Enhanced non-viral DNA delivery and expression

Abstract

The present invention features methods and compositions that can be used to facilitate intracellular delivery of DNA into a subject. The provided methods and compositions utilize nanoparticles for intracellular DNA delivery and a type 1 interferon receptor pathway inhibitor. The type 1 interferon receptor pathway inhibitor is provided to reduce an immune response in a subject that may be stimulated by the DNA.

Description

[Background technology]

[0001]

[0003] Gene therapy involves using nucleic acids to modify the DNA of a subject to achieve beneficial effects. Genetic modification can be performed using different strategies, including gene enhancement, gene suppression, and genome editing (Anguela and High Annu.Rev.Med. 2019, 70, 73; and Li et al., Signal Transduction and Targeted Therapy 2020, 5, 1).

[0002]

[0004] An effective delivery system for nucleic acids is critical to the success of gene therapy. Successful delivery of nucleic acids provides sufficient amounts to target cells to achieve a beneficial effect without unacceptable adverse reactions. The delivery system must protect the genetic material from enzymatic degradation, be sufficiently long-lived in the body, be able to reach the required site in the body, have tolerable toxicity, and be able to cross cell membranes.

[0003]

[0005] Gene therapy vectors can be broadly classified as viral and non-viral. Each type of vector has advantages and disadvantages. Viral vectors are generally more effective at delivering genetic material to cells, but have a higher potential for immunogenicity, toxin production, and insertional mutagenesis, and a more limited transgenic volume size. Advantages of non-viral vectors include a larger transgene capacity, the ability to administer to subjects with pre-existing antibodies to the vector capsid, and a greater ability to re-administer the subject. Challenges associated with non-viral delivery may include low transfection efficiency, potential for nucleic acid degradation, innate immunity, low efficiency of gene delivery to somatic cell targets, and lower in vivo gene expression levels than viral approaches (Hardee et al., Genes (2017) 8, 65; Nayerossadat et al., Adv. Biomed Res. (2012) 1, 27). Summary of the Invention

[0004]

[0006] The present invention features methods and compositions that can be used in methods involving intracellular delivery of DNA to a subject. The provided methods and compositions utilize nanoparticles for intracellular DNA delivery and a type 1 interferon receptor pathway inhibitor. The type 1 interferon receptor pathway inhibitor is provided to reduce an immune response in a subject that may be stimulated by the DNA.

[0005]

[0007] Thus, a first aspect of the present invention provides a method for intracellular delivery of DNA, comprising the steps of: a) a type 1 interferon receptor pathway inhibitor; and b) First nanoparticles containing DNA wherein step (b) can be performed before, simultaneously with, or after step (a).

[0006]

[0008] Another aspect of the present invention describes a nanoparticle comprising (a) DNA and (b) a type 1 interferon receptor pathway inhibitor.

[0007]

[0009] Additional aspects of the present invention include pharmaceutical compositions containing the nanoparticles, inhibitors, and DNA vectors described herein, pharmaceutical compositions for use as described herein, and preparations of medicaments for use as described herein. Pharmaceutical compositions for use as described herein can provide a DNA vector containing a transgene for use in a patient administered before, simultaneously with, or after a type 1 interferon receptor pathway inhibitor. Similarly, preparations of medicaments for use as described herein can include preparation of a pharmaceutical composition containing a DNA vector containing a transgene for use in a patient before, simultaneously with, or after a type 1 interferon receptor pathway inhibitor, or preparation of a pharmaceutical composition containing a type 1 interferon receptor pathway inhibitor DNA for use in a patient before, simultaneously with, or after a DNA vector containing a transgene.

[0008]

[0010] Other features and advantages of the present invention will be apparent from the additional description provided herein, including the different examples. The examples provided illustrate different components and methodologies useful in carrying out the invention. Such examples do not limit the claimed invention. Based on this disclosure, one skilled in the art will be able to identify and utilize different components and methodologies useful in carrying out the invention. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the survival rates of wild-type (WT) and interferon receptor alpha receptor (IFNAR)-deficient (IFNAR KO) mice administered systemically with 1.25 mpk (25 μg) of plasmid DNA encoding the human factor IX (hFIX) transgene encapsulated in lipid nanoparticles at t = 0, 7 weeks, and 12 weeks. [Figure 2] Figure 1 shows hFIX levels in WT and IFNAR-deficient (IFNAR KO) mice after systemic administration of 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles at t = 0, 7 weeks, and 12 weeks. [Figure 3A] Figure 3 shows cytokine levels in WT mice systemically dosed 6 weeks (day 41) after an initial dose of 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles, and either untreated or treated once with 15 mpk of an anti-mouse IFNAR blocking antibody ("+aIFNAR"). Plasma cytokine levels were assessed 4 hours after dosing on day 41 and compared to pooled plasma pre-dose levels ("baseline"). Figure 3A shows IL-6 levels, Figure 3B shows IFN-alpha levels, and Figure 3C shows IFN-gamma levels. "LLOQ" refers to the lower limit of quantification, and "ULOQ" refers to the upper limit of quantification. [Figure 3B]Figure 3 shows cytokine levels in WT mice systemically dosed 6 weeks (day 41) after an initial dose of 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles, and either untreated or treated once with 15 mpk of an anti-mouse IFNAR blocking antibody ("+aIFNAR"). Plasma cytokine levels were assessed 4 hours after dosing on day 41 and compared to pooled plasma pre-dose levels ("baseline"). Figure 3A shows IL-6 levels, Figure 3B shows IFN-alpha levels, and Figure 3C shows IFN-gamma levels. "LLOQ" refers to the lower limit of quantification, and "ULOQ" refers to the upper limit of quantification. [Figure 3C] Figure 3 shows cytokine levels in WT mice systemically dosed 6 weeks (day 41) after an initial dose of 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles, and either untreated or treated once with 15 mpk of an anti-mouse IFNAR blocking antibody ("+aIFNAR"). Plasma cytokine levels were assessed 4 hours after dosing on day 41 and compared to pooled plasma pre-dose levels ("baseline"). Figure 3A shows IL-6 levels, Figure 3B shows IFN-alpha levels, and Figure 3C shows IFN-gamma levels. "LLOQ" refers to the lower limit of quantification, and "ULOQ" refers to the upper limit of quantification. [Figure 4] Figure 1 shows survival rates in WT mice systemically dosed with 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles at t=0 and 2.5 mpk (50 μg) at day 41, and either untreated or treated once prior to each dose with 15 mpk of an anti-mouse IFNAR blocking antibody. [Figure 5] Figure 1 shows hFIX levels in WT mice systemically dosed with 1.25 mpk (25 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles at t=0 and 2.5 mpk (50 μg) at day 41, and either untreated or treated once with 15 mpk of an anti-mouse IFNAR blocking antibody. [Figure 6]Figure 1 shows the survival rate in WT mice that were systemically administered 2.5 mpk (50 μg) of plasmid DNA encoding the human factor IX (hFIX) transgene encapsulated in lipid nanoparticles and either untreated or orally treated with ruxolitinib or baricitinib 30 minutes before and 1, 2, and 3 days after systemic administration of the plasmid DNA encoding the hFIX transgene encapsulated in lipid nanoparticles. [Figure 7] Figure 1 shows FIX protein levels in the plasma of mice either untreated or orally treated with baricitinib ("Bar") 90 minutes before DNA-LNP dosing and then daily on days 1 through 6 after DNA-LNP dosing. Transgenic hFIX protein in the plasma of surviving mice was measured by ELISA 1 week after DNA-LNP dosing. [Figure 8] Figure 1 shows FIX protein levels in wild-type C57BL / 6 ("WT") mice and STING-deficient mice (i.e., mice with the Goldentiquet nonsense mutation ("STING(Gt)"). Mice were either untreated or treated IP once with 15 mpk of an anti-mouse IFNAR-blocking antibody ("anti-IFNAR") 3 hours prior to systemic (IV tail injection) dosing of 5 mpk (100 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles (DNA-LNP). Transgenic hFIX protein in the plasma of surviving mice was measured by ELISA 4 weeks after DNA-LNP dosing. "LLOQ" refers to the lower limit of quantification, and "ULOQ" refers to the upper limit of quantification. [Figure 9A] Figure 9 shows the effect of anti-IFNAR antibodies delivered by different LNPs on transgene FIX expression in C57BL / 6 mice. Figure 9A shows the results using GenVoy-ILM™. Figure 9B shows the results using LNPs containing Compound 9. "LLOQ" refers to lower limit of quantification. [Figure 9B]Figure 9 shows the effect of anti-IFNAR antibodies delivered by different LNPs on transgene FIX expression in C57BL / 6 mice. Figure 9A shows the results using GenVoy-ILM™. Figure 9B shows the results using LNPs containing Compound 9. "LLOQ" refers to lower limit of quantification. [Figure 10] 1 shows the effect of anti-IFNAR antibodies delivered by LNPs on transgene erythropoietin (EPO) expression in Balb / c mice. "LLOQ" refers to the lower limit of quantification, and "ULOQ" refers to the upper limit of quantification. DETAILED DESCRIPTION OF THE INVENTION

[0010]

[0021] The present invention features nanoparticles containing DNA and methods and compositions for intracellular DNA delivery to a subject utilizing a type 1 interferon receptor pathway inhibitor. Intracellular DNA delivery has different uses, including delivery of a DNA vector to a subject for transgene expression. As shown in the examples below, potential benefits of inhibiting a type 1 interferon receptor pathway inhibitor include improved transgene expression and reduced cytokine production, including IFN-gamma production.

[0011]

[0022] A "type 1 interferon receptor pathway inhibitor" refers to a compound that inhibits type 1 interferon receptor (IFNAR) activity and / or signal transduction from type 1 interferon receptor. Type 1 interferon receptor pathway inhibitors include compounds that inhibit IFNAR receptor activity and downstream activities, such as activities mediated by Janus-activated kinase 1, tyrosine kinase 2, or signal transducers and activators of transcription (STAT) proteins.

[0012]

[0023] "Nanoparticles" refer to small, non-viral particles that can encapsulate or bind DNA and facilitate DNA delivery to cells. Nanoparticles can also be used to deliver, for example, different DNA vectors, different transgenes, type 1 interferon receptor pathway inhibitors, cytosolic DNA sensing inhibitors, and immune cell modulators. Nanoparticles range in size from about 10 nm to about 1000 nm. In different embodiments, nanoparticles are about 50 nm to about 500 nm, or about 50 nm to about 200 nm.

[0013]

[0024] Reference to a "subject" refers to mammals, including humans; non-human primates, such as apes, gibbons, gorillas, chimpanzees, orangutans, and cynomolgus monkeys; livestock, such as dogs and cats; farm animals, such as poultry and ducks, horses, cows, goats, sheep, and pigs; and laboratory animals, such as mice, rats, rabbits, and guinea pigs. Preferred subjects are human subjects to be treated. However, subjects can also include animal disease models, such as mouse and other animal models of protein / enzyme deficiencies, such as Pompe disease (loss of GAA) and glycogen storage diseases (GSD).

[0014]

[0025] Reference to a "DNA vector" refers to a DNA polymer containing a transgene operably linked to another regulatory element that effects RNA expression from the transgene. The RNA produced can be functional by itself or can encode a protein. One type of regulatory element is a promoter, which binds RNA polymerase and the transcription factors necessary to initiate transcription. If encoding a protein, the RNA sequence produced also encodes a termination sequence at the end of the coding sequence. Additional regulatory elements include elements that affect RNA expression, RNA stability, and protein production. DNA vectors can be single-stranded, double-stranded, or contain a combination of single- and double-stranded regions. DNA vectors can also contain two or more transgenes and multiple regulatory elements of the same or different types.

[0015]

[0026] DNA refers to DNA polymers and includes double-stranded DNA, single-stranded DNA, and DNA with single- and double-stranded regions.

[0016]

[0027] Reference to DNA comprising a vector and "substantially comprising," "comprises," or "comprising" "double-stranded DNA" indicates that more than half, at least 75%, at least 90%, at least 95%, or at least 99% of the DNA is double-stranded, or that 100% of the DNA is double-stranded. Similarly, reference to DNA comprising a vector and "substantially comprising," "comprises," or "comprising" "single-stranded DNA" indicates that more than half, at least 75%, at least 90%, at least 95%, or at least 99% of the DNA is single-stranded, or that 100% of the DNA is single-stranded.

[0017]

[0028] The term "operably linked" refers to the association of two or more nucleic acid segments of a single DNA fragment so that the function of one is affected by the other.

[0018]

[0029] References to a "transgene" refer to a region of DNA capable of being expressed into RNA, regardless of the origin of the transgene sequence. A transgene is generally a portion of a larger length of DNA, which contains at least one region with which the transgene is not normally associated in nature.

[0019]

[0030] The singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0020]

[0031] As used herein, the connective term "and / or" between multiple mentioned elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the applicability of the first alternative without the second alternative, the second alternative refers to the applicability of the second alternative without the first, and the third alternative refers to the applicability of both the first and second alternatives. Any one of the alternatives is understood to be included within the meaning and therefore satisfies the requirements of the term "and / or." The simultaneous applicability of two or more alternatives is also understood to be included within the meaning of the term "and / or."

[0021]

[0032] Unless otherwise clearly indicated by the context in which they are used, the terms "or" and "and" have the same meaning as "and / or."

[0022]

[0033] Reference to terms such as "including," "for example," or "such as" used in conjunction with different members or examples is an open-ended description in which the listed members or examples are exemplary and other members or examples may be provided or used.

[0023]

[0034] The terms "polypeptide," "protein," and "peptide" can be used interchangeably to refer to amino acid sequences without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids. The amino acids provided include naturally occurring amino acids and amino acids obtained by cellular modification.

[0024]

[0035] References to "comprise" and variations such as "comprises" and "comprising," when used in reference to an element or group of elements, are open-ended and do not exclude additional unrecited elements and method steps. Terms such as "including," "containing," and "characterized by" are synonymous with comprising. In different aspects and embodiments described herein, references to open-ended terms such as "comprising" can be replaced with the terms "consisting" or "consisting essentially of."

[0025]

[0036] A reference to "consisting of" excludes any element, step, or ingredient not specified in the recited claim element, and such element, step, or ingredient relates to the claimed invention.

[0026]

[0037] References to "consisting essentially of" limit the scope of a claim to the particular materials or steps and to those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0027]

[0038] The term "about" refers to values ​​within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, "about 1:10" includes 1.1:10.1 or 0.9:9.9, and "about 5 hours" includes 4.5 hours or 5.5 hours. The term "about" at the beginning of a series of values ​​modifies each of the values ​​by 10%.

[0028]

[0039] All numerical values ​​or ranges of values ​​include integers within such ranges and fractions of values ​​or integers within ranges unless expressly stated otherwise. Thus, for purposes of illustration, reference to a reduction of 95% or greater includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc.; reference to a numerical range such as "1 to 4" includes 1, 2, 3, as well as 1.1, 1.2, 1.3, 1.4, etc. a reference to "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, and a reference to a numerical range such as "0.01 to 10" includes 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, 10, etc. For example, a dosage of "0.01 mg / kg to 10 mg / kg" of a subject's body weight includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0029]

[0040] Reference to a greater or lesser integer includes greater or lesser numbers, respectively, than the referenced number. Thus, for example, a reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and "two or more" administrations includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0030]

[0041] Various references, including articles and patent publications, are cited or discussed throughout the Background and Specification. Each of these references is incorporated herein by reference in its entirety. None of the references is admitted to be prior art with respect to any invention disclosed or claimed. In some cases, specific references are incorporated herein by reference to indicate the emphasis of incorporation.

[0031]

[0042] The definitions provided herein, including those in this section and other sections herein, apply throughout the specification.

[0032]

[0043] 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 invention pertains.

[0033]

[0044] The description is divided into various sections and paragraphs, providing various embodiments. These divisions should not be considered to separate a paragraph or section or embodiment entity from another paragraph or section or embodiment entity. The provided description has broad applicability and encompasses all combinations of the various sections, paragraphs, and sentences that may be contemplated. The discussion of any embodiments is meant to be illustrative only and is not intended to suggest that the scope of the present disclosure, including the claims (unless otherwise provided in the claims), is limited to these examples.

[0034]

[0045] Although certain combinations of features are highlighted herein, all of the features disclosed herein can be combined in any combination. I. Nanoparticles

[0046] A variety of different nanoparticles are available, including lipid nanoparticles (LNPs), polymeric nanoparticles, lipid-polymer nanoparticles (LPNPs), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide-cage nanoparticles, and exosomes (see, e.g., Riley and Vermerris Nanomaterials 2017, 7, 94; Thomas et al., Molecules 2019, 24, 3744; Bochicchio et al., Pharmaceutics 2021, 13, 198; Munagala et al., Cancer Letters 2021, 505, 58; Fu et al., 2020 NanoImpact 20, 100261; and Neshat et al. Current Opin. Biotechnol. 2020, 66:1-10).

[0035]

[0047] Optionally, the nanoparticles can be targeted to cell types using targeting ligands that recognize target cell receptors, for example. Examples of targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies (e.g., anti-HER2 antibodies and hD1), and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoprotein) and peptides (see, for example, Teo et al., Advanced Drug Delivery Reviews 2016, 98, 41).

[0036]

[0048] This application features the use of nanoparticles to deliver DNA. In different embodiments, the nanoparticles can deliver additional compounds, such as a type 1 interferon receptor pathway inhibitor, a cytosolic DNA sensing inhibitor, an immunosuppressant, a phagocyte-depleting compound, and an additional therapeutic compound, where the one or more additional compounds are provided on different nanoparticles, or the one or more additional compounds are provided on the same nanoparticle as the DNA vector, e.g., the DNA vector and the type 1 interferon receptor pathway inhibitor, or the DNA vector, the type 1 interferon receptor pathway inhibitor, the cytosolic DNA sensing inhibitor, and the immune cell modulator. Reference to "compound" includes small molecules and macromolecules (e.g., therapeutic proteins and antibodies), as well as nucleic acids.

[0037]

[0049] The production of different nanoparticles and the incorporation of nucleic acids and other compounds is well known in the art and is exemplified in different publications throughout the discussion in Section I. In general, the exposure kinetics of nanoparticle cargo (e.g., DNA and / or inhibitors) can be affected by generating different compounds and DNA that contain binding to different environments or different structures.

[0038]

[0050] Examples of publications demonstrating the incorporation of nucleic acids in certain nanoparticles, such as LPNPs and LNPs, include Teo et al., Advanced Drug Delivery Reviews 2016, 98, 41; Bochicchio et al., Pharmaceutics 2021, 13, 198; Mahzabin and Das, IJPSR 2021, 12(1), 65; and Teixeira et al., Progress in Lipid Research 2017, 1 (all of which are incorporated by reference in their entirety). Such references also point out the advantages of LPNPs in generating different structures that interact with nucleic acids and small molecules, which can affect the desired release kinetics. Factors that can influence the incorporation of small molecules into nanoparticles include the presence of hydrophobic and ionizable moieties (see, e.g., Nii and Ishii, International Journal of Pharmaceutics 2005, 298, 198; and Chen et al., Journal of Controlled Release 2018, 286, 46).

[0039]

[0051] In one embodiment, the compound (e.g., a type 1 interferon receptor pathway inhibitor, a cytosolic DNA sensing inhibitor, and / or an immune cell modulator) is conjugated with a fatty acid to increase its hydrophobicity. Examples of fatty acids that can be conjugated to small molecules include those described in Chen et al., Journal of Controlled Release 2018, 286, 46-54.

[0040] IA lipid-based delivery system

[0052] Lipid-based delivery systems involve the use of lipids as components. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0041]

[0053] "Lipid nanoparticle" or "LNP" refers to lipid-based vesicles with nanoscale dimensions that are useful for delivery of nucleic acid molecules. In different embodiments, the nanoparticles are about 10 nm to about 1000 nm, about 50 nm to about 500 nm, or about 50 nm to about 200 nm.

[0042]

[0054] DNA is negatively charged.Therefore, this can be beneficial for LNPs that contain cationic lipids, such as amino lipids.Exemplary amino lipids are described in U.S. Patent No. 9,352,042, U.S. Patent No. 9,220,683, U.S. Patent No. 9,186,325, U.S. Patent No. 9,139,554, U.S. Patent No. 9,126,966, U.S. Patent No. 9,018,187, U.S. Patent No. 8,999,351, U.S. Patent No. 8,722,082, U.S. Patent No. 8,642,076, U.S. Patent No. 8,569,256, U.S. Patent No. 8,466,122, and U.S. Patent No. 7,745,651 and U.S. Patent Application Publication No. 2016 / 02 No. 13785, U.S. Patent Application Publication No. 2016 / 0199485, U.S. Patent Application Publication No. 2015 / 0265708, U.S. Patent Application Publication No. 2014 / 0288146, U.S. Patent Application Publication No. 2013 / 0123338, U.S. Patent Application Publication No. 2013 / 0116307, U.S. Patent Application Publication No. 2013 / 0064894, U.S. Patent Application Publication No. 2012 / 0172411, and U.S. Patent Application Publication No. 2010 / 0117125, all of which are incorporated herein in their entireties. In certain embodiments, the LNP comprises an amino lipid as described in U.S. Patent No. 9,512,073, which is incorporated herein in its entirety.

[0043]

[0055] The terms "cationic lipid" and "amino lipid" are used interchangeably herein and include lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and pH-titratable amino groups (e.g., alkylamino or dialkylamino groups). Cationic lipids are typically protonated (i.e., positively charged) at pHs below the pKa of the cationic lipid and are substantially neutral at pHs above the pKa. Cationic lipids may also be titratable cationic lipids. In certain embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) group; a C18 alkyl chain, each alkyl chain of which may independently have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0044]

[0056] The cationic lipids were 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA, DLin-C2K-DMA, XTC2, and C2K). Also known as DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, also known as MC3), salts thereof, and mixtures thereof. Other cationic lipids also include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).

[0045]

[0057] Still further cationic lipids include 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleylcarbamoyloxy-3-dimethylamino ... -3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleyl)amino)-2-propanediol (DLin-EG-DMA ... N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ( DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]- These include 3-dimethyl-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-sperimine (DS), and disubstituted spermine (DSS), or mixtures thereof.

[0046]

[0058] Several commercially available preparations of cationic lipids can be used, such as LIPOFECTIN® (containing DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (containing DOSPA and DOPE, available from GIBCO / BRL).

[0047]

[0059] Additional ionizable lipids that can be used include C12-200, 306Oi10, MC3, cKK-E12, ATX-002, ATX-003, and Merck-32. U.S. Patent Application Publication No. 2017 / 0367988 describes Merck-32.

[0048]

[0060] In further embodiments, the cationic lipid may be present in an amount from about 10% by molar ratio of the LNP to about 85% by molar ratio of the LNP, or from about 50% by molar ratio of the LNP to about 75% by molar ratio of the LNP.

[0049]

[0061] LNPs can include neutral lipids. Neutral lipids can include lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by considerations including particle size and stability. In certain embodiments, the neutral lipid component can be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0050]

[0062] Lipids with various acyl chain groups of varying chain lengths and degrees of saturation are available or can be isolated or synthesized. In certain embodiments, lipids containing saturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In certain embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths ranging from C14 to C22 can be used. In addition, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids containing other head groups, such as serine and inositol.

[0051]

[0063] In further embodiments providing a neutral lipid, the neutral lipid may be present in an amount from about 0.1% by weight of the LNP to about 99% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0052]

[0064] LNPs may contain additional components such as sterols and polyethylene glycol. Sterols can impart fluidity to LNPs. As used herein, "sterol" refers to natural sterols derived from plants (phytosterols) or animals (zoosterols), as well as non-natural synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Suitable sterols include those conventionally used in the preparation of liposomes, lipid vesicles, or lipid particles, most commonly cholesterol. Plant sterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156. In different embodiments providing a sterol, the sterol is present in an amount ranging from about 1% by weight of the LNP to about 80% by weight of the LNP or from about 10% by weight of the LNP to about 25% by weight of the LNP.

[0053]

[0065] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units and can be linear or branched. PEGs are classified by their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. Commercially available PEGs from Sigma Chemical Co. and other companies include monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM).

[0054]

[0066] In certain embodiments of PEG, the PEG has an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted with alkyl, alkoxy, acyl, or aryl. In further embodiments, the PEG is substituted with methyl at the terminal hydroxyl positions. In further embodiments, the PEG has an average molecular weight of about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons.

[0055]

[0067] PEG-modified lipids include PEG-dialkyloxypropyl conjugates (PEG-DAAs) described in U.S. Patent Nos. 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) can have various "anchor" lipid moieties that anchor the PEG moiety to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) described in U.S. Patent No. 5,820,873, PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. In certain embodiments, PEG-modified lipids can be PEG-modified diacylglycerols and dialkylglycerols. In certain embodiments, PEG can be present in an amount ranging from about 0.1% by weight of the LNP to about 50% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

[0056]

[0068] In further embodiments regarding LNP size, prior to encapsulating nucleic acid, the LNPs have a size range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or 75 nm to about 125 nm.

[0057]

[0069] In certain embodiments of LNPs, the LNPs are described in Billingsley et al., Nano Lett. 2020, 20, 1578, or Billingsley et al., International Patent Publication No. 2021 / 077066 (both of which are incorporated herein by reference in their entireties). Billingsley et al. and International Patent Publication No. 2021 / 077066 describe LNPs containing a lipid anchor PEG, cholesterol, phospholipids, and ionizable lipids. In certain embodiments, the LNPs contain a C14-4 polyamine core and / or have a particle size of about 70 nm. The C14-4 has the following structure: [ka]

[0058]

[0070] In certain embodiments, the LNPs are comprised of cationic lipids or lipopeptides described in U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374, or WO 2021 / 077066 (each of which is incorporated by reference in its entirety). In certain embodiments, the LNPs contain cationic lipids, cholesterol-based lipids, and / or one or more PEG-modified lipids. In certain embodiments, the LNPs contain cKK-E12 (Dong et al., PNAS (2014) 111(11), 3955): [ka]

[0059]

[0071] In certain embodiments, the LNP comprises a modified form of cKK-E12, referred to herein as "bCKK-E12," having the following structure:

[0060] [ka]

[0072] Certain embodiments are directed to bCKK-E12 or a pharmaceutically acceptable salt thereof. In certain embodiments, the salt is an acid addition salt, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogensulfate, phosphate, acid phosphate, isonicotinate, carbonate, bicarbonate, acetate, lactate, salicylate, citrate, tartrate, propionate, butyrate, pyruvate, oxalate, malonate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0061]

[0073] In certain embodiments, the LNPs comprise lipid 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described in Sabnis et al., Molecular Therapy 2018, 26:6, 1509-1519, which is incorporated herein by reference in its entirety. In certain embodiments, the LNPs comprise lipid 5, 8, 9, 10, or 11 as described in Sabnis et al.

[0062]

[0074] Lipid 5 of Sabnis et al. has the structure:

[0063] [ka] It has.

[0064]

[0075] Lipid 9 of Sabnis et al. has the structure:

[0065] [ka] It has.

[0066]

[0076] Additional lipids include those described in U.S. Patent Application Publication No. 2022204439, which is incorporated herein by reference in its entirety. One of the lipids described in U.S. Patent Application Publication No. 2022204439 is Compound 9: [ka] is.

[0067]

[0077] Additional lipids that may be utilized include those described in Roces et al., Pharmaceutics, 2020, 12, 1095; Jayaraman et al., Angew. Chem. Int. Ed., 2012, 51, 8529-8533; Maier et al., www.moleculartherapy.org, 2013, 21, No. 8, 1570-1578; Liu et al., Adv. Mater. 2019, 31, 1902-575, e.g., BAMEA-O16B; Ch Eng et al., Adv. Mater., 2018, 30, 1805308, e.g., 5A2-SC8; Hajj and Ball, Small, 2019, 15, 1805097, e.g., 306Oi10; Du et al., U.S. Patent Application Publication No. 2016 / 0376224; and Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575, each of which is incorporated by reference in its entirety.

[0068] IB Additional Examples of Lipid-Based Delivery Systems

[0078] In further embodiments, the LNPs, by mol%, comprise, consist essentially of, or consist of the following components: (1) about 20% to about 65% of one or more cationic lipids, about 1% to about 50% of one or more phospholipid lipids, about 0.1% to about 10% of one or more PEG-conjugated lipids, and about 0% to about 70% of cholesterol; and (2) about 20% to about 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipid lipids, about 0.1% to about 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In further embodiments, the phospholipid lipid is a neutral lipid, and the phospholipid lipid is DOPE or DSPC.

[0069]

[0079] In further embodiments, the LNPs, by mole %, comprise, consist essentially of, or consist of the following components: (1) cKK-E12 (described further in Section IA above) about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12 (described further in Section IA above) about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) lipid 9 (Sabnis et al. and Section IA above). (4) lipid 5 (Sabnis et al. and further described in section IA above), about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; and (5) ionizable lipid, about 50%; DSPC, about 10%; cholesterol, about 37.5%; and stabilizer (PEG-lipid), about 2.5%; (6) Genvoi-ILM™ LNP (Precision NanoSystems); or (7) compound 9 (U.S. Patent Application Publication No. 2022204439) about 50%; C14-PEG2000, about 2.5%; cholesterol, about 37.5%; and DSPC, about 10%.

[0070] IC polymer-based nanoparticles

[0080] Polymer-based delivery systems can be composed of a variety of different natural and synthetic materials. DNA and other compounds can be entrapped in the polymer matrix of polymer nanoparticles or adsorbed or bound to the surface of the nanoparticles. Examples of polymers commonly used for nucleic acid delivery include poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI) and PEI derivatives, chitosan, dendrimers, polyanhydrides, polycaprolactone, polymethacrylate, poly-L-lysine, pullulan, dextran, as well as hyaluronic acid and poly-β-aminoesters (Thomas et al., Molecules 2019, 24, 3744).

[0071]

[0081] In certain embodiments, the polymeric nanoparticles have different sizes ranging from about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, from about 50 nm to about 200 nm, from about 100 nm to about 150 nm, and less than or equal to about 150 nm.

[0072] ID lipid polymer nanoparticles

[0082] Lipid-polymer nanoparticles are hybrid nanoparticles that contain both lipid and polymer components and can be considered LNPs or LPNPs. The relative configuration of LPNPs can result in an outer polymer and an inner lipid, or an outer lipid and an inner polymer. The presence of two different types of materials facilitates the design of nanoparticles that provide delayed release of components. Different lipid and polymer components can be selected taking into account the substance to be delivered (e.g., type 1 interferon receptor pathway inhibitors, cytosolic DNA sensing inhibitors, and DNA vectors) in line with the guidance provided herein and in the art (e.g., Teo et al., Advanced Drug Delivery Reviews 2016, 98, 41; Bochicchio et al., Pharmaceutics, 2021, 13, 198; Mahzabin and Das, IJPSR 2021, 12(1), 65; and Teixeira et al., Progress in Lipid Research, 2018, 1).

[0073] IE protein and peptide-based nanoparticles

[0083] Protein and peptide-based systems can utilize a variety of different proteins and peptides. Examples of proteins include gelatin and elastin. Peptide-based systems can utilize, for example, CPPs.

[0074]

[0084] CPPs are short peptides (6-30 amino acid residues) that may enable intracellular penetration for the delivery of therapeutic molecules. While the majority of CPPs consist primarily of arginine and lysine residues, which make them cationic and hydrophilic, CPPs can also be amphipathic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., Tat, HIV-1 protein) or obtained synthetically (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996-2006). Examples of CPPs include cationic CPPs (highly positively charged), such as Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine; amphipathic CPPs (chimeric or fusion peptides, constructed from different sources, containing both positively and negatively charged amino acid sequences), such as transportan, VT5, bactenecin-7 (Bac7), proline-rich peptides (PPRs), SAP (VRLPPP)3, TP10, pep-1, and MPG; membranotropic CPPs (which simultaneously exhibit both hydrophobic and amphipathic properties and contain both large aromatic and small residues), such as H625, SPION-PEG-CPP, and NP; and hydrophobic CPPs (containing only nonpolar motifs or residues), such as SG3, PFVYLI, pep-7, and fibroblast growth factor.

[0075]

[0085] Protein and peptide nanoparticles can be provided in different sizes, for example, in the range of about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or less than about 150 nm.

[0076] IF peptide cage nanoparticles

[0086] Peptide cage-based delivery systems can be generated from proteinaceous materials that can assemble into cage-like structures that form a confined internal environment. The peptide cage can include a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an internal cavity that is either naturally accessible to solvent or can be created by changing solvent concentration, pH, or equilibrium ratios). The monomers of the protein cage can be native or mutant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0077]

[0087] Different types of protein "shells" can be assembled and filled with different types of materials. Protein cages can be produced using viral coat protein(s) (e.g., from the protein coat of cowpea chlorotic mottle virus) as well as non-viral proteins (e.g., U.S. Pat. Nos. 6,180,389 and 6,984,386, U.S. Patent Application Publication No. 20040028694, and U.S. Patent Application Publication No. 20090035389, each of which is incorporated by reference in its entirety).

[0078]

[0088] Protein cages derived from non-viral proteins include ferritins and apoferritins from eukaryotes or prokaryotes, such as the 12- and 24-subunit ferritins; and heat shock proteins (HSPs), such as the 24-subunit heat shock proteins of the class that form the core interior space, the small HSP of Methanococcus jannaschii, the dodecamer Dsp HSP of E. coli, and the MrgA protein.

[0079]

[0089] In certain embodiments, the protein cages have different core sizes, such as in the range of about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or less than about 150 nm.

[0080] IG exosomes

[0090] Exosomes are small membrane vesicles that have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes generally range in size from about 30 nm to about 100 nm and can be taken up by cells to deliver their cargo. The cargo can be associated with the surface structure of the exosome or encapsulated within the exosome bilayer.

[0081]

[0091] Various modifications can be made to exosomes to facilitate cargo delivery and cellular targeting. Modifications that facilitate cargo delivery include structures for binding to cargo, such as protein backbones and polymers. Modifications for cellular targeting include targeting ligands and altering surface charge. Publications describing the production, modification, and use of exosomes for the delivery of different cargoes include Munagala et al., Cancer Letters 2021, 505, 58; Fu et al., 2020 NanoImpact 20, 100261; and Dooley et al., 2021 Molecular Therapy 29(5), 1729 (each of which is incorporated herein by reference).

[0082] II. Interferon Receptor Pathway Inhibitors

[0092] The interferon pathway, initiated by interferon binding, involves the activation of Janus-activated kinases, which can phosphorylate different proteins, including different signal transducers and activators of transcription (STAT) proteins. Interferons bind to interferon receptors, which initiate a cascade that results in the induction of IFN-stimulated gene transcription. Activation of certain proteins by the type 1 interferon receptor pathway, e.g., STAT1 and STAT2, activates the transcription of IFN-mimicking genes. For example, the STAT1-STAT2-IRF9 (IFN regulatory factor 9) complex can bind to IFN-stimulated response elements and initiate transcription, and the STAT1-STAT1 complex can bind to the IFN-γ active site and initiate transcription. There are a wide range of IFN-stimulated genes with different functions, including producing proteins involved in the suppression of viral gene expression (see, e.g., Yulantie et al., Acts Pharmaceutica Sinica B 2018, 86(6):889-899; Zanin et al., Frontiers in Immunology 2021, 11, Article 615603; Platanias Nature Review Immunology 2005, 5:370-386; and Schoggins Annual Review of Virology 2019, 6:567-84).

[0083]

[0093] The type 1 interferon receptor (IFNAR) is composed of the interferon alpha receptor 1 subunit (IFNAR1) and the interferon alpha receptor 2 subunit (IFNAR2). Type I interferons include IFN-α (which can be further divided into different subtypes), IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, and IFN-ω1, 2, and 3. IFN-α, IFN-β, IFN-e, IFN-κ, and IFN-ω are present in humans. Type 1 interferon receptors can phosphorylate different STATs, such as STAT1 and STAT2 (Platanias Nature Review Immunology 2005, 5:370-386).

[0084]

[0094] Type 1 interferon receptor pathway inhibitors include: (1) compounds that bind to the type 1 interferon receptor (IFNAR) and inhibit or block the binding of a ligand (e.g., interferon) to the IFNAR; (2) compounds that inhibit or block the activation of IFNAR; (3) compounds that inhibit or block downstream activities, such as Janus-activated kinase (JAK) activity and signal transducer and activator of transcription (STAT) activity; compounds that inhibit or block the expression of type 1 interferon pathway proteins (e.g., IFNAR, JAK2, tyrosine kinase 1, STAT1, and / or STAT2); (4) compounds that cause or result in the degradation of type 1 interferon pathway proteins (e.g., IFNAR, JAK2, tyrosine kinase 1, STAT1, and / or STAT2); and (5) compounds that inhibit IFNAR activation, generally by inhibiting the binding of a ligand to IFNAR (e.g., using a decoy). In certain embodiments, the type 1 interferon receptor pathway inhibitor is a small molecule, a polypeptide, including an antibody or antibody fragment, a peptide, a nucleic acid, or a degrading agent (e.g., a protease or degrader) targeting a protein, or an inhibitory nucleic acid, such as a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA i , ribozymes, antisense RNA, clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 constructs, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs).

[0085]

[0095] References providing examples of type 1 interferon receptor inhibitors include Gertenberger et al., J. Med. Chem. 2020, 63, 13561-13577; U.S. Patent No. 7,465,751; U.S. Patent Application Publication No. 2022 / 0144957; U.S. Patent No. 10,301,390; U.S. Patent No. 11,136,399; U.S. Patent No. 11,059,897; U.S. Patent No. 10,125,195; and International Patent Publication No. 2006 / 133426.

[0086]

[0096] In certain embodiments, the type 1 interferon receptor inhibitor is an antibody that binds to type 1 interferon receptor or comprises an antibody fragment that binds to type 1 interferon receptor. Different types of antibodies and antibody fragments can be used, including IgG-based, bispecific, human, and / or humanized. Binding fragments include FAb fragments, single-chain variable fragments (scFVs), single-domain fragments (dAbs), Fv fragments, camelid heavy chain variable domains (VHHs), minibodies, and bispecific antibodies. Examples of antibodies and polypeptides, including antibody fragments, are provided, for example, in Strohl, Protein Cell 2018, 9(1):86-129, and Chiu et al., Antibodies 2019, 8, 55 (both of which are incorporated herein by reference in their entirety).

[0087]

[0097] In certain embodiments, the type 1 interferon receptor inhibitor is an antibody that binds to the type 1 interferon receptor or is anifrolumab.

[0088]

[0098] In certain embodiments, the type 1 interferon receptor inhibitor binds to a type 1 interferon receptor intracellularly.

[0089]

[0099] In certain embodiments, the type 1 interferon receptor inhibitor binds to a type 1 interferon receptor extracellularly.

[0090]

[0100] References providing information on JAK inhibitors, JAK inhibitor scaffolds and motifs, and design considerations include Furumoto and Gadina BioDrugs 2013, 27(5); 431-438; Hu et al., Signal Transduction and Targeted Therapy 2021, 6:402; and WO 2022 / 81872, each of which is incorporated by reference in its entirety.

[0091]

[0101] In a different embodiment, the JAK inhibitor is as provided in Table 1, or a pharmaceutically acceptable salt thereof.

[0102]

[0092] [Table 1] JPEG2025531748000009.jpg193149

[0103] JAK inhibitors can differ in their selectivity: tofacitinib is selective for JAK1 and JAK3, baricitinib, ruxolitinib, and momelotinib are selective for JAK1 and JAK2, filgotinib, upadacitinib, abrocitinib, and itacitinib are selective for JAK1, and fedrantinib, pacritinib, and gandotinib are selective for JAK2. Decernotinib and peficitinib are selective for JAK3, gusacitinib is selective for JAK1, JAK2, JAK3 and TYK2, cerdulatinib is selective for JAK1, JAK2 and tyrosine kinase 2, compounds 1, 2 and 3 inhibit JAK1, JAK2, JAK3 and TYK2, and compounds PF-06826647, BMS-986165 and PF-06700841 inhibit TYK2.

[0093]

[0104] In different embodiments, the JAK inhibitor is selective for JAK1, selective for JAK2, selective for JAK3, or selective for tyrosine kinase 2. Reference to a selective JAK inhibitor refers to the ability to significantly inhibit a particular JAK (i.e., JAK1, JAK2, JAK3, or tyrosine kinase 2) over other JAK proteins within the JAK1, JAK2, JAK3, and tyrosine kinase 2 group. For example, an inhibitor that is selective for tyrosine kinase 2 inhibits tyrosine kinase 2 to a greater extent than it inhibits JAK1, JAK2, and JAK3. In different embodiments, selectivity refers to a 10-fold or 100-fold difference in activity (e.g., IC 50 ).

[0094]

[0105] References providing descriptions of STAT inhibitors, STAT inhibitor scaffolds and motifs, and design considerations include Yulantie et al., Acta Pharmaceutica Sinica B 2018, 8(6):889-899; Hu et al., Signal Transduction and Targeted Therapy 2021, 6:402; Miklossy et al., Nat Rev Drug Discov. 2013, 12(8):611-29; and Scully et al., J. Med. Chem. 2013, 56, 4125-4129, each of which is incorporated herein by reference in its entirety.

[0095]

[0106] In a different embodiment, the inhibitor is a STAT1 inhibitor provided in Table 2, or a pharmaceutically acceptable salt thereof.

[0107]

[0096] [Table 2]

[0097] III. DNA Vectors

[0108] A DNA vector contains a transgene and one or more regulatory elements that affect RNA expression or processing from the transgene. The RNA produced can be functional, for example, or can encode a specific protein. Regulatory elements can include, for example, elements that regulate transcription of functional RNA, production of the protein-encoding transgene, and protein processing. Regulatory elements that can be present include promoters, enhancer sequences, introns, Kozak sequences, post-transcriptional regulatory elements, polyadenylation signal sequences, regulatable sequences, cell-specific regulators, and internal ribosome entry sites. Depending on the DNA vector, the vector can contain elements in addition to regulatory elements, such as terminal inverted repeats, elements that facilitate plasmid replication and selection, and sequences that facilitate protein secretion. There can be multiple transgenes, which can be of the same or different types, and / or multiple elements, which can be of the same or different types.

[0098]

[0109] In one embodiment, DNA vectors are used in gene therapy. Gene therapy includes both loss-of-function and gain-of-function gene defects. The term "loss-of-function," with respect to gene defects, refers to a mutation in a gene in which the protein encoded by the gene exhibits either partial or complete loss of function normally associated with the wild-type protein. The term "gain-of-function," with respect to gene defects, refers to a mutation in a gene in which the protein encoded by the gene gains a function not normally associated with the wild-type protein, or which causes or contributes to a disease or disorder. A gain-of-function mutation can be a deletion, addition, or substitution of one or more nucleotides in a gene, resulting in a change in the function of the encoded protein. In certain embodiments, a gain-of-function mutation alters the function of the mutant protein or causes its interaction with other proteins. In certain embodiments, a gain-of-function mutation results in the reduction or elimination of the normal wild-type protein, for example, by allowing the altered mutant protein to interact with the normal wild-type protein.

[0099]

[0110] Different types of DNA vectors are available, including minicircles, nanoplasmids, open linear double-stranded DNA, closed linear double-stranded DNA (CELiD / ceDNA / Doggybone DNA), single-stranded circular DNA and single-stranded linear DNA.

[0100]

[0111] In one embodiment, the DNA vector takes into consideration the specific mammal selected as the target, motifs that promote gene expression, and sequences and motifs that induce immune stimulation. Gene expression in a specific mammal can be promoted, for example, by codon optimization, reduction of CpG, and reduction of RNA secondary structure and unstable motifs. Examples of immune stimulation motifs that can be reduced include CpG, pyrimidine-rich sequences, and palindromic sequences.

[0101]

[0112] In different embodiments, the transgene may be a viral antigen, a bacterial antigen, a therapeutic protein, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA i , encoding ribozymes, antisense RNAs, clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 constructs, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs).

[0102] III.A. Promoter

[0113] A promoter is typically located 5' to a polynucleotide sequence to be expressed and is operably linked to the polynucleotide sequence. For example, a promoter is operably linked to a polynucleotide sequence if it can affect the expression of the sequence (e.g., the sequence is under the transcriptional control of the promoter). A promoter binds RNA polymerase and necessary transcription factors to initiate transcription from the polynucleotide sequence. The promoter sequence determines the direction of transcription and which DNA strand is transcribed.

[0103]

[0114] Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. The term "heterologous promoter" refers to a promoter that is not found operably linked to a given coding sequence in nature.

[0104]

[0115] In certain embodiments, the promoter sequence is linked to an enhancer. An enhancer is a DNA region that increases promoter transcription. Typically, enhancers are located upstream of a promoter, but can also be located downstream of or within a promoter sequence. Enhancers can stimulate promoter activity and can be inherent elements of a promoter or heterologous elements inserted to improve the level or tissue specificity of a promoter.

[0105]

[0116] Promoters can be derived from different sources or generated from different elements, for example, a promoter can be derived entirely from a native gene, made up of different elements derived from different naturally occurring promoters, or can comprise a synthetic nucleotide sequence.

[0106]

[0117] Different promoters can be selected to direct the expression of nucleotide sequences in different tissues or cell types, at different stages of development, or in response to different environmental conditions or the presence or absence of drugs or transcriptional cofactors. Ubiquitous, cell-type-specific, tissue-specific, developmental stage-specific, and conditional promoters are well known in the art. Examples of promoters include the phosphoglycerate kinase (PKG) promoter, CAG (a composite of the CMV enhancer, chicken beta-actin promoter (CBA), and rabbit beta-globin intron), NSE (neuron-specific enolase), NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), SFFV promoter, Rous sarcoma virus (RSV) promoter, synthetic promoters, and hybrid promoters. Other promoters may be derived from humans or other species, including mice. Common promoters include the human cytomegalovirus (CMV) immediate-early gene promoter, the Rous sarcoma virus long terminal repeat, [beta]-actin, the rat insulin promoter, the human alpha-1 antitrypsin (hAAT) promoter, the transthyretin promoter, the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1-alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multiple tissue specificities, and neuron-specific promoters such as the synapsin and glyceraldehyde-3-phosphate dehydrogenase promoters. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, can also be used. Various promoter sequences are commercially available; see, for example, Stratagene (San Diego, CA).

[0107] III.B. Additional Features

[0118] Additional elements that may be present include introns, enhancers, polyadenylation signal sequences, Kozak sequences, post-translational regulatory elements, 5' and 3' inverted repeats (ITRs), regulatable elements, cell-specific regulators (e.g., microRNA (micoRNA) binding elements), internal ribosome entry sites, or other elements that affect the expression or stability or protein processing of the encoded sequence. Examples of different element arrangements, from 5' to 3', are promoters / enhancers, Kozak sequences, transgenes, post-transcriptional regulatory elements, and polyadenylation signal sequences. Polyadenylation signal sequences result in the formation of poly(A) tails, which promote nuclear export, translation, and / or mRNA stability, and may also be involved in transcription termination. Examples of polyadenylation signal sequences include the SV40 late polyadenylation signal, the bovine growth hormone polyA (bGHpA) signal sequence, synthetic polyA, mouse β-globin pA, rabbit β-globin pA, and H4-based pA (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197).

[0108]

[0119] The presence of an intron between the promoter and the transgene can improve gene expression and RNA processing (Powell et al., Discovery Medicine 2015, 19(102), 49). A variety of different introns can be used to improve gene expression. Examples of introns that can be used include the rabbit β-globin intron with splice donor / splice acceptor sites, the SV40 intron with splice donor / splice acceptor sites, the human β-globin intron, intron 2 of the human hemoglobin beta gene, hFIX int1 (intron 1 of the human clotting factor IX gene), CBA-rHHB (a synthetic intron derived from the fusion of intron 1 of the chicken beta actin gene and intron 2 of rabbit hemoglobin beta), CBA (intron 1 of the chicken beta actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (derived from different parts of the human clotting factor IX gene, and used in pLIVE vectors, Mirus synthetic introns present in Bio, Madison, WI); human hemoglobin subunit beta (HBB2) synthetic intron, and optimal HBB2; and chimeric introns, such as an intron consisting of a 5'-splice donor site and branch and 3'-acceptor site of the first human β-globin intron from the intron between the tip and body of the immunoglobulin gene heavy chain variable region (Buck et al., Int. J. Mol. Sci. 2020, 21, 4197; Ronzitti et al., Mol. Ther. Methods Clin Dev. 2016 Jul. 20; 3:16049; and the HBB-IGG intron provided in the pCMVNT™ vector).

[0109]

[0120] Optionally, the encoded polypeptide can be expressed with a secretory signal sequence that promotes extracellular secretion of the polypeptide. The term "secretory signal sequence" refers to an amino acid sequence that functions to enhance secretion of an operably linked polypeptide from a cell compared to the level of secretion observed for a polypeptide lacking a secretory signal sequence. Secretion of essentially the majority of the polypeptide is not necessary, as long as the level of secretion is enhanced compared to the native polypeptide. In different embodiments, at least 95%, 97%, 98%, or 99% of the polypeptide is secreted. Generally, the secretory signal sequence can be cleaved in the endoplasmic reticulum prior to secretion. The secretory signal sequence need not be cleaved, as long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional.

[0110]

[0121] The secretory signal sequence may be derived in whole or in part from the secretory signal (i.e., precursor) of the secreted polypeptide and / or may be wholly or partially synthetic. The length of the secretory signal sequence is not critical and may be, for example, from about 10-15 to 50-60 amino acids in length. The known secretory signal from the secreted polypeptide may be altered or modified (e.g., by amino acid substitution, deletion, truncation, or insertion) so long as the resulting secretory signal sequence functions to enhance secretion of the operably linked polypeptide. The secretory signal sequence may comprise, consist essentially of, or consist of a natural secretory signal sequence or a modification thereof. Examples of synthetic or artificial secretory signal peptides are provided in Barash et al., Biochem. Biophys. Res. Comm. 2002, 294, 835.

[0111]

[0122] The Kozak consensus sequence or a variant thereof is responsible for initiating translation. Kozak consensus sequences and variants are provided, for example, in McClements et al. (2021) Molecular Vision, 27, 233-242.

[0112]

[0123] Post-translational regulatory elements, such as the Woodchuck post-transcriptional regulatory element (WPRE) and the hepatitis B virus regulatory element, can increase gene expression (Buck et al., Int. J. Mol. Sci. 2020, 21, 4197; and Powell et al., Discovery Medicine 2015, 19(102), 49).

[0113]

[0124] Regulatable elements can be used to increase or decrease expression. Regulatable elements that increase expression of a transcribed nucleic acid in response to a signal or stimulus are also called "inducible elements" (i.e., induced by a signal). Regulatable elements include tissue-specific and drug-responsive transcription (promoter / enhancer) elements. Examples of regulatable elements include tetracycline-inducible elements, druggable ribozymes, druggable stepping stone switches, microRNA-responsive genes (e.g., mRNA stability or protein translation), morpholino-responsive mRNAs (e.g., splicing or mRNA stability), suppressor tRNA-regulated genes, alternative splicing-regulated genes, and druggable degrons.

[0114]

[0125] Typically, the increase or decrease imparted by the adjustable element is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionine (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 1998 / 10088); the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA 1882, 89:5547-5551); the tetracycline-inducible system (Gossen et al., Science 268:1995, 1766-1769); the RU486-inducible system (Harvey et al., Curr. Opin. Chem. Biol. 1998, 2:512-518; Wang et al., Nat. Biotech. 1997, 15:239-243; and Wang et al., Gene Ther. 1997, 4:432-441); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 1997, 100:2865-2872; and Rivera et al., Nat. Medicine 1996, 2:1028-1032). Other examples of regulatable control elements include those regulated by specific physiological conditions such as temperature, acute phase, or onset.

[0115] III.C. Therapeutic Proteins

[0126] DNA vectors can deliver a variety of different transgenes that can be expressed to result in proteins with desired activities. Examples of transgenes include those that provide healthy copies of genes in subjects where the original gene is defective, those that provide new or modified genes that can help treat a disease or disorder, or those that provide new genes that encode proteins that have a beneficial effect.

[0116]

[0127] In different embodiments, the transgene encodes GAA (acid alpha-glucosidase) for the treatment of Pompe disease; TPP1 (tripeptidyl peptidase-1) for the treatment of late infantile neuronal ceroid lipofuscinosis type 2 (CLN2); ATP7B (copper transporting ATPase 2) for the treatment of Wilson's disease; alpha galactosidase for the treatment of Fabry disease; ASS1 (arginosuccinate synthase) for the treatment of citrullinemia type 1; beta glucocerebrosidase for the treatment of Gaucher disease type 1; beta hexosaminidase A for the treatment of Tay-Sachs disease; SERPING1 (C1 protease inhibitor or C1 esterase inhibitor) for the treatment of hereditary angioedema (HAE), also known as C1 inhibitor deficiency types I and II; or glucose-6-phosphatase for the treatment of glycogen storage disease type I (GSDI).

[0117]

[0128] In different embodiments, the transgene is selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor (TF), and the like. The gene encoding the gene encoding transforming growth factor alpha (TGFα), platelet-derived growth factor (PDGF), insulin growth factor I or II (IGF-I or IGF-II), TGFβ, activin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 or NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 or netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, or tyrosine hydroxylase.

[0118]

[0129] In different embodiments, the transgene encodes thrombopoietin (TPO), interleukins (IL-1 through IL-36), monocyte chemotactic factors, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α or β, interferon α, β, or γ, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD, or IgE, chimeric immunoglobulins, antibodies, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, class I or class II MHC molecules. For example, antibodies and immunoglobulins can be provided that target cancer cells or cells causing other diseases or disorders.

[0119]

[0130] In different embodiments, the transgene is selected from the group consisting of CFTR (cystic fibrosis transmembrane conductance regulator), blood clotting (clotting) factors (Factor XIII, Factor IX (FIX), Factor VIII (FVIII), Factor X, Factor VII, Factor VIIa, or Protein C), gain-of-function blood clotting factors, erythropoietin, LDL receptor, lipoprotein lipase, ornithine carbamyl transferase, β-globin, α-globin, spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes involved in lysosomal storage diseases (ARSA), hypoxanthine guanine phosphoribosyltransferase, beta-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and 4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor α and β, cytokines, α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance protein, Tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (APC), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins, T-resitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), retinal pigment epithelium-specific 65 kDa protein (RPE65), Rab escort protein 1 (choroideremia), LCA5 (LCA-reversilin), ornithine ketoacid aminotransferase (gyrate atrophy), retinoschisin 1 (X-ray-associated retinoschisis), X-ray-associated retinitis pigmentosa GTPase (XLRP), MER proto-oncogene tyrosine kinase (MERTK) (autosomal recessive (AR) form of retinitis pigmentosa (RP)), ABCA4 (Stargardt disease), ACHM2, 3, and 4 (color blindness), anti-vascular endothelial growth factor (VEGF) agent polypeptides (e.g., bevacizumab, brolucizumab) , ranibizumab, aflibercept), DFNB1 (connexin 26 deafness), USH1C (Usher syndrome 1C), PKD-1 or PKD-2 (polycystic kidney disease), TPP1 (tripeptidyl peptidase-1), sulfatase, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator protein, or one or more donor sequences used as a repair template for genome editing.

[0120]

[0131] In different embodiments, the transgene is selected from the group consisting of erythropoietin (EPO) for the treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for the treatment of various immune disorders, viral infections, and cancer; interleukins (ILs) and corresponding receptors, including any one of IL-1 through IL-36, for the treatment of various inflammatory diseases or immune deficiencies; chemokines, including chemokine (C-X-C motif) ligand 5 (CXCL5), for the treatment of immune disorders; and granulocyte colony-stimulating factor (G-CSF) for the treatment of immune disorders such as Crohn's disease. granulocyte-macrophage colony-stimulating factor (GM-CSF) for the treatment of various human inflammatory diseases; macrophage colony-stimulating factor (M-CSF) for the treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for the treatment of epithelial tissue damage; chemokines such as monocyte chemoattractant protein-1 (MCP-1) for the treatment of recurrent miscarriage, HIV-related complications, and insulin resistance; tumor necrosis factor (TNF) and receptors for the treatment of various immune disorders; alpha 1-antitrypsin for the treatment of emphysema or chronic obstructive pulmonary disease (COPD); mucopolysaccharidosis I (MPS ornithine transcarbamoylase (OTC) for the treatment of OTC deficiency; phenylalanine hydroxylase (PAH) or phenylalanine ammonia-lyase (PAL) for the treatment of phenylketonuria (PKU); lipoprotein lipase for the treatment of lipoprotein lipase deficiency; apolipoprotein (Apo) AI for the treatment of apolipoprotein (Apo) AI deficiency; hypolipidemic lipase for the treatment of familial hypercholesterolemia (FH). Low-density lipoprotein receptor (LDL-R); albumin for the treatment of hypoalbuminemia; lecithin cholesterol acyltransferase (LCAT); carbamoyl synthetase I; argininosuccinate synthetase; argininosuccinate lyase; arginase; fumanylacetoacetate hydrolase; porphobilinogen deaminase; cystathionine beta-synthase for the treatment of homocystinuria; branched-chain ketoacid decarboxylase; isovaleryl-CoA dehydrogenase;Encoding propionyl-CoA carboxylase; methylmalonyl-CoA mutase; glutaryl-CoA dehydrogenase; insulin; pyruvate carboxylase; hepatic phosphorylase; phosphorylase kinase; glycine decarboxylase; H-protein; T-protein; cystic fibrosis transmembrane regulator (CFTR); ATP-binding cassette, subfamily A (ABC1), member 4 (ABCA4) for the treatment of Stargardt disease; or dystrophin;

[0121]

[0132] In further embodiments, the transgene encodes a protein for treating a disease or disorder selected from the group consisting of hereditary angioedema, Pompe disease, hemophilia A, hemophilia B, Fabry disease, wet macular degeneration, Leber's hereditary optic neuropathy, and Stargardt disease.

[0122] III.D. Inhibitory nucleic acids

[0133] DNA vectors include short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), and RNA iA variety of different transgenes may be provided that encode a variety of different inhibitory nucleic acids, such as ribozymes, and antisense RNA. In different embodiments, the inhibitory nucleic acid is a gene encoding the huntingtin (HTT) gene, a gene associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), the androgen receptor on the X chromosome in spinal and bulbar muscular atrophy, human ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA-binding protein, ataxin 8 opposite chain (ATXN8OS), serine / threonine-protein phosphatase 2A in spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, 17). 55 kDa regulatory subunit B beta isoform, FMR1 (Fragile X mental retardation 1) in fragile X syndrome, FMR1 (Fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (Fragile X mental retardation 2) in fragile XE mental retardation, or AF4 / FMR2 family member 2; myotonin protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; mutations in the superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, the transcriptional gene for human immunodeficiency virus transactivator, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene; CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute renal failure or delayed engraftment of a functional kidney transplant or kidney failure; protein kinase N3 (PKN3) in pre-recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB 8), metastatic melanoma;Proteasome subunit beta type 10 (PSMB 10), also known as MECL1 in metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitor B-cell lymphoma (BCL-2) in chronic myeloid leukemia (CLL) / lymphoma; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors; diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection; beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor in glaucoma; RTP801 / Redd1, also known as DNA damage-induced transcription 4 protein in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischemic optic neuropathy; and keratin 6A in pachyonychia congenita. N17K mutant protein; influenza A virus genome / gene sequence for influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequence for SARS infection; respiratory syncytial virus genome / gene sequence for respiratory syncytial virus infection; Ebola filovirus genome / gene sequence for Ebola infection; hepatitis B and C virus genome / gene sequence for hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequence for HSV infection; coxsackievirus B3 genome / gene sequence for coxsackievirus B3 infection; silencing of pathogenic alleles (allele-specific silencing) of genes such as torsin A (TOR1A) for primary dystonia, transplant-specific pan-class I and HLA-alleles; and mutant rhodopsin gene (RHO) for autosomal dominant retinitis pigmentosa (adRP);

[0123] III.E Gene Editing

[0134] The DNA vector can provide a variety of different transgenes encoding a variety of different gene-editing nucleic acids, such as ZFNs, TALENs, and CRISPR-Cas9. In different embodiments, the gene-editing nucleic acids edit the DNA of a subject to create a therapeutic protein, as described above in Section III.C., or to disrupt a gene, as described above in Section III.D.

[0124] IV. Cytosolic DNA Sensing Pathway Inhibitors

[0135] The cytosolic DNA sensing pathway detects foreign DNA and initiates an immune response that results in the production of proinflammatory cytokines, chemokines, and type I interferons (see, e.g., Hypertext Transfer Protocol: http: / / www.genome.jp / dbget-bin / www_bget?pathway+hsa04623, incorporated herein by reference in its entirety). Cytosolic DNA sensing pathway inhibitors can be provided to reduce the immune response triggered by the DNA. In different embodiments, cGAS-STING and / or inflammasome pathway inhibitors are utilized. Specific inhibitors that inhibit more than one target can be provided as inhibitors of each or any of the targets. Methods, compounds, and compositions for inhibiting the cGAS-STING pathway and / or the inflammasome pathway are described, for example, in International Publication No. WO2023004437, incorporated herein by reference in its entirety.

[0125] A variety of different types of compounds can inhibit the production or activity of proteins involved in the cytosolic DNA sensing pathway and can be used as inhibitors. In certain embodiments, the cytosolic DNA sensing pathway inhibitor is a small molecule, an antibody, a peptide, an inhibitory nucleic acid, or a target protein of a degrading agent (e.g., a protease inhibitor or degrader). The inhibitory nucleic acid can, for example, target a nucleic acid encoding a specific protein.

[0126] IV.A. cGAS-STING pathway inhibitors

[0136] cGAS-STING pathway inhibitors can directly affect cGAS-STING pathway proteins, such as cGAS, STING, or TBK1, or can affect agents that affect the cGAS-STING pathway. References describing the design of cGAS-STING pathway inhibitors and examples of inhibitors include Ding et al., Acta Pharmaceutica Sinica B 2020, 10(12), 2272; Fu et al., iScience 2020, 23, 101026; Konno et al., Cell Rep. 2018, 23(24), 1112; U.S. Patent Application Publication No. 2020 / 0291001; and Haag et al., Nature 2018, 559, 269-273, each of which is incorporated herein by reference.

[0127]

[0137] References providing STING inhibitors, STING inhibitor scaffolds and motifs, and design considerations include: Ding et al., Acta Pharmaceutica Sinica B 2020;10(12):2272; Decout et al., Nat. Rev. Immunol., September 2021;21(9):548-569; Dubensky et al., U.S. Pat. No. 10,189,873; Katibah et al., U.S. Pat. App. Pub. No. 2018 / 0369268; Seidel et al., WO 2020 / 150439; Roush et al., U.S. Pat. App. Pub. No. 2020 / 0172534; Roush et al., U.S. Pat. App. Pub. No. 2021 / 236466; Glick et al., WO 2022 / 140410; Hong et al., Proc. Natl. Acad. Sci. USA., June 15, 2021; 118(24); and Hong et al., Journal of Molecular Cell Biology 2022, 14(2), njac005, each of which is incorporated by reference herein in its entirety.

[0128]

[0138] In a different embodiment, the STING inhibitor is as provided in Table 3, or a pharmaceutically acceptable salt thereof.

[0129]

[0139] [Table 3] JPEG2025531748000012.jpg215149 JPEG2025531748000013.jpg215149

[0130]

[0140] GSK690693 is an AMP-activated protein kinase (AMPK) / AKT inhibitor and provides an example of an inhibitor that affects the cGAS-STING pathway. AMPK / AKT activity affects the cyclic cGAS-STING pathway, causing loss of ULK1 phosphorylation, releasing ULK1 to phosphorylate STING, thereby inhibiting STING activity. Examples of AMPK inhibitors, including GSK690693, are provided in Konno et al., Cell Rep. 2018, 23(4), 1112.

[0131]

[0141] References providing cGAS inhibitors, cGAS inhibitor scaffolds and motifs, and design considerations include: Ding et al., Acta Pharmaceutica Sinica B 2020;10(12):2272; Decout et al., Nat. Rev. Immunol., 2021 Sep;21(9):548-569; Vincent et al., Nat. Commun., 2017;8:750; Lama et al., Nat. Commun., 2019 May 21;10(1):2261; Obioma et al., U.S. Patent No. 10,738,056; Hong et al., Journal of Molecular Cell Biology, 2022, 14(2), njac005; Zhao et al., J. Chem. Inf. Model, 2020, 60, 3265-3276; and Padilla-Salinas et al., J. Org. Chem., 2020, 85, 1579-1600, each of which publications is incorporated by reference herein in its entirety.

[0132]

[0142] In a different embodiment, the cGAS inhibitor is as provided in Table 4, or a pharmaceutical salt thereof.

[0133]

[0143] [Table 4] JPEG2025531748000015.jpg215149 JPEG2025531748000016.jpg195149

[0134]

[0144] References providing TBK1 inhibitors, TBK1 inhibitor scaffolds and motifs, and design considerations include: Thomson et al., Expert Opinion on Therapeutic Patents, 2021, 31:9, pp. 785-794; Chekler et al., U.S. Patent Application Publication No. 2021 / 0214339; Newton and Stewart, U.S. Patent No. 11,058,686; Karra et al., International Patent Publication No. 2019 / 079373A1; Bigi et al., U.S. Patent No. 9,994,547; Schulze et al., U.S. Patent No. 10,894,784; Hassan and Yan, Parmacol. Res., 2016, 111:336-342; Li et al., Int. J. Cancer 2014, 134:1972-1980; Alam et al., International Journal of Biological Macromolecules, 2022, 2027:1022-1037; Perrior et al., U.S. Pat. No. 8,962,609; and Du et al., U.S. Pat. No. 10,316,049, each of which is incorporated by reference herein in its entirety.

[0135]

[0145] In certain embodiments, the TBK1 inhibitor is as provided in Table 5, or a pharmaceutical salt thereof.

[0136]

[0146] [Table 5] JPEG2025531748000018.jpg177149 JPEG2025531748000019.jpg199149 JPEG2025531748000020.jpg123149

[0137] IV.B. Inflammasome Pathway Inhibitors

[0147] Inflammasome pathway inhibitors can directly affect inflammasome pathway proteins, such as the absent in melanoma 2 (AIM2) protein, or can affect drugs that affect the inflammasome pathway. Certain DNA sequences, such as TTAGGG repeats commonly found in mammalian telomeric DNA, can bind to AIM2 and suppress innate immune activation (Kaminski et al., The Journal of Immunology, 2013, pp. 191-3876). Such sequences can also inhibit other innate responses, such as cGAS and STING. Examples of AIM2 inhibitors include A151, a synthetic oligonucleotide containing four repeats of the TTAGGG motif and having the following nucleotide sequence, where the bases are joined by phosphorothioate bonds: 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 1); and 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 2), which contains a phosphodiester bond. Additional oligonucleotide sequences include other types of modified SEQ ID NO: 2, such as those with the same nucleotide sequence but a different modified backbone. Such nucleotide sequences can also be used as cGAS and STING inhibitors and can be incorporated into DNA or DNA vectors.

[0138] V. Immune Cell Modulators

[0148] In some cases, administration of a DNA vector may result in an undesirable immune response, for example, due to DNA vector components, transgene products recognized as foreign, or edited genes that produce proteins considered foreign. In such cases, if desired, the host immune response can be reduced using, for example, immune cell modulators or immunosuppressants. In some cases, such as cancer treatment, viral treatment, and bacterial treatment, certain responses may be advantageous.

[0139] VA phagocyte depletor

[0149] In certain embodiments, phagocyte depletion agents are used. Methods, compounds, and compositions for the depletion of phagocyte immune cells are described, for example, in WO2022140788A1, which is incorporated herein by reference in its entirety.

[0140]

[0150] "Phagocyte-depleting agent" refers to an agent that depletes or destroys phagocytes in a subject and / or interferes with the function of one or more phagocytes. Phagocytes, also called phagocytic cells, phagocytic immune cells, phagocytes, or phagocytic immune cells, include macrophages, monocytes, neutrophils, and dendritic cells. Langerhans cells are dendritic cells found in the skin. Mast cells are found in many tissues, including the lungs or skin, and can act as phagocytes.

[0141]

[0151] "Monocyte and / or macrophage depleting agent" refers to an agent that depletes or destroys monocytes and / or macrophages in a subject and / or interferes with the function of one or more monocytes and / or macrophages. A monocyte and / or macrophage depleting agent can target monocytes and / or macrophages. Macrophages are mononuclear phagocytes that are differentiated monocytes. In different tissues, macrophages are called by different names. Examples of tissue-specific or resident macrophages include Kupffer cells in the liver, intestinal macrophages in the digestive tract, microglial cells in the brain, alveolar macrophages in the lungs, resident kidney macrophages, skin macrophages, red pulp macrophages in the spleen, and osteoclasts in bone. Examples of monocyte and / or macrophage depleting agents include agents targeting phagocytic immune cell markers, such as CD115 inhibitors, e.g., anti-CD115 antibodies or CD115 small molecule inhibitors; F4 / 80 inhibitors, e.g., anti-F4 / 80 antibodies or F4 / 80 small molecule inhibitors; CD68 inhibitors, e.g., anti-CD68 antibodies or CD68 small molecule inhibitors; CD11b inhibitors, e.g., anti-CD11b antibodies or CD11b small molecule inhibitors; the chemotherapeutic agent trabectedin; intralipid; empty liposomes; and bisphosphonates, including clodronate. In certain embodiments, the monocyte and / or macrophage depleting agent is not clodronate. In certain embodiments, clodronate and at least one additional monocyte and / or macrophage depleting agent are used together.

[0142]

[0152] "Neutrophil-depleting agent" refers to an agent that depletes or destroys neutrophils in a subject and / or interferes with one or more neutrophil functions. Neutrophil-depleting agents target neutrophils. Examples of neutrophil-depleting agents include agents that target phagocytic immune cell markers, such as Ly6G inhibitors, including anti-Ly6G antibodies or Ly6G small molecule inhibitors; CD177 inhibitors, including anti-CD177 antibodies or CD177 small molecule inhibitors; CD14 inhibitors, including anti-CD14 antibodies or CD14 small molecule inhibitors; CD15 inhibitors, including anti-CD15 antibodies or CD15 small molecule inhibitors; CD11b inhibitors, including anti-CD11b antibodies or CD11b small molecule inhibitors; CD16 inhibitors, including anti-CD16 antibodies or CD16 small molecule inhibitors; and anti-CD32 antibodies or CD32 small molecule inhibitors. CD32 inhibitors including anti-CD33 antibodies or CD33 small molecule inhibitors; CD44 inhibitors including anti-CD44 antibodies or CD44 small molecule inhibitors; CD45 inhibitors including anti-CD45 antibodies or CD45 small molecule inhibitors; CD66b inhibitors including anti-CD66b antibodies or CD66b small molecule inhibitors; CD18 or inhibitors including anti-CD18 antibodies or CD18 small molecule inhibitors; CD62L inhibitors including anti-CD62L antibodies or CD62L small molecule inhibitors; and Gr-1 inhibitors including anti-Gr-1 antibodies or Gr-1 small molecule inhibitors.

[0143]

[0153] A "dendritic cell depleting agent" refers to an agent that depletes or destroys dendritic cells in a subject and / or interferes with the function of one or more dendritic cells. Dendritic cell depleting agents can target any dendritic cell. Examples of dendritic cell depleting agents include agents that target phagocytic immune cell markers, such as PDCA1 inhibitors, including anti-PDCA1 antibodies or PDCA1 small molecule inhibitors; and CD11c inhibitors, including anti-CD11c antibodies or CD11c small molecule inhibitors.

[0144]

[0154] "Inhibitor" refers to any compound that can downregulate, reduce, decrease, suppress, or inactivate the amount and / or activity of a target protein. Inhibitors can be proteins, oligo- and polypeptides, nucleic acids, genes, or chemical molecules. Suitable protein inhibitors can be, for example, monoclonal or polyclonal antibodies that bind to the target protein; and small molecules.

[0145]

[0155] Examples of CD115 inhibitors include the CD115 small molecule inhibitors pexidartinib (PLX-3397), BLZ-945, linifanib (ABT-869), JNJ-28312141 (Johnson & Johnson), JNJ-40346527 (Johnson & Johnson), PLX7486 (Plexxikon), ARRY-382 (Array BioPharma), anti-CD115 antibodies such as AFS98 (Invitrogen or BioCell), 12-3A3-1B10 (Invitrogen), 6C7 (Bioss), cabilalizumab (FPA008), 25949-1-AP (Proteintech), 1G4 (Abnova), 3G12 (Abnova), 604B5 2E11 (Invitrogen), emactuzumab (RG-7155; Roche), AMG 820 (Amgen), IMC-CS4, and ROS8G11 (Invitrogen). In certain embodiments, the antibody or antigen-binding fragment thereof is AFS98 (e.g., BioCell BE0213 and Oncogene 1995;11(12):2469-2476).

[0146]

[0156] Any suitable Ly6G inhibitor, including those known to those of skill in the art, can be used in light of the present disclosure. Examples of anti-Ly6G antibodies include A8 (BioCell BP0075-1) and RB6-8C5 (ab25377).

[0147]

[0157] Intralipid and empty liposomes have been shown to interfere with one or more functions of monocytes and / or macrophages. See, e.g., Liu et al., Biochim Biophys Acta. 2013 Jun;1830(6):3447-53 and Saunders et al., Nano Lett. 2020 Jun;20(6):4264-4269. Pretreatment with intralipid or empty liposomes can effectively saturate monocyte / macrophage cells and prevent phagocytosis of non-viral therapeutic agents. Examples of intralipid and empty liposomes include I141-100ML (Sigma Aldrich), 2B6063 (Baxter), and those described in Liu et al., Biochim Biophys Acta. 2013 Jun;1830(6):3447-53 and Saunders et al., Nano Lett. 2020 Jun 10;20(6):4264-4269.

[0148]

[0158] Examples of bisphosphonates include clodronate, pamidronate, ibandronate, alendronate, and zoledronate.

[0149]

[0159] Other examples of "phagocyte-depleting agents" include palbociclib (Ibrance®; Pfizer), and cromolyn sodium (Nasalcrom®; Bausch & Lomb).

[0150] VB immunosuppressant

[0160] Immunosuppressants are compounds capable of slowing or halting the activity of the immune system in a subject. A variety of different immune responses can be produced, including various innate and humoral immune responses. For example, immune responses include detectable alterations in Toll receptor activation, lymphokine (e.g., cytokine or chemokine) expression and / or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and / or B cell activation (e.g., antibody production and / or secretion). Additional examples of immune responses include binding of an immunogen (e.g., an antigen) to an MHC molecule and induction of a cytotoxic T lymphocyte ("CTL") response, a B cell response (e.g., antibody production) and / or a T helper lymphocyte response, and / or a delayed-type hypersensitivity (DTH) response to the antigen from which the immunogenic polypeptide is derived, proliferation of cells of the immune system, and increased antigen processing and presentation by antigen-presenting cells.

[0151]

[0161] Examples of immunosuppressants include calcineurin inhibitors, such as cyclosporine, ISA(TX)247, tacrolimus or calcineurin; target of rapamycin, such as sirolimus, everolimus, FK778 or TAFA-93; interleukin-2 α-chain blockers, such as basiliximab and daclizumab; inosine monophosphate dehydrogenase inhibitors, such as mycophenolate mofetil; dihydrofolate reductase inhibitors, such as methotrexate; immunosuppressive antimetabolites, such as azathioprine; cytokine inhibitors, such as anti-cytokine antibodies, for example, siltuximab; or steroids.

[0152]

[0162] In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid, such as a corticosteroid, prednisone, prednisolone, cyclosporine (e.g., cyclosporine A), mycophenolate; a B cell-targeting antibody, such as rituximab; a proteasome inhibitor, such as bortezomib; a mammalian target of rapamycin (mTOR) inhibitor, such as rapamycin; a tyrosine kinase inhibitor, such as ibrutinib; an inhibitor of B cell-activating factor (BAFF); or an inhibitor of proliferation-inducing ligand (APRIL) or its derivative. In certain embodiments, the immunosuppressant is an anti-IL-1β agent (e.g., the anti-IL-1β monoclonal antibody canakinumab (Ilaris®)) or an anti-IL-6 agent (e.g., the anti-IL-6 antibody sirukumab or the anti-IL-6 receptor antibody tocilizumab (Actemra®)), or a combination thereof.

[0153]

[0163] The term "steroid" refers to a chemical compound containing three cyclohexane and cyclopentane rings. The rings are arranged to form a tetracyclic cyclopentaphenanthrene, or gonane. There are different types of steroids, such as corticosteroids and glucocorticosteroids.

[0154]

[0164] The term "corticosteroid" refers to a class of steroid hormones produced in the adrenal cortex or synthetically. In certain embodiments, the steroid can be a corticosteroid. Corticosteroids are involved in a wide range of physiological systems, including stress response, immune response and inflammation regulation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids are generally grouped into four classes based on their chemical structure. Group A corticosteroids (short- to medium-acting glucocorticoids) include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, and prednisone. Group B corticosteroids include triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, and halcinonide. Group C corticosteroids include betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, and fluocortolone. Group D corticosteroids include hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate.Non-limiting examples of corticosteroids include aldosterone, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone-21-phosphate disodium, betamethasone valerate, budesonide, clobetasol, clobetasol propionate, clobetasone butyrate, clocortolone pivalate, cortisol, cortisteron, cortisone, deflazacort, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, dihydroxycortisone, fluocinonide, fludrocortisone acetate, fluoxetine ... Includes methasone, flunisolide, fluocinolone acetonide, fluticasone furoate, fluticasone propionate, halcinonide, halpmetasone, hydrocortisone, hydrocortisone acetate, hydrocortisone succinate, 16α-hydroxyprednisolone, isoflupredone acetate, medrysone, methylprednisolone, prednacinolone, prednicarbate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisone, triamcinolone, triamcinolone, and triamcinolone diacetate.

[0155]

[0165] Cortisone (CORTONE™, ACETATE™, ADRESON™, ALTESONA™, CORTELANT™, CORTISTAB™, CORTISYL™, CORTOGEN™, CORTONE™, SCHEROSON™); dexamethasone oral (DECADRON™, DEXAMETH™, DEXONE™, HEXADROL™, DEXAMETHASONE™, INTENSOL™, DEXADROL 0.5™, DEXAMETHASONE 0.75™) ), Dexon 1.5™, Dexon 4™; hydrocortisone oral (CORTEF™, HYDROCORTONE™); hydrocortisone cypionate (CORTEF Oral Suspension™); methylprednisolone oral (MEDROL Oral™); prednisolone oral (PRELONE™, DELTA-CORTEF™, PEDIAPRED™, ADNISOLONE™, CORTALONE™, DELTACORTRIL™, DELTASOLONE™, DELTASTAB™, DI-ADRESON F F)(TM), ENCORTOLONE(TM), HYDROCORTANCYL(TM), MEDISOLONE(TM), METICORTELONE(TM), OPREDSONE(TM), PANAFCORTELONE(TM), PRECORTISYL(TM), PRENISOLONA(TM), SCHERISOLONA(TM), SCHERISOLONE(TM);Prednisone (DELTASONE™, LIQUID PRED™, METICORTENT™, ORASONE 1™, ORASONE 5™, ORASONE 10™, ORASONE 20™, ORASONE 50™, PREDNICEN-M™, PREDNISONE INTENSOL (PREDNISONE) INTENSOL™, STERAPRED™, STERAPRED DS™, ADASONE™, CORTANCYL (CARTANCYL)™, COLISONE™, CORDROL™, CORTAN™, DACORTIN™, DECORTIN™, DECORTISYL™, DELCORTIN™, DELLACORT™, DELTADOME™, DELTACORTENE™, DELTISONA™, DIADRESON™, ECONOSONE™, ENCORTON™, FERNISONE™, NISONA™, NOVOPREDNISONE™, PANAFCORT™, PANASOL™, PARACORT™, PARMENISON™, PEHACORT™, PREDELTIN™, PREDNICORT™, PREDNICOT™, PREDNIDIB™, PREDNIMENT™, RECTODELT™, ULTRACORTEN™, WINPRED™;Corticosteroids are available under a variety of generic brand names, including oral triamcinolone (KENACORT™, ARISTOCORT™, ATOLONE™, SHOLOG A™, TRAMACORT-D™, TRI-MED™, TRIAMCOT™, TRISTOPLEX™, TRYLONE D™, and U-TRI-LONE™). In certain embodiments, the corticosteroid can be dexamethasone, prednisone, prednisolone, triamcinolone, clobetasol propionate, betamethasone valerate, betamethasone dipropionate, or mometasone furoate. Methods for synthesizing steroids and corticosteroids are well known in the art, and many are commercially available.

[0156]

[0166] Corticosteroids such as dexamethasone, for example, can be delivered as free dexamethasone, as a separate LNP composition, or as part of the same LNP composition as DNA. Chen et al., Journal of Controlled Release 2018, 286, 46-54, includes a description of LNPs that deliver nucleic acids and dexamethasone that bind to fatty acids.

[0157] VI. Pharmaceutical Compositions

[0167] A pharmaceutical composition contains one or more active ingredients together with a pharmaceutically acceptable carrier. Reference to "pharmaceutical" or "pharmaceutically acceptable" refers to a non-toxic molecular entity that is suitable for administration and / or storage. A pharmaceutical composition may contain more than the therapeutically active agent. Examples of pharmaceutically acceptable carriers include non-toxic (in the amounts used) solid, semi-solid, or liquid fillers, excipients, encapsulating materials, or formulations.

[0158]

[0168] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen depend on the condition being treated, such as the severity of the disease, the age, weight and sex of the patient. Pharmaceutical compositions for the agents described herein can be formulated for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration.

[0159]

[0169] In one embodiment, the pharmaceutical composition contains a formulation that allows for injection into a subject. Examples of injectable formulation components include isotonic sterile saline (e.g., monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and mixtures of such salts), buffered saline, sugars (e.g., dextrose), and water for injection. The pharmaceutical composition optionally includes a dried, e.g., lyophilized, composition that allows for the constitution of an injectable solution upon addition of sterile water or physiological saline. The dosage used for administration can be adapted as a function of various parameters, such as the mode of administration, the associated pathology, and the duration of treatment.

[0160]

[0170] Other pharmaceutically acceptable forms include tablets or other solid forms for oral administration, including sustained release capsules.

[0161]

[0171] Pharmaceutical compositions containing DNA vectors carrying therapeutic transgenes can be administered to subjects at a dosage suitable for treating a particular disease or disorder. In different embodiments, suitable dosages may be about 0.01 mg / kg to about 10 mg / kg of vector per kg of subject body weight, about 0.01 mg / kg to about 0.1 mg / kg of vector per kg of subject body weight, about 0.1 mg / kg to about 1.0 mg / kg of vector per kg of subject body weight, and about 1.0 mg / kg to about 10 mg / kg of vector per kg of subject body weight.

[0162]

[0172] The small molecule cytosolic DNA sensing inhibitor, type 1 interferon receptor pathway inhibitor, or immune cell modulator inhibitor can be administered to a subject at a suitable dose taking into consideration the DNA vector and the disease or disorder being treated. In different embodiments, suitable dosages can be about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, about 0.1 mg / kg to about 1 mg / kg, about 1.0 mg / kg to about 10 mg / kg, and about 10.0 mg / kg to about 100 mg / kg.

[0163]

[0173] The antibody targeting the type 1 interferon receptor can be administered to a subject at a suitable dose taking into consideration the DNA vector and the disease or disorder to be treated. For example, a suitable dose can be about 0.01 mg / kg to about 5 mg / kg of the subject's body weight, administered in a total of 1 to 10 injections.

[0164]

[0174] Antibodies targeting phagocytic immune cell markers can be administered to a subject at a suitable dose, for example, from about 0.01 mg / kg to about 5 mg / kg of the subject's body weight, administered in a total of 1 to 10 injections.

[0165]

[0175] A CD115 inhibitor such as pexidartinib can be administered to a subject at a suitable dose. For example, suitable doses can be about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, about 0.1 mg / kg to about 1 mg / kg, about 1.0 mg / kg to about 10 mg / kg, and about 10.0 mg / kg to about 100 mg / kg.

[0166]

[0176] The bisphosphonate, such as clodronate, can be administered to a subject at a suitable dose. For example, suitable doses can be about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, about 0.1 mg / kg to about 1 mg / kg, about 1.0 mg / kg to about 10 mg / kg, and about 10.0 mg / kg to about 100 mg / kg.

[0167]

[0177] Corticosteroids, such as dexamethasone, can be administered to a subject at a suitable dose, for example, from about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, from about 0.1 mg / kg to about 1 mg / kg, from about 1.0 mg / kg to about 10 mg / kg, and from about 10.0 mg / kg to about 100 mg / kg.

[0168]

[0178] Various compounds described herein can be provided as pharmaceutically acceptable salts. Reference to a "pharmaceutically acceptable salt" refers to a salt that is suitable for administration to a subject. Depending on the compound, pharmaceutically acceptable salts include acid addition salts and base salts. Pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogensulfate, phosphate, acid phosphate, isonicotinate, carbonate, bicarbonate, acetate, lactate, salicylate, citrate, tartrate, propionate, butyrate, pyruvate, oxalate, malonate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Suitable base salts include ammonium, calcium, lithium, magnesium, potassium, sodium, zinc, bismuth, and diethanolamine salts. Additionally, various amino acids are available as pharmaceutically acceptable salts.

[0169] VII. Administration and Treatment

[0179] The different compounds and compositions described herein can be administered to a subject for different purposes, including research purposes and for treating a disease or disorder in a mammal. A preferred use is to treat a disease or disorder in a human.

[0170]

[0180] References to "treatment" or "treating" refer to both prophylactic and therapeutic treatment of patients with a disease or disorder. References to "prophylactic" treatment refer to a reduction in the likelihood of shortening the disease or disorder or reducing the potential severity of the disease or disorder. References to "treatment" refer to a clinically meaningful amelioration of at least one symptom or cause of the disease or disorder. Thus, treatment includes administration to subjects at risk of shortening the disease or disorder, subjects suspected of shortening the disease or disorder, as well as subjects who are ill or diagnosed with a disease or disorder, including the suppression of clinical recurrence.

[0171]

[0181] The terms "ameliorate" and "amelioration" refer to a detectable or measurable improvement in the symptoms or underlying cellular responses of a disease or disorder. Detectable or measurable improvement includes subjective or objective reduction, lowering, inhibition, suppression, limitation, or control of the onset, frequency, severity, progression, or duration of a disease or disorder, or a complication caused by or associated with a disease or disorder, or amelioration of a symptom or underlying cause or consequence of a disease or disorder, or reversal of a disease or disorder. For Pompe disease, an effective amount includes an amount that inhibits or reduces glycogen production or accumulation, improves or increases glycogen breakdown or removal, and improves muscle tone and / or strength and / or respiratory function. For HemA or HemB, an effective amount includes an amount that reduces the frequency or severity of acute bleeding episodes and shortens clotting time as measured in a clotting assay in a subject.

[0172]

[0182] The terms "effective amount" and "sufficient amount" refer to the amount required to achieve the desired effect. Treatment can be carried out by administering a therapeutically effective amount of a DNA vector to a subject. A therapeutically effective amount can be provided in a single or multiple doses to achieve a therapeutic or prophylactic effect.

[0173]

[0183] Different agents can be administered in an "effective amount" or a "sufficient amount" to achieve a desired effect. For example, an effective amount of a type 1 interferon receptor pathway inhibitor is an amount provided in a single or multiple doses that inhibits activity of the type 1 interferon receptor pathway, which results in a decrease in one or more activities of the innate immune response in response to the DNA, inhibits transcriptional activation of one or more interferon-stimulated genes, and / or increases the tolerability of DNA vector administration. In different embodiments, the amount is effective to reduce DNA-stimulated cytokine production or to reduce the expression of IL-6, IFN-alpha, and / or IFN-gamma induced by DNA administration.

[0174]

[0184] In different embodiments, an effective amount of a GAS-STING pathway inhibitor or an inflammasome pathway inhibitor is an amount provided in single or multiple doses that inhibits activity of the cGAS-STING pathway or the inflammasome pathway and results in a decrease in one or more activities of the innate immune response; an effective amount of an immune cell modulator is an amount provided in single or multiple doses that results in a detectable decrease in phagocytes and / or phagocyte function; and an effective amount of an immunosuppressant is an amount provided in single or multiple doses that inhibits immune system activity.

[0175]

[0185] An effective amount can be administered alone or in combination with another composition, treatment, protocol, or therapeutic regimen. The amount can be increased appropriately based on, for example, the need for treatment, the subject, type, condition, and severity of the disease or disorder being treated, or side effects.

[0176]

[0186] An effective or sufficient amount need not be effective in each and every subject treated, nor in the majority of treated subjects in a given group or population. An effective or sufficient amount refers to effectiveness or sufficiency in a particular subject, rather than an entire group or population. As is typical of such methods, some subjects will exhibit a greater, lesser, or no response to a given treatment or use.

[0177]

[0187] When administered separately, the DNA vector and the type 1 interferon receptor pathway inhibitor can be given in any order or at about the same time, hi certain embodiments, the inhibitor is administered at least 60 minutes, at least 90 minutes, or at least 120 minutes before the DNA vector.

[0178]

[0188] In certain embodiments, the type 1 interferon receptor pathway inhibitor is administered about simultaneously with, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2:00, up to about 3:00, up to about 4:00, up to about 5:00, up to about 6:00, up to about 7:00, up to about 8:00, up to about 9:00, up to about 10:00, up to about 12:00, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, or up to about 1 week prior to administration of the DNA vector. In certain embodiments, the type 1 interferon receptor pathway inhibitor is administered at about the same time as administration of the DNA vector, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2 o'clock, up to about 3 o'clock, or up to about 4 o'clock. In certain embodiments, the type 1 interferon receptor pathway inhibitor is administered about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 1 day after administration of the DNA vector.

[0179]

[0189] In certain embodiments, the type 1 interferon receptor pathway inhibitor is administered approximately simultaneously with administration of the DNA vector, such as about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2:00, about 2.5:00, about 3:00, about 3.5:00, or about 4:00.

[0180]

[0190] In certain embodiments, the DNA vector and the type 1 interferon receptor pathway inhibitor can be provided in the same nanoparticle for near simultaneous release, or the nanoparticle can be designed for delayed release of one of the components, hi one embodiment, the nanoparticle is designed to release the inhibitor prior to release of the DNA vector.

[0181]

[0191] In certain embodiments involving the use of a cytosolic DNA sensing inhibitor, the cytosolic DNA sensing inhibitor, when administered separately, can be given in any order or at about the same time as the type 1 interferon receptor pathway inhibitor and the DNA vector, hi certain embodiments, the cytosolic DNA sensing inhibitor is administered independently at least 60 minutes, at least 90 minutes, or at least 120 minutes (for the type 1 interferon receptor pathway inhibitor) before the DNA vector.

[0182]

[0192] In certain embodiments, the cytosolic DNA sensing inhibitor is independently administered approximately simultaneously with, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2:00, up to about 3:00, up to about 4:00, up to about 5:00, up to about 6:00, up to about 7:00, up to about 8:00, up to about 9:00, up to about 10:00, up to about 12:00, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, or up to about 1 week (for a type 1 interferon receptor pathway inhibitor) before administration of the DNA vector. In certain embodiments, the cytosolic DNA sensing inhibitor is administered independently at approximately the same time as, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2:00, up to about 3:00, or up to about 4:00 (for type 1 interferon receptor pathway inhibitors) before administration of the DNA vector.

[0183]

[0193] In certain embodiments, the cytosolic DNA sensing inhibitor is administered independently about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 1 day after the DNA, about simultaneously with, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, or about 4 hours (for type 1 interferon receptor pathway inhibitors) prior to DNA vector administration.

[0184]

[0194] In certain embodiments, the DNA vector, type 1 interferon receptor pathway inhibitor, and cytosolic DNA sensing inhibitor can be provided in the same nanoparticle for near simultaneous release, or the nanoparticle can be designed for delayed release of one of the components. In one embodiment, the nanoparticle is designed to release the type 1 interferon receptor pathway inhibitor and cytosolic DNA sensing inhibitor before release of the DNA vector.

[0185]

[0195] In certain embodiments, the additional immunosuppressant is not administered within two months prior to or within two months after the administration of the type 1 interferon receptor inhibitor. Reference to an "additional immunosuppressant" refers to one or more (including any combination or all) inhibitors selected from a JAK inhibitor, a JAK1 or JAK2 inhibitor, a STAT inhibitor, a cGAS inhibitor, a STING inhibitor, and / or an inflammasome pathway inhibitor. In further embodiments, the additional immunosuppressant is not administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months prior to the administration of the type 1 interferon receptor inhibitor; independently, the additional immunosuppressant is not administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of the type 1 interferon receptor inhibitor.

[0186]

[0196] Reference to "independently" with respect to two different lists, e.g., a time before and a time after, indicates that each member of one list can be combined with each member of the other list. For example, an additional immunosuppressant administered within 1 day before administration of a type 1 interferon receptor inhibitor cannot be combined without administering the additional immunosuppressant within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after administration of the type 1 interferon receptor inhibitor; an additional immunosuppressant administered within 2 days before administration of a type 1 interferon receptor inhibitor cannot be combined with the additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, 68 days, 69 An additional immunosuppressant cannot be administered within 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 3 days before the administration of a type 1 interferon receptor inhibitor cannot be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 4 days before the administration of a type 1 interferon receptor inhibitor cannot be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor. An additional immunosuppressant administered within 5 days prior to a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 5 days prior to a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor. An additional immunosuppressant administered within 6 days prior to the administration of a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 7 days prior to the administration of a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor.or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 2 weeks before the administration of a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 1 month before the administration of a type 1 interferon receptor inhibitor cannot be combined with an additional immunosuppressant administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after the administration of a type 1 interferon receptor inhibitor An additional immunosuppressant cannot be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after administration of a type 1 interferon receptor inhibitor without administering the additional immunosuppressant within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after administration of a type 1 interferon receptor inhibitor, and an additional immunosuppressant administered within 2 months or 2 days before administration of a type 1 interferon receptor inhibitor cannot be administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after administration of a type 1 interferon receptor inhibitor.

[0187]

[0197] The treatment dosage of the DNA vector will vary and may depend on the type, onset, progression, severity, frequency, duration, or probability of the disease or disorder being treated, the desired clinical goal, previous or concurrent treatments, the subject's general health, age, sex, race, or immunological competence, and other factors understood by those skilled in the art. The dosage, number, frequency, or duration can be increased or decreased as appropriate, as indicated by any adverse side effects, complications, or other risk factors of the treatment or therapy, and the condition of the subject.

[0188]

[0198] The dose that achieves a therapeutic effect, e.g., a dose of vector DNA in mg per kilogram of body weight (mg / kg), will also vary based on several factors, including the route of administration, the level of transgene expression necessary to achieve a therapeutic effect, the particular disease or disorder being treated, the host immune response to the DNA, the host immune response to the transgene expression product, and the stability of the expressed protein, peptide, or nucleic acid. Based on the guidance provided herein, one of skill in the art will be able to determine a suitable dose range of vector DNA for treating a patient with a particular disease or disorder.

[0189]

[0199] The overall level of transgene expression can vary depending on the use of the DNA vector and the target disease or disorder. In different embodiments of gene therapy providing a therapeutic protein, the expression or activity provided is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the normal expression of the corresponding protein of interest.

[0190]

[0200] In certain embodiments, methods according to the invention can result in a reduction in the expression or activity of a protein targeted by a therapeutic nucleic acid. In different embodiments, the reduction in expression or activity of a protein targeted by a therapeutic nucleic acid is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the normal expression of the target protein.

[0191]

[0201] The methods and uses of the present invention include delivery and administration systemically, locally, or locally, for example, by injection or infusion. Delivery of the composition in vivo can generally be achieved, for example, by injection using a conventional syringe, although other delivery methods, such as convection-enhanced drug delivery, are contemplated (see, for example, U.S. Pat. No. 5,720,720). For example, the composition can be delivered subcutaneously, transepidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally (IP), intravenously (IV), intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application.

[0192] VII.A. Exemplary Diseases and Disorders

[0202] Diseases and disorders that can be treated with vector DNA include pulmonary diseases (e.g., cystic fibrosis), blood disorders (e.g., anemia), CNS diseases and disorders, epilepsy, lysosomal storage diseases (e.g., aspartylglycosaminuria), Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2 gangliosidosis type I (Tay-Sachs disease), GM2 gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), type II (I-cell disease), type III (pseudo-infectious disease), and leukemia. and IV, mucopolysaccharidosis (Hurler disease and variants, Hunter disease, Sanfilippo disease types A, B, C, D, Morquio disease types A and B, Maroteaux-Lamy disease, and Sly disease), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), and diseases of solid organs (e.g., brain, liver, kidney, heart).

[0193]

[0203] Glycogen storage disease type II, also known as Pompe disease, is an autosomal recessive disorder caused by mutations in the gene encoding the lysosomal enzyme acid alpha-glucosidase (GAA), which catalyzes the breakdown of glycogen. The resulting enzyme deficiency leads to the pathological accumulation of glycogen and lysosomal alterations in body tissues, resulting in cardiac, respiratory, and skeletal muscle dysfunction.

[0194]

[0204] Blood clotting disorders that can be treated include hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency in any of the clotting factors: Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor V, Factor XII, Factor II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency.

[0195]

[0205] Other diseases and disorders that can be treated include bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulation disorders, disseminated intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or platelet disorders, such as Bernard-Lesurier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0196]

[0206] Other diseases and disorders that can be treated include proliferative diseases (e.g., cancer, tumors, and dysplasia), metabolic diseases of the liver such as Crigler-Najjar disease and metabolic diseases; Friedreich's ataxia; infectious diseases; viral diseases such as those induced by hepatitis B or hepatitis C virus, HIV, venom, and retroviruses; genetic diseases such as cystic fibrosis, dystroglycanopathy, myopathy such as Duchenne myomyopathies or dystrophy, myotubular myopathy, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubular myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy, or Charcot-Marie-Tooth disease; arthritis; severe combined immune deficiencies such as RS-SCID, ADA-SCID, or X- SCID; Wiskott-Aldrich syndrome; X-ray associated thrombocytopenia; X-ray associated congenital neutropenia; chronic granulomatous disease; coagulation factor deficiencies; cardiovascular diseases such as restenosis, ischemia, dyslipidemia, and homozygous familial hypercholesterolemia; eye or ocular diseases such as retinitis pigmentosa, X-ray associated retinitis pigmentosa, autosomal dominant retinitis pigmentosa, recessive retinitis pigmentosa, choroideremia, choroideremia, choroidal neovascularization, gyrate atrophy, retinoschisis, X-ray associated retinoschisis, macular degeneration, diabetic macular edema (DME), diabetic retinopathy associated with DME, wet age-related macular degeneration (wet AMD or wAMD), macular edema secondary to retinal vein occlusion, non-arteritic ischemic optic neuropathy, Leber congenital amaurosis, Leber hereditary optic neuropathy, color vision deficiency, and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia such as CN Glycogen storage diseases, including GSD-I, GSD-II (Pompe disease), GSD-III, GSD-IV, GSD-V, GSD-VI, GSD-VII, GSD-VIII, or fatal congenital glycogen storage disease of the heart.

[0197]

[0207] In certain embodiments, the subject has a disease or disorder that affects or originates in the central nervous system (CNS). In certain embodiments, the disease is a neurodegenerative disease. Non-limiting examples of CNS or neurodegenerative diseases include Alzheimer's disease, Huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy's disease, polyglutamine repeat disease, or Parkinson's disease. In certain embodiments, the disease is a psychiatric disorder, addiction (e.g., tobacco, alcohol, or drug), epilepsy, Canavan's disease, or adrenoleukodystrophy. In certain embodiments, the CNS or neurodegenerative disease is a polyglutamine repeat disease, such as spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).

[0198] VII.B. Dosage Examples for Different Diseases and Conditions

[0208] A wide variety of different diseases and disorders can be treated based on this application. Dosages for certain diseases or disorders are highlighted in this section. The examples provided, like other examples in this application, are for purposes of illustrating different embodiments.

[0199]

[0209] For Pompe disease, an effective amount is an amount of GAA that inhibits or reduces glycogen production or accumulation, improves or increases glycogen breakdown or removal, reduces lysosomal alterations in the subject's body tissue, or improves muscle tone and / or strength and / or respiratory function in the subject. An effective amount can be determined, for example, by determining the kinetics of GAA uptake by myoblasts from plasma. A myoblast GAA uptake rate (K uptake) of about 141-147 nM has been shown to be effective (e.g., Maga et al., J. Biol. Chem. 2012, 8;288(3), 1428). In animal models, plasma GAA activity levels of greater than about 1,000 nmol / hr / mL, e.g., about 1,000 to about 2,000 nmol / hr / mL, have been observed to be therapeutically effective.

[0200]

[0210] For HemA and HemB, it is generally expected that a blood clotting factor concentration greater than 1% of the factor concentration found in normal individuals is required to change from a severe to a moderate disease phenotype. The severe phenotype is characterized by joint damage and life-threatening bleeding. It is expected that a blood clotting factor concentration greater than 5% of normal is required to convert from a moderate to a mild disease phenotype.

[0201]

[0211] Normal levels of FVIII are approximately 100-200 ng / ml, and FIX levels in normal humans are 5000 ng / ml, but may be higher or lower and are considered normal for functional coagulation as determined, for example, by the activated partial thromboplastin time (aPTT) one-stage clotting assay. Thus, a therapeutic effect can be achieved such that the total amount of FVIII or FIX in a subject / human is greater than 1% of the FVIII or FIX present in a normal subject / human, e.g., 1% of 100-300 ng / mL.

[0202] VII.C. Combination Treatment

[0212] The DNA vectors described herein can be used in combination with other therapies for a particular disease or disorder.

[0203] VIII. Kit

[0213] Further provided herein is a kit providing, in separate containers, at least (a) a pharmaceutical composition comprising nanoparticles containing DNA; (b) a type 1 interferon receptor pathway inhibitor; (c) optionally a cGAS-STING pathway inhibitor; (d) optionally an inflammasome pathway inhibitor; and (e) optionally an immune cell modulator. The amounts of different components can be obtained taking into account different factors, such as the target disease or disorder, and the DNA vector. The kit also provides a label containing instructions for administration according to the methods described herein.

[0204] IX. Additional Aspects and Embodiments

[0214] Additional aspects, embodiments, and combinations thereof include:

[0215] A first aspect of the present invention is a method for intracellular delivery of DNA in a subject, comprising: a) a type 1 interferon receptor pathway inhibitor; and b) First nanoparticles containing DNA wherein step (b) is performed before, simultaneously with, or after step (a).

[0205]

[0216] Embodiment E1 further describes the first aspect, wherein the DNA is a DNA vector comprising a transgene operably linked to a regulatory element. In a further embodiment, the transgene is operably linked to a promoter, operably linked to a promoter / enhancer, operably linked to a promoter / enhancer, a polyadenylation signal sequence, and / or a regulatory element, and the DNA vector comprises, from 5' to 3', the promoter / enhancer, the transgene, and the polyadenylation signal sequence.

[0206]

[0217] The 5' to 3' reference to a particular element indicates the relative position of the different elements and does not require the different elements to be adjacent to each other, allowing for the presence of additional sequences. In some cases, additional sequences that confer additional activity can be located at different positions between the two identified elements, at the 3' end, or at the 5' end.

[0207]

[0218] Embodiment E2 further describes the first aspect and E1, wherein the type 1 interferon receptor pathway inhibitor is a type 1 interferon receptor inhibitor. In a different embodiment, the receptor inhibitor is an antibody that binds to the type 1 interferon receptor or comprises an antibody fragment that binds to the type 1 interferon receptor. The antibody binding fragment contains three complementarity determining regions in the variable region framework that allow antigen binding. In a further embodiment, the antibody is anifrolumab.

[0208]

[0219] A reference to a particular embodiment includes a reference to further and different embodiments provided herein, for example, a reference to a first embodiment in a second embodiment provides a reference to all of the embodiments provided in the first embodiment, including further and different embodiments provided herein.

[0209]

[0220] Embodiment E3 further describes the first aspect and E1, wherein the type 1 interferon receptor pathway inhibitor is a Janus activated kinase inhibitor. In different embodiments, the Janus activated kinase inhibitor is a Janus activated kinase 1 inhibitor or a tyrosine kinase 2 inhibitor, a selective Janus activated kinase 1 inhibitor, a selective tyrosine kinase 2 inhibitor, or a compound of Table 1 or a pharmaceutically acceptable salt thereof.

[0210]

[0221] Embodiment E4 further describes the first aspect and E1, wherein the type 1 interferon receptor pathway inhibitor is a signal transducer and activator of transcription (STAT) protein inhibitor. In a different embodiment, the STAT inhibitor is a STAT1 inhibitor or a STAT2 inhibitor, or is a compound of Table 2 or a pharmaceutically acceptable salt thereof.

[0211]

[0222] Embodiment E5 further describes the first aspect, E1, E2, E3, and E4, and in a different embodiment, the method inhibits cytokine production or inhibits IFN gamma, IFN alpha, and / or IL-6 production.

[0212]

[0223] Embodiment E6 further describes the first aspect, E1, E2, E3, E4, and E5, wherein the method further comprises administering a cGAS-STING pathway inhibitor. In a further embodiment, the cGAS-STING pathway inhibitor is any compound in Table 3, 4, or 5, or a pharmaceutically acceptable salt thereof.

[0213]

[0224] Embodiment E7 further describes the first aspect, E1, E2, E3, E4, E5, and E6, wherein the method further comprises administering an inflammasome pathway inhibitor. In a further embodiment, the inflammasome pathway inhibitor is a polynucleotide having the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0214]

[0225] Embodiment E8 further describes the first aspect, E1 and E2, wherein the JAK inhibitor is not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor; the JAK1 or JAK2 inhibitor is not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor; the STAT inhibitor is not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor; the cGAS inhibitor is not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor; the STING inhibitor is not administered within 2 months before or after the administration of the interferon receptor inhibitor; and / or the inflammasome pathway inhibitor is not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor, and the JAK inhibitor, STAT inhibitor, cGAS inhibitor, STING inhibitor and inflammasome pathway inhibitor are not administered within 2 months before or after the administration of the type 1 interferon receptor inhibitor. In a further embodiment, the inhibitor provided in E7 (other than a type 1 interferon receptor inhibitor) is not independently administered within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months before administration of the type 1 interferon receptor inhibitor and within 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months after administration of the type 1 interferon receptor inhibitor.

[0215]

[0226] Embodiment E9 further describes the first aspect, E2, E3, E4, E5, E6, E7, and E8, wherein the DNA vector is selected from the group consisting of a viral antigen, a bacterial antigen, a therapeutic protein, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA iIn further embodiments, the therapeutic protein comprises a transgene encoding a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In further embodiments, the therapeutic protein is as described in Section III.C. above.

[0216]

[0227] Embodiment E10 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, and E9, wherein the type 1 interferon receptor pathway inhibitor is provided in a second nanoparticle. In a further embodiment, the second nanoparticle has substantially the same composition as the first nanoparticle, and the first and second nanoparticles are lipid nanoparticles or lipid-polymer nanoparticles. In a further embodiment, the nanoparticle is an LNP, and the nanoparticle is described in Section IB, above.

[0217]

[0228] Embodiment E11 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, and E9, wherein the type 1 interferon receptor pathway inhibitor is provided with the DNA or DNA vector in a first nanoparticle. In further embodiments, the first nanoparticle is a lipid nanoparticle, the first nanoparticle is a lipid-polymer nanoparticle, the first nanoparticle is an exosome, the first nanoparticle is configured to release the inhibitor prior to release of the DNA or DNA vector, the first nanoparticle is a lipid nanoparticle configured to release the inhibitor prior to release of the DNA or DNA vector, the first nanoparticle is a lipid-polymer nanoparticle configured to release the inhibitor prior to release of the DNA or DNA vector, or the first nanoparticle is an exosome configured to release the inhibitor prior to release of the DNA or DNA vector.

[0218]

[0229] Embodiment E12 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, and E10, wherein the type 1 interferon receptor pathway inhibitor is administered about simultaneously with, prior to, or following administration of the DNA or DNA vector. In different embodiments, the Type 1 interferon receptor pathway inhibitor is administered at least 30 minutes, at least 60 minutes, at least 90 minutes, or at least 120 minutes before the DNA or DNA vector, and the Type 1 interferon receptor pathway inhibitor is administered at about the same time as the DNA or DNA vector, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2:00, up to about 3:00, up to about 4:00, up to about 5:00, up to about 6:00, up to about 7:00, up to about 8:00, up to about 9:00, up to about 10:00, up to about 12:00, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, or up to about 1 week. In additional embodiments, the Type 1 interferon receptor pathway inhibitor is administered at about the same time as the DNA or DNA vector, up to about 5 minutes, up to about 15 minutes, up to about 30 minutes, up to about 45 minutes, up to about 60 minutes, up to about 90 minutes, up to about 2 o'clock, up to about 3 o'clock, or up to about 4 o'clock.

[0219]

[0230] Embodiment E13 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, and E12, and employs two or more different type 1 interferon receptor pathway inhibitors. The different inhibitors can be provided without nanoparticles, can be provided on different nanoparticles, or can be provided on the same nanoparticles.

[0220]

[0231] Embodiment E14 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, and E13, wherein both the DNA and DNA vectors substantially comprise double-stranded DNA, or the DNA and DNA vectors substantially comprise single-stranded DNA. In different embodiments, more than half, at least 75%, at least 90%, at least 95%, or at least 99% of the DNA or DNA vector is double-stranded DNA, or 100% of the DNA is double-stranded.

[0221]

[0232] Embodiment E15 further describes the first aspect, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, and E14, wherein the subject is a human patient. In a further embodiment, the method provides a therapeutically effective amount of the transgene.

[0222]

[0233] A second aspect of the present invention is a) DNA; and b) Type 1 interferon receptor pathway inhibitors The nanoparticles are characterized, including:

[0223]

[0234] Embodiment E16 further describes the second aspect, wherein the DNA is a DNA vector comprising a transgene, the transgene operably linked to regulatory elements. In a further embodiment, the transgene is operably linked to a promoter, a promoter-enhancer, a promoter / enhancer, a polyadenylation signal sequence, and / or a regulatory element, and the DNA vector comprises, from 5' to 3', the promoter / enhancer, the transgene, and the polyadenylation signal sequence.

[0224]

[0235] Embodiment E17 further describes the second aspect and E16, wherein the type 1 interferon receptor pathway inhibitor is not a type 1 interferon receptor inhibitor.

[0225]

[0236] Embodiment E18 further describes the second aspect and E16, wherein the type 1 interferon receptor pathway inhibitor is a Janus activated kinase inhibitor. In different embodiments, the Janus activated kinase inhibitor is Janus activated kinase inhibitor 1 or a tyrosine kinase 2 inhibitor, or is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

[0226]

[0237] Embodiment E19 further describes the second aspect and E16, wherein the type 1 interferon receptor pathway inhibitor is a signal transducer and activator of transcription (STAT) protein inhibitor. In different embodiments, the STAT inhibitor is a STAT1 inhibitor or a STAT2 inhibitor, or is a compound of Table 2 or a pharmaceutically acceptable salt thereof.

[0227]

[0238]

[0023] Embodiment E20 further describes the second aspect, E16, E17, E18, and E19, wherein the composition further comprises a cGAS-STING pathway inhibitor. In a further embodiment, the cGAS-STING pathway inhibitor is any compound in Table 3, 4, or 5, or a pharmaceutically acceptable salt thereof.

[0228]

[0239] Embodiment E21 further describes the second aspect, E16, E17, E18, E19, and E20, wherein the composition further comprises an inflammasome pathway inhibitor. In a further embodiment, the inflammasome pathway inhibitor is a polynucleotide having the sequence of SEQ ID NO:1 or SEQ ID NO:21.

[0229]

[0240] Embodiment E22 further describes the second aspect, E16, E17, E19, E20, and E21, wherein the DNA is selected from the group consisting of a viral antigen, a bacterial antigen, a therapeutic protein, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA iIn a further embodiment, the therapeutic protein is a DNA vector comprising a transgene encoding a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In a further embodiment, the therapeutic protein is as described in Section III.C. above.

[0230]

[0241] Embodiment E23 further describes the second aspect, E16, E17, E18, E19, E20, E21, and E22, wherein the nanoparticles are lipid nanoparticles, lipid-polymer nanoparticles, or exosomes. In further embodiments, the nanoparticles are lipid nanoparticles, the nanoparticles are lipid-polymer nanoparticles, the nanoparticles are exosomes, the nanoparticles are lipid nanoparticles configured to release an inhibitor prior to release of the DNA or DNA vector, the nanoparticles are lipid-polymer nanoparticles configured to release an inhibitor prior to release of the DNA or DNA vector, or the nanoparticles are exosomes configured to release an inhibitor prior to release of the DNA or DNA vector. In further embodiments, the nanoparticles are LNPs, such as those described in Section IB, above.

[0231]

[0242] E24 further describes a second aspect, E16, E17, E18, E19, E20, E21, E22, and E23, wherein the DNA and DNA vector each substantially comprises double-stranded DNA, or the DNA and DNA vector each substantially comprises single-stranded DNA. In different embodiments, more than half, at least 75%, at least 90%, at least 95%, or at least 99% of the DNA or DNA vector is double-stranded DNA, or 100% of the DNA is double-stranded.

[0232]

[0243] A third aspect is directed to a pharmaceutical composition comprising the nanoparticle composition of the second aspect, E16, E17, E18, E19, E20, E21, E22, E23 and 24, and a pharmaceutically acceptable carrier.

[0233]

[0244] A fourth aspect is directed to a pharmaceutical composition for use in medicine (preferably gene therapy) comprising a DNA vector containing a transgene, the composition being for use before, simultaneously with, or after administration of a type 1 interferon receptor pathway inhibitor, and the DNA vector being provided in nanoparticles. Additional embodiments are provided in methods and components of the first aspect and related embodiments, compositions of the second aspect and related embodiments, and pharmaceutical compositions of the third aspect.

[0234]

[0245] A fifth aspect of the invention is directed to a method of making a medicament for medicine (preferably gene therapy) comprising nanoparticles comprising a DNA vector, wherein the medicament is for use before, simultaneously with, or after administration of a type 1 interferon receptor pathway inhibitor, wherein the DNA vector and / or the type 1 interferon receptor pathway inhibitor are combined with a pharmaceutically acceptable carrier, and wherein the nanoparticles and DNA vector are as described in the first aspect and related embodiments and the second aspect and related embodiments.

[0235]

[0246] A sixth aspect is a structure [ka] or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound is present in an LNP as provided in any of the first to fifth aspects, or in the accompanying embodiments (E1 to E23).

[0236]

[0247] Although several different aspects and embodiments of the present invention have been described throughout this application, those skilled in the art will nevertheless be able to make various changes and modifications to the invention to adapt it to various usages and conditions without departing from the spirit and scope of the invention. [Example]

[0237]

[0248] Examples are presented below to further illustrate different features of the invention and methodologies for practicing the invention. The presented examples do not limit the claimed invention. Example 1 - Ablation of Interferon Receptor Signaling

[0249] Viability and expression studies were performed in wild-type (WT) and IFNAR-deficient knockout (IFNAR KO) mice (n=5 per group) systemically dosed with 1.25 mpk (25 μg) of lipid nanoparticle-encapsulated plasmid DNA (DNA-LNP) encoding the human factor IX (hFIX) transgene at t=0, 7 weeks, and 12 weeks. The LNPs used in this study were formulated with cKK-E12.

[0238]

[0250] Elimination of IFNAR signaling improved DNA-LNP tolerability and transgene expression. Figure 1 shows the survival rates of WT and IFNAR KO mice up to 18 weeks. Figure 2 shows transgenic hFIX protein in plasma measured by ELISA at various time points after dosing in surviving mice from each group up to 16 weeks. Thus, IFNAR signaling may be an obstacle to the tolerability and efficacy of DNA-LNP gene therapy.

[0239] Example 2 - Inhibiting Interferon Receptor Signaling

[0251] Plasma cytokine levels, % survival, and expression levels were measured using WT mice (n=5 per group) either untreated or treated once with 15 mpk of an anti-mouse IFNAR blocking antibody. The aIFNAR used was MAR1-5A3 (Dunn et al., Nat Immunol. 2005 July;6(7):722-9). The blocking antibody was added 3 hours before systemic dosing with 1.25 mpk (25 μg) of hFIX DNA-LNP at t=0 and again with 2.5 mpk (50 μg) of hFIX DNA-LNP on day 41. Plasma cytokine levels were assessed 4 hours after dosing on day 41 and compared to pre-dose levels ("baseline") in pooled plasma. The LNPs used in this study were formulated with bCKK-E12. Figure 3A shows IL-6 levels, Figure 3B shows IFN-alpha levels, and Figure 3C shows IFN-gamma levels. Survival and hFIX expression were followed up to 70 days. Figure 4 shows survival up to 70 days. Figure 5 shows transgenic hFIX protein in plasma measured by ELISA at various time points post-dosing in surviving mice from each group up to 70 days.

[0240]

[0252] These data indicate that antibody-mediated inhibition of IFNAR signaling reduces acute inflammatory responses and improves survival in mice administered DNA-LNP gene therapy. Anti-IFNAR treatment improved the efficacy of DNA-LNP transgenes compared with mice not treated with anti-IFNAR. Blocking the IFNAR pathway may have the dual benefit of improving both the tolerability and efficacy of DNA-LNP-based gene therapy.

[0241] Example 3 - Targeting JAK1 and JAK2 kinases downstream of IFNAR

[0253] Wild-type mice were either untreated or orally treated with the JAK inhibitors ruxolitinib or baricitinib 30 minutes before and on days 1, 2, and 3 after systemic administration of 2.5 mpk (50 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles (DNA-LNPs) (n = 5 per group). The LNPs used in this study were formulated with bCKK-E12. Survival of mice in each group was followed for up to 30 days (Figure 6).

[0242]

[0254] Ruxolitinib and / or baricitinib, which are selective for JAK1 and JAK2, dramatically improved survival in mice treated with DNA-LNP gene therapy. JAK inhibitor treatment may act (at least in part) to improve DNA-LNP tolerability by blocking IFNAR signaling.

[0243] Example 4 - IFNAR neutralization improves transgene expression

[0255] Wild-type C57BL / 6 mice were either untreated or treated once IP with 15 mpk of an anti-mouse IFNAR-blocking antibody ("anti-IFNAR"; clone MAR1-5A3, described in Dunn et al., Nat Immunol. 2005 July;6(7):722-9) 3 hours before systemic (IV tail injection) dosing with 2.5 mpk (50 μg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles (DNA-LNP) (n=5 per group). The LNP used in this study was formulated with bCKK-E12. Mice were either untreated or orally treated with the JAK inhibitor baricitinib ("Bar") 90 minutes before DNA-LNP dosing and then daily on days 1 through 6 after DNA-LNP dosing. Transgenic hFIX protein in the plasma of surviving mice was measured by ELISA 1 week after DNA-LNP dosing (Fig. 7).

[0244]

[0256] Anti-IFNAR enhanced the efficacy of the DNA-LNP transgene. Baricitinib (selective for JAK1 and JAK2) did not enhance the efficacy of the transgene, and anti-IFNAR in combination with baricitinib did not enhance the efficacy of the transgene.

[0245] Example 5 - STING Removal

[0257] Wild-type C57BL / 6 ("WT") mice and STING-deficient mice (i.e., mice carrying the Goldentiquet nonsense mutation ("STING(Gt)")) were either untreated or treated once IP with 15 mpk of an anti-mouse IFNAR-blocking antibody ("anti-IFNAR") 3 hours before systemic (IV tail injection) dosing with 5 mpk (100 mg) of plasmid DNA encoding a human factor IX (hFIX) transgene encapsulated in lipid nanoparticles (DNA-LNP) (n = 5 per group). All WT mice not treated with anti-IFNAR died within 2 days of DNA-LNP dosing. Transgenic hFIX protein in the plasma of surviving mice was measured by ELISA 4 weeks after DNA-LNP dosing (Figure 8). The data suggest that complete ablation of STING signaling abolishes the positive effect that blocking IFNAR signaling has on transgene expression.

[0246]

[0258] Example 6 - Effect of anti-INFAR antibodies delivered by different LNPs

[0259] Wild-type C57BL / 6 mice (n=5 per group) were either untreated or treated IP once with 15 mpk of an anti-mouse IFNAR blocking antibody ("anti-IFNAR"; described in Example 2) 3 hours before systemic (IV tail injection) dosing with 2.5 mpk (50 mg) of plasmid DNA encoding a human factor IX ("FIX") transgene. Plasmid DNA encoding the FIX transgene encapsulated in lipid nanoparticles was formulated using different lipid formulations and ionizable lipids. Figure 9A shows the results using Genvoi-ILM™, and Figure 9B shows the results using the lipid of Compound 9. According to Roces et al., Pharmaceuticals, 2020, 12, 1095, Genvoi-ILM™ is composed of approximately 50% ionizable lipids; approximately 10% DSPC; approximately 37.5% cholesterol; and approximately 2.5% stabilizer (PEG-lipid). Transgenic FIX protein in plasma was measured by ELISA one week after DNA-LNP administration. The LNP containing Compound 9 in this example consisted of approximately 50% Compound 9 (US Patent Application Publication No. 2022204439), 2.5% C14-PEG2000, 37.5% cholesterol, and 10% DSPC. Different LNP compositions containing anti-IFNAR antibodies increased transgene expression.

[0247]

[0260] Example 7 - Effect of anti-INFAR antibodies on EPO expression in Babl / c mice

[0261] Wild-type Balb / c mice were either untreated or treated once IP with 15 mpk of an anti-mouse IFNAR-blocking antibody ("anti-IFNAR"; described in Example 2) 3 hours before systemic (IV tail injection) dosing with 2.5 mpk (50 mg) of human erythropoietin ("EPO")-expressing plasmid DNA encapsulated in lipid nanoparticles (DNA-LNP; the LNPs used in this study were formulated with bCKK-E12) (n=5 per group). Transgenic EPO protein in plasma was measured by ELISA 1 week after DNA-LNP dosing. The results are shown in Figure 10. Anti-IFNAR treatment resulted in increased expression of the EPO transgene derived from the DNA-LNP in Balb / c mice.

[0248]

[0262] Example 8 - bCKK-E12 synthesis

[0249]

[0263] bCKK-E12 was synthesized using the following procedure:

[0250]

[0264] Step A: [ka]

[0251]

[0265] A 1000 mL three-necked round-bottom flask was equipped with a magnetic stirrer, an addition funnel, and a thermometer. To a solution of compound 1 (50.0 g, 390 mmol, 1.0 equiv.) in dichloromethane (DCM) (300 mL) was added 4-toluenesulfonyl chloride (TsCl) (89.2 g, 468 mmol, 1.2 equiv.), triethylamine (TEA) (78.9 g, 780 mmol, 2.0 equiv.), and DMAP (2.38 g, 19.5 mmol, 0.05 equiv.). The resulting mixture was stirred at 15 °C for 15 h. Thin-layer chromatography (TLC) (petroleum ether / ethyl acetate = 10 / 1, R f =0.7), compound 1 was completely consumed and two new spots formed. The reaction was evident by TLC. The reaction mixture was quenched at 25 °C by the addition of HO (600 mL) and then extracted with DCM (500 mL, 300 mL). The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 50 / 1) to give compound 2 (100 g, 354 mmol, 90.8% yield) as a yellow oil.

[0266] Compound 2: 1H NMR: ET72382-2-P1B 400 MHz, CDCl3δ 7.80 (d, J = 8.4 Hz, 2 H), 7.35 (d, J = 8.0 Hz, 2 H), 5.43 - 7.81(m, 1 H), 4.88 - 5.05 (m, 2 H), 4.03 (t, J = 6.4 Hz, 2 H), 2.46 (s, 3 H), 2.01(q, J = 7.2 Hz, 2 H), 1.59 - 1.72 (m, 2 H), 1.15 - 1.39 (m, 6 H).

[0252]

[0267] Step B: [ka]

[0268] Step B: A 3000 mL three-neck round-bottom flask was equipped with a magnetic stirrer, an addition funnel, and a thermometer. A mixture of Mg (24.5 g, 1.01 mol, 3.0 equiv.) in tetrahydrofuran (THF) (400 mL) was added to a solution of compound 2A (138 g, 1.01 mol, 3.0 equiv.) in THF (100 mL) at 30 °C, and the resulting reaction mixture was stirred at 45 °C for 2 h. The reaction mixture was cooled to -60 °C, and compound 2 (95.0 g, 336 mmol, 1.0 equiv.) in THF (100 mL) and CuCl2.2LiCl (0.1 M, 101 mL, 0.03 equiv.) was added. The mixture was stirred at 15 °C for 12 h under a N2 atmosphere. TLC (petroleum ether:ethyl acetate = 1:0, Rf = 0.85) showed that compound 2 was completely consumed and one major new spot with low polarity was detected. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl (1000 mL) and then extracted with petroleum ether (1000 mL, 700 mL). The combined organic layers were concentrated under reduced pressure to give compound 3 (55.0 g, crude) as a colorless oil, which was used in the next step without purification.

[0253]

[0269] Compound 3: 1H NMR: ET72382-4-P1B 400 MHz, CDCl3δ 5.79 - 5.87 (m, 1 H), 4.89 - 5.07 (m, 2 H), 2.05 (q, J = 6.8 Hz, 2H), 1.47 - 1.61 (m, 2 H), 1.35 - 1.43 (m, 2 H), 1.13-1.30 (m, 9 H), 1.10 - 1.20(m, 2 H), 0.87 (d, J = 6.8 Hz, 8 H).

[0254]

[0270] Step C: [ka]

[0271] A 1000 mL three-necked round-bottom flask was equipped with a magnetic stirrer, an addition funnel, and a thermometer. To a solution of compound 3 (58.0 g, 345 mmol, 1.0 equiv.) in DCM (350 mL) was added m-CPBA (metachloroperbenzoic acid) (112 g, 517 mmol, 80% purity, 1.5 equiv.) at 15 °C. The resulting mixture was stirred at 15 °C for 15 h. TLC (petroleum ether / ethyl acetate = 5 / 1, R f =0.6), indicating the complete consumption of compound 3, with the detection of one major new, highly polar spot. The mixture was poured into NaHSO (1000 mL) and extracted with DCM (600 mL, 400 mL). The combined organic layers were washed with NaHCO (600 mL) and brine (100 mL). The combined organic layers were concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give compound 4 (37.0 g, 201 mmol, 58.3% yield) as a colorless oil.

[0272] Compound 4: 1H NMR: ET72382-5-P1B 400 MHz, CDCl3δ 2.87 - 2.98 (m, 1 H), 2.75 (t, J = 4.8 Hz, 1 H), 2.47 (dd, J = 4.8,2.8 Hz, 1 H), 1.40 - 1.61 (m, 5 H), 1.22 - 1.39 (m, 7 H), 1.22 - 1.39 (m, 1 H),1.08 - 1.19 (m, 2 H), 0.87 (d, J = 6.8 Hz, 6 H).

[0255]

[0273] Step D: [ka]

[0256]

[0274] To a solution of compound 5 (25.0 g, 47.6 mmol, 1.0 equiv.) in DCM (80 mL) and AcOH (80 mL) under N was added Pd / C (5.0 g, 10% purity). The resulting suspension was degassed under vacuum and purged with H several times. The mixture was stirred at 25 °C for 16 h under H (50 psi). TLC (dichloromethane / methanol = 10 / 1, R f =0.1), compound 5 was completely consumed and one new spot was formed. The reaction was clear by TLC. The mixture was filtered, and the cake was washed with MeOH (1000 mL). The filtrate was concentrated to give a residue. The residue was purified by recrystallization from ethyl acetate (EtOAc) (350 mL) at 25 °C. The solid was filtered, washed with EtOAc (100 mL), and dried in vacuo. The dried solid was dissolved in MeOH (150 mL), and the pH value of the mixture was adjusted to 8 twice with alkaline resin. Compound 6 (8.00 g, 31.2 mmol, 65.5% yield) was obtained as a white solid.

[0257]

[0275] Compound 6: 1H NMR: ET72382-1-P1B 400 MHz, MeOD δ 3.94 -4.07 (m, 2 H), 2.60 - 2.71 (m, 4 H), 1.73 - 1.94 (m, 4 H), 1.31 - 1.57 (m, 8H).

[0258]

[0276] Step E: [ka]

[0259]

[0277] A mixture of compound 6 (7.00 g, 27.3 mmol, 1.0 equiv.) and compound 4 (33.6 g, 182 mmol, 6.7 equiv.) in EtOH (280 mL) was degassed and purged with N2 three times, and the resulting mixture was stirred at 80 °C under N2 atmosphere for 24 h. LC-MS showed that compound 6 was completely consumed, with one major peak at the desired mass. The mixture was concentrated to give a residue. The residue was purified by column chromatography (SiO2, DCM:MeOH = 20 / 1 to 5 / 1) (TLC: DCM / MeOH = 10 / 1, R f =0.45) to give compound bCKK-E12 (10.5 g, 10.6 mmol, 38.7% yield) as a yellow gum.

[0260]

[0278] Compound bCKK-E12: 1 H NMR: ET72382-7-P1B 400 MHz, MeOD δ 3.93 - 4.05 (m, 2 H), 3.63 (br s, 4 H), 2.31 - 2.66 (m, 12 H), 1.74- 1.93 (m, 4 H), 1.28 - 1.58 (m, 60 H), 1.14 - 1.23 (m, 8 H), 0.88 (d, J = 6.8Hz, 24 H).

[0261]

[0279] Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various modifications, changes, alterations, substitutions, deletions, or additions to the procedures and protocols may be made without departing from the spirit and scope of the invention.

[0262] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 375,156, filed September 9, 2022, the disclosure of which is incorporated herein by reference in its entirety. REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING

[0263]

[0002] The contents of the electronic sequence listing (065830_17WO1.xml; size: 3,359 bytes; and creation date: September 6, 2023) are incorporated herein by reference in their entirety.

Claims

1. 1. A method for intracellular delivery of DNA in a subject, comprising: a) a type 1 interferon receptor pathway inhibitor; and b) first nanoparticles containing the DNA; wherein step (b) can be performed before, simultaneously with, or after step (a).

2. The method of claim 1 , wherein the DNA is a DNA vector comprising a transgene operably linked to a regulatory element.

3. 3. The method of claim 2, wherein the transgene is operably linked to a promoter, and the DNA vector comprises, from 5' to 3', the promoter, the transgene, and a polyadenylation signal sequence.

4. The method of any one of claims 1 to 3, wherein the type 1 interferon receptor pathway inhibitor is a type 1 interferon receptor inhibitor.

5. 5. The method of claim 4, wherein the type 1 interferon receptor inhibitor is an antibody that binds to a type 1 interferon receptor or comprises an antibody fragment that binds to a type 1 interferon receptor.

6. 6. The method of claim 5, wherein the antibody is anifrolumab.

7. The method of any one of claims 1 to 3, wherein the type 1 interferon receptor pathway inhibitor is a Janus activated kinase 1 inhibitor or a tyrosine kinase 2 inhibitor.

8. 8. The method of claim 7, wherein the type 1 interferon receptor pathway inhibitor is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

9. 4. The method of any one of claims 1 to 3, wherein the type 1 interferon receptor pathway inhibitor is a signal transducer and activator of transcription (STAT) protein inhibitor.

10. 10. The method of claim 9, wherein the STAT inhibitor is a STAT1 inhibitor or a STAT2 inhibitor.

11. 10. The method of claim 9, wherein the STAT inhibitor is a compound of Table 2 or a pharmaceutically acceptable salt thereof.

12. The method according to any one of claims 1 to 11, wherein IFN-gamma induced by DNA administration is inhibited.

13. 13. The method of any one of claims 1 to 12, further comprising administration of an interferon gene (cGAS-STING) pathway inhibitor cyclic GMP-AMP synthase stimulator.

14. 14. The method of claim 13, wherein the cGAS-STING pathway inhibitor is a compound of any of Tables 3, 4, or 5, or a pharmaceutically acceptable salt thereof.

15. The method of any one of claims 1 to 14, further comprising administration of an inflammasome pathway inhibitor.

16. The method of claim 14, wherein the inflammasome pathway inhibitor is a polynucleotide having the sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

17. 7. The method of any one of claims 4 to 6, wherein a JAK inhibitor, a STAT inhibitor, a cGAS inhibitor, a STING inhibitor and / or an inflammasome pathway inhibitor is not administered within 2 months before or within 2 months after the type 1 interferon receptor inhibitor.

18. The transgene may be a viral antigen, a bacterial antigen, a therapeutic protein, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA ｉ , a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

19. 19. The method of any one of claims 1 to 18, wherein the type 1 interferon receptor pathway inhibitor is provided in a second nanoparticle.

20. 20. The method of claim 19, wherein the second nanoparticles have substantially the same composition as the first nanoparticles.

21. The method of any one of claims 1 to 18, wherein the DNA or the DNA vector and the type 1 interferon receptor pathway inhibitor are provided together with the first nanoparticles.

22. The method of any one of claims 1 to 21, wherein the first nanoparticle is a lipid nanoparticle or a lipid-polymer nanoparticle.

23. 23. The method of claim 21 or 22, wherein the first nanoparticles are configured to release the type 1 interferon receptor pathway inhibitor prior to release of the DNA or DNA vector.

24. 24. The method of any one of claims 1 to 23, wherein the type 1 interferon receptor pathway inhibitor is administered at about the same time as, up to about 4 hours before, administration of the DNA or DNA vector.

25. 25. The method of any one of claims 1 to 24, wherein the DNA substantially comprises double-stranded DNA and the DNA vector substantially comprises double-stranded DNA.

26. The method of any one of claims 2 to 25, wherein the subject is a human patient and the method provides a therapeutically effective amount of the transgene.

27. a. DNA; and b. Type 1 interferon receptor pathway inhibitors 10. A nanoparticle composition comprising:

28. 28. The composition of claim 27, wherein the DNA is a DNA vector comprising a transgene operably linked to a regulatory element.

29. 29. The composition of claim 28, wherein the DNA vector comprises, from 5' to 3', a promoter, the transgene, and a polyadenylation signal sequence.

30. The composition of any one of claims 27 to 29, wherein the type 1 interferon receptor pathway inhibitor is a Janus activated kinase 1 inhibitor or a tyrosine kinase 2 inhibitor.

31. 31. The composition of claim 30, wherein the type 1 interferon receptor pathway inhibitor is a compound of Table 1 or a pharmaceutically acceptable salt thereof.

32. 30. The composition of any one of claims 27 to 29, wherein the type 1 interferon receptor pathway inhibitor is a signal transducer and activator of transcription (STAT) protein inhibitor.

33. 33. The composition of claim 32, wherein the STAT inhibitor is a STAT1 or STAT2 inhibitor.

34. 34. The composition of claim 33, wherein the STAT inhibitor is a compound of Table 2 or a pharmaceutically acceptable salt thereof.

35. The transgene may be a viral antigen, a bacterial antigen, a therapeutic protein, a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a microRNA (miRNA), an RNA ｉ , a ribozyme, an antisense RNA, a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 construct, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

36. The composition according to any one of claims 27 to 35, wherein the nanoparticles are lipid nanoparticles or lipid-polymer nanoparticles.

37. 37. The composition of claim 36, wherein the nanoparticle is a lipid polymer nanoparticle configured to release the type 1 interferon receptor pathway inhibitor prior to the DNA vector.

38. 38. The composition of any one of claims 27 to 37, wherein the DNA substantially comprises double-stranded DNA and the DNA vector substantially comprises double-stranded DNA.

39. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 27 to 38 and a pharmaceutically acceptable carrier.

40. 40. The pharmaceutical composition of claim 39, for use in medicine or gene therapy.

41. 27. A pharmaceutical composition for use in medicine, preferably gene therapy, comprising a first nanoparticle according to any one of claims 2 to 26 and a DNA vector for use together with a type 1 interferon receptor pathway inhibitor according to any one of claims 2 to 12.

42. [Catalog 1] or a pharmaceutically acceptable salt thereof.

43. The method of any one of claims 1 to 26 or the composition of any one of claims 27 to 41, wherein the nanoparticles comprise a compound of claim 42.

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