Compositions and methods for the treatment of lung damage and associated diseases and conditions

Abstract

Certain embodiments provide a method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Card) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy.

Description

[0001] RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0002] COMPOSITIONS AND METHODS FOR THE TREATMENT OF LUNG DAMAGE AND ASSOCIATED DISEASES AND CONDITIONS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to United States Provisional Application Number 63 / 725,196 that was filed on November 26, 2024. The entire content of the applications referenced above is hereby incorporated by reference herein.

[0005] GOVERNMENT FUNDING

[0006] This invention was made with government support under All 80644 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Lung diseases including asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), emphysema and pneumonia represent the leading causes of death and disability in the world (Collaborators GBDCRD (2020) Lancet Respir Med. 8(6): 585-96; Societies FoIR (2017), The Global Impact of Respiratory Disease - Second Addition, Sheffield, European Respiratory Society). Unfortunately, the prevalence of pulmonary fibrosis, emphysema, and other lung-related pathologies are expected to see sharp increases as a result of the COVID-19 pandemic and the long-term consequences of SARS-CoV-2 infection (George PM et al. (2020) Lancet Respir Med. 8(8): 807-15; Halpin DMG et al. (2021) Am J Respir Crit Care Med. 203(1): 24-36). Despite these alarming facts, many lung diseases remain difficult to treat and have extremely poor outcomes. It is imperative that we develop a better understanding of the cellular and molecular mechanisms that regulate immunity and inflammation in the lung to inform therapeutic strategies to treat these devastating diseases. Thus, a better understanding is needed to aid in the development of novel therapeutic strategies and improved treatment options for diverse forms of lung inflammation and damage.

[0009] SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

[0010] Certain embodiments provide a method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy, RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0011] wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0012] Certain embodiments provide a method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the lung damage is not caused by lung fibrosis and / or emphysema.

[0013] Certain embodiments provide a method for treating lung cancer comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal, wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0014] Certain embodiments provide a method for treating an alpha 1 antitrypsin (AAT) deficiency comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal, wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0015] Certain embodiments provide a method for treating bronchiectasis comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal, wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0016] Certain embodiments provide a method for treating lung fibrosis, emphysema, and / or COPD comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0017] (AAT) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0018] Certain embodiments provide a pharmaceutical composition comprising a Car4 therapy, an AAT therapy, and a pharmaceutically acceptable carrier, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0019] Certain embodiments provide a kit comprising a Car4 therapy, an AAT therapy, packaging material, and instructions for administering the Car4 therapy and the AAT therapy to a mammal in need thereof to treat lung damage, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended Figures. For the purpose of illustration, nonlimiting embodiments are shown in the Figures. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the Figures.

[0022] Figure 1. Fluorescent conjugated elastin was incubated withlU of elastase with or without 1.5 ug AAT or 1.5ug Car4 and elastase activity was measured overtime via absorbance at 405nm. Representative of at least 3 separate experiments.

[0023] Figure 2. Fluorescent conjugated elastin was incubated 1U of elastase with or without 1.5 ug AAT or 0.75ug Car4 combined with 0.75ug of AAT and elastase activity was measured over time via absorbance at 405nm. Representative of at least 3 separate experiments.

[0024] Figure 3. Fluorescent conjugated elastin was incubated with 1U of elastase with or without 1.5 ug AAT, 1.5ug Car4, or 0.75ug Car4 combined with 0.75ug of AAT and the area under the curve (AUC) was evaluated across three separate experiments. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0025] DETAILED DESCRIPTION

[0026] As described herein, the present inventors have demonstrated that carbonic anhydrase 4 (Car4) binds to alpha 1 antitrypsin (AAT). As described in Example 1, Car4 and AAT act synergistically to inhibit elastase activity. Inhibition of elastase protects the lung and prevents or mitigates lung damage.

[0027] Accordingly, the present invention provides methods for treating or preventing lung damage comprising administering to a mammal in need thereof (e.g., such as a human patient) an effective amount of a Car4 therapy in combination with an effective amount of an AAT therapy.

[0028] As used herein, the term “lung damage” refers to damage to the lung that prevents the organ from functioning properly. Lung damage may be caused by a variety of diseases or conditions, including but not limited to, lung (or pulmonary) fibrosis, bronchiectasis, chronic obstructive pulmonary disease (COPD) (e.g., emphysema and / or bronchitis), an autoimmune disease (e.g., rheumatoid arthritis, scleroderma, or Sjogren’s syndrome), a viral infection, gastroesophageal reflux disease (GERD), genetic factors (e.g., AAT deficiency), medication(s) (e.g., drug-induced pulmonary fibrosis), radiation treatment(s), COVID-19, Mycobacterium tuberculosis, fungal infections (e.g., Aspergillus fumigatus), cancer (e.g., lung cancer), a parasite infection, asthma, inhalation of noninfectious agent(s), hypersensitivity pneumonitis, nonspecific interstitial pneumonia, and pneumoconiosis. In certain embodiments, the lung damage is elastase-induced lung damage. In certain embodiments, the lung damage treated with the method described herein is M2 macrophage-mediated. Methods for evaluating lung damage are known in the art and described herein. For example, lung damage may be evaluated by investigating the architecture of the lung or by the measuring the lungs capacity to hold air, move air, absorb oxygen or by a method described herein (see, e.g., the Examples). Exemplary methods of evaluating lung damage include spirometry, pulse oximetry, lung volume test, lung diffusion capacity test, fractional exhaled nitric oxide (FeNO) tests, arterial blood gas test, chest X-ray, chest CT scan, chest MRI, and bronchoscopy.

[0029] Accordingly, in certain embodiments, the lung damage treated with a method described herein is caused by lung (or pulmonary) fibrosis, COPD (emphysema and / or bronchitis) bronchiectasis, an autoimmune disease (e.g., rheumatoid arthritis, scleroderma, or Sjogren’s syndrome), a viral infection, gastroesophageal reflux disease (GERD), genetic factors (e.g., AAT deficiency), medication(s) (e.g., drug-induced pulmonary fibrosis), radiation treatment(s), RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0030] COVID-19, Mycobacterium tuberculosis, fungal infections (e.g., Aspergillus fumigatus), cancer (e.g., lung cancer), a parasite infection, asthma, inhalation of noninfectious agent(s), hypersensitivity pneumonitis, non-specific interstitial pneumonia, and / or pneumoconiosis.

[0031] Thus, in certain embodiments, the mammal in need of treatment has or has had one or more of these diseases and / or conditions.

[0032] In certain embodiments, the lung damage treated with a method described herein is caused by COPD (emphysema and / or bronchitis). In certain embodiments, the lung damage treated with a method described herein is caused by COPD, lung fibrosis, and / or bronchiectasis. In certain embodiments, the mammal in need of treatment has COPD (emphysema and / or bronchitis). In certain embodiments, the mammal in need of treatment has COPD and / or lung fibrosis and / or bronchiectasis. Thus, certain embodiments also provide a method of treating COPD (emphysema and / or bronchitis), lung fibrosis, and / or bronchiectasis comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy. In certain embodiments, the mammal has emphysema. In certain embodiments, the mammal has bronchitis. In certain embodiments, the mammal has lung fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)). In certain embodiments, the mammal has bronchiectasis. In certain embodiments, the mammal has COPD (emphysema and / or bronchitis). In certain embodiments, the lung fibrosis and / or emphysema is M2 macrophage-mediated.

[0033] In certain embodiments, the lung damage treated with a method described herein is caused by an AAT deficiency. In certain embodiments, a mammal in need of treatment has an AAT deficiency, which results in the lung damage. Thus, certain embodiments also provide a method of treating an AAT deficiency comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy.

[0034] In certain embodiments, the lung damage treated with a method described herein is caused by lung cancer. In certain embodiments, a mammal in need of treatment has lung cancer, which results in the lung damage. Thus, certain embodiments also provide a method of treating lung cancer comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0035] In certain embodiments, the lung damage treated with a method described herein is caused by COVID-19, Mycobacterium tuberculosis, fungal infections (e.g., Aspergillus fumigatus), a parasite infection, or inhalation of noninfectious agent(s).

[0036] In certain embodiments, the lung damage treated with a method described herein is caused by COVID-19.

[0037] In certain embodiments, the lung damage treated with a method described herein is caused by Mycobacterium tuberculosis.

[0038] In certain embodiments, the lung damage treated with a method described herein is caused by fungal infections (e.g., Aspergillus fumigatus).

[0039] In certain embodiments, the lung damage treated with a method described herein is caused by a parasite infection, such as a helminth infection. In certain embodiments, the parasite infection is an infection caused by a human hookworm, such as Ancylostoma duodenale or Necator americanus.

[0040] In certain embodiments, the lung damage treated with a method described herein is caused by asthma.

[0041] In certain embodiments, the lung damage treated with a method described herein is caused by inhalation of noninfectious agents including at least one of a toxin, an irritant, or particulate matter. In certain embodiments, the toxin, irritant or particulate matter is silica dust, asbestos fibers, hard metal dusts, coal dust, grain dust, bird and animal droppings, cigarette smoke, and / or aspirated gastric acid.

[0042] In certain embodiments, the lung damage is prevented with a method described herein. Certain embodiments also provide a method of inhibiting elastase activity in a cell, the method comprising contacting the cell with an effective amount of a Car4 therapy in combination with an effective amount of an AAT therapy, wherein the cell expresses elastase. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is contacted in vitro. In certain embodiments, the cell is contacted in vivo. Accordingly, certain embodiments provide a method of inhibiting elastase activity in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a Car4 therapy in combination with an effective amount of an AAT therapy. In certain embodiments, the mammal is a human.

[0043] As described herein, a method of the invention may comprise administering an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy. Car4 and AAT therapies are described below. For example, RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0044] a Car4 therapy may comprise at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator and an AAT therapy may comprise at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0045] As used herein, the term “in combination” may refer to the sequential or simultaneous use of the agents, wherein the agents may be used separately or two or more of the agents may be formulated into a single composition. Accordingly, in certain embodiments, a Car4 therapy may be administered either simultaneously or sequentially with an AAT therapy. In certain embodiments, a Car4 therapy is administered simultaneously with an AAT therapy. In certain embodiments, a pharmaceutical composition comprising a Car4 therapy and an AAT therapy is administered. In certain embodiments, a Car4 therapy and an AAT therapy are administered sequentially. In certain embodiments, a Car4 therapy is administered first, and an AAT therapy is administered second. In certain embodiments, an AAT therapy is administered first and a Car4 therapy is administered second.

[0046] In certain embodiments, more than one Car4 therapy and / or more than one AAT therapy may be administered to the mammal. For example, in certain embodiments, a two-, three-, four-, or five-way combination selected from 1) at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 activator; and 2) at least one of an AAT protein or a nucleic acid encoding an AAT protein, is administered to the mammal. For example, in certain embodiments, one or more AAT therapies, a Car4 enzyme and a nucleic acid encoding a Car4 enzyme are administered in combination (e.g., the Car4 enzyme may be used to address acute disease aspects while the nucleic acid (e.g., expressed from a vector) may be used to promote long-term benefits). In certain other embodiments, one or more AAT therapies, a Car4 enzyme and a Car4 activator are administered in combination. In certain embodiments, one or more AAT therapies, a nucleic acid encoding a Car4 enzyme and a Car 4 activator are administered in combination. In certain embodiments, one or more AAT therapies, a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, and a Car4 enzyme activator are administered in combination. In certain embodiments, one or more Car4 therapies and an AAT protein are administered in combination. In certain embodiments, one or more Car4 therapies and a nucleic acid encoding AAT are administered in combination. In certain embodiments, one or more Car4 therapies, an AAT protein and a nucleic acid encoding AAT are administered in combination (e.g., the AAT protein may be used to address acute disease aspects while the nucleic acid (e.g., expressed from a vector) may be used to promote long-term benefits). RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0047] When a two-, three-, four-, or five-way combinations of the above agents are administered, they may be administered simultaneously, sequentially, or in a combination thereof, wherein some or all of the agents may be used separately and / or some or all of the agents may be formulated into a single composition. In certain embodiments, at least two of the agents (e.g., 2, 3, 4 or 5 agents) are administered simultaneously. In certain embodiments, a pharmaceutical composition comprising two or more of the agents (e.g., 2, 3, 4 or 5 agents) is administered. In certain embodiments, two or more of the agents (e.g., 2, 3, 4 or 5 agents) are administered sequentially.

[0048] In certain embodiments, a method described herein comprises intratracheally administering to a mammal in need thereof an effective amount of a Car4 therapy and / or an AAT therapy. In certain embodiments, a method described herein comprises administering to a mammal in need thereof an effective amount of a Car4 therapy and / or an AAT therapy by inhalation or by nebulization. In certain embodiments, a method described herein comprises nasally administering to a mammal in need thereof an effective amount of a Car4 therapy and / or an AAT therapy. In certain other embodiments, a method described herein comprises intravenously administering to a mammal in need thereof an effective amount of a Car4 therapy and / or an AAT therapy. In certain embodiments, a method described herein comprises orally administering to a mammal in need thereof an effective amount of a Car4 therapy and / or an AAT therapy.

[0049] Certain embodiments also provide a Car4 therapy and an AAT therapy for use in combination in medical therapy.

[0050] Certain embodiments provide a Car4 therapy and an AAT therapy for use in combination in the treatment of lung damage.

[0051] Certain embodiments provide a Car4 therapy and an AAT therapy for use in combination in the treatment of lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung cancer, and / or AAT deficiency.

[0052] Certain embodiments provide a Car4 therapy and an AAT therapy for use in combination in inhibiting elastase activity.

[0053] Certain embodiments provide the use of a Car4 therapy to prepare a medicament useful for treating lung damage in combination with an AAT therapy in a mammal in need thereof.

[0054] Certain embodiments provide the use of a Car4 therapy to prepare a medicament useful for treating lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0055] cancer, and / or AAT deficiency in combination with an AAT therapy in a mammal in need thereof.

[0056] Certain embodiments provide the use of a Car4 therapy to prepare a medicament useful for inhibiting elastase activity in combination with an AAT therapy in a mammal in need thereof.

[0057] Certain embodiments provide the use of an AAT therapy to prepare a medicament useful for treating lung damage in combination with an Car4 therapy in a mammal in need thereof.

[0058] Certain embodiments provide the use of an AAT therapy to prepare a medicament useful for treating lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung cancer, and / or AAT deficiency in combination with an Car4 therapy in a mammal in need thereof.

[0059] Certain embodiments provide the use of an AAT therapy to prepare a medicament useful for inhibiting elastase activity in combination with an Car4 therapy in a mammal in need thereof.

[0060] Certain embodiments of the invention provide a composition comprising a Car4 therapy and an AAT therapy for use in medical therapy.

[0061] Certain embodiments of the invention provide a composition comprising a Car4 therapy and an AAT therapy for use in treating lung damage.

[0062] Certain embodiments of the invention provide a composition comprising a Car4 therapy and an AAT therapy for use in treating lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung cancer, and / or AAT deficiency.

[0063] Certain embodiments of the invention provide a composition comprising a Car4 therapy and an AAT therapy for use in inhibiting elastase activity.

[0064] Certain embodiments of the invention provide the use of a Car4 therapy and an AAT therapy to prepare a medicament for treating lung damage in a mammal in need thereof.

[0065] Certain embodiments of the invention provide the use of a Car4 therapy and an AAT therapy to prepare a medicament for treating lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung cancer, and / or AAT deficiency in a mammal in need thereof.

[0066] Certain embodiments of the invention provide the use of a Car4 therapy and an AAT therapy to prepare a medicament for inhibiting elastase activity in a mammal in need thereof. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0067] Certain Additional Embodiments

[0068] Certain embodiments also provide a method of inhibiting elastase activity in a cell, the method comprising contacting the cell with a Car4 therapy, wherein the cell expresses elastase. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is contacted in vitro. In certain embodiments, the cell is contacted in vivo. Accordingly, certain embodiments provide a method of inhibiting elastase activity in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a Car4 therapy. In certain embodiments, the cell is not derived from a mammal that has fibrosis and / or emphysema. In certain embodiments, the cell is derived from a mammal having COPD, wherein the COPD is not associated with fibrosis and / or emphysema. In certain embodiments, the cell is not derived from a mammal having COPD. In certain embodiments, the mammal does not have lung fibrosis and / or emphysema. In certain embodiments, the mammal has COPD, wherein the COPD is not associated with fibrosis and / or emphysema. In certain embodiments, the mammal does not have COPD. In certain embodiments, the mammal has lung damage that has a certain etiology described herein. In certain embodiments, the mammal does not have lung damage having a certain etiology described herein (e.g., lung damage caused by a parasitic infection). In certain embodiments, the mammal is a human.

[0069] Certain embodiments also provide a method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a Car4 therapy, wherein the lung damage is not caused by lung fibrosis and / or emphysema. Accordingly, in such embodiments, the mammal does not have lung fibrosis and / or emphysema. In certain embodiments, the lung damage is caused by COPD / the mammal has COPD, wherein the COPD is not associated with lung fibrosis and / or emphysema. In certain embodiments, the lung damage is not caused by COPD / the mammal does not have COPD. In certain embodiments, the lung damage is elastase-induced lung damage. In certain embodiments, the lung damage is a lung damage described herein. In certain embodiments, the lung damage has a certain etiology described herein. In certain embodiments, the lung damage does not have a certain etiology described herein (e.g., lung damage caused by a parasitic infection).

[0070] Certain embodiments also provide a method for treating lung cancer comprising administering to a mammal in need thereof an effective amount of a Car4 therapy. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0071] Certain embodiments also provide a method for treating an AAT deficiency comprising administering to a mammal in need thereof an effective amount of a Car4 therapy. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal.

[0072] Certain embodiments also provide a method for treating bronchiectasis comprising administering to a mammal in need thereof an effective amount of a Car4 therapy. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal. Certain embodiments also provide a method for treating COPD, wherein the COPD is not associated with emphysema and / or lung fibrosis, the method comprising administering to a mammal in need thereof an effective amount of a Car4 therapy. In certain embodiments, the method further comprises administering an effective amount of an AAT therapy to the mammal.

[0073] In certain embodiments, more than one Car4 therapy is administered to the mammal. In certain embodiments, more than one AAT therapy is administered to the mammal. In such embodiments, any combination of Car4 therapies and / or AAT therapies as described herein may be administered (e.g., sequentially or simultaneously (e.g., separately or co-formulated)).

[0074] Certain embodiments provide a Car4 therapy for use in inhibiting elastase activity in a mammal in need thereof.

[0075] Certain embodiments provide the use of a Car4 therapy to prepare a medicament for inhibiting elastase activity in a mammal in need thereof.

[0076] Certain embodiments provide a Car4 therapy for use in the treatment of lung damage, wherein the lung damage is not caused by fibrosis and / or emphysema.

[0077] Certain embodiments provide the use of a Car4 therapy to prepare a medicament for the treatment of lung damage in a mammal in need thereof, wherein the lung damage is not caused by fibrosis and / or emphysema.

[0078] Certain embodiments provide a Car4 therapy for use in the treatment of lung cancer, bronchiectasis, or an AAT deficiency.

[0079] Certain embodiments provide the use of a Car4 therapy to prepare a medicament for the treatment of lung cancer, bronchiectasis, or an AAT deficiency in a mammal in need thereof. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0080] Car 4 Therapies

[0081] As discussed above, a method as described herein may comprise administering to a mammal in need thereof an effective amount of a Car4 therapy. The Car4 therapy may comprise at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, wherein each of these therapies are discussed in detail below. In certain embodiments, the Car4 therapy comprises a Car4 enzyme (e.g., recombinant Car4 enzyme). In certain embodiments, the Car4 therapy is a Car4 enzyme. In certain embodiments, the Car4 therapy comprises a nucleic acid encoding a Car4 enzyme. In certain embodiments, the Car4 therapy is a nucleic acid encoding a Car4 enzyme. In certain embodiments, the Car4 therapy comprises a Car4 enzyme activator, such as L-Histidine, D-Histidine, L-Phenylalanine, D-Phenylalanine, or a pharmaceutically acceptable salt thereof. In certain embodiments, the Car4 therapy is a Car4 enzyme activator.

[0082] In certain embodiments, more than one Car4 therapy may be administered to the mammal. In certain embodiments, a two- or three-way combination selected from a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, and a Car4 activator, is administered to the mammal. For example, in certain embodiments, a Car4 enzyme and a nucleic acid encoding a Car4 enzyme are administered to the mammal (e.g., the Car4 enzyme may be used to address acute disease aspects while the nucleic acid (e.g., expressed from a vector) may be used to promote long-term benefits). In certain other embodiments, a Car4 enzyme and a Car4 activator are administered. In certain embodiments, a nucleic acid encoding a Car4 enzyme and a Car 4 activator are administered. In certain embodiments, a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, and a Car4 enzyme activator are administered.

[0083] When a two- or three-way combination of the above agents are administered, they may be administered either simultaneously or sequentially. In certain embodiments, the agents are administered simultaneously. In certain embodiments, a pharmaceutical composition comprising the two or three agents is administered. In certain embodiments, the agents are administered sequentially. For example, in certain embodiments, the Car4 enzyme is administered first and the nucleic acid encoding the Car4 enzyme is administered second. In certain other embodiments, the nucleic acid encoding the Car4 enzyme is administered first and the Car4 enzyme is administered second. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0084] Car 4 Enzymes

[0085] As discussed above, in certain embodiments, a method as described herein may comprise administering to a mammal in need thereof an effective amount of a Car4 enzyme.

[0086] The terms “carbonic anhydrase 4 enzyme,” “Car4,” “Car4 enzyme,” “Car4 polypeptide,” and “Car4 amino acid sequence” are used interchangeably and refer to the metabolic enzyme, encoded by the CA4 gene, that catalyzes the conversion between carbon dioxide and bicarbonate (reversible hydration of carbon dioxide: CO2 + H2O = HCCh' + H+), or to its catalytically active fragments and variants. Car4 enzyme, or its active fragments, and variants thereof are known in the art and described herein. For example, in certain embodiments, a Car4 enzyme may comprise an amino acid (aa) sequence according to any one of NCBI / UniProt accession numbers P22748 (human), XP_005257696 (human), AAA35625.1 (human), AAA35626.1 (human), 5IPZ A (human), 1ZNC_A (human), XP_005257696.1 (human), or XP_011523485.1 (human), or active fragments and / or variants thereof (which are incorporated by reference herein). Car4 enzymes have also been characterized in a variety of other mammals. Accordingly, a Car4 enzyme may also comprise an amino acid (aa) sequence derived from, e.g., UniProt Q95323 (bovine), P48284 (rat), Q64444 (mouse), or P48283 (rabbit), or active fragments (e.g., such as Glu l8-Ser277 from Q64444) and / or variants thereof (which are incorporated by reference herein). Active site residues of Car4 enzyme, e.g., catalytic residue and zinc-metal binding residues are known in the art and described herein, e.g., T Stams et al., Proc Natl Acad Sci U S A. 1996 Nov 26;93(24): 13589-94, DOI: 10.1073 / pnas.93.24.13589, which is incorporated by reference herein. In certain embodiments, the Car4 enzyme may be expressed as a full-length preproprotein enzyme that could be further processed into a shorter, mature form or fragment. For example, the signal peptide (e.g., the first 18 aa residues in the N-terminus of full-length enzyme) and / or the pro-peptide (e.g., the last 28 aa residues in the C-terminal of full-length enzyme) may be removed in its mature form (e.g., see UniProt accession number P22748 or SEQ ID NO: 1 below for a full length Car4 enzyme sequence; see, e.g., SEQ ID NO:3 as an example of sequence for a mature form / fragment). The term “Car4 enzyme” also includes active fragments and / or variants that have, including but not limited to, one or more amino acid substitution(s), and / or N-terminal and / or C-terminal truncation or addition, such as tag (e.g., purification or affinity tag such as His6 tag), as compared to the full-length preproprotein, as long as the active fragment or variant is capable of catalyzing the conversion between carbon dioxide and bicarbonate (reversible hydration of carbon dioxide: CO2 + H2O = HCCh' + H+). As RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0087] used herein, the term “recombinant Car4 enzyme” refers to a recombinantly constructed or produced Car4 enzyme, active fragment, or variant, which may optionally lack the N-terminal signal peptide and / or the C-terminal pro-peptide.

[0088] In certain embodiments, the Car4 enzyme is a wild-type Car4 enzyme (e.g., a wild-type human Car4 enzyme). In some embodiments, the Car4 enzyme is a variant or a catalytically active fragment of a Car4 polypeptide or protein (e.g., comprises a Car4 amino acid sequence described herein). In certain embodiments, the Car4 enzyme is a Car4 enzyme variant (as compared to a wild-type Car4 enzyme). In certain embodiments, the Car4 enzyme is a catalytically active fragment of a Car4 enzyme or a Car4 enzyme variant. In some embodiments, a catalytically active variant or fragment of a Car4 enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater of the activity of the wild-type Car4 enzyme.

[0089] In certain embodiments, the Car4 enzyme is about 222 to 336, 246 to 312, 266 to 312, or 266 to 294 amino acids (aa) in length. In certain embodiments, the Car4 enzyme is about 266 to 312 aa in length. In certain embodiments, the Car4 enzyme is about 312 aa in length. In certain embodiments, the Car4 enzyme is about 266 aa in length. In certain embodiments, the Car4 enzyme is about 294 aa in length. In certain embodiments, the Car4 enzyme is about 267 aa in length. In certain embodiments, the Car4 enzyme is about 305 aa in length. In certain embodiments, the Car4 enzyme is about 260 aa in length. In certain embodiments, the Car4 enzyme is about 246 aa in length. In certain embodiments, the Car4 enzyme is about 222 aa in length.

[0090] In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:1, 2, or 3.

[0091] MRMLLALLALSAARPSASAESHWCYEVQAESSNYPCLVPVKWGGNCQKDRQSPINIVTTKAKVDKKLGRF FFSGYDKKQTWTVQNNGHSVMMLLENKASISGGGLPAPYQAKQLHLHWSDLPYKGSEHSLDGEHFAMEMH IVHEKEKGTSRNVKEAQDPEDEIAVLAFLVEAGTQVNEGFQPLVEALSNIPKPEMSTTMAESSLLDLLPK EEKLRHYFRYLGSLTTPTCDEKVVWTVFREPIQLHREQILAFSQKLYYDKEQTVSMKDNVRPLQQLGQRT VIKSGAPGRPLPWALPALLGPMLACLLAGFLR (SEQ ID NO: 1 ), MRMLLALLALSAARPSASAESHWCYEVQAESSNYPCLVPVKWGGNCQKDRQSPINIVTTKAKVDKKLGRF FFSGYDKKQTWTVQNNGHSVMMLLENKASISGGGLPAPYQAKQLHLHWSDLPYKGSEHSLDGEHFAMEMH IVHEKEKGTSRNVKEAQDPEDEIAVLAFLVEIGRMNWPPPLAPCRLSQDPSLPFQAGTQVNEGFQPLVEA LSNIPKPEMSTTMAESSLLDLLPKEEKLRHYFRYLGSLTTPTCDEKVVWTVFREPIQLHREQILAFSQKL YYDKEQTVSMKDNVRPLQQLGQRTVIKSGAPGRPLPWALPALLGPMLACLLAGFLR ( SEQ ID NO: 2 ), RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0092] AESHWCYEVQAESSNYPCLVPVKWGGNCQKDRQSPINIVTTKAKVDKKLGRFFFSGYDKKQTWTVQNNGH SVMMLLENKASISGGGLPAPYQAKQLHLHWSDLPYKGSEHSLDGEHFAMEMHIVHEKEKGTSRNVKEAQD PEDEIAVLAFLVEAGTQVNEGFQPLVEALSNIPKPEMSTTMAESSLLDLLPKEEKLRHYFRYLGSLTTPT CDEKVVWTVFREPIQLHREQILAFSQKLYYDKEQTVSMKDNVRPLQQLGQRTVIKS (SEQ ID NO: 3 ) In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 80% sequence identity to any one of SEQ ID NOs: 1 to 3.

[0093] In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:1. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:1. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:1. In certain embodiments, the Car4 enzyme comprises SEQ ID NO:1. In certain embodiments, the Car4 enzyme consists of SEQ ID NO: 1.

[0094] In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:2. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:2. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:2. In certain embodiments, the Car4 enzyme comprises SEQ ID NO:2. In certain embodiments, the Car4 enzyme consists of SEQ ID NO:2.

[0095] In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:3. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:3. In certain embodiments, the Car4 enzyme comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:3. In certain embodiments, the Car4 enzyme comprises SEQ ID NO:3. In certain embodiments, the Car4 enzyme consists of SEQ ID NO:3. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0096] In certain embodiments, an enzyme polypeptide as described herein is fused to a tag (e.g., affinity tag and / or detectable tag such as HIS tag, FLAG tag, or C-Myc tag) via an optional linker sequence, such as Glycine linker or Glycine-serine linker (e.g., GGGG, or GGGS, etc.).

[0097] In certain embodiments, the enzyme polypeptide as described herein is present in a fusion polypeptide or fusion protein. For example, the enzyme polypeptide may be joined either directly (i.e., through a peptide bond) or through a peptide / polypeptide linker to another polypeptide (e.g., a polypeptide tag or an antibody fragment, such as an immunoglobulin Fc domain). For example, in certain embodiments, the Car4 enzyme is linked either directly or through a peptide / polypeptide linker to a Fc domain, or fragment thereof (e.g., IgGl, IgG2, IgG3, or IgG4 Fc domain, or fragment thereof). In certain embodiments, the Car4 enzyme is linked either directly or through a peptide / polypeptide linker to an AAT protein sequence described herein. Accordingly, certain embodiments provide a fusion protein comprising a Car4 enzyme sequence as described herein linked either directly or through a peptide / polypeptide linker to an AAT protein sequence as described herein.

[0098] Nucleic Acids Encoding a Car 4 Enzyme

[0099] As discussed above, in certain embodiments, a method described herein comprises administering to a mammal in need thereof an effective amount of a nucleic acid encoding a Car4 enzyme.

[0100] Nucleic acids encoding a Car4 enzyme, or its active fragments, and variants are known in the art and described herein. For example, in certain embodiments, a nucleic acid encoding a Car4 enzyme, or its active fragments, and variants may comprise a nucleic acid sequence according to any one of NCBI accession number NM_000717.5, XM_047436653.1, XM_047436656.1, XM_005257639.4, XM_047436652.1, which are incorporated by reference herein.

[0101] In certain embodiments, the nucleic acid encodes a Car4 enzyme polypeptide described herein. For example, in certain embodiments, the nucleic acid encodes a Car4 enzyme polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1, 2, or 3.

[0102] In certain embodiments, the nucleic acid is DNA.

[0103] In certain embodiments, the nucleic acid is RNA, for example, mRNA. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0104] In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 or SEQ ID NO:5.

[0105] ACCCGCGGCGGCCTCCTCGGTGCGCGACCCCCGGCTCAGAGGACTCTTTGCTGTCCCGCAAGATGCGGAT GCTGCTGGCGCTCCTGGCCCTCTCCGCGGCGCGGCCATCGGCCAGTGCAGAGTCACACTGGTGCTACGAG GTTCAAGCCGAGTCCTCCAACTACCCCTGCTTGGTGCCAGTCAAGTGGGGTGGAAACTGCCAGAAGGACC GCCAGTCCCCCATCAACATCGTCACCACCAAGGCAAAGGTGGACAAAAAACTGGGACGCTTCTTCTTCTC TGGCTACGATAAGAAGCAAACGTGGACTGTCCAAAATAACGGGCACTCAGTGATGATGTTGCTGGAGAAC AAGGCCAGCATTTCTGGAGGAGGACTGCCTGCCCCATACCAGGCCAAACAGTTGCACCTGCACTGGTCCG ACTTGCCATATAAGGGCTCGGAGCACAGCCTCGATGGGGAGCACTTTGCCATGGAGATGCACATAGTACA TGAGAAAGAGAAGGGGACATCGAGGAATGTGAAAGAGGCCCAGGACCCTGAAGACGAAATTGCGGTGCTG GCCTTTCTGGTGGAGGCTGGAACCCAGGTGAACGAGGGCTTCCAGCCACTGGTGGAGGCACTGTCTAATA TCCCCAAACCTGAGATGAGCACTACGATGGCAGAGAGCAGCCTGTTGGACCTGCTCCCCAAGGAGGAGAA ACTGAGGCACTACTTCCGCTACCTGGGCTCACTCACCACACCGACCTGCGATGAGAAGGTCGTCTGGACT GTGTTCCGGGAGCCCATTCAGCTTCACAGAGAACAGATCCTGGCATTCTCTCAGAAGCTGTACTACGACA AGGAACAGACAGTGAGCATGAAGGACAATGTCAGGCCCCTGCAGCAGCTGGGGCAGCGCACGGTGATAAA GTCCGGGGCCCCGGGTCGGCCGCTGCCCTGGGCCCTGCCTGCCCTGCTGGGCCCCATGCTGGCCTGCCTG CTGGCCGGCTTCCTGCGATGATGGCTCACTTCTGCACGCAGCCTCTCTGTTGCCTCAGCTCTCCAAGTTC CAGGCTTCCGGTCCTTAGCCTTCCCAGGTGGGACTTTAGGCATGATTAAAATATGGACATATTTTTGGAG AAA ( SEQ ID NO: 4 )

[0106] GCAGAGTCACACTGGTGCTACGAGGTTCAAGCCGAGTCCTCCAACTACCCCTGCTTGGTGCCAGTCAAGT GGGGTGGAAACTGCCAGAAGGACCGCCAGTCCCCCATCAACATCGTCACCACCAAGGCAAAGGTGGACAA AAAACTGGGACGCTTCTTCTTCTCTGGCTACGATAAGAAGCAAACGTGGACTGTCCAAAATAACGGGCAC TCAGTGATGATGTTGCTGGAGAACAAGGCCAGCATTTCTGGAGGAGGACTGCCTGCCCCATACCAGGCCA AACAGTTGCACCTGCACTGGTCCGACTTGCCATATAAGGGCTCGGAGCACAGCCTCGATGGGGAGCACTT TGCCATGGAGATGCACATAGTACATGAGAAAGAGAAGGGGACATCGAGGAATGTGAAAGAGGCCCAGGAC CCTGAAGACGAAATTGCGGTGCTGGCCTTTCTGGTGGAGGCTGGAACCCAGGTGAACGAGGGCTTCCAGC CACTGGTGGAGGCACTGTCTAATATCCCCAAACCTGAGATGAGCACTACGATGGCAGAGAGCAGCCTGTT GGACCTGCTCCCCAAGGAGGAGAAACTGAGGCACTACTTCCGCTACCTGGGCTCACTCACCACACCGACC TGCGATGAGAAGGTCGTCTGGACTGTGTTCCGGGAGCCCATTCAGCTTCACAGAGAACAGATCCTGGCAT TCTCTCAGAAGCTGTACTACGACAAGGAACAGACAGTGAGCATGAAGGACAATGTCAGGCCCCTGCAGCA GCTGGGGCAGCGCACGGTGATAAAGTCC ( SEQ ID NO: 5 )

[0107] In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 4. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:4. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO:4. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises SEQ ID NO:4. In certain embodiments, the nucleic acid encoding a Car4 enzyme consists of SEQ ID NO:4. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0108] In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 5. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO: 5. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO: 5. In certain embodiments, the nucleic acid encoding a Car4 enzyme comprises SEQ ID NO:5. In certain embodiments, the nucleic acid encoding a Car4 enzyme consists of SEQ ID NO:5.

[0109] In certain embodiments, the nucleic acid is comprised within an expression cassette, wherein the nucleic acid is operably linked to a promoter.

[0110] In certain embodiments, the nucleic acid encoding the Car4 enzyme or expression cassette is comprised within a vector. Any suitable vector may be used for introducing a nucleic acid / expression cassette described herein into a mammalian cell. Examples of suitable vectors include plasmids, cosmids, phage, liposomes, molecular conjugates, and viruses. In certain embodiments, the vector is a plasmid. In certain embodiments, the vector is a viral vector, for example, an adeno-associated virus vector (AAV).

[0111] Thus, in certain embodiments, a method described herein comprises administering an effective amount of an expression cassette or vector (e.g., a viral vector, such as AAV viral particles) comprising the nucleic acid encoding the Car4 enzyme to the mammal.

[0112] In certain embodiments, the nucleic acid (e.g., mRNA) is comprised within a liposome or nanoparticle (e.g., lipid nanoparticle). Thus, in certain embodiments, a method described herein comprises administering an effective amount of a liposome or nanoparticle comprising the nucleic acid encoding the Car4 enzyme to the mammal.

[0113] Car 4 Enzyme Activators

[0114] As discussed above, in certain embodiments, a method as described herein may comprise administering to a mammal in need thereof an effective amount of a Car4 enzyme activator.

[0115] As used herein, the term “carbonic anhydrase 4 enzyme activator (Car4 enzyme activator)” includes any therapeutic agent or compound or treatment capable of enhancing the expression and / or function of a Car4 enzyme (e.g., enhances transcription, RNA maturation, RNA translation, post-translational modification, or enzymatic activity (e.g., enhances activation of the enzyme)). For example, in certain embodiments, the activator detectably enhances the RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0116] expression level or enzymatic activity of a Car4 enzyme as measured, e.g., using an assay described herein. In certain embodiments, the activator enhances the expression level or enzymatic activity of a Car4 enzyme by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0117] The activator may be of natural or synthetic origin. For example, it may be a polypeptide, a protein, a peptide, or small molecule (e.g., an organic compound).

[0118] The term “small molecule” includes organic molecules having a molecular weight of less than about 1000 amu. In one embodiment a small molecule can have a molecular weight of less than about 800 amu. In another embodiment a small molecule can have a molecular weight of less than about 500 amu.

[0119] In certain embodiments, the activator is a selective Car4 activator. For example, the Car4 activator may be at least 5, at least 10, at least 50, at least 100, at least 500, or at least 1,000 fold selective for Car4 over another Car in a selected assay.

[0120] Non-limiting examples of Car4 enzyme activators that may be used in the present invention include L-phenylalanine, D-phenylalanine, L-histidine, D-histidine, or a pharmaceutically acceptable salt thereof. Thus, in certain embodiments, the Car4 enzyme activator comprises at least one of L-phenylalanine, D-phenylalanine, L-histidine, D-histidine, or a pharmaceutically acceptable salt thereof. In certain embodiments, a combination of Car4 enzyme activators are administered.

[0121] AAT Therapies

[0122] As discussed above, a method as described herein may comprise administering to a mammal in need thereof an effective amount of an AAT therapy. The AAT therapy may comprise at least one of AAT protein or a nucleic acid encoding an AAT protein, wherein each of these therapies are discussed in detail below. In certain embodiments, the AAT therapy comprises an AAT protein (e.g., recombinant AAT protein). In certain embodiments, the AAT therapy is an AAT protein. In certain embodiments, the AAT therapy comprises a nucleic acid encoding an AAT protein. In certain embodiments, the AAT therapy is a nucleic acid encoding an AAT protein.

[0123] In certain embodiments, more than one AAT therapy may be administered to the mammal. In certain embodiments, an AAT protein and a nucleic acid encoding an AAT protein RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0124] are administered to the mammal (e.g., the AAT enzyme may be used to address acute disease aspects while the nucleic acid (e.g., expressed from a vector) may be used to promote long-term benefits). When two agents are administered, they may be administered either simultaneously or sequentially. In certain embodiments, the agents are administered simultaneously. In certain embodiments, a pharmaceutical composition comprising the two agents is administered. In certain embodiments, the agents are administered sequentially. For example, in certain embodiments, the AAT protein is administered first and the nucleic acid encoding the AAT protein is administered second. In certain other embodiments, the nucleic acid encoding the AAT protein is administered first and the AAT protein is administered second.

[0125] AAT Proteins

[0126] As discussed above, in certain embodiments, a method for treating lung damage comprises administering to a mammal in need thereof an effective amount of an AAT protein.

[0127] As used herein, the term “Alpha- 1 -Antitrypsin” or “AAT” refers to the protein, encoded by the serpin family A member 1 (SERPINA1) gene, that is a serine protease inhibitor that may inhibits one or more targets of elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator (see, Gene ID 5265). The term AAT also includes its active fragment and variant that is serine protease inhibitor that may inhibits one or more targets of elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator. In certain embodiments, AAT or its active fragments, and variants may inhibit elastase, plasmin, and thrombin. In certain embodiments, AAT or its active fragments, and variants may irreversibly inhibit trypsin, chymotrypsin, and plasminogen activator. AAT, or its active fragments, and variants are known in the art and described herein, for example, in certain embodiments, AAT, or its active fragments, and variants may comprise the amino acid (aa) sequence according to any one of NCBI accession numbers P01009, 1EZX A, 4PYW A, 1OPH A, KAI2572619, and AAA40788.1. In certain embodiments, the AAT may be expressed as a full-length precursor protein that could be further processed into a shorter, mature form or fragment. For example, the signal peptide (e.g., the first 24 aa residues in the N-terminus of full-length protein such as in NCBI accession number P01009) may be removed in its mature form. The term “AAT” also includes active fragments or variants that have, including but not limited to, one or more amino acid substitution(s), or N-terminal and / or C-terminal truncation or addition such as a tag (e.g., purification or affinity tag such as His6 tag), as compared to full-length precursor protein, as RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0128] long as the active fragment or variant is a serine protease inhibitor that may inhibit one or more targets of elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator.

[0129] In certain embodiments, the AAT is about 326 to 418, 335 to 404, or 343 to 394 aa in length. In certain embodiments, the AAT is about 418 aa in length. In certain embodiments, the AAT is about 404 aa in length. In certain embodiments, the AAT is about 343 aa in length. In certain embodiments, the AAT is about 335 aa in length. In certain embodiments, the AAT is about 359 aa in length. In certain embodiments, the AAT is about 394 aa in length.

[0130] In certain embodiments, the AAT comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:6, 7, 8, or 10.

[0131] MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSN STNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGN GLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVN YIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPD EGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAP LKLSKAVHKAVLTIDEKGTEAAGAMFLEAI PMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKWNPTQK

[0132] ( SEQ ID NO: 6 ), EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEIL EGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAK KQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQH CKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGI TKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAI PMSIPPEVKFNKPFVFLMIEQNTKSPLFM GKWNPTQK ( SEQ ID NO: 7 ), NKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIH EGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEK GTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCK KLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVL GQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPM ( SEQ ID NO: 8 ) MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFS PVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKF LEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEED FHVDQATTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKVRSP ( SEQ ID NO: 10 )

[0133] In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 80% sequence identity to any one of SEQ ID NOs:6, 7, 8 or 10.

[0134] In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:6. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:6. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 90% RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0135] sequence identity to SEQ ID NO:6. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:6. In certain embodiments, the AAT protein comprises SEQ ID NO:6. In certain embodiments, the AAT protein consists of SEQ ID NO: 6.

[0136] In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:7. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:7. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:7. In certain embodiments, the AAT protein comprises SEQ ID NO:7. In certain embodiments, the AAT protein consists of SEQ ID NO: 7.

[0137] In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 8. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 8. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 8. In certain embodiments, the AAT protein comprises SEQ ID NO:8. In certain embodiments, the AAT protein consists of SEQ ID NO: 8.

[0138] In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 10. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 10. In certain embodiments, the AAT protein comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 10. In certain embodiments, the AAT protein comprises SEQ ID NO: 10. In certain embodiments, the AAT protein consists of SEQ ID NO: 10. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0139] In certain embodiments, an active AAT fragment is about 44 aa in length (e.g., the last 44 aa residues in the C-terminus of full-length protein such as in NCBI accession number P01009). In certain embodiments, an active AAT fragment has an amino acid sequence of MFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:6), which is also referred to as “Short Peptide from AAT” that is a reversible chymotrypsin inhibitor, which also inhibits elastase, but not trypsin.

[0140] In certain embodiments, the AAT is derived and purified from human blood.

[0141] Accordingly, in certain embodiments, the AAT is Aralast, Glassia, Prolastin, Prolastin-C, or Zemaira. In certain embodiments, the AAT is a recombinant AAT.

[0142] In certain embodiments, an AAT protein is fused to a tag (e.g., affinity tag and / or detectable tag such as HIS tag, FLAG tag, or C-Myc tag) via an optional linker sequence, such as Glycine linker or Glycine-serine linker (e.g., GGGG, or GGGS, etc.).

[0143] In certain embodiments, the AAT protein is present in a fusion polypeptide or fusion protein. For example, the AAT protein may be joined either directly (i.e., through a peptide bond) or through a peptide / polypeptide linker to another polypeptide (e.g., a polypeptide tag or an antibody fragment, such as an immunoglobulin Fc domain). For example, in certain embodiments, the AAT protein is linked either directly or through a peptide / polypeptide linker to a Fc domain, or fragment thereof (e.g., IgGl, IgG2, IgG3, or IgG4 Fc domain, or fragment thereof). In certain embodiments, the AAT protein is linked either directly or through a peptide / polypeptide linker to a Car4 enzyme sequence described herein. Accordingly, certain embodiments provide a fusion protein comprising an AAT sequence as described herein linked either directly or through a peptide / polypeptide linker to a Car4 enzyme sequence as described herein.

[0144] Nucleic Acids encoding an AAT Protein

[0145] As discussed above, in certain embodiments, a method as described herein comprises administering to a mammal in need thereof an effective amount of a nucleic acid encoding an AAT protein, such as mRNA encoding an AAT protein.

[0146] Nucleic acids encoding AAT, or its active fragments, and variants are known in the art and described herein, for example, in certain embodiments, nucleic acids encoding AAT, or its active fragments, and variants may comprise the nucleic acid sequence according to any one of NCBI accession number X01683.1, XM_054328987.1, XM_054328986.1 and NM_000295.5. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0147] In certain embodiments, the nucleic acid encodes an AAT protein as described herein. For example, in certain embodiments, the nucleic acid encodes an AAT protein comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:6, 7, 8, or 10.

[0148] In certain embodiments, the nucleic acid is DNA.

[0149] In certain embodiments, the nucleic acid is RNA, for example, mRNA.

[0150] In certain embodiments, the nucleic acid comprises nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9.

[0151] CTGCAGGGGGGGGGGGGGGCTGGGACAGTGAATCGACAATGCCGTCTTCTGTCTCGTGGGGCATCCTCCT GCTGGCAGGCCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTGAGGATCCCCAGGGAGATGCTGCCCAGAAG ACAGATACATCCCACCATGATCAGGATCACCCAACCTTCAACAAGATCACCCCCAACCTGGCTGAGTTCG CCTTCAGCCTATACCGCCAGCTGGCACACCAGTCCAACAGCACCAATATCTTCTTCTCCCCAGTGAGCAT CGCTACAGCCTTTGCAATGCTCTCCCTGGGGACCAAGGCTGACACTCACGATGAAATCCTGGAGGGCCTG AATTTCAACCTCACGGAGATTCCGGAGGCTCAGATCCATGAAGGCTTCCAGGAACTCCTCCGTACCCTCA ACCAGCCAGACAGCCAGCTCCAGCTGACCACCGGCAATGGCCTGTTCCTCAGCGAGGGCCTGAAGCTAGT GGATAAGTTTTTGGAGGATGTTAAAAAGTTGTACCACTCAGAAGCCTTCACTGTCAACTTCGGGGACACC GAAGAGGCCAAGAAACAGATCAACGATTACGTGGAGAAGGGTACTCAAGGGAAAATTGTGGATTTGGTCA AGGAGCTTGACAGAGACACAGTTTTTGCTCTGGTGAATTACATCTTCTTTAAAGGCAAATGGGAGAGACC CTTTGAAGTCAAGGACACCGAGGAAGAGGACTTCCACGTGGACCAGGTGACCACCGTGAAGGTGCCTATG ATGAAGCGTTTAGGCATGTTTAACATCCAGCACTGTAAGAAGCTGTCCAGCTGGGTGCTGCTGATGAAAT ACCTGGGCAATGCCACCGCCATCTTCTTCCTGCCTGATGAGGGGAAACTACAGCACCTGGAAAATGAACT CACCCACGATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGTCTGCCAGCTTACATTTACCCAAA CTGTCCATTACTGGAACCTATGATCTGAAGAGCGTCCTGGGTCAACTGGGCATCACTAAGGTCTTCAGCA ATGGGGCTGACCTCTCCGGGGTCACAGAGGAGGCACCCCTGAAGCTCTCCAAGGCCGTGCATAAGGCTGT GCTGACCATCGACGAGAAAGGGACTGAAGCTGCTGGGGCCATGTTTTTAGAGGCCATACCCATGTCTATC CCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACAAAATACCAAGTCTCCCCTCT TCATGGGAAAAGTGGTGAATCCCACCCAAAAATAACTGCCTCTCGCTCCTCAACCCCTCCCCTCCATCCC TGGCCCCCTCCCTGGATGACATTAAAGAAGGGTTGAGCTGG ( SEQ ID NO: 9 )

[0152] In certain embodiments, the nucleic acid encoding an AAT protein comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 9. In certain embodiments, the nucleic acid encoding an AAT protein comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO:9. In certain embodiments, the nucleic acid encoding an AAT protein comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO:9. In certain embodiments, the nucleic acid encoding an AAT protein comprises SEQ ID NO:9. In certain embodiments, the nucleic acid encoding an AAT protein consists of SEQ ID NO:9.

[0153] In certain embodiments, the nucleic acid is comprised within an expression cassette, wherein the nucleic acid is operably linked to a promoter. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0154] In certain embodiments, the nucleic acid encoding the AAT protein or expression cassette is comprised within a vector. Any suitable vector may be used for introducing a nucleic acid / expression cassette described herein into a mammalian cell. Examples of suitable vectors include plasmids, cosmids, phage, liposomes, molecular conjugates, and viruses. In certain embodiments, the vector is a plasmid. In certain embodiments, the vector is a viral vector, for example, an adeno-associated virus vector (AAV).

[0155] Thus, in certain embodiments, a method described herein comprises administering an effective amount of an expression cassette or vector (e.g., a viral vector, such as AAV viral particles) comprising the nucleic acid encoding the AAT protein to the mammal.

[0156] In certain embodiments, the nucleic acid (e.g., mRNA) is comprised within a liposome or nanoparticle (e.g., lipid nanoparticle). Thus, in certain embodiments, a method described herein comprises administering an effective amount of a liposome or nanoparticle comprising the nucleic acid encoding the AAT protein to the mammal.

[0157] Additional Therapeutic Agents

[0158] In certain embodiments, a method as described herein further comprises administering one or more additional therapeutic agents or therapies to the mammal.

[0159] In certain embodiments, the one or more additional therapeutic agents or therapies are useful for treating lung damage.

[0160] In certain embodiments, the one or more additional therapeutic agents or therapies are useful for treating lung fibrosis (e.g., Nintebanib or Pirfenidone). In certain embodiments, the one or more additional therapeutic agents or therapies is Nintedanib. In certain embodiments, the one or more additional therapeutic agents is Pirfenidone.

[0161] In certain embodiments, the one or more additional therapeutic agent or therapies are useful for treating emphysema.

[0162] In certain embodiments, the one or more additional therapeutic agent or therapies are useful for treating bronchiectasis.

[0163] In certain embodiments, the one or more additional therapeutic agents or therapies is an anti-inflammatory agent. In certain embodiments, the one or more additional therapeutic agents or therapies is a corticosteroid, an anticholinergic, and / or a beta2 -adrenergic agonist.

[0164] In certain embodiments, the one or more additional therapeutic agents or therapies are useful for treating cancer (e.g., an anti-cancer therapeutic agent or therapy). Anti-cancer RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0165] therapies include but are not limited to cislatin, carboplatin, pemetrexed, paclitaxel, docetaxel, gemcitabine, and vinorelbine. Anti-cancer therapies also include immunotherapies (e.g., Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab).

[0166] In certain embodiments, the one or more additional therapeutic agents or therapies are useful for treating an AAT deficiency.

[0167] In certain embodiments, the one or more additional therapeutic agents or therapies may be administered either simultaneously or sequentially with a Car4 therapy and / or AAT therapy. In certain embodiments, the one or more additional therapeutic agents is administered simultaneously with the Car4 enzyme, nucleic acid encoding Car4 enzyme, Car4 enzyme activator, AAT protein, and / or a nucleic acid encoding AAT protein. In certain embodiments, a pharmaceutical composition comprising the Car4 enzyme, nucleic acid encoding Car4 enzyme, Car4 enzyme activator, AAT protein, and / or a nucleic acid encoding AAT protein and the at least one other therapeutic agent is administered. In certain embodiments, the Car4 enzyme, nucleic acid encoding Car4 enzyme, Car4 enzyme activator, AAT protein, and / or a nucleic acid encoding AAT protein and the one or more additional therapeutic agents are administered sequentially. In certain embodiments, the Car4 enzyme, nucleic acid encoding Car4 enzyme, Car4 enzyme activator, AAT protein, and / or a nucleic acid encoding AAT protein is administered first and one or more additional therapeutic agents is administered second. In certain embodiments, the one or more additional therapeutic agents is administered first and the Car4 enzyme, nucleic acid encoding Car4 enzyme, Car4 enzyme activator, AAT protein, and / or a nucleic acid encoding AAT protein is administered second.

[0168] Compositions and Kits

[0169] Certain embodiments provide a composition (e.g., pharmaceutical composition) comprising a Car4 therapy, an AAT therapy, and a diluent or carrier (e.g., a pharmaceutically acceptable diluent or carrier). In certain embodiments, the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein. In certain embodiments, the composition comprises more than one Car4 therapy and / or more than one AAT therapy. Accordingly, in certain embodiments, the composition comprises a two-, three-, four-, or five-way combination RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0170] selected from 1) at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 activator; and 2) at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0171] Certain embodiments also provide a kit comprising a Car4 therapy, an AAT therapy, packaging material, and instructions for administering the Car4 therapy and the AAT therapy in combination to a mammal in need thereof to treat lung damage. Also provided is a kit comprising a Car4 therapy, an AAT therapy, packaging material, and instructions for administering the Car4 therapy and the AAT therapy to a mammal in need thereof to treat lung fibrosis, COPD (e.g., emphysema and / or bronchitis), bronchiectasis, lung cancer, and / or AAT deficiency. In certain embodiments, the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0172] Certain embodiments provide a kit comprising a Car4 therapy, packaging material, and instructions for administering the Car4 therapy to a mammal in need thereof to treat lung damage, wherein the lung damage is not caused by lung fibrosis and / or emphysema. Certain embodiments provide a kit comprising a Car4 therapy, packaging material, and instructions for administering the Car4 therapy to a mammal in need thereof to treat lung cancer, an AAT deficiency, or bronchiectasis. In certain embodiments, the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator. In certain embodiments, the kit further comprises an AAT therapy as described herein and instructions for administering the Car4 therapy and the AAT therapy in combination. In certain embodiments, the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

[0173] Administration

[0174] Compounds and agents described herein (e.g., Car4 enzymes, Car4 enzyme activators, nucleic acids encoding Car4 enzymes, AAT proteins, nucleic acids encoding AAT proteins, and additional therapeutic agents) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical, sub-lingual, optical (e.g., eye drops), nasal (i.e., nasal spray) or subcutaneous routes. In certain embodiments, the compounds / agents may be delivered via delayed administration. In RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0175] certain embodiments, the compounds / agents may be administered via nasal or pulmonary delivery, for example, via intratracheal delivery or via inhalation through an inhaler or nebulizer. Delivery of protein or nucleic acids to the lung, or pulmonary delivery of protein or nucleic acids are known in the art and described herein, for example, U. S. Patent 6,737,045; U. S. Patent 8,007,780; U. S. Patent 9,597,413; and U. S. Patent 9,725,522 are incorporated by reference herein.

[0176] Thus, the present compounds / agents may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound / agent may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound / agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound / agent in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0177] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound / agent, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound / agent may be incorporated into sustained-release preparations and devices. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0178] The active compound / agent may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound / agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0179] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0180] Sterile injectable solutions are prepared by incorporating the active compound / agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0181] For topical administration, the compounds / agents may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0182] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the compounds / agents can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0183] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0184] Useful dosages of the compounds / agents (e.g., Car4 enzyme, Car4 enzyme activator, nucleic acid encoding a Car4 enzyme, AAT protein, or nucleic acid encoding AAT protein) can be determined by comparing their in vitro activity, and in vivo activity in animal models.

[0185] Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Pat. No. 4,938,949.

[0186] The amount of the compound / agent, or an active salt or derivative thereof, required for use in treatment will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0187] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator.

[0188] As described herein, the Car4 enzymes, Car4 enzyme activators, nucleic acids encoding a Car4 enzyme, AAT proteins, and / or nucleic acids encoding an AAT protein can also be administered in combination with other therapeutic agents, for example, other therapeutic agents described herein. Accordingly, in one embodiment the invention also provides a composition comprising 1) a Car4 enzyme, Car4 enzyme activator, and / or a nucleic acid encoding a Car4 enzyme; 2) an AAT protein, and / or a nucleic acid encoding an AAT protein; 3) at least one other therapeutic agent; and 4 a pharmaceutically acceptable diluent or carrier. The invention also provides a kit comprising 1) a Car4 enzyme, Car4 enzyme activator and / or a nucleic acid RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0189] encoding a Car4 enzyme; 2) an AAT protein, and / or a nucleic acid encoding an AAT protein; 3) at least one other therapeutic agent, 4) packaging material, and 5) instructions for administering the Car4 enzyme, Car4 enzyme activator, and / or nucleic acid encoding a Car4 enzyme, the AAT protein, and / or a nucleic acid encoding an AAT protein, and the other therapeutic agent or agents to a mammal to treat lung damage (e.g., lung fibrosis, bronchiectasis, COPD (e.g., emphysema and / or bronchitis), lung cancer, and / or alpha 1 antitrypsin (AAT) deficiency).

[0190] Certain Definitions

[0191] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein. Polypeptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right. A protein molecule (e.g., enzyme) may exist in an isolated or purified form, for example, an isolated or purified form as an active ingredient of a drug dosage form. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0192] The invention encompasses isolated or substantially purified protein compositions. In the context of the present invention, an "isolated" or "purified" polypeptide is a polypeptide that exists apart from its native environment. A polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell such as a bacterium or a mammalian expression system for the production of the polypeptide. For example, an "isolated" or "purified" protein, or biologically active fragment thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active fragment thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0193] In certain embodiments, one or more amino acid residues are mutated within the polypeptide as described herein. For example, the mutation is conducted via error-prone PCR or site directed mutagenesis. In certain embodiments, an amino acid residue is mutated into one RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0194] that allows the properties of the amino acid side-chain to be conserved. Examples of the properties of amino acid side chains comprise: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising the following side chains: aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containing sidechains (S, T, Y); sulfur atom-containing side-chains (C, M); carboxylic acid- and amide-containing side-chains (D, N, E, Q); base-containing side-chains (R, K, H); and aromatic-containing side-chains (H, F, Y, W). The letters within parenthesis indicate the one-letter amino acid codes. Amino acid substitutions within each group are called conservative substitutions. It is well known that a polypeptide comprising a modified amino acid sequence in which one or more amino acid residues is deleted, added, and / or substituted can retain the original biological activity (Mark D. F. et al., Proc. Natl. Acad. Sci. U. S. A. 81:5662-5666 (1984); Zoller M. J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. et al., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl. Acad. Sci. U. S. A. 79: 6409-6413 (1982)). The number of mutated amino acids is not limited, but in general, the number falls within 40% of amino acids, and specifically within 35%, and still more specifically within 30% (e.g., within 25%). The identity of amino acid sequences can be determined as described herein. In certain embodiments, one or more amino acid residue is mutated into one that is a non-conservative substitution.

[0195] The polypeptides obtained can be purified to homogeneity. The polypeptides can be isolated and purified by a method routinely used to isolate and purify proteins. The polypeptides can be isolated and purified by the combined use of one or more methods appropriately selected from column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectro-focusing, for example (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Such methods are not limited to those listed above. Chromatographic methods include affinity chromatography (e.g., metal affinity chromatography), ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography. These chromatographic methods can be practiced using liquid phase chromatography, such as HPLC and FPLC. The polypeptides can also be purified by utilizing target binding, using carriers on which targets have been immobilized. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0196] The polypeptides of the present invention can be formulated according to standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U. S. A), and may comprise pharmaceutically acceptable carriers and / or additives. The present invention relates to compositions (including reagents and pharmaceuticals) comprising the polypeptides of the invention, and pharmaceutically acceptable carriers and / or additives. Other exemplary carriers include surfactants (for example, PEG and Tween), excipients, antioxidants (for example, ascorbic acid), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (for example, phosphoric acid, citric acid, and other organic acids), chelating agents (for example, EDTA), suspending agents, isotonizing agents, binders, disintegrators, lubricants, fluidity promoters, and corrigents. However, the carriers that may be employed in the present invention are not limited to this list. In fact, other commonly used carriers can be appropriately employed: light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salt, and so on. The composition may also comprise other low-molecular-weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine, and lysine. When the composition is prepared as an aqueous solution for injection, it can comprise an isotonic solution comprising, for example, physiological saline, dextrose, and other adjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride, which can also contain an appropriate solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol and PEG), and non-ionic detergent (polysorbate 80 and HCO-50).

[0197] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0198] of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

[0199] Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art.

[0200] Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0201] As used herein, "comparison window" makes reference to a contiguous and specified segment of an amino acid or polynucleotide sequence, wherein the sequence in the comparison window may comprise additions or deletions ( / .<., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least about 20 contiguous amino acid residues or nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.

[0202] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0203] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, and at least about 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0204] by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least about 70%, at least about 80%, 90%, or at least about 95%.

[0205] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

[0206] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity or complementarity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0207] The term "amino acid" includes the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L form, as well as unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4, -tetrahydroi soquinoline-3 -carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine,

[0208] para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxy carbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (Ci-Ce)alkyl, phenyl or benzyl ester or amide; or as an a-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Synthesis,' Wiley: New York, 1981, and references cited therein) The term also comprises natural and unnatural amino acids bearing a cyclopropyl side chain or an ethyl side chain. RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0209] The term "nucleic acid" and “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

[0210] " Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0211] exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

[0212] Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0213] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0214] " Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. " Promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. " Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0215] The "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e.

[0216] 31 RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0217] further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0218] Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g, a TATA box and / or an initiator.

[0219] As used herein, the term "operably linked" refers to a linkage of two elements in a functional relationship. For example, “operably linked” may refer to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence ( / .<., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. “Operably-linked” also refers to the association of two chemical moieties so that the function of one is affected by the other, e.g, an arrangement of elements wherein the components so described are configured so as to perform their usual function.

[0220] " Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0221] The term “effective amount” or “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of a polypeptide either alone or as contained in a pharmaceutical composition that produces therapeutic effect or is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0222] The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder, such as lung damage (e.g., lung fibrosis, bronchiectasis, COPD (e.g., RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0223] emphysema and / or bronchitis), lung cancer, and / or alpha 1 antitrypsin (AAT) deficiency). For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized ( / .<., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. " Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0224] As used herein, the terms “for use in combination” may refer to the sequential or simultaneous use of specified agents, wherein the agents may be used separately or in a single composition for simultaneous use.

[0225] As used herein, the term “therapeutic agent” refers to any agent or material that has a beneficial effect on the mammalian recipient.

[0226] The term “mammal” refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like. Accordingly, in certain embodiments, the mammal is a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit or livestock. In certain embodiments, the mammal is a patient (e.g., a human patient). In certain embodiments, the mammal is a pet, such a dog, cat, hamster, guinea pig or rabbit. In certain embodiments, the mammal is a livestock mammal (e.g., a cow, sheep, horse, pig, chicken, etc.).

[0227] " Wild-type" refers to the normal gene, or organism found in nature without any known mutation.

[0228] The invention will now be illustrated by the following non-limiting Example.

[0229] EXAMPLE 1: Carbonic Anhydrase 4 Cooperates with Alpha- 1 Antitrypsin to Inhibit Elastase Activity

[0230] Emphysema, a type of chronic obstructive pulmonary disease (COPD), is characterized by destruction of the tissue architecture of the lung that results in a permanent dilation of the airspaces. The damage associated with emphysema results in a loss of alveolar and capillary surface area that is required for efficient gas exchange. As a result, patients suffering from emphysema present with numerous symptoms including shortness of breath, coughing, RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0231] wheezing, reoccurring infections, fatigue and chest pain (NCBI BOOK NBK482217). The most common causes of emphysema include smoking or chronic exposure to industrial pollutants or dust that promote immune cell activation and the release of proteases such as elastase that can damage lung tissue.

[0232] Under normal conditions, innate immune cells such as neutrophils and macrophages produce elastase with enzymatic activity that promotes protective responses to several pathogens including bacteria and fungi (PMID: 16982831, PMID: 34234752 ). While beneficial in these contexts, dysregulated and unchecked elastase production can also result in emphysema and the debilitating symptoms described above. Specifically, overactive elastase responses promote the breakdown of elastin which is an important part of the extracellular matrix (ECM) that maintains the alveolar structures of the lung. Degradation of the ECM results in direct damage to the alveoli and the initiation of additional tissue remodeling responses that cause emphysema and subepithelial pulmonary fibrosis (PMID: 28848433, PMID: 26741177). Therefore, it is critical that expression of elastase remains tightly regulated to prevent chronic lung disease.

[0233] To prevent dysregulated elastase responses, the protein alpha- 1 antitrypsin (AAT) is produced in the liver where it can enter the blood stream, travel to the lung and inhibit elastase activity (PMID: 32268028). The importance of AAT is evident by individuals that have genetic mutations that result in reduced AAT production and / or transport to the lung. Individuals with these AAT deficiencies are at a higher risk of lung diseases such as emphysema, bronchiectasis, and other forms of COPD (PMID: 27465791). Patients suffering from AAT deficiency are often treated with injections of recombinant AAT to reduce elastase activity and lung damage.

[0234] Additionally, gene therapies are also being investigated as a therapeutic method to restore AAT levels in the context of emphysema (PMID: 27564673).

[0235] Despite the advances described above, treatments for emphysema and other forms of COPD remain limited and no cure is known. Patients suffering from chronic lung diseases are often treated with bronchodilators, inhaled steroids, supplemental oxygen, antibiotics, surgery, and in severe cases lung transplants. Given that chronic lung diseases represent the 4thleading cause of death worldwide, better treatments options are urgently needed.

[0236] Carbonic anhydrase (Car) enzymes are a family of metalloenzymes that are known to regulate pH and carbon dioxide homeostasis (PMID: 37108175). Despite their well described enzymatic activity, emerging data demonstrates that Car isozymes have diverse expression patterns and unique functions that regulate the development of immunity and inflammation RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0237] (PMID: 27526715, PMID: 32421753). Carbonic anhydrase 4 is a membrane bound isozyme that is known to be expressed by alveolar macrophages and alveolar capillaries in the lung (PMID: 36103853, PMID: 35511435). However, a further understanding of how Car4 operates in the lung is needed.

[0238] Methods and Results

[0239] To better understand the functions of Car4, we sought to determine whether it interacted with any binding partners. To determine potential binding partners of Car4, we utilized recombinant mouse rCar4 (R& D Systems, 2414-CA-010) with a lOx His tag in combination with Ni-NTA column (ThermoScientific, Cat# 88224). Briefly, alveolar macrophage native whole cell extract was incubated with or without lOng / ul rCar4 for two hours at 37C. The lysates were then resuspended in equilibration buffer and incubated in pre-equilibrated Ni-NTA column in an end-over-end mixer at 4C for one hour. After washing, elutions were collected and combined. Elute containing equal volumes of protein were combined with Lamelli loading buffer, reduced and denatured by boiling at 95C for ten minutes. Equal amounts of protein were loaded onto a 4-20% Tris-glycine gel and run at 90V for 3 hours. Whole lanes were excised and evaluated via Liquid chromatography-mass spectrometry (LC-MS / MS) (Table 1). The resulting peptides were analyzed by LC-MS / MS on Orbitrap Fusion Lumos MS instrument using the DIA method. MS / MS spectra were searched against Uniprot mouse database using Spectronaut software. The protein false discovery rate was less than 1%. The protein relative quantitation is calculated based on the protein group quantity of the two samples. The results are listed in the Table 1.

[0240] Table 1. Comparison of the identified proteins and their relative quantitation based on Data Independent Acquisition (DIA) (sorted by Ratio)

[0241] PG. Protein Descriptions PG. Genes Relative abundance

[0242] (rCar4 treated / untreated) Carbonic anhydrase 4 Ca4 33997

[0243] Alpha- 1 -antitrypsin 1-6 Serpinalf 21042

[0244] Fibrinogen alpha chain Fga 23473

[0245]

[0246] Fibrinogen gamma chain Fgg _ 8172

[0247] These data indicate that Car4 binds to alpha- 1 -antitrypsin (AAT) (Table 1). As described above, ATT is a protease inhibitor that is primarily produced by the liver and is known to protect the lung from proteolytic damage caused by molecules such as elastase (PMID 27465791). RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0248] These data also demonstrate that Car4 was bound to fibrinogen, which is also a known binder of ATT (Table 1) (PMID 35269582). These studies strongly suggest that membrane bound Car4 may protect the lung by binding soluble AAT from the circulation and thereby enhancing its presence in the alveolar space where it can best protect from proteolytic cleavage. These data also suggest that Car4 may operate in a similar manner as AAT and / or enhancing the elastase-inhibitory functions of ATT in the lung.

[0249] To first test whether Car4 operates in a similar manner as AAT and is sufficient to inhibit elastase activity we employed a colorimetric elastase kit (Milipore Sigma MAK246-1KT) and treated the substrate with elastase alone, elastase plus AAT, or elastase plus Car4. The substrate provided is an elastin conjugate that is highly labeled so that the fluorescence signal is quenched until enzymatic digestion yields highly fluorescent fragments that can be detected at 405nm. As expected, treatment of substrate with 1 enzyme unit (U) of elastase resulted in the rapid breakdown of elastin conjugate and an increased 405nm signal over time (Fig. 1). Consistent with its known ability to inhibit elastase, treatment with 1.5ug of ATT resulted in a decreased breakdown of elastin coagulate as illustrated by a reduced 405nm signal over time (Fig. 1). Additionally, treatment with 1.5ug of Car4 also resulted in a decreased breakdown of elastin coagulate as illustrated by a reduced 405nm signal over time (Fig. 1). These data are the first to demonstrate the elastase inhibitory abilities of Car4 and illustrates its therapeutic potential to prevent chronic lung disease.

[0250] Next, we sought to determine whether Car4 can cooperate with and enhance the inhibitory functions of AAT. In this assay we sought to evaluate whether treatment with equal amounts of Car4 and AAT inhibit elastase better than the same total amount of AAT alone. Surprisingly, treatment with 0.75ug of Car4 and 0.75ug of ATT showed a stronger ability to inhibit elastase than 1.5ug of AAT alone as indicated by a reduced 405nm signal (Fig. 2) and statistically confirmed by analyzing the area under the cure (Fig. 3). These studies demonstrate that Car4 can directly inhibit elastase activation and can act cooperatively with AAT to inhibit elastase more effectively than AAT alone.

[0251] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be RU Docket #T2024-142 VHPM Docket #08035.150WO1

[0252] understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

RU Docket #T2024-142 VHPM Docket #08035.150WO1CLAIMSWhat is claimed is:

1. A method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

2. The method of claim 1, wherein the lung damage is caused by chronic obstructive pulmonary disease (COPD).

3. The method of claim 1 or 2, wherein the lung damage is caused by emphysema, lung fibrosis and / or bronchiectasis.

4. The method of claim 3, wherein the lung damage is caused by lung fibrosis and / or emphysema, and wherein the lung fibrosis and / or emphysema is M2 macrophage-mediated.

5. The method of claim 3 or 4, wherein the lung damage is caused by lung fibrosis, and wherein the lung fibrosis is idiopathic pulmonary fibrosis (IPF).

6. The method of claim 1, wherein the lung damage is caused by COVID-19, Mycobacterium tuberculosis, fungal infections (e.g., Aspergillus fumigatus), a parasite infection, asthma, or inhalation of noninfectious agent(s) (e.g., a toxin, an irritant and / or particulate matter).

7. The method of claim 1, wherein the lung damage is caused by lung cancer.

8. The method of claim 1, wherein the lung damage is caused by an AAT deficiency.

9. A method for treating lung damage comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapyRU Docket #T2024-142 VHPM Docket #08035.150WO1comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the lung damage is not caused by lung fibrosis and / or emphysema.

10. A method for treating lung cancer comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

11. A method for treating an alpha 1 antitrypsin (AAT) deficiency comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

12. A method for treating bronchiectasis comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

13. The method of any one of claims 10-12, further comprising administering an effective amount of an AAT therapy to the mammal, wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

14. A method for treating lung fibrosis, emphysema, and / or COPD comprising administering to a mammal in need thereof an effective amount of a carbonic anhydrase 4 (Car4) therapy in combination with an effective amount of an alpha 1 antitrypsin (AAT) therapy, wherein the Car4 therapy comprises at least one of a Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.RU Docket #T2024-142 VHPM Docket #08035.150WO115. The method of any one of claims 1-14, wherein the Car4 therapy comprises the Car4 enzyme.

16. The method of claim 15, wherein the Car4 enzyme comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 1 to 3.

17. The method of any one of claims 1-16, wherein the Car4 therapy comprises a nucleic acid encoding a Car4 enzyme.

18. The method of claim 17, wherein the nucleic acid encoding a Car4 enzyme is comprised within an expression cassette comprising a promoter, and wherein the nucleic acid is operably linked to the promoter.

19. The method of claim 18, wherein the expression cassette is comprised within a vector (e.g., a viral vector).

20. The method of any one of claims 17-19, wherein the nucleic acid encoding a Car4 enzyme comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NOs: 4 or 5.

21. The method of any one of claims 1-20, wherein the Car4 therapy comprises the Car4 enzyme activator.

22. The method of claim 21, wherein the Car4 enzyme activator comprises at least one of L-phenylalanine, D-phenylalanine, L-histidine, D-histidine, or a pharmaceutically acceptable salt thereof.

23. The method of any one of claims 1-8 and 13-22, wherein the AAT therapy comprises an AAT protein.RU Docket #T2024-142 VHPM Docket #08035.150WO124. The method of claim 23, wherein the AAT protein comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs:6, 7, 8 or 10.

25. The method of any one of claims 1-24, wherein the AAT therapy comprises a nucleic acid encoding an AAT protein.

26. The method of claim 25, wherein the nucleic acid encoding an AAT protein is comprised within an expression cassette comprising a promoter, and wherein the nucleic acid is operably linked to the promoter.

27. The method of claim 26, wherein the expression cassette is comprised within a vector (e.g., a viral vector).

28. The method of any one of claims 25-27, wherein the nucleic acid encoding an AAT protein comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO:9.

29. The method of any one of claims 1-28, further comprising administering one or more additional therapeutic agents or therapies to the mammal.

30. The method of claim 29, wherein the one or more additional therapeutic agents or therapies is an anti-inflammatory agent, a corticosteroid, an anticholinergic, a beta2 -adrenergic agonist, and / or an agent useful for treating fibrosis (e.g., Nintedanib or Pirfenidone).

31. The method of claim 29, wherein the one or more additional therapeutic agents or therapies is an anti-cancer therapeutic agent or therapy.

32. The method of claim 31, wherein the anti -cancer therapeutic agent or therapy comprises at least one of cislatin, carboplatin, pemetrexed, paclitaxel, docetaxel, gemcitabine, vinorelbine, Nivolumab, Pembrolizumab, Atezolizumab, and Durvalumab.RU Docket #T2024-142 VHPM Docket #08035.150WO133. The method of any one of claims 1-32, wherein the Car4 therapy is comprised in a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.

34. The method of any one of claims 1-8 and 13-32, wherein the AAT therapy is comprised in a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.

35. The method of any one of claims 1-8 and 13-32, wherein the Car4 therapy and the AAT therapy are comprised in a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.

36. The method of any one of claims 1-35, wherein the Car4 therapy and / or AAT therapy is administered intratracheally, by inhalation, by nebulization, nasally, intravenously, or orally.

37. A pharmaceutical composition comprising a Car4 therapy, an AAT therapy, and a pharmaceutically acceptable carrier, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

38. A kit comprising a Car4 therapy, an AAT therapy, packaging material, and instructions for administering the Car4 therapy and the AAT therapy to a mammal in need thereof to treat lung damage, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

39. A Car4 therapy and an AAT therapy for use in combination in the treatment of lung damage, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.RU Docket #T2024-142 VHPM Docket #08035.150WO140. The use of a Car4 therapy to prepare a medicament useful for treating lung damage in combination with an AAT therapy in a mammal in need thereof, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator; and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

41. The use of an AAT therapy to prepare a medicament useful for treating lung damage in combination with an Car4 therapy in a mammal in need thereof, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator; and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

42. A kit comprising a Car4 therapy, an AAT therapy, packaging material, and instructions for administering the Car4 therapy and the AAT therapy to a mammal in need thereof to treat lung fibrosis, emphysema, COPD, bronchiectasis, lung cancer, and / or AAT deficiency, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

43. A Car4 therapy and an AAT therapy for use in combination in the treatment of lung fibrosis, emphysema, COPD, bronchiectasis, lung cancer, and / or AAT deficiency, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator, and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

44. The use of a Car4 therapy to prepare a medicament useful for treating lung fibrosis, emphysema, COPD, bronchiectasis, lung cancer, and / or AAT deficiency in combination with an AAT therapy in a mammal in need thereof, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator; and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.RU Docket #T2024-142 VHPM Docket #08035.150WO145. The use of an AAT therapy to prepare a medicament useful for treating lung fibrosis, emphysema, COPD, bronchiectasis, lung cancer, and / or AAT deficiency in combination with an Car4 therapy in a mammal in need thereof, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator; and wherein the AAT therapy comprises at least one of an AAT protein or a nucleic acid encoding an AAT protein.

46. A kit comprising a Car4 therapy, packaging material, and instructions for administering the Car4 therapy to a mammal in need thereof to treat lung damage, wherein the lung damage is not caused by lung fibrosis and / or emphysema, and wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

47. A Car4 therapy for use in the treatment of lung damage, wherein the lung damage is not caused by lung fibrosis and / or emphysema, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

48. The use of a Car4 therapy to prepare a medicament for the treatment of lung damage in a mammal in need thereof, wherein the lung damage is not caused by lung fibrosis and / or emphysema, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

49. A kit comprising a Car4 therapy, packaging material, and instructions for administering the Car4 therapy to a mammal in need thereof to treat lung cancer, an AAT deficiency, or bronchiectasis, wherein the Car4 therapy comprises at least one of a carbonic anhydrase 4 (Car4) enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

50. A Car4 therapy for use in the treatment of lung cancer, an AAT deficiency, or bronchiectasis, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.RU Docket #T2024-142 VHPM Docket #08035.150WO151. The use of a Car4 therapy to prepare a medicament for the treatment of lung cancer, an AAT deficiency, or bronchiectasis in a mammal in need thereof, wherein the Car4 therapy comprises at least one of Car4 enzyme, a nucleic acid encoding a Car4 enzyme, or a Car4 enzyme activator.

52. The method, composition, or use of any one of claims 1-51, wherein the mammal is human.

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