol molecule may be directly bound to the cationic lipopolymer backbone, and the polyethylene glycol molecule is indirectly bound to the lipopolymer via a linker or spacer.

[0126] A specific linker between the polyethylene glycol and the cationic polymer backbone is an alkylene group bearing a terminal carboxyl group, preferably a linear alkylene group of 1 to 20 carbon atoms, more preferably about 2 to about 4 carbon atoms. When the terminal carboxyl group on the linker is attached to an amino group on the cationic polymer backbone, it forms an amide bond between the cationic lipopolymer and the polyethylene glycol. A suitable starting polyethylene glycol for reaction with the cationic polymer backbone molecule is a polyethylene glycol bearing a linker molecule terminated with an activating group, such as N-hydroxysuccinimidyl ester. One example of such a polyethylene glycol is methoxypolyethylene glycol-propionic acid N-hydroxysuccinimidyl ester.

[0127] Some examples of cationic lipopolymer structures resulting from the reaction between polyethyleneimine, cholesteryl chloroformyl carbonate (stereochemistry omitted), and methoxypolyethyleneglycol-propionic acid N-hydroxysuccinimidyl ester are the following structures: The schematic convention reflects the approximate distribution of primary, secondary, and tertiary amino groups in polyethyleneimine, and for clarity, the absence of regularity in polyethyleneimine chains is assumed here. In some embodiments, the gene delivery agent has the following structure: [ka]

[0128] In various embodiments of the present disclosure, n is typically about 8 to about 20, more specifically about 10 to about 15, and even more specifically about 12. x is typically about 2 to about 3, and more specifically about 2.5. y is typically about 6 to about 10, more specifically about 7 to about 9, and even more specifically 7.5. z is typically about 0.4 to about 0.8, more specifically about 0.5 to about 0.7, and even more specifically about 0.6.

[0129] Furthermore, in some embodiments, nucleic acids previously condensed using a secondary condensation system can be further condensed using the techniques presented herein to achieve greater stability of nucleic acids at high concentrations.Thus, prior to condensation according to embodiments of the present disclosure, nucleic acids can be in a partially condensed or non-condensed form.The secondary condensation system can include any condensation material or technique known to those skilled in the art, including, but not limited to, cationic lipids, cationic peptides, cyclodextrins, cationized gelatin, dendrimers, chitosan, and combinations thereof.

[0130] Various degrees of nucleic acid condensation can be achieved for compositions according to embodiments of the present disclosure. In one embodiment, all or a substantial portion of the nucleic acids in the composition are condensed by complexing with cationic polymers. In another embodiment, about 30% by weight of the nucleic acids in the composition are condensed. In yet another embodiment, about 50% by weight of the nucleic acids in the composition are condensed. In a further embodiment, about 70% by weight of the nucleic acids in the composition are condensed. In yet a further embodiment, about 90% by weight of the nucleic acids are condensed. In a further embodiment, 90% by weight of the nucleic acids are condensed.

[0131] Furthermore, the concentration of nucleic acid in the composition varies depending on the materials used in the composition, the concentration method, and the intended use of the nucleic acid. However, in one embodiment, the concentration of nucleic acid is at least about 0.5 mg / mL. In another embodiment, the concentration of nucleic acid is at least about 1 mg / mL. In yet another embodiment, the concentration of nucleic acid is at least about 3 mg / mL. In a further embodiment, the concentration of nucleic acid can be at least about 5 mg / mL, or is at least about 5 mg / mL. In a further embodiment, the concentration of nucleic acid can be at least about 10 mg / mL, or is at least about 10 mg / mL. In another embodiment, the concentration of nucleic acid can be at least about 20 mg / mL, or is at least about 20 mg / mL. In yet another embodiment, the concentration of nucleic acid can be from about 10 mg / mL to about 40 mg / mL, or is from about 10 mg / mL to about 40 mg / mL.

[0132] Various methods can be used to determine the degree of condensation of a nucleic acid composition. For example, in one embodiment, the composition can be electrophoresed to determine the extent to which the nucleic acid in the composition has formed complexes with the cationic polymer added to the composition. The electrostatic attraction of the negatively charged nucleic acid to the positively charged cationic lipopolymer inhibits the nucleic acid from migrating through the agarose gel. Thus, after electrophoresis, nucleic acids condensed by complexation with the cationic polymer remain immobile in the gel, while non-condensed nucleic acids (nucleic acids not associated with the cationic polymer) migrate any distance relative to the intensity of the current in the gel. In another example, nucleic acid condensation can be determined by particle size within the composition. Particle size can be measured by dynamic light scattering. Typically, condensed nucleic acids have smaller particle sizes than non-condensed nucleic acids. Preferred condensed nucleic acids are those in nanoparticles of nucleic acid and cationic lipopolymer with sizes of about 50 nm to about 300 nm, more preferably about 50 to 200 nm, and even more preferably about 50 to 150 nm.

[0133] Any known nucleic acid can be used in the compositions and methods according to the embodiments of the present disclosure, including the examples described above. Thus, the nucleic acids described herein should not be considered limiting. In one embodiment, for example, the nucleic acid can include a plasmid encoding a protein, polypeptide, or peptide. Many peptides are known that will prove beneficial when formulated as pharmaceutical compositions according to the embodiments of the present disclosure. In some embodiments, the nucleic acid can be a plasmid containing a nucleic acid encoding interleukin-12.

[0134] As described above, the cationic lipopolymer may comprise a cationic polymer backbone having covalently bound cholesterol and polyethylene glycol. The cationic polymer backbone may comprise any cationic polymer known to those skilled in the art that can be used to condense and concentrate nucleic acids according to various aspects of the present disclosure. However, in one aspect, the cationic polymer backbone may comprise polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimer, chitosan, and combinations thereof. In one specific aspect, the cationic polymer backbone may be polyethyleneimine.

[0135] In certain embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol. In this embodiment, the average PEG:PEI:cholesterol molar ratio in the cationic lipopolymer is about 1.4-3:1:0.25-1, and preferably about 1.41-2.42:1:0.5-0.9. In certain embodiments, such lipopolymers have a molecular weight (as the free base) of about 3-4 kD, preferably about 3.25-3.75 kD, and more preferably about 3.54 kD. The corresponding hydrochloride salt has a molecular weight of about 4-5 kD, preferably about 4.5 kD.

[0136] Furthermore, the molecular weight of the cationic polymer backbone can vary depending on numerous factors, such as the properties of the nucleic acid and the intended use of the composition. However, in one embodiment, the cationic polymer backbone can have a molecular weight of about 100 to about 500,000 daltons. Furthermore, the molecular weights of the various other components of the cationic lipopolymer can also vary. In one embodiment, for example, polyethylene glycol can have a molecular weight of about 50 to about 20,000 daltons.

[0137] In constructing the pharmaceutical compositions of the present disclosure, it was discovered that the molar ratio of amine nitrogens in the functionalized cationic lipopolymer to phosphates in the nucleic acid (N:P ratio) can affect the degree to which the nucleic acid can be condensed and / or condensed. The optimal N:P ratio may vary somewhat depending on the chemical characteristics of the nucleic acid, but in one embodiment, the ratio of amine nitrogens in the cationic polymer backbone to phosphates in the nucleic acid is about 0.1:1 to about 100:1. In another embodiment, the ratio of amine nitrogens in the cationic polymer backbone to phosphates in the nucleic acid is about 3:1 to about 20:1. In yet another embodiment, the ratio of amine nitrogens in the cationic polymer backbone to phosphates in the nucleic acid is about 6:1 to about 15:1. In other embodiments, the ratio of amine nitrogens to phosphates in the nucleic acid is about 3:1 to about 100:1, or about 5:1 to about 100:1, or about 7:1 to about 100:1. In yet another embodiment, the ratio is from about 10:1 to about 100:1, or more preferably from 10:1 to about 20:1. In one particular embodiment, the ratio of amine nitrogens in the cationic polymer backbone to phosphate in the nucleic acid is about 11:1.

[0138] It is also contemplated that bulking agents may be included in the pharmaceutical composition. Such bulking agents may provide various beneficial properties to the formulation, such as cryoprotection, binding, isotonic balance, and stabilization during lyophilization and reconstitution. It should be understood that bulking agent materials may vary between compositions, and the specific bulking agent used should not be considered limiting. In one aspect, for example, the bulking agent may include various sugars, sugar alcohols, starches, celluloses, and combinations thereof. In another aspect, the bulking agent may include lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, ficoll, sodium chloride, calcium phosphate, calcium carbonate, polyethylene glycol, and combinations thereof. In yet another aspect, the filler may include lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, and any combination thereof. In one aspect, the filler may include sucrose. In another aspect, the filler may include lactose.

[0139] The concentration of the filler in the composition can be from about 0.01% to about 10%, more specifically from about 0.1% to about 5.0%, and even more specifically from about 1% to about 3%.

[0140] In some embodiments, it may be beneficial to functionalize the cationic lipopolymer to enable targeting of specific cells or tissues in a subject or culture. Such targeting is well known, and the examples described herein should not be considered limiting. In one embodiment, for example, the cationic lipopolymer may include a targeting moiety covalently attached to either the cationic lipopolymer or the polyethylene glycol molecule. Such a targeting moiety may enable the cationic lipopolymer to circulate systemically in a subject and locate and specifically target specific cell types or tissues. Examples of such targeting moieties include transferrin, asialoglycoprotein, antibodies, antibody fragments, low-density lipoprotein, cell receptors, growth factor receptors, cytokine receptors, folate, transferrin, insulin, asialoorosomucoid, mannose 6-phosphate, mannose, interleukins, GM-CSF, G-CSF, M-CSF, stem cell factor, erythropoietin, epidermal growth factor (EGF), insulin, asialoorosomucoid, mannose 6-phosphate, mannose, Lewis X and Cialil Lewis X , N-acetyllactosamine, folate, galactose, lactose, and thrombomodulin, fusogenic agents such as polymyxin B and hemagglutinin HA2, lysosomotropic agents, nuclear localization signals (NLS) such as T antigens, and combinations thereof. The selection and attachment of a particular targeting moiety is well within the knowledge of one of ordinary skill in the art.

[0141] The present disclosure also provides a lyophilized pharmaceutical composition that can be stored for long periods of time and reconstituted prior to use. In one embodiment, the lyophilized pharmaceutical composition may comprise a lyophilized mixture of a bulking agent and a cationic lipopolymer condensed with a nucleic acid, the cationic lipopolymer comprising a cationic polymer backbone having cholesterol and polyethylene glycol covalently attached thereto, the molar ratio of cholesterol to cationic polymer backbone being within the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to cationic polymer backbone being within the range of about 0.1 to about 10. The lyophilized pharmaceutical composition may be in a variety of forms, ranging from a dry powder to a partially reconstituted mixture.

[0142] The present disclosure also includes methods for making various pharmaceutical compositions containing condensed nucleic acids. In one embodiment, for example, a method for making a pharmaceutical composition is provided, comprising concentrating condensed nucleic acids to at least 10 mg / mL in an isotonic solution. Such methods may include mixing nucleic acids and a cationic lipopolymer in a bulking agent, wherein the cationic lipopolymer comprises a cationic polymer backbone having cholesterol and polyethylene glycol covalently attached thereto, the molar ratio of cholesterol to cationic polymer backbone being in the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to cationic polymer backbone being in the range of about 0.1 to about 10. The mixture can be lyophilized to a powder to concentrate the nucleic acid mixture, which can then be reconstituted with a diluent to form a solution containing at least about 10 mg / mL of condensed nucleic acid in an isotonic solution.

[0143] Those skilled in the art can understand that different methods can be used to condense nucleic acids. Each of these methods may vary in efficiency, purification speed, cost, and effort, but are within the knowledge of those skilled in the art. Some embodiments of the present disclosure include a method of condensing nucleic acids using tangential flow filtration. Tangential flow filtration (TFF) refers to cross-flow filtration in which the majority of the feed stream moves tangentially across the surface of the filter rather than entering the filter. In some embodiments of the present disclosure, TFF is performed before lyophilization of the formulation. In some embodiments of the present disclosure, the condensed nucleic acid concentration is at least 0.3 mg / mL, or at least 0.45 mg / mL, or at least 0.6 mg / mL, or at least 0.75 mg / mL. In some embodiments of the present disclosure, the condensed nucleic acid concentration is at least about 0.3 mg / mL, or at least about 0.45 mg / mL, or at least about 0.6 mg / mL, or at least about 0.75 mg / mL.

[0144] The condensed nucleic acid recovery rate after TFF can be calculated by calculating the ratio of the cationic lipopolymer / nucleic acid concentration before TFF and the cationic lipopolymer / nucleic acid concentration after TFF.The condensed nucleic acid recovery rate of the present disclosure is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 100%.The condensed nucleic acid recovery rate of the present disclosure is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or about 100%.

[0145] In some aspects of the present disclosure, the condensed nucleic acid after TFF is frozen and stored at about -20°C. In some embodiments, the condensed nucleic acid after TFF is frozen and stored at about -30°C, about -40°C, about -50°C, about -60°C, about -70°C or about -80°C. In some embodiments, the condensed nucleic acid after TFF is quick-frozen. In some embodiments, the condensed nucleic acid after TFF is quick-frozen using dry ice or liquid nitrogen.

[0146] In some embodiments of the present disclosure, the condensed nucleic acids are frozen for more than 1 week, more than 1 month, more than 3 months, more than 6 months, more than 9 months, more than 12 months, more than 15 months, more than 18 months, or more than 24 months, after which the frozen condensed nucleic acids are thawed and have the same or substantially the same assay performance as the condensed nucleic acids that were not frozen. In some embodiments of the present disclosure, the condensed nucleic acids are frozen for more than about 1 week, more than about 1 month, more than about 3 months, more than about 6 months, more than about 9 months, more than about 12 months, more than about 15 months, more than about 18 months, or more than about 24 months, after which the frozen condensed nucleic acids are thawed and have the same or substantially the same assay performance as the condensed nucleic acids that were not frozen.

[0147] Generally, compositions can be obtained by mixing a nucleic acid solution with a cationic lipopolymer solution in the presence of a disaccharide, followed by lyophilization and reconstitution in an isotonic solution. This process is scalable, producing shelf-stable, highly concentrated nucleic acid formulations in quantities ranging from a few milligrams (bench scale) to thousands of milligrams (GMP scale). As described above, cationic lipopolymers have a cationic polymer backbone to which polyethylene glycol and cholesterol are covalently attached. In the case of a polyethyleneimine backbone, in one embodiment, the stoichiometries between polyethylene glycol and polyethyleneimine and between cholesterol and polyethyleneimine are in the ranges of 0.5-10 and 0.1-10, respectively. In the case of a polyethyleneimine backbone, in one embodiment, the stoichiometries between polyethylene glycol and polyethyleneimine and between cholesterol and polyethyleneimine are in the ranges of about 0.5-10 and about 0.1-10, respectively. The chemical composition of the cationic polymer can be important for obtaining highly concentrated, stable nucleic acid formulations. As shown in the examples below, cationic polymers that do not exhibit cholesterol or PEG linkages tend not to produce stable, highly concentrated formulations.

[0148] The compositions according to embodiments of the present disclosure can also be combined with other condensation complexes of nucleic acids to achieve even greater stability of the complexes at high nucleic acid concentrations. For example, various amounts of PEG-PEI-cholesterol can be added to enhance the stability of other nucleic acid delivery systems that are generally unstable at high nucleic acid concentrations.

[0149] In various embodiments, the synthetic delivery system comprises a nucleic acid and a cationic carrier, which can be prepared by various techniques available in the art. Several cationic carriers for nucleic acids are known, such as polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, cationic lipids, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3 N-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristoyloxypropan-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), and combinations thereof. When these delivery systems are combined with PEG-PEI-cholesterol, the stability of the nucleic acid delivery system is increased.

[0150] The biological activity of condensed nucleic acid after TFF can be calculated by calculating the biological activity of cationic nucleic acid concentration before TFF and the biological activity of condensed nucleic acid after TFF.The biological activity percentage of the present disclosure is at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 100%.The biological activity percentage of the present disclosure is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

[0151] Higher concentration lyophilized formulations prepared by the methods disclosed herein are stable (eg, particle size and osmolality).

[0152] III. Gene Delivery Polymers Certain aspects of the present disclosure relate to methods for the preparation and use of concentrated nucleic acid formulations comprising the gene delivery polymers disclosed herein.

[0153] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) covalently attached independently to a cholesterol group and a polyethylene glycol (PEG) group.

[0154] The present disclosure provides a polymer system, PEG-PEI-cholesterol (PPC), which differs from WSLP (PEI-cholesterol) in that it contains a PEG moiety, resulting in significantly higher transfection efficiency within tumors. The addition of PEG is designed to enhance the stability of the nucleic acid / polymer complex in biological environments, circumventing this deficiency in the prior art (WSLP). Furthermore, the addition of PEG chains allows for the incorporation of ligands onto the PPC chains to improve tissue selectivity of delivery. For example, the cholesterol moiety directly linked to the PEI backbone of the prior art (WSLP) can be extended further from the PEI backbone to create a more flexible geometry for cell receptor interaction. Controlling the number of PEG molecules per PEI backbone unit is important to achieve optimal enhancement of transfection activity. A preferred composition range was a PEG:PEI molar ratio of 2 to 4 at a constant cholesterol content. In some embodiments, the optimal PEI-to-cholesterol ratio was 1:0.5 to 1:1. In some embodiments, the optimal PEI-to-cholesterol ratio was approximately 1:0.5 to 1:1.

[0155] In some embodiments, the polynucleotide encodes human IL-12.

[0156] In some embodiments, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0157] In some embodiments, the nucleic acid vector (eg, a plasmid) comprises an intron, a 3'UTR (eg, an hGH 3'UTR), an antibiotic resistance gene, or any combination thereof.

[0158] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) covalently attached independently to a cholesterol group and a polyethylene glycol (PEG) group.

[0159] In some embodiments, the nanoparticles disclosed herein comprise a DNA plasmid encoding human IL-12.

[0160] In some embodiments, the nanoparticles comprise a synthetic polymer that facilitates plasmid delivery that is a lipopolymer.

[0161] In some embodiments, the lipopolymer further comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group.

[0162] In some embodiments, the gene delivery polymer is a cationic polymer or a non-condensing polymer. The cationic polymer is selected from the group consisting of polylysine, polyethyleneimine, functionalized derivatives of polyethyleneimine (PEI), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, and spermidine. One example of a cationic gene delivery polymer suitable for the present disclosure is a PEI derivative comprising a PEI backbone, a lipid, and a hydrophilic polymer spacer, where the lipid is either directly attached to the polyethyleneimine backbone or covalently attached to the polyethylene glycol spacer, and the polyethylene glycol spacer is attached to the PEI via a biocompatible bond.

[0163] The cationic gene delivery polymers of the present disclosure are suitable for use in the delivery of antibodies or antibody fragments, cell receptors, growth factor receptors, cytokine receptors, folate, transferrin, epidermal growth factor (EGF), insulin, asialoorosomucoid, mannose-6-phosphate (monocytes), mannose (macrophages, some B cells), Lewis X and Cialil Lewis XThe gene delivery polymer may further comprise a targeting moiety including a fusion agent such as polymyxin B and hemagglutinin HA2, a lysosomotrophic agent, a nuclear localization signal (NLS), such as T antigen, etc. Another gene delivery polymer is a non-condensing polymer selected from the group including polyvinylpyrrolidone, polyvinyl alcohol, poly(lactide-co-glycolide) (PLGA), and a triblock copolymer of PLGA and PEG. The gene delivery polymer may also be a non-condensing polymer. Examples of such non-condensing polymers include polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, polyglutamate, gelatin, polyphosphoester, silk-elastin-like hydrogels, agarose hydrogels, lipid microtubules, poly(lactide-co-glycolide), and polyethylene glycol-conjugated poly(lactide-co-glycolide).

[0164] The gene delivery polymer is a cationic polymer or a non-condensing polymer. The cationic polymer is selected from the group consisting of polylysine, polyethyleneimine, functionalized derivatives of polyethyleneimine, polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, and spermidine. One example of a cationic gene delivery polymer suitable for the present invention is a polyethyleneimine derivative comprising a polyethyleneimine (PEI) backbone, a lipid, and a polyethylene glycol spacer, wherein the lipid is directly bound to the polyethyleneimine backbone or covalently bound to the polyethylene glycol spacer, and the polyethylene glycol spacer is bound to the PEI via a biocompatible bond.

[0165] In some embodiments, the gene delivery polymer comprises a lipopolyamine having the following formula: [ka]

[0166] In some embodiments, the gene delivery polymer comprises a mixture of lipopolyamine and an alkylated derivative of lipopolyamine. In some embodiments, the alkylated derivative of lipopolyamine is polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinyl ether maleic anhydride. In some embodiments, the alkylated derivative of lipopolyamine has the following formula: [ka]

[0167] wherein n represents an integer of 10 to 100 repeating units each containing 2 to 5 carbon atoms. In some embodiments, the alkylated derivatives of lipopolyamines have the formula: [ka]

[0168] wherein n=11 (stalamine-mPEG515). In some embodiments, the alkylated derivative of lipopolyamine has the formula: [ka]

[0169] In some embodiments, the ratio of lipopolyamine to alkylated derivative of lipopolyamine in the mixture is 1:1 to 10:1. In some embodiments, the lipopolyamine is present in an amount sufficient to provide a ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector of about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 1:10 to about 10:1.

[0170] In some embodiments, the gene delivery polymer comprises a lipopolyamine having the following formula: [ka]

[0171] In some embodiments, the gene delivery polymer comprises a mixture of lipopolyamine and an alkylated derivative of lipopolyamine. In some embodiments, the alkylated derivative of lipopolyamine is polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinyl ether maleic anhydride. In some embodiments, the ratio of lipopolyamine to alkylated derivative of lipopolyamine in the mixture is 1:1 to 10:1. In some embodiments, the ratio of lipopolyamine to alkylated derivative of lipopolyamine in the mixture is about 1:1 to 10:1. In some embodiments, the lipopolyamine is present in an amount sufficient to provide a ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector of about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some embodiments, the ratio of amine nitrogens in the lipopolyamine to phosphate in the nucleic acid vector is about 1:10 to about 10:1.

[0172] In some embodiments, the gene delivery polymer comprises a poloxamer backbone having a metal chelator covalently attached to at least one end of the poloxamer backbone. In some embodiments, the metal chelator is attached to at least two ends of the poloxamer backbone. In some embodiments, the poloxamer backbone is a poloxamer backbone disclosed in U.S. Patent Publication No. 2010 / 0004313, the entire contents of which are incorporated herein by reference. In some embodiments, the metal chelator is a metal chelator disclosed in U.S. Patent Publication No. 2010 / 0004313. In some embodiments, the gene delivery polymer is a polymer having the formula: [ka]

[0173] and pharmaceutically acceptable salts thereof, wherein:

[0174] A represents an integer from 2 to 141,

[0175] B represents an integer from 16 to 67,

[0176] C represents an integer from 2 to 141;

[0177] R A and R C are the same or different and are R′-L-, or H, and at least one of R A and R C is R′-L-;

[0178] L is a bond, —CO—, —CH—O—, or —O—CO—;

[0179] R' is a metal chelator.

[0180] In some embodiments, the metal chelator is RNNH—, RN2N—, or (R″—(N(R″—CH2CH2)x)2-N—CH2CO—, where each x is independently 0 to 2, and R″ is HO2C—CH2—. In some embodiments, the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24-crown-8. In some embodiments, the crown ether is a substituted crown ether, wherein the substituted crown ether has the following:

[0181] (1) one or more crown ether oxygens independently substituted by NH or S;

[0182] (2) one or more crown ether -CH-CH- moieties substituted by -C6H4-, -C10H6-, or -C6H10-;

[0183] (3) one or more crown ether -CH2-O-CH2- moieties substituted by -C4H2O-, or -C5H3N-, or

[0184] (4) Any combination thereof.

[0185] In some embodiments, the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of a (1,2,2) cryptand, a (2,2,2) cryptand, a (2,2,3) cryptand, and a (2,3,3) cryptand. In some embodiments, the cryptand is a substituted cryptand, wherein the substituted cryptand has:

[0186] (1) one or more of the cryptand ether oxygens independently substituted by NH or S;

[0187] (2) one or more crown ether -CH-CH- moieties substituted by -C6H4-, -C10H6-, or -C6H10-;

[0188] (3) one or more crown ether -CH2-O-CH2- moieties substituted by -C4H2O-, or -C5H3N-, or

[0189] (4) Any combination thereof.

[0190] In some embodiments, the gene delivery polymer is a crown poloxamer (aza-crown linked poloxamer), which includes a polymer having the formula of the present disclosure. In some embodiments, the gene delivery polymer is a crown poloxamer (aza-crown linked poloxamer), which includes a polymer having the formula: [ka]

[0191] or a pharmaceutically acceptable salt thereof, wherein:

[0192] a represents an integer of approximately 10 units,

[0193] b represents an integer of approximately 21 units;

[0194] The total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.

[0195] In some embodiments, the crown poloxamer is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0196] In some embodiments, the gene delivery polymer is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0197] In some embodiments, the gene delivery polymer is a β-amino ester. In some embodiments, the polymer is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0198] In some embodiments, the gene delivery polymer is polyinosinic-polycytidylic acid, which is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0199] In some embodiments, the gene delivery polymer further comprises benzalkonium chloride.

[0200] In some embodiments, the gene delivery polymer comprises BD15-12. In some embodiments, the ratio of nucleotide to BD15-12 polymer (N:P) is 5:1.

[0201] In some embodiments, the gene delivery polymer comprises Omnifect. In some embodiments, the ratio of nucleotides to Omnifect polymer (N:P) is 10:1.

[0202] In some embodiments, the gene delivery polymer comprises a crown poloxamer (aza-crown linked poloxamer). In some embodiments, the ratio of nucleotide to crown poloxamer (N:P) is 5:1. In some embodiments, the gene delivery polymer comprises a crown poloxamer and a PEG-PEI-cholesterol (PPC) lipopolymer. In some embodiments, the gene delivery polymer comprises a crown poloxamer and benzalkonium chloride. In some embodiments, the gene delivery polymer comprises a crown poloxamer and Omnifect. In some embodiments, the gene delivery polymer comprises a crown poloxamer and linear polyethyleneimine (LPEI). In some embodiments, the gene delivery polymer comprises a crown poloxamer and BD15-12.

[0203] In some embodiments, the gene delivery polymer comprises stalamine and mPEG-modified stalamine. In some embodiments, the mPEG-modified stalamine is stalamine-mPEG515. In some embodiments, the mPEG-modified stalamine is stalamine-mPEG11. In some embodiments, the ratio of stalamine to mPEG-modified stalamine is 10:1. In some embodiments, the nucleotide to polymer (N:P) ratio is 5:1. In some embodiments, the gene delivery polymer comprises stalamine, mPEG-modified stalamine, and crown poloxamer. In some embodiments, the gene delivery polymer comprises stalamine, stalamine-mPEG515, and crown poloxamer. In some embodiments, the gene delivery polymer comprises stalamine, stalamine-mPEG11, and crown poloxamer.

[0204] In some embodiments, the gene delivery polymer comprises a poloxamer backbone as disclosed in WO 2022 / 072910(A1), which is incorporated by reference in its entirety.

[0205] In some embodiments, nanoparticles comprising a DNA plasmid encoding interleukin-12 (IL-12) and a synthetic gene delivery polymer (eg, PPC) that facilitates delivery of the plasmid, for example, into the peritoneal cavity.

[0206] IV.GEN-1 formulation GEN-1 is a gene-based immunotherapy that includes a human IL-12 gene expression plasmid and a synthetic lipopolymer delivery system (Thaker, Premal H. et al., Future Oncol. (2019) 15(4), 421-438). GEN-1 contains a plasmid vector encoding the p35 and p40 subunits of the human IL-12 gene, each under the control of a cytomegalovirus (CMV) promoter, and a synthetic lipopolymer delivery system called polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC). In some embodiments, GEN-1 is formulated as a lyophilized powder that can be reconstituted at the bedside, for example, at a dose of up to 0.5 mg / mL. In some embodiments, the GEN-1 DNA plasmid is delivered using a lipopolymer, a synthetic polymer that facilitates plasmid delivery.

[0207] In some embodiments, the GEN-1 formulation is about 35 mg / m 2 ~about 120mg / m 2 In some embodiments, the GEN-1 formulation is prepared in a dosage form of about 35 mg / m 2 ~about 110mg / m 2 In some embodiments, the GEN-1 formulation is prepared in a dosage form of about 50 mg / m 2 ~about 100mg / m 2 In some embodiments, the GEN-1 formulation is prepared in a dosage form of about 100 mg / m 2 In some embodiments, the GEN-1 formulation is prepared in a dosage form of about 60 mg / m 2 It is prepared in the following dosage form.

[0208] In some embodiments, the GEN-1 formulation comprises about 180 mg of DNA (a plasmid containing the coding sequence for hIL-12) at about 100 mg / m 2 It is prepared in the following dosage form.

[0209] The present disclosure provides methods for preparing GEN-1 formulations that are at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more concentrated than GEN-1 formulations prepared using methods known in the art. For example, using known methods, a dose containing approximately 180 mg of DNA requires approximately 30 vials (50 mL vials) to be reconstituted (i.e., each vial holds approximately 6 mg of DNA for reconstitution). This is a commercially difficult preparation. The disclosed methods allow for DNA preparations that contain approximately 5 times more material per vial. For a dose containing approximately 180 mg, the methods disclosed herein reduce the number of vials required for reconstitution from approximately 30 vials to approximately 6 vials (50 mL vials) (i.e., each vial holds approximately 30 mg of DNA for reconstitution). In some embodiments, for a dose containing about 180 mg of plasmid DNA (GEN-1), the methods disclosed herein reduce the number of vials to be reconstituted from about 30 vials to about 2 vials (250 mL vials) (e.g., each vial carrying about 150 mg of DNA for reconstitution).

[0210] In some embodiments, the higher concentration GEN-1 lyophilized formulations prepared by the methods disclosed herein are stable (eg, particle size and osmolality).

[0211] V. How to use Certain aspects of the present disclosure relate to methods using concentrated nucleic acid compositions prepared according to the methods disclosed herein. For example, in one aspect, a method for transfecting mammalian cells can include contacting the mammalian cells with a composition described herein and incubating the mammalian cells under conditions that allow the composition to enter the cells and induce the biological activity of the nucleic acid. Such transfection techniques are known to those skilled in the art. In yet another aspect, target tissues can be transfected by delivering the composition to a warm-blooded organism or subject. Such delivery can be by administration modes such as intratumoral, intraperitoneal, intravesicular, intravenous, intraarterial, intratracheal, intrahepatic portal vein, oral, intracranial, intramuscular, intraarticular, and combinations thereof. Such target tissues can include any tissue or subset of tissues that benefit from transfection. For example, but without limitation, such target tissues may include the ovaries, uterus, stomach, colon, rectum, bone, blood, intestines, pancreas, breast, head, neck, lungs, spleen, liver, kidneys, brain, thyroid, prostate, bladder, thyroid gland, skin, abdominal cavity, thoracic cavity, and combinations thereof.

[0212] The present disclosure provides a method for treating mammalian cancer or hyperproliferative disorders by intratumoral, intraperitoneal, intravesicular, intravenous, intravesicular, intratracheal, intracranial, or systemic administration of a pharmaceutical composition comprising a plasmid-based gene expression system and a gene delivery polymer without the use of chemotherapy. The mammalian cancer is selected from the group consisting of primary or metastatic ovarian tumors. In some embodiments, the nucleic acid is a plasmid-based gene expression system containing a DNA sequence encoding human interleukin-12 (e.g., GEN-1).

[0213] Treatment of tumors with pharmaceutical compositions (e.g., GEN-1) prepared according to the methods disclosed herein results in tumor shrinkage and extended lifespan. In some embodiments, the treatment method includes combination therapy.

[0214] In some embodiments, GEN-1 can be delivered intraperitoneally (ip) to produce localized and sustained levels of IL-12 at the tumor site in patients with advanced ovarian cancer. GEN-1 can be administered alone or in combination with chemotherapy.

[0215] In some embodiments, the combination therapy involves the use of a nucleic acid composition (e.g., GEN-1) prepared according to the methods disclosed herein and chemotherapy (chemotherapeutic agent). Efficacy of the disclosed methods is defined as, but not limited to, a reduction in tumor size or tumor density, an increase in lymphocyte count or neutrophil count, or improved survival, or all of the above. In some embodiments, the combination of GEN-1 and chemotherapy (chemotherapeutic agent) according to the methods of the present invention can reduce the toxicity of the chemotherapy agent and reverse tumor resistance to chemotherapy. Toxicity, as used herein, is defined as any treatment-related adverse effect on clinical findings, including, but not limited to, abnormal hematology or serum chemistry or organ toxicity. Furthermore, the combination of GEN-1 with a suboptimal dose of chemotherapy (chemotherapeutic agent) according to the methods of the present invention enhances anti-cancer efficacy to levels above those achieved by an optimal dose of the chemotherapy agent, but with reduced toxicity.

[0216] In some embodiments, a nucleic acid composition (e.g., GEN-1) prepared according to the methods disclosed herein has a concentration of about 35 mg / m 2 ~about 120mg / m 2 In some embodiments, the nucleic acid compositions (e.g., GEN-1) prepared according to the methods disclosed herein are administered at a dose of about 35 mg / m 2 ~about 110mg / m 2 In some embodiments, the nucleic acid compositions (e.g., GEN-1) prepared according to the methods disclosed herein are administered at a dose of about 50 mg / m 2 ~about 100mg / m 2 In some embodiments, the nucleic acid compositions (e.g., GEN-1) prepared according to the methods disclosed herein are administered at a dose of about 100 mg / m 2In some embodiments, the nucleic acid compositions (e.g., GEN-1) prepared according to the methods disclosed herein are administered at a dose of about 60 mg / m 2 is administered at a dose of

[0217] In some embodiments, the GEN-1 formulation prepared according to the methods disclosed herein contains about 180 mg of DNA (a plasmid containing the coding sequence for hIL-12) at about 100 mg / m 2 In some embodiments, the dose is administered at a dose of 1.8 m2 of the average surface area of ​​the subject. 2 is administered.

[0218] The present disclosure provides methods for preparing GEN-1 formulations that are at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more concentrated than GEN-1 formulations prepared using methods known in the art. For example, using known methods, a dose containing approximately 180 mg of DNA requires approximately 30 vials (50 mL vials) to be reconstituted (i.e., each vial holds approximately 6 mg of DNA for reconstitution). This is a commercially difficult preparation. The disclosed methods allow for DNA preparations that contain approximately 5 times more material per vial. For a dose containing approximately 180 mg, the methods disclosed herein reduce the number of vials required for reconstitution from approximately 30 vials to approximately 6 vials (50 mL vials) (i.e., each vial holds approximately 30 mg of DNA for reconstitution). In some embodiments, for a dose containing about 180 mg of plasmid DNA (GEN-1), the methods disclosed herein reduce the number of vials to be reconstituted from about 30 vials to about 2 vials (250 mL vials) (i.e., each vial carries about 150 mg of DNA for reconstitution).

[0219] Certain aspects of the present disclosure relate to combination therapies comprising (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide prepared according to the methods disclosed herein, formulated with a lipopolymer (e.g., a nanoparticle), and (ii) an anti-cancer agent. In some aspects, the polynucleotide encodes human interleukin-12 (hIL-12).

[0220] In some embodiments, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL-12.

[0221] In some embodiments, the nucleic acid vector (eg, a plasmid) comprises an intron, a 3'UTR (eg, an hGH 3'UTR), an antibiotic resistance gene, or any combination thereof.

[0222] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) covalently attached independently to a cholesterol group and a polyethylene glycol (PEG) group.

[0223] In some embodiments, the anti-cancer agent is a chemotherapeutic agent.

[0224] In some embodiments, the chemotherapeutic agent is selected from the group consisting of topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin, epirubicin, idarubicin), anti-microtubule agents (e.g., paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, dacarbizine), platinum-based drugs (cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., gemcitabine, methotrexate, 5-fluorouracil), or combinations thereof.

[0225] In some embodiments, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0226] In some embodiments, the anti-cancer agent is doxorubicin. In some embodiments, the anti-cancer agent is paclitaxel. In some embodiments, the anti-cancer agent is carboplatin. In some embodiments, the anti-cancer agent is docetaxel. In some embodiments, the anti-cancer agent is nab-paclitaxel. In some embodiments, the anti-cancer agent is olaparib.

[0227] In some embodiments, the nucleic acid vector formulated with lipopolymer is administered prior to, concurrently with, or after the anti-cancer agent.

[0228] In some embodiments, the treatment method further comprises surgery to remove all or part of the tissue or tumor in question (eg, interval cytoreductive surgery).

[0229] In some embodiments, the nucleic acid vector formulated with lipopolymer is administered intratumorally or intraperitoneally.

[0230] In some aspects, the nucleic acid vector formulated with lipopolymer is administered intravenously.

[0231] In some embodiments, surgery to remove all or part of the tissue or tumor (e.g., cytoreductive surgery) is performed (e.g., first), followed by administration of an anti-cancer agent, followed by administration of a nucleic acid vector formulated with a lipopolymer prepared according to the methods disclosed herein.

[0232] example The following examples are provided to facilitate a clearer understanding of certain embodiments of the present invention and are not meant to be limiting in any way.

[0233] Example 1: Small-scale preparation of liquid formulations of condensed nucleic acids with cationic lipopolymers This example demonstrates the preparation of fully condensed nucleic acid formulations for bench-scale production. This involves the preparation of nucleic acid complexes with cationic polymers, followed by lyophilization and reconstitution into an isotonic solution. The nucleic acids used contained plasmid DNA encoding IL-12 or luciferase genes, and the polymer contained a polyethyleneimine (PEI) backbone covalently linked to polyethylene glycol (PEG) and cholesterol (Chol) (PEG-PEI-Chol, also known as PPC). The molar ratios of PEG to PEI and cholesterol to PEI were 0.5-10 and 0.1-10, respectively. DNA and PPC solutions were first prepared separately at 5 mg / mL in water for injection, and then diluted to 0.15 mg / mL (DNA) and 0.554 mg / mL (PPC) in 3% lactose. DNA in lactose solution was added to PPC in lactose solution using a micropipette until the nitrogen to phosphate (N:P) ratio was 11:1, and the preparation was incubated at room temperature for 15 minutes to allow complex formation.

[0234] The PPC / DNA complex in 3% lactose was lyophilized using a FREEZONE freeze-drying system (LABCONCO Corp., Kansas City, MO.) 500 μl of the prepared formulation was added to a 2 mL borosilicate glass vial and then lyophilized using a freeze-drying program that included the following segments: (1) a freezing segment (ramp 0.25°C / min, hold 34°C for 4 hours), (2) a primary drying segment (hold 34°C for 24 hours), (3) a secondary drying segment (ramp to 20°C, hold 24 hours), and (4) a ramp to 4°C at 0.25°C / min.

[0235] The resulting lyophilized powder was reconstituted with water for injection to various concentrations ranging from 0.1 mg / mL to 20 mg / mL DNA. A typical batch of small-scale preparation amounted to 100-200 mg of fully formulated DNA.

[0236] Example 2: Scale-up preparation of liquid formulations of condensed nucleic acids containing cationic lipopolymers This example demonstrates the preparation of a condensed nucleic acid formulation to produce up to 6 mg of DNA per vial (e.g., 100 mL vial), as shown in Figure 1A. This process can produce at least 6,000 mg of fully formulated DNA and can be scaled up to even higher production volumes (compared to the 100–200 mg of DNA produced from the small-scale preparation described in Example 1). The scaled-up method involves mixing bulk DNA and polymer solution via a peristaltic pump to achieve online mixing and complex formation, followed by a freeze-dry cycle compatible with large-scale processing. Briefly, DNA and PPC solutions were prepared at 0.3 mg / mL and 1.1 mg / mL in 3% lactose, respectively. These two components were combined at a constant flow rate of 225 ± 25 mL / min using a peristaltic pump (WATSON MARLOW, SCI400) with a 0.89 mm inner diameter silicone tubing (WATSON MARLOW, Z982-0088). The two mixtures were joined at the ends of each tube using polypropylene T-connectors. Mixing the polymer and DNA solutions resulted in the rapid formation of nanoparticles at a concentration of 0.15 mg / mL formulation. Forty milliliters of the formulated complex was placed in a 100 mL glass vial and lyophilized using a freeze-drying program consisting of the following segments: (1) prefreezing at -50°C for up to 720 minutes; (2) primary drying at -40°C for up to 180 minutes, followed by -34°C for up to 1980 minutes at 65 μmHg; (3) secondary drying at -25°C for up to 720 minutes, -15°C for up to 3180 minutes, -10°C for up to 1500 minutes, and 4°C for up to 1440 minutes at 65 μmHg.

[0237] Each 100 mL glass vial contained 6 mg of lyophilized DNA powder. The resulting lyophilized DNA powder was reconstituted with water for injection to various concentrations ranging from 0.1 mg / mL to 20 mg / mL DNA. A typical batch at this scale would yield approximately 6000 mg of fully formulated DNA.

[0238] Example 3: Preparation of concentrated liquid formulations of condensed nucleic acids with cationic lipopolymers using tangential flow filtration (TFF) This example demonstrates the preparation of a highly concentrated formulation of condensed nucleic acids to produce up to 30 mg of DNA per vial (e.g., 100 mL vial), as shown in Figure 1B. After adding a tangential flow filtration (TFF) step to the process, the DNA was lyophilized using a freeze-drying program. After mixing the DNA and PPC solutions (as described in Example 2), the 0.15 mg / mL formulated complex solution was processed through a KrosFlow KMPi system. This resulted in a DNA and PPC complex formulation (0.75 mg / mL) that was up to 5-fold more concentrated than the original 0.15 mg / mL formulation.

[0239] After the TFF process, the concentrated formulations were either lyophilized using a freeze-drying program or stored frozen below -20°C.

[0240] KMPi system configuration options for TFF are shown in Table 1. [Table 1]

[0241] Example 4: Measurement of particle size and osmolality of concentrated liquid formulations of condensed nucleic acids containing cationic lipopolymers Formulations of plasmid DNA containing cationic lipopolymer (PPC) were prepared as described in Examples 1-3. For polymer / nucleic acid particle size measurements, aliquots of the liquid formulations were analyzed using a 90Plus / BI-MAS Particle Sizer manufactured by Brookhaven Instruments Corp., Holtsville, NY. Specifically, for analysis, 50 μl of the formulation was added to 950 μl of Milli-Q water in a polystyrene cuvette. For polymer / nucleic acid particle osmolality measurements, a Pharmacopeia for Osmolality and Mass Osmolality was used. <785> Followed the law.

[0242] Figures 2A-2E show the particle size and osmolality of DNA / PPC complexes from lyophilized formulations at concentrations of 1x (0.5 mg / mL DNA), 2x (1 mg / mL DNA), 3x (1.5 mg / mL DNA), 4x (2 mg / mL DNA), and 5x (2.5 mg / mL DNA). Higher concentrations of the lyophilized formulations did not significantly affect particle size and osmolality, indicating that the complexes were stable.

[0243] Example 5: Measurement of nucleic acid concentration and PPC in concentrated liquid formulations of nucleic acids containing cationic lipopolymers The amount of nucleic acid in high-concentration formulations of DNA and PPC complexes was quantified using an AGILENT 8453 spectrophotometer (AGILENT TECHNOLOGIES, Inc., Santa Clara, CA). 50 μl of the formulation was diluted with 950 μl of water for injection (WFI) in a quartz cuvette, and absorbance was measured at a wavelength of 260 nm. DNA concentration was determined assuming 1 optical density (260 nm) = 50 μg / mL of DNA. The amount of PPC in high-concentration formulations of DNA and PPC complexes was measured using TNBSA (2,4,6-trinitrobenzenesulfonic acid) assay reagent (catalog number TS-28997) from Fisher Scientific. TNBSA was used to measure free amino groups in PPC, which form a highly chromogenic derivative and can be measured at 335 nm using UV / Vis spectrophotometry.

[0244] Figures 3A-3D show the DNA / PPC concentration and ratio of lyophilized formulations without (1x) and with (2x-5x) TFF processing. The high-concentration lyophilized formulations produced using the TFF process did not significantly affect the DNA / PPC concentration ratio or recovery rate after the TFF process.

[0245] Example 6: Analysis of nucleic acid condensation with concentrated liquid formulations of nucleic acids and cationic lipopolymers In this example, the ability of PPC polymer to condense plasmid DNA was evaluated. Plasmid DNA formulations containing cationic lipopolymer (PPC) were prepared as described in Examples 1-3. The nucleic acid / polymer complexes (1x-5x) were electrophoresed through a 1% agarose gel. The electrostatic attraction of the negatively charged plasmid DNA to the positively charged PPC polymer prevented the DNA from passing through the agarose gel. As shown in Figures 4A-4C, the DNA present in the high-concentration formulations that underwent the TFF process (2x-5x) condensed and released in a manner similar to that of the 1x concentration formulations with added dextran sulfate.

[0246] Example 7: Measurement of transfection activity of concentrated liquid formulations of nucleic acids using cationic lipopolymers The transfection activity of the DNA and PPC complex formulations was determined in vitro. Cos-1 cells (1.5 × 10 5 Cells (1000 cells / well) were seeded into 12-well tissue culture plates in 10% fetal bovine serum (FBS). Each well was incubated with 4 μg of complexed DNA in a total volume of 500 μl of Dulbecco / Vogt modified Eagle's minimum essential medium (DMEM) in the absence of FBS for 6 hours. Upon completion of the additional 40-hour incubation period, the medium was replaced with 1 mL of fresh DMEM supplemented with 10% FBS. At the end of the incubation period, transfection activity was measured in the cell culture medium (IL-12). For IL-12 levels, the cell culture medium was analyzed directly by an IL-12 ELISA assay. Direct comparisons were made with a 1x concentrated formulation. Transfection complexes containing IL-12 plasmids were prepared by the methods described in Examples 1-3 and reconstituted at 1x to 5x concentrations with DNA concentrations ranging from 600 ng / well to 6400 ng / well. IL-12 expression levels from concentrated preparations of IL-12 plasmid / PPC complexes are shown in Figures 5A-5D. The data demonstrate that transfection biological activity of the highly concentrated forms of the nucleic acid complexes was retained.

[0247] Example 8: IL-12 expression in normal brain parenchyma after intracranial expression of concentrated liquid formulations of nucleic acids containing cationic lipopolymers Direct administration of IL-12 plasmids containing the cationic polymer PPC in normal brain tissue was investigated to determine whether highly concentrated formulations of nucleic acid and cationic lipopolymer were biologically active in vivo. Immunohistochemical staining for IL-12 was performed on brain sections from animals euthanized 14 days or 1 month after treatment. The brain parenchyma of animals treated with PPC alone did not show any IL-12 staining. In contrast, the brain parenchyma of mice intracranially injected with pmIL-12 / PPC stained positive for IL-12. This experiment demonstrates that the biological activity of the nucleic acid complex with the cationic polymer is preserved during the concentration process. Furthermore, IL-12 was shown to persist for at least 1 month after injection. Furthermore, the presence of this cytokine in the brains of animals that survived to euthanasia suggests that actual expression of IL-12 did not cause lethal toxicity in the brain.

[0248] Example 9: Biological activity of concentrated liquid formulations of nucleic acids containing cationic lipopolymers in tumor-bearing mice As shown in Figures 6A and 6B, the biological activity of formulations (1- to 5-fold) of fully condensed nucleic acid expressing the IL-12 gene was examined in the ascites and serum of tumor-bearing mice. C57BL / 6 female mice were injected with 2.5 x 10 6 Tumors were implanted by intraperitoneal injection of ID8 ovarian cancer cells. On day 41 after tumor implantation, once the animals had achieved a significant increase in body weight relative to their starting weight, the mice were randomized into treatment groups. Forty-three days after tumor implantation, GEN-1 was administered intraperitoneally at a dose of 10 mg / kg. Approximately 24 hours after GEN-1 administration, the animals were euthanized, and serum and ascites were collected. Animals were weighed before and after complete removal of ascites, and the volume of ascites was calculated. hIL-12 expression levels in both serum and ascites were measured using a human IL-12p70 ELISA kit. These data demonstrated that a highly concentrated formulation of IL-12 nucleic acid maintained gene expression for in vivo application compared to a 1x formulation.

[0249] Example 10: Biological activity of concentrated liquid formulations of nucleic acids containing cationic lipopolymers in tumor-bearing mice As shown in Figures 7A and 7B, the biological activity of the fully condensed nucleic acid expressing the IL-12 gene in the 1x and 5x formulations after TFF and in formulations from two separate lots without TFF was examined in the ascites and serum of tumor-bearing mice. C57BL / 6 female mice were injected with 2.5 x 10 6 Tumors were implanted by intraperitoneal injection of ID8 ovarian cancer cells. On day 41 after tumor implantation, once the animals had achieved a significant increase in body weight relative to their starting weight, the mice were randomized into treatment groups. Forty-three days after tumor implantation, GEN-1 was administered intraperitoneally at a dose of 10 mg / kg. Approximately 24 hours after GEN-1 administration, the animals were euthanized, and serum and ascites were collected. Animals were weighed before and after complete removal of ascites, and the volume of ascites was calculated. hIL-12 expression levels in both serum and ascites were measured using a human IL-12p70 ELISA kit. These data demonstrated that a highly concentrated formulation of IL-12 nucleic acid maintained gene expression for in vivo application compared to a 1x formulation.

[0250] Example 11: Long-term stability of lyophilized or concentrated liquid formulations of nucleic acids containing cationic polymers Lyophilized IL-12 / PPC complexes were prepared using the methods outlined in Examples 1–3 and stored at -80°C, -20°C, 4°C, and 25°C. At the time of analysis, vials were removed from the storage container and 2.4 mL of water for injection (WFI) was added. For each sample, pH, DNA concentration, osmolality, particle size, and biological activity were measured. As shown in Figures 8A–8F, the DNA particle size, DNA concentration, PPC concentration, PPC / DNA ratio, osmolality, and pH of the 5x concentrated IL-12 / PPC complexes were maintained after up to 30 months of storage at the indicated temperatures when compared to the reference standard of 1x concentrated IL-12 / PPC complexes. The gene transfer activity of pIL-12 / PPC in COS-1 cells was quantified as described in Example 7. COS-1 cells were transfected with 4 μg of DNA biomaterial. 48 hours after transfection, the levels of IL-12 in the cell culture medium were quantified using a commercially available ELISA kit. The bioactivity results from the 30-month stability study are shown in Figure 8G. The bioactivity of the 5x concentrated IL-12 / PPC complex biological product did not change significantly during storage at -20°C or 4°C.

[0251] Relative potency results up to 30 months are shown in Figures 8H and 8I. The relative potency of the 5x concentrated IL-12 / PPC complex also showed no significant decline in DNA particle size, DNA concentration, PPC concentration, PPC / DNA ratio, osmolality, and pH after up to 30 months of storage when compared to the 1x concentrated IL-12 / PPC complex stored at -80°C during storage at -20°C or 4°C. Thus, these results indicate that the 5x concentration was able to maintain stability of particle size, DNA concentration, PPC concentration, PPC / DNA ratio, osmolality, pH, and biological activity at 4°C and -20°C during the 30-month storage period. Furthermore, the results demonstrate superior relative potency of the 5x formulation compared to the 1x formulation after 30 months of storage at 4°C.

[0252] It should be understood that the above-described compositions and application modes are merely illustrative of embodiments of the present invention. Numerous modifications and alternative configurations may be devised by those skilled in the art without departing from the spirit and scope of the present invention, and the appended claims are intended to cover all such modifications and configurations. Thus, while specific aspects of the present invention have been described above, it will be apparent to those skilled in the art that many modifications, including but not limited to changes in size, material, shape, form, function and mode of operation, assembly and use, can be made without departing from the principles and concepts described herein.

Claims

1. 1. A method of making a concentrated nucleic acid composition, comprising: (a) combining (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide, (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC), and (iii) a filler excipient in an aqueous medium to complex the nucleic acid with the cationic lipopolymer, thereby forming a nucleic acid mixture having a concentration of at least 0.1-0.2 mg / mL; (b) concentrating the nucleic acid mixture of (a) by tangential flow filtration to form a concentrated nucleic acid composition having a concentration of at least 0.7-0.8 mg / mL, wherein the recovery of nucleic acid complexed with the cationic lipopolymer after the tangential flow filtration is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, and wherein the concentrated nucleic acid composition is suitable for pharmaceutical use, storage at about -20°C or below, storage at about 4°C or below, and / or lyophilization.

2. 10. The method of claim 1, further comprising storing the concentrated nucleic acid composition at about -20°C or below, or at about 4°C or below, for at least 24 hours, at least 1 week, at least 1 month, at least 1 year, or at least 30 months.

3. 3. The method of claim 1 or 2, wherein the nucleic acid mixture has a concentration of at least 0.15 mg / mL.

4. The method of any one of claims 1 to 3, wherein the concentrated nucleic acid composition has a concentration of at least 0.75 mg / mL.

5. 5. The method of any one of claims 1-4, further comprising: (c) lyophilizing the condensed nucleic acid composition having a concentration of at least 0.75 mg / mL to a volume of 20-40 mL, thereby forming a lyophilized formulation comprising at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, or at least 30 mg of the DNA plasmid.

6. The method of claim 5, wherein the lyophilized formulation comprises about 10 to 50 mg of the DNA plasmid.

7. 7. The method of claim 5 or 6, further comprising (d) reconstituting the lyophilized formulation in a diluent, thereby forming a reconstituted composition.

8. 8. The method of claim 7, wherein the reconstituted composition comprises about 0.1 mg / mL to about 20 mg / mL of the DNA plasmid.

9. 8. The method of claim 7, wherein the diluent is water, about 5% dextrose, or saline.

10. The method of any one of claims 1 to 9, wherein the DNA plasmid further comprises a promoter, preferably a cytomegalovirus (CMV) promoter.

11. The method of any one of claims 1 to 10, wherein the nucleic acid mixture comprises GEN-1 nanoparticles.

12. 12. The method of any one of claims 1 to 11, wherein the ratio of amine nitrogens in the cationic polymer backbone to phosphate in the nucleic acid is from about 10:1 to about 100:

1.

13. The method of any one of claims 1 to 12, wherein the filler comprises a sugar, a sugar alcohol, a starch, a cellulose, or a combination thereof.

14. 14. The method of any one of claims 1 to 13, wherein the filler comprises one or more of lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, ficoll, sodium chloride, calcium phosphate, calcium carbonate, and / or polyethylene glycol.

15. The method of any one of claims 1 to 14, wherein the filler comprises lactose.

16. A pharmaceutical composition comprising the concentrated nucleic acid composition of any one of claims 1 to 15 or the reconstituted composition of any one of claims 7 to 15.

17. 17. The pharmaceutical composition of claim 16, comprising more than 6 mg of DNA plasmid in a volume of less than 50 mL.

18. 18. The pharmaceutical composition of claim 16 or 17, comprising 10 to 40 mg of DNA plasmid in a volume of less than 50 mL.

19. 19. The pharmaceutical composition of any one of claims 16 to 18, comprising about 30 mg of DNA plasmid in a volume of 20 to 45 mL.

20. 20. The pharmaceutical composition according to any one of claims 16 to 19, which is used to prepare a dose of 100 to 200 mg of plasmid DNA in less than 300 mL of aqueous solution.

21. 21. The pharmaceutical composition of any one of claims 16 to 20, wherein the DNA particle size, concentration, osmolality and pH are maintained after storage at about -20°C or about 4°C for at least up to 30 months.

22. A method for treating cancer in a subject, comprising administering to a subject in need thereof the pharmaceutical composition of any one of claims 16 to 21.

23. 23. The method of claim 22, wherein the pharmaceutical composition is formulated for intratumoral, intraperitoneal, intravesicular, intravenous, intraarterial, intratracheal, intrahepatic portal, intracranial, intramuscular, or intraarticular administration.

24. 24. The method of claim 22 or 23, wherein the cancer is ovarian cancer.

25. The method of any one of claims 22 to 24, further comprising administering a chemotherapeutic agent to the subject.

26. 1. A kit comprising: a first vial containing a nucleic acid mixture comprising: (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium; The nucleic acid is complexed with the cationic lipopolymer, and the nucleic acid mixture comprises at least 25 mg to 50 mg of the DNA plasmid, and optionally further comprises a second vial containing a diluent.

27. 27. The kit of claim 26, wherein the first vial and / or the second vial can hold up to about 100 mL, about 150 mL, about 200 mL, about 250 mL, or about 500 mL.

28. 28. The kit of claim 26 or 27, wherein the second vial contains a volume of diluent of about 200-300 mL.

29. 29. The kit of any one of claims 26-28, wherein the first vial contains about 10-50 mg, about 10-40 mg, about 20-50 mg, about 20-25 mg, about 20-45 mg, about 20-40 mg, or about 25-35 mg of the DNA plasmid.

30. 1. A kit comprising: a vial containing a nucleic acid mixture comprising: (i) a DNA plasmid comprising a nucleic acid encoding a human IL-12 polypeptide; (ii) a cationic lipopolymer comprising polyethylene glycol (PEG)-polyethyleneimine (PEI)-cholesterol (PPC); and (iii) a bulking agent in an aqueous medium, wherein the nucleic acid is complexed with the cationic lipopolymer, and the nucleic acid mixture is contained in an aqueous solution at a concentration of at least 0.7 mg / mL to 0.8 mg / L (e.g., at least 0.75 mg / mL).

31. 31. The kit of claim 30, wherein the nucleic acid mixture is contained in an aqueous solution at a concentration of at least 0.75 mg / mL.

32. 32. The kit of claim 30 or 31, wherein the first and / or second of the vials can hold up to about 100 mL, about 150 mL, about 200 mL, about 250 mL, or about 500 mL.

33. 33. The kit of any one of claims 30 to 32, wherein the vial further comprises a diluent in a volume of about 200 to 300 mL.

34. 34. The kit of any one of claims 30 to 33, wherein the vial contains 10 to 50 mg, 10 to 40 mg, 20 to 50 mg, 20 to 45 mg, 20 to 40 mg, 20 to 25 mg, or 25 to 35 mg of the DNA plasmid.

35. The kit according to any one of claims 26 to 34, which can be stored at -20°C or below.

36. The kit according to any one of claims 26 to 35, wherein the composition is prepared according to the method according to any one of claims 1 to 15 or the pharmaceutical composition according to any one of claims 16 to 21.

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