Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
  • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the invention relates to the field of gene therapy. In particular, it relates to methods of enhancing non-viral gene therapy.
• Gene therapy involves delivery of foreign nucleic acids to cells to provide a therapeutic benefit.
• Genetic diseases for which a causative abnormality is known can potentially be treated using gene therapy, either by blocking a gene that is being either mis- or overexpressed, or by providing a working copy of a malfunctioning gene.
• Such gene therapy approaches are currently being developed to treat or cure a number of diseases that arise from genetic abnormalities.
• An effective delivery system for the therapeutic genetic material is required for successful gene therapy.
• Successful delivery of the genetic material requires that, for example, the gene delivery system be deliverable to the subject, be able to protect the genetic material from enzymatic degradation, have a long lifetime in the body, be able to reach the site within the body where it is needed, be biocompatible and biodegradable or have tolerable toxicity, and be able to cross the cell membrane and transit through the cytosol and/or cross the nuclear membrane to release the genetic material at the desired point of action.
• Gene therapy can be delivered by viral and non-viral vectors.
• Viral vectors demonstrate high transduction efficiency and exhibit stable long-term expression of a foreign gene when the recombinant DNA remains episomal or is integrated into the chromosome.
• their in vivo efficacy may be limited due to concerns of toxicity, random integration of genetic materials into the genome, disruption of proper gene function, cancer formation, potential replication, cell division-dependent dilution, limited DNA carrying capacity, severe immune and inflammatory responses due, in part, to humoral and cell- mediated immune responses to the vector capsid, and the inability to re-dose subjects due to the presence of pre-existing antibodies to the vector capsid in a subset of subjects.
• Non-viral gene delivery approaches are being developed to overcome the deficiencies of viral vectors. Compared to viral vectors, certain types of non-viral vectors are easier to make, do not require replication, and are less likely to produce adaptive immune responses due to the lack of a protein-based vector capsid. In addition, non-viral vectors offer some invaluable advantages, including the lack of transgene size restriction associated with viral vectors, the ability to dose subjects with pre-existing antibodies to vector capsid, and the ability to re-dose a subject. However, non-viral delivery approaches suffer from challenges such as toxicity, transfection efficiency, nucleic acid degradation, and innate immunity. In addition, non-viral approaches have demonstrated low efficiency of gene delivery to somatic targets and lower in vivo gene expression levels than viral approaches.
• the liver is a central organ in metabolism, and it is a target for gene therapy of many inherited metabolic disease and disorders, including inborn errors of metabolism and hemophilia, as well as acquired diseases such as liver cancer and hepatitis.
• the particular cell targets for liver-directed gene therapies are the parenchymal liver cells, or hepatocytes.
• nanoparticles are predominantly taken up by non-parenchymal cells including Kupffer cells (-50%) and liver sinusoidal endothelial cells (-30%), limiting the efficacy of hepatocyte-directed gene therapy (Shi et al., J Histochem Cytochem. 2011 59(8):727-40; Jacobs et al., Pharmaceuticals 2012, 5(12): 1372-1392).
• tissue-specific macrophages also referred to as resident macrophages
• resident macrophages include intestinal macrophages in the gut, microglial cells in the brain, alveolar macrophages in the lung, resident kidney macrophages, skin macrophages, red pulp macrophages in the spleen, and osteoclasts in bone.
• Site-specific improved efficiency of gene therapy could be achieved through enhancing the entry of non-viral vectors into the target cells and reducing the uptake of the vectors by nontarget cells.
• Methods for delivering a transgene to a subject in need thereof can be used to treat a subject in need of treatment for a disease caused by a loss of function or activity of a protein, or to treat a subject in need of treatment for a disease caused by a gain of function activity or expression of a protein.
• depletion of phagocytic immune cells by administration of a phagocytedepleting agent reduces the unproductive uptake of non-viral vectors by non-target cells, enabling enhanced entry of the non-viral vectors into target cells and resulting in improved transgene expression.
• the invention relates to methods of delivering a transgene to a subject in need thereof comprising: a. administering to the subject a phagocyte-depleting agent; and b. administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises: a. a non-viral delivery nanoparticle; and b. the non-viral vector, wherein the non-viral vector comprises the transgene operably linked to a promoter.
• the phagocyte-depleting agent is a monocyte and/or macrophage-depleting agent.
• the monocyte and/or macrophage-depleting agent is a CD115 inhibiting agent.
• the CD115 inhibiting agent is an antibody or an antigen binding fragment thereof that specifically binds to CD115.
• the anti-CDl 15 antibody or antigen binding fragment thereof is emactuzumab, AMG820, or cabiralizumab.
• the CD115 inhibiting agent is a small molecule inhibitor of CD115.
• the small molecule inhibitor of CD115 is pexidartinib.
• the monocyte and/or macrophage-depleting agent is not clodronate, or the monocyte and/or macrophage-depleting agent comprises clodronate and one or more additional phagocyte-depleting agent such as a CD115 inhibiting agent or a CD177, CD14, CD15, CDllb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibiting agent.
• additional phagocyte-depleting agent such as a CD115 inhibiting agent or a CD177, CD14, CD15, CDllb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibiting agent.
• the phagocyte-depleting agent is a neutrophil-depleting agent.
• the neutrophil-depleting agent is a CD177, CD14, CD15, CDl lb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibiting agent, or an agent that inhibits the corresponding or equivalent human protein and/or functional cell type.
• the CD177, CD14, CD15, CDllb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibiting agent is an antibody or an antigen binding fragment thereof that specifically binds to CD177, CD14, CD15, CDllb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L.
• the CD177, CD14, CD15, CDl lb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L inhibiting agent is a small molecule inhibitor of CD177, CD14, CD15, CDllb, CD16, CD32, CD33, CD44, CD45, CD66b, CD18, or CD62L.
• the phagocyte-depleting agent is a dendritic cell-depleting agent.
• the non-viral vector and phagocyte-depleting agent are coadministered.
• the phagocyte-depleting agent is administered at least one day before the non-viral vector is administered, optionally no more than 1 year before the non-viral vector is administered.
• the method further comprises administering to the subject a bisphosphonate, preferably together with one or more other phagocyte-depleting agents.
• the bisphosphonate is clodronate, pamidronate, ibandronate, or zoledronate, optionally clodronate.
• the method further comprises administering to the subject an immunosuppressant.
• the immunosuppressant is a steroid.
• the steroid is a corticosteroid, optionally dexamethasone.
• the method results in expression of the transgene in the subject.
• the subject has a minimal or absent undesirable immune response induced by the non-viral vector.
• the non-viral delivery particle is selected from the group consisting of a lipid nanoparticle, a polymer nanoparticle, a protein-based nanoparticle, and a peptide cage.
• the non-viral vector is a dsDNA molecule, wherein the dsDNA molecule is selected from the group consisting of a minicircle, a plasmid, an open linear duplex DNA, and a closed-ended linear duplex DNA (CELiD/ceDNA/doggybone DNA).
• the non-viral vector is an ssDNA molecule, wherein the ssDNA molecule is a closed circular or an open linear DNA.
• the transgene encodes a therapeutic or prophylactic protein or peptide.
• the transgene encodes a therapeutic or prophylactic nucleic acid.
• a package or kit has disposed therein: (a) a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier; (b) a phagocyte-depleting agent; and (c) a label with instructions for performing a method as disclosed herein.
• (a) and (b) are in separate or the same container.
• the combination, package or kit can further comprise an immunosuppressant.
• FIG. 1 shows the immune response results of the study described in Example 1, specifically, FIG. 1A is a graph showing levels of IFN-y cytokine production in the plasma of animals 6 hours following dosing with 50 pg of DNA-LNP, with or without anti-CD115 or anti-Ly6G antibodies or large ODN or H-151; and FIG. IB is a graph showing levels of IFN- y cytokine production in the plasma of animals 6 hours following dosing with 10 pg of DNA- LNP, with or without anti-CD115 or anti-Ly6G antibodies or large ODN or H-151.
• FIG. 2 shows the efficacy results of the study described in Example 1, specifically, FIG. 2A is a graph showing expression levels of the FIX transgene in the plasma of animals 1, 8, 14, 28, 63, 84, and 98 days following dosing with 50 pg of DNA-LNP, with or without anti-CDl 15 or anti-Ly6G antibodies or large ODN or H-151; and FIG. 2B is a graph showing expression levels of the FIX transgene in the plasma of animals 1, 8, 14, 28, 63, 84, and 98 days following dosing with 10 pg of DNA-LNP, with or without anti-CDl 15 or anti-Ly6G antibodies or large ODN or H-151.
• FIG. 3 shows the immune response results of the study described in Example 2, specifically, FIG. 3 A is a graph showing levels of IFN-y cytokine production in the plasma of animals 6 hours following dosing with 50 pg of DNA-LNP, with or without anti-CDl 15 and/or clodronate; and FIG. 3B is a graph showing levels of IL-6 cytokine production in the plasma of animals 6 hours following dosing with 50 pg of DNA-LNP, with or without anti- CDl 15 and/or clodronate.
• FIG. 4 shows the efficacy results of the study described in Example 2, specifically, FIG. 4 is a graph showing expression levels of the FIX transgene in the plasma of animals 1 week following dosing with 50 pg of DNA-LNP, with or without anti-CDl 15 and/or clodronate.
• FIG. 5 shows depletion efficacy resulting from treatment with clodronate, anti- CDl 15, and pexidartinib.
• FIG. 5A provides results measuring CLEC4F+ cells.
• FIG. 5B provides results measuring CD68+ cells.
• FIG. 6 shows depletion efficacy resulting from treatment with pexidartinib mixed with chow.
• FIG. 6A provides results measuring CD68+ cells.
• FIG. 6B provides results measuring CLEC4F+ cells.
• FIG. 7 shows hFIX expression in mouse treated with pexidartinib and dosed with DNA-LNP-5. Mice were pretreated with pexidartinib for 7 or 21 days. hFIX levels in mouse plasma were quantified using ELISA 1, 2, 3 or 4 weeks after dosing.
• FIG. 8 shows hFIX expression in mouse treated with anti-CD-115 antibodies and dosed with DNA-LNP-5. Mice were pretreated with anti-CD-115 antibodies three times (days -5, -3, and -1) before DNA-LNP-5 dosing. hFIX levels in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing.
• “ns” denotes not significant and *P ⁇ 0.05 by t-test; and LLOQ denotes lower limit of quantitation.
• FIG. 9 shows hFIX expression in mouse treated with anti-CD-115 antibodies and dosed with DNA-LNP-3. Mice were pretreated with anti-CD-115 antibodies four times (days -10, -8, -6, and -2) before DNA-LNP-3 dosing. hFIX levels in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing.
• “ns” denotes not significant, *P ⁇ 0.05, and **P ⁇ 0.01 by t-test; and LLOQ denotes lower limit of quantitation.
• FIG. 10 shows hFIX expression in mouse treated with anti-CD-115 antibodies and dosed with DNA-LNP-4. Mice were pretreated with anti-CD-115 antibodies four times (days -10, -8, -6, and -2) before DNA-LNP-4 dosing. hFIX levels in mouse plasma were quantified using ELISA 1, 2, 4 or 12 weeks after dosing.
• “ns” denotes not significant, *P ⁇ 0.05, and ***P ⁇ 0.001 by t-test; and LLOQ denotes lower limit of quantitation.
• the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
• a dosage of about “0.01 mg/kg to about 10 mg/kg” body weight of a subject 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., and so forth.
• Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively.
• reference to more than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., and so forth.
• administration of a non-viral vector and/or a phagocyte-depleting agent “two or more” times includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more times.
• reference to trolley range such as “1 to 90” includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc., and so forth.
• “between about 1 minute to about 90 days” includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as one day, 2 days, 3 days, 4 days, 5 days .... 81 days, 82 days, 83 days, 84 days, 85 days, etc., and so forth.
• Methods to improve gene therapy comprising administration of an agent that modulates immune cells.
• Methods according to the instant invention can be used to treat a subject in need of treatment for a disease, such as one caused by a loss of function or activity of a protein, or to treat a subject in need of treatment for a disease caused by a gain of function activity or expression of a protein.
• a transgene to a subject in need thereof comprising: a. administering to the subject a phagocyte-depleting agent; and b. administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises: a. a non-viral delivery nanoparticle; and b. a non-viral vector, wherein the non-viral vector comprises the transgene operably linked to a promoter.
• vector refers to a nucleic acid molecule comprising a gene of interest.
• vectors include, but are not limited to, viral vectors delivered by viral particles or virus-like particles (VLPs) that resemble viral particles but are non-infectious, such as retroviral, adenoviral, adeno-associated viral, and lenti viral particles or VLPs; and non-viral vectors delivered by non-viral gene transfer systems, such as microinjection, electroporation, liposomes, large natural polymers, large synthetic polymers, and polymers comprised of both natural and synthetic components.
• VLPs virus-like particles
• non-viral vectors delivered by non-viral gene transfer systems such as microinjection, electroporation, liposomes, large natural polymers, large synthetic polymers, and polymers comprised of both natural and synthetic components.
• a “non-viral vector” refers to a vector that is not delivered by viral particles or by viral-like particles (VLPs).
• a non-viral vector is a vector that is not delivered by a capsid.
• the vector can be encapsulated, admixed, or otherwise associated with the non-viral delivery nanoparticle.
• a gene transfer system refers to any means of delivering a composition comprising a nucleic acid sequence (e.g., transgene) to a cell or tissue.
• a gene transfer system can be a viral gene transfer system, e.g., intact viruses, modified viruses and VLPs to facilitate delivery of a viral vector to a desired cell or tissue.
• a gene transfer system can also be a non-viral delivery system that does not comprise viral coat protein or form a viral particle or VLP, e.g., liposome-based systems, polymer-based systems, protein-based systems, metallic particle-based systems, peptide cage systems, etc.
• non-viral delivery nanoparticle can be, for example, a lipid-based nanoparticle, a polymer-based nanoparticle, a protein-based nanoparticle, a microparticle, a microcapsule, a metallic particle-based nanoparticle, a peptide cage nanoparticle, etc.
• a non-viral delivery nanoparticle of the instant invention can be constructed by any method known in the art, and a non-viral vector of the instant invention comprising the non- viral delivery nanoparticle and nucleic acid molecule comprising a therapeutic transgene can be constructed by any method known in the art.
• Lipid-based delivery systems are well known in the art, and any suitable lipid-based delivery system known to those skilled in the art in view of the present disclosure can be used in the invention.
• lipid-based delivery systems include, e.g., liposomes, lipid nanoparticles, micelles, or extracellular vesicles.
• a “lipid nanoparticle” or “LNP” refers to a lipid-based vesicle useful for delivery of nucleic acid molecules and having dimensions on the nanoscale, i.e., from about 10 nm to about 1000 nm, or from about 50 nm to about 500 nm, or from about 50 nm to about 200 nm.
• DNA is negatively charged.
• the LNP can be beneficial for the LNP to comprise a cationic lipid such as, for example, an amino lipid.
• a cationic lipid such as, for example, an amino lipid.
• Exemplary amino lipids have been described in U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651 and U.S. Patent Publication Nos.
• the LNP comprises amino lipids such as any of those described in WO2013/063468, incorporated herein in its entirety.
• cationic lipid and “amino lipid” are used interchangeably herein to include those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino group (e.g., an alkylamino or dialkylamino group).
• the cationic lipid is typically protonated (i. e. , positively charged) at a pH below the pKa of the cationic lipid and is substantially neutral at a pH above the pKa.
• the cationic lipids can also be titratable cationic lipids.
• the cationic lipids comprise: a protonatable tertiary amine (e.g., pH-titratable) group; Cl 8 alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains.
• a protonatable tertiary amine e.g., pH-titratable
• Cl 8 alkyl chains wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds
• ether, ester, or ketal linkages between the head group and alkyl chains e.g., 1, 2, or 3
• Cationic lipids can include, without limitation, l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (y-DLenDMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA, also known as DLin-C2K-DMA, XTC2, and C2K), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-Dilin
• cationic lipids also include, but are not limited to, 1 ,2-distearyloxy- N,N-dimethyl -3 -aminopropane (DSDMA), l,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(3-dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K- DMA, y-DLen-C2K-DMA, and (DLin-MP-DMA) (also known as 1-B11).
• DSDMA 1 ,2-distearyloxy- N,N-dimethyl -3 -aminopropane
• Still further cationic lipids can include, without limitation, 2,2-dilinoleyl-5- dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [l,3]-dioxolane (DLin-K-MPZ), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2- dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l-
• a number of commercial preparations of cationic lipids can be used, such as, LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE® (comprising DOSPA and DOPE, available from GIBCO/BRL).
• LIPOFECTIN® including DOTMA and DOPE, available from GIBCO/BRL
• LIPOFECTAMINE® comprising DOSPA and DOPE, available from GIBCO/BRL
• Other commercially available ionizable lipids include, e.g., SS-OP (NOF American Corporation), C12-200 (described in Kauffman et al., Nano Lett.
• cationic lipid can be present in an amount from about 10% by molar ratio of the LNP to about 85% by molar ratio of the lipid nanoparticle, or from about 50 % by molar ratio of the LNP to about 75% by molar ratio of the LNP.
• Sterols can confer fluidity to the LNP.
• “sterol” refers to any naturally occurring sterol of plant (phytosterols) or animal (zoosterols) origin as well as non-naturally occurring synthetic sterols, all of which are characterized by the presence of a hydroxyl group at the 3-position of the steroid A-ring.
• the sterol can be any sterol conventionally used in the field of liposome, lipid vesicle or lipid particle preparation, most commonly cholesterol.
• Phytosterols can include campesterol, sitosterol, and stigmasterol.
• Sterols also include sterol- modified lipids, such as those described in U.S. Patent Application Publication
• a sterol can be present in an amount from about 5% by weight of the LNP to about 50% by weight of the lipid nanoparticle or from about 10% by weight of the LNP to about 25% by weight of the LNP.
• LNP can comprise a neutral lipid.
• Neutral lipids can comprise any lipid species which exists either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, without limitation, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is generally guided by consideration of, inter aha, particle size and the requisite stability.
• the neutral lipid component can be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
• Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or can be isolated or synthesized by well-known techniques.
• lipids containing saturated fatty acids with carbon chain lengths in the range of C14 to C22 can be used.
• lipids with mono or diunsaturated fatty acids with carbon chain lengths in the range of C14 to C22 are used.
• lipids having mixtures of saturated and unsaturated fatty acid chains can be used.
• Exemplary neutral lipids include, without limitation, l,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), or any related phosphatidylcholine.
• DOPE dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine
• DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
• POPC 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine
• the neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups, such as serine and ino
• the neutral lipid can be present in an amount from about 0.1% by weight of the lipid nanoparticle 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%.
• LNP encapsulated nucleic acid molecules can be incorporated into pharmaceutical compositions, e.g., a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for, among other things, administration and delivery of LNP encapsulated nucleic acid molecules to a subject in vivo or ex vivo.
• Preparations of LNP can be combined with additional components, which can include, for example and without limitation, polyethylene glycol (PEG) and sterols.
• PEG refers to a polyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; 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. PEGs are commercially available from Sigma Chemical Co.
• PEGs monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol- succinimidyl succinate (MePEG-S -NHS), monomethoxypolyethylene glycol-amine (MePEG- NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).
• MePEG-OH monomethoxypolyethylene glycol
• MePEG-S monomethoxypolyethylene glycol-succinate
• MePEG-NHS monomethoxypolyethylene glycol- succinimidyl succinate
• MePEG-NH2 monomethoxypolyethylene glycol-amine
• MePEG-TRES monomethoxypolyethylene glycol-tresylate
• MePEG-IM mono
• PEG can be a polyethylene glycol with an average molecular weight of about 550 to about 10,000 daltons and is optionally substituted by alkyl, alkoxy, acyl or aryl. In certain embodiments, the PEG can be substituted with methyl at the terminal hydroxyl position. In certain embodiments, the PEG can have an average molecular weight from about 750 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons, or from about 1,500 to about 3,000 daltons or from about 2,000 daltons or of about 750 daltons. The PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl. In certain embodiments, the terminal hydroxyl group can be substituted with a methoxy or methyl group.
• PEG-modified lipids include the PEG-dialkyloxypropyl conjugates (PEG-DAA) described in U.S. Patent Nos. 8,936,942 and 7,803,397.
• PEG-modified lipids (or lipidpolyoxyethylene conjugates) that are useful can have a variety of “anchoring” lipid portions to secure the PEG portion to the surface of the lipid vesicle.
• suitable PEG- modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which are described in U.S. Patent No.
• the PEG-modified lipid can be PEG-modified diacylglycerols and dialkylglycerols.
• the PEG can be in an amount from about 0.5% by weight of the LNP to about 20% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.
• LNP can be a PEG-modified and/or a sterol-modified LNP.
• the LNPs, combined with additional components, can be the same or separate LNPs.
• the same LNP can be PEG modified and sterol modified or, alternatively, a first LNP can be PEG modified and a second LNP can be sterol modified.
• the first and second modified LNPs can be combined.
• prior to encapsulating LNPs can have a size in a range from about 10 nm to 500 nm, or from about 50 nm to about 200 nm, or from 75 nm to about 125 nm.
• the LNP is described by Billingsley et al., Nano Lett. 2020, 20, 1578 or International Patent Publication WO 2021/077066 (both of which are hereby incorporated by reference herein in their entirety).
• Billingsley et al., and WO 2021/077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipid and ionizable lipids.
• the LNP contains a Cl 4-4 polyamine core and/or has a particle size of about 70 nm. Cl 4-4 has the following structure.
• the LNP is made up of a cationic lipid or lipopeptide described by U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374 or WO2021/077066 (each of which is hereby incorporated by reference herein in its entirety).
• the LNP contains a cationic lipid, a cholesterol-based lipid, and/or one or more PEG-modified lipid.
• the LNP contains cKK-E12 (Dong et al., PNAS 2014, 111(11), 3955):
• the LNP comprises Lipid 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described by Sabnis et al., Molecular Therapy 2018, vol. 26, No. 6, 1509- 1519 (hereby incorporated by reference herein in its entirety).
• the LNP comprises Lipid 5, 8, 9, 10, or 11 described in Sabnis et al.
• Lipid 9 has the structure:
• Additional lipids that may be utilized include those described by Roces et al., Pharmaceutics 2020, 12,1095; Jayaraman et al; Angew. Chem. Int. Ed. 2012, 51, 8529-8533; and Maier et al., www.moleculartherapy.org Vol.21, No. 8, 1570-1578, 2013; Liu et al., Adv.
• the LNP contains one or more lipids as provided in Table 1 or a pharmaceutically acceptable salt thereof.
• the LNP comprises mol% of the following components: one or more cationic lipids from about 20% to 65%, one or more phospholipid lipids from about 1% to about 50%, one or more PEG-conjugated lipid from about 0.1 % to 10%, and cholesterol from about 0% to about 70%; one or more cationic lipids from about 20% to 50%, one or more phospholipid lipids from about 5% to about 20%, one or more PEG-conjugated lipid from about 0.1 % to 5%, and cholesterol from about 20% to about 60%; in additional embodiments the phospholipid lipid is a neutral lipid; and the phospholipid lipid is DOPE or DSPC.
• Polymer-based delivery systems are well known in the art, and any suitable polymer- based delivery system or polymeric nanoparticle known to those skilled in the art in view of the present disclosure can be used in the invention.
• DNA can be entrapped into the polymeric matrix of polymeric nanoparticles or can be adsorbed or conjugated on the surface of the nanoparticles.
• Examples of commonly used polymers for gene delivery include, e.g., poly(lactic-co-gly colic acid) (PLGA), poly lactic acid (PLA), poly(ethylene imine) (PEI), chitosan, dendrimers, polyanhydride, polycaprolactone, and poly methacrylates.
• the polymeric-based non-viral vectors can have different sizes, ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally about 100 nm to about 150 nm, optionally about 150 nm or less.
• Protein-based delivery systems are well known in the art, and any suitable proteinbased delivery system or cell-penetrating peptide (CPP) known to those skilled in the art in view of the present disclosure can be used in the invention.
• CPP cell-penetrating peptide
• CPPs are short peptides (6-30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules.
• the majority of CPPs consists mainly of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic.
• CPPs can be derived from natural biomolecules (e.g., Tat, an HIV-1 protein), or obtained by synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., DrugDeliv. 2018;25(l):1996-2006).
• CPPs include, e.g., cationic CPPs (highly positively charged) (e.g., the Tat peptide, penetratin, protamine, poly-L-lysine, polyarginine, etc.); amphipathic CPPs (chimeric or fused peptides, constructed from different sources, contain both positively and negatively charged amino acid sequences) (e.g., transportan, VT5, bactenecin-7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPPjs, TP 10, pep-1, MPG, etc.); membranotropic CPPs (exhibit both hydrophobic and amphipathic nature simultaneously, and comprise both large aromatic residues and small residues) (e.g., gH625, SPIONs-PEG-CPP NPs, etc.); and hydrophobic CPPs (contain only non-polar motifs or residues) (e.g., SG3, PFVYLI, pep
• the protein-based non-viral vectors can have different sizes, ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally about 100 nm to about 150 nm, optionally about 150 nm or less.
• Peptide cage-based delivery systems are well known in the art, and any suitable peptide cage-based delivery system known to those skilled in the art in view of the present disclosure can be used in the invention.
• any proteinaceous material that is able to be assembled into a cage-like structure, forming a constrained internal environment, can be used.
• protein “shells” can be assembled and loaded with different types of materials.
• protein cages comprising a shell of viral coat protein(s) (e.g., from the Cowpea Chlorotic Mottle Virus (CCMV) protein coat) that encapsulate a non-viral material, as well as protein cages formed from non-viral proteins have been described (see, e.g., U.S.
• CCMV Cowpea Chlorotic Mottle Virus
• Peptide cages can comprise a proteinaceous shell that self-assembles to form a protein cage (e.g., a structure with an interior cavity which is either naturally accessible to the solvent or can be made to be so by altering solvent concentration, pH, equilibria ratios).
• protein cages derived from non-viral proteins include, e.g., ferritins and apoferritins, derived from both eukaryotic and prokaryotic species, e.g., 12 and 24 subunit ferritins; and protein cages formed from heat shock proteins (HSPs), e.g., the class of 24 subunit heat shock proteins that form an internal core space, the small HSP of Methanococcus jannaschii, the dodecameric Dsp HSP of E. coli, the MrgA protein, etc.
• HSPs heat shock proteins
• the monomers of the protein cages can be naturally occurring or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments) that can be made.
• the protein cages can have different core sizes, ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally about 100 nm to about 150 nm, optionally about 150 nm or less.
• Therapeutic nucleic acids ranging from about 1 nm to about 1000 nm, optionally from about 10 nm to about 500 nm, optionally from about 50 nm to about 200 nm, optionally about 100 nm to about 150 nm, optionally about 150 nm or less.
• nucleic acid and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• Nucleic acids include genomic DNA, cDNA, antisense DNA/RNA, plasmid DNA, linear DNA, (poly- and oligo-nucleotide), chromosomal DNA, spliced or unspliced mRNA, rRNA, tRNA inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, transsplicing RNA, or antisense RNA), locked nucleic acid analogue (LNA), oligonucleotide DNA (ODN) single and double stranded, immunostimulating sequence (ISS), riboswitches and ribozymes.
• RNAi e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, transsplicing RNA, or antisense RNA
• LNA locked nucleic acid analogue
• Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single, double, or triplex, linear or circular, and can be of any length. In discussing nucleic acids, a sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5’ to 3’ direction.
• the nucleic acid agent is a single-stranded (ssDNA) or a double-stranded DNA (dsDNA) molecule.
• the nucleic acid agent is for therapeutic use, e.g. an ssDNA or dsDNA encoding a therapeutic transgene.
• the dsDNA molecule is a minicircle, a nanoplasmid, open linear duplex DNA or a closed-ended linear duplex DNA (CELiD/ceDNA/doggybone DNA).
• the ssDNA molecule is a closed circular or an open linear DNA.
• ssDNA and dsDNA molecules comprising therapeutic transgenes can be produced by conventional techniques that are well known to those of skill in the art.
• transgene is used herein to conveniently refer to a nucleic acid that is intended or has been introduced into a cell or organism.
• Transgenes include any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid encoding a protein or peptide or a nucleic acid (e.g., miRNA, etc.).
• the term transgene and heterologous nucleic acid/polynucleotide sequences are used interchangeably herein.
• “therapeutic transgene” of the instant invention comprises an expression cassette.
• expression cassette refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.
• an expression cassette comprises the nucleic acid molecule of the instant invention operatively linked to a promoter sequence.
• operatively linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
• a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter).
• Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation.
• the promoter is a heterologous promoter.
• heterologous promoter refers to a promoter that is not found to be operatively linked to a given encoding sequence in nature.
• operably linked means that regulatory sequences having an effect on the expression of a transgene are placed in the appropriate positions relative to the sequence so as to affect expression of the transgene. This same definition is sometimes applied to the arrangement of transgenes and transcription control elements (e.g., promoters, enhancers, promoter/enhancers, and termination elements) in an expression vector.
• Transgenes can be operably linked to regulatory sequences in sense or antisense orientation.
• a wide variety of regulatory sequences that can be operably linked with transgenes are known in the art and can be used in the methods of the present invention.
• the operably linked regulatory sequence is a promoter, such as a liver specific promoter, or a promoter/enhancer, such as ApoE/hAAT.
• an expression cassette can comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and/or other elements known to affect expression levels of the encoding sequence.
• promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
• the nucleic acid molecule of the instant invention is located 3’ of a promoter sequence.
• a promoter sequence consists of proximal and more distal upstream elements and can comprise an enhancer element.
• An “enhancer” is a nucleotide sequence that can stimulate promoter activity.
• Enhancer elements are typically located upstream of a promoter element but can also be located downstream or within a promoter and can, for example, be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
• an expression cassette comprises a tissue-specific enhancer.
• the promoter is derived in its entirety from a native gene.
• the promoter is composed of different elements derived from different naturally occurring promoters.
• the promoter comprises a synthetic nucleotide sequence.
• promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor.
• Ubiquitous, cell-type-specific, tissuespecific, developmental stage-specific, and conditional promoters for example, drug- responsive promoters (e.g., tetracycline-responsive promoters) are well known to those of skill in the art.
• promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron), NSE (neuronal specific enolase), synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
• PKG phosphoglycerate kinase
• CAG composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron
• NSE neurospecific en
• promoters can be of human origin or from other species, including from mice.
• Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, beta-actin promoter, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha- 1 antitrypsin (hAAT) promoter, the transthyretin promoter, the thyroxine binding globulin (TBG) promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EFl -alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in
• Operably linking the transgene to tissue specific regulatory elements provides for at least partial tissue tropism for transgene expression.
• the transgene is operably linked to a liver specific promoter and/or liver specific enhancer.
• liver specific indicates at least partial tissue tropism for transgene expression in the liver.
• promoter/enhancer indicates operably linked to an enhancer and promoter. Examples of promoters active in the liver include, without limitation, the transthyretin (TTR) gene promoter and mutant versions thereof (Anguela and Shen, US Patent No. 11,168,124, Anguela and Shen, International Publication No.
• TTR transthyretin
• telomeres apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem, 1997, 272:29113-19).
• the promoter/enhancer is ApoE/hAAT (Okuyama et al., 1996, Human Gene Therapy 7:637-645, and Miao et al., 2000, Molecular Therapy Vol. 1, No. 6). (Each of the references in the present paragraph are hereby incorporated by reference herein.)
• the expression cassette comprises an appropriate secretory signal sequence that will allow the secretion of the polypeptide encoded by the nucleic acid molecule of the instant invention.
• secretory signal sequence or variations thereof are intended to refer to amino acid sequences that function to enhance (as defined above) secretion of an operably linked polypeptide from the cell as compared with the level of secretion seen with the native polypeptide.
• enhanced secretion it is meant that the relative proportion of the polypeptide synthesized by the cell that is secreted from the cell is increased; it is not necessary that the absolute amount of secreted protein is also increased. In certain embodiments, essentially all (i.e.
• secretory signal sequences are cleaved within the endoplasmic reticulum and, in certain embodiments, the secretory signal sequence is cleaved prior to secretion. It is not necessary, however, that the secretory signal sequence is cleaved as long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional. Thus, in certain embodiments, the secretory signal sequence is partially or entirely retained.
• the secretory signal sequence can be derived in whole or in part from the secretory signal of a secreted polypeptide (i.e., from the precursor) and/or can be in whole or in part synthetic.
• the length of the secretory signal sequence is not critical; generally, known secretory signal sequences are from about 10-15 to 50-60 amino acids in length.
• known secretory signals from secreted polypeptides can be altered or modified (e.g., by substitution, deletion, truncation or insertion of amino acids) as long as the resulting secretory signal sequence functions to enhance secretion of an operably linked polypeptide.
• the secretory signal sequences of the instant invention can comprise, consist essentially of or consist of a naturally occurring secretory signal sequence or a modification thereof (as described above). Numerous secreted proteins and sequences that direct secretion from the cell are known in the art.
• the secretory signal sequence of the instant invention can further be in whole or in part synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art, see, e.g., Barash et al., Biochem. Biophys. Res. Comm. 294:835-42 (2002).
• the instant invention includes a method of treating a disease by gene therapy using a therapeutic transgene.
• a heterologous polynucleotide encodes GAA (acid alphaglucosidase) for treatment of Pompe disease or another glycogen storage disease; ATP7B (copper transporting ATPase2) for treatment of Wilson’s disease; alpha galactosidase A for treatment of Fabry’s disease; ASS1 (arginosuccinate synthase) for treatment of Citrullinemia Type 1; beta-glucocerebrosidase for treatment of Gaucher disease Type 1; betahexosaminidase A for treatment of Tay Sachs disease; SERPING1 (Cl protease inhibitor or Cl esterase inhibitor) for treatment of hereditary angioedema (HAE), also known as Cl inhibitor deficiency type I and type II); or glucose-6-phosphatase for treatment of glycogen storage disease type I (GSDI).
• GAA acid alphaglucosidase
• ATP7B copper transporting ATPase2
• Wilson alpha galactos
• a heterologous polynucleotide encodes 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), angiopoietins, 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 a (TGFa), platelet- derived growth factor (PDGF), insulin growth factors I or II (IGF-I or IGF-II), TGFP, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain- derived neurotrophic factor
• a heterologous polynucleotide thrombopoietin TPO
• an interleukin IL-1 through IL-36, etc.
• monocyte chemoattractant protein monocyte chemoattractant protein
• leukemia inhibitory factor granulocyte-macrophage colony stimulating factor
• Fas ligand tumor necrosis factors a or P
• interferons a a tumor necrosis factors a or P
• interferons a or y
• stem cell factor flk-2/flt3 ligand
• IgG, IgM, IgA, IgD or IgE chimeric immunoglobulins
• humanized antibodies single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I or class II MHC molecules.
• a heterologous polynucleotide encodes CFTR (cystic fibrosis transmembrane regulator protein), a blood coagulation (clotting) factor (Factor XIII, Factor IX (FIX), Factor VIII (FVIII), Factor X, Factor VII, Factor Vila, protein C, etc.) a gain of function blood coagulation factor, an antibody, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, P-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphorib
• CFTR cy
• a heterologous polynucleotide encodes erythropoietin (EPO) for treatment of anemia; interferon-alpha, interferon-beta, and interferon-gamma for treatment of various immune disorders, viral infections and cancer; an interleukin (IL), including any one of IL-1 through IL-36, and corresponding receptors, for treatment of various inflammatory diseases or immuno-deficiencies; a chemokine, including chemokine (C-X-C motif) ligand 5 (CXCL5) for treatment of immune disorders; granulocyte-colony stimulating factor (G-CSF) for treatment of immune disorders such as Crohn’s disease; granulocyte-macrophage colony stimulating factor (GM-CSF) for treatment of various human inflammatory diseases; macrophage colony stimulating factor (M-CSF) for treatment of various human inflammatory diseases; keratinocyte growth factor (KGF) for treatment of epithelial tissue damage;
• EPO erythrop
• the protein encoded by the heterologous polynucleotide comprises a gene editing nuclease.
• the gene editing nuclease comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN).
• the gene editing nuclease comprises a functional Type II CRISPR-Cas9.
• polypeptides proteins and peptides
• polypeptides include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full-length protein.
• polypeptides, proteins, and peptides encoded by the polynucleotide sequences can be, but are not required to be, identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.
• the heterologous polynucleotide encodes an inhibitory nucleic acid selected from the group consisting of a siRNA, an antisense molecule, miRNA, RNAi, a ribozyme and an shRNA.
• an inhibitory nucleic acid binds to a gene, a transcript of a gene, or a transcript of a gene associated with a polynucleotide repeat disease selected from the group consisting of a huntingtin (HTT) gene, a gene associated with dentatorubropallidoluysian atrophy (atrophin 1, ATN1), androgen receptor on the X chromosome in spinobulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P/Q voltage-dependent calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 opposite strand (ATXN8OS), Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMRI (fragile X mental retardation 1) in fragile X syndrome, FMRI (fragile X mental retardation 1) in fragile X syndrome
• the instant invention relates to a method of producing a therapeutically effective non-viral gene therapy in a subject comprising administration to the subject at least one immune cell modulator, e.g., at least one phagocyte-depleting agent, or at least one immunosuppressant.
• at least one immune cell modulator e.g., at least one phagocyte-depleting agent, or at least one immunosuppressant.
• phagocyte-depleting agent refers to any agent that depletes or destroys phagocytes in a subject and/or interferes with one or more phagocyte functions.
• the phagocyte-depleting agents can target any phagocytes.
• Phagocytes also referred to herein as phagocytic cells, phagocytic immune cells, phagocyte cells, or phagocyte immune cells, include, e.g., macrophages, monocytes, neutrophils, and dendritic cells. Langerhans cells are dendritic cells found in the skin. Mast cells, found in many tissues, including, e.g., lung or skin, can also act as phagocytes.
• monocyte and/or macrophage-depleting agent refers to any agent that depletes or destroys monocytes and/or macrophages in a subject and/or interferes with one or more monocyte and/or macrophage functions.
• the monocyte and/or macrophagedepleting agents can target any monocytes and/or macrophages. Macrophages are mononuclear phagocytes that are differentiated monocytes. In different tissues, macrophages are referred to by different names.
• tissue-specific, or resident, macrophages include, e.g., Kupffer cells in the liver, intestinal macrophages in the gut, microglial cells in the brain, alveolar macrophages in the lung, resident kidney macrophages, skin macrophages, red pulp macrophages in the spleen, and osteoclasts in bone.
• monocyte- and/or macrophage-depleting agents include, e.g., agents that target phagocytic immune cell markers, e.g., CD115 inhibiting agents, including but not limited to anti-CDl 15 antibodies or CD115 small molecule inhibitors; F4/80 inhibiting agents, including but not limited to anti- F4/80 antibodies or F4/80 small molecule inhibitors; CD68 inhibiting agents, including but not limited to anti-CD68 antibodies or CD68 small molecule inhibitors; CD1 lb inhibiting agents, including but not limited to anti-CDl lb antibodies or CD1 lb small molecule inhibitors; the chemotherapeutic agent Trabectedin; intralipids; empty liposomes; and bisphosphonates, including but not limited to clodronate.
• CD115 inhibiting agents including but not limited to anti-CDl 15 antibodies or CD115 small molecule inhibitors
• F4/80 inhibiting agents including but not limited to anti- F4/80 antibodies or F4/80 small molecule inhibitors
• 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 in a method of the invention.
• neutrophil-depleting agent refers to any agent that depletes or destroys neutrophils in a subject and/or interferes with one or more neutrophil functions. The neutrophil-depleting agents can target any neutrophils.
• neutrophil-depleting agents include, e.g., agents that target phagocytic immune cell markers, e.g., Ly6G inhibiting agents, including but not limited to anti-Ly6G antibodies or Ly6G small molecule inhibitors; CD177 inhibiting agents, including but not limited to anti-CD177 antibodies or CD177 small molecule inhibitors; CD14 inhibiting agents, including but not limited to anti-CD14 antibodies or CD14 small molecule inhibitors; CD15 inhibiting agents, including but not limited to anti-CDl 5 antibodies or CD 15 small molecule inhibitors; CDllb inhibiting agents, including but not limited to anti-CDl lb antibodies or CD1 lb small molecule inhibitors; CD 16 inhibiting agents, including but not limited to anti-CDl 6 antibodies or CD 16 small molecule inhibitors; CD32 inhibiting agents, including but not limited to anti-CD32 antibodies or CD32 small molecule inhibitors; CD33 inhibiting agents, including but not limited to anti-CD33 antibodies or CD33 small molecule inhibitors; CD44 inhibiting agents,
• dendritic cell-depleting agent refers to any agent that depletes or destroys dendritic cells in a subject and/or interferes with one or more dendrite functions.
• the dendritic cell-depleting agents can target any dendritic cell.
• dendritic cell-depleting agents include, e.g., agents that target phagocytic immune cell markers, e.g., PDCA1 inhibiting agents, including but not limited to anti-PDCAl antibodies or PDCA1 small molecule inhibitors; and CDllc inhibiting agents, including but not limited to anti-CDl 1c antibodies or CD11c small molecule inhibitors.
• the term “inhibiting agent” refers to any compound capable of downregulating, decreasing, reducing, suppressing, or inactivating the amount and/or activity of the targeted protein.
• Inhibiting agents can be proteins, oligo- and polypeptides, nucleic acids, genes, or chemical molecules.
• Suitable protein inhibitors can be, for example, monoclonal or polyclonal antibodies which bind to the targeted protein.
• Any suitable CD115 inhibiting agent, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• CD115 small molecule inhibitors include, e.g., pexidartinib (PLX-3397), BLZ-945, Linifanib (ABT-869), JNJ-28312141 (Johnson & Johnson), JNJ-40346527 (Johnson & Johnson), PLX7486 (Plexxikon), and ARRY-382 (Array BioPharma).
• any suitable anti-CD115 antibody including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• commercial anti- CD115 antibodies include, e.g., AFS98 (Invitrogen or BioCell), 12-3A3-1B10 (Invitrogen), 6C7 (Bioss), cabiralizumab (FPA008), 25949-1-AP (Proteintech), 1G4 (Abnova), 3G12 (Abnova), 604B5 2E11 (Invitrogen), emactuzumab (RG-7155; Roche), AMG 820 (Amgen), IMC-CS4, and ROS8G11 (Invitrogen).
• the antibody or antigenbinding fragment thereof is AFS98 (e.g., BioCell BE0213), see also Oncogene. 1995;l l(12):2469-2476.
• Ly6G inhibiting agent including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• any suitable anti-Ly6G antibody including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Examples of commercial anti- Ly6G antibodies include, e.g., 1A8 (BioCell BP0075-1) and RB6-8C5 (ab25377).
• any suitable anti-CD177 antibodies or CD177 small molecule inhibitors can be used in the invention.
• Any suitable anti-CD14 antibodies or CD 14 small molecule inhibitors including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CD15 antibodies or CD 15 small molecule inhibitors including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CDl lb antibodies or CDllb small molecule inhibitors, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CD16 antibodies or CD 16 small molecule inhibitors, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CD32 antibodies or CD32 small molecule inhibitors, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CD33 antibodies or CD33 small molecule inhibitors, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• Any suitable anti-CD44 antibodies or CD44 small molecule inhibitors, including those known to those skilled in the art, in view of the present disclosure can be used in the invention.
• any suitable anti-CD45 antibodies or CD45 small molecule inhibitors can be used in the invention.
• Any suitable anti-CD66b antibodies or CD66b small molecule inhibitors can be used in the invention.
• Any suitable anti-CD18 antibodies or CD 18 small molecule inhibitors can be used in the invention.
• Any suitable anti-CD62L antibodies or CD62L small molecule inhibitors can be used in the invention.
• 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 10;20(6):4264-4269.
• Pretreatment with intralipid or empty liposomes can effectively saturate monocyte/macrophage cells and prevent phagocytosis of a non-viral therapeutic agent.
• Any suitable intralipid or empty liposome known to those skilled in the art in view of the present disclosure can be used in the invention.
• intralipids and empty liposomes examples include, e.g., 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.
• any suitable bisphosphonate known to those skilled in the art in view of the present disclosure can be used in the invention.
• bisphosphonates include, e.g., clodronate, pamidronate, ibandronate, alendronate, and zoledronate.
• phagocyte-depleting agent examples include, for example, palbociclib (Ibrance®; Pfizer), cromolyn sodium (Nasalcrom®; Bausch & Lomb), which are known to inhibit mast cells.
• immunosuppressant refers to any compound capable of slowing or halting immune system activity in a subject.
• immunosuppressants include, but are not limited to, a calcineurin inhibitor, such as, but not limited to, cyclosporine, ISA(TX) 247, tacrolimus or calcineurin, a target of rapamycin, such as, but not limited to, sirolimus, everolimus, FK778 or TAFA-93, an interleukin-2 a-chain blocker, such as, but not limited to, basiliximab and daclizumab, an inhibitor of inosine monophosphate dehydrogenase, such as my cophenolate mofetil, an inhibitor of dihydrofolic acid reductase, such as, but not limited to, methotrexate, an immunosuppressive antimetabolite, such as, but not limited to, azathioprine, a JAK
• the term “steroid” is used to encompass both corticosteroids and glucocorticosteroids.
• the steroid can be a corticosteroid. Any suitable corticosteroid known to those skilled in the art in view of the present disclosure can be used in the invention.
• the term “steroid” refers to a chemical substance comprising three cyclohexane rings and a cyclopentane ring. The rings are arranged to form tetracyclic cyclopentaphenanthrene, i.e., gonane.
• corticosteroid refers to a class of steroid hormones that are produced in the adrenal cortex or produced synthetically. Corticosteroids are involved in a wide range of physiologic systems such as stress response, immune response and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids are generally grouped into four classes, based on 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-val erate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol- 17- propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate.
• corticosteroids include, aldostemone, beclomethasone, beclomethasone dipropionate, betametahasone, betametahasone-21 -phosphate disodium, betametahasone valerate, budesonide, clobetasol, clobetasol propionate, clobetasone butyrate, clocortolone pivalate, cortisol, cortisteron, cortisone, deflazacort, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, dihydroxy cortison, flucinonide, fludrocortisones acetate, flumethasone, flunisolide, flucionolone acetonide, fluticasone furate, fluticasone propionate, halcinonide, halpmetasone, hydrocortisone, hydroconrtisone
• corticosteroid can include, but is not limited to, the following generic and brand name corticosteroids: cortisone (CORTONETM ACETATETM, ADRESONTM, ALTESONATM, CORTELANTTM, CORTISTABTM, CORTISYLTM, CORTOGENTM, CORTONETM, SCHEROSONTM); dexamethasone-oral (DECADRON ORALTM, DEXAMETHTM, DEXONETM, HEXADROL-ORALTM, DEXAMETHASONETM INTENSOLTM, DEXONE 0.5TM, DEXONE 0.75TM, DEXONE 1.5TM, DEXONE 4TM); hydrocortisone-oral (CORTEFTM, HYDROCORTONETM); hydrocortisone cypionate (CORTEF ORAL SUSPENSIONTM); methylprednisolone-oral (MEDROL-ORALTM); prednisolone-oral (PRELONETM, DELTA
• a corticosteroid can be dexamethasone, prednisone, prednisolone, triamcinolone, clobetasol propionate, betamethasone valerate, betamethasone dipropionate, or mometasone furoate.
• Methods of synthesizing steroids and corticosteroids are well known in the art and such compounds are also commercially available, e.g. dexamethasone (Cat. No. D4902, Sigma- Aldrich; St. Louis, Mo.) and prednisone (Cat. No. P6254, Sigma-Aldrich; St. Louis, Mo.).
• a corticosteroid e.g., dexamethasone
• a corticosteroid e.g., dexamethasone
• the instant invention relates to a therapeutic composition comprising a phagocyte-depleting agent, according to the instant invention, for use in the treatment of a disease treated by gene therapy using a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier, according to the instant invention, in a patient in need thereof.
• Any therapeutic agent or pharmaceutical composition of the instant invention can be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
• “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
• compositions of the instant invention can be formulated for topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
• compositions of the instant invention can contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These can be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
• Reference to pharmaceutically acceptable salt indicate the salt at the provided amount is suitable for administration to a mammal, preferable a human.
• compositions of the instant invention can comprise a further therapeutically active agent.
• a pharmaceutical composition comprising a non-viral delivery nanoparticle and a non-viral vector comprising a therapeutic transgene, operably linked to a promoter, can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.01 mg/ml to about 10 mg/ml of the non-viral vector per kg body weight of a subject.
• An antibody that targets a phagocytic immune cell marker can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.01 mg/kg to about 5 mg/kg body weight of a subject, wherein the dosage is administered in 1 to 10 total injections.
• An anti-CD115 antibody can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.01 mg/kg to about 5 mg/kg body weight of a subject, wherein the dosage is administered in 1 to 10 total injections.
• the dose can be from about 0.01 mg/kg to about 0. 1 mg/kg, about 0.
• the dose is less than about any of the following doses (in mg/kg): 5, 4.9, 4.8,
• the dose is greater than about any of the following doses (in mg/kg): 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,
• the dose can be any of a range of doses (in mg/ml) having an upper limit of 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9,
• An anti-Ly6G, anti-CD177, anti-CD14, anti-CD15 , anti-CDl lb, anti-CD16, anti- CD32, anti-CD33, anti-CD44, anti-CD45, anti-CD66b, anti-CD18, or anti-CD62L antibody can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.01 mg/kg to about 5 mg/kg body weight of a subject, wherein the dosage is administered in 1 to 10 total injections.
• the dose can be from about 0.01 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg, about 0.3 mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 0.6 mg/kg, about 0.6 mg/kg to about 0.7 mg/kg, about 0.7 mg/kg to about 0.8 mg/kg, about 0.8 mg/kg to about 0.9 mg/kg, about 0.9 mg/kg to about 1 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 3 mg/kg to about 3.5 mg/kg, about 3.5 mg/kg to about 4 mg/kg, about 4 mg/kg to about 4.5 mg/kg, about 4.5 mg/kg to about 5 mg/kg.
• the dose is less than about any of the following doses (in mg/kg): 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, or 0.02.
• the dose is greater than about any of the following doses (in mg/kg): 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,
• the dose can be any of a range of doses (in mg/ml) having an upper limit of 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4,
• a small molecule inhibitor that targets a phagocytic immune cell marker can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.1 mg/kg to about 15 mg/kg body weight of a subject.
• the dose can be from about 0.1 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, about 14 mg/kg to about 15 mg/kg.
• the dose is less than about any of the following doses (in mg/kg): 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2. In certain embodiments, the dose is greater than about any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. That is, the dose can be any of a range of doses (in mg/ml) having an upper limit of 15, 14, 13, 12, 11,
• a CD115 inhibitor such as pexidartinib can be administered to a subject at any suitable dose.
• a suitable dosage can be from about 0.1 mg/kg to about 15 mg/g body weight of a subject. In certain embodiments, the dose can be from about 0.
• 1 mg/kg to about 1 mg/kg about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, about 14 mg/kg to about 15 mg/kg.
• the dose is less than about any of the following doses (in mg/kg): 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2. In certain embodiments, the dose is greater than about any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
• the dose can be any of a range of doses (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, wherein the lower limit is less than the upper limit.
• a bisphosphonate e.g., clodronate
• a suitable dosage can be from about 0.
• the dose can be from about 0.1 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, about 14 mg/kg to about 15 mg/kg.
• the dose is less than about any of the following doses (in mg/kg): 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2. In certain embodiments, the dose is greater than about any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
• the dose can be any of a range of doses (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, wherein the lower limit is less than the upper limit.
• a corticosteroid e.g., dexamethasone
• a suitable dosage can be from about 0.
• the dose can be from about 0.1 mg/kg to about 1 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 9 mg/kg, about 9 mg/kg to about 10 mg/kg, about 10 mg/kg to about 11 mg/kg, about 11 mg/kg to about 12 mg/kg, about 12 mg/kg to about 13 mg/kg, about 13 mg/kg to about 14 mg/kg, about 14 mg/kg to about 15 mg/kg.
• the dose is less than about any of the following doses (in mg/kg): 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2. In certain embodiments, the dose is greater than about any of the following doses (in mg/kg): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
• the dose can be any of a range of doses (in mg/ml) having an upper limit of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2, and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, wherein the lower limit is less than the upper limit.
• the instant invention relates to methods of delivering a transgene to a subject in need thereof comprising (a) administering to the subject a monocyte- and/or macrophage-depleting agent; and (b) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier.
• a disease treated by gene therapy using a non-viral vector denotes a disease wherein a polynucleotide (encoding at least one polypeptide or inhibitory nucleic acid) is delivered into the cell(s) of a patient as a drug to treat said disease (gene therapy).
• the vector encodes at least one specific polypeptide or inhibitory nucleic acid useful to treat a disease.
• the vector encodes/comprises a therapeutic polynucleotide appropriate for treating a disease.
• the vector encodes/comprises a therapeutic polynucleotide appropriate for treating a disease using gene therapy.
• the terms “patient” and “subject” interchangeably refer to an animal, typically a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject or patient according to the instant invention is a human.
• treatment refers to both prophylactic or preventive treatment like gene therapy as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
• the treatment can be administered to a subject having a medical disorder or who ultimately can acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
• a method can comprise first (a) administering to a subject a phagocyte-depleting agent, and then (b) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier. In certain embodiments, (b) administering to the subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier, is performed between about 1 minute to about 1 year after (a) administering to the subject a phagocyte-depleting agent.
• a method can comprise first (a) administering to a subject a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier, and then (b) administering to the subject a phagocytedepleting agent.
• (b) administering to the subject a phagocytedepleting agent is performed between about 1 minute to about 1 year after (a) administering to a subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier. In certain embodiments, (a) administering to a subject a pharmaceutical composition comprising a non-viral vector comprising the transgene and a pharmaceutically acceptable carrier, and (b) administering to the subject a phagocyte-depleting agent, are performed at about the same time.
• the phagocyte-depleting agent is administered between about one minute to about 1 hour, 2 hours, 3 hours, or 4 hours before the non-viral vector comprising the transgene and a pharmaceutically acceptable carrier is administered.
• the non-viral vector comprising the transgene and a pharmaceutically acceptable carrier is administered between about one minute to about 1 hour, 2 hours, 3 hours, or 4 hours before the phagocyte-depleting agent is administered.
• the phagocyte-depleting agent is administered at least one day before the non-viral vector is administered, optionally no more than 1 year before the non-viral vector is administered, such as no more than 52 weeks, 51 weeks, 50 weeks, 49 weeks, 48 weeks, 47 weeks, 46 weeks, 45 weeks, 44 weeks, 43 weeks, 42 weeks, 41 weeks,
• a non-viral vector comprising a heterologous polynucleotide can be administered to a subject any number of times.
• a non-viral vector comprising a heterologous polynucleotide can be administered once, 2 to 15 times to a subject.
• the non-viral vector is administered multiple times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.
• the spacing between the times can vary, for example, every 1 hour, 6 hours, 24 hours, 1-7 days, or 2-10 weeks; or a combination thereof.
• a non-viral vector comprising a heterologous polynucleotide can be administered to a subject for any duration of time on a regular basis, such as consecutive days, or alternating days, or an irregular basis.
• a non-viral vector comprising a heterologous polynucleotide is administered from about 1 to 52 weeks, after administration of an immune cell modulator.
• a phagocyte-depleting agent can be administered to a subject any number of times.
• a phagocyte-depleting agent can be administered once, 2 to 5 times, 2 to 10 times, 2 to 15 times to a subject, per each administration of a non-viral vector comprising a heterologous polynucleotide.
• An anti-CD115 antibody can be administered to a subject any number of times.
• an anti-CD115 antibody can be administered once, 2 to 5 times, 2 to 10 times, 2 to 15 times to a subject, per each administration of a non-viral vector comprising a heterologous polynucleotide.
• An anti-CD115 antibody can be administered to a subject for any duration of time on a regular basis, such as consecutive days, or alternating days, or an irregular basis. In certain embodiments, an anti-CD115 antibody is administered from about 1 to 52 weeks before administration of a non-viral vector.
• a small molecule CD115 inhibitor can be administered to a subject any number of times.
• a small molecule CD115 inhibitor can be administered once, 2 to 5 times, 2 to 10 times, 2 to 15 times to a subject, per each administration of a non-viral vector comprising a heterologous polynucleotide.
• a small molecule CD115 inhibitor can be administered to a subject for any duration of time on a regular basis, such as consecutive days, or alternating days, or an irregular basis.
• a small molecule CD115 inhibitor is administered from about 1 to 52 weeks before administration of a non-viral vector.
• An anti-Ly6G, anti-CD177, anti-CD14, anti-CD15 , anti-CDl lb, anti-CD16, anti- CD32, anti-CD33, anti-CD44, anti-CD45, anti-CD66b, anti-CD18, or anti-CD62L antibody can be administered to a subject any number of times.
• an anti-Ly6G, anti- CD177, anti-CD14, anti-CD15 , anti-CDllb, anti-CD16, anti-CD32, anti-CD33, anti-CD44, anti-CD45, anti-CD66b, anti-CD18, or anti-CD62L antibody can be administered once, 2 to 5 times, 2 to 10 times, 2 to 15 times to a subject, per each administration of a non-viral vector comprising a heterologous polynucleotide.
• An anti-Ly6G, anti-CD177, anti-CD14, anti-CD15 , anti-CDl lb, anti-CD16, anti- CD32, anti-CD33, anti-CD44, anti-CD45, anti-CD66b, anti-CD18, or anti-CD62L antibody can be administered to a subject for any duration of time on a regular basis, such as consecutive days, or alternating days, or an irregular basis.
• an anti- Ly6G, anti-CD177, anti-CD14, anti-CD15 , anti-CDl lb, anti-CD16, anti-CD32, anti-CD33, anti-CD44, anti-CD45, anti-CD66b, anti-CD18, or anti-CD62L antibody is administered from about 1 to 52 weeks before administration of a non-viral vector.
• a bisphosphonate e.g., clodronate
• a bisphosphonate can be administered once, 2 to 5 times, 2 to 10 times, 2 to 15 times to a subject, per each administration of a non-viral vector comprising a heterologous polynucleotide.
• a bisphosphonate e.g., clodronate
• a bisphosphonate, e.g., clodronate is administered from about 1 to 52 weeks before administration of a non-viral vector.
• a corticosteroid e.g., dexamethasone
• a corticosteroid e.g., dexamethasone
• a corticosteroid e.g., dexamethasone
• a corticosteroid e.g., dexamethasone
• the non-viral vector comprising a heterologous polynucleotide can be administered alone or in a combination.
• the non-viral vector is administered to a subject separately from the at least one immune cell modulator, e.g. phagocyte-depleting agent, bisphosphonate; and/or immunosuppressant.
• the non-viral vector is administered to a subject in combination with the at least one immune cell modulator, e.g. phagocyte-depleting agent, bisphosphonate, and/or immunosuppressant.
• the bisphosphonate and/or immunosuppressant is included within the non-viral delivery particle, e.g., is encapsulated within a lipid nanoparticle, a polymer nanoparticle, a protein-based nanoparticle, or a peptide cage, along with the non-viral vector.
• a mixture of at least one immune cell modulator e.g. phagocyte-depleting agent, bisphosphonate, and/or immunosuppressant, is administered to a subject, one or more times. In certain embodiments, two or more immune cell modulators are administered to a subject, one or more times.
• administration of at least one immune cell modulator to a subject reduces the dose of a non-viral vector comprising a heterologous polynucleotide required to be effective for treatment of a subject. In certain embodiments, administration of at least one immune cell modulator to a subject allows for administration of an increased dose of a non-viral vector comprising a heterologous polynucleotide.
• Doses can vary and depend upon the type, onset, progression, severity, frequency, duration, or probability of the disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan.
• the dose amount, number, frequency or duration can be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that can influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
• the dose to achieve a therapeutic effect e.g., the dose in dsDNA mg per kilogram of body weight (mg/kg) will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the non-viral vector, any host immune response to the heterologous polynucleotide or expression product, and the stability of the protein, peptide, or nucleic acid expressed.
• route of administration e.g., the level of heterologous polynucleotide expression required to achieve a therapeutic effect
• the specific disease treated any host immune response to the non-viral vector, any host immune response to the heterologous polynucleotide or expression product, and the stability of the protein, peptide, or nucleic acid expressed.
• One skilled in the art can determine a non-viral vector genome dose range to treat a patient having a particular disease or disorder based on the aforementioned
• an “effective amount” or “sufficient amount” refers to an amount that provides, in single or multiple doses, alone or in combination, with one or more other compositions, treatments, protocols, or therapeutic regimens agents, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).
• an “effective amount” or “sufficient amount” for treatment typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is a satisfactory outcome.
• the methods of the instant invention produce a therapeutically effective non-viral gene therapy in a subject in need thereof, wherein the therapeutically effective non-viral gene therapy is characterized by expression of the therapeutic protein or nucleic acid and/or minimal or absent undesirable immune response induced by the non-viral vector.
• “expression of the therapeutic protein or nucleic acid” refers to an adequate level of protein or nucleic acid to result in a therapeutic effect, as discussed herein.
• a method according to the instant invention results in expression of the trans gene in the subject.
• a method according to the instant invention can result in expression or activity of a therapeutic protein at a therapeutically effective level.
• a method according to the instant invention can result in expression or activity of a therapeutic protein at a level that 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%, at least 100% of normal expression of the heterologous protein.
• a method according to the instant invention can result in reduction of expression or activity of a protein targeted by a therapeutic nucleic acid.
• a method according to the instant invention can result in reduction of expression or activity of a protein targeted by a therapeutic nucleic acid by 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%, at least 100% of normal expression of the target protein.
• Non-limiting examples of a biological sample from a subject that can be analyzed include whole blood, serum, plasma, the like, and a combination thereof.
• a biological sample can be devoid of cells, or can include cells (e.g., red blood cells, platelets and/or lymphocytes).
• Any suitable method for measuring expression of a therapeutic protein or nucleic acid known to those skilled in the art in view of the present disclosure can be used in the invention.
• Exemplary methods for analyzing and measuring heterologous protein or nucleic acid expression levels in a biological sample include, e.g., ELISA.
• immune response refers to a response by the immune system of a subject.
• immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) 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 generation and/or secretion).
• lymphokine e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine
• macrophage activation e.g., dendritic cell activation
• T cell activation e.g., CD4+ or CD8+ T cells
• NK cell activation e.g., antibody generation and/or secreti
• immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte (“CTL”) response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells.
• an immunogen e.g., antigen (e.g., immunogenic polypeptide)
• CTL cytotoxic T lymphocyte
• B cell response e.g., antibody production
• T-helper lymphocyte response e.g., T-helper lymphocyte response
• DTH delayed type
• an immune response can be to immunogens that the subject’s immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign).
• immunogens that the subject’s immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign).
• immune response refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) cell- mediated immune responses (e.g., responses mediated by T cells (e.g., antigen- specific T cells) and non-specific cells of the immune system) and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
• innate immune responses e.g., activation of To
• minimal or absent undesirable immune response induced by the non-viral vector refers to a safe or therapeutically tolerable immune response within a human subject.
• the undesirable immune response induced by the vector is distinct from a desirable immune response in the subject that is induced by an antigen encoded by a non-viral vector.
• the safe or therapeutically tolerable immune response relates to cytokine release levels in a subject upon administration of the non-viral vector comprising the heterologous polynucleotide.
• methods described herein decrease incidence of toxic cytokine release or “cytokine release syndrome” (CRS) or “severe cytokine release syndrome” (sCRS) or “cytokine storm” that can occur in a subject.
• CRS cytokine release syndrome
• sCRS severe cytokine release syndrome
• decreasing toxic cytokine release or toxic cytokine levels comprises decreasing or inhibiting production of toxic cytokine levels in a subject, or inhibiting or reducing the incidence of cytokine release syndrome or a cytokine storm in a subject.
• the toxic cytokines comprise pro-inflammatory cytokines.
• pro-inflammatory cytokines comprise IL-6, IFN-y, IL-ip, or TNF-a, or any combination thereof.
• cytokine release syndrome is characterized by elevated levels of several inflammatory cytokines and adverse physical reactions in a subject such as low blood pressure, high fever and shivering.
• CRS is characterized by elevated levels of IL-6, IFN-y, IL-ip, or TNF-a, or any combination thereof.
• measurement of cytokine levels or concentration, as an indicator of cytokine storm can be expressed as fold increase, percent (%) increase, net increase or rate of change in cytokine levels or concentration.
• absolute cytokine levels or concentrations above a certain level or concentration can be an indication of a subject undergoing or about to experience a cytokine storm.
• absolute cytokine levels or concentration at a certain level or concentration for example a level or concentration normally found in a control subject not undergoing non- viral gene therapy, can be an indication of a method for inhibiting or reducing the incidence of a cytokine storm in a subject undergoing non- viral gene therapy.
• cytokine level can encompass a measure of concentration, a measure of fold change, a measure of percent (%) change, or a measure of rate change. Further, the methods for measuring cytokines in blood, saliva, serum, urine, and plasma are well known in the art.
• INF-y levels can be used as a common measure of cytokine storm and/or as a common measure of the effectiveness of a treatment for cytokine storms.
• IL-6 levels can be used as a common measure of cytokine storm and/or as a common measure of the effectiveness of a treatment for cytokine storms.
• Levels of cytokines in a subject can be analyzed, measured or determined before and/or after administration of the non-viral vector comprising a heterologous polynucleotide.
• Levels of cytokines in a subject can also be analyzed, measured or determined before and/or after the administration of the immune cell modulator.
• Levels of cytokines in a subject can also be analyzed or measured multiple times, before and/or after administration of the non- viral vector comprising a heterologous polynucleotide as well as before and/or after administration of the immune cell modulator.
• cytokine levels in a biological sample include, e.g., mesoscale delivery platform (MSD).
• MSD mesoscale delivery platform
• An effective amount or a sufficient amount can but need not be provided in a single administration, can require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen.
• another composition e.g., agent
• the amount can be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment.
• an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens can be included in order to be considered effective or sufficient in a given subject.
• a second composition e.g., another drug or agent
• additional doses, amounts or duration above and beyond such doses, or additional compositions e.g., drugs or agents
• treatments, protocols or therapeutic regimens can be included in order to be considered effective or sufficient in a given subject.
• Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol, such as administration of recombinant GAA for treatment of a lysosomal storage disease (e.g., Pompe disease), or administration of a recombinant clotting factor protein (e.g., FVIII or FIX) for treatment of a clotting disorder (e.g., hemophilia A (HemA) or hemophilia B (HemB)).
• a lysosomal storage disease e.g., Pompe disease
• a recombinant clotting factor protein e.g., FVIII or FIX
• a clotting disorder e.g., hemophilia A (HemA) or hemophilia B (HemB)
• an effective amount would be an amount of GAA that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, reduces lysosomal alterations in tissues of the body of a subject, or improves muscle tone and/or muscle strength and/or respiratory function in a subject, for example.
• Effective amounts can be determined, for example, by ascertaining the kinetics of GAA uptake by myoblasts from plasma. Myoblasts GAA uptake rates (K uptake) of about 141 - 147 nM can appear to be effective (see, e.g., Maga et al., J. Biol. Chem. 2012)
• GAA activity levels in plasma of greater than about 1,000 nmol/hr/mL, for example, about 1,000 to about 2,000 nmol/hr/mL have been observed to be therapeutically effective.
• a blood coagulation factor concentration that is greater than 1% of factor concentration found in a normal individual is needed to change a severe disease phenotype to a moderate one.
• a severe phenotype is characterized by joint damage and life-threatening bleeds.
• a blood coagulation factor concentration greater than 5% of normal is needed.
• FVIII and FIX levels in normal humans are about 150-200 ng/mL plasma, but can be less (e.g., range of about 100-150 ng/mL) or greater (e.g., range of about 200-300 ng/mL) and still considered normal, due to functional clotting as determined, for example, by an activated partial thromboplastin time (aPTT) one-stage clotting assay.
• aPTT activated partial thromboplastin time
• the composition can be administered to a subject as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of a non-viral vector comprising a heterologous polynucleotide.
• the instant invention provides combinations in which a method or use of the instant invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art.
• the compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of a non-viral vector comprising a heterologous polynucleotide, to a subject.
• the instant invention includes methods and uses that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy.
• a method of treatment according to the instant invention has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement for the deficient or defective (abnormal or mutant) endogenous clotting factor in the subject.
• a methods of treatment according to the instant invention has a therapeutic benefit even if a less frequent or reduced dose of a recombinant viral vector comprising GAA has been previously administered, or continues to be administered to a subject.
• reducing the need for, or the use of, another treatment or therapy is included in the instant invention.
• An effective amount or a sufficient amount need not be effective in each and every subject treated, nor a majority of treated subjects in a given group or population.
• An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use.
• a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease.
• an effective amount would be an amount that inhibits or reduces glycogen production or accumulation, enhances or increases glycogen degradation or removal, improves muscle tone and/or muscle strength and/or respiratory function, for example.
• HemA or HemB an effective amount would be an amount that reduces frequency or severity of acute bleeding episodes in a subject, for example, or an amount that reduces clotting time as measured by a clotting assay, for example.
• compositions of the instant invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using techniques and guidance known in the art and using the teachings provided herein.
• Therapeutic doses will depend on, among other factors, the age and general condition of the subject, the severity of the aberrant phenotype, and the strength of the control sequences regulating expression levels. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that can be determined by a medical practitioner based on the response of an individual patient to a vector-based treatment.
• compositions such as pharmaceutical compositions can be delivered to a subject, so as to allow transgene expression and optionally production of encoded protein.
• pharmaceutical compositions comprising sufficient genetic material to enable a subject to produce a therapeutically effective amount of a therapeutic protein or nucleic acid.
• a therapeutic effect in a subject is sustained for a desirable period of time. Accordingly, in certain embodiments, a non-viral vector provides a therapeutic effect.
• compositions can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
• the compositions can be administered to a patient alone, or in combination with other agents, which influence dosage amount, administration frequency and/or therapeutic efficacy.
• Methods and uses of the instant invention include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion. Delivery of the compositions in vivo can generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery are envisioned (see, e.g., U.S. Pat. No. 5,720,720).
• compositions can be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally (IP), intravenously (IV), intra-pleurally, intraarterially, orally, intrahepatically, via the portal vein, or intramuscularly.
• Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications.
• a clinician specializing in the treatment of patients can determine the optimal route for administration of the compositions based on a number of criteria, including, but not limited to: the condition of the patient and the purpose of the treatment.
• the compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of a non-viral vector.
• the instant invention therefore provides combinations in which a method of treatment according to the instant invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art.
• the compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of a non-viral vector to a patient according to the instant invention.
• Methods according to the instant invention are applicable to both loss of function and gain and function genetic defects.
• loss-of-function in reference to a genetic defect as used herein, refers to any mutation in a gene in which the protein encoded by said gene (i.e., the mutant protein) exhibits either a partial or a full loss of function that is normally associated with the wild-type protein.
• gain-of-function in reference to a genetic defect as used herein, refers to any mutation in a gene in which the protein encoded by said gene (i.e., the mutant protein) acquires a function not normally associated with the protein (i.e., the wild type protein) causes or contributes to a disease or disorder.
• the gain-of- function mutation can be a deletion, addition, or substitution of a nucleotide or nucleotides in the gene, which gives rise to the change in the function of the encoded protein.
• the gain-of-function mutation changes the function of the mutant protein or causes interactions with other proteins.
• the gain-of-function mutation causes a decrease in or removal of normal wild-type protein, for example, by interaction of the altered, mutant protein with said normal, wild-type protein.
• Diseases and disorders that can be treated by methods according to the instant invention include, for example and without limitation, lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, a blood disorder (e.g., anemia), Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), epilepsy, a lysosomal storage disease (e.g., aspartylglucosaminuria, Batten disease, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis types I (sialidosis),
• Glycogen storage disease type II also called Pompe disease can be treated by methods according to the instant invention.
• Pompe disease is an autosomal recessive disorder caused by mutations in the gene encoding the lysosomal enzyme acid a-glucosidase (GAA), which catalyzes the degradation of glycogen.
• GAA acid a-glucosidase
• the resulting enzyme deficiency leads to pathological accumulation of glycogen and lysosomal alterations in all tissues of the body, resulting in cardiac, respiratory, and skeletal muscle dysfunction (van der Ploeg & Reuser, 2008).
• Blood clotting disorders which can be treated by methods according to the instant invention, include, for example and without limitation, hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, a deficiency in any coagulation Factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or a combined FV/FVIII deficiency, thalassemia, vitamin K epoxide reductase Cl deficiency or gammacarboxylase deficiency.
• Other diseases and disorders that can be treated by methods according to the instant invention include, for example and without limitation, anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors), or a platelet disorder such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.
• DIC disseminated intravascular coagulation
• FXa inhibitors small molecule antithrombotics
• a platelet disorder such as, Bernard Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.
• Other diseases and disorders that can be treated by methods according to the instant invention include, for example and without limitation, proliferative diseases (cancers, tumors, dysplasias, etc.), Crigler-Najjar and metabolic diseases like metabolic diseases of the liver, Friedreich ataxia, infectious diseases, viral diseases (induced, e.g., by hepatitis B or C viruses, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, dystroglycanopathies, myopathies such as Duchenne muscular myopathy or dystrophy, myotubular myopathy, sickle-cell anemia, sickle cell disease, Fanconi’s anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubularin myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinobulbar muscular atrophy, or Charcot-Marie-Tooth disease, arthritis, severe combined immunodeficiencies (such as RS-SCID, ADA-SCID or X
• the subject has a disease that affects or originates in the central nervous system (CNS).
• the disease is a neurodegenerative disease.
• CNS or neurodegenerative disease include Alzheimer’s disease, Huntington’s disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, Kennedy’s disease, a poly glutamine repeat disease, or Parkinson’s disease.
• the disease is a psychiatric disease, an addition (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan’s disease, or adrenoleukodystrophy.
• the CNS or neurodegenerative disease is a polyglutamine repeat disease such as, for example and without limitation, spinocerebellar ataxia (SCA1, SCA2, SCA3, SCA6, SCA7, or SCA17).
• the instant invention can be used in human and veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals.
• the term “subject” refers to an animal, typically a mammal, such as humans, non- human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat, rabbit, guinea pig).
• Human subjects include fetal, neonatal, infant, juvenile and adult subjects.
• Subjects also include animal disease models, for example, mouse and other animal models of protein/enzyme deficiencies such as Pompe disease (loss of GAA), and glycogen storage diseases (GSDs) and others known to those of skill in the art.
• compositions or combination for example, as a package or kit, having (a) a pharmaceutical composition comprising a non-viral vector comprising a transgene and a pharmaceutically acceptable carrier; (b) a phagocyte-depleting agent; and (c) a label with instructions for performing the method described herein, wherein (a) and (b) are in separate or the same container.
• Any suitable phagocyte-depleting agent can be included in the combination or kit, including but not limited to CD115 inhibiting agents, such as an antibody or an antigen binding fragment thereof that specifically binds to CD115, or a small molecule inhibitor of CD115; Ly6G inhibiting agents, including but not limited to, anti-Ly6G antibodies or anti-Ly6G small molecule inhibitors; CD177 inhibiting agents, including but not limited to anti-CD177 antibodies or CD177 small molecule inhibitors; CD14 inhibiting agents, including but not limited to anti-CD14 antibodies or CD14 small molecule inhibitors; CD 15 inhibiting agents, including but not limited to anti-CD15 antibodies or CD 15 small molecule inhibitors; CD1 lb inhibiting agents, including but not limited to anti-CDl lb antibodies or CDllb small molecule inhibitors; CD 16 inhibiting agents, including but not limited to anti-CDl 6 antibodies or CD 16 small molecule inhibitors; CD32 inhibiting agents, including but not limited to anti-CD32 antibodies or CD32 small molecule inhibitors; CD33
• the pharmaceutical composition comprises (a) a non-viral delivery nanoparticle; and (b) the non-viral vector, wherein the non-viral vector comprises the transgene operably linked to a promoter.
• Example 1 Immune cell depletion with anti-CD115 improved transgene expression
• the effects of immune depletion on cytokine release and on transgene expression were studied using an anti-CDl 15 antibody (BioXcell, clone AFS98), which targets monocytes and macrophages; using an anti-Ly6G antibody (BioXcell, clone 1A8), which targets myeloid-derived cells; using large ODNs, e.g., ODN oligo formulated in LNPs with a size of ⁇ 200nm, which inhibit TLR-9, AIM2, and other innate immune sensors; or using IT- 151, which is a small molecule stimulator of interferon genes (STING) inhibitor.
• STING small molecule stimulator of interferon genes
• transgene DNA plasmid nano-plasmid encoding human coagulation factor IX (hFIX)
• LNP lipid nanoparticle
• DNA-LNP lipid nanoparticle
• IV intravenous
• IP intraperitoneal
• anti-CDl 15 antibody anti-Ly6G antibody
• H-151 purchased from InvivoGen
• 50 pg large ODN purchased from InvivoGen
• FIX expression As shown in FIG. 2A, essentially no FIX expression was detected in the plasma of animals administered with 50 pg of DNA-LNP, without anti-CD115 or anti-Ly6G antibodies, while FIX expression of about 250 ng/mL (about 5% or more of normal expression level) was observed in the plasma of animals for over 14 weeks following dosing with 50 pg of DNA-LNP in combination with an anti-CD115 antibody, and FIX expression of about 100- 200 ng/mL (less than 5% normal expression level) was observed in the plasma for over 14 weeks following dosing with 50 gg of DNA-LNP in combination with an anti-Ly6G antibody.
• FIX expression As shown in FIG. 2B, essentially no FIX expression was detected in the plasma of animals administered with 10 pg of DNA-LNP, without anti-CD115 or anti-Ly6G antibodies, while FIX expression of about 250 ng/mL (about 5% or more of normal expression level) was observed in the plasma of animals for over 14 weeks following dosing with 10 pg of DNA-LNP in combination with an anti-CD115 antibody, and FIX expression of about 100- 300 ng/mL was observed in the plasma for over 14 weeks following dosing with 10 pg of DNA-LNP in combination with an anti-Ly6G antibody.
• Example 2 Immune cell depletion with anti-CD115 and another immunosuppressant improved transgene expression and reduced cytokine production
• FIX transgene DNA plasmid 50 pg
• lipid nanoparticle (LNP) delivery vehicles SPK-LNP1
• SPK-LNP-1 lipid nanoparticle delivery vehicles
• anti-CDl 15 antibody and/or clodronate purchased from Liposoma
• Table 3 SPK-LNP-1 had the following composition: C12-200 lipid at 35%; DOPE at 16%; cholesterol at 46.5%; and C14-PEG2000 (Avanti® Polar Lipids) at 2.5%.
• DNA and anti-CDl 15 antibody were as described in Example 1.
• FIX expression of about 500 ng/mL (-10% of normal expression levels) was observed in the plasma of animals for one week following dosing with 50 pg of SPK- LNP1 in combination with an anti-CDl 15 antibody and clodronate, which is significantly higher than that when SPK-LNP1 was administered alone, or in combination with only one of the anti-CDl 15 antibody and clodronate.
• Example 3 Immune cell depletion with clodronate, anti-CDl 15 and pexidartinib
• Balb/C mice were treated with the immune cell depletion agents clodronate, anti- CDl 15 antibody (as described in Example 1), or pexidartinib.
• Clodronate was provided by IV administration at 50 mpk (1.25 mg) or 17 mpk (425 pg), anti-CDl 15 antibody was provided by IP administration at 12 mpk (300 pg) 1 dose vs 4 doses.
• Anti-CDl 15 antibody was as described in Example 1.
• Pexidartinib was provided by oral gavage administration at 100 mpk (2.5 mg) 4 doses.
• a no-treatment mice group was used as a control. Each group contained 5 male BALB/c mice.
• mice were euthanized and the liver samples were harvested for histology. Liver samples were stained with either anti-CLEC4F or anti-CD68 mouse antibody. Individual scores of the treatment groups were normalized by the no-treatment group to calculate depletion efficacy.
• Table 4 The specific treatment dose levels and schedules are shown in Table 4: Table 4
• Clodronate and anti-CD115 antibodies had a significant effect on Kuffer and macrophage depletion.
• Pexidartinib had a small impact on Kupffer cell depletion (CLEC4F+ staining) and no impact on CD68+ macrophage depletion.
• the results with pexidartinib were attributed to the mode of administration.
• mice chow was mixed with mice chow at 400 mg Pex/kg chow.
• mice chow was replaced with Pex chow at 21 days and 7 days before DNA-LNP-5 dosing.
• the experimental design is shown in Table 5.
• mice in group 1 were fed with regular chow throughout the study and served as controls. Mice were dosed with DNA-LNP-5 (10 pg per mouse). LNP-5 (GenVoyTM LNP) was obtained from Precision NanoSystems Inc.) and contained: ionizable lipid at 50%; DSPC at 10%; cholesterol 37.5%; and stabilizer (PEG-Lipid) at 2.5% (see Roces et al., Pharmaceutics, 2020 12, 1095.) DNA was as described in Example 1. hFIX levels in mouse plasma were quantified using ELISA 1-4 weeks after dosing. The results are shown in Figure 7. After DNA-LNP-5 dosing, a few mice in Pex-treated groups showed detectable expression. By contrast, no detectable hFIX levels was observed in mice fed with regular chow.
• mice were treated with anti-CDl 15 antibody (300 pg anti-CD115 per injection) three times (days -5, -3, and -1) before DNA-LNP-5 dosing (10 pg per mouse). Mice in the control group were treated with DNA-LNP-5 only. DNA and anti-CDl 15 antibody were as described in Example 1. The experimental design is shown in Table 6.
• mice were treated with anti-CDl 15 antibody (300 pg anti-CDl 15 per injection) four times (days -10, -8, -6, and -2) before DNA-LNP-3 dosing (25 pg per mouse). Mice in the control group were treated with DNA-LNP-3 only.
• DNA-LNP-3 had the following composition: cKK-E12 at 35%; DOPE at 16%; cholesterol at 46.5%; and PEGylated lipid C14-PEG2000 at 2.5%.
• DNA and anti-CDl 15 antibody were as described in Example 1. [0259] The experimental design is shown in Table 7.
• mice were treated with anti-CD115 antibody (300 pg anti-CD115 per injection) four times (days -10, -8, -6, and -2) before DNA-LNP-4 dosing. Mice in the control group was treated with DNA-LNP-4 only.
• LNP-4 had the following composition: Lipid 9 (Sabnis et al., Molecular Therapy 2018, vol. 26, No. 6, 1509-1519) at 50%; DSPC at 10%; cholesterol at 38.5%; and C-14-PEG2000 at 1.5%.
• the experimental design is shown in Table 8.
• the instant invention is generally disclosed herein using affirmative language to describe the numerous embodiments of the instant invention.
• the instant invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
• materials and/or method steps are excluded.

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