In vivo degradation of toxins

Abstract

This invention generally relates to a technique for degrading systemic toxins in vivo by enhancing the metabolic function of the lungs. Such a technique involves introducing a composition into the lung tissue of a subject, the composition comprising at least one enzyme known to enzymatically break down at least one toxin systemically present in the subject's body. The provided technique enables the lungs to act as a modulated metabolic organ, thereby promoting the removal of toxins from the systemic circulation of a subject, such as a subject suffering from a condition leading to the accumulation of toxins or endogenous metabolites, or a subject who has ingested toxic substances.

Description

[0001] Cross-references to related applications This invention is based on Priority is claimed to U.S. Provisional Application 63 / 541,229, filed September 28, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] Many human diseases and conditions are caused by the accumulation of toxins in the body. Certain genetic disorders can cause the body to express enzymes that function abnormally or are not functioning properly. These enzymes can act on certain natural metabolites, leading to the toxic accumulation of those metabolites. Other diseases and conditions are caused by intentional or accidental poisoning events. What all these diseases have in common is the excessive accumulation of a certain chemical substance in the subject's body. In many cases, the toxic accumulation of this type of molecule occurs in the subject's systemic circulation.

[0003] Hereditary diseases can lead to the accumulation of toxic macromolecules in the body, the degradation or metabolism of which is essential for normal biological function and health. For example, phenylketonuria (PKU) is a hereditary metabolic disorder caused by the inactivation or deficiency of phenylalanine hydroxylase (PAH). PAH is an enzyme essential for the breakdown of phenylalanine (Phe), an amino acid found in all protein-containing foods. When PAH is deficient or inactivated, the level of Phe in the blood becomes abnormally high. The clinical manifestations of persistently high Phe levels include a variety of serious neurological and neuropsychiatric complications. Dietary modifications are often the first-line approach to disease management, including low-protein diets and specially formulated Phe-free medical foods. However, dietary modifications are often insufficient to mitigate the negative health effects of persistently elevated systemic Phe levels.

[0004] Systemic accumulation of toxic molecules can also result from exogenous substances entering the human circulatory system. For example, parathion is an organophosphate insecticide with an oral LD50 of 8 mg / kg, readily absorbed through the skin, mucous membranes, and oral ingestion. Parathion covalently binds to the active site of acetylcholinesterase, directly and stoichiometrically inactivating the enzyme. Without intervention, parathion poisoning can be fatal. It is estimated that 3 million or more people worldwide are exposed to organophosphates annually, with approximately 300,000 deaths. Therefore, exposure to organophosphates and other types of toxins remains a serious public health problem.

[0005] US Patent US20220047682A1 describes an amino acid sequence encoding ADH / KRED that binds to at least one long-acting molecule or complex molecule. The disclosed long-acting alcohol dehydrogenase exhibits a longer circulating half-life, a higher area under the curve (AUC), a lower clearance rate, a lower elimination rate, and a higher t1 / 2 value in blood and serum compared to wild-type ADH. Summary of the Invention

[0006] On one hand, the present invention provides a method for mitigating one or more negative effects of a toxin in the body, the method comprising: introducing at least one preselected enzyme into the lung tissue of a subject, said at least one preselected enzyme being known to enzymatically break down at least one toxin that produces one or more negative effects on the subject; and allowing said at least one toxin to undergo enzymatic breakdown in the body, thereby mitigating said one or more negative effects. In some embodiments, the subject suffers from one or more hereditary diseases. In some embodiments, said one or more hereditary diseases prevent the breakdown of said at least one toxin. In some embodiments, said one or more hereditary diseases are selected from phenylketonuria, gout, cystinuria, ornithine transcarbamate deficiency (OTCD), galactosemia, maple syrup urine disease, tumor suppression disorder, cocaine use disorder, urea cycle disorder, tobacco use disorder, Pompe disease, sucrase-isomaltase deficiency, arginase deficiency, hyperargininemia, and combinations thereof. In some embodiments, the subject suffers from a disease that leads to the accumulation of said at least one toxin. In some embodiments, the subject has been exposed to said at least one toxin. In some embodiments, the at least one toxin is selected from phenylalanine, uric acid, cystine, arginine, lysine, ornithine, leucine, isoleucine, valine, amino acids, galactose, kynurenine, cocaine, ammonia, nicotine, cyanide, organophosphates, and combinations thereof.In some embodiments, the at least one preselected enzyme is selected from: phenylalanine ammonia lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase α-subunit mitochondria (BCKDHA), 2-oxoisovalerate dehydrogenase β-subunit mitochondria (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthase, arginine succinate lyase, arginine succinate synthase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine carbamoyltransferase, ornithine translocase, nicotinic acid oxidoreductase, uricase, acid α-glucosidase (GAA), thiosulfate. Thiotransferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (tPA), alteplase, bovine pegase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase; lipase; protease), sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idolesulfatase, aglucosidase α, thrombin, verasidase α, pegologase, asparaginase, tadalasidase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A. Elosulfate esterase α, coagulation factor X, asfortaase α, cybebe esterase α, seripa esterase α, vetranesase α-vjbk, pevaricase-pqpz, and combinations thereof. In some embodiments, the at least one toxin is an amino acid (phenylalanine or Phe) or an acid (uric acid). In some embodiments, the lung tissue is contacted with the at least one toxin via the action of the subject's circulatory system. In some embodiments, the introduction step includes contacting the lung tissue with a polypeptide having an amino acid sequence corresponding to the at least one preselected enzyme, a polynucleotide encoding the at least one polypeptide, or both. In some embodiments, the introduction step includes transducing multiple cells present in the lung tissue to express the at least one preselected enzyme. In some embodiments, the multiple cells are transduced by DNA or a construct thereof. In some embodiments, the multiple cells are transduced by mRNA or a construct thereof. In some embodiments, allowing the enzymatic degradation of the at least one toxin includes allowing the at least one toxin that has diffused or migrated from the subject's circulatory system into the subject's lungs and / or lung mucus to undergo degradation.

[0007] On one hand, the present invention provides a method for mitigating one or more negative effects of at least one toxin in the body, the method comprising introducing at least one preselected enzyme into the lung tissue of a subject, said at least one preselected enzyme being known to enzymatically break down at least one toxin that produces one or more negative effects on said subject. In some embodiments, said at least one preselected enzyme is selected from phenylalanine aminolyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase α-subunit mitochondria (BCKDHA), 2-oxoisovalerate dehydrogenase β-subunit mitochondria (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthase, arginine succinate lyase, arginine succinate synthase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine carbamoyltransferase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), thiosulfate. Thiotransferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (tPA), alteplase, bovine pegase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase; lipase; protease), sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idolesulfatase, aglucosidase α, thrombin, verasidase α, pegologase, asparaginase, tadalafilase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A. Elosulfate esterase α, coagulation factor X, asfortaase α, secbease α, seripaase α, vetranesase α-vjbk, pevaricase-pqpz, and combinations thereof. In one embodiment of the invention, the at least one preselected enzyme is not an ADH / KRED bound to at least one long-acting molecule or complex molecule. In some embodiments, the method further includes allowing the at least one toxin that has diffused or migrated from the subject's circulatory system to the subject's lungs and / or pulmonary mucus to degrade in vivo, thereby mitigating the one or more negative effects.

[0008] On one hand, the present invention provides a method for converting the lungs of a subject to include at least one enzymatic degradation function. The method includes introducing at least one preselected enzyme into the lungs of the subject, the enzyme being known to promote the enzymatic degradation of at least one toxin, which, if present, is present in the subject's systemic system. In some embodiments, the at least one toxin circulates within the subject's body (including in the subject's lungs). In some embodiments, the method further includes contacting the at least one preselected enzyme with the at least one toxin to promote its enzymatic degradation in the body. In some embodiments, the enzymatic degradation occurs in the lungs and / or lung mucus of the subject.

[0009] On one hand, the present invention provides a transformed lung capable of enzymatically breaking down at least one toxin systemically present in a subject's body (including the subject's lungs), said transformed lung comprising lung tissue transformed to carry at least one preselected enzyme known to be capable of enzymatically breaking down said at least one toxin. In some embodiments, said transformed lung tissue carries said at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to said preselected enzyme, a polynucleotide encoding said polypeptide, or both.

[0010] On one hand, the present invention provides transformed lung cells comprising alveolar cells carrying at least one preselected enzyme, said preselected enzyme being known to enzymatically break down at least one toxin. In some embodiments, the transformed alveolar cells carry the at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to said preselected enzyme, a polynucleotide encoding said polypeptide, or both.

[0011] On one hand, the present invention provides a lung cell population comprising a plurality of alveolar cells carrying at least one preselected enzyme, the preselected enzyme being known to enzymatically break down at least one toxin.

[0012] On one hand, the present invention provides a method for mitigating one or more negative effects of leukemia in a subject, the method comprising: introducing an asparaginase into the lung tissue of a subject diagnosed with leukemia, the asparaginase being known to enzymatically break down asparagine; and allowing the asparagine to undergo enzymatic breakdown in vivo, thereby mitigating the one or more negative effects. In some embodiments, the leukemia includes acute lymphoblastic leukemia (ALL). In some embodiments, the asparaginase includes L-asparaginase.

[0013] On one hand, the present invention provides a method for mitigating one or more negative effects caused by excessive oxalate in a subject's body, the method comprising: introducing an oxalate decarboxylase into the lung tissue of a subject diagnosed with oxalate deposition, primary hyperoxaluria, or secondary hyperoxaluria, said oxalate decarboxylase being known to enzymatically break down oxalate; and causing said oxalate to be enzymatically broken down. In some embodiments, said oxalate includes endogenously produced oxalate or dietaryly ingested oxalate.

[0014] On one hand, the present invention provides a method for mitigating one or more negative effects of phenylketonuria in a subject, the method comprising: introducing phenylalanine ammonia lyase (PAL) into the lung tissue of a patient diagnosed with phenylketonuria, said PAL being known to enzymatically break down phenylalanine; and causing said phenylalanine to undergo enzymatic breakdown. In some embodiments, the patient has a mutation in the phenylalanine hydroxylase (PAH) gene.

[0015] On one hand, the present invention provides a method for mitigating one or more negative effects of hyperuricemia in a subject, the method comprising: introducing uricase, known to be capable of enzymatically breaking down uric acid, into the lung tissue of a subject diagnosed with hyperuricemia; and causing the uric acid to undergo enzymatic breakdown. In some embodiments, the subject has excessive uric acid production. In some embodiments, the subject has insufficient uric acid excretion. In some embodiments, the subject is diagnosed with or suspected of having gout.

[0016] On one hand, the present invention provides a method for mitigating one or more negative effects of cyanide poisoning in a subject, the method comprising: introducing thiosulfate-transferase (thiocyanate) into the lung tissue of a patient diagnosed with cyanide poisoning, said thiocyanate being known to enzymatically decompose cyanide; and causing said cyanide to undergo enzymatic decomposition. In some embodiments, the patient has ingested cyanide salts, taken liquid hydrogen cyanide, absorbed hydrogen cyanide through the skin, received intravenous injection of sodium nitroprusside, or inhaled hydrogen cyanide gas. In some embodiments, the introduction further includes introducing sodium thiosulfate into the lung tissue of the subject.

[0017] On one hand, the present invention provides a method for mitigating one or more negative effects of hyperammonemia in a subject, the method comprising: introducing glutamine synthase into the lung tissue of a subject diagnosed with hyperammonemia, said glutamine synthase being known to enzymatically remove ammonia; and causing said ammonia to undergo enzymatic removal. In some embodiments, the subject has excessive ammonia production. In some embodiments, the subject has insufficient ammonia excretion. In some embodiments, the subject is diagnosed with or suspected of having ornithine carbamoyltransferase deficiency (OTCD).

[0018] On one hand, the present invention provides a method for mitigating one or more negative effects of hyperarginemia in a subject, the method comprising: introducing an arginase, known to be capable of enzymatically breaking down arginine, into the lung tissue of a subject diagnosed with hyperarginemia; and causing the arginine to undergo enzymatic breakdown. In some embodiments, the subject has an excess of arginine. In some embodiments, the subject has been diagnosed with or is suspected of having arginase deficiency.

[0019] In some implementations, the at least one preselected enzyme, its variants, or combinations thereof are primarily present in the lung tissue of the subject.

[0020] In some embodiments, the at least one preselected enzyme, its variants, or combinations thereof are introduced under conditions that inhibit or do not support the systemic delivery of the enzyme, its variants, or combinations thereof to the subject.

[0021] In some embodiments, the introduction of the at least one preselected enzyme, its variants, or combinations thereof does not involve delivering the at least one preselected enzyme, its variants, or combinations thereof to the subject via the pulmonary mucosa.

[0022] In some embodiments, the at least one preselected enzyme, its variants, or combinations thereof are introduced into at least a portion of the lungs of the subject in a manner that minimizes systemic introduction.

[0023] The above description of the invention, along with the accompanying drawings and detailed embodiments below, are exemplary and illustrative, intended to provide further details of the invention, and should not be construed as limiting the scope of the invention. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

[0024] It should be understood that all combinations of the above-described invention and other concepts discussed in more detail below are considered part of the inventive subject matter disclosed herein and can be used in any combination to achieve the beneficial effects described herein. Brief description of the attached figures

[0025] Figure 1 This is a graph showing the effect of the route of administration of phenylalanine ammonia-lyase (PAL) on the change of circulating phenylalanine (Phe) levels over time in a chemically induced phenylketonuria (PKU) rat model, with measurements in µg / mL serum.

[0026] Figure 2A This is a graph showing the effect of the route of administration of phenylalanine ammonia-lyase (PAL) on the level of circulating phenylalanine (Phe) in a chemically induced phenylketonuria (PKU) mouse model, measured in µg / mL serum.

[0027] Figure 2B This is a graph showing the change in circulating Phe levels in mice as a function of PAL administration, measured in µg / mL serum. The y-axis shows the difference between the Phe value measured after PAL administration (reported in µg / mL) and the Phe value measured before PAL administration.

[0028] Figure 3 This is a graph showing the effect of the administration route of phenylalanine ammonia-lyase (PAL) on the circulating phenylalanine (Phe) level in a PKU gene-producing mouse model. The measurement unit is µM. The gray box indicates PAL administered intratracheally; the blue box indicates PAL administered subcutaneously (SQ).

[0029] Figure 4 This is a graph showing the change in serum uric acid concentration (µM) in mice over time after administration of uricase. The measurement at time 0 was obtained immediately before administration of uricase. IT = intratracheal administration; IV = intravenous administration; black circle = no uricase administration; red circle = intravenous uricase administration; blue circle = intratracheal uricase administration.

[0030] Figure 5A This is a graph showing the change in serum uric acid concentration (µM) over time in mice given uricase. Each healthy female mouse received 2 mg of aflatoxin-based uricase via intravenous (green; n=5), intratracheal (blue; n=5), or intratracheal injection of saline (black; n=2). The graph shows the time progression data of serum uric acid (UA) after administration. Both intravenous and intratracheal administration of uricase significantly reduced uric acid levels. Uric acid levels remained unchanged in mice injected with saline throughout the study. Statistical analysis: Data are expressed as mean ± standard deviation; multiple t-tests were used to compare the control and treatment groups.

[0031] Figure 5B yes Figure 5A The graph shows the area under the curve (AUC) of the time-course data. Statistical analysis: Data are expressed as mean ± standard deviation; one-way ANOVA was used to compare the control group and the treatment group (*). Black arrows indicate the percentage decrease compared to the control group. An unpaired t-test was used to compare the treatment group (^).

[0032] Figure 6A This is a graph showing the change in uric acid concentration (µM) over time in serum samples from cynomolgus non-human primates (NHP) given a variant of uricase. At Cmax, the serum UA concentrations were: 74 µM ± 24.5 SD (UA control group), 59 µM ± 12 SD (low-dose uricase + UA group), and 46 µM ± 20 SD (high-dose uricase + UA group).

[0033] Figure 6B yes Figure 6A The figure shows the area under the curve (AUC) of the time-course data. Based on the AUC analysis of the time-course data (60–515 minutes), serum uric acid decreased by 36.6% (low-dose uricase group) and 56.8% (high-dose uricase group) compared to the UA control group, p<0.01. Data are expressed as mean ± SD and were compared with the uric acid control group using one-way ANOVA. **p<0.01, ***p<0.001.

[0034] Figure 7A This is a graph showing the change in uric acid concentration (µM) over time in serum samples from mice administered wild-type or variant uricase. Each healthy female mouse received 2 mg of either aflatoxin-derived uricase or an engineered variant uricase, or a vector (black, n=3). The graph shows the time progression data of serum uric acid (UA) after administration. Statistical analysis: Data are expressed as mean ± SD; multiple t-tests were used to compare the control and treatment groups.

[0035] Figure 7B yes Figure 7A A graph of the area under the curve (AUC) of the time-course data. Based on the AUC analysis of the time-course data (0-240 minutes), serum uric acid decreased by 29.8% (wild-type uricase group) and 34.4% (variant uricase group) compared to the UA control group, p<0.01. Statistical analysis: Data are expressed as mean ± SD; one-way ANOVA was used to compare the control group and the treatment group (*). Black directional arrows indicate the percentage decrease compared to the control group. Unpaired t-tests were used to compare the treatment group (^).

[0036] Figure 8A This is a graph showing the change in plasma ammonia (µM) levels over time in samples collected from mice after drug administration. These mice were administered saline followed by NH4Cl (triangle), wild-type glutamine synthase (GS) followed by NH4Cl (cross), or wild-type glutamine synthase (GS) plus glutamate followed by NH4Cl (circle).

[0037] Figure 8B This is a graph showing the change in plasma glutamine (µM) levels over time in samples collected from mice. These mice were administered saline followed by NH4Cl (triangle), wild-type glutamine synthase (GS) followed by NH4Cl (cross), or wild-type glutamine synthase (GS) plus glutamate followed by NH4Cl (circle).

[0038] Figure 8CThis is a graph showing the change in plasma glutamate (µM) levels over time in samples collected from mice after drug administration. These mice were administered saline followed by NH4Cl (triangle), wild-type glutamine synthase (GS) followed by NH4Cl (cross), or wild-type glutamine synthase (GS) plus glutamate followed by NH4Cl (circle).

[0039] Figure 9 The graphs show the changes in plasma ammonia levels (µM) over time in samples collected from the following groups of mice: wild-type (WT) mice given NH4Cl after saline administration (cross-shaped), OTCD model mice given NH4Cl after saline administration (round), and OTCD model mice given NH4Cl after wild-type glutamine synthase (GS) administration (triangle).

[0040] Figure 10A This is a graph showing the change in plasma L-arginine levels (µM) over time in samples collected from mice that were given either saline (square) or arginase (round).

[0041] Figure 10B This is a graph showing the change in plasma urea levels (µM) over time in samples collected from mice that were given either saline (square) or arginase (round).

[0042] Figure 11A This is a graph showing the change in plasma asparagine levels (µM) over time in samples collected from mice that were given either saline (square) or asparaginase (round).

[0043] Figure 11B This is a graph showing the change in plasma aspartate levels (µM) over time in samples collected from mice that were given either saline (square) or asparaginase (round).

[0044] Figure 11C This is a graph showing the change in plasma ammonia levels (µM) over time in samples collected from mice that were given either saline (square) or asparaginase (round). Detailed Implementation

[0045] Systemic toxicity refers to a condition caused by the accumulation and / or absorption of a substance and its distribution throughout the body (rather than in a localized area). For example, systemic toxicity may be caused by one or more genetic diseases in which the enzymes responsible for breaking down or clearing endogenous molecules or metabolites are impaired or lost, leading to the accumulation of the substance in the systemic circulation and consequently impairing certain physiological processes. Systemic toxicity can also be caused by a subject ingesting a toxic substance and subsequently absorbing it into the systemic circulation. Therefore, toxicity in the systemic circulation can be considered an important factor in the etiology of many diseases and conditions. In one embodiment of the invention, the toxic substance does not include alcohols, such as ethanol.

[0046] Given the fundamental role of systemic toxicity in the pathophysiology of a variety of diseases and conditions, new compositions, methods, and techniques are still needed to degrade toxic substances present in the systemic circulation of subjects in vivo.

[0047] This invention generally relates to techniques for degrading systemic toxins in a subject in vivo. Such techniques include introducing a composition into the lung tissue of a subject, said composition comprising at least one enzyme known to enzymatically break down at least one toxin systemically present in the subject's body (e.g., reducing the level of toxins in the subject, such as reducing the level of endogenous toxic metabolites in the subject, or reducing the level of toxins exogenously ingested by the subject). It should be understood that introducing at least one preselected enzyme includes introducing at least one preselected variant, or a combination of at least one preselected enzyme and at least one preselected variant thereof.

[0048] The embodiments of the present invention will now be described in more detail. However, various aspects of the invention may be embodied in different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided to make the present invention exhaustive and to fully elucidate the scope of the invention to those skilled in the art. The terminology used in this description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

[0049] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, it should be understood that terms such as those defined in common dictionaries shall be interpreted as having the same meaning in the context of this application and related technical fields, and should not be interpreted in an idealized or overly formal manner unless expressly defined herein. Although not expressly defined below, such terms shall be interpreted according to their ordinary meaning.

[0050] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail. Other aspects are set forth in the following claims.

[0051] Unless otherwise stated, the practice of this technique will employ conventional techniques such as tissue culture, immunology, molecular biology, microbiology, chemical engineering, and cell biology, all of which fall within the skill scope of this field.

[0052] Unless the context otherwise requires, the features described herein may be used in any combination. Furthermore, this application contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For example, if the specification indicates that a complex comprises components A, B, and C (or A, B, and / or C), it expressly indicates that any one of A, B, or C, or any combination thereof, may be omitted or excluded individually or in any combination.

[0053] Unless otherwise expressly stated, all specified implementations, features and terms are intended to include the described implementations, features or terms and their biological equivalents.

[0054] All values, such as pH, temperature, time, concentration, and molecular weight (including range), are approximate and can be adjusted positively or negatively in increments of 1.0 or 0.1, or in increments of + / - 15%, 10%, 5%, or 2%, depending on the specific circumstances. It is important to note that, although not always explicitly stated, all values ​​are preceded by the word "approximately."

[0055] definition Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this specification and appended claims also include the plural forms.

[0056] The terms "substantially" and "approximately" used in this document are to describe and indicate minor variations. When used in conjunction with an event or situation, these terms can refer to either the event or situation occurring completely or very close to occurring. When used with a numerical value, these terms can refer to a deviation of less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. When referring to a first and second numerical value as "substantially" or "approximately" the same, these terms can refer to a deviation of less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. When terms such as “acceptable,” “effective,” or “sufficient” are used to describe any selection of components, ranges, dosage forms, etc. disclosed herein, it means that the components, ranges, dosage forms, etc. are suitable for the disclosed purpose.

[0057] In addition, this document sometimes presents quantities, ratios, and other values ​​in range form. It should be understood that this range form is used for convenience and brevity, and its meaning should be flexibly interpreted to include both the values ​​explicitly specified as range boundaries and all individual values ​​or subranges covered within that range, as if each value and subrange had been explicitly specified. For example, a ratio of approximately 1 to approximately 200 should be understood to include the explicitly listed ranges “approximately 1” and “approximately 200”, as well as individual ratios such as “approximately 2”, “approximately 3”, and “approximately 4”, and subranges such as “approximately 10 to approximately 50”, “approximately 20 to approximately 100”, and so on.

[0058] Furthermore, the term “and / or” as used herein refers to and covers any and all possible combinations of one or more of the related listed items, as well as cases where no combination exists when interpreted in an alternative manner (i.e., “or”).

[0059] The term "comprising" as used herein is intended to mean that a composition and method includes the listed elements, but does not exclude other elements. "Substantially consisting of" when used to define a composition and method should mean excluding other elements that have any substantial significance to the composition or method. "Constitutes of" should mean excluding trace amounts of other ingredients for the claimed composition and substantial method steps. Examples and embodiments defined by each of the foregoing transitional terms are within the scope of this invention. Therefore, the methods and compositions may comprise other steps and components (comprising), or alternatively include steps and compositions that are not substantial (substantially consisting of), or consist only of the method steps or compositions (consisting of).

[0060] As used herein, “optional” or “optionally” means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which the event or situation does not occur.

[0061] As used herein, the terms “administration” or “application,” “introduction” or “extraction” refer to any route by which a product is introduced or delivered to a subject to perform its intended function. Administration or introduction of a product into the lung tissue of a subject may be performed via any suitable route, but must be performed without causing significant or substantial exudation of the product from the lung tissue (i.e., minimizing systemic absorption). Administration or introduction of a product may be performed by inhalation. However, to avoid significant or substantial systemic absorption, inhalation of a nebulized solution of the product is preferred. Avoiding or minimizing systemic absorption means that the majority of the introduced product remains in the lung tissue. In some embodiments of the invention, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the introduced drug permeates through mucosal tissue (i.e., introduction is not via mucosa) into the blood or serum of the subject. Administration or introduction of a product may be performed via an intratracheal route, with the introduced product ultimately entering the lung tissue of the subject. Alternatively, the administration or introduction of the agent may be performed locally, intranasally, but preferably not via intraperitoneal, intradermal, ocular, intrathecal, intraventricular, iontophoresis, transmucosal, intravitreal, or intramuscular routes. Administration methods include self-administration, administration by another person, or administration using a device (e.g., a nebulizer, dry powder inhaler, or vapor pump, but preferably not an infusion pump). In one embodiment of the invention, "introduction" does not include delivery of the drug via the pulmonary mucosa, as this would result in systemic administration of the drug to the subject. In some embodiments, "introduction" does not include ADH / KRED bound to at least one long-acting molecule or complexing molecule. In other embodiments, "administration" does not include inhalation of a nebulized solution of ADH / KRED bound to at least one long-acting molecule or complexing molecule.

[0062] As used in this article, “improvement” or “relief” of a disease, disorder, or condition means that, in a statistical sample or specific group of subjects, the occurrence of the disease, disorder, or condition (or its signs, symptoms, or status) is improved or more tolerable compared to a control sample or group of subjects.

[0063] The term "amino acid" as used herein includes both naturally occurring and non-natural amino acids. Unless otherwise stated, the term "amino acid" includes both isolated amino acid molecules (i.e., molecules containing both a hydrogen atom attached to the amino group and a hydroxyl group attached to the carbonyl carbon) and amino acid residues (i.e., molecules with the hydrogen atom attached to the amino group or the hydroxyl group attached to the carbonyl carbon removed, or both). The amino group can be α-amino, β-amino, etc. For example, the term "amino acid alanine" can refer to isolated alanine H—Ala-OH, or any of the alanine residues H-Ala, -Ala-OH, or -Ala-. Unless otherwise stated, all amino acids found in the reagents described herein may be in the D or L configuration. D-configured amino acids can be written by adding "D" before the amino acid abbreviation. For example, "D-Arg" indicates arginine in the D configuration. By convention, if an amino acid name is not preceded by "D" or "L", it is assumed to be in the "L" configuration. It is worth noting that many amino acid residues are commercially available in both D and L configurations.

[0064] The term "amino acid" includes its salts, including pharmaceutically acceptable salts. Any amino acid may be protected or unprotected. Protecting groups may be attached to an amino group (e.g., α-amino), a main-chain carboxyl group, or any functional group on the side chain. For example, phenylalanine protected by a benzyloxycarbonyl (Z) group on its α-amino group may be represented as Z-Phe-OH. Amino acid protecting groups are well known in the art. For a comprehensive review of amino acid protecting groups, see: Isidro-Llobet et al., Chem. Rev. (2009) 109: 2455-2504.

[0065] Except for the N-terminal amino acid, all amino acid abbreviations in this application (e.g., Phe) represent the structure —NH—C(R)(R′)—CO—, where R and R′ are each independently a hydrogen atom or an amino acid side chain (e.g., for Phe, R = benzyl, R′ = H). Accordingly, phenylalanine is H-Phe-OH. For these amino acids or peptides (e.g., Lys-Val-Leu-OH), “OH” indicates that its C-terminus is a free acid. For example, “NH2” in H-Phe-D-Arg-Phe-Lys-NH2 indicates that the C-terminus of the protected peptide fragment is amidated. In each case, the “H” before the amino acid or peptide indicates that the N-terminal amine of the amino acid or peptide is unmodified (i.e., -NH2). Furthermore, certain R and R', individually or in combination, forming cyclic structures, may contain functional groups that may require protection in liquid-phase or solid-phase synthesis.

[0066] As used herein, the terms “carrier,” “pharmaceutically acceptable carrier,” “physiologically acceptable carrier,” or “appearance-acceptable carrier” refer to diluents, adjuvants, excipients, or carriers used for administration or formulation of pharmaceutical preparations / compositions. Such pharmaceutically acceptable carriers include, but are not limited to, liquids (e.g., water, physiological saline), oils, and solids (e.g., gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, silica particles (nanoparticles or microparticles), urea, etc.). Additionally, excipients, stabilizers, thickeners, lubricants, flavoring agents, and coloring agents may also be used. Other suitable examples of pharmaceutical carriers can be found in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference in its entirety.

[0067] As used herein, the term "effective amount" refers to an amount of composition / pharmaceutical product sufficient to achieve the intended therapeutic and / or preventative effect, such as an amount capable of treating, preventing, inhibiting, improving, or delaying the onset of a disease, disorder, or condition, or improving the physiological signs, symptoms, or condition of a disease or disorder. In therapeutic or preventative applications, in some embodiments, the amount of composition / pharmaceutical product given to the subject depends on the type and severity of the disease and individual characteristics, such as general health status, age, sex, weight, and tolerance to the drug. In some embodiments, it also depends on the extent, severity, and type of the disease. Those skilled in the art can determine an appropriate dosage based on these and other factors. The composition / pharmaceutical product may also be used in combination with one or more other therapeutic agents (i.e., so-called "combination therapy," where additional or other therapeutic agents may be administered simultaneously, sequentially, or separately).

[0068] As used herein, the term "pharmaceutical composition" refers to a combination of a therapeutic agent and a carrier (inert or active carrier) that makes the composition particularly suitable for in vivo treatment. Similarly, dietary supplements, food, and cosmetic compositions also fall into this category.

[0069] As used in this article, “prevention” or “control” of a disease, disorder, or condition refers to a reduction in the incidence of the disease, disorder, or condition in a sample or subject receiving one or more therapeutic agents compared to a control group. This prevention is sometimes referred to as preventative treatment.

[0070] As used in this article, "simultaneous" therapeutic use refers to the administration of at least two active ingredients (e.g., therapeutic agents) via the same route and at the same or substantially the same time.

[0071] As used herein, the term "subject" refers to a live animal. In various embodiments, the subject is a mammal. In various embodiments, the subject is a non-human mammal, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, sheep, goats, cats, dogs, pigs, miniature pigs, horses, cattle, or non-human primates. In some embodiments, the subject is a human.

[0072] As used herein, the terms “treatment” or “under treatment” refer to therapeutic interventions aimed at reducing, alleviating, or slowing down (reducing) an existing disease or condition, or its associated signs, symptoms, or status. For example (but not limited to), a subject is considered successfully “treated” if, after receiving an effective amount of the composition / pharmaceutical product or its pharmaceutically acceptable salt, the subject exhibits an observable and / or measurable reduction or disappearance of one or more signs, symptoms, or statuses associated with the disease, disorder, or condition. It should also be understood that the various modes of treatment for medical conditions are intended to represent “substantial” relief, including complete remission of the symptoms, signs, or status of the disease or condition, as well as “partial” relief, i.e., achieving some biologically or medically relevant therapeutic effect.

[0073] As used herein, the term "toxin" refers to a toxic chemical substance whose accumulation or exposure can have adverse effects on a subject. Toxic chemicals include discrete small molecules, macromolecules, synthetic or natural products, amino acids, peptides, polypeptides, or proteins of any size that are present in the subject's body. Because toxins are discrete, they can be identified or isolated from mixtures. As used herein, the term "toxin" does not include microorganisms and viruses, but includes toxins produced by microorganisms or viruses (if present). Microorganisms can be bacteria, fungi, archaea, or protozoa. Microorganisms do not include viruses and prions, which are generally classified as abiotic. Nucleotides, polynucleotides, and nucleic acids (including ribonucleic acid (RNA)) are thought to be toxic in certain circumstances; these toxic molecules are considered to be toxins herein. In one embodiment, a toxin is defined as a metabolite that accumulates in the body to a level sufficient to produce a toxic effect. In one embodiment, a toxin is present in the circulatory system of the affected subject. It should be noted that this term does not include non-systemic toxic substances—that is, substances that do not circulate in the affected subject but are primarily confined to or accumulate in specific tissues (such as lung tissue).

[0074] Drug composition, route of administration and dosage The methods, uses, and compositions of the present invention utilize a therapeutically effective amount of the composition to degrade or metabolize systemic toxins in vivo. In some embodiments, the composition comprises an enzyme or a polynucleotide encoding the enzyme, which may deposit in the lung tissue of a subject to degrade or metabolize toxins capable of diffusing between the lung tissue and the subject's circulatory system. This deposition of the enzyme or polynucleotide in the subject's lung tissue enhances the subject's metabolic efficiency and may alleviate symptoms caused by systemic toxins in the subject.

[0075] The pharmaceutical compositions used herein refer to compositions comprising a therapeutic agent and a carrier (inert or active), particularly suitable for in vivo therapy. For example, a pharmaceutical composition may comprise an enzyme known to catalyze the breakdown or degradation of toxins. For example, a pharmaceutical composition may comprise a polynucleotide encoding an enzyme known to catalyze the breakdown or degradation of toxins. In some embodiments, a pharmaceutical composition may comprise an enzyme known to catalyze the breakdown or degradation of toxins and a polynucleotide encoding that enzyme. In some preferred embodiments, the pharmaceutical composition is particularly suitable for delivering and depositing a therapeutic agent (e.g., an enzyme or a polynucleotide encoding that enzyme) into the tissues of a subject. In some preferred embodiments, the pharmaceutical composition is particularly suitable for delivering and depositing an enzyme into the lung tissue of a subject.

[0076] The pharmaceutical compositions disclosed in this invention may comprise enzymes known to catalyze the breakdown or degradation of at least one systemic toxin. Non-limiting examples of enzymes known to catalyze the breakdown or degradation of systemic toxins include phenylalanine ammonia-lyase (PAL) (e.g., Genbank accession numbers NP_181241.1, NP_187645.1, NP_190894.1, NP_001190223.1, and NP_196043.2), phenylalanine hydroxylase (PAH) (e.g., Genbank accession numbers NP_000268 and NP_001341233), galactose-degrading enzymes, galactose-1-phosphate uridine dihydrogenase (GALT) (e.g., Genbank accession numbers NP_000146.2 and NP_001245261.1), and 2-oxoisovalerate dehydrogenase subunit α-mitochondrial (BCKDHA) (e.g., Genbank accession numbers NP_181241.1, NP_187645.1, NP_190894.1, NP_001190223.1, and NP_196043.2), Genbank accession numbers NP_181241.1, NP_187645.1, NP_18 ...7645.1, NP_187645.1, NP_187645.1, NP_187645.1, Accession numbers NP_001158255.1 and NP_000700.1), 2-oxoisovalerate dehydrogenase β subunit mitochondria (BCKDHB) (e.g., Genbank accession numbers NP_000047.1, NP_001305904.1 and NP_898871.1), BDT Complex enzymes, kynurenase (e.g., Genbank accession numbers NP_001028170.1, NP_001186170.1, and NP_003928.1), cocaine esterase (e.g., Genbank accession numbers NP_001352334.1, NP_003860.3, NP_932327.2, NP_001352335.1, NP_001352336.1, and NP_001352337.1), arginase (e.g., Genbank accession numbers NP_001163.1, NP_000036.2, and NP_001231367.1), glutamine... Amide synthases (e.g., GenBank accession number NP_003866.1), arginine succinate lyases (e.g., GenBank accession numbers NP_000039.2, NP_001020117.1, NP_001020114.1 and NP_001020115.1), arginine succinate synthases (e.g., GenBank accession numbers NP_000041.2 and NP_446464.1), carbamoyl phosphate synthase I (e.g., GenBank accession numbers NP_001116105.2, NP_001866.2), N-acetylglutamate synthases (e.g., GenBank... Accession number NP_694551.1), ornithine transcarbamate (e.g., GenBank accession numbers NP_000522.3 and NP_001394021.1), ornithine translocase (e.g., GenBank accession numbers NP_055067.1 and NP_114153).1) Nicotine oxidoreductase, uricase (e.g., GenBank accession number NP_180191.1), acid α-glucosidase (GAA) (e.g., GenBank accession numbers NP_000143.2, NP_001073271.1, and NP_001073272.1), thiosulfate-thiotransferase (thiocyanate) (e.g., GenBank accession numbers NP_001257412.1 and NP_003303.2), collagenase (e.g., GenBank accession numbers NP_002412.1, NP_001291370.1, NP_001291371.1, and NP_001291371.1). Asparaginase (e.g., Genbank accessions NP_001077395.1 and NP_079356.3), anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA) (e.g., alteplase; tissue plasminogen activator), bovine peggenase (e.g., PEG-adenosine deaminase), vidarase (e.g., a modified form of human β-glucocerebrosidase), imiglucerase (e.g., a form of recombinant human β-glucocerebrosidase), factor IX (e.g., Genbank accessions NP_000124.1 and NP_001300842.1), deoxyribonuclease, pancreatic lipase (e.g., amylase; lipase; proteinase). Albuminase, sucrase (e.g., a sucrase substitute), truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa (e.g., Genbank accession numbers NP_000122.1, NP_001254483.1, NP_062562.1), tissue plasminogen activator, antihemophilic factor (AHF) (e.g., coagulation activator), laronidase (e.g., a form of recombinant human α-L-idurosidase), agalsidase β (e.g., a form of recombinant human α-galactosidase), hyaluronidase (e.g., an enzyme that reversibly depolymerizes hyaluronic acid), sulfidase (e.g., polymorphic human enzyme N-acetylgalactosamine-4- Variant forms of sulfatase), idoosulfatase (e.g., purified lysosomal enzyme), alglucosidase α (e.g., acid α-glucosidase (GAA) derivative), thrombin (e.g., serine protease), vilazinase α (e.g., gene-activated human recombinant glucocerebrosidase), polyethylene glycol uricase (e.g., polyethylene glycolated recombinant uricase), asparaginase, tadalafilase α (e.g., recombinant glucocerebrosidase), plasminogen activator (e.g., GenBank accession numbers NP_000292.1 and NP_001161810.1), carboxypeptidase g2 (e.g., UniProt accession number P06621), glutapeptidase (e.g., recombinant carboxypeptidase G2), coagulation factor XIII A (e.g., GenBank accession number NP_000120).2) Elosulfate esterase α (e.g., a synthetic version of N-acetylgalactosamine-6-sulfate esterase), coagulation factor X (e.g., Genbank accession numbers NP_001299604.1, NP_000495.1, and NP_001299603.1), asfortaase α (e.g., a recombinant glycoprotein containing a tissue-nonspecific alkaline phosphatase catalytic domain (active site), secbease α (e.g., a recombinant lysosomal acid lipase), seripaase α (e.g., hydrolyzing lysosomal N-terminal tripeptidyl peptidase-1 (TPP1)), vetranesase α-vjbk (e.g., a recombinant form of human β-glucuronidase), pevaricase-pqpz (e.g., recombinant phenylalanine ammonia-lyase (PAL)), and combinations thereof. The enzymes may be isolated human enzymes. The enzymes may be non-human enzymes. The enzymes may be recombinant enzymes. The enzymes may be wild-type enzymes or variants thereof. In some embodiments, the variant enzyme contains an amino acid sequence that shares at least 65%, 70%, 80%, 90%, 95%, or 99% sequence identity with the corresponding wild-type enzyme.

[0077] In some embodiments, the enzyme, its variants, or combinations thereof are substantially present in the lung tissue of the subject after introduction. In some embodiments, the enzyme, its variants, or combinations thereof are introduced under conditions that inhibit or do not support systemic delivery of the enzyme, its variants, or combinations thereof to the subject. In some embodiments, the enzyme, its variants, or combinations thereof are introduced into at least a portion of the lungs of the subject in a manner that minimizes systemic delivery to the subject. In some embodiments, the introduction of the enzyme, its variants, or combinations thereof does not involve delivery of the at least one preselected enzyme, its variant, or combinations thereof to the subject via the pulmonary mucosa.

[0078] In some embodiments, an enzyme known to enzymatically break down at least one toxin, used according to the technology of the invention, is linked to a polypeptide, polypeptide domain, or fragment thereof, which is absorbed by the lungs via a receptor-mediated endocytic transport pathway. While not wishing to be bound by any single theory, it is understood that absorption via such a receptor-mediated endocytic transport pathway can increase the proportion of the enzyme known to enzymatically break down at least one toxin remaining in the lung tissue of the subject, and / or minimize the systemic introduction of the enzyme into the subject.

[0079] In some embodiments, an enzyme known to enzymatically break down at least one toxin, used according to the technology of the present invention, is PEGylated. While not wishing to be bound by any single theory, it is understood that PEGylation of the enzyme can reduce the clearance of such enzyme in the blood (e.g., increase the proportion of enzymes known to enzymatically break down at least one toxin remaining in the lung tissue of a subject, and / or minimize the systemic introduction of said enzyme into the subject).

[0080] The enzymes used in this invention, whether used alone or included in a pharmaceutical composition, are preferably enzymes capable of degrading or breaking down systemic toxins that can diffuse between the circulatory system and the lung tissue of a subject. Therefore, toxins that can be targeted by the methods of this invention are toxins capable of diffuse between the circulatory system and the lung tissue of a subject. Without wishing to be bound by any theory, upper limits for the size of circulating toxins may be considered in some embodiments of this disclosure. For example, these upper limits may be 60 kDa and above, 100 kDa and above, 500 kDa and above, 1000 kDa and above, or 2000 kDa and above. Therefore, in some embodiments, toxins with molecular weights reaching or exceeding these upper limits may not be degraded by degrading enzymes in the pulmonary environment.

[0081] In some embodiments, the pharmaceutical composition may comprise one or more cofactors, coenzymes, or cosubstrates. In some embodiments, the pharmaceutical composition comprises at least one enzyme known to enzymatically break down at least one systemic toxin, and further comprises one or more cofactors, coenzymes, cosubstrates, or combinations thereof. It will be apparent to those skilled in the art to select a suitable cofactor based on an enzyme known to enzymatically break down at least one toxin. While not wishing to be bound by any single theory, it is understood that the relative amounts of reduced and oxidized cofactors play an important role in the balance of biochemical reactions. Therefore, cofactor regeneration is crucial for metabolic efficiency. In some embodiments, the pharmaceutical composition of the present invention comprises an additional enzyme for cofactor regeneration.

[0082] A drug is generally considered to be a composition or preparation specifically designed to treat a disease, disorder, or symptom (e.g., the presence of systemic toxins or the systemic accumulation of toxic metabolites).

[0083] In some embodiments, the therapeutic agent (e.g., an enzyme or a polynucleotide encoding an enzyme) may be formulated to contain little or no excipients or carriers. In some embodiments, the therapeutic agent may also be formulated to consist primarily of excipients or carriers. In short, those skilled in the art will adjust the content of excipients or carriers in the formulation based on the needs / condition of the subject, the type and severity of the disease to be treated, the characteristics of the therapeutic agent to be delivered, and the chosen route of administration.

[0084] In some embodiments, the pharmaceutical composition may further comprise, in addition to containing at least one enzyme known to enzymatically break down at least one toxin, at least one other therapeutic agent. For example, the pharmaceutical composition may further comprise one or more antiemetics, analgesics, naltrexone, acampalic acid, disulfiram, gabapentin, topiramate, antifungals, antibiotics, and probiotics. In some embodiments, the pharmaceutical composition may further comprise, in addition to containing at least one enzyme known to enzymatically break down at least one toxin (or a polynucleotide encoding that enzyme), at least one agent that promotes the absorption of the enzyme in the lungs. Agents that promote the absorption of the enzyme in the lungs include, but are not limited to, agents that promote endocytic transport (e.g., receptor-mediated endocytosis), liposomes, cyclodextrins, and low molecular weight amino acids.

[0085] The pharmaceutical composition may contain an effective amount of one or more therapeutic agents described herein, and may selectively disperse them in a pharmaceutically acceptable carrier (e.g., dissolved, suspended, or otherwise). The components of the pharmaceutical composition should also be miscible with other therapeutic agents or active agents and with each other, provided that such miscibility does not produce interactions that significantly impair the intended therapeutic effect.

[0086] As stated above, "effective amount" refers to any dose of a specific therapeutic agent sufficient to achieve the intended biological effect. In conjunction with the content described herein, by selecting different therapeutic agents and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse effects, and route of administration, effective prophylactic or therapeutic regimens can be developed that effectively treat a specific condition, disorder, or disease in a particular subject without causing substantial undesirable toxicity. The effective amount of a therapeutic agent for any specific indication can vary depending on factors such as the disease, disorder, or condition being treated, the specific drug administered, the subject's body type, age, overall health status, and / or the severity of the disease, disorder, or disease. Effective amounts can be determined using methods familiar to physicians and clinicians in preclinical and / or clinical trials. Those skilled in the art can determine the effective amount of a particular therapeutic agent or multiple therapeutic agents empirically without extensive experimentation. A maximum dose, i.e., the highest safe dose determined based on certain medical judgments, can be used. Multiple daily dosing may be considered to achieve an appropriate systemic level of administration. An appropriate systemic level of administration can be determined, for example, by measuring the patient's peak or steady-state plasma levels. "Dosage" and "administration volume" are used interchangeably herein. The dosage can be administered by the patient, by someone else, or through a device (such as an inhaler or nebulizer).

[0087] For any therapeutic composition described herein, its effective therapeutic dose may be determined, for example, first through animal models. For drugs already tested in humans and those known to have similar pharmacological activity (e.g., other relevant active drugs), the effective therapeutic dose may also be determined based on human data. Parenteral administration may require higher doses. Dosage can be adjusted based on the relative bioavailability and potency of the administered drug. Adjusting the dose to achieve maximum efficacy according to the methods described above and other methods known in the art is entirely within the capabilities of those skilled in the art.

[0088] Therapeutic agents (used alone or formulated into pharmaceutical compositions / drugs) for treatment or prevention can be tested in suitable animal model systems. Suitable animal model systems include, but are not limited to, rats, mice, chickens, cattle, monkeys, rabbits, pigs, miniature pigs, etc., prior to human trials. Therapeutic agents can be tested in vivo using any animal model system known in the art before administration to human subjects. In some embodiments, the dosing regimen can be tested directly in humans.

[0089] The dosage, toxicity, and therapeutic effect of any therapeutic agent or composition (e.g., formulations or drugs comprising a therapeutic composition as described herein), other / additional therapeutic agents, or mixtures thereof can be determined by standard pharmaceutical procedures in cell culture or laboratory animals, such as determining the LD50 (the dose that would be lethal to 50% of the population) and ED50 (the dose that would be effective to 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Drugs exhibiting a high therapeutic index are advantageous.

[0090] For example, exemplary treatment regimens may involve administration once daily, twice daily, three times daily, once weekly, or once monthly. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until the progression of the disease or condition is slowed, reduced, or terminated, or until the subject's symptoms of the disease or condition are partially or completely relieved. In some embodiments, a single dose is sufficient to slow, reduce, terminate, or relieve the symptoms of the disease or condition.

[0091] When used for treatment, an effective amount of the therapeutic composition (used alone or as a formulation) may be administered to the subject by any means that delivers the composition to a target surface (e.g., lung tissue). Administration may be achieved by any means known to those skilled in the art. Routes of administration include, but are not limited to: oral, topical, intranasal, intratracheal, inhalation, systemic, intravenous, subcutaneous, intraperitoneal, intradermal, intraocular, intrathecal, intraventricular, iontophoresis, transmucosal, intravitreal, or intramuscular administration. In some preferred embodiments, delivery of the composition is accomplished by inhalation. Administration methods include patient self-administration, administration by another person, and administration via a device (e.g., an inhaler or nebulizer).

[0092] The therapeutic agents disclosed in this invention (e.g., enzymes, peptides, or polynucleotides encoding them) can be delivered to a subject in the form of a formulation or a pharmaceutical (i.e., a pharmaceutical composition). Formulations and pharmaceuticals can be prepared, for example, by dissolving or suspending the therapeutic agents disclosed in this invention (e.g., enzymes, peptides, polynucleotides encoding them, and combinations thereof) in water, a solvent, a pharmaceutically acceptable carrier, a salt (e.g., NaCl or sodium phosphate), a buffer, a preservative, a compatible carrier, an adjuvant, and optionally other therapeutically acceptable components.

[0093] Pharmaceutical compositions (e.g., formulations or drugs) may contain a carrier (e.g., a pharmaceutically acceptable carrier), which may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained by using, for example, coatings (e.g., lecithin), maintaining the desired particle size in the case of dispersions, and using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Glutathione and other antioxidants may be added to prevent oxidation. In many cases, the addition of isotonic agents (e.g., sugars (e.g., trehalose), polyols (e.g., mannitol, sorbitol), or sodium chloride) to the composition is advantageous.

[0094] When systemic administration is required, therapeutic agents or pharmaceutical compositions can be formulated as parenteral preparations for injection, such as by bolus or continuous infusion (e.g., by intravenous injection or by metered administration via a pump over a defined time). Injectable formulations can be provided in single-dose form, for example, in ampoules or multi-dose containers, with added preservatives. Pharmaceutical compositions can be in the form of suspensions, solutions, or emulsions in oily or aqueous carriers, and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Furthermore, suspensions of therapeutic agents can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils (e.g., sesame oil), synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the therapeutic agent to prepare a high-concentration solution.

[0095] Systemic formulations include those designed for injection, such as subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, oral administration via mucosal membranes, or pulmonary administration.

[0096] For intravenous and other parenteral routes of administration, formulations can be prepared as lyophilized preparations, lyophilized therapeutic agents with liposome embedding or lipid encapsulation, lipid complexes in aqueous suspensions, or salt complexes. Lyophilized preparations are typically reconstituted with a suitable aqueous solution (e.g., sterile water or physiological saline) shortly before administration.

[0097] Pharmaceutical compositions suitable for injection (e.g., formulations or drugs) may include sterile aqueous solutions (if water-soluble) or dispersions, as well as sterile powders for the provisional preparation of sterile injections or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Pasipani, NJ), or phosphate-buffered saline (PBS). Compositions intended for injection should generally be sterile formulations and should have sufficient flowability for easy injection manipulation. The composition should remain stable under manufacturing and storage conditions and should be protected against contamination by microorganisms such as bacteria and fungi.

[0098] Sterile injectable solutions (e.g., formulations or drugs) can be prepared by dissolving the desired amount of a therapeutic agent in a suitable solvent in combination with one or more of the components listed above, followed by sterile filtration. Typically, dispersions are prepared by adding the therapeutic agent to a sterile carrier containing a base dispersion medium and the other desired components listed above. For sterile powders used to prepare sterile injectable solutions, typical preparation methods include vacuum drying and freeze-drying, both of which yield powders of the active ingredient and any other desired components from a pre-filtered sterile solution.

[0099] For oral administration, the therapeutic agent can be readily formulated with a pharmaceutically acceptable carrier known in the art. Such carriers enable the therapeutic agent to be formulated into dosage forms such as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral administration to a subject to be treated. Dosage forms such as tablets, pills, capsules, and lozenges may contain any of the following components or agents with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogelâ, or corn starch; lubricants, such as magnesium stearate or stearates; gliding agents, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring.

[0100] Oral pharmaceutical formulations can be obtained in the form of solid excipients. The resulting mixture can be ground and, if necessary, added with appropriate excipients to form tablets or sugar-coated tablet cores. Suitable excipients include, in particular, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as crospyvinylpyrrolidone, agar, or alginate or its salts, such as sodium alginate, can be added. Optionally, oral formulations can also be prepared with physiological saline or buffer solutions, for example, with EDTA to neutralize the acidic environment in the body, or can be used directly without any carrier.

[0101] In addition, oral dosage forms of the aforementioned drugs are particularly considered, which can be chemically modified to make them effective orally. Typically, the chemical modifications considered involve attaching at least one moiety to the therapeutic agent, ingredient, and / or excipient, said moiety being capable of (a) inhibiting acid hydrolysis and (b) promoting the absorption of the drug from the gastrointestinal tract into the bloodstream. Furthermore, it is desirable to improve the overall stability of the therapeutic agent, ingredient, and / or excipient and prolong its circulation time in vivo. Examples of such moiety include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. See Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” in: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, pp. 367–383 (1981); Newmark et al., J Appl Biochem 4:185–9 (1982). Other available polymers include poly-1,3-dioxolane and poly-1,3,6-trioxane. As mentioned above, polyethylene glycol (PEG) of various molecular weights is suitable for pharmaceutical applications.

[0102] For formulations of therapeutic agents, ingredients, and / or excipients, the site of release can be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. Those skilled in the art possess formulations that do not dissolve in the stomach but release substances into the duodenum or other intestinal sites. Preferably, this mode of release avoids harmful effects on the gastric environment by protecting the therapeutic agent or releasing the bioactive substance outside the gastric environment (e.g., in the intestines).

[0103] For tablets not intended to protect the stomach, coatings or coating mixtures may also be used. This may include sugar coatings or coatings that make the tablets easier to swallow. Capsules may consist of a hard shell (e.g., gelatin) used for delivering dry therapeutic agents (e.g., powders); for liquid dosage forms, a soft gelatin shell may be used. The shell material for flat capsules may be thick starch or other edible paper. For tablets, lozenges, molded tablets, or tablet mills, wet forming techniques may be used.

[0104] Colorants and flavorings can be added. For example, therapeutic or pharmaceutical compositions can be formulated into formulations and further added to edible products, such as chilled beverages containing colorants and flavorings.

[0105] Inert substances can be used to dilute or increase the volume of therapeutic agents or pharmaceutical compositions. These diluents include carbohydrates, particularly mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts can also be used as fillers, such as tricalcium phosphate, magnesium carbonate, and sodium chloride. Commercially available diluents include Fast-Flo®, Emdex®, STARCH 1500®, Emcompress®, and Avicel®.

[0106] Disintegrants can be included in formulations that conjugate therapeutic agents or pharmaceutical compositions into solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, such as the commercially available starch-based disintegrant Explotab. Other available disintegrants include sodium carboxymethyl starch, Amberlite®, sodium carboxymethyl cellulose, hyperbranched starch, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponges, and bentonite. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as disintegrants and binders, such as agar, carrageenan, or tragacanth gum. Alginic acid and its sodium salts can also be used as disintegrants.

[0107] Adhesives can be used to bind therapeutic agents together to form hard tablets; their components include natural materials such as gum arabic, tragacanth, starch, and gelatin. Other adhesives include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) are both soluble in alcohol solutions and are used to form therapeutic agents into granules.

[0108] To prevent adhesion during formulation, anti-friction agents may be added to the formulation of therapeutic agents or pharmaceutical compositions. Lubricants may be used as a separating layer between the therapeutic agent and the mold wall. Lubricants include, but are not limited to, stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Soluble lubricants such as sodium dodecyl sulfate, magnesium dodecyl sulfate, polyethylene glycol (PEG) of various molecular weights, and Carbowax™ 4000 and 6000 may also be used.

[0109] To improve the flowability of drugs during formulation and promote rearrangement during tableting, gliding agents can be added. Gliding agents may include starch, talc, pyrolytic silica, and hydrated aluminosilicates.

[0110] To aid in the dissolution of therapeutic compositions / reagents or pharmaceutical compositions in an aqueous environment, surfactants may be added as wetting agents. Surfactants may include anionic surfactants such as sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic surfactants that may be used include benzalkonium chloride and benzyl chloride. Potential nonionic surfactants that may be added as surfactants to formulations or pharmaceuticals include lauryl ether-400, polyoxyethylene stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present individually or in mixtures in varying proportions in the formulations or pharmaceuticals or their derivatives disclosed herein.

[0111] Orally administered pharmaceutical compositions include push-in capsules made of gelatin and soft-sealable capsules made of gelatin and plasticizers (such as glycerin or sorbitol). Push-in capsules may contain an active ingredient and fillers (such as lactose), binders (such as starch), and / or lubricants (such as talc or magnesium stearate), and optionally, stabilizers. In soft capsules, the therapeutic agent may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Stabilizers may also be added. Microspheres formulated specifically for oral administration may also be used. Such microspheres are well defined in the art. All orally administered formulations should have a dosage suitable for such administration.

[0112] For oral administration, the composition can be formulated into tablets or lozenges and prepared in a conventional manner.

[0113] For topical administration, therapeutic agents or pharmaceutical compositions can be formulated into dosage forms known in the art, such as solutions, gels, ointments, creams, and suspensions. Solutions, gels, ointments, creams, or suspensions can all be administered topically. These drugs can also be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

[0114] For administration via inhalation (introduction), the therapeutic composition / reagent or pharmaceutical composition used in this invention can be conveniently aspirated from a pressurized package or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In some embodiments, the formulation, pharmaceutical product, and / or therapeutic composition / reagent can be aspirated from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide), or aspirated via a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798. For pressurized aerosols, the dosage can be controlled by setting a valve, thereby achieving metered delivery. For pressurized aerosols, the dosage unit can be controlled by setting a valve, thereby achieving metered delivery. For example, gelatin capsules and cartridges for inhalers or blowpipes can be formulated as a powder mixture comprising the therapeutic composition / reagent and a suitable powder matrix such as lactose or starch. Alternatively, the therapeutic composition / reagent or pharmaceutical composition may be in powder form and prepared with a suitable carrier (e.g., sterile pyrogen-free water or a suitable buffer solution) before use.

[0115] For administration by inhalation (introduction), the reagents (e.g., enzymes known to enzymatically break down at least one toxin) or compositions used according to the present invention can be formulated as dry powder inhalers. Dry powder inhalers of macromolecules (e.g., enzymes of the present disclosure) can be formulated using a biodegradable polymer carrier comprising polyethylene glycol-copolymer-poly(glycerol-adipic acid-copolymer-ω-pentadecanol), as described by Tawfeek HM et al. (Int J Pharm. 2013 Jan 30; 441(1-2):611-9; this document is incorporated herein by reference in its entirety).

[0116] Nasal administration of therapeutic compositions / reagents or pharmaceutical compositions is also a viable treatment option. Nasal administration allows the therapeutic composition / reagent or pharmaceutical composition to enter the bloodstream directly after nasal administration, without deposition in the lungs. Formulations for nasal administration include those containing dextran or cyclodextrin.

[0117] For nasal administration, a practical device is a small hard bottle with a metered-dose nebulizer. In some embodiments, a dose is delivered by inhaling a pharmaceutical composition (in solution form) into a chamber of a specific volume, the chamber having an orifice sized such that when a liquid within the chamber is compressed, a spray is formed, thereby atomizing and aerosolizing the formulation. Therapeutic agents or pharmaceutical compositions are administered by compressing the chamber. In one specific embodiment, the chamber employs a piston-like structure. Such devices are commercially available.

[0118] Alternatively, a plastic squeeze bottle with an orifice or opening can be used, the size of which is designed to atomize the aerosol formulation by forming a spray when the bottle is squeezed. This opening is typically located at the top of the bottle and is usually tapered to partially fit the nasal cavity, thereby effectively administering the aerosol formulation. Preferably, the nasal inhaler provides a metered dose of the aerosol formulation for administering a metered dose of a therapeutic agent or pharmaceutical composition.

[0119] A wide variety of mechanical devices can be used in this technical practice. These devices are designed for the inhaled delivery of therapeutic products, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are well known to those skilled in the art.

[0120] Specific examples of commercially available devices applicable to this technical practice include: the Ultravent™ nebulizer manufactured by Mallinckrodt, Inc. (located in St. Louis, Missouri); the Acorn II® nebulizer manufactured by Marquest Medical Products (located in Englewood, Colorado); the Ventolin® metered-dose inhaler manufactured by Glaxo Inc. (located in Research Triangle Park, North Carolina); and the Spinhaler® powder inhaler manufactured by Fisons Corp. (located in Bedford, Massachusetts).

[0121] All such devices require the use of formulations suitable for delivering therapeutic compositions / reagents or pharmaceutical compositions. Typically, each formulation is designed for the specific device used and may require a suitable propellant in addition to diluents, adjuvants, and / or carriers commonly used in treatment. Furthermore, liposomes, microcapsules, microspheres, nanoparticles, nanospheres, inclusion complexes, or other types of carriers may also be considered.

[0122] Formulations suitable for nebulizers (jet or ultrasonic) may, for example, contain a water-soluble therapeutic composition / reagent or pharmaceutical composition at a concentration of approximately 0.01 to 50 mg of bioactive ingredient per milliliter of solution. The formulation may also contain a buffer and may optionally contain monosaccharides (e.g., for stabilizing inhibitors and regulating osmotic pressure). Nebulizer formulations may also contain surfactants to reduce or prevent surface-induced aggregation of the therapeutic or pharmaceutical compositions disclosed herein during aerosol formation.

[0123] Formulations for metered-dose inhalation devices typically comprise a fine powder containing the therapeutic compositions / reagents or pharmaceutical compositions disclosed herein, suspended in a propellant by means of a surfactant. The propellant can be any conventional material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include trioleate sorbitol and soy lecithin. Oleic acid can also be used as a surfactant.

[0124] Formulations for powder inhalation devices may comprise a finely dispersed dry powder containing a therapeutic composition / active ingredient or pharmaceutical composition, and may also include a filler, such as lactose, sorbitol, sucrose, or mannitol, in an amount that facilitates the dispersion of the powder from the device, for example, 50% to 90% by weight of the formulation. The therapeutic composition / pharmaceutical composition may preferably be prepared in granular or nanoparticle form with an average particle size of less than 10 micrometers (μm), more preferably 0.5 to 5 μm, for most effective delivery to deep into the lungs.

[0125] Preparations and drugs Any formulation described in the section “Pharmaceutical Compositions, Routes of Administration, and Dosage” above (which may also be referred to as a drug or composition when formulated for the treatment of a subject suffering from a specific disease or requiring medical care) can be used to prepare a composition (i.e., a formulation or drug) suitable for the treatment of a subject in need. Therefore, in some embodiments, the present invention relates to compositions, formulations, and drugs suitable for the treatment of a subject suffering from or suspected of suffering from a disease or condition in which systemic toxins (e.g., exogenous or foreign toxins, or endogenous toxins (e.g., accumulated metabolites)) are having a negative effect on the subject.

[0126] Treatment methods and detoxification in the body Toxins that have adverse effects on subjects can enter the systemic circulation, thereby acting on multiple systems in the body. It is understood that such systemic toxins can be transported from the systemic circulation to the pulmonary circulation and can easily diffuse from the pulmonary bloodstream to the lung tissue. While not wishing to be bound by any single theory, it is understood that, using the methods described herein, introducing a composition containing at least one enzyme known to enzymatically break down at least one systemic toxin into at least a portion of the lungs of a subject can promote the degradation of systemic toxins in the body, thereby effectively transforming the lungs into a regulated metabolic organ whose metabolic capacity is enhanced by the exogenously added enzyme.

[0127] This invention provides a method for mitigating one or more negative effects of toxins in the body. In some embodiments, the method for mitigating one or more negative effects of toxins in the body includes introducing at least one preselected enzyme into the lung tissue of a subject, said preselected enzyme preferably being an enzyme known to be capable of enzymatically breaking down at least one toxin that produces one or more negative effects on said subject.

[0128] In some implementations, methods for mitigating one or more negative effects of toxins in the body include allowing at least one toxin to undergo enzymatic breakdown in the body, thereby mitigating the one or more negative effects.

[0129] In some embodiments, a method for mitigating one or more negative effects of a toxin in the body includes: (a) introducing at least one preselected enzyme into the lung tissue of a subject, said at least one preselected enzyme being known to enzymatically break down at least one toxin that produces one or more negative effects on said subject; and (b) causing said at least one toxin to undergo enzymatic breakdown in the body, thereby mitigating said one or more negative effects.

[0130] The introduction step can be achieved by any means that exposes the subject's lung tissue to a substance having an amino acid sequence corresponding to the at least one preselected enzyme, a polynucleotide encoding the at least one peptide, or both the peptide and the polynucleotide encoding the peptide. In some embodiments, the introduction step is achieved by transducing multiple cells present in the lung tissue to express the at least one preselected enzyme. In some embodiments, the multiple cells are transduced using DNA or a construct thereof. In some embodiments, the multiple cells are transduced using mRNA or a construct thereof. The DNA, mRNA, or a construct thereof may be formulated with or without a pharmaceutically acceptable vector.

[0131] In some embodiments, the introduction step includes contacting lung tissue with a polypeptide having an amino acid sequence corresponding to the at least one preselected enzyme, a polynucleotide encoding the at least one polypeptide, or both.

[0132] In some embodiments, the subject suffers from one or more hereditary diseases. In some preferred embodiments, the subject suffers from a hereditary disease that leads to the systemic accumulation of toxins (e.g., toxic metabolites) in the body. In some embodiments, the disease is caused by a deficiency of an enzyme that catalyzes the breakdown or transformation of biomolecules or metabolites in subjects without the disease. In some cases, the toxin is a metabolite that exerts its toxic effect only when it is systemically present in the body at a level sufficient to have a negative effect on the subject, and is non-toxic when it is systemically present in the body and at a level maintained at normal levels by one or more metabolic enzymes. Non-limiting examples include phenylketonuria, gout, cystinuria, ornithine carbamoyltransferase deficiency (OTCD), galactosemia, maple syrup urine disease, tumor suppression disorder, cocaine use disorder, urea cycle disorder, tobacco use disorder, Pompe disease, sucrase-isomaltase deficiency, arginase deficiency, and hyperargininemia. These hereditary diseases and conditions involve the toxicity and systemic accumulation of toxins (e.g., metabolites). In some embodiments, one or more hereditary diseases are selected from phenylketonuria, gout, cystinuria, ornithine carbamoyltransferase deficiency (OTCD), galactosemia, maple syrup urine disease, tumor suppressor disorder, cocaine use disorder, urea cycle disorder, tobacco use disorder, Pompe disease, sucrase-isomaltase deficiency, arginase deficiency, hyperargininemia, and combinations thereof. The hereditary diseases that the method disclosed in this invention can address are preferably hereditary diseases that prevent the breakdown of at least one toxin.

[0133] In some embodiments, the subject has been exposed to at least one toxin. Toxins that the subject may have been exposed to include, but are not limited to, phenylalanine, uric acid, cystine, arginine, lysine, ornithine, leucine, isoleucine, valine, amino acids, galactose, kynurenine, cocaine, ammonia, nicotine, cyanide, and organophosphates. In some embodiments, at least one toxin is selected from phenylalanine, uric acid, cystine, arginine, lysine, ornithine, leucine, isoleucine, valine, amino acids, galactose, kynurenine, cocaine, ammonia, nicotine, cyanide, organophosphates, and combinations thereof.

[0134] According to the disclosure of this invention, the enzymes used may include those known to catalyze the decomposition or degradation of toxins. Non-limiting examples of known enzymes capable of catalyzing the decomposition or degradation of toxins include: phenylalanine ammonia lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondrial (BCKDHA), 2-oxoisovalerate dehydrogenase β-subunit mitochondrial (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthase, arginine succinate lyase, arginine succinate synthase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotine oxidoreductase, and uricase. In some embodiments of the methods described herein, the preselected enzymes are selected from: phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondria (BCKDHA), 2-oxoisovalerate dehydrogenase β-subunit mitochondria (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), and combinations thereof. Other known enzymes that can catalyze the breakdown or degradation of toxins include: collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine pegase, vidarabinease, imiglucerase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase; lipase; protease), sucrase, truncated (non-glycosylated) tPA (357 of 527 amino acids), coagulation factor VIIa, and tissue plasminogen activator. Enzyme activator variants, uricase (Aspergillus flavus), antihemophilic factor, laronidase, agarsinase β, hyaluronidase (sheep), hyaluronidase (bovine), thiolated enzyme, hyaluronidase (human), idoxurase, aglucosidase α, thrombin (human), thrombin (bovine), verasidase α, verasidase α, asparaginase (Erwinia chrysogenum), talidase α, recombinant truncated human plasmin, recombinant carboxypeptidase g2, glutamate, coagulation factor XIII A, ilometasidase α, coagulation factor X, asfortaase α, cybebeta esterase α, selexipasin α, vertranesinase α-vjbk, pevaricase-pqpz and combinations thereof.

[0135] Methods for mitigating one or more negative effects of at least one toxin in vivo can also be used to treat a subject's hereditary disease, preferably a hereditary disease in which the natural breakdown of one or more toxins in the subject's body is impaired. For example, in some embodiments, methods for mitigating one or more negative effects of at least one toxin in vivo can also be used to treat a subject with a hereditary condition (e.g., an enzyme deficiency) that leads to the accumulation of endogenous metabolites. Non-limiting examples of hereditary conditions involving enzyme deficiencies include phenylketonuria, hyperuricemia, gout, cystinuria, ornithine transcarbamate deficiency (OTCD), galactosemia, maple syrup urine disease, and urea cycle disorders. In some embodiments, methods for mitigating one or more negative effects of at least one toxin in vivo can be used to treat a subject with a condition of secondary accumulation of endogenous metabolites. In some embodiments, methods for mitigating one or more negative effects of at least one toxin in vivo can be implemented on a subject with an autoimmune disease. In some embodiments, methods for mitigating one or more negative effects of at least one toxin in vivo can be implemented on a subject with cancer (e.g., leukemia). In some embodiments, the methods of this disclosure can be implemented on a subject with a substance use disorder. Non-restrictive examples of substance use disorders include cocaine use disorder, tobacco use disorder, nicotine use disorder, stimulant use disorder, sedative use disorder, and opioid use disorder.

[0136] Methods for mitigating one or more negative effects of at least one toxin in the body can also be used to treat subjects who have ingested or otherwise been exposed to a toxin and which has entered the subject's systemic circulation. In some embodiments, the methods of the present invention can be implemented in subjects who have been poisoned. In some embodiments, the methods of the present invention can be used in subjects who are considering ingesting, are ingesting, or have ingested one or more solid or liquid preparations containing a toxin (e.g., a drug or other toxic substance). For example, in some embodiments, methods for mitigating one or more negative effects of at least one toxin in the body can be implemented in subjects who have ingested or otherwise been exposed to cocaine and which has entered the subject's systemic circulation. In some embodiments, methods for mitigating one or more negative effects of at least one toxin in the body can be implemented in subjects who have ingested or otherwise been exposed to cyanide and which has entered the subject's systemic circulation.

[0137] This invention provides a method for mitigating one or more negative effects in patients with leukemia. In some embodiments, the method for mitigating one or more negative effects of leukemia includes introducing an asparaginase into the lung tissue of a patient diagnosed with or suspected of having leukemia, said asparaginase being known to enzymatically break down asparagine. In some embodiments, said asparaginase is an L-asparaginase. In some embodiments, the method for mitigating one or more negative effects of leukemia includes allowing the enzymatic breakdown of asparagine in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that, at least in some types of leukemia, leukemia cells are unable to synthesize L-asparagine and therefore these leukemia cells depend on systemic L-asparagine for survival. The methods disclosed herein can lead to the breakdown of asparagine, thereby depriving leukemia cells of the nutrients necessary for survival and mitigating one or more negative effects in leukemia subjects. In some embodiments, the method for mitigating one or more negative effects of leukemia includes (a) introducing an asparaginase into the lung tissue of a subject; and (b) allowing the enzymatic breakdown of asparagine in vivo, thereby mitigating said one or more negative effects. In some implementations, the leukemia includes acute lymphoblastic leukemia (ALL).

[0138] This invention discloses a method for mitigating one or more negative effects caused by oxalate excess in a subject. In some embodiments, the method for mitigating one or more negative effects caused by oxalate excess includes introducing an oxalate decarboxylase into the lung tissue of a subject diagnosed or suspected of having oxalate deposition, primary hyperoxaluria, or secondary hyperoxaluria, said oxalate decarboxylase being known to enzymatically break down oxalate. In some embodiments, the method for mitigating one or more negative effects caused by oxalate deposition, primary hyperoxaluria, or secondary hyperoxaluria includes enzymatically breaking down oxalate in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that oxalate deposition may occur due to impaired renal function, including in patients with hyperoxaluria due to primary and intestinal-related causes, leading to the accumulation of oxalate in the blood. The method disclosed herein can lead to the breakdown of oxalate, thereby reducing circulating oxalate levels and mitigating one or more negative effects of oxalate deposition, primary hyperoxaluria, or secondary hyperoxaluria in patients. In some embodiments, methods for mitigating one or more adverse effects of oxalate deposition include: (a) introducing oxalate decarboxylase into the lung tissue of a subject; and (b) enzymatically breaking down oxalate in vivo, thereby mitigating said one or more negative effects. In some embodiments, the oxalate is endogenously produced oxalate. In some embodiments, the oxalate is dietaryly ingested oxalate.

[0139] This invention provides a method for mitigating one or more negative effects of phenylketonuria (PKU) in a subject. In some embodiments, the method for mitigating one or more negative effects of PKU includes introducing phenylalanine ammonia-lyase (PAL) into the lung tissue of a subject diagnosed with or suspected of having PKU, said PAL being known to enzymatically break down phenylalanine. In some embodiments, the method for mitigating one or more negative effects of PKU includes causing the enzymatic breakdown of phenylalanine in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that PKU may occur due to the loss of function of the phenylalanine hydroxylase (PAH) gene, leading to the accumulation of phenylalanine in the blood. The methods disclosed herein can cause the breakdown of phenylalanine, thereby reducing circulating phenylalanine levels and mitigating one or more negative effects of PKU in a subject. In some embodiments, the method for mitigating one or more negative effects of PKU includes (a) introducing PAL into the lung tissue of a subject; and (b) causing the enzymatic breakdown of phenylalanine in vivo, thereby mitigating said one or more negative effects.

[0140] This invention provides a method for mitigating one or more negative effects of hyperuricemia in a subject. In some embodiments, the method for mitigating one or more negative effects of hyperuricemia in a subject includes introducing uricase into the lung tissue of a subject diagnosed with or suspected of having hyperuricemia, said uricase being known to enzymatically break down uric acid. In some embodiments, the method for mitigating one or more negative effects of hyperuricemia in a subject includes allowing uric acid to be enzymatically broken down in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that hyperuricemia may occur due to insufficient uric acid excretion or excessive uric acid production, leading to the accumulation of uric acid in the blood. Untreated hyperuricemia can impair kidney function and lead to gout. The methods disclosed herein are capable of breaking down uric acid, thereby reducing circulating uric acid levels and mitigating one or more negative effects in subjects with hyperuricemia. In one embodiment, a method for mitigating one or more negative effects of hyperuricemia includes: (a) introducing uricase into the lung tissue of a subject; and (b) allowing uric acid to be enzymatically broken down in vivo, thereby mitigating said one or more negative effects.

[0141] This invention provides a method for mitigating one or more negative effects of ornithine transcarbamate deficiency (OTCD) on a subject. In some embodiments, the method for mitigating one or more negative effects of ornithine transcarbamate deficiency includes introducing an ornithine transcarbamate known to enzymatically break down ammonia into the lung tissue of a subject diagnosed with or suspected of having ornithine transcarbamate deficiency. In some embodiments, the method for mitigating one or more negative effects of ornithine transcarbamate deficiency includes allowing the enzymatic breakdown of ammonia in vivo, thereby mitigating the one or more negative effects. While not wishing to be bound by any single theory, it is understood that ornithine transcarbamate deficiency leads to the accumulation of ammonia in the blood. The methods disclosed herein are capable of breaking down ammonia, thereby reducing circulating ammonia levels and mitigating one or more negative effects of ornithine transcarbamate deficiency on a subject. In one embodiment, a method for mitigating one or more negative effects of ornithine transcarbamate deficiency includes: (a) introducing ornithine transcarbamate into the lung tissue of a subject; and (b) allowing ammonia to be enzymatically broken down in vivo, thereby mitigating the one or more negative effects.

[0142] In some embodiments, methods for mitigating one or more negative effects of ornithine transcarbamate deficiency (OTCD) include introducing glutamine synthase into the lung tissue of a subject diagnosed with ornithine transcarbamate deficiency, said glutamine synthase being known to enzymatically remove ammonia. In some embodiments, methods for mitigating one or more negative effects of ornithine transcarbamate deficiency include allowing enzymatic removal of ammonia in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that ornithine transcarbamate deficiency leads to the accumulation of ammonia in the blood. The methods disclosed herein are capable of removing ammonia, thereby reducing circulating ammonia levels and mitigating one or more negative effects of ornithine transcarbamate deficiency in the subject. In some embodiments, methods for mitigating one or more negative effects of ornithine transcarbamate deficiency include: (a) introducing glutamine synthase into the lung tissue of a subject; and (b) allowing enzymatic removal of ammonia in vivo, thereby mitigating said one or more negative effects.

[0143] This invention provides a method for mitigating one or more negative effects of hyperammonemia in a subject. In some embodiments, the method for mitigating one or more negative effects of hyperammonemia includes introducing a glutamine synthase into the lung tissue of a subject diagnosed with or suspected of having hyperammonemia, said glutamine synthase being known to enzymatically remove ammonia. In some embodiments, the method for mitigating one or more negative effects of hyperammonemia includes allowing the enzymatic removal of ammonia to occur in vivo, thereby mitigating said one or more negative effects. The methods disclosed herein are capable of removing ammonia, thereby reducing circulating ammonia levels and mitigating one or more negative effects of hyperammonemia in a subject. In some embodiments, the method for mitigating one or more negative effects of hyperammonemia includes: (a) introducing a glutamine synthase into the lung tissue of a subject; and (b) allowing the enzymatic removal of ammonia to occur in vivo, thereby mitigating said one or more negative effects.

[0144] This invention provides a method for mitigating one or more negative effects of galactosemia in a subject. In some embodiments, the method for mitigating one or more negative effects of galactosemia includes introducing galactose-1-phosphate uridine transferase (GALT) into the lung tissue of a subject diagnosed with or suspected of having galactosemia, said GALT being known to enzymatically break down galactose. In some embodiments, the method for mitigating one or more negative effects of galactosemia includes allowing the enzymatic breakdown of galactose in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that galactosemia leads to the accumulation of galactose in the blood. The methods disclosed herein are capable of breaking down galactose, thereby reducing circulating galactose levels and mitigating one or more negative effects of galactosemia in a subject. In one embodiment, the method for mitigating one or more negative effects of galactosemia includes: (a) introducing GALT into the lung tissue of a subject; and (b) allowing the enzymatic breakdown of galactose in vivo, thereby mitigating said one or more negative effects.

[0145] This invention provides a method for mitigating one or more negative effects of Pompe disease in a subject. In some embodiments, the method for mitigating one or more negative effects of Pompe disease includes introducing an acidic α-glucosidase (GAA) into the lung tissue of a subject diagnosed with or suspected of having Pompe disease, said GAA being known to enzymatically break down glycogen. In some embodiments, the method for mitigating one or more negative effects of Pompe disease includes allowing the enzymatic breakdown of glycogen in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that Pompe disease can lead to the accumulation of glycogen in the body. The methods disclosed herein can lead to glycogen breakdown, thereby reducing glycogen levels and mitigating one or more negative effects of Pompe disease in a subject. In some embodiments, the method for mitigating one or more negative effects of Pompe disease includes: (a) introducing a GAA into the lung tissue of a subject; and (b) allowing the enzymatic breakdown of glycogen in vivo, thereby mitigating said one or more negative effects.

[0146] This invention provides a method for mitigating one or more negative effects of isomaltase deficiency in a subject. In some embodiments, the method for mitigating one or more negative effects of isomaltase deficiency includes introducing isomaltase into the lung tissue of a subject diagnosed with or suspected of having isomaltase deficiency, said isomaltase being known to enzymatically break down disaccharides and oligosaccharides. In some embodiments, the method for mitigating one or more negative effects of isomaltase deficiency includes allowing the enzymatic breakdown of disaccharides and oligosaccharides in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that isomaltase deficiency leads to the accumulation of disaccharides and oligosaccharides in the blood. The methods disclosed herein can lead to the breakdown of disaccharides and oligosaccharides, thereby reducing the levels of disaccharides and oligosaccharides in the blood and mitigating one or more adverse effects of isomaltase deficiency on the subject. In some implementations, methods for mitigating one or more adverse effects of sucrase isomaltase deficiency include (a) introducing sucrase isomaltase into the lung tissue of a subject; and (b) causing enzymatic breakdown of disaccharides and oligosaccharides in vivo, thereby mitigating the one or more adverse effects.

[0147] This invention provides a method for mitigating one or more negative effects of arginase deficiency in a subject. In some embodiments, the method for mitigating one or more negative effects of arginase deficiency includes introducing an arginase into the lung tissue of a subject diagnosed with or suspected of having arginase deficiency, said arginase being known to enzymatically break down arginine. In some embodiments, the method for mitigating one or more negative effects of arginase deficiency includes allowing the enzymatic breakdown of arginine in vivo, thereby mitigating said one or more negative effects. While not wishing to be bound by any single theory, it is understood that arginase deficiency leads to the accumulation of arginine in the blood. The methods disclosed herein result in the breakdown of arginine, thereby reducing the level of arginine in the blood and mitigating one or more negative effects in patients with arginase deficiency. In some embodiments, the method for mitigating one or more negative effects of arginase deficiency includes: (a) introducing an arginase into the lung tissue of a subject; and (b) allowing the enzymatic breakdown of arginine in vivo, thereby mitigating said one or more negative effects.

[0148] This invention provides a method for mitigating one or more negative effects of hyperarginemia in a subject. In some embodiments, the method for mitigating one or more negative effects of hyperarginemia includes introducing an arginase into the lung tissue of a subject diagnosed with or suspected of having hyperarginemia, said arginase being known to enzymatically break down arginine. In some embodiments, the method for mitigating one or more negative effects of hyperarginemia includes allowing the enzymatic breakdown of arginine in vivo, thereby mitigating said one or more negative effects. The methods disclosed herein can lead to the breakdown of arginine, thereby reducing the level of arginine in the blood and mitigating one or more negative effects of hyperarginemia in a subject. In some embodiments, the method for mitigating one or more negative effects of hyperarginemia includes: (a) introducing an arginase into the lung tissue of a subject; and (b) allowing the enzymatic breakdown of arginine in vivo, thereby mitigating said one or more negative effects.

[0149] This invention provides a method for mitigating one or more negative effects of cyanide poisoning in a subject. In some embodiments, the method for mitigating one or more negative effects of cyanide poisoning includes introducing thiosulfate-transferase (thiocyanate oxidase) into the lung tissue of a subject with confirmed or suspected cyanide poisoning. In some embodiments, the method for mitigating one or more negative effects of cyanide poisoning includes causing enzymatic degradation of cyanide in vivo, thereby mitigating said one or more negative effects. While not wishing to be limited to any single theory, it is understood that cyanide poisoning can result from ingestion of cyanide salts, drinking pure liquid hydrogen cyanide, skin absorption of hydrogen cyanide, intravenous infusion of sodium nitroprusside due to hypertensive crisis, or inhalation of hydrogen cyanide gas, leading to the accumulation of cyanide in the blood. The method disclosed herein causes the degradation of cyanide, thereby reducing circulating cyanide levels and mitigating one or more negative effects of cyanide poisoning on the subject. In some embodiments, methods for mitigating one or more negative effects of cyanide poisoning include: (a) introducing thiocyanate into the lung tissue of a subject; and (b) enzymatically breaking down cyanide in vivo, thereby mitigating said one or more negative effects. In some embodiments, thiocyanate may be introduced into the lung tissue of a subject together with a cofactor (e.g., sodium thiosulfate).

[0150] In some embodiments, causing the toxin to undergo enzymatic breakdown or degradation includes: causing at least one toxin that has diffused or migrated from the subject's circulatory system into the subject's lungs and / or lung mucus to undergo enzymatic breakdown or degradation in vivo. In some embodiments, the enzymatic breakdown or degradation of the toxin is promoted in the subject's lungs and / or lung mucus. In some embodiments, the enzymatic breakdown or degradation of the toxin is promoted in the lungs and / or lung mucus within the subject.

[0151] The method of the present invention can reduce the level of toxins (e.g., toxic metabolites, endogenous molecules, or ingested substances) in the blood of a subject. For example, the level of toxins in the blood of a subject can be reduced by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 75% or more, about 90% or more, about 95% or more, or about 99% or more compared to the level before the application of one or more of the techniques disclosed herein. In some embodiments, after introducing a composition comprising at least one enzyme known to enzymatically break down at least one toxin into the lung tissue of a subject, the toxin level in the subject's blood may be reduced by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 75% or more, or about 90% or more, after about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 68 hours, about 72 hours, or about 96 hours or more, or for a period of time longer, wherein the reduction is relative to a level existing prior to the application of one or more of the techniques disclosed herein. In some embodiments, after introducing a composition comprising at least one enzyme known to enzymatically break down at least one toxin into the lung tissue of a subject, the toxin level in the subject's blood may be reduced by about 10% or more, about 25% or more, about 50% or more, about 75% or more, about 90% or more, about 95% or more, or about 99% or more, wherein the reduction is relative to a level existing prior to the application of one or more of the techniques disclosed herein.

[0152] The techniques for measuring toxin levels in the blood of subjects are common knowledge in the art, and the use of these techniques in accordance with this disclosure is entirely within the skill of a person of ordinary skill in the art.

[0153] This invention provides a method for converting the lungs of a subject to include at least one enzyme with degradative function. The method comprises introducing at least one preselected enzyme or a polynucleotide encoding at least one preselected enzyme into the lungs of the subject. The preselected enzyme is known to promote the enzymatic breakdown of at least one toxin, which, if present, is present in the subject. The conversion can be achieved by depositing at least one enzyme into the lung tissue (i.e., lung tissue) of the subject, or by transducing cells of the lung tissue to express said at least one enzyme.

[0154] This invention provides a transformed lung capable of enzymatically breaking down at least one toxin systemically present in a subject's body (including the subject's lungs). The transformed lung comprises lung tissue transformed to carry at least one preselected enzyme, the preselected enzyme being known to enzymatically break down the at least one toxin. In some embodiments, the transformed lung tissue carries at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to the preselected enzyme, a polynucleotide encoding the polypeptide, or both. Methods of transforming tissue by introducing an enzyme or a polynucleotide encoding the enzyme are common knowledge in the art. The transformed lung of this invention can be prepared by one or more transformation methods known in the art. In some embodiments, the transformed lung comprises at least a portion of the lung containing an exogenously introduced enzyme or a polynucleotide encoding the enzyme. In some embodiments, the transformed lung contains an exogenously introduced enzyme selected from phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondrial (BCKDHA), 2-oxoisovalerate dehydrogenase subunit β-mitochondrial (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), and thiosulfate. Thiocyanate transferase, collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine pegase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase; lipase; protease), sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idolesulfatase, aglucosidase α, thrombin, verasidase, pegologase, asparaginase, tadalafilase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A, elostase α, coagulation factor X, asfortaase α, cybepiperase α, seripaase α, vetranesase α-vjbk, pevaricase-pqpz, or combinations thereof, or polynucleotides encoding said enzymes.

[0155] This invention provides transformed lung cells comprising alveolar cells carrying at least one preselected enzyme known to enzymatically break down at least one toxin. In some embodiments, the transformed alveolar cells carry at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to the preselected enzyme, a polynucleotide encoding the polypeptide, or both. Methods for transforming lung cells by introducing an enzyme or a polynucleotide encoding the enzyme are known in the art. The transformed lung cells of this invention can be prepared by one or more transformation methods known in the art. In some embodiments, the transformed lung cells comprise alveolar cells containing an exogenously introduced enzyme or a polynucleotide encoding the enzyme. In some embodiments, the transformed lung cells contain exogenously introduced enzymes selected from: phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondrial (BCKDHA), 2-oxoisovalerate dehydrogenase subunit β-mitochondrial (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), and thiocyanate. Thiosulfate-transferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine peglucosidase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase, lipase, protease), sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idoxuronidase, aglucosidase α, thrombin, verasidase α, pegologase, asparaginase, tadalasidase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A. Elosulfate esterase α, coagulation factor X, asfortaase α, cybebeta esterase α, seripa esterase α, vetranesase α-vjbk, pevaricase-pqpz, and combinations thereof, or polynucleotides encoding said enzymes. In some embodiments, the present invention provides a lung cell population comprising a plurality of alveolar cells carrying at least one preselected enzyme known to enzymatically break down at least one toxin.In some embodiments, the lung cell population includes multiple alveolar cells carrying exogenously introduced enzymes selected from: phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondrial (BCKDHA), 2-oxoisovalerate dehydrogenase subunit β-mitochondrial (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (…). GAA), thiosulfate thiotransferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine pegase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase (amylase; lipase; protease), sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idoxurase, aglucosidase α, thrombin, verasidase, pegologase, asparaginase, tadalafilase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A, elostase α, coagulation factor X, asfortaase α, cybepi esterase α, seripa esterase α, vetranesase α-vjbk, pevaricase-pqpz, or combinations thereof, or polynucleotides encoding said enzymes.

[0156] Subject group In some embodiments, the subject of the present invention (e.g., a subject receiving the methods described herein to mitigate the negative effects of one or more toxins) may be, but is not limited to, humans or non-human vertebrates. In some embodiments, the subject of the methods according to the present invention is a mammal. Mammals include, but are not limited to, domestic pets (e.g., dogs, cats, rabbits, ferrets, hamsters, etc.), livestock or farm animals (e.g., cattle, pigs, sheep, goats, pigs, chickens, or other poultry), horses (e.g., thoroughbred horses), monkeys, laboratory animals (e.g., mice, rats, rabbits, etc.), etc. In a preferred embodiment, the subject of the present invention is a human. The technology of the present invention can be implemented in any subject who requires treatment and is suitable for delivery of exogenous enzymes via the lungs.

[0157] In some embodiments, the method of the present invention can be performed in subjects suffering from a hereditary condition (e.g., an enzyme deficiency) that leads to the accumulation of endogenous metabolites. Non-limiting examples of hereditary conditions involving enzyme deficiency include phenylketonuria, hyperuricemia, gout, cystinuria, ornithine transcarbamate deficiency (OTCD), galactosemia, maple syrup urine disease, and urea cycle disorders. In some embodiments, the method of the present invention can be performed in subjects suffering from a disease with secondary endogenous metabolite accumulation. In some embodiments, the method of the present invention can be performed in subjects suffering from an autoimmune disease. In some embodiments, the method of this disclosure can be performed in subjects suffering from cancer (e.g., leukemia). In some embodiments, the method of this disclosure can be performed in subjects suffering from a substance use disorder. Non-limiting examples of substance use disorders include cocaine use disorder, tobacco use disorder, nicotine use disorder, stimulant use disorder, sedative use disorder, and opioid use disorder.

[0158] In some embodiments, the method of the present invention can be performed in subjects who have ingested or otherwise been exposed to a toxin and said toxin has entered the subject's systemic circulation. In some embodiments, the method of the present invention can be performed in subjects who have been poisoned. In some embodiments, the method of the present invention can be performed in subjects who are considering ingesting, are ingesting, or have ingested one or more solid or liquid preparations containing a toxin (e.g., a drug or other toxic substance). For example, in some embodiments, the method of the present invention can be performed in subjects who have ingested or otherwise been exposed to cocaine and said cocaine has entered the subject's systemic circulation. In some embodiments, the method of the present invention can be performed in subjects who have ingested or otherwise been exposed to cyanide and said cyanide has entered the subject's systemic circulation.

[0159] In some implementations, the subjects of this disclosure are human newborns (e.g., from birth to 1 month old), human infants (e.g., from 1 month to 1 year old), human children (from 1 year to 12 years old), human adolescents (from 13 years to 17 years old), or human adults (18 years or older).

[0160] Combination therapy In some aspects, the method of the present invention further includes administering one or more additional therapeutic agents and / or methods (e.g., "combination therapy") to a subject. The additional therapeutic agent may be introduced or delivered to the subject via any route to perform its intended function. In some embodiments, a composition comprising at least one enzyme known to enzymatically break down at least one toxin and the additional therapeutic agent are administered via the same route of administration. In some embodiments, a composition comprising at least one enzyme known to enzymatically break down at least one toxin and the additional therapeutic agent are administered via different routes of administration. For example, in some embodiments, the composition comprising at least one enzyme known to enzymatically break down at least one toxin is administered by inhalation or intratracheal administration, while the additional therapeutic agent is administered orally or intravenously.

[0161] In some aspects, the method of the present invention further includes administering one or more additional therapeutic agents and / or methods that can reduce the level of toxins in the subject's systemic circulation (e.g., the subject's blood) and / or reduce the negative effects of toxin intake, such as nausea, vomiting, confusion, seizures, and slowed breathing. In some embodiments, the method of the present invention is used in conjunction with one or more methods (e.g., gastric lavage, intravenous infusion of saline, antiemetics, and analgesics) on the subject.

[0162] In some embodiments, the method of the present invention further includes administering one or more additional therapeutic agents and / or methods for treating autobrewing syndrome. Such additional therapeutic agents and / or methods may, for example, include antifungal drugs, antibiotics, probiotics, and low-carbohydrate diets. In some embodiments, the method of the present invention does not include the administration of antibiotics.

[0163] Example These embodiments are provided for illustrative purposes only and do not limit the scope of protection of the claims set forth herein.

[0164] Example 1. Reducing circulating phenylalanine levels in vivo by depositing PAL in the lungs of rats and mice. This example describes the effect of depositing phenylalanine aminolyase (PAL) in the lung tissue of mice or rats on the level of circulating phenylalanine (Phe).

[0165] To establish a chemically induced phenylketonuria (PKU) disease state model, mice and rats were administered p-chlorophenylalanine methyl ester (pCP) (a known in vivo phenylalanine hydroxylase (PAH) inhibitor) and Phe to simulate persistently elevated circulating Phe levels.

[0166] Each experimental group consisted of 4 rats, who received a single dose of pCP-Phe (300 mg / kg + 100 mg / kg) via intraperitoneal injection (IP) for 3 consecutive days. Blood samples were collected in the morning before pCP-Phe injection on day 1 to determine baseline Phe levels. Additional blood samples were collected on days 3 and 4. On day 4, phenylalanine ammonia-lyase (PAL) was administered via intravenous (IV) (35 mg / kg body weight) or intratracheal (IT) (55 mg / kg body weight), respectively. Blood samples were then collected at 1, 2, 3, 6, and 19 hours after PAL administration. Blood samples were analyzed using the Abcam PAL assay kit.

[0167] like Figure 1 As shown, Phe accumulation was observed in all three groups of rats due to the inhibition of natural PAH by pCP and the additional administration of Phe. As expected, serum Phe levels gradually decreased in the untreated groups on day 4 as PAH inhibition waned over time. However, the decrease in circulating Phe levels was significantly greater in the PAL-treated groups. Surprisingly, the decrease in Phe levels after intratracheal administration of PAL was similar to that observed after intravenous administration. This suggests that even if PAL does not enter the systemic circulation as it does after intravenous administration, PAL deposited in lung tissue can still promote the breakdown of Phe.

[0168] A similar experiment was conducted using a mouse PKU model. Mice were first treated with pCP / Phe to increase circulating Phe levels in their blood, followed by administration of PAL via subcutaneous (SQ) or intratracheal (IT) administration. In short, mice were divided into three groups (n=4 per group) and administered pCP / Phe via intraperitoneal (IP) for two consecutive days to increase Phe levels in their blood. On day 3 after pCP / Phe administration, one group received pCP alone for two days, another group received pCP combined with PAL (subcutaneous administration), and the third group received pCP combined with PAL (intratracheal administration). On day 4, blood samples were collected to measure circulating Phe levels. A fourth group consisted of two mice that received neither pCP nor PAL (untreated control group). Phe levels in the collected blood samples were determined using LC-MS / MS analysis.

[0169] like Figure 2A and Figure 2BAs shown, animals treated with PAL via pulmonary delivery exhibited lower circulating phenylalanine levels compared to animals that did not receive PAL treatment and animals that received PAL via subcutaneous administration. These results indicate that pulmonary deposition of PAL enzymes is sufficient to reduce phenylalanine levels in subjects, thereby protecting them from the physiological effects of elevated phenylalanine levels. This treatment strategy could be applicable to subjects with PAL deficiency and / or diseases caused by elevated blood phenylalanine levels, including phenylketonuria.

[0170] In another set of experiments, a PAH-deficient gene mouse model was used to investigate the efficacy of intratracheal administration of PAL in reducing circulating Phe levels. In short, homozygous enu2 / enu2 mice were bred from heterozygous mice obtained from the Jackson Laboratory (jax.org). Due to the PAH gene defect, homozygous enu2 / enu2 mice accumulate Phe.

[0171] During the week-long experiment, mice were administered PAL via intratracheal or subcutaneous administration on days 2, 3, and 4, respectively. Blood samples were collected daily from the mice during the 7-day experiment and analyzed by LC-MS / MS.

[0172] like Figure 3 As shown, administration of PAL via intravenous (IV) and intratracheal (IT) administration reduced circulating phenylalanine (Phe) levels in PKU model mice. PKU mice administered PAL twice daily via both intratracheal and subcutaneous administration showed reduced plasma phenylalanine levels. Similar to the chemically induced PKU mouse model, intratracheal delivery of PAL was significantly more effective than subcutaneous delivery. On each day following treatment, intratracheal delivery was significantly more effective than subcutaneous delivery in reducing PAL levels. Figure 3 The levels decreased by 51-59% (p<0.001). By day 5 (after a 1-day washout period), Phe levels in both groups of mice returned to baseline levels before treatment (day 1). Surprisingly, intratracheal administration of PAL significantly reduced circulating PAL levels more than subcutaneous administration. This confirms previous data that PAL deposited in lung tissue can promote Phe breakdown even if it fails to enter systemic circulation via the SQ pathway.

[0173] Example 2. Reducing circulating uric acid levels in mice by depositing uricase in the lungs. This example describes the effect of uricase deposition in mouse lung tissue on circulating uric acid levels.

[0174] Gout is an inflammatory arthritis characterized by recurrent redness, tenderness, heat, and swelling in the joints caused by the deposition of needle-shaped uric acid crystals called monosodium urate crystals. Gout is a condition caused by persistently elevated levels of uric acid (urate crystals) in the blood (a condition known as hyperuricemia).

[0175] Uric acid oxidase (UO), uricase, or factor-independent uricase (which are absent in humans) catalyze the oxidation of uric acid to 5-hydroxyisouric acid. Therefore, it is hypothesized that the deposition of uricase in mouse lung tissue could reduce circulating uric acid levels.

[0176] In summary, mice were administered uricase suspended in a uricase stock solution five times via intratracheal (IT) or intravenous (IV) administration. The uricase stock solution consisted of recombinant aflatoxin uricase from *E. coli* (prospecbio.com / urate_oxidase). 20 mg of the enzyme was suspended in 0.45 ml of 50 mM borate buffer at pH 8.5, containing 0.001% Triton X-100 and 1.0 mM EDTA. Each mouse was given 40 µL of the stock solution, resulting in an intake of approximately 2 mg of enzyme per mouse. Control mice were given saline without the enzyme.

[0177] Following administration of uricase, blood samples were collected from mice at 30, 60, 120, 180, and 240 minutes. Blood samples were also collected immediately before uricase administration (time point 0). Blood samples were analyzed using a uric acid assay kit (abcam.com / products / assay-kits / uric-acid-assay-kit-ab65344.html) to quantitatively determine uric acid levels in the blood.

[0178] like Figure 4 As shown, administration of uricase via either intrapulmonary inhalation (IT) or intravenous (IV) routes was sufficient to significantly reduce circulating uric acid levels at all test time points. Surprisingly, although IT administration, unlike IV administration, does not deliver uricase systemically, the reduction in circulating uric acid levels achieved by IT administration was comparable to that achieved by IV administration. Conversely, IT administration resulted in uricase deposition in lung tissue, and the inventors of this invention believe that uricase acts on uric acid that diffuses from the pulmonary blood supply into the lung tissue. These data suggest that the lungs can be transformed into a modulated metabolic organ capable of metabolizing systemic circulating toxins to a degree sufficient to detect a significant reduction in systemic toxin circulating levels. These data also suggest that pulmonary administration of uricase may be suitable for treating diseases involving systemic uric acid accumulation, such as hyperuricemia.

[0179] In the second experiment, mice (n=5 per group) were administered aflatoxin uricase suspended in sterile water via either intratracheal (IT) or intravenous (IV) administration. The prepared stock solution concentration was 40 mg / mL. Each mouse was given 50 µL of the stock solution, resulting in an intake of approximately 2 mg of enzyme per mouse. The control group (n=2) was given physiological saline (IT) without enzyme. Mice were fed commercially available feed (LabDiet 5K52) and reverse osmosis purified water, which were freely available. The uricase was commercially available lyophilized aflatoxin uricase (1 mg / vial, Prospec Bio), stored at -20°C before the scheduled experiment date. The experimental mice were 8-week-old female C57BL / 6 mice purchased from Charles River Labs.

[0180] Blood samples were collected from mice at 30, 60, 120, 180, and 240 minutes after administration of uricase. Blood samples were also collected immediately before uricase administration (time point 0). Uric acid levels in the blood were quantitatively determined using a uric acid assay kit (AbCam #ab65344) via fluorescence or colorimetric methods.

[0181] Animals treated with 2 mg of commercially available aflatoxin uricase showed a significant reduction in circulating uric acid levels. Figure 5A Compared with the untreated control group, IT administration reduced uric acid levels by 31.2%, while IV administration reduced uric acid levels by 43.1% (AUC, ...). Figure 5B The reduction in efficacy was statistically significant compared to that of intravenous administration (IT administration) (p<0.0001).

[0182] It was also noted that mice receiving uricase via IV administration were more sluggish after administration, while animals receiving IT administration appeared to be in good condition and no behavioral changes were observed.

[0183] These data further confirm the potential of pulmonary delivery of therapeutic enzymes and demonstrate that intratracheal administration offers better safety compared to systemic administration. In summary, this suggests that pulmonary drug delivery therapy holds great promise for the treatment of gout and other metabolic diseases.

[0184] Example 3. Reducing circulating uric acid levels in vivo by depositing variant uricase in the lungs of mice and monkeys. This example describes the effect of variant uricase deposition in mouse and monkey lung tissue on circulating uric acid levels.

[0185] A hyperuricemia-inducing model was established in cynomolgus nonhuman primates (NHPs) by intravenous injection of uric acid (UA; 8.15 mg / kg) to evaluate the activity of an inhaled engineered variant of uricase in response to serum uric acid.

[0186] In summary, on the morning of the experiment, 12 male non-human primates (NHP, not first-time uric acid exposure) weighing 4-5 kg ​​were randomly divided into four groups of three animals each: 1) Negative control group: no treatment and no uric acid administration; 2) Uric acid control group: no treatment but uric acid administration; 3) Low-dose uricase + uric acid; 4) High-dose uricase + uric acid. All animals were fasted overnight. Baseline blood samples were collected at time 0, and inhalation therapy was administered within 15 minutes of the first blood collection using the Aeroneb® Solo vibrating mesh nebulizer system (equipped with a spacer and oral / nasal mask). Nebulization therapy lasted for 20 minutes (uricase buffer, 60 mg / mL uricase, or 20 mg / mL uricase), with target inhaled uricase doses of 25 mg / kg and 8.3 mg / kg for the treatment groups, respectively. At 75 minutes, uric acid (to induce hyperuricemia) or 0.9% saline (control group) was administered intravenously.

[0187] Blood samples were collected at 60 and 65 minutes (after inhalation therapy and before UA injection), and at 95, 155, 275, and 515 minutes (after inhalation therapy and before UA administration), corresponding to 1, 2, 4, and 8 hours after the end of nebulization therapy, respectively. Animals were fed after the last blood collection. A crossover design was used to maximize the number of non-human primates per group (n=6 in total), with a 7-day washout period between each administration day. Serum was separated from the blood samples using a standard protocol and frozen at -80 °C until uric acid content was quantified using tandem mass spectrometry.

[0188] Prior to inhalation therapy and uric acid injection, the baseline uric acid concentration in healthy individuals was 4 µM ± 3.4 SD, a concentration maintained in Group 1 (negative control group) throughout the experiment. Following uric acid injection, serum uric acid concentration rapidly increased (C... max =75 minutes), then gradually decreased, with the uricase treatment group returning to baseline levels at approximately 275 minutes, while in the uricase control group, uric acid remained elevated at the end of the experiment.

[0189] Variant uricase therapy targeting the lungs showed dose-dependent reductions in circulating uric acid at all time points. For example, in C... max At that time, serum uric acid concentrations were: 74 µM ± 24.5 SD (UA control group), 59 µM ± 12 SD (low-dose uricase + UA), and 46 µM ± 20 SD (high-dose uricase + UA). Figure 6ABased on the area under the curve analysis of the time-course data (60-515 minutes), compared with the UA control group, serum uric acid levels decreased by 36.6% (low-dose uricase) and 56.8% (high-dose uricase, p<0.01), respectively. Figure 6B No adverse effects were observed throughout the study.

[0190] In the second experiment, the ability of the variant uricase to reduce uric acid levels in mice after lung deposition was evaluated by directly comparing the activities of the variant uricase with wild-type uricase in mice. Briefly, three mice per group were administered 2 mg of uricase (Aspergillus flavus wild-type uricase or engineered Arthroblastus globulus variant uricase) or a carrier (uricase buffer) via intratracheal administration (IT). Mice were fed commercial feed (LabDiet 5K52) and reverse osmosis purified water on an ad libitum basis. The uricase was prepared on the day of administration. Each mouse received 2 mg of uricase via direct lung deposition (IT) in a 50 µL volume.

[0191] Blood samples were collected from mice at 30, 60, 120, 180, and 240 minutes after administration of uricase. Blood samples were also collected immediately before uricase administration (time point 0). Uric acid levels in the blood were quantitatively determined using a uric acid assay kit (AbCam # ab65344) via fluorescence or colorimetric methods.

[0192] A direct comparison was made between commercially available wild-type Aspergillus flavus uricase and engineered Arthroblastus variant uricase, with 2 mg of the enzyme delivered to the lungs of healthy female mice. Serum uric acid levels were then measured to determine efficacy compared to an untreated control group.

[0193] Circulating uric acid levels were significantly reduced in all treated animals (Figure 7). Time-course analysis showed that the reduction in uric acid levels was similar regardless of the enzyme used. Figure 7A Compared with the untreated control group, administration of wild-type uricase (WT) reduced uric acid levels by 29.8%, while administration of variant uricase reduced uric acid levels by 34.4% (AUC). The results of variant uricase administration were statistically significantly better than those of the wild-type uric acid group (p<0.01). Figure 7B ).

[0194] These data further confirm the potential benefits of delivering therapeutic enzymes to the lungs, and these benefits can be enhanced by using protein engineering techniques to improve the stability of the enzymes in the lungs, which has broad application prospects in the treatment of gout and other metabolic diseases.

[0195] Example 4. Reducing circulating cyanide levels in vivo by depositing thiosulfate-transferase (thiocyanate) in mouse lungs. This example describes the effect of thiocyanate deposition in mouse lung tissue on circulating cyanide levels in a mouse cyanide poisoning model.

[0196] In summary, this embodiment establishes an animal model of cyanide poisoning by intraperitoneal (IP) administration of potassium cyanide (KCN) dissolved in Na₂CO₃ to mice. Possible dosages include 200 µL of 10 mM or 20 mM KCN. The effect of thiocyanate enzyme + sodium thiosulfate on cyanide-related lethality was assessed by measuring changes in mouse mortality associated with KCN administration, particularly the change in the median lethal dose (LD50) of KCN. Thiocyanate enzyme + sodium thiosulfate can be administered intratracheally (IT) before or after intraperitoneal administration of KCN (e.g., at least 5 minutes before or after intraperitoneal administration of KCN). In some experiments, thiocyanate was administered intratracheally only, without concomitant administration of sodium thiosulfate. Control groups included a negative control group without KCN and a positive control group with intraperitoneal administration of KCN only. In another experimental setting, thiocyanate enzyme + sodium thiosulfate could be administered intravenously (IV) to compare its efficacy with that observed after IT administration.

[0197] Thiocyanate enzyme can be commercially available (e.g., sigmaaldrich.com / US / en / product / sigma / r1756). Sodium thiosulfate is also available from commercial sources (e.g., sigmaaldrich.com / US / en / product / sial / phr2690).

[0198] It is expected that intratracheal administration of thiocyanate enzyme + sodium thiosulfate will improve the survival rate of mice given KCN and increase the KCN LD50 in these mice.

[0199] Example 5. Reducing circulating ammonia levels in mouse lungs by depositing glutamine synthase. This example describes the effect of glutamine synthase deposition in mouse lung tissue on circulating ammonia levels.

[0200] Hyperammonemia is a metabolic disorder characterized by elevated levels of ammonia (nitrogenous compounds) in the body. Normal ammonia levels vary with age. Hyperammonemia can be caused by a variety of congenital and acquired diseases, in which ammonia may be the primary toxin.

[0201] Glutamine synthase (GS) (EC 6.3.1.2) is an enzyme that plays an important role in nitrogen metabolism by catalyzing the condensation of glutamate and ammonia to form glutamine: glutamate + ATP + NH3 → glutamine + ADP + phosphate.

[0202] Glutamine synthase utilizes ammonia produced by nitrate reduction, amino acid degradation, and photorespiration. The amide group of glutamate is the nitrogen source for the synthesis of metabolites in the glutamine pathway.

[0203] The enzyme used in the following experiments is glutamine synthase, also known as glutamate-amino ligase.

[0204] In the first set of experiments, the applicant conducted experiments in an induced hyperammonemia mouse model established by injecting ammonium chloride (NH4Cl) into wild-type mice. In short, ammonia levels in these mice were increased by injecting NH4Cl, and glutamate and NH4Cl levels were analyzed after glutamine synthase (GS) deposition in the lungs. Ten minutes before intraperitoneal (IP) administration of NH4Cl, mice were administered saline, GS, or GS-glutamate via intratracheal (IT) administration. The glutamine synthase stock solution consisted of L-glutamine synthase from *E. coli* (sigmaaldrich.com / US / en / product / sigma / g1270). 300 μg of GS enzyme was suspended in 300 μL of water to obtain a stock solution with a concentration of 1 U / μL. For the control group, each mouse was given 40 μL of saline. For the GS IT NH4Cl group, each mouse was given 20 µL of stock solution plus 20 µL of water, resulting in an intake of approximately 20 units of enzyme per mouse. For the GS-glutamate IT NH4Cl group, each mouse was given 20 µL of stock solution plus 20 µL of glutamate stock solution, so that each mouse ingested about 20 units of enzyme and 11.8 μg of glutamate.

[0205] Ten minutes after administration of glutamine synthase, NH4Cl was injected intraperitoneally at a dose of 7 mg / kg. Blood samples were collected from mice at 5, 15, 30, 45, 60, 80, and 120 minutes post-injection. Blood samples were also collected immediately before NH4Cl administration (time point 0). Blood samples were analyzed using an ammonia assay kit (sigmaaldrich.com / US / en / product / sigma / mak538) to quantify blood ammonia levels, a glutamine assay kit (abcam.com / en-us / products / assay-kits / glutamine-assay-kit-colorimetric-ab197011) to quantify blood glutamine levels, and a glutamate assay kit (abcam.com / en-us / products / assay-kits / glutamate-assay-kit-ab83389) to quantify blood glutamate levels.

[0206] like Figure 8A As shown, intratracheal administration of glutamine synthase (GS) was sufficient to significantly reduce circulating ammonia levels. Compared to the control group, glutamate supplementation further enhanced the reduction in circulating plasma ammonia levels. Figure 8B As shown in Figure 8C, in the presence of GS and GS+glutamate, glutamine levels increase while glutamate levels decrease over time, which is consistent with the biochemical activity of glutamine synthase (i.e., glutamate-amino ligase).

[0207] In the second set of experiments, the applicant evaluated the ability of IT-administered GS enzymes to reduce circulating ammonia levels in an ornithine carbamoyltransferase deficiency (OTCD) mouse model.

[0208] Ornithine transcarbamoylase deficiency (OTCD) is an X-linked liver disease caused by partial or complete loss of OTC enzyme activity, characterized by elevated plasma ammonia levels.

[0209] In summary, OTCD mice were purchased from Jackson Laboratories (strain #001811, jax.org / strain / 001811). A wild-type (WT) mouse control group was also established. Ten minutes before intraperitoneal (IP) administration of NH4Cl, mice were administered either saline or glutamine synthase (GS) via intratracheal (IT) administration. The glutamine synthase stock solution consisted of L-glutamine synthase (GS) from *E. coli*, with a sequence identical to that of Sigma-Aldrich (sigma.com / US / en / product / sigma / g1270). The GS enzyme was suspended in water and diluted to obtain a stock solution with a concentration of 1 μg / μL. For the WT and OTC control groups, each mouse received 20 μL of saline. For the OTC GS-IT treatment group, each mouse received 20 μL of the GS stock solution, resulting in an ingestion of approximately 20 μg of enzyme per mouse.

[0210] Ten minutes after administration of glutamine synthase, NH4Cl was injected intraperitoneally at a dose of 7 mg / kg. Blood samples were collected from mice at 5, 15, 30, 45, 60, 80, and 120 minutes post-injection. Blood samples were also collected immediately before NH4Cl administration (time point 0). Blood ammonia levels were quantified using an ammonia assay kit (sigmaaldrich.com / US / en / product / sigma / mak538), glutamine levels were quantified using a glutamine assay kit (abcam.com / en-us / products / assay-kits / glutamine-assay-kit-colorimetric-ab197011), and glutamate levels were quantified using a glutamate assay kit (abcam.com / en-us / products / assay-kits / glutamate-assay-kit-ab83389).

[0211] like Figure 9 As shown, intratracheal administration of GS enzyme was sufficient to significantly reduce the accumulation of ammonia in the plasma of ammonia-stimulated mice.

[0212] In summary, these data suggest that pulmonary delivery of glutamate synthase may be suitable for treating diseases involving systemic ammonia accumulation, such as hyperammonemia and ornithine transcarbamate deficiency (OTCD).

[0213] Example 6. Reducing circulating arginine levels in mice by depositing argininase in the lungs. This example describes the effect of arginase deposition in mouse lung tissue on circulating arginine levels.

[0214] Arginase deficiency is a genetic disorder that causes a gradual accumulation of the amino acid arginine (hyperarginemia) and ammonia in the blood. Ammonia is a product of protein breakdown in the body and is toxic if its concentration is too high. The nervous system is particularly sensitive to the effects of excessive ammonia.

[0215] Arginine deficiency typically begins to appear around age 3. The most common symptom is muscle stiffness, especially in the legs, caused by abnormal muscle tension (spasm). Other symptoms may include slower than normal growth, developmental delays and eventual failure to reach developmental milestones, intellectual disability, seizures, tremors, and difficulty with balance and coordination (ataxia). Sometimes, a high-protein diet, stress from illness, or fasting can cause a rapid buildup of ammonia in the blood. This rapid increase in ammonia can lead to symptoms such as irritability, refusal to eat, and vomiting.

[0216] In some affected individuals, the signs and symptoms of arginase deficiency may be mild and may not appear until later in life.

[0217] L-arginase hydrolyzes L-arginine into L-ornithine and urea, leading to the accumulation of ammonia.

[0218] In summary, mice were administered arginase suspended in an arginase stock solution via intratracheal administration (IT) four times. This arginase stock solution consisted of L-arginase derived from bovine liver. (sigmaaldrich.com / US / en / product / sigma / a3233?utm_source=google&utm_medium=cpc&utm_campaign=8691857242&utm_content=98395646591&gbraid=0AAAAAD8kLQQ4EXPn2pu6CMF4WyzF4-npu&gclid=CjwKCAjwnK60BhA9EiwAmpHZw-waI-letyOPj_OY-AbkQpqEoIIpgOT4c6KsFjgF_le-M1MGCm8f1RoCSUQQAvD_BwE). 8 mg of the enzyme was suspended in 105 µL of water. Each mouse was given 25 µL of the stock solution, resulting in an intake of approximately 2 mg of the enzyme per mouse. Control mice were given saline solution and no enzyme was administered.

[0219] Blood samples were collected from mice at 15, 30, 60, 120, 180, and 240 minutes after administration of arginase. Blood samples were also collected immediately before arginase administration (time point 0). Blood samples were analyzed using an L-arginine assay kit (abcam.com / en-us / products / assay-kits / l-arginine-assay-kit-ab241028) to quantify L-arginine levels in the blood, and blood urea levels were quantified using a urea assay kit (sigmaaldrich.com / US / en / product / sigma / mak471).

[0220] like Figure 10A As shown, intratracheal administration of arginase was sufficient to significantly reduce circulating L-arginine levels. L-arginine levels decreased for up to 3 hours after arginase administration. Figure 10B As shown, urea (a byproduct of arginase degradation of arginine) accumulated in the plasma of mice treated with arginase. These data also suggest that pulmonary delivery of arginase may be suitable for treating diseases involving systemic accumulation of L-arginine, such as hyperarginemia and arginase deficiency.

[0221] Example 7. Reducing circulating asparagine levels in mice by depositing asparaginase in the lungs This example describes the effect of asparaginase deposition in mouse lung tissue on circulating asparagine levels.

[0222] Acute lymphoblastic leukemia (ALL) is a rare hematologic malignancy that leads to the production of abnormal lymphocyte precursor cells. ALL can be divided into B-cell and T-cell subtypes and is more common in children, accounting for approximately 30% of childhood malignancies, but also accounting for 1% of adult cancer diagnoses. Prognosis is age-related, with a five-year overall survival rate exceeding 90% in children and less than 20% in the elderly. L-asparaginase reduces serum L-asparagine levels. Because leukemia cells cannot synthesize this amino acid, its deficiency leads to cell death. Asparaginase-containing therapies have been successful in treating childhood ALL, while traditional cytotoxic regimens have been less effective in adults. Therefore, trials of pediatric or child-targeted therapies containing asparaginase have begun in adolescents, young adults, and adults. See Juluri et al., *Blood and Lymphatic Cancer: Targets and Therapy*, 2022.

[0223] Asparaginase converts asparagine into aspartic acid and releases ammonia.

[0224] In summary, mice were administered asparaginase suspended in a stock solution via intratracheal administration (IT) four times. The stock solution consisted of asparaginase derived from *E. coli* (sigmaaldrich.com / US / en / product / sigma / a3809). 2 mg of enzyme was suspended in 100 µL of water. Each mouse was given 25 µL of the stock solution, receiving approximately 100 units of enzyme (100–300 units / mg). Control mice were given saline and no enzyme. Mice were fed a standard diet before and during the experiment.

[0225] Following administration of asparaginase to mice, blood samples were collected at 5, 15, 30, 60, 120, 180, and 240 minutes post-administration. Additionally, blood samples were collected immediately before asparaginase administration (time point 0). Blood samples were analyzed using an asparagine assay kit (abcam.com / en-us / products / assay-kits / asparagine-assay-kit-fluormetric-ab273333) to quantify blood asparagine levels, aspartate assay kit (abcam.com / en-us / products / assay-kits / aspartate-assay-kit-ab102512) to quantify blood aspartate levels, and ammonia assay kit (sigmaaldrich.com / US / en / product / sigma / mak538) to quantify blood ammonia levels.

[0226] like Figure 11A As shown, intratracheal administration of asparaginase was sufficient to significantly reduce circulating asparagine levels. Asparagine levels decreased at 4 hours post-administration of asparaginase (the longest time point measured). Aspartate (a protein) transiently accumulated in the plasma of mice treated with asparaginase. Figure 11B ) and ammonia ( Figure 11C These substances are byproducts of asparaginase degradation of asparagine. These data also suggest that lung delivery of asparaginase may be suitable for treating certain diseases requiring lowered circulating asparagine levels, such as acute lymphoblastic leukemia (ALL).

[0227] These embodiments are provided for illustrative purposes only and do not limit the scope of the claims appended herein.

[0228] While certain embodiments have been described and illustrated, it should be understood that those skilled in the art can make changes and modifications thereto without departing from the broader aspects of the art as defined in the following claims.

[0229] The embodiments described herein can be suitably implemented in the absence of any elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and not as limiting. Furthermore, the terminology and expressions used herein are for descriptive purposes only and not for limitation, and their use is not intended to exclude equivalents of the features shown and described or portions thereof, but it should be understood that various modifications can be made within the scope of the claimed technology. Additionally, the phrase “consisting primarily of…” should be understood to include the elements expressly listed as well as other elements that do not materially affect the essential and novel features of the claimed technology. The phrase “composed of…” excludes any unspecified elements.

[0230] This invention is not limited to the specific embodiments described herein. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to the methods and compositions listed herein, functionally equivalent methods and compositions exist within the scope of this disclosure, as will be readily understood by those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This disclosure is limited only by the appended claims and the full scope of their equivalents. It should be understood that this disclosure is not limited to specific methods, reagents, formulations, or compositions, which may, of course, vary. It should be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the invention.

[0231] Furthermore, when features or aspects of the invention are described in the form of a Markush group, those skilled in the art will recognize that this disclosure is therefore also described in the form of any single component or subgroup of components within that Markush group.

[0232] Those skilled in the art will understand that, for any and all purposes, particularly when provided in writing, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof, including endpoints. Any listed scope can be readily considered sufficient to describe and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, middle third, and upper third, etc. Those skilled in the art will also understand that all expressions such as “up to,” “at least,” “greater than,” “less than,” etc., include the listed numbers and refer to scopes that can be further decomposed into subscopes as described above. Finally, those skilled in the art will understand that a scope includes each of its constituent parts.

[0233] All publications, patent applications, granted patents, and other documents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent application, granted patent, or other document were expressly and separately indicated to be incorporated herein by reference in its entirety. Definitions contained in the referenced text that conflict with definitions in this disclosure are excluded.

[0234] Other embodiments are set forth in the claims.

Claims

1. A method for mitigating one or more negative effects of toxins in the body, comprising: (a) Introducing at least one preselected enzyme into the lung tissue of a subject, said at least one preselected enzyme being known to enzymatically break down at least one toxin that produces one or more negative effects on the subject; as well as (b) To cause the enzymatic breakdown of the at least one toxin in vivo, thereby mitigating the one or more negative effects.

2. The method according to claim 1, wherein, The subject had one or more genetic diseases.

3. The method according to claim 2, wherein, The one or more hereditary diseases mentioned are selected from phenylketonuria, gout, cystinuria, ornithine transcarbamylase deficiency (OTCD), galactosemia, maple syrup urine disease, tumor suppression disorder, cocaine use disorder, urea cycle disorder, tobacco use disorder, Pompe disease, sucrase-isomaltase deficiency, arginase deficiency, hyperarginemia, and combinations thereof.

4. The method according to claim 1, wherein, The subject suffered from a condition that led to the accumulation of at least one toxin.

5. The method according to claim 1, wherein, The subject had been exposed to at least one toxin.

6. The method according to claim 1, wherein, The at least one toxin is selected from: phenylalanine, uric acid, cystine, arginine, lysine, ornithine, leucine, isoleucine, valine, amino acids, galactose, kynurenine, cocaine, ammonia, nicotine, cyanide, organophosphates, and combinations thereof.

7. The method according to claim 1, wherein, The at least one preselected enzyme is selected from: phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondria (BCKDHA), 2-oxoisovalerate dehydrogenase subunit β-mitochondria (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), thiosulfate. Thiotransferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine peglucosidase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase, sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idolesulfatase, aglucosidase α, thrombin, verasidase α, pegologase, asparaginase, tadalafilase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A. Elosulfate esterase α, coagulation factor X, asfortaase α, cybepi esterase α, seripa esterase α, vetranesase α-vjbk, pevaricase-pqpz, and combinations thereof.

8. The method according to claim 1, wherein, The at least one toxin is an amino acid (phenylalanine or Phe) or an acid (uric acid).

9. The method according to claim 1, wherein, The lung tissue comes into contact with the at least one toxin through the action of the subject's circulatory system.

10. The method according to claim 1, wherein, The introduction step includes contacting lung tissue with a polypeptide having an amino acid sequence corresponding to the at least one preselected enzyme, a polynucleotide encoding the at least one polypeptide, or both.

11. The method according to claim 1, wherein, The introduction step includes transducing multiple cells present in lung tissue to express the at least one preselected enzyme.

12. The method according to claim 11, wherein, The multiple cells are transduced using DNA or its constructs.

13. The method according to claim 11, wherein, The multiple cells were transduced using mRNA or its constructs.

14. A method for mitigating one or more negative effects of at least one toxin in the body, comprising introducing at least one preselected enzyme into the lung tissue of a subject, wherein, The at least one preselected enzyme is known to be capable of enzymatically breaking down at least one toxin that produces one or more negative effects on the subject.

15. The method of claim 14, wherein, The at least one preselected enzyme is selected from: phenylalanine ammonia-lyase (PAL), phenylalanine hydroxylase (PAH), galactose-degrading enzyme, galactose-1-phosphate uridine dihydrogenase (GALT), 2-oxoisovalerate dehydrogenase subunit α-mitochondria (BCKDHA), 2-oxoisovalerate dehydrogenase subunit β-mitochondria (BCKDHB), BDT complex enzyme, kynurenase, cocaine esterase, arginase, glutamine synthetase, arginine succinate lyase, arginine succinate synthetase, carbamoyl phosphate synthase I, N-acetylglutamate synthase, ornithine transcarbamoylase, ornithine translocase, nicotinic oxidoreductase, uricase, acid α-glucosidase (GAA), thiosulfate. Thiotransferase (thiocyanate), collagenase, asparaginase, anti-inhibitory coagulation complex, tissue plasminogen activator (t-PA), alteplase, bovine peglucosidase, alglucosidase, imiglucosidase, coagulation factor IX, deoxyribonuclease, pancreatic lipase, sucrase, truncated (non-glycosylated) t-PA (357 of 527 amino acids), coagulation factor VIIa, tissue plasminogen activator, antihemophilic factor (AHF), laronidase, agalsidase β, hyaluronidase, thioglucosidase, idolesulfatase, aglucosidase α, thrombin, verasidase α, pegologase, asparaginase, tadalafilase α, plasmin, carboxypeptidase g2, glutamate, coagulation factor XIII A. Elosulfate esterase α, coagulation factor X, asfortaase α, cybepi esterase α, seripa esterase α, vetranes esterase α-vjbk, pevaricase-pqpz, and combinations thereof.

16. The method of claim 14, further comprising degrading in vivo the at least one toxin that has diffused from the subject's circulatory system into or migrated into the subject's lungs and / or lung mucus, thereby mitigating the one or more negative effects.

17. A method for converting the lungs of a subject to include at least one enzymatic decomposition function, the method comprising introducing at least one preselected enzyme into the lungs of the subject, the preselected enzyme being known to promote the enzymatic decomposition of at least one toxin, which, if present, is systemically present in the subject.

18. The method of claim 17, wherein the at least one toxin circulates in the subject's body (including the subject's lungs).

19. The method of claim 18, further comprising contacting the at least one preselected enzyme with the at least one toxin to promote its enzymatic degradation in vivo.

20. A converted lung capable of enzymatically breaking down at least one toxin present systemically in the body of a subject (including the lungs of the subject), said converted lung comprising lung tissue converted to carry at least one preselected enzyme, said preselected enzyme being known to enzymatically break down at least one toxin.

21. The transformed lung according to claim 20, wherein, The transformed lung tissue carries at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to the preselected enzyme, a polynucleotide encoding the polypeptide, or both.

22. A transformed lung cell comprising alveolar cells carrying at least one preselected enzyme known to enzymatically break down at least one toxin.

23. The transformed lung cells according to claim 22, wherein, The transformed alveolar cells carry at least one preselected enzyme by introducing a polypeptide having an amino acid sequence corresponding to the preselected enzyme, a polynucleotide encoding the polypeptide, or both.

24. A lung cell population comprising a plurality of alveolar cells carrying at least one preselected enzyme, said preselected enzyme being known to enzymatically break down at least one toxin.

25. The method according to claim 2, wherein, One or more of the genetic diseases prevent the breakdown of at least one of the toxins.

26. A method for mitigating one or more negative effects of leukemia in a subject, comprising: (a) Introducing an asparaginase into the lung tissue of a subject diagnosed with leukemia, wherein the asparaginase is known to enzymatically break down asparagine; as well as (b) To induce the enzymatic degradation of asparagine in vivo, thereby mitigating one or more of the aforementioned negative effects.

27. The method according to claim 26, wherein, The leukemia mentioned includes acute lymphoblastic leukemia (ALL).

28. The method according to claim 26, wherein, The asparaginases include L-asparaginases.

29. A method for mitigating one or more negative effects caused by excessive oxalate in a subject, comprising: (a) Introducing an oxalate decarboxylase into the lung tissue of a subject diagnosed with oxalate deposition, primary hyperoxaluria, or secondary hyperoxaluria, wherein the oxalate decarboxylase is known to enzymatically break down oxalate; and (b) To cause the oxalate to undergo enzymatic decomposition.

30. The method according to claim 29, wherein, The oxalates include endogenously produced oxalates or oxalates ingested through diet.

31. A method for mitigating one or more negative effects of phenylketonuria in a subject, comprising: (a) Introducing phenylalanine ammonia-lyase (PAL) into the lung tissue of a subject diagnosed with phenylketonuria, wherein the PAL is known to enzymatically break down phenylalanine; and (b) To cause the phenylalanine to undergo enzymatic decomposition.

32. The method according to claim 31, wherein, The subjects contained a mutation in the phenylalanine hydroxylase (PAH) gene.

33. A method for mitigating one or more negative effects of hyperuricemia in a subject, comprising: (a) Introducing uricase into the lung tissue of a subject diagnosed with hyperuricemia, wherein the uricase is known to enzymatically break down uric acid. as well as (b) To cause the uric acid to undergo enzymatic decomposition.

34. The method according to claim 33, wherein, The subject had excessive uric acid production.

35. The method according to claim 33, wherein, The subjects had insufficient uric acid excretion.

36. The method according to claim 33, wherein, The subjects were diagnosed with or suspected of having gout.

37. A method for mitigating one or more negative effects of cyanide poisoning in a subject, comprising: (a) Introducing thiosulfate-thiotransferase (thiocyanate) into the lung tissue of a subject diagnosed with cyanide poisoning, wherein the thiocyanate is known to enzymatically break down cyanide; and (b) Cause the cyanide to undergo enzymatic decomposition.

38. The method according to claim 37, wherein, The subjects ingested cyanide salts, drank liquid hydrogen cyanide, absorbed hydrogen cyanide through the skin, received sodium nitroprusside intravenously, or inhaled hydrogen cyanide gas.

39. The method according to claim 37, wherein, The introduction also includes introducing sodium thiosulfate into the lung tissue of the subject.

40. The method according to claim 1, 14 or 17, wherein, The at least one preselected enzyme is primarily found in the lung tissue of the subject.

41. The method according to claim 1, 14 or 17, wherein, The at least one preselected enzyme is introduced under conditions that inhibit or do not support the systemic delivery of the at least one preselected enzyme to the subject.

42. The method according to claim 1, 14 or 17, wherein, The method of introducing the at least one preselected enzyme does not include delivery of the at least one preselected enzyme to the subject via the lung mucosa.

43. The method according to claim 1, 14 or 17, wherein, The at least one preselected enzyme is introduced into at least a portion of the lungs of the subject in a manner that minimizes systemic exposure to the subject.

44. The method according to claim 1, 14 or 17, wherein, The at least one preselected enzyme, its variants, or combinations thereof do not include ADH / KRED bound to at least one long-acting molecule or complex molecule.

45. The method according to claim 1, 14 or 17, wherein, Introduce a nebulized solution that does not contain ADH / KRED that binds to at least one long-acting molecule or complex molecule.

46. ​​The method according to claim 1, wherein, Enzymatically degrading the at least one toxin includes degrading the at least one toxin that has diffused or migrated from the subject's circulatory system to the subject's lungs and / or lung mucus.

47. The method of claim 19, wherein the enzymatic degradation occurs in the lungs and / or lung mucus of the subject.

48. A method for mitigating one or more negative effects of hyperammonemia in a subject, comprising: (a) Introducing glutamine synthase into the lung tissue of a subject diagnosed with hyperammonemia, wherein the glutamine synthase is known to enzymatically remove ammonia; and (b) The ammonia is enzymatically removed.

49. The method according to claim 48, wherein, The subject produced excessive ammonia.

50. The method according to claim 48, wherein, The subjects had insufficient ammonia excretion.

51. The method according to claim 48, wherein, The subjects were diagnosed with or suspected of having ornithine carbamoyltransferase deficiency (OTCD).

52. A method for mitigating one or more negative effects of hyperarginineemia in a subject, comprising: (a) Introducing arginase into the lung tissue of a subject diagnosed with hyperarginemia, wherein the arginase is known to enzymatically break down arginine; and (b) Cause the arginine to undergo enzymatic decomposition.

53. The method according to claim 52, wherein, The subjects produced excessive amounts of arginine.

54. The method according to claim 52, wherein, The subjects were diagnosed with or suspected of having arginase deficiency.

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