Biliary delivery methods, compositions and kits for use therewith

Abstract

The present disclosure provides compositions and methods for the delivery of therapeutic biologics to locations where bile is present, including the biliary system, and compositions and kits for use in such methods. Also provided are compositions comprising biliary therapeutic enhancers for the delivery of therapeutic biologics, and methods using biliary therapeutic enhancers. The methods of biliary delivery of therapeutic biologics described herein generally include one or more administrations of the associated biologics and biliary therapeutic enhancers. Biliary delivery compositions generally include one or more biliary therapeutic enhancers and therapeutic biologics. Compared to the reduced activity of therapeutic biologics in the presence of bile, the compositions, methods, and kits of the present disclosure provide an increase, enhancement, and / or rescue of the activity of the therapeutic agent in the presence of bile. Also provided are, for example, the use of biliary therapeutic enhancers in the described methods, compositions, and kits.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 236,574, filed August 24, 2021, U.S. Provisional Patent Application No. 63 / 175,693, filed April 16, 2021, and U.S. Provisional Patent Application No. 63 / 128,983, filed December 22, 2020, which applications are incorporated by reference herein in their entireties. [Background technology]

[0002] In the United States alone, there are over 100,000 patients with acute liver disease and over 500,000 with decompensated cirrhosis. Liver disease causes 62,000 deaths annually in the United States and approximately 2 million deaths worldwide, with 1.3 million deaths due to cirrhosis. As of 2019, cirrhosis was the 11th leading cause of death worldwide and the 12th leading cause of death in the United States. Approximately 2 billion people worldwide consume alcohol, an additional 2 billion adults are obese or overweight, and 400 million adults suffer from diabetes. Alcohol consumption, hepatic lipid deposition, and insulin resistance are all believed to be major risk factors in the development of fibrosis and ultimately cirrhosis. Furthermore, drug-induced liver injury continues to increase as a leading cause of acute hepatitis, while the global prevalence of viral hepatitis remains high.

[0003] Liver transplantation, when available and successful, is a life-changing treatment that represents the second most common solid organ transplant. However, suitable livers are often not available in the quantities or time required for subjects with rapidly declining conditions such as acute liver failure. Compared to the widespread disease prevalence described above, fewer than 9,000 liver transplants have been performed in the United States.

[0004] Many other therapies, including numerous biologics, have shown promise either mechanistically or in preclinical studies, but few have progressed through clinical trials or demonstrated significant positive outcomes in human subjects. For example, targeting collagen cross-linking, an essential process in fibrosis, by monoclonal antibody inhibition of lysyl oxidase (LOX) family enzymes has shown promise in vitro and in preclinical studies (see, e.g., Ikenaga et al. Gut (2017) 66:1697-1708). However, clinical trials of selonsertib administered by either subcutaneous injection or intravenous infusion did not demonstrate clinical efficacy (Harrison et al. Gastroenterology (2018) 155(4):1140-1153). Other treatments targeting liver disease, or its pathological features, such as fibrosis and cirrhosis, have shown a similar pattern of initial promise but unsatisfactory clinical outcomes. Summary of the Invention

[0005] The present disclosure provides methods for the delivery of therapeutic biologics to locations where bile is present, including the biliary system, as well as compositions and kits for use in such methods. Also provided are compositions (e.g., pharmaceutical compositions) that include biliary therapeutic enhancers for the delivery of therapeutic biologics, and methods of using biliary therapeutic enhancers. Biliary delivery compositions (e.g., pharmaceutical compositions) include one or more therapeutic biologics and / or one or more biliary therapeutic enhancers (e.g., sequestration agents), which may be included in separate compositions and / or combined in a single composition (e.g., pharmaceutical composition). The biliary delivery methods of therapeutic biologics described herein generally include one or more administrations of the relevant biologics, biliary therapeutic enhancers, and / or compositions described herein. Compared to the decreased activity of therapeutic biologics in the presence of bile, the compositions, methods, and kits of the present disclosure provide a surprising and unexpected increase, enhancement, and / or rescue of the activity of the therapeutic agent in the presence of bile. For example, the use of biliary therapeutic enhancers in the described methods, compositions and kits is also provided.

[0006] In some aspects, compositions comprising one or more therapeutic biologics and / or one or more biliary therapeutic enhancers (e.g., one or more sequestration agents and / or one or more transduction enhancers) are described herein. The therapeutic biologics and / or biliary therapeutic enhancers may be formulated in the same composition or in one or more different compositions (e.g., one or more separate compositions comprising one or more therapeutic biologics and one or more separate compositions comprising one or more biliary therapeutic enhancers, or one or more combined compositions comprising one or more therapeutic biologics and one or more biliary therapeutic enhancers). In some embodiments, a composition comprises a combination of at least one therapeutic biologic and at least one biliary therapeutic enhancer. In certain embodiments, In some embodiments, the at least one biliary therapeutic enhancer of the composition comprises at least one bile sequestration agent. In other embodiments, the at least one biliary therapeutic enhancer comprises a transduction enhancer. In further embodiments, the composition comprises a combination of at least one sequestration agent and at least one transduction enhancer. The compositions of the present invention may be pharmaceutical compositions and may further comprise one or more pharma- ceutically acceptable excipients, buffers, and / or other reagents. Any amount (dosage) of therapeutic biologics and biliary therapeutic enhancers may be used in the separate or combined compositions described herein. Useful dosages of therapeutic biologics and / or biliary therapeutic enhancers administered separately or together may be readily determined.

[0007] Thus, aspects of the present disclosure also include pharmaceutical compositions, including those that include a pharma- ceutically acceptable carrier configured as a liquid for delivery to the biliary system, an effective amount of one or more therapeutic biologics, and one or more biliary therapeutic enhancers. The pharmaceutical composition (formulation) may include one or more biliary therapeutic enhancers in the same or different formulation as the therapeutic biologics. In certain embodiments, one or more biliary therapeutic enhancers may be formulated with additional components (e.g., with one or more therapeutic biologics). In other embodiments, separate pharmaceutical compositions (formulations) of any of the components may be used, e.g., one or more therapeutic biologics may be formulated in one or more formulations different from one or more biliary therapeutic enhancers.

[0008] In some aspects, the composition may include a therapeutic biologic that is susceptible to inactivation by bile. In some aspects, the therapeutic biologic of the composition may include, but is not limited to, a gene therapy (e.g., a nucleic acid construct encoding one or more therapeutic proteins or portions thereof, a non-coding nucleic acid, etc.) and / or a protein, including but not limited to, a non-viral vector, a viral vector, a lipid nanoparticle (LNP), an enveloped viral vector, a lentiviral vector, an adenovirus, an adeno-associated viral (AAV) vector, a therapeutic peptide, and / or an antibody. In certain embodiments, the virus (e.g., lentivirus or AAV) or non-viral vector includes one or more polynucleotides that encode one or more proteins (e.g., a therapeutic protein, an antibody, etc.), including but not limited to, a protein described herein, including, for example, a protein that is lacking (i.e., missing and / or absent) in liver disease.

[0009] In some embodiments, the biliary therapeutic enhancer of the composition may include, but is not limited to, a polyamine or polyether polymer. In some examples, the biliary therapeutic enhancer includes one or more bile acid sequestrants, including, but not limited to, colesevelam, cholestyramine, colestipol, and sevelamer. In some examples, the biliary therapeutic enhancer may be, for example, a cationic or non-ionic amphiphilic transduction enhancer, including, but not limited to, polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108. Combinations of any biliary therapeutic enhancer (e.g., one or more sequestration agents and / or one or more transduction enhancers) may be used.

[0010] Aspects of the present disclosure also include the use of a liquid composition comprising a biliary therapeutic enhancer to deliver a therapeutic biologic to the biliary system of a subject to treat a hepatic condition of the subject. In some aspects, the biliary therapeutic enhancer of such uses is a bile acid sequestrant, and optionally, the bile acid sequestrant is a polyamine or polyether polymer. In some aspects, the biliary therapeutic enhancer of such uses is a cationic or non-ionic amphipathic transduction enhancer, and optionally, the cationic or non-ionic amphipathic transduction enhancer is a polyamine or polyether polymer.

[0011] The bile acid secretion inhibitor may also be included in a separate composition and / or with a composition that includes one or more therapeutic biologics and / or biliary therapeutic enhancers. Bile acid secretion inhibitors include, but are not limited to, agonists of the bile acid receptor (BAR), also known as farnesoid X receptor (FXR) or NR1H4 (nuclear receptor subfamily 1, group H, member 4). In certain embodiments, the bile acid secretion inhibitor is an FXR agonist.

[0012] Aspects of the disclosure include methods of treating a condition in a subject, including, for example, using one or more compositions described herein (compositions including a therapeutic biologic and / or a biliary therapeutic enhancer) to administer an effective amount of one or more therapeutic biologics and an effective amount of one or more biliary therapeutic enhancers directly to the biliary system of a subject, thereby treating the condition in the subject.

[0013] In some embodiments, the condition treated by the methods and compositions described herein may be, but is not limited to, a genetic disease, a monogenic disease, a metabolic disease, or a hepatic condition, including, for example, acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha-1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug-induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end-stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver disease, focal nodular hyperplasia, hepatic hydatid disease, hepatic encephalopathy, hepatic infarction, liver failure, hepatic porphyria. , hepatic tuberculosis, hepatic veno-occlusive disease, hepatitis, hepatocellular carcinoma, hepatic erythropoietic porphyria, hepatolenticular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, hereditary coproporphyria, liver abscess, hepatocellular adenoma, liver cirrhosis, liver failure, liver tumor, massive hepatic necrosis, nonalcoholic fatty liver disease, parasitic liver disease, peliosis hepatis, porphyria cutanea tarda, portal hypertension, pyogenic liver abscess, Reye's syndrome, variant porphyria, viral hepatitis, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, Zellweger syndrome. In some embodiments, the condition may be a condition other than hyperlipidemia, secondary dyslipidemia, bile acid malabsorption condition, and / or diabetic condition.

[0014] In some aspects, retroductal delivery of therapeutic biologics, biliary therapeutic enhancers and / or compositions described herein is used to deliver therapeutic agents to the liver of a subject. In some instances, direct retroductal delivery of a therapeutic biologic results in enhanced local liver concentrations of the therapeutic biologic. In some instances, the therapeutic biologic is subject to (susceptible to) inactivation by bile.

[0015] In some aspects of the methods, the therapeutic biologic may include, but is not limited to, a gene therapy agent, or a protein, including, but not limited to, a non-viral vector, a viral vector, a lipid nanoparticle, an enveloped viral vector, a lentiviral vector, an adenovirus, an adeno-associated virus (AAV), a therapeutic peptide, or an antibody in a subject with liver disease (i.e., that is missing and / or absent).

[0016] In some embodiments, the biliary therapeutic enhancer may include, but is not limited to, polyamines and / or polyether polymers. In some examples, the biliary therapeutic enhancer may be a bile acid sequestrant, including, but not limited to, colesevelam, cholestyramine, colestipol, and / or sevelamer. In some examples, the biliary therapeutic enhancer may be a cationic or non-ionic amphipathic transduction enhancer, including, but not limited to, polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and / or F108.

[0017] In some aspects, the therapeutic biologic and the biliary therapeutic enhancer may be co-administered. In some aspects of the method, the therapeutic biologic and the biliary therapeutic enhancer may be co-formulated in a single pharmaceutical composition. In some aspects of the method, the biliary therapeutic enhancer may be administered before the therapeutic biologic. In some aspects of the method, two or more biliary therapeutic enhancers may be administered, including, for example, when the two or more biliary therapeutic enhancers include a bile acid sequestrant and a cationic or non-ionic amphiphilic transduction enhancer. In some aspects of the method where two or more biliary therapeutic enhancers may be administered, the two or more biliary therapeutic enhancers may be co-administered or co-formulated in a single pharmaceutical composition.

[0018] In some aspects, the method may further include administering to the subject a bile acid secretion inhibitor, including, but not limited to, for example, where the bile acid secretion inhibitor is an FXR agonist, optionally wherein the FXR agonist is selected from obeticholic acid or an FGF19 analog.

[0019] Aspects of the present disclosure also include kits that may include any of the compositions and / or reagents described herein, including, for example, a liquid pharma- ceutically acceptable carrier; a therapeutic biologic; and / or a biliary therapeutic enhancer. The kits may further include instructions for using the compositions and / or for practicing the methods described herein.

[0020] In some aspects, the biliary therapeutic enhancer of the kit and the liquid pharma- ceutically acceptable carrier of the kit are formulated together in a container of the kit. In some aspects, the therapeutic biologic of the kit is susceptible to inactivation by bile. In some aspects, the therapeutic biologic may include, but is not limited to, a gene therapy agent, or a protein, including but not limited to, a non-viral vector, a viral vector, a lipid nanoparticle, an enveloped viral vector, a lentiviral vector, an adenovirus, an adeno-associated virus (AAV), a therapeutic peptide, or an antibody. In certain embodiments, the viral or non-viral vector includes one or more polynucleotides encoding one or more proteins (e.g., therapeutic proteins, antibodies, etc.), for example, one or more proteins described herein that are lacking (i.e., missing and / or absent) in one or more liver conditions.

[0021] In some embodiments, the biliary therapeutic enhancer of the kit may include, but is not limited to, a polyamine or polyether polymer. In some examples, the biliary therapeutic enhancer may be a bile acid sequestrant, including, but not limited to, colesevelam, cholestyramine, colestipol, and sevelamer. In some examples, the biliary therapeutic enhancer may be a cationic or non-ionic amphipathic transduction enhancer, including, but not limited to, polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108.

[0022] In some aspects, the kit may include a device, including but not limited to where the device is configured for retrograde ductal delivery of a therapeutic biologic to the liver, hi some aspects, the kit may be used for the treatment of conditions described herein.

[0023] Aspects of the disclosure include a method of transducing or transfecting a cell comprising contacting the cell with a biliary transduction enhancer in the presence of bile to produce a transduction / transfection composition; and contacting the transduction / transfection composition with a gene therapy agent comprising an exogenous nucleic acid under conditions sufficient for transduction or transfection of the exogenous nucleic acid into the cell, thereby transducing or transfecting the cell with the exogenous nucleic acid.

[0024] In some aspects, the cells are hepatocytes, optionally hepatocytes. In some aspects, the gene therapy agent may comprise a non-viral or viral vector, including lipid nanoparticles or enveloped viral vectors. In some aspects, the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[0025] In some embodiments, the exogenous nucleic acid comprises a coding sequence, the coding sequence comprises encoding a transcription factor, a therapeutic peptide, or an antibody. In some embodiments, the biliary therapeutic enhancer comprises a polyamine or polyether polymer. In some embodiments, the biliary therapeutic enhancer is a bile acid sequestrant, such as colesevelam, cholestyramine, colestipol, or sevelamer. In some embodiments, the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer, such as polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, or F108. In some embodiments, contacting the cell with the biliary transduction enhancer may include contacting the cell with both the bile acid sequestrant and the cationic or non-ionic amphipathic transduction enhancer. In some embodiments, the biliary transduction enhancer and the gene therapy agent are present together in a formulation prior to contacting. [Brief description of the drawings]

[0026] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0027] [Figure 1] FIG. 13 is a graph showing results from a lentiviral transduction assay in HeLaRC32 cells demonstrating that addition of a bile acid sequestrant to the transduction reaction rescues the activity of a lentiviral vector ("LV-SFFV-LUC2-P2A-EmGFP" containing luciferase and Emerald GFP transgenes linked via a 2A peptide driven by the spleen focus forming virus (SFFV) promoter) as measured by relative luciferase units ("RLU") that are otherwise substantially reduced in the presence of bile.

[0028] [Diagram 2] Graph showing results from lentiviral transduction assays performed in a series of bile dilutions in the presence of different transduction enhancers (polyethylenimine "PEI" or polyethylene glycol), demonstrating that the transduction enhancer alone can rescue lentiviral vector activity that is otherwise lost in the presence of bile.

[0029] [Diagram 3] 1 is a graph showing results from a bile time series demonstrating the temporal kinetics of lentiviral vector ("LVV") inactivation by exposure to 15% rat bile.

[0030] [Figure 4A]4A-4B are graphs showing the effect of bile sequestration agents and / or enhancers on the transduction efficiency of LNPs carrying mRNA encoding a green fluorescent protein marker (GFP). FIG. 4A shows the percentage of cells expressing GFP at the indicated concentrations of sequestration agent (colesevelam) or transduction enhancer (F108). FIG. 4B is a graph showing the results of FIG. 4A in logarithmic format. "Bile" refers to samples containing only 30% rat bile. "Bile+colesevelam" refers to samples containing bile and colesevelam (at the indicated concentrations). "Bile+F108" refers to samples containing bile and F108, and "No treatment" refers to a control without bile, sequestration agent, or enhancer. [Figure 4B] 4A-4B are graphs showing the effect of bile sequestration agents and / or enhancers on the transduction efficiency of LNPs carrying mRNA encoding a green fluorescent protein marker (GFP). FIG. 4A shows the percentage of cells expressing GFP at the indicated concentrations of sequestration agent (colesevelam) or transduction enhancer (F108). FIG. 4B is a graph showing the results of FIG. 4A in logarithmic format. "Bile" refers to samples containing only 30% rat bile. "Bile+colesevelam" refers to samples containing bile and colesevelam (at the indicated concentrations). "Bile+F108" refers to samples containing bile and F108, and "No treatment" refers to a control without bile, sequestration agent, or enhancer.

[0031] [Figure 5A] Representative luciferase stained liver sections from animals treated with vehicle or lentiviral vector (LVV) carrying a luciferase transgene are shown. Figure 5A shows a liver section from an animal treated with vehicle. Figure 5B shows a section from an animal treated with LVV. As shown, only animals treated with LVV expressed luciferase in their livers in vivo (Figure 5B). [Figure 5B]Representative luciferase stained liver sections from animals treated with vehicle or lentiviral vector (LVV) carrying a luciferase transgene are shown. Figure 5A shows a liver section from an animal treated with vehicle. Figure 5B shows a section from an animal treated with LVV. As shown, only animals treated with LVV expressed luciferase in their livers in vivo (Figure 5B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides compositions and methods for enhancing the delivery of therapeutic biologics to locations where bile is present, including the biliary system, and compositions and kits for use in such methods. Also provided are compositions comprising biliary therapeutic enhancers for the delivery of therapeutic biologics, and methods using biliary therapeutic enhancers. The methods of biliary delivery of therapeutic biologics described herein generally include one or more administrations of the associated biologics and biliary therapeutic enhancers. Biliary delivery compositions generally include one or more biliary therapeutic enhancers and therapeutic biologics. Compared to the reduced activity of therapeutic biologics in the presence of bile, the compositions, methods, and kits of the present disclosure provide an increase, enhancement, and / or rescue of the activity of the therapeutic agent in the presence of bile. For example, the use of biliary therapeutic enhancers in the described methods, compositions, and kits is also provided.

[0033] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0034] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0035] Certain ranges are described herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the near or approximate unrecited number may be a number that, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0037] All publications and patents cited herein are incorporated by reference herein as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0038] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0039] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0040] Although the apparatus and methods have been described for grammatical fluidity with functional descriptions, it is to be clearly understood that the claims should not be construed as necessarily limited by the construction of "means" or "step" limitations unless expressly formulated under 35 U.S.C. §112, but rather should be given the full scope of meaning and equivalents of the definitions provided by the claims under the legal theory of equivalents, and that if a claim is expressly formulated under 35 U.S.C. §112, the full legal equivalents under 35 U.S.C. §112 should be given.

[0041] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are intended to include their various grammatical forms, where applicable. As used herein, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to an "agent" includes reference to one or more agents known to those of ordinary skill in the art, and so forth.

[0042] The term "about" with respect to a reference numerical value can include a range of values ​​from that value plus or minus 10%. For example, the amount "about 10" includes values ​​from 9 to 11, including the values ​​9, 10, and 11. The term "about" with respect to a reference numerical value can also include a range of values ​​from that value plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0043] Before describing particular embodiments of the present disclosure, it will be useful to set forth definitions that will be used in describing the present disclosure.

[0044] The term "biliary therapeutic enhancer" refers to any compound, molecule, or formulation of one or more compounds and molecules that enhances the delivery of a biologic to one or more hepatocytes in the presence of bile. Hepatocytes may be, but are not limited to, isolated (e.g., isolated populations of mature or hepatic stem cells) in a living subject (in vivo as a whole liver), or obtained from a subject and introduced into the same or a different subject (ex vivo). The term includes, but is not limited to, one or more bile sequestrants (also referred to as "sequestration agents") and / or one or more transduction enhancers, which may be administered simultaneously (in the same or different formulations) or sequentially in any order (in the same or different formulations).

[0045] The term "estimating" includes any form of measurement, including determining whether an element is present or not. The terms "determining," "measuring," "assessing," "estimating," and "assaying" are used interchangeably and include quantitative and qualitative determinations. Estimating can be relative or absolute.

[0046] The terms "control," "control assay," "control sample," and the like refer to a sample, test, or other part of an experimental or diagnostic procedure or experimental design in which the expected outcome is known with a high degree of certainty, for example, to indicate whether the results obtained from the associated experimental sample are reliable, to indicate the confidence that the associated experimental results indicate a true outcome, and / or to allow calibration of the experimental results. For example, in some instances, a control may be a "negative control" assay in which essential components of the assay are omitted so that the experimenter can have a high degree of certainty that the negative control assay will not produce a positive outcome. In some instances, a control may be a "positive control" assay in which all components of a particular assay are characterized and known to produce a particular outcome in the assay being performed when combined, so that the experimenter can have a high degree of certainty that the positive control assay will not produce a positive outcome. Controls may also include "blank" samples, "standard" samples (e.g., "gold standard" samples), validated samples, and the like.

[0047] The terms "specific binding," "specifically binds," and the like refer to the non-covalent or covalent preferential binding to a molecule compared to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope compared to other available polypeptides or epitopes). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is greater than or equal to 10 5 Characterized by a KD (dissociation constant) of less than or equal to M (e.g., 10 6 M or less, 10 7 M or less, 10 8 M or less, 10 9 M or less, 10 10 M or less, 10 11 M or less, 10 12 M or less, 10'' 13 M or less, 10 14 M or less, 10 15 M or less, or 10 16 "Affinity" refers to the strength of binding, with increased binding affinity correlated with a lower KD.

[0048] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, e.g., a human subject, for which diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, and the like. In some embodiments, the mammal is a human. In some cases, the methods of the invention are used in laboratory animals, in veterinary applications, and / or in the development of animal models, including, but not limited to, rodents, including mice, rats, and hamsters; rabbits, dogs, cats, non-human primates, and other animals.

[0049] The terms "treatment", "treating", "treating" and the like are used herein generally to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in that it completely or partially prevents the disease or its symptoms, and / or may be therapeutic in that it partially or completely stabilizes or cures the disease and / or the adverse effects caused by the disease. For example, preventative treatment, i.e., prophylactic treatment, may include treatment that effectively prevents a condition (e.g., a liver condition) or treatment that effectively prevents or controls the progression of a condition (e.g., a liver condition). In some instances, treatment may result in a therapeutic response, such as a complete or partial remission. The term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease and / or symptoms from occurring in a subject who may be susceptible to the disease or symptoms but has not yet been diagnosed as suffering from the disease; (b) inhibiting the disease and / or symptoms, i.e., halting the development of the disease and / or associated symptoms; and (c) relieving the disease and associated symptoms, i.e., causing regression of the disease and / or symptoms.

[0050] Those in need of treatment can include those already affected (e.g., those with a liver condition (e.g., acute liver condition, chronic liver condition, etc.), those with cirrhosis, those with fibrosis, etc.), as well as those in whom prevention is desired (e.g., those with increased susceptibility to a liver condition; those suspected of having a liver condition; those at increased risk of developing a liver condition; those with increased environmental exposure to practices or agents that cause a liver condition; those suspected of having a genetic or behavioral predisposition to a liver condition; those with a liver condition; those with screening results indicating an increased risk of a liver condition; those who have tested positive for a liver condition; those who have tested positive for one or more biomarkers of a liver condition, etc.).

[0051] A therapeutic treatment is one in which the subject is afflicted prior to administration, and a prophylactic treatment is one in which the subject is not afflicted prior to administration. In some embodiments, the subject is likely to be afflicted or suspected of being likely to be afflicted (e.g., compared to the norm, e.g., compared to the average individual, e.g., the subject may have a family history indicating a genetic predisposition and / or increased risk for the condition), in which case the treatment may be a prophylactic treatment.

[0052] As used herein, the term "small molecule" refers to a small organic or inorganic compound having a molecular weight of more than 50 Daltons and less than about 2,500 Daltons. The agent may contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may contain at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two functional chemical groups. Small molecule agents may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more functional groups. Small molecule agents are also found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0053] The term "recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which is not related by its origin or manipulation to all or a portion of the polynucleotide sequence associated in nature. The term recombinant, as used with respect to a protein or polypeptide, means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant, as used with respect to a host cell or virus, means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to a material (e.g., a cell, nucleic acid, protein, or vector) to refer to the material having been altered by the introduction of heterologous material (e.g., a cell, nucleic acid, protein, or vector).

[0054] The terms "nucleic acid" and "polynucleotide", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, including analogs thereof. These terms refer only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, triple-stranded DNA, and double- and single-stranded RNA. It also includes modified forms, for example, by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also intended to include molecules containing non-naturally occurring or synthetic nucleotides and nucleotide analogs. Non-limiting examples of nucleic acids and polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, primers, single-, double-, or multiple-stranded DNA or RNA, genomic DNA, DNA-RNA hybrids, chemically or biochemically modified, non-natural, or derivatized nucleotide bases, oligonucleotides containing modified or non-natural nucleotide bases (e.g., locked nucleic acid (LNA) oligonucleotides), and interfering RNA. In some instances, the polynucleotide may be a contiguous open reading frame polynucleotide that excludes at least some non-coding sequences from the corresponding sequence present in the genome of the organism.

[0055] The term "nucleic acid therapeutic agent" as used herein generally refers to a nucleic acid that can be administered in the context of therapy to treat a condition of a subject, for example, as applied in the context of gene therapy as described herein.Non-limiting examples of nucleic acid therapeutic agents include interfering nucleic acids for suppressing the expression of genes associated with a condition, coding sequences for replacing defective or abnormal gene expression (e.g., placed in an expression cassette, plasmid, vector genome, etc.), coding sequences for the expression of heterologous gene products that provide a therapeutic effect, and the like.

[0056] The term "polypeptide" is used interchangeably with the terms "polypeptide" and "protein" and refers to a polymer of amino acid residues. Polypeptides include functional protein fragments of essentially any length as well as full-length proteins. The term "peptide" as used herein generally refers to a polypeptide chain of 40 amino acids or less. A "peptide therapeutic" is a peptide with an established therapeutic function.

[0057] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins that comprise the antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies, are also encompassed by the term. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which were produced from a single cell by repeated cell replication. That is, a clone of a cell produces only a single antibody species. Monoclonal antibodies may be produced using hybridoma production techniques, although other production methods known to those skilled in the art may also be used (e.g., antibodies derived from an antibody phage display library). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

[0058] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies useful in the context of the present disclosure can be prepared using a wide variety of techniques, including, but not limited to, the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0059] The term "transduction", as used herein, generally refers to the introduction of an exogenous nucleic acid into a cell using a viral vector, and the term "transfection", as used herein, generally refers to the process of introducing a nucleic acid into a cell by a non-viral method. However, in some instances throughout this disclosure that will be readily apparent to one of skill in the art, the terms "transduction" and "transfection" may be used interchangeably. In some instances, the use of the term transduction may exclude non-viral delivery of a nucleic acid. In some instances, the use of the term transfection may exclude viral delivery of a nucleic acid.

[0060] The term "vector copy number" or "VCN" refers to the number of copies of a vector or a portion thereof introduced into a cell. The average VCN may be determined from a population of cells or from individual cell colonies. Exemplary methods for determining VCN include polymerase chain reaction (PCR) and flow cytometry.

[0061] The terms "viral particle," "virus," and the like refer to infectious viral agents including, for example, baculovirus particles, lentivirus particles, adenovirus particles, etc. Viruses and viral particles may be naturally occurring, recombinant, engineered, or synthetic.

[0062] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, or "insert," can be attached so as to bring about the replication of the attached segment in a cell.

[0063] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. Retroviruses are a common tool for gene delivery. Exemplary retroviruses include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumaretrovirus, Friend murine leukemia virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV)) and lentivirus.

[0064] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) may be used.

[0065] Retroviral vectors, and more specifically, lentiviral vectors, may be used in the practice of the present invention. Thus, as used herein, the terms "retrovirus" or "retroviral vector" are meant to include "lentivirus" and "lentiviral vector," respectively.

[0066] Compositions and methods The present disclosure includes compositions and methods comprising biliary therapeutic enhancers for delivering therapeutic biologics in situations where bile may be present at the target delivery location, thereby providing improved therapeutic benefit from the biologic.

[0067] As described and demonstrated herein, the presence of bile has a destructive effect on the activity of therapeutic biologics, reducing, inhibiting, or eliminating the therapeutic effect of such biologics in living subjects. Thus, when a biologic is delivered to a target location within a subject where bile is present, the therapeutic effect may be reduced, for example, compared to the activity expected in the absence of bile, or may be lost entirely. This detrimental effect of bile on biologics limits the in vivo locations to which a biologic therapeutic may be administered while still achieving the desired therapeutic effect.

[0068] Numerous biologic therapeutics have been developed for the treatment of liver conditions, which have shown promise in in vitro and preclinical studies, but many of these biologics do not demonstrate clinical efficacy when administered systemically to human subjects in clinical trials. One alternative approach to improve liver-directed therapy and / or increase the clinical efficacy of liver-directed therapeutic biologics is to concentrate the therapeutic in the liver, thus achieving a high local concentration of the therapeutic in and / or around the liver, for example, compared to the relatively low concentration of the biologic systemically and / or at locations other than the liver. As described herein, increasing the local concentration of the relevant biologic therapeutic in the liver can be achieved by delivering the biologic directly to the biliary system and allowing retrograde movement of the biologic to the liver. This approach offers various advantages, including increased liver concentration, lower doses, more rapid exposure, etc., but the presence of bile in the biliary system can have a detrimental effect on the activity of the delivered biologic, as described herein.

[0069] The present disclosure is based, at least in part, on the discovery that the addition of certain reagents to a mixture containing bile can reduce the deleterious effects of bile on the activity of a biologic therapeutic that may come into contact with the bile-containing mixture at the time or thereafter, or can otherwise increase the activity of the biologic when present in an environment in which bile is present. Thus, such reagents enhance the effect of a therapeutic delivered in the presence of bile, and are referred to herein as biliary therapeutic enhancers. The methods and compositions described herein provide a variety of advantages, including, but not limited to, unexpectedly superior results following direct delivery of a biologic therapeutic to the biliary system, such as increased local concentration in the liver, resulting in increased therapeutic efficacy.

[0070] Described herein are compositions comprising one or more therapeutic biologics and / or one or more biliary therapeutic enhancers. In certain embodiments, the compositions comprise a combination of at least one therapeutic biologic and at least one biliary therapeutic enhancer. The compositions of the present invention may be pharmaceutical compositions and may further comprise one or more pharma- ceutically acceptable excipients, buffers, and / or other reagents. Additional agents may also be included in separate compositions and / or with compositions comprising one or more therapeutic biologics and / or biliary therapeutic enhancers. For example, in some instances, bile acid secretion inhibitors may also be included in separate compositions and / or with compositions comprising one or more therapeutic biologics and / or biliary therapeutic enhancers. Bile acid secretion inhibitors include, but are not limited to, agonists of the bile acid receptor (BAR), also known as farnesoid X receptor (FXR) or NR1H4 (nuclear receptor subfamily 1, group H, member 4). In certain embodiments, the bile acid secretion inhibitor is an FXR agonist. Any amount (dosage) of therapeutic biologic and biliary therapeutic enhancer can be used in the separate or combined compositions described herein. Such dosages can be easily determined.

[0071] Aspects of the present disclosure include pharmaceutical compositions for carrying out one or more of the methods described herein, which may include a therapeutic biologic and / or a biliary therapeutic enhancer appropriately formulated for administration as described herein. In some examples, a pharmaceutical composition may include a therapeutic biologic and two or more biliary therapeutic enhancers (e.g., a bile acid sequestrant and a cationic or nonionic amphipathic transduction enhancer) appropriately formulated for administration as described herein. In some examples, a pharmaceutical composition may include two or more therapeutic biologics and a biliary therapeutic enhancer appropriately formulated for administration as described herein.

[0072] The active agents of the pharmaceutical composition may be combined (i.e., as a composition of two or more active agents) or may be formulated individually in separate compositions. In some instances, the pharmaceutical compositions formulated individually with each active agent may be provided in the form of a kit for treating a subject with a combination treatment of two or more compositions, each having one or more active agents (e.g., a biologic therapeutic agent, a biliary therapeutic enhancer, a bile acid sequestrant, a cationic or non-ionic amphiphilic transduction enhancer, etc.), as described herein.

[0073] Pharmaceutical compositions containing one or more compounds may be administered to a patient alone or in combination with other co-active agents. Pharmaceutical compositions can be manufactured using any of a variety of processes, including but not limited to conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, encapsulating, and lyophilizing. Pharmaceutical compositions can take any of a variety of forms, including but not limited to sterile solutions, suspensions, emulsions, lyophilisates, tablets, pills, pellets, capsules, powders, syrups, elixirs, or any other dosage forms suitable for administration.

[0074] In some embodiments, the pharmaceutical compositions may be configured as liquid preparations or liquid suspensions that allow for delivery to the biliary system, including, for example, by injection or infusion. Suitable liquid preparations may be prepared just prior to use or delivery, or may be prepared well in advance and stored prior to use, depending on the desired formulation.

[0075] The compositions described herein that contain one or more compounds can be administered to the host using any convenient means that can bring about the desired reduction of disease symptoms or symptoms.Therefore, the compounds can be incorporated into various preparations for therapeutic administration.More specifically, the compounds can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable carriers or diluents, and can be formulated into various preparations.

[0076] Guidance for the formulation of pharmaceutical compositions is readily available. For example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and their components) suitable for pharmaceutical delivery of the disclosed compounds. Pharmaceutical compositions containing at least one of the compounds can be formulated for use in human or veterinary medicine. The particular formulation of the disclosed pharmaceutical compositions can depend, for example, on the mode of administration and / or the location of the affected area to be treated. In some embodiments, the formulation comprises a pharma- ceutically acceptable carrier in addition to at least one active ingredient. In other embodiments, other pharmaceuticals or agents, e.g., having similar, related, or complementary effects on the affliction to be treated, can also be included as active ingredients in the pharmaceutical composition.

[0077] Pharmaceutically acceptable carriers useful for the disclosed methods and compositions are readily available. The nature of the pharmaceutical carrier will depend on the particular mode of administration used. For example, liquid formulations will usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (e.g., water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc.) as excipients. For solid compositions (e.g., in powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may optionally contain minor amounts of non-toxic auxiliary substances (e.g., pharmaceutical excipients), such as wetting or emulsifying agents, preservatives, and pH buffering agents; for example, sodium acetate or sorbitan monolaurate. Other non-limiting pharmaceutical excipients include non-ionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations.

[0078] Some examples of substances which can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) pharmaceutical excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil. , soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0079] The disclosed pharmaceutical compositions and / or components thereof may be formulated as pharma- ceutically acceptable salts of the disclosed compounds. Pharmaceutically acceptable salts are non-toxic salts of the free base form of the compounds that have the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methylsulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists of other suitable pharma- ceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, PA, 1985. Pharmaceutically acceptable salts may also serve to adjust the osmolality of the composition.

[0080] Pharmaceutical compositions containing one or more component compounds can be formulated into injectable preparations by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, and, if desired, can be formulated together with conventional additives, such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0081] In some embodiments, the compositions described herein can be delivered by a continuous delivery system. The term "continuous delivery system" is used interchangeably herein with "controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are readily available.

[0082] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount (or a predetermined amount thereof) of the compositions described herein calculated in an amount sufficient to produce the desired effect in association with a pharma- ceutically acceptable diluent, carrier, or excipient. The specifications of the compositions described herein depend on the particular compounds used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dosage form of the disclosed pharmaceutical compositions is determined by the mode of administration selected.

[0083] Each therapeutic compound or individual component of the composition may be independently in any dosage form, such as those described herein, and may be administered in various ways, as described herein. For example, the compounds may be formulated together in a single dosage unit (i.e., combined together in one form, such as capsule, tablet, powder, liquid, or suspension) as a combination product. Alternatively, when not formulated together in a single dosage unit, an individual compound may be administered simultaneously with another therapeutic compound, or sequentially in any order. In some instances, for liquid delivery of an amount or dosage provided in a solid form (e.g., capsule, tablet, powder, etc.), the solid form may be dissolved, suspended, emulsified, rehydrated, etc., prior to use.

[0084] As described in more detail herein, the order of delivery of the compositions (steps in the described methods) may vary. For example, but not limited to, in some instances, the method may include contacting a biliary therapeutic enhancer with bile followed by adding or administering a biologic therapeutic. In some embodiments, a biliary therapeutic enhancer composition may be delivered directly to the biliary system, and then a biologic composition may be administered directly to the biliary system, for example, by the same route. Thus, the biliary therapeutic enhancer and the biologic may be combined in vivo. In some instances, the biliary therapeutic enhancer and the biologic therapeutic may be contacted with bile simultaneously or essentially simultaneously. In some embodiments, the biliary therapeutic enhancer may be mixed with the biologic prior to administration (ex vivo) and prior to direct delivery to the biliary system. In some instances, a composition including both a biliary therapeutic enhancer and a biologic therapeutic may be contacted with bile together. The compositions described herein, e.g., combination compositions, can be prepared at any time prior to administration, including immediately before, seconds, minutes, hours, days, or months prior to administration. In certain embodiments, the compositions can be prepared well before administration, and the compositions can then be administered directly to the biliary system. Furthermore, the sequential and / or simultaneous administration of the compositions described herein can be performed in any order and at any number of times, including, but not limited to, one or more sequential and / or simultaneous administrations of the same or different compositions.

[0085] As summarized above, the methods of the present disclosure may include contacting a bile-containing solution with a biliary therapeutic enhancer, therapeutic biologic, and / or one or more compositions as described herein, including when the solution is subsequently or simultaneously contacted with a therapeutic biologic, biliary therapeutic enhancer, therapeutic biologic, and / or one or more compositions. Bile-containing solutions vary and include essentially any solution of bile, or a liquid containing bile or primary bile components. Typically, the bile-containing solution includes bile from a subject treated with a composition or according to a method described herein. Bile is primarily composed of conjugated bile salts, cholesterol, phospholipids, bilirubin, and electrolytes, resulting in a clear, yellow, or orange liquid. Bile is produced by the liver, stored as a concentrate in the gallbladder, and released into the small intestine via the bile duct when needed for digestion. Bile aids in alkalinizing intestinal contents and emulsifying, absorbing, and digesting fats. Bile salts produced in the liver are made from bile acids conjugated with glycine or taurine. Glyco- and tauro-bile acids are also called conjugated bile acids. Glycine or taurine conjugation increases the water solubility of bile salts. Bile acids are steroid acids derived from cholesterol and can be classified as primary (i.e., those synthesized in the liver, e.g., cholic acid and chenodeoxycholic acid) and secondary (i.e., those produced from primary bile acids by intestinal bacteria and returned to the liver by the enterohepatic circulation, e.g., deoxycholic acid and lithocholic acid). The human liver synthesizes approximately 200-600 mg of bile acids per day.

[0086] Non-limiting examples of bile-containing solutions include bile (e.g., naturally occurring in the bile duct; extracted from a subject;), diluted bile (e.g., present in the bile duct after addition of or washing with a diluent, buffer, medium, or other solution; prepared ex vivo from bile;), and the like. The bile solution may vary, for example, 0.01% or less bile, 0.01% or more bile, 0.1% or less bile, 0.1% or more bile, 1% or less bile, 1% or more bile, 2% or less bile, 2% or more bile, 3% or less bile, 3% or more bile, 4% or less bile, 4% or more bile, 5% or less bile, 5% or more bile, 6% or less bile, 6% or more bile, 7% or less bile, 7% or more bile, 8% or less bile, 8% or more bile, 9% or less bile, 9% or more bile, 10% or less bile, 10% or more bile, 15% or less bile, 15% or more bile, 20% or less bile, 20% or more bile, 25% or less bile. , 25% or more bile, 30% or less bile, 30% or more bile, 40% or less bile, 40% or more bile, 50% or less bile, 50% or more bile, 60% or less bile, 60% or more bile, 70% or less bile, 70% or more bile, 75% or less bile, 75% or more bile, 80% or less bile, 80% or more bile, 90% or less bile, 90% or more bile, 95% or less bile, 95% or more bile, 97% or less bile, 97% or more bile, 98% or less bile, 98% or more bile, 99% or less bile, 99% or more bile, or 100% bile.

[0087] Useful biliary therapeutic enhancers are varied and may in some instances include polymers or resins, hi some instances, useful biliary therapeutic enhancers include polyamine polymers or polyether polymers.

[0088] Useful biliary therapeutic enhancers include, but are not limited to, bile acid sequestrants. In some examples, useful bile acid sequestrants include polymeric and / or resinous bile acid sequestrants, such as polyamine polymeric bile acid sequestrants and / or polyether polymeric bile acid sequestrants.

[0089] Bile acid sequestrants include drugs that belong to a class of polymeric resins that bind to bile acids in the gastrointestinal tract and prevent their recirculation. Traditionally, this class of drugs is administered orally and used to treat high serum cholesterol levels. Bile acid sequestrants are not significantly absorbed from the intestine into the bloodstream. Thus, the bile acid sequestrants, along with any bile acids bound to the drug, are excreted via the fecal route after passing through the gastrointestinal tract. Non-limiting examples of useful bile acid sequestrants include colesevelam, colestyramine (also known as cholestyramine), colestipol, sevelamer, and various salts and any derivatives thereof. In some instances, a binding enhancer, such as but not limited to oleic acid, may be used or combined with one or more of the subject bile acid sequestrants. In general, such binding enhancers increase the bile acid binding capacity of the subject bile acid sequestrant when present together in solution, for example, but not limited to, enhance the binding of sevelamer when present together with oleic acid.

[0090] Useful bile acid sequestrants also include those described in U.S. Pat. Nos. 5,607,669, 5,679,717, and 7,229,613, U.S. Patent Publication No. 201000331516, and Camilleri & Gores, Am J Physiol Gastrointerst Liver Physiol, (2015) 309(4) G209-G 215, the disclosures of which are incorporated herein by reference in their entireties.

[0091] Bile acid sequestrants include crosslinked polymers or cationic hydrogels with ammonium groups, whose sequestration activity is mainly based on the electrostatic interaction between the cationically charged polymer and the anionically charged bile acid.Suitable bile acid sequestrants include, for example, amphiphilic polymers based on poly(meth)acrylates, poly(meth)acrylamides, polyalkylamines and polyarylamines containing quaternary ammonium groups; cyclodextrins and other sugar-based sequestrants; and sequestrants prepared via molecular imprinting methods that selectively bind bile acids and salts.

[0092] Useful biliary therapeutic enhancers include, but are not limited to, transduction enhancers. The term "transduction enhancer" as used herein generally refers to an agent used to enhance the efficiency of in vitro transduction (or transfection) reactions. This term includes agents useful in both viral and non-viral transduction of nucleic acid into target cells, so useful transduction enhancers include agents that enhance both viral and non-viral transduction and transfection reactions. Transduction enhancers differ in their mechanism of enhancing transduction and / or transfection. Any amount of one or more enhancers can be used, and such amounts can be easily determined.

[0093] Non-limiting examples of transduction enhancers include 16,16-dimethyl prostaglandin E2, 1-oleoyl lysophosphatidylcholine, AdenoBlast™ (Nordic Diagnostica AB), AdenoBOOST™ (Sirion-Biotech GmbH), Akti-1 / 2, BX795, cholesterol-linked oligonucleotides, chondroitan-based proteoglycans, cyclosporin A, cyclosporin H, daunorubicin hydrochloride, DEAE-dextran, dexamethasone, Eeyarestatin I, etoposide, F108, F127, glycerol, integrin-binding peptides, LentiBlast™ (Nordic Diagnostica AB), Lenti ... GmbH), linear dextran nonasaccharide, lysophosphatide, lysophosphatidylcholine, lysophosphatidylethanolamine, lysosomal disrupting peptide, MG132, PF03814735, poly(ethylene glycol), polybrene, polyamine (PEI), poly-L-lysine, prostaglandin E2, protamine sulfate, Protransduzin™ (JPT Peptide Technologies Inc.), rosuvastatin calcium, SAHA, staurosporine, sulfated proteoglycan, Transdux™ MAX (System Biosciences, LLC.), TransPlus™ (ALSTEM, INC.), and the like.

[0094] Transduction enhancers useful as biliary therapeutic enhancers include, for example, cationic or non-ionic amphipathic transduction enhancers. In some embodiments, a cationic transduction enhancer or a combination of multiple different cationic transduction enhancers may be used. In some embodiments, a non-ionic amphipathic transduction enhancer or a combination of multiple different non-ionic amphipathic transduction enhancers may be used. In some embodiments, a combination of a cationic transduction enhancer and a non-ionic amphipathic transduction enhancer, a combination of two or more different cationic transduction enhancers and a non-ionic amphipathic transduction enhancer, or a combination of a cationic transduction enhancer and two or more different non-ionic amphipathic transduction enhancers may be used. In some instances, useful transduction enhancers, e.g., cationic or non-ionic amphipathic transduction enhancers, may include polymeric transduction enhancers, e.g., cationic polymers or non-ionic polymeric amphipathic transduction enhancers, e.g., polyamine polymeric transduction enhancers and / or polyether polymeric transduction enhancers. In some instances, useful cationic or non-ionic amphipathic transduction enhancers may be polybrene (1,5-dimethyl-1,5-diaza-undeca-methyl-polymethobromide), protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, or F108. In some examples, useful transduction enhancers include those described in U.S. Pat. Nos. 9,771,599 and 10,815,498, U.S. Patent Publication Nos. 20020019358, 20150064788, 20180016600, and 20200124505, and PCT Publication Nos. WO2020197400 and WO2018208960, the disclosures of which are incorporated by reference in their entireties.

[0095] In some instances, useful transduction enhancers are poloxamers, including but not limited to poloxamers having molecular weights of, for example, 12.8 kDa to about 15 kDa. The term "poloxamer" is easily understood and refers to a non-ionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. Suitable poloxamer transduction enhancers include triblock copolymers (Pluronics) containing various ratios of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). Examples of poloxamer transduction enhancers are Pluronic® F108 or Synperonic® F108 (HO-[CH2CH 20 ] x -[CH2C2H 40 ] Z -[CH2CH 20 ] y where: x+y =265.45 and z =50.34).

[0096] In some embodiments, the bile acid sequestrant comprises a compound having formula (I): [ka] During the ceremony, p is an integer from 1 to 3 X is an electrophilic leaving group; R a is hydrogen, C1-C 20 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C 20 alkyl ammonium groups; The alkyl is optionally substituted with OH or alkylammonium.

[0097] In some embodiments, for compounds of formula (I), X is selected from Cl or Br. In some embodiments, X is Cl. In some embodiments, for compounds of formula (I), Ra is H or C1-C 20 In some embodiments, R a is hydrogen or optionally substituted C1-C 20 is selected from alkyl.

[0098] In some embodiments, the bile acid sequestrant comprises a compound of formula (I) having structure (Ia): [ka] wherein (a) represents an allylamine monomer unit that is not alkylated with either 1-bromodecane or (6-bromohexyl)-trimethylammonium bromide alkylating agents and is not crosslinked with epichlorohydrin, (b) represents an allylamine unit crosslinked with epichlorohydrin, (c) represents an allylamine unit alkylated with a decyl group, and (d) represents an allylamine unit alkylated with a (6-trimethylammonium)hexyl group. In some embodiments, a, b, c, and d are each independently present or absent.

[0099] In some embodiments, the bile acid sequestrant comprises a compound having the formula (II): [ka] During the ceremony, R 1 is C6~C 10 Aryl or C2-C 10 and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with 1 to 3 substituents selected from the group consisting of C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, or an alkyl ammonium halide group. In some embodiments, for a compound of formula (II), R 1 is a C6-C alkyl or halogenated alkyl ammonium group optionally substituted with 10 In some embodiments, R 1is benzyl optionally substituted with a C1-C6 alkyl or alkyl ammonium halide group. 1 is benzyl substituted with a C1-C3 alkyl group. 1 is CH2N + (CH3)3Cl - Benzyl substituted with a group.

[0100] In some embodiments, the bile acid sequestrant comprises a compound of formula (II) having the structure (IIa): [ka]

[0101] In some embodiments, the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer, including a compound having formula (III): [ka] During the ceremony, Each of x, y, and z is independently an integer of 1 to 250.

[0102] In some embodiments, the bile acid sequestrant comprises a compound having the formula (III), where x is an integer between 10 and 150, y is an integer between 10 and 100, and z is an integer between 10 and 150. In some embodiments, the bile acid sequestrant comprises a compound having the formula (III), where x and z are each between 130 and 140, and y is between 40 and 60.

[0103] In some embodiments, the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer, including a compound having formula (IVa) or formula (IVb): [ka] During the ceremony, x is an integer from 1 to 6; R 1is an amino group optionally substituted with one or more C1-C6 alkyl groups.

[0104] In some embodiments, the bile acid sequestrant comprises a compound having the formula (V): [ka] During the ceremony, R 1 and R 2 Each of is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group.

[0105] In some embodiments, the bile acid sequestrant comprises a compound of formula (V) having structure (Va): [ka]

[0106] In some embodiments, the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer, including compounds having formula (VI): [ka] During the ceremony, x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R a are independently hydrogen or a C1 to C6 alkyl group.

[0107] In some embodiments, for compounds of formula (I), X is selected from Cl or Br. In some embodiments, X is Br. In some embodiments, each Ra is hydrogen. In some embodiments, each Ra is C1-C3 alkyl. In some embodiments, each Ra is methyl.

[0108] In some embodiments, the bile acid sequestrant comprises a compound of formula (VI) having structure (VIa): [ka]

[0109] In some embodiments, the bile acid sequestrant comprises a compound having formula (VII): [ka] During the ceremony, a is an integer from 1 to 6; b is an integer from 1 to 6; and c is an integer from 1 to 3.

[0110] In some embodiments, the bile acid sequestrant comprises a compound of formula (VII), where a and b represent the number of primary amine groups, c represents the number of crosslinking groups, and m represents a large number indicating an extended polymer network. In some embodiments, a, b, and c are each independently present or absent. In some embodiments, a+b=9 and c=1.

[0111] In all of the above compounds of formula (I)-(VII), the total number of repeating units can be varied to achieve the desired molecular weight depending on the desired biological activity. Thus, the value of n or m in the compounds of formula (I)-(VII) can vary from 3 to 10,000, 3 to 1,000, 3 to 500, 3 to 300, or 3 to 100, or any value between and including these values. The desired average molecular weight (MW) of each of the compounds of formula (I), (Ia), (II), (IIa), (III), (IVa), (IVb), (V), (Va), (VI), (VIa), and (VII) can be in the range of about 100 to about 500,000 g / mol or Daltons (including about 100 to about 1000, about 1000 to about 5000, about 5000 to about 10000, about 10000 to about 50,000, about 50,000 to about 100,000, about 100,000 to about 300,000, about 300,000 to about 500,000), and can be in the range between and including any two of these values. In some embodiments, the compound of formula (I) can have values ​​of m and n corresponding to a MW of about 500 to about 1000. In some embodiments, the compound of formula (Ia) may have a value of m corresponding to a MW of about 500 to about 1000. In some embodiments, the compound of formula (II) and (IIa) may have a value of n corresponding to a MW of about 100 to about 500. In some embodiments, the compound of formula (III) may have values ​​of x, y, and z corresponding to a MW of about 10,000 to about 15,000. In some embodiments, the compound of formula (IVa) and (IVb) may have a value of n corresponding to a MW of about 50,000 to about 200,000. In some embodiments, the compound of formula (V) may have values ​​of m and n corresponding to a MW of about 100 to about 500. In some embodiments, the compound of formula (VI) and (VIa) may have a value of n corresponding to a MW of about 100 to about 600. In some embodiments, the compound of formula (VII) may have a value of m corresponding to a MW of about 100 to about 500.

[0112] As summarized above, in some embodiments of the methods described herein, the biliary therapeutic enhancer may be administered prior to the therapeutic biologic. Correspondingly, in some embodiments, the target cells of the biologic may be contacted with the biliary therapeutic enhancer prior to contact with the therapeutic biologic. In some embodiments of the methods described herein, the biliary therapeutic enhancer may be administered together (including simultaneously or essentially simultaneously) with the therapeutic biologic. Correspondingly, in some embodiments, the target cells of the biologic may be contacted simultaneously or essentially simultaneously with the biliary therapeutic enhancer and the therapeutic biologic. As described herein, in some embodiments, the biliary therapeutic enhancer and the therapeutic biologic may be co-administered. In some embodiments, the methods of the present disclosure may use co-formulation, including, for example, when the biliary therapeutic enhancer and the therapeutic biologic are co-formulated.

[0113] In some embodiments, two or more biliary therapeutic enhancers may be used, including, but not limited to, when, for example, 2-10, 3-10, 4-10, 5-10, 2-5, 3-5, 2, 2 or more, 3, 3 or more, 4, 4 or more, 5, 5 or more, 6, 6 or more, 7, or 7 or more biliary therapeutic enhancers are used. In some embodiments, at least one bile acid sequestrant and at least one transduction enhancer may be used. In some examples, one bile acid sequestrant and at least two transduction enhancers may be used. In some examples, two or more bile acid sequestrants may be used. When a combination of two or more biliary therapeutic enhancers is used, the individual elements of the combination may be administered and / or contacted with the target cell simultaneously, essentially simultaneously, or sequentially. Thus, a combination of two or more biliary therapeutic enhancers may be co-administered and / or co-formulated.

[0114] In some instances, a combination of at least colesevelam and at least polybrene may be used, including, for example, when colesevelam and polybrene are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colesevelam and at least protamine sulfate may be used, including, for example, when colesevelam and protamine sulfate are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colesevelam and at least polyethyleneimine (PEI) may be used, including, for example, when colesevelam and polyethyleneimine (PEI) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colesevelam and at least poly(ethylene glycol) (PEG) may be used, including, for example, when colesevelam and poly(ethylene glycol) (PEG) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colesevelam and at least poly-L-lysine may be used, including, for example, when colesevelam and poly-L-lysine are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colesevelam and at least F108 may be used, including, for example, when colesevelam and F108 are co-administered, co-formulated, or administered sequentially.

[0115] In some instances, a combination of at least cholestyramine and at least polybrene may be used, including, for example, when cholestyramine and polybrene are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least cholestyramine and at least protamine sulfate may be used, including, for example, when cholestyramine and protamine sulfate are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least cholestyramine and at least polyethyleneimine (PEI) may be used, including, for example, when cholestyramine and polyethyleneimine (PEI) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least cholestyramine and at least poly(ethylene glycol) (PEG) may be used, including, for example, when cholestyramine and poly(ethylene glycol) (PEG) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least cholestyramine and at least poly-L-lysine may be used, including, for example, when cholestyramine and poly-L-lysine are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least cholestyramine and at least F108 may be used, including, for example, when cholestyramine and F108 are co-administered, co-formulated, or administered sequentially.

[0116] In some examples, a combination of at least colestipol and at least polybrene may be used, including, for example, when colestipol and polybrene are co-administered, co-formulated, or administered sequentially. In some examples, a combination of at least colestipol and at least protamine sulfate may be used, including, for example, when colestipol and protamine sulfate are co-administered, co-formulated, or administered sequentially. In some examples, a combination of at least colestipol and at least polyethyleneimine (PEI) may be used, including, for example, when colestipol and polyethyleneimine (PEI) are co-administered, co-formulated, or administered sequentially. In some examples, a combination of at least colestipol and at least poly(ethylene glycol) (PEG) may be used, including, for example, when colestipol and poly(ethylene glycol) (PEG) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colestipol and at least poly-L-lysine may be used, including, for example, when colestipol and poly-L-lysine are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least colestipol and at least F108 may be used, including, for example, when colestipol and F108 are co-administered, co-formulated, or administered sequentially.

[0117] In some instances, a combination of at least sevelamer and at least polybrene may be used, including, for example, when sevelamer and polybrene are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least sevelamer and at least protamine sulfate may be used, including, for example, when sevelamer and protamine sulfate are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least sevelamer and at least polyethyleneimine (PEI) may be used, including, for example, when sevelamer and polyethyleneimine (PEI) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least sevelamer and at least poly(ethylene glycol) (PEG) may be used, including, for example, when sevelamer and poly(ethylene glycol) (PEG) are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least sevelamer and at least poly-L-lysine may be used, including, for example, when sevelamer and poly-L-lysine are co-administered, co-formulated, or administered sequentially. In some instances, a combination of at least sevelamer and at least F108 may be used, including, for example, when sevelamer and F108 are co-administered, co-formulated, or administered sequentially.

[0118] The above combinations are merely representative and non-limiting. As provided above, the present disclosure expressly contemplates and describes all combinations of the biliary therapeutic enhancers described herein, as well as such combinations with all therapeutic biologics described herein.

[0119] A variety of therapeutic biologics are used in the methods, compositions, and kits described herein. The term "therapeutic biologic" is used interchangeably herein with "biologic therapeutic agent," "biologic," and "therapeutic agent," and generally refers to a therapeutically active agent that is derived from, consists of, or at least partially comprises modified or unmodified polynucleotides or polypeptides, and combinations / compositions thereof. Biologies may be isolated from a variety of sources, including, but not limited to, human, animal, or microbial, or may be produced by biotechnology or recombinant methods. Biologies may be wholly or partially synthetic, and may include, for example, modified forms of naturally occurring polynucleotides or polypeptides, etc.

[0120] In some embodiments, the therapeutic biologics used in the methods described herein may be susceptible to inactivation by bile. Such bile-labile activity of a biologic may be partially or completely inactivated by the presence of bile. The mechanism of inactivation of a particular biologic by bile varies depending on various factors, including the nature of the biologic, the mechanism of therapeutic action of the biologic, the compositional makeup of the biologic, and the like. For example, in some instances, a bile-labile biologic may be degraded, including partially or completely degraded, in the presence of bile. As a non-exclusive and non-limiting example, a biologic that includes a lipid component, such as, but not limited to, an enveloped viral vector, lipid nanoparticles, and the like, may be degraded in the presence of bile, thus rendering such bile-labile biologics inactive. As another non-exclusive and non-limiting example, the activity of the biologic, or some events necessary for the activity of the biologic, such as binding to a receptor, ligand, cell, and the like, may be blocked, slowed down, or otherwise inhibited by one or more components of bile, such that the biologic is functionally inactive in the presence of bile. As another non-exclusive and non-limiting example, the chemical characteristics of a biologic may be altered by the presence of bile, rendering the biologic inactive.

[0121] In some embodiments, a therapeutic biologic useful in the methods described herein may be a gene therapy agent. The term "gene therapy" as used herein refers to the delivery of a nucleic acid therapeutic, sometimes referred to as a gene payload, to a cell of a subject to treat a condition of the subject. Thus, as used herein, "gene therapy agent" generally refers to a therapeutic nucleic acid and any associated components, if any, used in the delivery of the nucleic acid to a cell, such as a delivery vehicle and vector. The nucleic acid of a gene therapy agent can vary and provide a variety of functions, including, for example, correcting a defective gene in a host cell, encoding and / or expressing a heterologous gene product in a cell, encoding and / or expressing one or more additional copies of an endogenous gene product in a cell, inhibiting expression of a gene or gene product in a cell, and the like. Gene therapy agents include, but are not limited to, viral and / or non-viral vectors carrying one or more polynucleotides encoding one or more proteins (e.g., therapeutic proteins, antibodies, etc.). Gene therapy can be administered in vitro to cells within an organism or ex vivo to cells removed from the organism.

[0122] Examples of nucleic acid therapeutics useful in gene therapy include, but are not limited to, expression cassettes, recombinant mRNA, recombinant vector genomes (e.g., recombinant viral genomes, etc.), recombinant plasmids, minicircle plasmids, minigenes, microgenes, artificial chromosomes, interfering nucleic acids (e.g., siRNAs, shRNAs, etc.). In carrying out gene therapy, nucleic acid therapeutics can be delivered in a variety of ways, including, but not limited to, as naked nucleic acids (e.g., naked DNA, naked plasmids, etc.), as nucleic acid-protein complexes (e.g., DNA-protein complexes, RNA-protein complexes such as ribonucleoprotein (RNP) complexes, etc.), as nucleic acids in viral vectors, as nucleic acids in non-viral vectors, etc. In some examples, proteins can be incorporated into gene therapy agents, such as, but not limited to, DNA-binding proteins, RNA-binding proteins, enzymes such as nucleases, and combinations thereof.

[0123] Useful gene therapy agents include viral vectors. In some embodiments, useful viral vectors include non-replicating (i.e., replication-defective) viral vectors. Viral vectors can be integrating or non-integrating. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, and the like. Useful gene therapy agents also include non-viral vectors, which are delivery vehicles that do not use viral particles and can generally be considered to be classified into three categories: naked nucleic acid, particle-based (e.g., nanoparticles), or chemical-based. Non-limiting examples of non-viral vectors include lipoplexes (e.g., cationic lipid-based lipoplexes), emulsions (e.g., lipid nanoemulsions, etc.), lipid nanoparticles (LNPs), solid lipid nanoparticles, peptide-based vectors, polymer-based vectors (e.g., polymersomes, polyplexes, polyethyleneimine (PEI)-based vectors, chitosan-based vectors, poly(DL-lactide) (PLA) and poly(DL-lactide-co-glycoside) (PLGA)-based vectors, dendrimers, vinyl-based polymers (e.g., polymethacrylate-based vectors), etc.), inorganic nanoparticles, etc.

[0124] Gene therapy agents (for delivery of nucleic acid therapeutics) may further include promoter sequences (e.g., constitutive, tissue-specific, etc.), terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and / or locus control regions. Additionally, multiple nucleic acid therapeutics may be expressed from one gene therapy agent by linking the individual components (transgenes) in one open reading frame separated by, for example, a self-cleaving 2A peptide or an IRES sequence.

[0125] Vectors, including retroviral vectors, e.g., lentiviral vectors, may include (or optionally exclude) a variety of elements, including cis-acting elements such as promoters, long terminal repeats (LTRs) and / or elements thereof, including 5'LTR and 3'LTR and elements thereof, central polypurine tract (cPPT) elements, DNA flap (FLAP) elements, export elements (e.g., rev response element (RRE), Hepatitis B virus post-transcriptional regulatory element (HPRE), etc.), post-transcriptional regulatory elements (e.g., Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE), Hepatitis B virus Regulatory Element (HPRE), etc.), polyadenylation sites, transcription termination signals, insulator elements (e.g., the β-globin insulator, e.g., chicken HS4), and the like. Other elements that may or may not be present in various vectors include, but are not limited to, enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, regions of homology useful in homology directed repair (HDR), and the like.

[0126] Useful LTRs include, but are not limited to, LTRs that include, for example, U3, R and / or U5 regions and portions thereof. LTRs provide functions for retroviral gene expression (e.g., promoting, initiating and polyadenylation of gene transcripts) and viral replication. LTRs can include multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. U3 regions can include enhancer and promoter elements. U5 regions can include polyadenylation sequences. R (repeat) regions are generally adjacent to U3 and U5 regions. LTRs composed of U3, R and U5 regions can appear at both the 5' and 3' ends of the viral genome. Viral genomes can include sequences adjacent to the 5'LTR that function in reverse transcription of the genome, such as for efficient packaging of viral RNA into particles (e.g., Psi site).

[0127] In various embodiments, the vector comprises a modified 5'LTR and / or 3'LTR. Modification of the 3'LTR is often done to improve the safety of lentiviral or retroviral systems by making the virus replication-deficient. As used herein, the term "replication-deficient" refers to a virus that is unable to replicate completely and efficiently, such that infectious virions are not produced (e.g., replication-deficient lentiviral progeny). The term "replication-competent" refers to a wild-type or mutant virus that can replicate, such that viral replication of the virus can produce infectious virions (e.g., replication-competent lentiviral progeny).

[0128] In some embodiments, useful vectors may be self-inactivating. The term "self-inactivating" (SIN) with respect to vectors refers to replication-deficient vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, including the U3 region, has been modified (e.g., by deletion and / or substitution) to prevent viral transcription beyond the first round of viral replication. In further embodiments, the 3'LTR may be modified such that the U5 region is replaced with, e.g., a heterologous or synthetic poly(A) sequence, one or more insulator elements, and / or an inducible promoter. It is readily apparent to one of skill in the art that reference to an LTR, e.g., a 3'LTR or a 5'LTR, may include modified LTRs or modifications to LTRs, e.g., modifications to the 3'LTR, 5'LTR, or both the 3'LTR and the 5'LTR.

[0129] In some instances, the retroviral vector may include a heterologous promoter. For example, the U3 region of the 5'LTR may be replaced with a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of useful heterologous promoters include, for example, viral Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), Herpes Simplex Virus (HSV), Spleen Focus Forming Virus (SFFV) promoters, and the like. In certain embodiments, the heterologous promoter may be inducible, such that transcription of all or part of the viral genome occurs only in the presence of one or more inducers. Inducers include, but are not limited to, one or more compounds or physiological conditions under which the host cells are cultured, such as temperature or pH.

[0130] In some embodiments, the viral vector may contain a TAR element. The term "TAR" refers to the "transactivation response" genetic element located in the R region of lentivirus (e.g., HIV) LTR. This element interacts with the lentivirus transactivator (tat) genetic element to enhance viral replication. In some embodiments, the vector may not contain a TAR element, including, for example, when the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0131] In some instances, the vector may be a pseudotyped vector. The term "pseudotype" or "pseudotyping" as used herein refers to a virus having one or more viral envelope proteins replaced with an envelope protein of another virus having preferred characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G protein (VSV-G) envelope protein. In some embodiments, lentivirus envelope protein is pseudotyped with VSV-G. In some embodiments, packaging cells can be used to produce recombinant retroviruses, e.g., lentiviruses, pseudotyped with VSV-G envelope glycoprotein.

[0132] Vectors (both viral and non-viral) may contain structural and / or genetic elements, or portions thereof, from viruses. Retroviral vectors may contain structural and / or genetic elements, or portions thereof, from retroviruses. Lentiviral vectors may contain structural and functional genetic elements, or portions thereof, including LTRs, primarily from lentiviruses. In some instances, hybrid vectors may be used, including, for example, where the hybrid vector contains LTRs or other nucleic acids that contain both retroviral, e.g., lentiviral, and non-retroviral, e.g., non-lentiviral, viral sequences. In some embodiments, the hybrid vector may contain vectors that contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0133] In some examples, the vectors used in the compositions and methods described herein may include lipid components, such as, but not limited to, enveloped viral vectors, lipid nanoparticles, etc. In some examples, the vectors may not have lipid components. In some examples, one or more biliary therapeutic enhancers used in the compositions and methods described herein may not include any components of the vector (such as lipids or polymers present in the non-viral vectors also used in the methods) (or may exclude all components). In some examples, one or more vectors may not include any components of the biliary therapeutic enhancers (or may exclude all components), such as transduction enhancers or components thereof present in the enhancers also used in the methods.

[0134] Any vector used in the compositions and methods described herein may be recombinant, comprising one or more heterologous coding sequences. For example, in some instances, the vector may comprise a coding sequence for a heterologous protein or peptide useful for treating a condition in a subject. In some instances, the heterologous protein or peptide encoded by the vector may be a liver-directed therapeutic, such as a liver transcription factor, a ligand for a receptor expressed on hepatocytes, an antibody (such as a monoclonal antibody) that specifically binds to an epitope of a protein expressed by hepatocytes, an enzyme active on hepatocytes or within the liver microenvironment, etc. In some instances, the vector may comprise a therapeutic nucleic acid that inhibits expression of a liver-expressed gene, such as, but not limited to, an interfering nucleic acid that inhibits expression of a gene product in hepatocytes. In some instances, hepatocytes may be used to express, by the delivered vector, a heterologous gene product useful in treating a condition other than a hepatic condition. As a non-limiting example, a vector encoding a hormone, such as insulin, may be delivered to hepatocytes such that the hormone is expressed and secreted by hepatocytes to treat a non-hepatic condition, such as diabetes.

[0135] A variety of payloads can be delivered by gene therapy agents, including, but not limited to, non-coding nucleic acids, as well as nucleic acids encoding one or more proteins and / or peptides, including, but not limited to, one or more of the therapeutic proteins and / or peptides described herein. In some embodiments, the payload can include a nucleic acid sequence encoding an enzyme, such as, for example, a nuclease, a DNA base editor, an RNA editor, etc. In some embodiments, the payload can include a non-coding nucleic acid, such as, for example, a microRNA (i.e., miRNA), shRNA, siRNA, piRNA, snoRNA, snRNA, exRNA, sCaRNA, lncRNA, guide RNA (gRNA, sgRNA, etc.), alone or in combination with other payload elements.

[0136] In some embodiments, the gene therapy payload may include elements for editing the target locus. For example, the gene therapy vector may include an exogenous template nucleic acid that includes regions of homology to the target site flanking the sequence that includes the desired edit. The gene editing payload may repair or otherwise introduce the desired edit at the target locus by various mechanisms, including, for example, homology-directed repair (HDR). Thus, in some instances, the gene therapy payload may not include a coding sequence, such as, for example, a coding sequence that encodes a transgene, a therapeutic polypeptide or peptide, etc. Correspondingly, in some instances, the gene therapy payload may exclude one or more elements for expression, including, for example, but not limited to, those described herein (e.g., promoters, terminators, enhancers, polyadenylation sequences, etc.). In some instances, the gene therapy does not express a protein or polypeptide from a coding sequence and / or does not express a non-coding nucleic acid.

[0137] The homology regions targeted to a genomic locus may be referred to as "homology arms" or separately as "5' homology arms" and "3' homology arms," ​​which share homology to the endogenous nucleic acid 5' and 3' of the target site, respectively. Homology arms may vary and range in size from 200 nt or less to 2000 nt or more, including but not limited to 200 nt or more, 500 nt or more, 500 nt to 1000 nt, etc. Essentially any nucleic acid edit can be introduced, and useful edits can include a single nucleotide edit (i.e., a change of one base for another, e.g., a base change of A to C, A to T, A to G, C to A, C to G, C to T, G to A, G to C, G to T, T to A, T to C, or T to G), a change of two or more nucleotides (i.e., a change of another base at two or more sites), a single nucleotide insertion, an insertion of two or more nucleotides, a single codon insertion (i.e., an insertion of three nucleotides), an insertion of two or more codons, an insertion of a heterologous coding sequence, a single nucleotide deletion, a deletion of two or more nucleotides, a deletion of one or more codons, a deletion of one or more exons, a deletion of a coding region or a portion thereof, a deletion of a non-coding region or a portion thereof, a gene replacement, a replacement of a portion of a gene, and the like.

[0138] Gene therapy agents containing a gene editing payload can include a variety of other elements, including, but not limited to, for example, one or more guide RNAs (e.g., gRNA, sgRNA, etc.), a nuclease (e.g., a Cas nuclease (e.g., Cas9), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, etc.) or a sequence encoding one or more nucleases, a nickase or a sequence encoding one or more nickases, a base editing enzyme (e.g., a cytosine base editor, an adenine base editor, a dual deaminase editor, etc.) or a sequence encoding one or more base editing enzymes, etc.

[0139] In some embodiments, the therapeutic biologics useful in the methods described herein may be therapeutic proteins or peptides. Useful therapeutic proteins and peptides include, but are not limited to, secreted factors, hormones, chemokines, cytokines, transcription factors, ligands for receptors, receptor-blocking proteins and peptides, enzymes, extracellular matrix proteins, signal transduction proteins, antibodies, and the like. In certain embodiments, the therapeutic proteins include functional proteins that are lacking (i.e., missing and / or absent) in liver disease.

[0140] Non-limiting examples of therapeutic proteins and peptides include lepirudin, cetuximab, dornase alfa, denileukin diftitox, etanercept, bivalirudin, leuprolide, peginterferon alfa-2a, alteplase, interferon alfa-n1, darbepoetin alfa, reteplase, epoetin alfa, salmon calcitonin, interferon alfa-n3, pegfilgrastim, sargramostim, secretin, peginterferon alfa-2b, asparaginase, thyrotropin alfa, antihemophilic factor, anakinra, gramicidin D, intravenous immunoglobulin, anistreplase, insulin regular, tenecteplase, menotropin, interferon gamma-1b, interferon alpha-2a, recombinant, coagulation factor VIIa, oprelvekin, palifermin, glucagon recombinant, aldesleukin, botulinum toxin type B, omalizumab, lutropin alfa, insulin lispro, insulin glargine, collagenase, rasburicase, adalimumab, imiglucerase, abciximab, alpha-1 proteinase inhibitor, pe Guasparagase, interferon beta-1a. Pegademase, bovine, human serum albumin, eptifibatide, iodized human serum albumin, infliximab, follitropin beta, vasopressin, interferon beta-1b, interferon alfacon-1, hyaluronidase, insulin, porcine, trastuzumab, rituximab, basiliximab, muromonab, digoxin immune Fab (ovine), ibritumomab, daptomycin, tositumomab, pegvisomant, botulinum toxin type A, pancrelipase, streptokinase, alemtuzumab , alglucerase, capromab, laronidase, urofollitropin, efalizumab, serum albumin, choriogonadotropin alfa, antithymocyte globulin, filgrastim, coagulation factor IX, becapremin, agalsidase beta, interferon alfa-2b, oxytocin, enfuvirtide, palivizumab, daclizumab, bevacizumab, arcitumomab, eculizumab, panitumumab, ranibizumab, idursulfase, alglucosidase alfa, exenatide, mecasermin, pramlintide, galsulfase, abatacept,Cosyntropin, corticotropin, insulin aptamer, insulin detemir, insulin glulisine, pegaptanib, nesiritide, thymalfasin, defibrotide, natural alpha interferon OR multiferon, glatiramer acetate, preotactic acid, teicoplanin, canakinumab, ipilimumab, sulodexide, tocilizumab, teriparatide, pertuzumab, rilonacept, denosumab, liraglutide, golimumab, benil Latacept, Buserelin, Velaglucerase alfa, Tesamorelin, Brentuximab vedotin, Taliglucerase alfa, Belimumab, Aflibercept, Asparaginase Erwinia chrysanthemi, Ocriplasmin, Glucarpidase, Teduglutide, Raxibacumab, Certolizumab pegol, Insulin, Isophene, Epoetin zeta, Obinutuzumab, Fibrinolysin plasmin (Fibrinolysin aka plasmin), follitropin alfa, romiplostim, lucinactant, natalizumab, aliskiren, ragweed pollen extract, secukinumab, somatotropin recombinant, drotrecogin alfa, alefacept, OspA lipoprotein, urokinase, abarelix, sermorelin, aprotinin, gemtuzumab ozogamicin, satumab pendetide, albiglutide, alirocumab, anthesim, antithrombin alfa, antithrombin III human, asfotase alfa, atezolizumab, autologous cultured chondrocytes, beractant, blinatumomab, C1 esterase inhibitor (human), coagulation factor XIII A subunit (recombinant), conestat a LUFA, Daratumumab, Desirudin, Dulaglutide, Elosulfase alfa, Elotuzumab, Evolocumab, Fibrinogen concentrate (human), Filgrastim-sndz, Gastric intrinsic factor. Hepatitis B immunoglobulin, Human calcitonin, Human clostridial tetanytoxoid immunoglobulin, Human rabies virus immunoglobulin, Human Rho(D) immunoglobulin, Hyaluronidase (human recombinant), Idarucizumab, Immunoglobulin human, Vedolizumab, Ustekinumab, Turoctocog alfa, Tuberculin purified protein derivative, Simoctocog alfa, Sirtoximab, Sebelipase alfa, Saclosidase, Ramucirumab, Prothrombin complex concentrate,Poractant alfa, pembrolizumab, peginterferon beta1a, ofamumab, obeltoxakimab, nivolumab, necitumumab, metreleptin, methoxypolyethylene glycol-epoetin beta, mepolizumab, ixekizumab, insulin pork, insulin degludec, insulin beef, thyroglobulin, anthrax immunoglobulin human, anticoagulant complex, antithymocyte globulin (horse), antithymocyte globulin (rabbit), brodalumab, C1 esterase inhibitor Inhibitor (recombinant), canakinumab, chorionic gonadotropin (human), chorionic gonadotropin (recombinant), coagulation factor X human, dinutuximab, efmoloctocog alfa, factor IX complex (human), hepatitis A vaccine, human varicella zoster immunoglobulin, ibritumomab tiuxetan, lenograstim, pegloticase, protamine sulfate, protein S human, sipuleucel T, somatropin recombinant, susocog alfa, thrombomodulin alpha, and the like. The methods of the disclosure may include administering proteins and peptides such as those identified above by contacting a target cell with the protein or peptide, or by contacting a target cell with a gene therapy agent that encodes the protein or peptide.

[0141] In some examples, useful therapeutic proteins include proteins that specifically bind to a target protein, such binding resulting in a therapeutic effect (e.g., therapeutic antibodies that bind to a target protein and result in a therapeutic effect). Non-limiting examples of therapeutic antibodies include 9E10, 8H9, abagonomab, abatacept, abciximab, avituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afelimumab, afetuzumab, alactizumab pegol, ALD518, alefacept, alemtuzumab, alirocumab, altumomab pentatate, amatuximab, anatumab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, arbutinib ... Polizumab, arcitumomab, asclinbacumab, acelizumab, atacicept, atezolizumab, atinumab, atlizumab / tocilizumab, atrolimumab, AVE1642, bapineuzumab, basiliximab, bavituximab, bectomomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bevacizumab / ranibizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, brosozumab , BMS-936559, bococizumab, brentuximab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, bronticizumab, canakinumab, cantuzumab metansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CDP791, cedelizumab, certolizumab, cetuximab, Ch.14.18, sitatuzumab bogadox, cixutumumab, clazam Kizumab, crenolixumab, clivatumab tetraxetan, codrituzumab, cortuximab ravtansine, conatumab, concizumab, CP-751871, CR6261, crenesumab, CS-1008, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denituzumab mafodotin, denosumab, dellotuximab biotin, detumomab, dinutuximab, dilidabumab, dorlimomab aritox, dorozitumab,Durigotumab, dupilumab, durvalumab, ducidizumab, eclomaximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, ergemtumab, elotuzumab, ersilimomab, emactutuzumab, emibetuzumab, enavatuzumab, enformab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enotizumab, enocituximab, ensituximab, epitumomab situsetan, epratuzumab, erlizumab, ertumaxomab, etanercept, etaracizumab, etrolizumab, ebina Kumab, evolocumab, exvilumab, F19, fanolesomab, faralimomab, faretuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fullanumab, futuximab, galixumab, ganitumab, gantenerumab, gavilimomab, gemtuzumab, gevokizumab, girentuximab, glevatumumab vedotin, golimumab, gomiliximab, guselkumab, H GS-ETR2, Imalizumab, Ibritumomab, Icrucumab, Idarucizumab, Igomomab, IIIA4, IM-2C6, IMAB362, Imalumab, IMC-A12, Imciclomab, Imgatuzumab, Incracumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab, Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, KB004, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Remaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rilotomab satetraxetan, lintuzumab, lirilumab, roderucizumab, loxivetumab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, mapatumumab, margetuximab, masilimomab, matuzumab, mavilililimumab, MEDI4736, mepolizumab, metelimuab, milatuzumab, minretumomab, mirvetuximab soravtansine, mitumomab, MK-0646,Mocamulizumab, morolimumab, morolimumab immuno, motavizumab, moxetumomab pasudotox, MPDL33280A, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafetox, narutumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab perpentane, obitoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onal Tuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregomab, Olticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pantumumab, Pancomab, Pambacumab, Palsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placumab, Polatuzumab vedotin, Ponezumab, Prunezumab ... Liliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, R1507, Racotumomab, Ladolezumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Rafanezumab, Ragavirumab, Resilizumab, Rilotumumab, Linukumab, Rituximab, Lobatumumab, Loredumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SG N-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, dadocizumab, talizumab, tanezumab, taplitumomab paptox, talexuzumab, tefavazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesilolumab, tetulomab, TGN1412,Ticilimumab / tremelimumab, tigatuzumab, tidrakizumab, TNX-650, tocilizumab, toralizumab, tosatoxumab, tobetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, trevoglumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, usu Examples of such antibodies include tekinumab, bundurumab vedotin, vanticutumab, vanticutumab, vancizumab, bapaliximab, valilumab, batelizumab, vedolizumab, veltuzumab, pevalimomab, besenkumab, vicilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, dillalimumab, and zolimomab. The methods of the present disclosure may include administering an antibody such as those identified above by contacting a target cell with the antibody or by contacting a target cell with a gene therapy agent that encodes the antibody.

[0142] Any amount (dosage) of the therapeutic biologic may be used and can be readily determined by one of skill in the art. In certain embodiments, the therapeutic biologic comprises a viral gene vector, the dosage of which can be readily determined.

[0143] In some embodiments, the disclosed methods may further include administration of one or more additional agents that inhibit the secretion of bile acids. Such additional agents, referred to herein as bile acid secretion inhibitors, may be administered by essentially any route, including the biliary route described herein. In some examples, such bile acid secretion inhibitors may be administered before, simultaneously, or after administration of the biliary therapeutic enhancers described herein. In some examples, such bile acid secretion inhibitors may be administered before, simultaneously, or after administration of the biologic therapeutics described herein. Useful bile acid secretion inhibitors are varied and may include, but are not limited to, bile acid analogs and derivatives (e.g., chenodeoxycholic acid and its derivatives), agonists (e.g., FXR agonists), growth factors, growth factor analogs, and the like. In some examples, useful FXR agonists may include, but are not limited to, obeticholic acid, FGF19 analogs, bile acid analogs, or derivatives thereof.

[0144] As summarized above, the method of the present disclosure includes contacting a biologic therapeutic agent and a biliary therapeutic enhancer with bile acids. Thus, the method described herein may include administering the agent described herein to an in vivo location that contains bile, such as the biliary system. In some examples, the agent described herein is administered directly to the biliary system, either individually or together as a composition. In some examples, the agent described herein is administered to the left hepatic duct, the right hepatic duct, the cystic duct, the common hepatic duct, the common bile duct, or some combination thereof (including all of the left hepatic duct, the right hepatic duct, the cystic duct, the common hepatic duct, and the common bile duct).

[0145] The compositions and methods described herein may employ retrograde ductal delivery of therapeutic biologics to the liver via retrograde ductal delivery of the biologic to the biliary system, including, for example, retrograde ductal delivery through the left hepatic duct, right hepatic duct, common hepatic duct, and / or common bile duct. In some instances, retrograde ductal delivery to the liver via the biliary system may involve blocking the outflow from the bile duct to the duodenum. If used, any convenient method of blocking the outflow from the bile duct may be used, including, but not limited to, occluding, for example, the ampulla of Vater or another point along the biliary system, for example, through the use of a catheter, such as a balloon catheter.

[0146] Any direct delivery to the biliary system may be used in the practice of the present invention. For clarity, direct delivery to the biliary system does not include oral or systemic delivery of any agent. In some instances, an endoscope may be used for guided delivery of an agent to a target site, such as the biliary system or a site therein. Useful endoscopes may include a camera and one or more additional endoscopic instruments, such as, but not limited to, catheters, balloons, probes, stents, and the like. In some embodiments, an endoscope may be guided through the subject's esophagus, stomach, and duodenum, and at the ampulla, an injection device is navigated into the common bile duct, and an agent is injected directly into the biliary system using the injection device in a retrograde direction toward the liver. In some instances, components of the endoscopic device may occlude the ampulla or other portions of the biliary system, preventing outflow. In some instances, outflow is prevented by inflating a balloon catheter. If necessary, the biliary system or a portion thereof may be flushed prior to injecting an agent. Any suitable flushing medium may be used, including, but not limited to, buffered saline.

[0147] In some instances, endoscopic retrograde cholangiopancreatography (ERCP) or techniques and / or devices may be used for direct delivery of agents to the biliary system. In some instances, imaging techniques such as, but not limited to, magnetic resonance cholangiopancreatography (MRCP) and endoscopic ultrasound may be used individually, together, and with or without ERCP. In some instances, the methods of the present disclosure include minimally invasive direct delivery of agents to the biliary system. In some instances, invasive procedures may be used. In some embodiments, direct delivery of agents to the biliary system may include or be performed as part of a surgical common bile duct exploration. In some instances, a laparotomy may be performed to access the biliary system or adjacent organs. In some instances, a catheter may be inserted through the gallbladder and navigated to the desired delivery site. In some instances, the subject may be cannulated for delivery of agents to the biliary system.

[0148] Direct delivery of active therapeutic biologics to the biliary system (e.g., via retrograde ductal delivery) offers many advantages. For example, such methods result in increased local concentrations of therapeutic agents within the biliary system and / or liver. Thus, increased local concentrations of therapeutic agents provide increased therapeutic efficacy and / or the ability to deliver less therapeutic agent while still achieving sufficient therapeutic efficacy at the target location, e.g., liver, or within target cells, e.g., hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs), hepatic sinusoidal endothelial cells (LSECs), ductal cells, or combinations thereof.

[0149] As summarized above, subjects with various conditions, including liver conditions as well as conditions other than those related to the liver, can be treated according to the methods described herein. In some examples, subjects can be treated for genetic diseases, such as single gene diseases. For example, but not limited to, subjects with genetic diseases, such as single gene diseases, can be administered gene therapy targeting genetic diseases via the methods described herein. In some examples, subjects can be treated for metabolic diseases, including cases where the metabolic disease may or may not be genetic diseases. Treatment of metabolic diseases according to the methods described herein can include gene therapy and / or delivery of proteins or peptides to treat metabolic diseases of subjects, such as via administration of replacement enzymes or metabolites.

[0150] As summarized above, in some instances, subjects treated according to the methods described herein and / or with the compositions and / or kits described herein may be treated for a hepatic condition. Non-limiting examples of hepatic conditions that may be treated include acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha 1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug-induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end-stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver disease, focal nodular hyperplasia, hepatic hydatid disease, hepatic encephalopathy, hepatic infarction, liver failure, hepatic porphyria, hepatic porphyria, hepatic encephalopathy, hepatic infarction, hepatic failure, hepatic porphyria ... fibrosis, hepatic tuberculosis, hepatic veno-occlusive disease, hepatitis, hepatocellular carcinoma, hepatic erythropoietic porphyria, hepatolenticular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, hereditary coproporphyria, liver abscess, hepatocellular adenoma, liver cirrhosis, liver failure, liver tumor, massive hepatic necrosis, non-alcoholic fatty liver disease, parasitic liver disease, peliosis hepatis, porphyria cutanea tarda, portal hypertension, pyogenic liver abscess, Reye's syndrome, variant porphyria, viral hepatitis, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, and Zellweger syndrome. In some examples, the subject can be treated for fibrosis or fibrosis symptoms. In some examples, the subject can be treated for cirrhosis or cirrhosis symptoms.

[0151] In some instances, for example, when a bile acid sequestrant is used as a biliary therapeutic enhancer, the condition for which a subject is treated according to the methods of the present disclosure may be a condition for which a bile acid sequestrant is not traditionally administered, including, but not limited to, hyperlipidemia, secondary dyslipidemia, a bile acid malabsorption condition, or a diabetic condition.

[0152] In some instances, the condition that the subject is treated for is not a hepatic condition.The method of the present disclosure can be used to treat various diseases other than hepatic conditions.For example, the liver or its cells can be utilized for the expression of cell-exogenous factors that treat non-hepatic conditions, for example, in some embodiments, the biologic used can be a gene therapy agent that causes hepatic cells to express insulin or another hormone to treat diabetic conditions or other endocrine disorders.

[0153] The method may include co-administration of one or more agents. The terms "co-administration" and "in combination with" include administration of two or more agents simultaneously, in parallel, or sequentially without specific time restrictions. In one embodiment, the agents are present in a solution, body fluid, target tissue, cell, cellular environment, and / or in the subject's body at the same time or exert their chemical, biological, or therapeutic effects at the same time. In one embodiment, the agents are in the same composition or unit dosage form. In other embodiments, the agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., seconds, minutes, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly, or after (e.g., minutes, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second agent.

[0154] The treatments described herein may be performed chronically (i.e., continuously) or non-chronically (i.e., discontinuously) and may include administration of one or more agents chronically (i.e., continuously) or non-chronically (i.e., discontinuously). Chronic administration of one or more agents according to the methods described herein may be used in various cases, including, for example, when a subject has a chronic condition, including, for example, a chronic liver condition (e.g., chronic liver disease, cirrhosis, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD / NASH), chronic viral hepatitis, etc.), a chronic genetic liver condition (alpha 1-antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, etc.), a chronic liver-associated autoimmune condition (e.g., primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), etc.), etc. Administration of one or more agents for a chronic condition may include, but is not limited to, administration of the agent for several months, a year or more, over several years. Such chronic administration may be on any convenient and suitable dosing schedule, including, but not limited to, daily, twice daily, weekly, twice weekly, monthly, twice monthly, etc. In some instances, for example, in the case of genetic conditions or other chronic gene therapies, the chronic condition may be treated by one or several (e.g., 2, 3, 4, or 5) treatments. Non-chronic administration of one or more agents may include, but is not limited to, administration for a month or less, for example, for a few weeks, a week, a few days, including a limited number of doses (e.g., less than 10 doses, e.g., 9 doses or less, 8 doses or less, 7 doses or less, etc., including a single dose).

[0155] The route of administration may be selected according to a variety of factors, including, but not limited to, the condition being treated, the formulation and / or device being used, the patient being treated, etc. Useful routes of administration include, but are not limited to, oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ocular, nasal, and transdermal. Pharmaceutical compositions formulated for a particular delivery route, as described herein, may be used.

[0156] In some embodiments, biliary or intraductal routes of administration can be used, including where the biliary system is accessed by any convenient method, including but not limited to surgical laparotomy, ERCP, and the like.

[0157] The effective amount of a subject compound will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, the manner of administration of the therapeutic composition, and the mechanism of action of the therapeutic agent. A "therapeutically effective amount" of a composition is that amount of a particular compound sufficient to achieve a desired effect in the subject being treated.

[0158] A therapeutically effective dose of one or more compositions described herein can be determined by one of skill in the art with the goal of achieving a local (e.g., tissue) concentration at least as high as the EC50 or IC50 of the applicable compound disclosed herein. In the case of gene therapy, depending on the context, a therapeutically effective dose can in some instances include transducing or transfecting some desired percentage of target cells (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, more than 90%, etc.) with one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, etc.) copies of viral or non-viral particles or exogenous genetic material.

[0159] The specific dose level and dosing frequency for any particular subject may vary and will depend on a variety of factors, including the activity of the composition, the stability and length of action of the compound, the age, weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and the severity of the symptoms of the host being treated. Non-limiting examples of doses that may be used include one or more doses of the composition and / or formulation in the range of 1 ng to 1 gram (or any value therebetween including any nanogram, microgram or gram amount).

[0160] The examples of treatments listed above should not be construed as limiting, and essentially any suitable treatment that produces the desired therapeutic outcome in a subject identified as described may be used.

[0161] kit Aspects of the present disclosure also include kits, and in some examples, devices for use with or in the kits. The kits may, for example, include one or more of any of the components described above with respect to the compositions and methods of the present disclosure. The agents may be in separate containers, or may be combined in a common container according to any combination described or suitable combination. Useful containers include vials, tubes, syringes, bottles, bags, ampoules, etc.

[0162] As summarized above, in some examples, the kits of the present disclosure may include compositions of the present disclosure, which may be pharmaceutical compositions, including, for example, pharmaceutical compositions comprising effective amounts of a therapeutic biologic and a biliary therapeutic enhancer. Such pharmaceutical compositions may include a pharma- ceutical acceptable carrier configured for delivery, e.g., as a liquid, to the biliary system.

[0163] Also provided are kits that include one or more compositions and / or kits for use in the methods described herein. The kits include any combination of components and compositions for carrying out the methods. The kits may include, for example, any one or more of the components described above with respect to the methods. The agents may be in separate containers or may be combined in a common container according to any combination described or suitable combination. Useful containers include vials, tubes, syringes, bottles, bags, ampoules, etc.

[0164] In some embodiments, the useful kit may further include a device. Useful devices are various and may include an injection or infusion device for delivering one or more agents or compositions to a subject. In some examples, the kit of the present disclosure may include a delivery device that is a component of, or is compatible with, an endoscope for endoscopic delivery of one or more agents or compositions of the present disclosure to the biliary system of a subject. In some examples, the delivery device of the present disclosure will be adapted to deliver agents during an ERCP procedure according to the methods described herein.

[0165] In addition to the above components, the kit may further include (in certain embodiments) instructions for carrying out the method. These instructions may be present in the kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is on a suitable medium or substrate, such as information printed on one or more sheets of paper on which the information is printed, within the kit packaging, within a package insert, etc. Yet another form in which these instructions may be present is a computer readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a flash drive, etc. Yet another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote location.

[0166] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following embodiments: 1. A method of treating a condition in a subject, the method comprising: administering an effective amount of a therapeutic biologic and an effective amount of a biliary therapeutic enhancer directly to the biliary system of a subject, thereby treating a condition in the subject. A method comprising: 2. The method of embodiment 1, wherein the condition is a genetic disease, and optionally, the genetic disease is a monogenic disease. 3. The method of embodiment 1 or embodiment 2, wherein the condition is a metabolic disorder. 4. The method of any one of embodiments 1-3, wherein the condition is a liver condition. 5. Liver manifestations include acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha 1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug-induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end-stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver disease, focal nodular hyperplasia, hepatic hydatid disease, hepatic encephalopathy, hepatic infarction, hepatic failure, hepatic porphyria, hepatic tuberculosis, hepatic veno-occlusive 5. The method of embodiment 4, wherein the disease is selected from the group consisting of hepatitis, hepatocellular carcinoma, hepatic erythropoietic porphyria, hepatolenticular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, hereditary coproporphyria, liver abscess, hepatocellular adenoma, cirrhosis, liver failure, liver tumor, massive hepatic necrosis, nonalcoholic fatty liver disease, parasitic liver disease, peliosis hepatis, porphyria cutanea tarda, portal hypertension, suppurative liver abscess, Reye's syndrome, variant porphyria, viral hepatitis, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, and Zellweger syndrome. 6. The method of any one of embodiments 1-5, wherein the administering comprises retrograde ductal delivery of the therapeutic biologic to the liver of the subject, thereby resulting in an increased local liver concentration of the therapeutic biologic. 7. The method of any one of embodiments 1-6, wherein the therapeutic biologic is susceptible to inactivation by bile. 8. The method of any one of the preceding embodiments, wherein the therapeutic biologic comprises a gene therapy agent or a protein. 9. The method of embodiment 8, wherein the gene therapy agent comprises a non-viral vector or a viral vector. 10. The method of embodiment 9, wherein the gene therapy agent comprises a lipid nanoparticle or an enveloped viral vector. 11. The method of embodiment 10, wherein the enveloped viral vector is a lentiviral vector, and optionally, the lentiviral vector comprises a 5'LTR, a promoter, a coding sequence, and a 3'LTR. 12. The method of embodiment 10, wherein the lipid nanoparticles do not contain a biliary therapeutic enhancer. 13. The method of embodiment 9, wherein the viral vector is an adenoviral vector or an adeno-associated viral (AAV) vector. 14. The method of embodiment 8, wherein the protein is a therapeutic peptide. 15. The method of embodiment 8, wherein the protein is an antibody. 16. The method of any one of embodiments 1-15, wherein the biliary therapeutic enhancer comprises a polyamine or a polyether polymer. 17. The method of any one of embodiments 1-16, wherein the biliary therapeutic enhancer comprises one or more bile acid sequestrants. 18. The method of embodiment 17, wherein the one or more bile acid sequestrants comprises a compound of formula (I): [ka] During the ceremony, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3 X is an electrophilic leaving group; R a is hydrogen, C1-C 20 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C 20 alkyl ammonium groups; The alkyl is optionally substituted with OH or alkylammonium. 19. The method of embodiment 17, wherein the one or more bile acid sequestrants comprise a compound of formula (II): [ka] During the ceremony, n corresponds to MW of about 100 to about 500, R 1 is C6~C 10 Aryl or C2-C 10and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with 1 to 3 substituents selected from the group consisting of C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, or an alkyl ammonium halide group. 20. The method of embodiment 17, wherein the one or more bile acid sequestrants comprises a compound of formula (V): [ka] During the ceremony, m and n correspond to MW of about 100 to about 500; R 1 and R 2 Each of is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group. 21. The method of embodiment 17, wherein the one or more bile acid sequestrants comprises a compound of formula (VII): [ka] During the ceremony, m corresponds to MW of about 100 to about 500, a is an integer from 1 to 6; b is an integer from 1 to 6; and c is an integer from 1 to 3. 22. The method of any one of embodiments 17-21, wherein the one or more bile acid sequestrants are selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer. 23. The method of any one of embodiments 1-22, wherein the biliary therapeutic enhancer comprises a cationic or non-ionic amphipathic transduction enhancer. 24. The method of embodiment 23, wherein the cationic or non-ionic amphipathic transduction enhancer is a compound of formula (III): [ka] During the ceremony, Each of x, y, and z is independently an integer of 1 to 250. 25. The method of embodiment 23, wherein the cationic or non-ionic amphipathic transduction enhancer is a compound of formula (IVa) or (IVb): [ka] During the ceremony, n corresponds to MW of about 50,000 to about 200,000, x is an integer from 1 to 6; R 1 is an amino group optionally substituted with one or more C1-C6 alkyl groups. 26. The method of embodiment 23, wherein the cationic or non-ionic amphipathic transduction enhancer is a compound of formula (VI): [ka] During the ceremony, m corresponds to MW of about 100 to about 600, x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R a are independently hydrogen or a C1 to C6 alkyl group. 27. The method of any one of embodiments 23 to 26, wherein the cationic or non-ionic amphipathic transduction enhancer is selected from the group consisting of polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108. 28. The method of any one of embodiments 1-27, wherein the therapeutic biologic and the biliary therapeutic enhancer are co-administered. 29. The method of any one of embodiments 1-28, wherein the therapeutic biologic and the biliary therapeutic enhancer are co-formulated in a single pharmaceutical composition. 30. The method of any one of embodiments 1-29, wherein the biliary therapeutic enhancer is administered prior to the therapeutic biologic. 31. The method of any one of embodiments 1-30, comprising administering two or more biliary therapeutic enhancers. 32. The method of embodiment 31, wherein the two or more biliary therapeutic enhancers comprise a bile acid sequestrant and a cationic or non-ionic amphipathic transduction enhancer. 33. The method of embodiment 31 or 32, wherein two or more biliary therapeutic enhancers are co-administered. 34. The method of embodiment 33, wherein the two or more biliary therapeutic enhancers are co-formulated in a single pharmaceutical composition. 35. The method of any one of embodiments 1-34, further comprising administering a bile acid secretion inhibitor to the subject. 36. The method of embodiment 35, wherein the bile acid secretion inhibitor is an FXR agonist, optionally wherein the FXR agonist is selected from obeticholic acid or an FGF19 analogue. 37. The method of any one of the preceding embodiments, wherein the condition is not a hyperlipidemia, secondary dyslipidemia, bile acid malabsorption condition, and / or a diabetic condition. 38. A pharmaceutical composition comprising: a pharma- ceutically acceptable carrier configured as a liquid for delivery to the biliary system; an effective amount of a therapeutic biologic; and / or Biliary Therapeutic Enhancers 13. A pharmaceutical composition comprising: 39. The composition of embodiment 38, wherein the therapeutic biologic is susceptible to inactivation by bile. 40. The composition of embodiment 38 or 39, wherein the therapeutic biologic comprises a gene therapy agent or a protein. 41. The composition of embodiment 40, wherein the gene therapy agent comprises a non-viral vector or a viral vector. 42. The composition of embodiment 41, wherein the gene therapy agent comprises a lipid nanoparticle or an enveloped viral vector. 43. The composition of embodiment 42, wherein the enveloped viral vector is a lentiviral vector. 44. The composition of embodiment 42, wherein the lipid nanoparticles do not contain a biliary therapeutic enhancer. 45. The composition of embodiment 41, wherein the viral vector is an adenoviral vector or an adeno-associated viral (AAV) vector. 46. ​​The composition of embodiment 40, wherein the protein is a therapeutic peptide. 47. The composition of embodiment 40, wherein the protein is an antibody. 48. The composition of any one of embodiments 38 to 40, wherein the biliary therapeutic enhancer comprises a polyamine or a polyether polymer. 49. The composition of any one of embodiments 38-48, wherein the biliary therapeutic enhancer is a bile acid sequestrant. 50. The composition of embodiment 49, wherein the bile acid sequestrant is a compound of formula (I): [ka] During the ceremony, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3 X is an electrophilic leaving group; R a is hydrogen, C1-C 20 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C 20 alkyl ammonium groups; The alkyl is optionally substituted with OH or alkylammonium. 51. The composition of embodiment 49, wherein the bile acid sequestrant is a compound of formula (II): [ka] During the ceremony, n corresponds to MW of about 100 to about 500, R 1 is C6~C 10 Aryl or C2-C 10and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with 1 to 3 substituents selected from the group consisting of C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, or an alkyl ammonium halide group. 52. The composition of embodiment 49, wherein the bile acid sequestrant is a compound of formula (V): [ka] During the ceremony, m and n correspond to MW of about 100 to about 500; R 1 and R 2 Each of is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group. 53. The composition of embodiment 49, wherein the bile acid sequestrant is a compound of formula (VII): [ka] During the ceremony, m corresponds to MW of about 100 to about 500, a is an integer from 1 to 6; b is an integer from 1 to 6; and c is an integer from 1 to 3. 54. The composition of any one of embodiments 49-53, wherein the bile acid sequestrant is selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer. 55. The composition of any one of embodiments 49-54, wherein the bile acid sequestrant is present in the pharmaceutical composition in a subtherapeutic amount. 56. The composition of any one of embodiments 38 to 48, wherein the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer. 57. The composition according to embodiment 56, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (III): [ka] During the ceremony, Each of x, y, and z is independently an integer of 1 to 250. 58. The composition according to embodiment 56, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (IVa) or (IVb): [ka] During the ceremony, n corresponds to MW of about 50,000 to about 200,000, x is an integer from 1 to 6; R 1 is an amino group optionally substituted with one or more C1-C6 alkyl groups. 59. The composition according to embodiment 56, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (VI): [ka] During the ceremony, m corresponds to MW of about 100 to about 600, x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R a are independently hydrogen or a C1 to C6 alkyl group. 60. The composition of any one of embodiments 56 to 59, wherein the cationic or non-ionic amphipathic transduction enhancer is selected from the group consisting of polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108. 61. A method of treating a condition in a subject, comprising: Administering an effective amount of the pharmaceutical composition of any one of embodiments 38-60 directly to the biliary system of a subject, thereby treating a condition in the subject. A method comprising: 62. The method of embodiment 61, wherein the condition is a genetic disease, and optionally, the genetic disease is a monogenic disease. 63. The method of embodiment 61 or embodiment 62, wherein the condition is a metabolic disorder. 64. The method of any one of embodiments 61-63, wherein the condition is a hepatic condition. 65. Liver manifestations include acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha 1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug-induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end-stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver disease, focal nodular hyperplasia, hepatic hydatid disease, hepatic encephalopathy, hepatic infarction, hepatic failure, hepatic porphyria, hepatic tuberculosis, hepatic veno-occlusive 65. The method of embodiment 64, wherein the disease is selected from the group consisting of hepatitis, hepatocellular carcinoma, hepatic erythropoietic porphyria, hepatolenticular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, hereditary coproporphyria, liver abscess, hepatocellular adenoma, cirrhosis, liver failure, liver tumor, massive hepatic necrosis, nonalcoholic fatty liver disease, parasitic liver disease, peliosis hepatis, porphyria cutanea tarda, portal hypertension, suppurative liver abscess, Reye's syndrome, variant porphyria, viral hepatitis, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, and Zellweger syndrome. 66. The method of any one of embodiments 61-65, wherein the administering comprises retrograde ductal delivery of the therapeutic biologic to the liver of the subject, thereby resulting in an increased local liver concentration of the therapeutic biologic. 67. A kit comprising: a liquid pharma- ceutically acceptable carrier; A therapeutic biologic; Biliary Therapeutic Enhancers and Including the kit 68. The kit of embodiment 67, wherein the biliary therapeutic enhancer and a liquid pharma- ceutically acceptable carrier are formulated together in a container. 69. The kit of embodiment 67 or 68, wherein the therapeutic biologic is susceptible to inactivation by bile. 70. The kit of any one of embodiments 67-69, wherein the therapeutic biological agent comprises a gene therapy agent or a protein. 71. The kit of embodiment 70, wherein the gene therapy agent comprises a non-viral vector or a viral vector. 72. The kit of embodiment 71, wherein the gene therapy agent comprises a lipid nanoparticle or an enveloped viral vector. 73. The kit of embodiment 72, wherein the enveloped viral vector is a lentiviral vector. 74. The kit of embodiment 72, wherein the lipid nanoparticles do not contain a biliary therapeutic enhancer. 75. The kit of embodiment 71, wherein the viral vector is an adenoviral vector or an adeno-associated viral (AAV) vector. 76. The kit of embodiment 70, wherein the protein is a therapeutic peptide. 77. The kit of embodiment 70, wherein the protein is an antibody. 78. A kit according to any one of embodiments 67 to 77, wherein the biliary therapeutic enhancer comprises a polyamine or a polyether polymer. 79. The kit of any one of embodiments 67-78, wherein the biliary therapeutic enhancer is a bile acid sequestrant. 80. The kit of embodiment 79, wherein the bile acid sequestrant is a compound of formula (I): [ka] During the ceremony, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3 X is an electrophilic leaving group; R a is hydrogen, C1-C 20 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C 20 alkyl ammonium groups; The alkyl is optionally substituted with OH or alkylammonium. 81. The kit of embodiment 79, wherein the bile acid sequestrant is a compound of formula (II): [ka] During the ceremony, n corresponds to MW of about 100 to about 500, R 1 is C6~C 10 Aryl or C2-C 10 and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with 1 to 3 substituents selected from the group consisting of C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, or an alkyl ammonium halide group. 82. The kit of embodiment 79, wherein the bile acid sequestrant is a compound of formula (V): [ka] During the ceremony, m and n correspond to MW of about 100 to about 500; R 1 and R 2 Each of is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group. 83. The kit according to embodiment 79, wherein the bile acid sequestrant is a compound of formula (VII): [ka] During the ceremony, m corresponds to MW of about 100 to about 500, a is an integer from 1 to 6; b is an integer from 1 to 6; and c is an integer from 1 to 3. 84. The kit of any one of embodiments 79-83, wherein the bile acid sequestrant is selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer. 85. A kit according to any one of embodiments 67 to 78, wherein the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer. 86. The composition according to embodiment 85, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (III): [ka] During the ceremony, Each of x, y, and z is independently an integer of 1 to 250. 87. The composition according to embodiment 85, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (IVa) or (IVb): [ka] During the ceremony, n corresponds to MW of about 50,000 to about 200,000, x is an integer from 1 to 6; R 1 is an amino group optionally substituted with one or more C1-C6 alkyl groups. 88. The composition according to embodiment 85, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (VI): [ka] During the ceremony, m corresponds to MW of about 100 to about 600, x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R a are independently hydrogen or a C1 to C6 alkyl group. 89. A kit according to any one of embodiments 85 to 88, wherein the cationic or non-ionic amphipathic transduction enhancer is selected from the group consisting of polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108. 90. The kit of any one of embodiments 67-89, further comprising a device configured for retrograde ductal delivery of a therapeutic biologic to the liver. 91. Use of one or more compositions and / or one or more kits according to any one of embodiments 38-60 or 67-90 for treating a condition in a subject, optionally wherein the condition is a hepatic condition, and optionally wherein the hepatic condition is acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha 1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver. 20. The method of claim 1, wherein the patient is a patient with a chronic hepatitis B disease, a patient with a chronic hepatitis C disease, ... 92. Use of a liquid composition comprising one or more compositions and / or biliary therapeutic enhancers according to any one of embodiments 38-60 or 86-88 to deliver a therapeutic biologic to the biliary system of a subject to treat a hepatic condition in the subject. 93. The use according to embodiment 92, wherein the biliary therapeutic enhancer is a bile acid sequestrant, optionally, the bile acid sequestrant is a polyamine or polyether polymer. 94. The use according to embodiment 92, wherein the biliary therapeutic enhancer is a cationic or non-ionic amphipathic transduction enhancer, optionally wherein the cationic or non-ionic amphipathic transduction enhancer is a polyamine or polyether polymer. 95. A method for transducing or transfecting a cell, comprising: contacting the cell with a biliary transduction enhancer in the presence of bile to produce a transduction / transfection composition; contacting the transduction / transfection composition with a gene therapy agent comprising an exogenous nucleic acid under conditions sufficient to transduce or transfect the exogenous nucleic acid into the cell, thereby transducing or transfecting the cell with the exogenous nucleic acid; A method comprising: 96. The method of embodiment 95, wherein the cells are hepatocytes, and optionally, the hepatocytes are hepatocytes. 97. The method of embodiment 95 or 96, wherein the gene therapy agent comprises a non-viral vector or a viral vector. 98. The method of embodiment 97, wherein the gene therapy agent comprises a lipid nanoparticle or an enveloped viral vector. 99. The method of embodiment 98, wherein the enveloped viral vector is a lentiviral vector. 100. The method of embodiment 98, wherein the lipid nanoparticles do not contain a biliary therapeutic enhancer. 101. The method of embodiment 97, wherein the viral vector is an adenoviral vector or an adeno-associated viral (AAV) vector. 102. The method of any one of embodiments 95 to 101, wherein the exogenous nucleic acid comprises a coding sequence or a portion thereof, and optionally the coding sequence encodes a transcription factor, a therapeutic peptide, an antibody or a portion thereof. 103. The method of any one of embodiments 95 to 101, wherein the exogenous nucleic acid comprises a non-coding sequence. 104. The method of any one of embodiments 95-103, wherein the biliary therapeutic enhancer comprises a polyamine or a polyether polymer. 105. The method of any one of embodiments 95-104, wherein the biliary therapeutic enhancer comprises one or more bile acid sequestrants. 106. The method of embodiment 105, wherein the one or more bile acid sequestrants comprise a compound of formula (I): [ka] During the ceremony, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3 X is an electrophilic leaving group; R a is hydrogen, C1-C 20 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C 20 alkyl ammonium groups; The alkyl is optionally substituted with OH or alkylammonium. 107. The method of embodiment 105, wherein the one or more bile acid sequestrants comprise a compound of formula (II): [ka] During the ceremony, n corresponds to MW of about 100 to about 500, R 1 is C6~C 10 Aryl or C2-C 10 and heteroaryl, wherein the aryl or heteroaryl is optionally substituted with 1 to 3 substituents selected from the group consisting of C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, or an alkyl ammonium halide group. 108. The method of embodiment 105, wherein the one or more bile acid sequestrants comprise a compound of formula (V): [ka] During the ceremony, m and n correspond to MW of about 100 to about 500; R 1 and R 2 Each of is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group. 109. The method of embodiment 105, wherein the one or more bile acid sequestrants comprise a compound of formula (VII): [ka] During the ceremony, m corresponds to MW of about 100 to about 500, a is an integer from 1 to 6; b is an integer from 1 to 6; and c is an integer from 1 to 3. 110. The method of any one of embodiments 105-109, wherein the one or more bile acid sequestrants are selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer. 111. The method of any one of embodiments 95-110, wherein the biliary therapeutic enhancer comprises a cationic or non-ionic amphipathic transduction enhancer. 112. The method according to embodiment 111, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (III): [ka] During the ceremony, Each of x, y, and z is independently an integer of 1 to 250. 113. The method according to embodiment 111, wherein the cationic or non-ionic amphipathic transduction enhancer is a compound of formula (IVa) or (IVb): [ka] During the ceremony, n corresponds to MW of about 50,000 to about 200,000, x is an integer from 1 to 6; R 1 is an amino group optionally substituted with one or more C1-C6 alkyl groups. 114. The method according to embodiment 111, wherein the cationic or non-ionic amphiphilic transduction enhancer is a compound of formula (VI): [ka] During the ceremony, m corresponds to MW of about 100 to about 600, x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R a are independently hydrogen or a C1 to C6 alkyl group. 115. The method of any one of embodiments 111 to 114, wherein the cationic or non-ionic amphipathic transduction enhancer is selected from the group consisting of polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108. 116. The method of any one of embodiments 105-115, wherein contacting with a biliary transduction enhancer comprises contacting the cell with both a bile acid sequestrant and a cationic or non-ionic amphipathic transduction enhancer. 117. The method of any one of embodiments 95 to 116, wherein the biliary transduction enhancer and the gene therapy agent are present together in a formulation prior to contacting. EXAMPLES

[0167] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. They are not intended to limit the scope of what the inventors perceive as the invention. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0168] General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 4th Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2012); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley&Sons 1999); Protein Methods (Bollag et al., John Wiley&Sons 1996); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle&Griffiths, John Wiley&Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, antibodies, cells, tissue samples, etc., and kits referred to in this disclosure are available from commercial suppliers, such as, but not limited to, those identified herein.

[0169] Example 1: Effect of bile on lentiviral transduction An assay was designed to evaluate the effect of the presence of bile on lentiviral transduction of human cells, including human hepatocytes. A lentiviral vector (LV-SFFV-Luc2-P2A-EmGFP) encoding a firefly luciferase (Luc2) and Emerald GFP (EmGFP) reporter, separated by a self-cleaving peptide (P2A) under the control of the spleen focus forming virus (SFFV) promoter, was used to facilitate the detection of successfully transduced HepG2 or HeLa-RC32 cells in the absence or presence of various concentrations of Sprague Dawley rat bile. Briefly, 10 MOI of LV-SFFV-Luc2-P2A-EmGFP vector was incubated with 100% PBS, 99.9% PBS / 0.1% bile, 99% PBS / 1% bile, 90% PBS / 10% bile, 50% PBS / 50% bile, or 100% bile in separate containers at 37° C. for 1 h. The cells and cell medium containing the transduction enhancer were then mixed with the vector solution and incubated for 72 hours to allow transduction. The ONE-glo luciferase assay system (Promega, Madison, WI) was used to quantitatively estimate transduction and lentiviral vector activity after bile exposure. To assay cell viability and bile cytotoxicity, cells were incubated in the bile, cell medium, and transduction enhancer mixture described above without the lentiviral vector for 72 hours, and plates were assayed using the CellTiter-Glo assay system (Promega, Madison, WI).

[0170] The reporter was readily detected in samples treated with 100% PBS positive control, indicating successful transduction and expression from the lentivirus-delivered transgene in the transduced cells. However, samples treated with as little as 0.1% bile gave low RLU measurements, demonstrating that the presence of bile, even at low concentrations, is detrimental to successful transduction with lentiviral vectors. These data indicate that transduction of target cells with lentiviral vectors is a bile-labile process and clearly demonstrate the negative impact that the presence of bile can have on therapeutic biologics delivered to locations where bile is present.

[0171] Example 2: Bile acid sequestrants rescue lentiviral transduction in the presence of bile Bile acid sequestrants, also called bile acid resins, are a class of antilipidemic agents that bind to bile acids in the intestine and inhibit bile acid lipid-solubilizing activity, thus blocking cholesterol absorption and inhibiting bile acid reabsorption, which causes contraction of the bile acid pool and subsequently increases bile acid synthesis, which competes with cholesterol synthesis in the liver. This process ultimately results in a reduction in serum cholesterol levels. Various bile acid sequestrants are available for clinical use. All are administered only by the oral route.

[0172] Bile acid sequestrants were investigated as candidate reagents for improving transduction of lentiviral vectors in the presence of bile. In this example, 60 μg / mL of the sequestrant, colesevelam or cholestyramine, was added to mixtures containing 100% PBS, 99.9% PBS / 0.1% bile, 99.7% PBS / 0.3% bile, 99% PBS / 1% bile, 97% PBS / 3% bile, 90% PBS / 10% bile, or 70% PBS / 30% bile, and the mixtures were incubated at room temperature for 30 minutes. The lentiviral vectors described above were added to each mixture, and the mixtures were incubated at 37° C. for 1 hour. For viability estimation, the corresponding mixtures of sequestrant and bile were similarly incubated at room temperature for 30 minutes and then at 37° C. for 1 hour, but in this case, no lentiviral vector was added. After incubation, the solution was added to the cells along with cell medium and transduction enhancer, and the cells were transduced / incubated for 72 hours. As described above, ONE-glo and CellTiter-Glo assays were used to estimate transduction and viability, respectively.

[0173] The results of the viability assay are shown in Table 1. These results demonstrate that the presence of sequestration agents improves the viability of cells treated with bile. For analysis of the effect of sequestration agents on transduction efficiency in the presence of bile, luciferase levels were measured in HeLa-RC32 cells 72 hours after treatment with LV-SFFV-Luc2-P2A-EmGFP in the presence of various bile concentrations, in the presence or absence of sequestration agents. These measurements were corrected for cell viability, and the results of this analysis are shown in Figure 1. [Table 1]

[0174] As shown, in the absence of bile (0% rat bile), all conditions tested demonstrated efficient transduction of lentiviral vectors. In the absence of sequestration agents ("no sequestration agents"), a significant reduction in transduction levels approaching a complete lack of transduction was observed at bile levels above 1%, again indicating that at bile concentrations of approximately 1% bile, over 60% transduction was lost. However, addition of either sequestration agent, colesevelam or cholestyramine, rescued transduction and achieved viability-adjusted luciferase levels at 1% bile comparable to those observed in the 0% bile condition. Moreover, both sequestration agents unexpectedly rescued transduction at bile levels above 1% and achieved efficient transduction at 30% bile, the highest bile level tested. The bile acid sequestrant sevelamer was tested separately using similar methods and also showed enhanced transduction compared to the level of transduction observed in bile in the absence of the sequestrant.

[0175] Taken together, these data demonstrate that the addition of a bile acid sequestration agent to a transduction composition in the presence of bile effectively rescues lentiviral transduction that would otherwise be reduced or eliminated by even small amounts of bile. Thus, this study indicates that the use of a bile acid sequestration agent enhances the resulting activity of bile-labile biologics when such a sequestration agent is included in a delivery composition or treatment method targeted to locations where bile is present.

[0176] Example 3: Sequestration agents and transduction enhancers individually enhance and rescue lentiviral transduction in the presence of bile. Previous examples used transduction enhancers in the transduction medium. To better evaluate the transduction enhancing effect of bile acid sequestrants in bile-containing medium, the roles of sequestration agents and transduction enhancers were evaluated separately. Briefly, transduction of HeLa-RC32 cells with LV-SFFV-Luc2-P2A-EmGFP vector was performed for 72 hours essentially as described above using transduction enhancer (F108, 1000 μg / mL), sequestration agent (colesevelam, 250 μg / mL), or a combination of transduction enhancer and sequestration agent (1000 μg / mL F108 + 250 μg / mL colesevelam) in a bile dilution series spanning 0.1% to 100% rat bile. Additives, transduction enhancers and / or sequestration agents were co-formulated with the vector and incubated for 60 minutes prior to transduction. Controls were also evaluated, including a no bile (0%) control and a "no additive" (i.e., no sequestration agent or transduction enhancer) control. Quantification was performed essentially as described herein, and measurements were corrected for cell viability.

[0177] The results demonstrated that the bile acid sequestrant provided significantly increased transduction of lentiviral vectors in the presence of various bile concentrations compared to transduction reactions containing bile but neither the sequestration agent nor the transduction enhancer. Specifically, while the "no additive" control showed decreasing levels of transfection with increasing levels of bile, the addition of the combination of the transduction enhancer and sequestration agent resulted in increased levels of transfection at all bile dilutions ranging from 0-100%, thus reinforcing the findings described in the previous examples.

[0178] Furthermore, bile acid sequestration agent compositions that did not contain a transduction enhancer nevertheless demonstrated enhanced transduction at various bile levels compared to the "no additive" control. Thus, this example demonstrated that the transduction enhancing effect of sequestration agents on bile-labile lentiviruses is not dependent on the presence of a transduction enhancer. Rather, the sequestration agent alone demonstrated enhanced transduction in the presence of bile.

[0179] Furthermore, transduction enhancer F108 was unexpectedly found to rescue transduction even in the absence of sequestration agents across a range of bile concentrations, indicating that the transduction enhancer alone can restore the otherwise bile-labile activity of lentiviral vectors. Other transduction enhancers were also tested. As demonstrated herein, despite the deleterious effect of 0.1% bile on lentiviral transduction, other transduction enhancers were also found to have a positive effect on the transduction of the LV-SFFV-Luc2-P2A-EmGFP vector in the presence of bile concentrations greater than 1%. For example, as shown in Figure 2, transduction compositions containing polyethylenimine (PEI, 15 μg / mL) or polyethylene glycol (PEG, 15 μg / mL) rescued vector transduction at rat bile concentrations of 10% or less, as measured by viability-corrected luciferase expression in transduced HeLa-RC32 cells 72 hours after vector transduction. In comparison, the negative control containing no transduction enhancer ("no transduction enhancer") showed significantly reduced levels of transduction in the presence of bile at all concentrations tested.

[0180] Taken together, these results demonstrate that transduction enhancers and bile acid sequestrants, individually or in combination, can rescue transduction and enhance the activity of bile-labile lentiviral vectors when bile is present in the transduction milieu. This finding was particularly unexpected given that even small amounts of bile (e.g., less than 1%) have been observed to profoundly affect the ability of lentiviruses to transduce target cells and result in expression of reporter transgenes.

[0181] Example 4: Kinetics of lentiviral vector inactivation and rescue of vector activity in the presence of human bile To estimate the temporal dynamics of lentiviral vector exposure to bile, a bile exposure time series was performed. Specifically, the LV-SFFV-Luc2-P2A-EmGFP vector was exposed to 15% bile prepared from a fresh lot of rat bile in culture medium containing 500 μg / mL of F108 for various times ranging from 1 min to 1 h. After incubating the vector in bile medium solution for the indicated times, HeLaRC32 cells were transduced for 72 h, and the resulting effect of different bile exposure times on vector transduction efficiency was estimated by measuring the luciferase reporter transgene as described herein. Figure 3 provides the results of this bile exposure time series, along with vector activity expressed as a percentage of vector activity observed in the absence of bile ("no bile control") corrected for viability. As shown, after 15 min exposure to 15% bile, 97% of the vector activity was lost, and when the bile exposure time was increased to periods greater than 15 min, more than 99% of the activity was lost. In contrast, when the vector was exposed to 15% bile for 1 min, only about 5% activity was lost.

[0182] These findings demonstrate that inactivation of lentiviral vectors by bile occurs rapidly but progressively over time, and add to the previously described findings that increasing concentrations of bile decrease vector activity. These findings highlight the opportunity to optimize transduction compositions, as well as treatment times and other transduction parameters, to achieve enhanced transduction in the presence of bile.

[0183] In addition to optimizing the transduction composition and other transduction parameters, the bile-containing environment can also be adjusted in vivo to affect the amount of bile present locally during viral transduction performed in a subject. For example, prior to delivery of the transduction composition to the bile duct, the duct may be flushed to reduce the concentration of bile in the local environment, thus allowing the subsequent introduction of the transduction composition containing the viral vector into a bile-poor environment. However, it remains unlikely that, for example, flushing the bile duct can completely deplete the bile duct of bile in a living subject, since at least bile is constantly produced.

[0184] Considering the above, the activity of lentiviral vector was evaluated in human bile dilution series.Briefly, HeLa-RC32 cells were transduced with LV-SFFV-Luc2-P2A-EmGFP vector in the presence of various concentrations of bile ranging from less than 0.01% to more than 10% for 72 hours, and vector activity was estimated as the viability-corrected percentage of reporter measured in control without bile.It was observed that human bile levels above 10% are toxic to cells.However, the results showed that there is a viable window in which the enhancement described herein can provide efficient transduction at human bile levels below 10% that can be achieved in an in vivo setting.

[0185] Transduction was performed to evaluate the ability of bile acid sequestrants or transduction enhancers to enhance lentiviral vector activity in the presence of 1% and 0.1% human bile concentrations. Briefly, LV-SFFV-Luc-P2A-EmGFP vectors were incubated for 1 h with various concentrations of colesevelam or F108 (ranging from less than 10 μg / mL to more than 1000 μg / mL final well concentrations) at the respective human bile concentrations. HeLa-RC32 cells were then transduced and vector activity was estimated by reporter expression at 72 h. Results are reported and compared as viability-corrected reporter activity expressed as a percentage of that measured in control transduction with vectors not exposed to bile. Results demonstrated that increasing amounts of either sequestrants or transduction enhancers enhanced and at least partially rescued lentiviral vector activity after exposure to 0.1% or 1% human bile.

[0186] Taken together, these findings indicate that what was observed in rat bile is transferable to the human bile environment, and further demonstrate that bile acid sequestrants and transduction enhancers, both individually and in combination, function to enhance the activity of bile-labile therapeutics, such as lentiviral vectors, in the presence of bile. Thus, the examples provided herein demonstrate that biologics that are normally inactivated in the presence of bile can be effectively administered to environments where bile is present (such as the biliary tract) when reagents such as bile acid sequestrants and / or transduction enhancers are also used, which reduce the deleterious effects of bile on the biologic in the presence of bile and / or otherwise enhance the activity of the biologic.

[0187] Example 5: Bile acid sequestrant and transduction enhancer formulations improve transfection of cells with bile-labile non-viral vectors The effect of bile on non-viral vectors was also estimated. Specifically, HeLa-RC32 cells were plated overnight at a density of 40,000 cells / well in 24-well plates. Lipid nanoparticle (LNP) non-viral vectors containing EGFP mRNA were pre-incubated with a solution of 30% rat (Sprague Dawley) bile in PBS or a positive control solution containing PBS without bile for 1 h at 37°C. The mixture was then diluted 10-fold with cell culture medium and cells were incubated with the vector-bile solution at 37°C for 24 h with 5% CO2. Thus, cells were exposed to a final bile concentration of 3% in the test group. After incubation, EGFP expression and cell viability were estimated by flow cytometry.

[0188] In the positive control, 90% of the cells showed EGFP expression, indicating successful transfection and EGFP expression from the introduced mRNA. However, cells treated with LNP vector exposed to 30% bile showed 100% loss of LNP-driven EGFP fluorescence compared to the positive control, indicating complete impairment of the activity of the LNP EGFP mRNA vector by the presence of bile. Thus, these data demonstrate that the activity of non-viral vectors such as the LNP vector is susceptible to degradation in the presence of bile.

[0189] The ability of the combination of bile acid sequestrant and transduction enhancer to increase the activity of non-viral vectors in the presence of bile was also estimated. Specifically, in parallel with the above assay, LNP non-viral vectors containing EGFP mRNA were pre-incubated with a solution of 30% rat bile and a formulation containing bile acid sequestrant and transduction enhancer (250 μg / mL colesevelam and 10 mg / mL F108 in DPBS) for 1 hour at 37°C. The mixture was then diluted 10-fold in cell culture medium as described above (resulting in a final concentration of 3% bile, 25 μg / mL colesevelam and 1 mg / mL F108), and cells were incubated with the vector-bile solution at 37°C for 24 hours in 5% CO2. Cells were then estimated for EGFP expression and cell viability by flow cytometry, and the results were compared to those obtained from cells treated with bile-exposed LNPs as described above.

[0190] Samples treated with the sequestration agent and transduction enhancer showed an unexpected increase in EGFP expression, indicating successful transfection and partial rescue of LNP vector activity lost in the presence of bile. Specifically, 11% of cells treated with medium containing LNP, bile, sequestration agent, and transduction enhancer showed LNP-driven EGFP expression compared to the complete lack of LNP-driven EGFP expression observed in corresponding samples containing bile but not the sequestration agent or transduction enhancer.

[0191] Taken together, these results demonstrate that bile sequestration agents and transduction enhancers can rescue transfection of non-viral vectors and increase the activity of bile-labile non-viral vectors when the vectors are exposed to bile and / or when bile is present in the transfection environment. This finding was particularly unexpected given that the profound effect of bile on LNP vectors resulted in a complete impairment of activity of vectors that otherwise yielded 90% transfection and expression in positive controls.

[0192] Example 6: Bile acid sequestrant and transduction enhancer formulations individually improve the efficiency of LNP-mediated transduction in the presence of bile The effect of individual formulation components on the transduction of non-viral vectors in the presence of bile was estimated. The amount of individual formulation components, the sequestration agent colesevelam or the transduction enhancer F108, was titrated in LNP-mediated transduction reactions of GFP-encoding nucleic acid essentially as described in Example 5.

[0193] HeLa-RC32 cells were plated overnight at a density of 40,000 cells / well in 24-well plates. Transduction reactions of mRNA encoding GFP in LNPs were carried out for 30 min at 37° C. in the presence of various concentrations of 30% rat bile alone ("bile"), bile and colesevelam ("bile+colesevelam"), or bile and F108 ("bile+F108"). LNP transduction in the absence of bile was also performed as a control ("no treatment"). 24 h after the various transduction reactions, GFP expression was assayed by flow cytometry.

[0194] As can be readily seen in Figure 4A, the sequestration agent improved LNP transduction as evidenced by the increased number of GFP-expressing cells compared to the number of GFP-expressing cells seen in bile-containing transduction reactions that did not contain a sequestration agent. Furthermore, various colesevelam treatments resulted in a near-complete rescue of LNP transduction to levels observed in the absence of bile (e.g., compare % GFP+ cells measured at colesevelam concentrations of 6.1 μg / mL, 2.0 μg / mL, and 0.68 μg / mL to % GFP+ cells measured in the "no treatment" control).

[0195] Figure 4B provides a log-scale rendering of the data presented in Figure 4A. As can be seen in Figure 4B, the presence of the F108 enhancer in the transduction reaction also improved the transduction efficiency in the presence of bile compared to the transduction efficiency observed in the presence of bile without the addition of F108.

[0196] This example demonstrates that inclusion of a transduction enhancer and / or a bile acid sequestration agent in a transduction reaction in the presence of bile each individually improves the transduction efficiency of a non-viral vector, and that addition of a sequestration agent can rescue transduction efficiency to levels observed when transduction is performed in the absence of bile.

[0197] Example 7: In vivo marking of the liver following intraductal delivery of lentiviral vectors and biliary therapeutic enhancer formulations Marking of hepatocytes was estimated by intraductal delivery of LVV encoding luciferase in a bile acid sequestrant and transduction enhancer-containing composition. Briefly, C67 / Bl6 mice surgically implanted with an external bile duct catheter were purchased from a commercial supplier. Retrograde injection was performed into the bile duct with 150 μL of a transduction composition containing a diluent containing a 7.5E7 TU / mouse lentiviral vector with a luciferase expression cassette (i.e., a ubiquitous constitutive promoter such as a synthetic MND promoter operably linked to a sequence encoding luciferase), bile acid sequestrant, and a transduction enhancer at a final concentration of 50 μg / mL colesevelam and 500 μg / mL F108, according to standard techniques. The injection was performed over a period of 30 seconds. After injection, the injection syringe was held in place for 5 minutes to prevent vector runoff. Control animals were injected with a vehicle composition that did not contain a vector carrying a luciferase expression construct. After treatment, the animals were maintained under standard conditions.

[0198] Injected mice were necropsied 4 days after LVV or vehicle injection. Left, middle, and right liver samples were collected from necropsied animals and fixed in 10% neutral buffered formalin for 24-48 hours, then transferred to 70% ethanol. Blocked tissues were sectioned and 3,3'-diaminobenzidine (DAB) stained immunohistochemistry was performed using an anti-luciferase antibody to detect the presence of luciferase expressed by the transduced cells.

[0199] Figures 5A and 5B provide representative sections of liver from vehicle and vector (LVV-luciferase) treated animals, respectively. The presence of DAB positive cells (dark brown) in Figure 5B demonstrates successful transduction and expression of luciferase in hepatocytes of mice administered intraductally with a composition containing LVV encoding luciferase, a bile acid sequestrant, and a transduction enhancer. As shown in Figure 5A, no positive cells were detected in control tissue.

[0200] This example demonstrates successful in vivo transduction of hepatocytes by intraductal LVV delivery using the described composition containing a bile acid sequestrant and a transduction enhancer, and expression of the delivered transgene. Using the delivery composition of the present disclosure, hepatocytes in living animals were successfully transduced with a bile-labile vector, despite the presence of bile in the delivery route used.

[0201] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made without departing from the spirit or scope of the appended claims.

[0202] Thus, the above merely describes the principles of the invention. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the invention and are within the spirit and scope of the invention, although not expressly described or shown herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts that the inventors have contributed to advancing the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly recited in the claims.

[0203] Therefore, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is expressly defined as being invoked for a limitation in a claim only if the precise phrase "means for" or the precise phrase "step for" is recited at the beginning of such limitation in the claim. If such precise phrase is not used in a limitation in a claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

Claims

1. 1. A composition for use in a method of treating a condition in a subject, comprising: The composition comprises an effective amount of a therapeutic biologic; the method comprising administering an effective amount of the therapeutic biologic directly to the biliary system of the subject, the method further comprising administering an effective amount of a biliary therapeutic enhancer directly to the biliary system of the subject, thereby treating a condition in the subject. composition.

2. The composition of claim 1 , wherein the condition is a liver condition.

3. The liver condition is selected from the group consisting of acute intermittent porphyria, acute liver failure, Alagille syndrome, alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, alcoholic liver disease, alpha 1-antitrypsin deficiency, amebic liver abscess, autoimmune hepatitis, biliary cirrhosis, Budd-Chiari syndrome, chemical and drug induced liver injury, cholestasis, chronic hepatitis, chronic hepatitis B, chronic hepatitis C, chronic hepatitis D, end stage liver disease, erythroblastic protoporphyria, fascioliasis, fatty liver disease, focal nodular hyperplasia, hepatic hydatid disease, hepatic encephalopathy, hepatic infarction, liver failure, hepatic porphyria, hepatic tuberculosis, hepatic veno-occlusive 3. The composition of claim 2, wherein the disease is selected from the group consisting of hepatitis, hepatocellular carcinoma, hepatic erythropoietic porphyria, hepatolenticular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, hereditary coproporphyria, liver abscess, hepatocellular adenoma, cirrhosis, liver failure, liver tumor, massive hepatic necrosis, nonalcoholic fatty liver disease, parasitic liver disease, peliosis hepatis, porphyria cutanea tarda, portal hypertension, suppurative liver abscess, Reye's syndrome, variant porphyria, viral hepatitis, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, and Zellweger syndrome.

4. 4. The composition of any one of claims 1-3, wherein said administering comprises retrograde ductal delivery of said therapeutic biologic to the liver of said subject, thereby resulting in an increased local liver concentration of said therapeutic biologic.

5. The composition of any one of claims 1 to 4, wherein the therapeutic biologic comprises a gene therapy agent or a protein.

6. 6. The composition of claim 5, wherein the gene therapy agent comprises a non-viral or viral vector, and optionally, the gene therapy agent comprises a lipid nanoparticle of an enveloped viral vector.

7. The composition of any one of claims 1 to 6, wherein the biliary therapeutic enhancer comprises one or more bile acid sequestrants.

8. the one or more bile acid sequestrants being Compounds of formula (I): 【Chemistry 1】 (In the formula, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3; X is an electrophilic leaving group; R ａ is hydrogen, C １ ~C ２０ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Alkynyl, or C １ ~C ２０ alkylammonium groups; wherein said alkyl is optionally substituted with OH or alkylammonium; Compound of formula (II): 【Chemistry 2】 (In the formula, n corresponds to a MW of about 100 to about 500; R １ is C ６ ~C １０ Aryl or C ２ ~C １０ heteroaryl, wherein said aryl or heteroaryl is selected from the group consisting of １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ optionally substituted with 1 to 3 substituents selected from the group consisting of alkynyl, alkynyl, or alkyl ammonium halide groups; Compound of formula (V): 【Chemistry 3】 (In the formula, m and n correspond to MW from about 100 to about 500; R １ and R ２ each is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group; and Compound of formula (VII): 【Chemistry 4】 (In the formula, m corresponds to MW from about 100 to about 500; a is an integer from 1 to 6 b is an integer from 1 to 6 c is an integer from 1 to 3.

8. The composition of claim 7, comprising a compound selected from the group consisting of:

9. 9. The composition of claim 7 or 8, wherein the one or more bile acid sequestrants are selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer.

10. The composition of any one of claims 1 to 9, wherein the biliary therapeutic enhancer comprises a cationic or non-ionic amphipathic transduction enhancer.

11. The cationic or non-ionic amphipathic transduction enhancer comprises: Compound of formula (III): 【Chemistry 5】 (In the formula, Each of x, y and z is independently an integer from 1 to 250; Compound of formula (IVa) or (IVb): 【Chemistry 6】 (In the formula, n corresponds to a MW of about 50,000 to about 200,000; x is an integer from 1 to 6 R １ is one or more C １ ~C ６ an amino group optionally substituted with an alkyl group; and Compound of formula (VI): 【Chemistry 7】 (In the formula, m corresponds to MW from about 100 to about 600; x is an integer from 1 to 10; y is an integer from 1 to 6; X is an electrophilic leaving group; Each R ａ are independently hydrogen or C １ ~C ６ is an alkyl group) The composition of claim 10, wherein the compound is selected from the group consisting of:

12. 12. The composition of claim 10 or 11, wherein the cationic or non-ionic amphipathic transduction enhancer is selected from the group consisting of polybrene, protamine sulfate, polyethyleneimine (PEI), poly(ethylene glycol) (PEG), poly-L-lysine, and F108.

13. 13. The composition of any one of claims 1-12, wherein the therapeutic biologic and the biliary therapeutic enhancer are co-administered, optionally wherein the therapeutic biologic and the biliary therapeutic enhancer are co-formulated in a single pharmaceutical composition.

14. The composition of any one of claims 1 to 13, wherein the biliary therapeutic enhancer is administered prior to the therapeutic biologic.

15. 1. A pharmaceutical composition comprising: a pharma- ceutically acceptable carrier configured as a liquid for delivery to the biliary system; an effective amount of a therapeutic biologic; Biliary Therapeutic Enhancers and 13. A pharmaceutical composition comprising:

16. 16. The composition of claim 15, wherein the therapeutic biologic comprises a gene therapy agent or a protein, optionally wherein the gene therapy agent comprises a non-viral or viral vector, optionally wherein the gene therapy agent comprises a lipid nanoparticle of an enveloped viral vector.

17. The biliary therapeutic enhancer comprises a bile acid sequestrant, and optionally, the bile acid sequestrant comprises: Compounds of formula (I): 【Chemistry 8】 (In the formula, m and n correspond to MW of about 500 to about 1000; p is an integer from 1 to 3; X is an electrophilic leaving group; R ａ is hydrogen, C １ ~C ２０ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ Alkynyl, or C １ ~C ２０ alkylammonium groups; wherein said alkyl is optionally substituted with OH or alkylammonium; Compound of formula (II): 【Chemistry 9】 (In the formula, n corresponds to a MW of about 100 to about 500; R １ is C ６ ~C １０ Aryl or C ２ ~C １０ heteroaryl, wherein said aryl or heteroaryl is selected from the group consisting of １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ optionally substituted with 1 to 3 substituents selected from the group consisting of alkynyl, alkynyl, or alkyl ammonium halide groups; Compound of formula (V): 【Chemistry 10】 (In the formula, m and n correspond to MW from about 100 to about 500; R １ and R ２ each is independently selected from the group consisting of an amino, an alkylamino, or an alkylammonium halide group; and Compound of formula (VII): 【Chemistry 11】 (In the formula, m corresponds to MW from about 100 to about 500; a is an integer from 1 to 6, b is an integer from 1 to 6; c is an integer from 1 to 3.

17. The composition of claim 15 or 16, wherein the compound is selected from the group consisting of:

18. The composition of any one of claims 15 to 17, wherein the bile acid sequestrant is selected from the group consisting of colesevelam, cholestyramine, colestipol, and sevelamer.

19. The composition of any one of claims 15 to 18, wherein the biliary therapeutic enhancer comprises a cationic or non-ionic amphipathic transduction enhancer.

20. A kit comprising: a liquid pharma- ceutically acceptable carrier; A therapeutic biologic; Biliary Therapeutic Enhancers and Including the kit.

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