WO2023225619A2 - Delivery of focal adhesion kinase for treating vascular and/or capillary tissue injury - Google Patents

Delivery of focal adhesion kinase for treating vascular and/or capillary tissue injury Download PDF

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Publication number
WO2023225619A2
WO2023225619A2 PCT/US2023/067201 US2023067201W WO2023225619A2 WO 2023225619 A2 WO2023225619 A2 WO 2023225619A2 US 2023067201 W US2023067201 W US 2023067201W WO 2023225619 A2 WO2023225619 A2 WO 2023225619A2
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vector
fak
lung
construct
cells
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PCT/US2023/067201
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French (fr)
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WO2023225619A3 (en
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Sherwood Russ Lehrman
James P. BRIDGES
Elizabeth F. REDENTE
Ehud Goldin
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Biosuperior Technology, Inc.
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Publication of WO2023225619A2 publication Critical patent/WO2023225619A2/en
Publication of WO2023225619A3 publication Critical patent/WO2023225619A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the subject matter described herein relates to delivery of Focal Adhesion Kinase (FAK) or a biologically active fragment, derivative, or variant thereof to treat vascular and/or capillary cell injury.
  • FAK Focal Adhesion Kinase
  • Focal adhesion kinase is a multifunctional integrin-associated protein tyrosine kinase.
  • FAK is encoded by the PTK2 gene. When activated via auto- phosphorylation at Tyr- 397, FAK localizes to focal adhesion complexes of endothelial cells in association with paxillin, vinculin leading to the extracellular association of integrin and E-cadherin. The interactions of integrin and E-cadherin on neighboring cells promotes tight association. FAK also appears to enhance association of neighboring epithelial cells.
  • FAK plays a key role in the formation of a complex in which actinomyosin stress fibers are part of actin cytoskeleton and regulate cell spreading and migration, proliferation, and cell survival (Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol. 1996;134(3):793-799. doi: 10.1083/jcb. l34.3.793).
  • This enzyme plays a pivotal role in remodeling adherens junctions leading to the resealing of barriers in epithelial and microvascular endothelial tissues in multiple organs (Quadri SK, Bhattacharj ee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelial barrier strengthening by activation of focal adhesion kinase. J Biol Chem. Apr 11, 2003;278(15): 13342-9. doi:10.1074/jbc.M209922200; Schmidt TT, Tauseef M, Yue L, et al. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rael activities.
  • FAK has additional signaling capacities through cell surface receptors and FAK interacting proteins including Src family kinases and phosphoinostitide-3 kinase (PI3K) resulting in the regulation of multiple cellular functions including migration, proliferation and cell survival (Zhao X, Guan J-L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Del Rev. 2011;63(8):610-615. doi: 10.1016/j.addr.2010.11.001; Owen KA, Abshire MY, Tilghman RW, Casanova JE, Bouton AH. FAK Regulates Intestinal Epithelial Cell Survival and Proliferation during Mucosal Wound Healing. PLoS ONE. 201 l;6(8):e23123. doi: 10.1371/joumal. pone.0023123).
  • Acute lung injury or the more severe acute respiratory distress syndrome (ARDS) is reduction of functional breathing due to the degradation of lung tissue as a result of inflammation.
  • ARDS acute respiratory distress syndrome
  • therapies available for treatment (Matthay MA, Zemans RL. The Acute Respiratory Distress Syndrome: Pathogenesis and Treatment. Annual Review of Pathology: Mechanisms of Disease. 2011;6(1): 147-163. doi:10. 1146/annurev- pathol-011110-130158).
  • ALI/ ARDS is defined clinically with the following Berlin criteria: bilateral pulmonary opacities on chest radiographs, acute onset, and arterial hypoxemia (PaCh/FiCh ratio ⁇ 300 mm Hg) 2 , which is an indicator of poor oxygenation, pulmonary infiltrates, and acuity of onset (Matthay MA, Zemans RL, Zimmerman GA, et al. Acute respiratory distress syndrome. Nature Reviews Disease Primers. 2019;5(l)doi: 10.1038/s41572- 019-0069-0).
  • ARDS affects more than 200,000 patients annually in the United States, resulting in about 75,000 deaths (Fan E, Brodie D, Slutsky AS. Acute Respiratory Distress Syndrome. JAMA. 2018;319(7):698. doi: 10.1001/jama.2017.21907; Blank R, Napolitano LM.
  • ALI/ARDS leads to alveolar epithelial cell apoptosis, denudation of the epithelium, and destruction of the underlying basement membranes (Wheaton AK, Agarwal M, Jia S, Kim KK. Lung epithelial cell focal adhesion kinase signaling inhibits lung injury and fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2017;312(5):L722-L730.
  • PA Pseudomonas aeruginosa
  • human activated focal adhesion kinase was modified to include an N-terminal His-tag which indirectly assisted with cellular uptake by forming a complex with Cu 2+ and TAT, a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • a construct comprising a nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof is provided.
  • FAK Focal Adhesion Kinase
  • the construct may be an engineered mRNA encoding the FAK protein, fragment derivative, or variant, or an expression vector encoding the FAK protein, fragment derivative, or variant.
  • the expression vector may be a viral vector.
  • the viral vector may be selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno- associated viral (AAV) vector.
  • the vector may be an AAV vector.
  • the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
  • the nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof may be at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be at least 90% identical to SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 1.
  • FAK Focal Adhesion Kinase
  • the construct may comprise a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 2. In certain embodiments, the construct may comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2. In certain embodiments, the construct may be AAV6.2FF-PTK2 comprising the nucleic acid sequence as set forth in SEQ ID NO: 2
  • composition comprising any of the constructs disclosed above and herein is provided.
  • the pharmaceutical composition may further comprise a reagent capable of assisting delivery of the construct to a recipient cell.
  • a reagent capable of assisting delivery of the construct to a recipient cell.
  • the reagent is polyethylenimine.
  • the pharmaceutical composition may further comprise a stabilizing excipient.
  • the stabilizing excipient may be a pharmaceutically acceptable excipient, including but not limited to, citrate, glycine, phosphate, tris, histidine leucine, raffinose, trehalose, mannitol, lactose and trileucine.
  • the pharmaceutical composition may be in a form of a dry powder or a liquid.
  • the dry powder may be produced by lyophilization or spray-drying.
  • kits comprising any of the constructs or any of the pharmaceutical compositions disclosed above and herein and instructions for use is provided.
  • a method of preventing or repairing an injured vascular and/or capillary tissue in a subject in need thereof may comprise administering to the subject an effective amount of any of the pharmaceutical compositions disclosed above or herein or using the kit disclosed above and herein.
  • the subject may suffer from an injury that involves endothelial cells and/or epithelial cells.
  • the injury may involve endothelial cells found in the capillary tissue.
  • the injury involves epithelial cells that make up alveolar cells in the deep lung.
  • the injury may be a lung injury, a hemorrhagic stroke, an acute or chronic kidney disease, or a capillary leak syndrome.
  • the lung injury may be ALI/ARDS, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, or asthma.
  • COPD chronic obstructive pulmonary disease
  • the lung injury may be caused by SARS-CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury.
  • the lung pathogen(s) include pseudomonas aeruginosa.
  • the internal organ injury includes acute pancreatitis.
  • the pharmaceutical composition may be administered to the subject intranasally, intratracheally, or parenterally.
  • the pharmaceutical composition may be administered via intratracheal or intranasal instillation of a liquid, nebulization of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol.
  • the effective amount of the pharmaceutical composition administered to the subject is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK.
  • a method of preventing or treating ALI/ARDS in a subject in need thereof may comprise administering to the subject an effective amount of any of the pharmaceutical compositions disclosed above or herein or using the kit disclosed above and herein.
  • the lung injury may be caused by SARS- CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury.
  • the lung pathogen(s) include pseudomonas aeruginosa.
  • the internal organ injury includes acute pancreatitis.
  • the pharmaceutical composition may be administered to the subject intranasally, mtratracheally, or parenterally.
  • the pharmaceutical composition may be administered via intratracheal or intranasal instillation of a liquid, nebulization of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol.
  • DPI dry powder inhaler
  • the effective amount of the pharmaceutical composition administered to the subject is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK.
  • a method of treating lung vascular leak in a COVID-19 patient may comprise expressing an intracellular, barrier-enhancing therapeutic agent in the lung endothelium of the patient through a gene delivery tool.
  • the therapeutic agent may be Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof.
  • FAK Focal Adhesion Kinase
  • the gene delivery tool may be an engineered mRNA encoding the FAK protein, fragment derivative, or variant, or an expression vector encoding the FAK protein, fragment derivative, or variant.
  • the expression vector may be a viral vector.
  • the viral vector may be selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno-associated viral (AAV) vector.
  • the vector may be an AAV vector.
  • the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
  • the AAV vector comprises a nucleic acid sequence encoding FAK protein or a biologically active fragment, derivative, or variant thereof.
  • the nucleic acid sequence may be at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1.
  • the nucleic acid sequence may be at least 90% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1.
  • the nucleic acid sequence may be identical to SEQ ID NO: 1.
  • the construct may compnse a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 2.
  • the construct may comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2.
  • the construct may be AAV6.2FF-PTK2 vector comprising the nucleic acid sequence as set forth in SEQ ID NO: 2.
  • FIGS. 1A-1E demonstrate in vivo targeting of mouse alveolar type II (AT2) cells by AAV6.2FF.
  • FIG. 1A shows luciferase expression of AAV6.2FF-luciferrase in lung field of a C57BL/6 mouse by whole body IVIS imaging.
  • FIG. IB shows quantitative longitudinal analysis of AAV6.2FF-luciferase expression in live mice following a single administration of the virus.
  • N 4 mice/group.
  • FIG. 1C shows FACS analysis of wild-type AT2 cells (EpCAM+/MCHII+) and AT2 cells 7 days post-intratracheal administration of AAV6.2FF- GFP.
  • FIG. 1A shows luciferase expression of AAV6.2FF-luciferrase in lung field of a C57BL/6 mouse by whole body IVIS imaging.
  • FIG. IB shows quantitative longitudinal analysis of AAV6.2FF-luciferase expression in live mice following a single administration of the virus.
  • N 4 mice/group.
  • FIG. 1C shows
  • FIGS. 2A-2B demonstrate immunofluorescent staining of GFP (green) and the AT2- specific marker pro-SPC (red) in AAV6.2FF-GFP transduced control lungs and in influenza A virus (1AV) infected/ AAV6.2FF-GFP transduced lungs.
  • FIG. 2A shows abundance of GFP/pro-SPC double positive AT2 cells in AAV6.2-GFP transduced lung.
  • FIG. 3A is a diagram of FAK-FLAG construct for AAV-driven expression in AT2 cells.
  • FIG. 3B illustrates the ability of AAV6.2FF-PTK2 to transfect AT2 cells and produce the FAK protein.
  • FIGS. 4A-4B show dose-dependent weight loss and increased inflammation in acute IAV infection mouse model.
  • FIGS. 5A-5C show increased tissue density in lAV-infected lungs.
  • FIG. 5A shows the images of air-inflated, perfusion-fixed lungs in naive animals or after IAV infection by microCT (9 mm images).
  • FIG. 5B shows widespread tissue thickening and loss of alveolar structure in 3D-reconstructions.
  • FIG. 5C shows increased lung tissue volume (top) and the increased percentage of total lung tissue/total lung volume (bottom).
  • FIGS. 6A-6D show increased permeability of alveolar barrier following acid inhalation injury.
  • FIG. 6A shows the albumin levels in the bronchoalveolar lavage (BAL) fluid.
  • FIG. 6B shows quantification of differential cell counts from the BAL.
  • FIG. 6C shows static lung compliance as measured by flexiVent.
  • FIG. 7 illustrates the cDNA sequence of the hFAK gene (SEQ ID NO: 1).
  • FIG. 8 illustrates the complete nucleic acid sequence of the AAV6.2FF-PTK2 construct (SEQ ID NO: 2).
  • FIGS. 9A-9C illustrate AAV6.2FF -driven FAK expression in vitro and in vivo.
  • FIG. 9B shows anti-FLAG immunohistochemistry (arrows show FAK-FLAG in pro-SPC+ AT2 cells).
  • FIG. 9C shows flow cytometry of lung AT2 cells after 1 wk from non-transduced or transduced mice with AAV6.2FF-FAK-FLAG virions.
  • FIGS. 10A-10D illustrate similar immune response to LPS challenge in AAV6.2FF- FAK and AAV6.2FF-GFP mice.
  • FIG. 10A shows total cell counts in BAL fluid of mice transduced with AAV6.2FF-GFP or AAV6.2FF-FAK (2xl0 12 vg, intranasally) on Day 0 followed by LPS (50 pg, intratracheal) challenge on Day 14. Mice were harvested on Day 17, 3 days post LPS challenge.
  • FIGS. 10B-10D show differential cell counts in BAL fluid of the AAV6.2FF-FAK and the AAV6.2FF-GFP mice.
  • FIG. 10B neutrophils.
  • FIG. 10C macrophages.
  • FIG. 10D lymphocytes.
  • FIGS. 11A-11B illustrate AAV6.2FF-GFP expression in vivo following LPS challenge.
  • FIG. 11A representative FACS histogram plots showing GFP expression in lung cell types 10 days after LPS challenge.
  • FIG. 1 IB is a graph showing frequency of EpCAM+/MHCII+ AT2 cells that are GFP+ in AAV -GFP treated mice (AAV -GFP) or AAV-GFP treated mice dosed 3 days post-LPS (LPS + AAV-GFP).
  • the word “about” when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation.
  • “about 49, about 50, about 55” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.
  • compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.
  • FAM Focal adhesion kinase
  • Activated FAK means the phosphorylated form of FAK that is referred to herein as “FAKp”, including any biologically active fragment, modification, derivative, or variant thereof.
  • biologically active fragment means a fragment of a protein that retains the biological activity of the protein or that exhibits a similar, but not necessarily identical, activity to the protein, preferably FAK or FAKp.
  • the biological activity of the fragment may include an improved desired activity such as enhancing barrier function and decreasing undesirable activity.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, salts, compositions, dosage forms, etc., which are-within the scope of sound medical judgment— suitable for use in contact with the tissues of human beings and/or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly, in humans.
  • treating is used herein, for instance, in reference to methods of treating capillary tissue injury and/or vascular tissue injury, and generally includes the administration of a compound or composition which repairs damaged epithelial tissue in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition (e.g., strengthen epithelial barrier function to protect and/or accelerate alveolar repair following acute lung injury).
  • a therapeutically effective amount of a protein or polypeptide is an amount that achieves the desired therapeutic result.
  • a therapeutically effective amount is an amount that ameliorates one or more symptoms of the disease, including ALI or ARDS, or that decreases lung endothelial cell (EC) permeability, i.e., increases the EC barrier in an animal, preferably a human, has or is at nsk of developing ALI or ARDS.
  • EC lung endothelial cell
  • a meaningful change to inform a treatment decision may be a change from baseline, a change from matched controls, a change from a reference value from a healthy individual, a change from a non-mvolved area or some other change.
  • the change may be more or less than 1%, 10%, 25% 50%, 75%, 90% or 99%.
  • the change may be based on an average, a standard deviation, a cutoff, an interquartile range, or some other meaningful change.
  • the changes may be based on meaningful changes in a database, a clinical trial, previous experiments, or intra-experimental controls. The changes may be based on high or low values. The changes may or may not be statistically significant.
  • the present disclosure relates a gene delivery tool to deliver FAK protein, a biologically active fragment, derivative, or variant thereof, to a subject in need thereof, e.g., to prevent or treat vascular and/or capillary tissue injury, to prevent or repair an injured vascular and/or capillary tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a COVID-19 patient, etc.
  • a construct comprising a nucleic acid sequence encoding FAK protein or a biologically active fragment, denvative, or variant thereof.
  • the construct may be an engineered mRNA encoding the FAK protein, fragment derivative or variant.
  • An engineered mRNA may be designed to address infrastructural barrier to clinical and translational research in gene therapy. In the wake of COVID- 19 pandemic, mRNA technology has played an important role in developing vaccines to combat the virus. It is anticipated that an engineered mRNA could be an effective tool to deliver FAK to injured capillary tissues and/or injured vascular tissues for repair.
  • the construct may be an expression vector encoding the FAK protein, fragment derivative, or variant.
  • the expression vector may be a viral vector such as a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, or an adeno-associated viral (AAV) vector.
  • the vector may be an AAV vector.
  • the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
  • the nucleic acid sequence encoding the FAK protein, fragment derivative, or variant may be naked DNA or cDNA sequence.
  • the nucleic acid sequence is at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1 (human FAK cDNA sequence; NCBI reference sequence NM_153831.4; see, FIG. 7).
  • the nucleic acid sequence may be 90% identical to SEQ ID NO: 1.
  • the nucleic acid sequence may be 100% identical to SEQ ID NO: 1.
  • AAV gene delivery for therapeutic applications has increased, largely due to its non-pathogenicity to humans, alterations of recombinant AAV preparations that prevent integration into the genome, and minimal host immune response to the vector.
  • LUXTURNA® is the first AAV gene delivery product to be FDA approved for Leber congenital amaurosis, a genetic retinal disease.
  • 11 of 30 AAV capsid therapeutic candidates that were approved to proceed into clinical trials have reached the stage of new drug application (ND A), close to 4 times greater than standard success rates (Kuzmin DA, Shutova MV, Johnston NR, et al.
  • AAV vector that expresses full length human FAK is to be generated.
  • AAV6 is an AAV serotype that has good specificity for lung tissue.
  • AAV6.2FF is a modified AAV serotype that is even more specific for lung tissue.
  • AAV6.2FF virus encoding aN-terminal FLAG-tagged version of human FAK cDNA (NCBI reference sequence NM_153831.4; SEQ ID NO: 1; FIG. 7) is constructed as described in Example 2 below.
  • Diagram of the AAV6.2FF-PTK2 vector is shown in FIG. 3A.
  • this vector may comprise the nucleic acid sequence as set forth in SEQ ID NO: 2 (FIG. 8). Such vector is designed for growth in adherent cells.
  • the nucleic acid sequence encoding FAK may be optimized for production in suspended HEK293 cells.
  • the AAV6.2FF-PTK2 vector includes the FLAG®-tag at the N- terminus, which may be removed when constructing a modified AAV-FAK vector for clinical trials.
  • the FLAG® tag also known as the DYKDDDDK-tag (SEQ ID NO: 9), is a popular protein tag that is commonly used in affinity chromatography and protein research.
  • the nucleic acid sequence of the AAV6.2FF-PTK2 vector as shown in FIG. 8 contains 3 x FLAG-tags in a tandem formation right next to each other (DYKDHDGDYKDHDIDYKDDDDK; SEQ ID NO: 10).
  • the FLAG®-tag is used for protein purification from mammalian expression systems or general immunostaining and immunoprecipitation assays.
  • it is desirable to remove the FLAG®-tag once the purification of protein is complete because the tag may interfere with the biophysical and biochemical properties of the purified protein.
  • tags can cause undesired changes in protein structure and function and further cause adverse immunological events which can be toxic.
  • the FLAG®-tag may be removed from the AAV6.2FF-PTK2 vector.
  • a protease cleavage site needs to be engineered between the tag and the protein.
  • the sequence that codes for FLAG® can be removed from the cDNA.
  • compositions for use in accordance with the present disclosure may be formulated in the conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or parenteral administration.
  • the compounds for use are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • FAK In the case of delivery of FAK, it may be done either by nebulization of aqueous droplets using a CP AP -type device or by a dry powder inhaler (DPI) using a mask. Masks are needed to avoid dispersal of the treatment to the surrounding environment.
  • a physician may deliver FAK by intratracheal instillation of an aqueous suspension of the virions. While unacceptable for ambulatory care of asthmatics, this mode of delivery ensures that the virions are not dispersed to the environment and is more accepted for the treated of acute illness such as ALI/ARDS. It is expected that the patient would be under anesthetics at the time of treatment.
  • Capsules and cartridges of gelatin e.g., for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a pharmaceutical composition may comprise any one of the constructs described above and herein, and optionally together with a reagent capable of assisting delivery' of the construct to a recipient cell.
  • the reagent is polyethylenimine.
  • a pharmaceutical composition may comprise any one of the constructs described above and herein, and optionally together with a stabilizing excipient or at least one earner.
  • excipient or “carrier” includes solutions, emulsions, suspensions, gels, sols, colloids, and solids, designed for delivery of the aforementioned agents to capillary tissues and/or vascular tissues, e.g., alveolar epithelial Type II (AT2) cells.
  • solution refers to a liquid mixture in which the minor component e.g., the AAV6.2FF-PTK2 construct) is uniformly distributed within the major component (e.g., buffer).
  • Embodisions refer to a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible (e.g., oil and water).
  • Small refer to solid jelly-like material that can have properties ranging from soft and weak to hard and tough and are defined as a substantially dilute cross-linked system, which exhibits no flow.
  • Sols refer to colloidal suspensions of very small solid particles in a continuous liquid medium.
  • the term “colloid” may be used interchangeably with the terms “gel,” “sol,” and “suspension” and refers to homogeneous mixtures of ultramicroscopic particles of one substance dispersed through a second substance.
  • the carrier is a liquid.
  • the liquid carrier may include an excipient suitable for application to the capillary and/or vascular tissues.
  • Suitable carriers and/or excipients include aqueous or non-aqueous diluents or combination thereof.
  • the excipient may be a pharmaceutically acceptable excipient, including but not limited to, citrate, glycine, phosphate, tns, histidine leucine, raffinose, trehalose, mannitol, lactose and trileucine.
  • aqueous carriers and/or excipients include, but are not limited to, saline, water, dextrose or combinations thereof.
  • Non-aqueous carriers and/or excipients include, but are not limited to, alcohols, particularly polyhydroxy alcohols such as propylene glycol, polyethylene glycol, glycerol, and vegetable and mineral oils. These aqueous and/or non-aqueous carriers and/or excipients can be added in various concentrations and combinations to form solutions, suspensions, oil-in-water emulsions or water-in-oil emulsions.
  • the carrier and/or excipient is a polar solvent material selected from the group consisting of C3-C4 diols, C3-C6 triols, and mixtures thereof, and/or a polar lipid material selected from the group consisting of fatty alcohol esters, fatty acid esters.
  • a mixture of the polar solvent material and the lipid material for example, in a weight ratio of solvent material to the lipid material of about 60:40 to about 99: 1, may also be used.
  • Other suitable carriers are provided in U.S. Pat. No. 5,026,556 (Drust etal.
  • kits comprising any of the constructs or any of the pharmaceutical compositions disclosed above and herein, optionally together with instructions for administering the construct or pharmaceutical composition are described herein.
  • the components of the kit e.g., the construct containing the active agent and the carrier, optionally together with other ingredients, e.g., gelling agents, emollients, surfactants, humectants, viscosity enhancers, emulsifiers, etc., in one or more compartments.
  • the kits may optionally comprise instructions for using the components, either individually or together, to practice the various prophylactic and/or therapeutic applications described below.
  • kits may comprise single or multi-dose amounts of the active agents.
  • the kit comprises a minimal number of doses as the treatment is intended to be an acute treatment. It is not well-suited to chronic treatment of an ambulatory individual. As such, “take home” kits with drug administration by the patient is unlikely in this case.
  • the kit can be packaged and shipped.
  • the compounds disclosed above and herein may be formulated and administered to a subject in need thereof, e.g., to prevent or treat capillary tissue injury, to prevent or repair injured vascular tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a CO VID-19 patient, etc.
  • the administration produces contact of the active ingredient with the site of action in the body of the subject, e.g., the lungs.
  • Any injury that involves vascular and/or capillary tissues may be treated with the methods disclosed herein.
  • the injury may be a lung injury, a hemorrhagic stroke, an acute or chronic kidney disease, or a capillar ⁇ ' leak sy ndrome.
  • the lung injury may be ALI or ARDS, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, or asthma.
  • COPD chronic obstructive pulmonary disease
  • the lung injury could be caused by SARS-CoV-2, lung pathogens (e.g., pseudomonas aeruginosa), inhalation of excess smoke or water, or internal organ injury (e.g., acute pancreatitis).
  • a therapeutically effective amount of the drug FAK (expressed and delivered via an AAV vector) is administered to a patient suffering from ALI, ARDS or other disease related to reduced E-cadherin at EC junctions or decreased EC barrier function.
  • the route of administration can be any route that delivers the therapeutic agent to the intended target, e.g., by inhalation or injection.
  • FAK expressed and delivered via an AAV vector is administered with a selected pharmaceutical carrier based on the chosen route of administration and standard pharmaceutical practice.
  • the pharmaceutical composition is administered intranasally or intratracheally.
  • the pharmaceutical composition is administered via intratracheal or intranasal instillation of a liquid, nebuhzation of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol.
  • DPI dry powder inhaler
  • a method of treating lung vascular leak in a COVID-19 patient may comprise expressing an intracellular, barrier-enhancing therapeutic agent in the lung endothelium of the patient through a gene delivery tool.
  • the therapeutic agent is Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative or variant thereof.
  • the gene delivery tool may be an engineered mRNA or an expression vector as disclosed above and herein.
  • the pharmaceutical composition disclosed above and herein may be delivered via various routes and to various sites in an animal body to achieve a particular effect, e.g., repair injured capillary and/or vascular tissues, strengthen epithelial barrier function to protect and/or accelerate alveolar repair following acute lung injury.
  • a particular route can provide a more immediate and more effective reaction than another route depending on the pathological conditions to be treated.
  • Local or sy stemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation, or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • the dosage administered is a therapeutically effective amount of the compound sufficient to result in amelioration of one or more symptoms of the ARDS or ALI or other disease as described herein, and varies depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
  • Treatment of a subject with a therapeutically effective amount of FAK can include a single treatment or, preferably a series of treatments. Previously 500 micrograms/kg (or 0.5 mg/kg) isolated and purified FAKp was used in the in vivo experiments, which offers a good starting place for determining the ideal dose in humans or other mammals.
  • the effective amount of the pharmaceutical composition is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK, as needed to normalize or maintain lung function.
  • the therapeutic embodiments are carried out by administering the kits to a subject, e.g., a patient suffering from vascular and/or capillary tissue injury.
  • a subject e.g., a patient suffering from vascular and/or capillary tissue injury.
  • administering means applying as a remedy, such as by the placement of a drug in a manner in which such drug would be received, e.g., intranasally or intratracheally, and be effective in carrying out its intended purpose.
  • the drug may be administered systemically (e g., parenteral administration).
  • a “subject” or “patient” in whom administration of the therapeutic compound is an effective therapeutic regimen for a disease or disorder is preferably a human, but can be any animal, including a laboratory animal in the context of a trial or screening or activity experiment.
  • the methods, compounds and compositions are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, humans, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, non-human primates (e.g.
  • the “subject” is a human patient afflicted with a disease to be treated, e.g., AL1 or ARDS.
  • the delivery' tool delivers a therapeutically effective amount of the active agent to the subject.
  • therapeutically effective amount refers to the amount of an active agent that is non-toxic but sufficient to provide the desired therapeutic effect.
  • the amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like as known to those skilled in the art.
  • Treatment of a subject with the pharmaceutical composition or the kit disclosed above and herein may be monitored using methods known in the art. See, e.g., Schmidt et al., Trials, 18:116, 2017 (PMID: 28274276).
  • the efficacy of treatment using the combination is preferably evaluated by examining the subject’s symptoms in a quantitative way, e.g., by noting a decrease in the frequency of adverse symptoms, behaviors, or attacks, or an increase in the time for sustained worsening of symptoms.
  • the subject’s status w ill have improved (i.e., frequency of relapses will have decreased or the time to sustained progression will have increased).
  • the pharmaceutical composition or the kit disclosed above and herein are used to treat various pathological conditions that are associated with capillary cell injury, e.g., to prevent or repair an injured vascular and/or capillary tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a COVID- 19 patient.
  • the term “treating” is used herein generally includes the administration of the compound or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition (e.g., ALI or ARDS) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition (e.g., repair damaged lung epithelial tissue in the alveoli).
  • the pharmaceutical composition may be administered with other pharmaceutically active materials for combination therapy, e.g., anti-inflammatory agents (e.g., dexamethasone, budesonide, formoterol, aspirin, acetaminophen and ibuprofen, or a combination thereof), or anti-infectives (e.g., paxlovid, molnupiravir, remdesivir, (l-lactam antibiotics, fluoroquinoline antibiotics, tetracyclines, aminoglycosides, or a combination thereof).
  • anti-inflammatory agents e.g., dexamethasone, budesonide, formoterol, aspirin, acetaminophen and ibuprofen, or a combination thereof
  • anti-infectives e.g., paxlovid, molnupiravir, remdesivir, (l-lactam antibiotics, fluoroquinoline antibiotics, tetra
  • intranasal (i.n.) administration was utilized to transfect C57BL/6 mice with AAV6.2FF capsids encoding genes that express luciferase, mCherry, GFP, or Cre recombinase.
  • Albino C57BL/6 mouse received 1 x io 11 viral genomes (vg) by i.n. delivery of AAV6.2FF capsids.
  • Luciferin was administered via intraperitoneal (z.p.) injection to mice 1 hour prior to imaging. Luciferase expression in lung field by whole body IVIS imaging 1 day post transduction is shown in FIG. 1A.
  • Luciferin was administered via i.p. injection to mice 1 hour prior to imaging at each time point.
  • the results of the quantitative longitudinal analysis of AAV6.2FF-luciferase expression in live mice following a single administration of virus are shown in FIG. IB.
  • FIG. 2A persistence of AAV6.2FF-GFP signal in pro-SPC positive cells of IAV+AAV6.2FF- GFP lung
  • FIG. 2B persistence of AAV6.2FF-GFP signal in pro-SPC positive cells of IAV+AAV6.2FF- GFP lung
  • AAV6.2FF virus encoding aN-terminal FLAG-tagged version of human FAK cDNA (NCBI reference sequence NM_153831.4; SEQ ID NO: 1) is to be amplified from human AT2 cells via PCR, sub-cloned into the pGEM®-T Easy shuttle vector (Promega), and sequenced. The sequence is to be confirmed afterwards.
  • Diagram of a FAK-FLAG construct for AAV- driven expression in AT2 cells is shown in FIG. 3A. In this construct, human FAK cDNA is tagged with FLAG in frame atN-terminus. Known phosphorylation sites and associated interacting proteins are also shown in FIG. 3A.
  • FIG. 3B demonstrates the ability of AAV6.2FF-PTK2 to transfect AT2 cells and produce the FAK protein, wherein expression of FAK-FLAG protein driven from the pCASI AAV vector in transiently transfected HEK293 cells is shown.
  • FAK-transfected cells stained with anti-FLAG antibody are shown as pointed by the arrows, non-transfected cells are designated with asterisks (*).
  • Enrichment of FAK-FLAG subtending the plasma membranes is noted in this transformed cell line that lacks bona fide focal adhesion complexes.
  • hAT2 primary human AT2
  • Human AT2 cells are to be isolated by elastase digestion and panning on IgG plates as previously described (Wang J, Edeen K, Manzer R, et al. Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro. American Journal of Respiratory Cell and Molecular Biology. 2007;36(6):661-668. doi: 10.1165/rcmb.2006-041 Ooc; Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010; 120(5): 1736-1748. doi: 10.
  • AAV6.2FF-PTK2 virus is to be generated by co-transfection of the pCASI FAK-FLAG WPRE plasmid with the AAV6.2FF packaging plasmid into HEK293 cells as previously described (Rindler TN, Brown KM, Stockman CA, et al. Efficient Transduction of Alveolar Type 2 Cells with Adeno- associated Virus for the Study of Lung Regeneration. American Journal of Respiratory Cell and Molecular Biology. 2021;65(l):l 18-121. doi:10.1165/rcmb.2021-00491e).
  • AAV6.2FF- PTK2 particles are to be purified, and titers are to be determined via qPCR.
  • all experiments are to be conducted with male and female mice to assess the possibility of sex being a relevant biological variable.
  • Experimental readouts to be performed at each time point include: 1) FACS analysis of enzymatically dispersed lung cells w ith identification of AT2 cells (EpCAM/CD326+ (CD326 (EpCAM) Antibody, APC (17-5791-82). htps://www.thermofisher.com/antibody/product/CD326-EpCAM-Antibody-clone-G8-8- Monoclonal/17-5791-82) MHCII+ (MHC Class II (I-A/I-E) Antibody, Alexa Fluor® 700 (56- 5321-82).
  • Enzymatic lung cell dispersion, multiparameter flow cytometry, and co-localization immunostaining are routine procedures and can be performed from whole mouse lungs by utilizing the left lung for immunostaining and the right lung for FACS analysis (Rindler TN, Brown KM, Stockman CA, et al.
  • mice Efficient Transduction of Alveolar Type 2 Cells with Adeno-associated Virus for the Study of Lung Regeneration. American Journal of Respiratory Cell and Molecular Biology. 2021;65(l):118- 121. doi: 10.1165/rcmb.2021-00491e; Redente EF, Chakraborty S, Sajuthi S, et al. Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. JCI Insight. Dec 8 2020;6(l)doi: 10.1172/jci.insight. l41618). 160 mice are included in this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 4 timepoints x 2 experimental replicates.
  • Readouts for the microCT cohort include measurement of aerated lung volume, tissue volume, Hounsfield Units and, if necessary', hyperplastic lesion counting (Redente EF, Black BP, Backos DS, et al. Persistent, Progressive Pulmonary' Fibrosis and Epithelial Remodeling in Mice. Am J Respir Cell Mol Biol. Jan 6 2021;doi:10.1165/rcmb.2020-0542MA). 40 mice are included in this study: 10 mice/sex x 2 sexes x 2 treatments/group x 1 harvest timepoint x 1 experimental replicate.
  • AAV6,2FF-Mediated Targeting of AT2 Cells to Repair Lung Epithelial Tissue AAV6.2FF is to be instilled into mouse lungs to selectively target expression of FAK to alveolar type 11 (AT2) cells.
  • AAV-delivered FAK is likely to have the appropriate post-translational modifications.
  • the viral vector includes the FAK cDNA encoding FAK protein. This pharmacologically active protein strengthens cell-cell junctions and enhances cell survival and is therefore expected to enhance epithelial repair in the context of lung injury. The overall goal is to develop a genedelivery treatment to repair damaged lung epithelial tissue in the alveoli.
  • Treatment administration includes two delivery methods: intranasal (i.n.) instillation in liquid suspension, which is an established method, compared to a dry nanoparticulate aerosol, which would be particularly suitable for efficient deposition in the deep lung.
  • intranasal (i.n.) instillation in liquid suspension which is an established method, compared to a dry nanoparticulate aerosol, which would be particularly suitable for efficient deposition in the deep lung.
  • the specific goal of this study is to develop an AAV6.2FF-PTK2 vector, test its efficacy in preclinical animal models of acute lung infection (ALI) and manufacture large-scale dry powder formulations of the vector that can ultimately be delivered in non-cold chain conditions for clinical use in the field.
  • ALI acute lung infection
  • AAV6.2FF Two distinct models of ALI are to be tested with the AAV6.2FF-PTK2 vector.
  • AAV6.2FF is to be used to deliver FAK to AT2 cells in mice before and after lung injury using two preclinical mouse models: 1) an influenza A virus (I AV) infection model that mimics severe viral infection in humans; and 2) an acid instillation model that mimics gastric acid aspiration in humans.
  • IAV infectious model results in ALI/ ARDS 6-7 days postinfection characterized by decreased body weight (see, FIG. 4A), oxygen saturation levels and static lung compliance, increased inflammation (see, FIG. 4B) and widespread alveolar epithelial cell injury and death (see, FIGS. 5A-5C).
  • FIGS. 5A- 5C Images of air-inflated, perfusion-fixed lungs in naive animals or after IAV infection by microCT (9 mm images) demonstrate increased tissue opacity in transverse sections (FIG. 5A), widespread tissue thickening and loss of alveolar structure in 3D-reconstructions (FIG. 5B), and increased lung tissue volume (top; FIG. 5C) and the increased percentage of total lung tissue/total lung volume (bottom; FIG. 5C).
  • FAK Association of FAK with Focal Adhesion Complexes in AT2 Cells
  • One of the functions of FAK is the association with paxillin and vinculin in focal adhesion complexes (Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM. Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility. J Cell Biol. 2004;165(3):371-381. doi: 10.1083/jcb.200308011).
  • AT2 cells are to be isolated using a dispase-based enzymatic lung digestion protocol that is well established (Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010;120(5): 1736-1748.
  • FAK-FLAG is to be immunoprecipitated from AT2 cell lysates with anti-FLAG antibody and immunoblotted for active FAK (phospho-FAK Y397, Cell Signaling (Phospho-FAK (Tyr397) Antibody. https://www.cellsignal.com/products/primary-antibodies/phospho-fak-tyr397-antibody/3283)). Then, it is to be determined if AAV-driven FAK associates with focal adhesion complex proteins (e.g., paxillin and vinculin) in AT2 cells similar to its well described role in endothelial cells.
  • focal adhesion complex proteins e.g., paxillin and vinculin
  • the functional effect of FAK expression is to be determined in mock treated and AAV6.2FF-PTK2 infected primary mouse AT2 (mAT2) cell cultures by 1) measuring trans- epithelial resistance (TER) in cell monolayers, at baseline and following monolayer perturbation, 2) determining their ability to repair a monolayer after a scratch wound, and 3) measuring the susceptibility to bleomycin-induced apoptosis.
  • TER trans- epithelial resistance
  • primary mAT2 cells are to be isolated and immediately transduced with AAV6.2FF-PTK2 or AAV6.2FF- mCherry control (1000 vg/cell).
  • the transduced cells will then be seeded on Matngel/collagen coated transwell filters (Millicell-PCF) as previously described (Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010;120(5): 1736-1748. doi: 10. 1172/j ci38061 ). mAT2 cells will be grown to confluence and the TER values will be measured using a Millicell ERS-2 epithelial volt-ohm meter (Merck Millipore) (Ma Y, Semba S, Khan MRI, et al.
  • Millicell ERS-2 epithelial volt-ohm meter Merck Millipore
  • AT2 cell monolayers will be subjected to a calcium switch assay (Zheng B, Cantley LC. Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase. Proceedings of the National Academy of Sciences. 2007;104(3):819-822. doi: 10. 1073/pnas.0610157104; Playford M, Vadali K, Cai X, Burridge K, Schaller M. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho. Exp Cell Res.
  • transduced mAT2 cells will be grown to confluence on Matrigel/collagen-coated transwells and will undergo scratch wounding with a pipette tip on the apical surface. Cells will be imaged immediately after wound induction and 24 hours postwound. Wound width will be calculated using ImageJ software (NIH) and wound repair will be expressed as the percentage of initial wound remaining after 24 hours, as previously described (McClendon J, Jansing NL, Redente EF, et al. Hypoxia-Inducible Factor la Signaling Promotes Repair of the Alveolar Epithelium after Acute Lung Injury. The American Journal of Pathology. 2017;187(8):1772-1786. doi: 10. 1016/j.ajpath.2017.04.012).
  • AAV6.2FF-PTK2 and AAV6.2FF-mCherry control transduced cells will be treated with bleomycin sulfate, an apoptotic mimetic drug (100 pg/ml for 48 hours) (Tounekti O, Pron G, J. B, Mir LM. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Journal of Cancer Research. 1993;88(22):5462-5469.; Karam H, Hurbain-Kosmath I, Housset B.
  • Apoptosis will be determined by caspase 3/7 and 8 activation (Caspase- Glo®, Promega (Caspase-Gio® 3/7 Assay System
  • Apoptosis Detection, https://worldwide.promega.com/products/cell-health-assays/apoptosis- assays/caspase_glo-3_7-assay-systems/?catNum G8090)) as previously described (Bamberg A, Redente EF, Groshong SD, et al.
  • Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis. Am J Respir Crit CareMed. 2018;198(7):914-927. doi: 10.1164/rccm.201707-1497oc).
  • hAT2 Primary human AT2 cells will be isolated from lungs of deidentified organ donors whose lungs are not suitable for transplantation. The cells may be frozen down for subsequent thawing and culturing. After thawing, hAT2 cells will be transduced with AAV6.2FF-PTK2 or AAV6.2FF-mCherry control (1000 vg/cell) and cultured on Matrigel/collagen-coated inserts for two days for adherence and then six days under air/liquid (ALI) interface conditions in the presence of FGF7 and dexamethasone (Wang J, Edeen K, Manzer R, et al. Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro. American Journal of Respiratory Cell and Molecular Biology.
  • mice are needed to isolate primary AT2 cells: 3 mice/sex x 2 sexes x 2 treatments/group x 3 readouts (TER, scratch wound and apoptosis) x 3 experimental replicates.
  • AAV6.2FF capsids containing the FAK transgene will be produced in 200 mL shake flasks by co-transfection of HEK293 cells with same plasmids used to generate AAV in adherent cells, i.e., the pCASI FAK-FLAG WPRE plasmid with the AAV6.2FF packaging plasmid.
  • HEK293 cells from Mass Biologies will be expanded in Hy clone protein-free and animal-derived component-free (ADCF) cell culture media. pH, temperature, and stir rate will be altered in order to determine the optimal conditions for FAK transgene packaging into the viral capsid.
  • Shake flask conditions designed to generate 10 14 viral capsids per flask will be repeated 20 times and purified from HEK293 using cell lysis, filtration, affinity chromatography, and anion exchange chromatography. The later two column chromatographic steps will reduce empty capsids, nucleic acid contaminants, host cell protein, host cell DNA and media components.
  • the purified AAV6.2FF-PTK2 viral capsids will be characterized by droplet digital PCR (ddPCR), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), Western bloting, and analytical ultracentrifugation. These analytical methods will assess transgene vector production levels, viral capsid quality, and the percent of empty capsids, respectively.
  • ddPCR droplet digital PCR
  • CE-SDS capillary electrophoresis sodium dodecyl sulfate
  • Western bloting Western bloting
  • analytical ultracentrifugation will assess transgene vector production levels, viral capsid quality, and the percent of empty capsids, respectively.
  • DLS dynamic light scatering
  • AF4 asymmetrical flow field-flow fractionation
  • Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy. ACS Omega. 2021 ;6(25): 16428- 16437. doi:10.1021/acsomega.lc01443). Denaturing and non-denaturmg agarose gels will be used to determine any variability in the transgene DNA.
  • Formulations will be prepared according to methods known in the art (e.g., Zhang et al., International Journal of Pharmaceutics, 2021(606): 120912) and evaluated for their stability as a function of pH and buffer composition. Samples will be evaluated at time zero (tO) for content, osmolality, and pH. The purities of each composition will be measured using asymmetric-Flow Field Flow Fractionation monitored with a multi-angle light scatering detector (AF4-MALS) and anion exchange chromatography (AEX-HPLC). Samples will also be tested after one week at 40°C, two weeks at 25°C, and four weeks at 5°C.
  • AF4-MALS multi-angle light scatering detector
  • AEX-HPLC anion exchange chromatography
  • lyophilized formulations will be identified for testing. Each of these will contain 10 12 vg/mL and will include pharmaceutically acceptable excipients that are capable of forming completely amorphous matrices (using sucrose and/or trehalose). In addition, the effects of the following will be checked: 1) crystalline bulking agent (e.g., mannitol), 2) buffer (e.g., TRIS), 3) tonicity modifier (e.g., glycine, or sodium chloride) and 4) plasticizing agent (e.g., glycerol). Each selected formulation will be lyophilized using conditions that allow all preparations to fully dry and will be evaluated for moisture content, glass transition temperature (Tg), crystallinity, and reconstitution time at time zero (tO).
  • Tg glass transition temperature
  • tO reconstitution time at time zero
  • AAV6.2FF-PTK2 Male and female wild-type C57BL/6J mice will be used in both models.
  • AAV6.2FF-PTK2 or AAAV6.2FF-mCherry as a control will be delivered using two strategies: (1) by intranasal (/. «.) delivery, and (2) by aerosolization (1 x 10 12 viral genomes/mouse). Mice will be sacrificed at the indicated endpoints and assessed for established ALI outcomes.
  • Experimental readouts include: 1) assessment of clinical parameters in live animals, and 2) assessment of injury and inflammation markers in post-mortem samples.
  • mice/sex will be used with two experimental replicates, to provide a >80% power to detect an effect size (between experimental groups and sex of mouse) of 1.6 with a Gaussian distribution (2 -sided t-test, p ⁇ 0.05). Differences will be analyzed between conditions with parametric (t-test and ANOVA with Newman-Keuls post hoc analysis) or nonparametric (Wilcoxon Signed Rank) statistical analyses as appropriate. To determine an effect of sex, a 2 way-ANOVA will be used. All data will be presented as the mean ⁇ SEM. A p-value of ⁇ 0.05 will be considered significant.
  • the left lung can be tied off and inflated for histology/immunostaining, while the right lung will undergo BAL. Post BAL, the upper right lobe will be processed for protein, and the lower right lobes will be digested for FACS and cell sorting. Utilizing the lungs in this manner will aid in reducing animal use and allow for a comprehensive, multi-parameter study of each animal.
  • BAL bronchoalveolar lavage
  • IL-6 pro-inflammatory cytokine levels in BAL fluid
  • IL-6 pro-inflammatory cytokine levels in BAL fluid
  • lung permeability assays IgM (IgM Mouse Uncoated ELISA Kit with Plates - Invitrogen. https://www.thermofisher.com/elisa/product/IgM-Mouse-Uncoated-ELISA-Kit-with-Plates/88- 50470-22), albumin (Mouse Albumin ELISA Kit (Colorimetric).
  • RNA sequencing identifies TGFbeta as a key regenerative cue following LPS-induced lung injury. JCI Insight. Mar 26 2019;5doi: 10.1172/jci. insight. 123637) and multi-parameter flow cytometry protocols have been established using cell surface markers analyzed on a BD FortessaTM instrument.
  • the flow strategy involves initial identification immune (CD45 and Ly6G) and endothelial (CD31) cells, AT2 cells (EpCAM+MHCII+) and fibroblasts (PDGFRa) along with mCherry or FLAG detection of the AAV6.2FF-PTK2.
  • mice will be administered AAV6.2FF-PTK2 or AAV -mCherry by the i.n. route (1 x 10 12 viral genomes/mouse) Iweek prior to disease initiation.
  • mice will be included for i.n. administration (moderate dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV doses x 2 endpoints x 2 expenmental replicates.
  • 40 mice will be included for i.n. administration (lethal dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoints x 2 experimental replicates.
  • mice will be included for aerosol administration model: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoints x 2 experimental replicates.
  • mice will first be administered AAV-FAK or AAV-mCherry by the i.n. route (1 x 10 12 viral genomes/mouse).
  • mice will be administered HC1 (2 ml/kg, 0. IN) in saline by i.t. instillation.
  • mice will be euthanized 1-week and 3-week post-HCl administration and tissues will be harvested for experimental readouts detailed above. 80 mice will be included in this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF- mCherry) x 1 acid dose x 2 endpoints x 2 experimental replicates.
  • mice Two independent doses of IAV will model moderate (1 x 10 2 PFU) and lethal (1 x 10 4 PFU) ALI injury as described in Example 5 above.
  • Mice will be euthanized 1- week and 3-week post-IAV administration and tissues will be harvested for experimental readouts. These experiments will be repeated with the final formation and development from the large scale AAV6.2FF-PTK2 formulation via dry aerosolization exposure based on the time and dose determined from the i.n. delivery experiments that provides the most protection. Additionally, it will be determined if overexpression of FAK initiates de novo development of cancer or pulmonary fibrosis in the context of ALI.
  • mice will be used for i.n. administration (moderate dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 2 endpoints x 2 experimental replicates.
  • 40 mice will be used for i.n. administration (lethal dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCheny) x 1 IAV dose x 1 endpoint x 2 experimental replicates.
  • mice will be used for aerosol administration model: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoint x 2 experimental replicates. 20 mice will be used for moderate dose longitudinal CT analysis: 5 mice/sex x 2 sexes x 2 treatments/group.
  • mice will first be administered HC1 at 2 ml/kg (0.1N) in saline by i.t. instillation. Three days after receiving HC1, AAV6.2FF-PTK2 or AAV-mCherry will be given by the i.n. route (1 x 10 12 viral genomes/mouse). Mice will be euthanized 1-week and 3-week post-HCl administration and tissues will be harvested for experimental readouts.
  • mice will be included for this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 acid dose x 2 endpoints x 2 experimental replicates.
  • Protein was eluted off of the beads with 2X laemmli buffer. Lysates were run on a 10-20% tn cine polyacrylamide gel, transferred to PVDF membrane, and immunoblotted with primary antibodies against phospho- FAK (Y397) followed by incubated with an HRP-conjugated secondary antibody. The blot was then incubated with HRP substrate and imaged. Following imaging, the blot was stripped with stripping buffer, blocked in milk, and incubated with primary antibodies against total FAK followed by incubation with an HRP-conjugated secondary antibody. Blot was then incubated with HRP substrate and imaged. Each lane represents AT2 cells from a single animal.
  • FIG. 9A The results are shown in FIG. 9A, wherein FLAG immunoprecipitation followed by Western blot of FAK was detected by anti-phospho FAK (Y397) and total FAK antibodies in whole AT2 cell lysates of AAV6.2FF-FAK-FLAG mouse (transduced) vs. no signal was detected in non-transduced mouse.
  • Sections were rehydrated, blocked, and incubated with primary antibodies against the FLAG epitope (FLAG), pro-surfactant protein C (pro-SPC) and nuclei w ere counterstained with DAPI. Sections were then incubated with fluorophore-conjugated secondary antibodies and mounted in imaging media. Images were obtained on an EVOS7000 fluorescent microscope. Images are representative of one section from a single animal. The results are shown in FIG. 9B, with the arrows pointing to FAK-FLAG in pro-SPC+ AT2 cells from a non-transduced mouse (top panel) and a transduced mouse (bottom panel).
  • Non-permeabilized cells were stained with anti-EpCAM and anti-MHCII antibodies conjugated to fluorophores, then permeabilized with saponin, blocked and stained with anti-FLAG antibody.
  • Cells were washed and immediately analyzed by FACS analysis. Gates were set based on no stain and single-stained controls; 100,000 events were collected and analyzed for the triple-stained experimental samples. Data shown are representative plots from one sample per group. The results are shown in FIG. 9C. Therein, the dashed boxes represent lack of FLAG expression in non-EpCAM+ AT2 cells.
  • cytospins were prepared for each sample, fixed, and stained with the DiffQuik kit. Samples were analyzed under a light microscope and immune cells were manually differentiated by size and morphology, and were stained by two lab personnel and data were averaged. A total of 250-300 cells were counted per slide, with 1 slide analyzed per mouse. The results are shown in FIGS. 10B-10D (neutrophils, macrophages, and lymphocytes counts, respectively). Similar immune cell profiles were observed in both groups, dominated primarily by a neutrophilic response. Similar immune response was observed in both groups. EXAMPLE 15
  • the experimental group was challenged with LPS (50 pg intratracheally) and transduced with AAV6.2FF-GFP (2xl0 12 vg, intranasally) 3 days post-LPS.
  • the control group did not receive LPS and were also transduced with AAV6.2FF-GFP.
  • mice were harvested, and lung cells were isolated via antibody and magnetic bead isolation.
  • Non-permeabilized cells were stained with anti- CD45, anti-CD31, anti-PDGFRa, anti-EpCAM, and anti-MHCII antibodies. Cells were washed and immediately analyzed by FACS analysis. Gates were set based on no stain and singlestained controls; 100,000 events were collected and analyzed for the triple-stained experimental samples. Each data point represents AT2 cells from a single animal analyzed by enzyme-based dispersion of the lung, follow ed by antibody staining and FACS analysis. [0139] The results are shown in FTG. 11 A-l IB, wherein the data illustrated in FIG. 11 A are representative FACS histogram plots from one sample per group, and the data in FIG.
  • FIG. 11B are quantitative data for GFP expression in EpCAM+/MHCII+ AT2 cells with each data point representing cells from a single animal.
  • the data in FIG. 11A show that similar GFP expression level in AT2 cells with respect to cell number (counts) and GFP intensity (GFP fluorescence) was observed in naive and LPS-inflamed lungs, and minimal targeting of AAV- GFP in immune cell (CD45+), fibroblasts (PDGFRa+), and endothelial cells (CD31+) was observed in the LPS inflamed lung.
  • the data in FIG. 11B show that no difference in AT2 targeting of AAV was observed in naive or LPS treated mice.

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Abstract

Constructs comprising a nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof, and pharmaceutical compositions comprising the constructs, and various uses thereof are provided. Methods of using FAK protein or a biologically active fragment, derivative, or variant thereof for treating lung vascular leak are also provided.

Description

DELIVERY OF FOCAL ADHESION KINASE FOR TREATING VASCULAR AND/OR
CAPILLARY TISSUE INJURY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/343,334, filed May 18, 2022, the content of which is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing that has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 18, 2023, is named ‘T 17313-0018-8001WO00_SEQ.xml” and is 28,871 bytes in size.
TECHNICAL FIELD
[0003] The subject matter described herein relates to delivery of Focal Adhesion Kinase (FAK) or a biologically active fragment, derivative, or variant thereof to treat vascular and/or capillary cell injury.
BACKGROUND
[0004] Focal adhesion kinase (FAK) is a multifunctional integrin-associated protein tyrosine kinase. FAK is encoded by the PTK2 gene. When activated via auto- phosphorylation at Tyr- 397, FAK localizes to focal adhesion complexes of endothelial cells in association with paxillin, vinculin leading to the extracellular association of integrin and E-cadherin. The interactions of integrin and E-cadherin on neighboring cells promotes tight association. FAK also appears to enhance association of neighboring epithelial cells. Within epithelial and endothelial cells, FAK plays a key role in the formation of a complex in which actinomyosin stress fibers are part of actin cytoskeleton and regulate cell spreading and migration, proliferation, and cell survival (Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol. 1996;134(3):793-799. doi: 10.1083/jcb. l34.3.793). This enzyme plays a pivotal role in remodeling adherens junctions leading to the resealing of barriers in epithelial and microvascular endothelial tissues in multiple organs (Quadri SK, Bhattacharj ee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelial barrier strengthening by activation of focal adhesion kinase. J Biol Chem. Apr 11, 2003;278(15): 13342-9. doi:10.1074/jbc.M209922200; Schmidt TT, Tauseef M, Yue L, et al. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rael activities. Am J Physiol Lung Cell Mol Physiol. Aug 15 2013;305(4):L291-300. doi: 10.1152/ajplung.00094.2013; Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesion kinase. Am J Physiol Lung Cell Mol Physiol. Jan 2007;292(l):L334-42. doi: 10.1152/ajplung.00228.2006; Holinstat M, Knezevic N, Broman M, Samarel AM, Malik AB, Mehta D. Suppression of RhoA Activity by Focal Adhesion Kinase-induced Activation of pl90RhoGAP. J Biol Chem. 2006;281(4):2296-2305. doi: 10.1074/jbc.m511248200; Ma Y, Semba S, Khan MRI, et al. Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2013:1832(1): 151-159. doi: 10.1016/j.bbadis.2012.10.006; Thomas KS, Owen KA, Conger K, Llewellyn RA, Bouton AH, Casanova JE. Non-redundant functions of FAK and Pyk2 in intestinal epithelial repair. Scientific Reports. 2019;9(l)doi: 10. 1038/s41598-019-41116- 1). FAK has additional signaling capacities through cell surface receptors and FAK interacting proteins including Src family kinases and phosphoinostitide-3 kinase (PI3K) resulting in the regulation of multiple cellular functions including migration, proliferation and cell survival (Zhao X, Guan J-L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Del Rev. 2011;63(8):610-615. doi: 10.1016/j.addr.2010.11.001; Owen KA, Abshire MY, Tilghman RW, Casanova JE, Bouton AH. FAK Regulates Intestinal Epithelial Cell Survival and Proliferation during Mucosal Wound Healing. PLoS ONE. 201 l;6(8):e23123. doi: 10.1371/joumal. pone.0023123).
[0005] Acute lung injury (ALI), or the more severe acute respiratory distress syndrome (ARDS), is reduction of functional breathing due to the degradation of lung tissue as a result of inflammation. There are currently no effective therapies available for treatment (Matthay MA, Zemans RL. The Acute Respiratory Distress Syndrome: Pathogenesis and Treatment. Annual Review of Pathology: Mechanisms of Disease. 2011;6(1): 147-163. doi:10. 1146/annurev- pathol-011110-130158). ALI/ ARDS is defined clinically with the following Berlin criteria: bilateral pulmonary opacities on chest radiographs, acute onset, and arterial hypoxemia (PaCh/FiCh ratio < 300 mm Hg)2, which is an indicator of poor oxygenation, pulmonary infiltrates, and acuity of onset (Matthay MA, Zemans RL, Zimmerman GA, et al. Acute respiratory distress syndrome. Nature Reviews Disease Primers. 2019;5(l)doi: 10.1038/s41572- 019-0069-0). ARDS affects more than 200,000 patients annually in the United States, resulting in about 75,000 deaths (Fan E, Brodie D, Slutsky AS. Acute Respiratory Distress Syndrome. JAMA. 2018;319(7):698. doi: 10.1001/jama.2017.21907; Blank R, Napolitano LM.
Epidemiology of ARDS and ALL Crit Care Clin. Jul 2011;27(3):439-58. doi: 10. 1016/j.ccc.2011.05.005). [0006] ALI/ARDS leads to alveolar epithelial cell apoptosis, denudation of the epithelium, and destruction of the underlying basement membranes (Wheaton AK, Agarwal M, Jia S, Kim KK. Lung epithelial cell focal adhesion kinase signaling inhibits lung injury and fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2017;312(5):L722-L730. doi: 10.1152/ajplung.00478.2016; Hungerford JE, Compton MT, Matter ML, Hoffstrom BG, Otey CA. Inhibition of ppl25FAK in cultured fibroblasts results in apoptosis. J Cell Biol.
1996; 135(5): 1383-1390. doi: 10.1083/jcb, 135.5.1383; Gilmore AP, Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Molecular Biology of the Cell. 1996;7(8): 1209-1224. doi:10.1091/mbc.7.8.1209; Zhao J-H, Reiske H, Guan J-L. Regulation of the Cell Cycle by Focal Adhesion Kinase. J Cell Biol. 1998;143(7): 1997-2008. doi:10.1083/jcb, 143.7.1997; Richardson A, Parsons JT. A mechanism for regulation of the adhesion-associated protein tyrosine kinase ppl25FAK. Nature. 1996;380(6574):538-540. doi:10.1038/380538a0; Llic D, FurutaY, Kanazawa S, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK- deficient mice. Nature. 1995;377(6549):539-544. doi:10.1038/377539a0; Cary LA, Chang JF, Guan JL. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci. 1996;109(7):1787-1794. doi: 10.1242/jcs.109.7.1787). Additionally, endothelial cells of blood capillaries and epithelial cells of alveoli become permeable due to loss of intercellular junctions leading to inflammation. While the extent and timing to which each process contributes to the pathophysiology is not clear, the result is leakage of plasma into the deep lung, which inhibits the surface tension and lowers properties of the pulmonary surfactant resulting in reduced lung function (Matthay MA, Zemans RL, Zimmerman GA, et al. Acute respiratory distress syndrome. Nature Reviews Disease Primers. 2019;5(l)doi: 10.1038/s41572-019-0069-0).
[0007] Previous studies have shown that FAK enhanced the barrier strength of microvascular endothelial cells, likely due to its ability to recruit paxillin, vinculin, and a-actinin to a macromolecular structure that strengthened intercellular VE-cadherin interactions at the cellsurface, contributing to repair of microvascular damage (Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesion kinase. Am J Physiol Lung Cell Mol Physiol. Jan 2007;292(l):L334-42. doi:10.1152/ajplung.00228.2006; Quadri SK, Sun L, Islam MN, Shapiro L, Bhattacharya J. Cadherin selectivity filter regulates endothelial sieving properties. Nature Communications. 2012;3(l): 1099. doi: 10.1038/ncomms2107; Quadri SK, Bhattacharjee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelial Barrier Strengthening by Activation of Focal Adhesion Kinase. J Biol Chem. 2003;278(15): 13342-13349. doi: 10. 1074/jbc.m209922200). This led to the demonstration that injected cell-permeating FAK protein protected mice from serious illness and death caused by Pseudomonas aeruginosa (PA) infection (U.S. Patent 8,420,080). In those studies, human activated focal adhesion kinase (FAKp) was modified to include an N-terminal His-tag which indirectly assisted with cellular uptake by forming a complex with Cu2+ and TAT, a cell penetrating peptide (CPP). Although the tnmenc complex TAT, Cu2+, and FAKp (TAT-FAKp) was identified as a potential treatment for ARDS, using His-Tags in therapeutic proteins is discouraged by the FDA due to adverse immunological events. In addition, the FDA may be concerned not only with the safety of TAT-FAKp, but also the individual components that make up the complex.
[0008] Accordingly, it would be advantageous to develop an effective means of delivering FAK to injured epithelial and microvascular endothelial tissues to reseal the barriers. Such delivery systems may be advantageously employed for various applications, e.g., treatment of ALI/ARDS.
BRIEF SUMMARY
[0009] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.
[0010] In one aspect, a construct comprising a nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof is provided.
[0011] In some embodiments, the construct may be an engineered mRNA encoding the FAK protein, fragment derivative, or variant, or an expression vector encoding the FAK protein, fragment derivative, or variant.
[0012] In some embodiments, the expression vector may be a viral vector. By way of nonlimiting example, the viral vector may be selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno- associated viral (AAV) vector. In certain embodiments, the vector may be an AAV vector. By way of non-limiting example, the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
[0013] In certain embodiments, the nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof may be at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be at least 90% identical to SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 1.
[0014] In some embodiments, the construct may comprise a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 2. In certain embodiments, the construct may comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2. In certain embodiments, the construct may be AAV6.2FF-PTK2 comprising the nucleic acid sequence as set forth in SEQ ID NO: 2
[0015] In another aspect, a pharmaceutical composition comprising any of the constructs disclosed above and herein is provided.
[0016] In some embodiments, the pharmaceutical composition may further comprise a reagent capable of assisting delivery of the construct to a recipient cell. By way of non-limiting example, the reagent is polyethylenimine.
[0017] In some embodiments, the pharmaceutical composition may further comprise a stabilizing excipient. In certain embodiments, the stabilizing excipient may be a pharmaceutically acceptable excipient, including but not limited to, citrate, glycine, phosphate, tris, histidine leucine, raffinose, trehalose, mannitol, lactose and trileucine.
[0018] In some embodiments, the pharmaceutical composition may be in a form of a dry powder or a liquid. In certain embodiments, the dry powder may be produced by lyophilization or spray-drying.
[0019] In another aspect, a kit comprising any of the constructs or any of the pharmaceutical compositions disclosed above and herein and instructions for use is provided.
[0020] In still another aspect, a method of preventing or repairing an injured vascular and/or capillary tissue in a subject in need thereof is provided. This method may comprise administering to the subject an effective amount of any of the pharmaceutical compositions disclosed above or herein or using the kit disclosed above and herein.
[0021] In some embodiments, the subject may suffer from an injury that involves endothelial cells and/or epithelial cells. In certain embodiments, the injury may involve endothelial cells found in the capillary tissue. In certain embodiments, the injury involves epithelial cells that make up alveolar cells in the deep lung. In certain embodiments, the injury may be a lung injury, a hemorrhagic stroke, an acute or chronic kidney disease, or a capillary leak syndrome. By way of non-limiting example, the lung injury may be ALI/ARDS, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, or asthma. In certain embodiments, the lung injury may be caused by SARS-CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury. By way of non-limiting example, the lung pathogen(s) include pseudomonas aeruginosa. By way of non-limiting example, the internal organ injury includes acute pancreatitis.
[0022] In some embodiments, the pharmaceutical composition may be administered to the subject intranasally, intratracheally, or parenterally. In certain embodiments, the pharmaceutical composition may be administered via intratracheal or intranasal instillation of a liquid, nebulization of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol. [0023] In some embodiments, the effective amount of the pharmaceutical composition administered to the subject is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK.
[0024] In still another aspect, a method of preventing or treating ALI/ARDS in a subject in need thereof is provided. This method may comprise administering to the subject an effective amount of any of the pharmaceutical compositions disclosed above or herein or using the kit disclosed above and herein. In some embodiments, the lung injury may be caused by SARS- CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury. By way of non-limiting example, the lung pathogen(s) include pseudomonas aeruginosa. By way of non-limiting example, the internal organ injury includes acute pancreatitis.
[0025] In some embodiments, the pharmaceutical composition may be administered to the subject intranasally, mtratracheally, or parenterally. In certain embodiments, the pharmaceutical composition may be administered via intratracheal or intranasal instillation of a liquid, nebulization of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol. [0026] In some embodiments, the effective amount of the pharmaceutical composition administered to the subject is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK.
[0027] In still yet another aspect, a method of treating lung vascular leak in a COVID-19 patient is provided. This method may comprise expressing an intracellular, barrier-enhancing therapeutic agent in the lung endothelium of the patient through a gene delivery tool.
[0028] In some embodiments, the therapeutic agent may be Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof.
[0029] In some embodiments, the gene delivery tool may be an engineered mRNA encoding the FAK protein, fragment derivative, or variant, or an expression vector encoding the FAK protein, fragment derivative, or variant. In certain embodiments, the expression vector may be a viral vector. In certain embodiments, the viral vector may be selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno-associated viral (AAV) vector. In certain embodiments, the vector may be an AAV vector. By way of non-limiting example, the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
[0030] In some embodiments, the AAV vector comprises a nucleic acid sequence encoding FAK protein or a biologically active fragment, derivative, or variant thereof. In certain embodiments, the nucleic acid sequence may be at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be at least 90% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be identical to SEQ ID NO: 1.
[0031] In some embodiments, the construct may compnse a nucleic acid sequence that is at least 75% identical to SEQ ID NO: 2. In certain embodiments, the construct may comprise a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 2. In certain embodiments, the construct may be AAV6.2FF-PTK2 vector comprising the nucleic acid sequence as set forth in SEQ ID NO: 2.
[0032] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.
[0033] Additional embodiments of the present compositions and methods will be apparent from the following description, drawings, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Additional aspects and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the disclosure will be apparent from the drawings/tables and detailed description, and from the claims.
[0035] FIGS. 1A-1E demonstrate in vivo targeting of mouse alveolar type II (AT2) cells by AAV6.2FF. FIG. 1A shows luciferase expression of AAV6.2FF-luciferrase in lung field of a C57BL/6 mouse by whole body IVIS imaging. FIG. IB shows quantitative longitudinal analysis of AAV6.2FF-luciferase expression in live mice following a single administration of the virus. N = 4 mice/group. FIG. 1C shows FACS analysis of wild-type AT2 cells (EpCAM+/MCHII+) and AT2 cells 7 days post-intratracheal administration of AAV6.2FF- GFP. FIG. ID shows immunostaining of wild-type lung sections 2 weeks post intranasal delivery of AAV6.2FF-mCherry virus. FIG. IE shows FACS analysis of wild-type lung cells 2 weeks post intranasal delivery of AAV6.2FF-mCherry virus. N = 4-6 mice/group. #p < 0.01. [0036] FIGS. 2A-2B demonstrate immunofluorescent staining of GFP (green) and the AT2- specific marker pro-SPC (red) in AAV6.2FF-GFP transduced control lungs and in influenza A virus (1AV) infected/ AAV6.2FF-GFP transduced lungs. FIG. 2A shows abundance of GFP/pro-SPC double positive AT2 cells in AAV6.2-GFP transduced lung. FIG. 2B shows persistence of AAV6.2FF-GFP signal in pro-SPC positive cells of IAV+AAV6.2FF-GFP lung. [0037] FIG. 3A is a diagram of FAK-FLAG construct for AAV-driven expression in AT2 cells. FIG. 3B illustrates the ability of AAV6.2FF-PTK2 to transfect AT2 cells and produce the FAK protein.
[0038] FIGS. 4A-4B show dose-dependent weight loss and increased inflammation in acute IAV infection mouse model. FIG. 4A shows body weight measurements over time following IAV administration. N = 5 mice/group. FIG. 4B shows total bronchoalveolar (BAL) cell counts 7 days post IAV administration. N = 2 mice/group.
[0039] FIGS. 5A-5C show increased tissue density in lAV-infected lungs. FIG. 5A shows the images of air-inflated, perfusion-fixed lungs in naive animals or after IAV infection by microCT (9 mm images). FIG. 5B shows widespread tissue thickening and loss of alveolar structure in 3D-reconstructions. FIG. 5C shows increased lung tissue volume (top) and the increased percentage of total lung tissue/total lung volume (bottom).
[0040] FIGS. 6A-6D show increased permeability of alveolar barrier following acid inhalation injury. FIG. 6A shows the albumin levels in the bronchoalveolar lavage (BAL) fluid. FIG. 6B shows quantification of differential cell counts from the BAL. FIG. 6C shows static lung compliance as measured by flexiVent. FIG. 6D shows representative Tri chrome staining of lung sections. N = 6/group. **p<0.01, ***p<0.00I.
[0041] FIG. 7 illustrates the cDNA sequence of the hFAK gene (SEQ ID NO: 1).
[0042] FIG. 8 illustrates the complete nucleic acid sequence of the AAV6.2FF-PTK2 construct (SEQ ID NO: 2).
[0043] FIGS. 9A-9C illustrate AAV6.2FF -driven FAK expression in vitro and in vivo. FIG. 9A shows FLAG immunoprecipitation followed by Western blot of FAK detected by anti- phospho FAK (Y397) and total FAK antibodies in whole AT2 cell lysates of AAV6.2FF-FAK- FLAG transduced vs. non-transduced mice (n = 3 mice/group). FIG. 9B shows anti-FLAG immunohistochemistry (arrows show FAK-FLAG in pro-SPC+ AT2 cells). FIG. 9C shows flow cytometry of lung AT2 cells after 1 wk from non-transduced or transduced mice with AAV6.2FF-FAK-FLAG virions. Dashed boxes show lack of FLAG expression in non- EpCAM+ AT2 cells. [0044] FIGS. 10A-10D illustrate similar immune response to LPS challenge in AAV6.2FF- FAK and AAV6.2FF-GFP mice. FIG. 10A shows total cell counts in BAL fluid of mice transduced with AAV6.2FF-GFP or AAV6.2FF-FAK (2xl012 vg, intranasally) on Day 0 followed by LPS (50 pg, intratracheal) challenge on Day 14. Mice were harvested on Day 17, 3 days post LPS challenge. FIGS. 10B-10D show differential cell counts in BAL fluid of the AAV6.2FF-FAK and the AAV6.2FF-GFP mice. FIG. 10B: neutrophils. FIG. 10C: macrophages. FIG. 10D: lymphocytes.
[0045] FIGS. 11A-11B illustrate AAV6.2FF-GFP expression in vivo following LPS challenge. FIG. 11A: representative FACS histogram plots showing GFP expression in lung cell types 10 days after LPS challenge. FIG. 1 IB is a graph showing frequency of EpCAM+/MHCII+ AT2 cells that are GFP+ in AAV -GFP treated mice (AAV -GFP) or AAV-GFP treated mice dosed 3 days post-LPS (LPS + AAV-GFP).
DETAILED DESCRIPTION
I. Definitions
[0046] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.
[0047] Where a range of values is provided, it is intended that each intervening value betw een the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as w ell as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm.
[0048] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “polymer” includes a single polymer as w ell as two or more of the same or different polymers, reference to an “excipient” includes a single excipient as well as two or more of the same or different excipients, and the like.
[0049] The word “about” when immediately preceding a numerical value means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.
5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein.
[0050] The compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.
[0051] All percentages, parts and ratios are based upon the total weight of the topical compositions and all measurements made are at about 25°C, unless otherwise specified. [0052] Focal adhesion kinase (FAK) is a 125 KDa non-receptor protein-tyrosine kinase, including any biologically active fragment, modification, derivative, or variant thereof. Activated FAK means the phosphorylated form of FAK that is referred to herein as “FAKp”, including any biologically active fragment, modification, derivative, or variant thereof.
[0053] The term “biologically active fragment” means a fragment of a protein that retains the biological activity of the protein or that exhibits a similar, but not necessarily identical, activity to the protein, preferably FAK or FAKp. The biological activity of the fragment may include an improved desired activity such as enhancing barrier function and decreasing undesirable activity.
[0054] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, salts, compositions, dosage forms, etc., which are-within the scope of sound medical judgment— suitable for use in contact with the tissues of human beings and/or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some aspects, “pharmaceutically acceptable” means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly, in humans.
[0055] The term “treating” is used herein, for instance, in reference to methods of treating capillary tissue injury and/or vascular tissue injury, and generally includes the administration of a compound or composition which repairs damaged epithelial tissue in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition (e.g., strengthen epithelial barrier function to protect and/or accelerate alveolar repair following acute lung injury).
[0056] The terms “inhibit”, “elevate”, “increase”, “decrease” or the like, e.g., which denote quantitative differences between two states, refer to a difference, e.g., a statistically significant difference, between the two states. [0057] A therapeutically effective amount of a protein or polypeptide (i.e., an effective dosage of FAKp) is an amount that achieves the desired therapeutic result. For instance, a therapeutically effective amount is an amount that ameliorates one or more symptoms of the disease, including ALI or ARDS, or that decreases lung endothelial cell (EC) permeability, i.e., increases the EC barrier in an animal, preferably a human, has or is at nsk of developing ALI or ARDS.
[0058] By reserving the right to proviso out or exclude any individual members of any such group, including any sub-ranges or combinations of sub-ranges within the group, that can be claimed according to a range or in any similar manner, less than the full measure of this disclosure can be claimed for any reason. Further, by reserving the right to proviso out or exclude any individual substituents, analogs, compounds, ligands, structures, or groups thereof, or any members of a claimed group, less than the full measure of this disclosure can be claimed for any reason.
[0059] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference to describe the state of the art more fully as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.
[0060] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
[0061] The magnitude of change in the examples is not meant to be rate limiting. The meaningful change will depend on the specific diagnostic assay. A meaningful change to inform a treatment decision may be a change from baseline, a change from matched controls, a change from a reference value from a healthy individual, a change from a non-mvolved area or some other change. The change may be more or less than 1%, 10%, 25% 50%, 75%, 90% or 99%. The change may be based on an average, a standard deviation, a cutoff, an interquartile range, or some other meaningful change. The changes may be based on meaningful changes in a database, a clinical trial, previous experiments, or intra-experimental controls. The changes may be based on high or low values. The changes may or may not be statistically significant.
II. Delivery Tool
[0062] The present disclosure relates a gene delivery tool to deliver FAK protein, a biologically active fragment, derivative, or variant thereof, to a subject in need thereof, e.g., to prevent or treat vascular and/or capillary tissue injury, to prevent or repair an injured vascular and/or capillary tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a COVID-19 patient, etc.
[0063] In one embodiment, disclosed herein is a construct comprising a nucleic acid sequence encoding FAK protein or a biologically active fragment, denvative, or variant thereof.
[0064] In some embodiments, the construct may be an engineered mRNA encoding the FAK protein, fragment derivative or variant. An engineered mRNA may be designed to address infrastructural barrier to clinical and translational research in gene therapy. In the wake of COVID- 19 pandemic, mRNA technology has played an important role in developing vaccines to combat the virus. It is anticipated that an engineered mRNA could be an effective tool to deliver FAK to injured capillary tissues and/or injured vascular tissues for repair.
[0065] In some embodiments, the construct may be an expression vector encoding the FAK protein, fragment derivative, or variant. In certain embodiments, the expression vector may be a viral vector such as a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, or an adeno-associated viral (AAV) vector. In certain embodiments, the vector may be an AAV vector. By way of non-limiting example, the viral vector may be an AAV2 vector, an AAV6 vector, or a combination thereof.
[0066] In some embodiments, the nucleic acid sequence encoding the FAK protein, fragment derivative, or variant may be naked DNA or cDNA sequence. In some cases, the nucleic acid sequence is at least 75% identical to the nucleic acid sequence of PTK2 as set forth in SEQ ID NO: 1 (human FAK cDNA sequence; NCBI reference sequence NM_153831.4; see, FIG. 7). In certain embodiments, the nucleic acid sequence may be 90% identical to SEQ ID NO: 1. In certain embodiments, the nucleic acid sequence may be 100% identical to SEQ ID NO: 1. [0067] The use of AAV gene delivery for therapeutic applications has increased, largely due to its non-pathogenicity to humans, alterations of recombinant AAV preparations that prevent integration into the genome, and minimal host immune response to the vector. LUXTURNA® is the first AAV gene delivery product to be FDA approved for Leber congenital amaurosis, a genetic retinal disease. At this time, there are -150 clinical programs utilizing AAV vectors that deliver pharmaceutically active agents to specific organs. As of this year, 11 of 30 AAV capsid therapeutic candidates that were approved to proceed into clinical trials have reached the stage of new drug application (ND A), close to 4 times greater than standard success rates (Kuzmin DA, Shutova MV, Johnston NR, et al. The clinical landscape for AAV gene therapies. Nat Rev Drug Discov. Mar 2021;20(3): 173-174. doi:10.1038/d41573-021-00017-7). To date, no ND As have been submitted for gene-delivered treatments for lung disease. An initial attempt to treat cystic fibrosis by delivering AAV2 to distal lung epithelial cells failed (Moss RB, Milla C, Colombo J, et al. Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial. Hum Gene Ther. Aug 2007;18(8):726-32. doi:10.1089/hum.2007.022) in part due to its inability to infect airway cells, resulting in poor protein translation (Yan Z, Lei -Butters DC, Liu X, et al. Unique biologic properties of recombinant AAV1 transduction in polarized human airway epithelia. J Biol Chem. Oct 6 2006;281(40):29684-92. doi: 10.1074/jbc.M604099200).
A. Construction of AAV6 2FF-PTK2
[0068] An AAV vector that expresses full length human FAK is to be generated. AAV6 is an AAV serotype that has good specificity for lung tissue. AAV6.2FF is a modified AAV serotype that is even more specific for lung tissue. AAV6.2FF virus encoding aN-terminal FLAG-tagged version of human FAK cDNA (NCBI reference sequence NM_153831.4; SEQ ID NO: 1; FIG. 7) is constructed as described in Example 2 below. Diagram of the AAV6.2FF-PTK2 vector is shown in FIG. 3A. In some embodiments, this vector may comprise the nucleic acid sequence as set forth in SEQ ID NO: 2 (FIG. 8). Such vector is designed for growth in adherent cells. In some embodiments, the nucleic acid sequence encoding FAK may be optimized for production in suspended HEK293 cells.
[0069] As shown in FIG. 3 A, the AAV6.2FF-PTK2 vector includes the FLAG®-tag at the N- terminus, which may be removed when constructing a modified AAV-FAK vector for clinical trials. The FLAG® tag, also known as the DYKDDDDK-tag (SEQ ID NO: 9), is a popular protein tag that is commonly used in affinity chromatography and protein research. The nucleic acid sequence of the AAV6.2FF-PTK2 vector as shown in FIG. 8 contains 3 x FLAG-tags in a tandem formation right next to each other (DYKDHDGDYKDHDIDYKDDDDK; SEQ ID NO: 10). Typically, the FLAG®-tag is used for protein purification from mammalian expression systems or general immunostaining and immunoprecipitation assays. However, in some applications, it is desirable to remove the FLAG®-tag once the purification of protein is complete because the tag may interfere with the biophysical and biochemical properties of the purified protein. For instance, tags can cause undesired changes in protein structure and function and further cause adverse immunological events which can be toxic. As such, the FLAG®-tag may be removed from the AAV6.2FF-PTK2 vector. To allow cleavage of the FLAG®-tag, a protease cleavage site needs to be engineered between the tag and the protein. Alternatively, the sequence that codes for FLAG® can be removed from the cDNA.
B. Pharmaceutical Compositions [0070] Pharmaceutical compositions for use in accordance with the present disclosure may be formulated in the conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or parenteral administration. For administration by inhalation, a preferred embodiment for delivering drugs to the lungs, the compounds for use are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. In the case of delivery of FAK, it may be done either by nebulization of aqueous droplets using a CP AP -type device or by a dry powder inhaler (DPI) using a mask. Masks are needed to avoid dispersal of the treatment to the surrounding environment. Alternatively, a physician may deliver FAK by intratracheal instillation of an aqueous suspension of the virions. While unacceptable for ambulatory care of asthmatics, this mode of delivery ensures that the virions are not dispersed to the environment and is more accepted for the treated of acute illness such as ALI/ARDS. It is expected that the patient would be under anesthetics at the time of treatment. Capsules and cartridges of gelatin, e.g., for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0071] In some embodiments, a pharmaceutical composition may comprise any one of the constructs described above and herein, and optionally together with a reagent capable of assisting delivery' of the construct to a recipient cell. By way of non-limiting example, the reagent is polyethylenimine.
[0072] In some embodiments, a pharmaceutical composition may comprise any one of the constructs described above and herein, and optionally together with a stabilizing excipient or at least one earner. As used herein, the term “excipient” or “carrier” includes solutions, emulsions, suspensions, gels, sols, colloids, and solids, designed for delivery of the aforementioned agents to capillary tissues and/or vascular tissues, e.g., alveolar epithelial Type II (AT2) cells. The term “solution” refers to a liquid mixture in which the minor component e.g., the AAV6.2FF-PTK2 construct) is uniformly distributed within the major component (e.g., buffer). “Emulsions” refer to a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible (e.g., oil and water). “Suspensions” refer to heterogeneous mixtures in which the solute particles do not dissolve but get suspended throughout the bulk of the medium. “Gels” refer to solid jelly-like material that can have properties ranging from soft and weak to hard and tough and are defined as a substantially dilute cross-linked system, which exhibits no flow. “Sols” refer to colloidal suspensions of very small solid particles in a continuous liquid medium. The term “colloid” may be used interchangeably with the terms “gel,” “sol,” and “suspension” and refers to homogeneous mixtures of ultramicroscopic particles of one substance dispersed through a second substance.
[0073] In some embodiments, the carrier is a liquid. The liquid carrier may include an excipient suitable for application to the capillary and/or vascular tissues. Suitable carriers and/or excipients include aqueous or non-aqueous diluents or combination thereof. In certain embodiments, the excipient may be a pharmaceutically acceptable excipient, including but not limited to, citrate, glycine, phosphate, tns, histidine leucine, raffinose, trehalose, mannitol, lactose and trileucine. Examples of aqueous carriers and/or excipients include, but are not limited to, saline, water, dextrose or combinations thereof. Non-aqueous carriers and/or excipients include, but are not limited to, alcohols, particularly polyhydroxy alcohols such as propylene glycol, polyethylene glycol, glycerol, and vegetable and mineral oils. These aqueous and/or non-aqueous carriers and/or excipients can be added in various concentrations and combinations to form solutions, suspensions, oil-in-water emulsions or water-in-oil emulsions. In certain embodiments, the carrier and/or excipient is a polar solvent material selected from the group consisting of C3-C4 diols, C3-C6 triols, and mixtures thereof, and/or a polar lipid material selected from the group consisting of fatty alcohol esters, fatty acid esters. A mixture of the polar solvent material and the lipid material, for example, in a weight ratio of solvent material to the lipid material of about 60:40 to about 99: 1, may also be used. Other suitable carriers are provided in U.S. Pat. No. 5,026,556 (Drust etal.
C. Kits
[0074] In certain aspects, kits comprising any of the constructs or any of the pharmaceutical compositions disclosed above and herein, optionally together with instructions for administering the construct or pharmaceutical composition are described herein. The components of the kit, e.g., the construct containing the active agent and the carrier, optionally together with other ingredients, e.g., gelling agents, emollients, surfactants, humectants, viscosity enhancers, emulsifiers, etc., in one or more compartments. The kits may optionally comprise instructions for using the components, either individually or together, to practice the various prophylactic and/or therapeutic applications described below.
[0075] Usually, a kit may comprise single or multi-dose amounts of the active agents. In the case of delivery of FAK protein, the kit comprises a minimal number of doses as the treatment is intended to be an acute treatment. It is not well-suited to chronic treatment of an ambulatory individual. As such, “take home” kits with drug administration by the patient is unlikely in this case. In some embodiments, the kit can be packaged and shipped.
D. Methods of Use
[0076] The compounds disclosed above and herein may be formulated and administered to a subject in need thereof, e.g., to prevent or treat capillary tissue injury, to prevent or repair injured vascular tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a CO VID-19 patient, etc. The administration produces contact of the active ingredient with the site of action in the body of the subject, e.g., the lungs. Any injury that involves vascular and/or capillary tissues may be treated with the methods disclosed herein. In some embodiments, the injury may be a lung injury, a hemorrhagic stroke, an acute or chronic kidney disease, or a capillar}' leak sy ndrome. By way of non-limiting example, the lung injury may be ALI or ARDS, chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, or asthma. For instance, the lung injury could be caused by SARS-CoV-2, lung pathogens (e.g., pseudomonas aeruginosa), inhalation of excess smoke or water, or internal organ injury (e.g., acute pancreatitis).
[0077] In some embodiments, a therapeutically effective amount of the drug FAK (expressed and delivered via an AAV vector) is administered to a patient suffering from ALI, ARDS or other disease related to reduced E-cadherin at EC junctions or decreased EC barrier function. The route of administration can be any route that delivers the therapeutic agent to the intended target, e.g., by inhalation or injection. In some embodiments, FAK expressed and delivered via an AAV vector is administered with a selected pharmaceutical carrier based on the chosen route of administration and standard pharmaceutical practice. In some embodiments, the pharmaceutical composition is administered intranasally or intratracheally. In certain embodiments, the pharmaceutical composition is administered via intratracheal or intranasal instillation of a liquid, nebuhzation of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol.
[0078] In some embodiments, a method of treating lung vascular leak in a COVID-19 patient is provided herein. This method may comprise expressing an intracellular, barrier-enhancing therapeutic agent in the lung endothelium of the patient through a gene delivery tool. In some embodiments, the therapeutic agent is Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative or variant thereof. In some embodiments, the gene delivery tool may be an engineered mRNA or an expression vector as disclosed above and herein. [0079] Accordingly, the pharmaceutical composition disclosed above and herein may be delivered via various routes and to various sites in an animal body to achieve a particular effect, e.g., repair injured capillary and/or vascular tissues, strengthen epithelial barrier function to protect and/or accelerate alveolar repair following acute lung injury. Although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route depending on the pathological conditions to be treated. Local or sy stemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation, or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
[0080] The dosage administered is a therapeutically effective amount of the compound sufficient to result in amelioration of one or more symptoms of the ARDS or ALI or other disease as described herein, and varies depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired. Treatment of a subject with a therapeutically effective amount of FAK can include a single treatment or, preferably a series of treatments. Previously 500 micrograms/kg (or 0.5 mg/kg) isolated and purified FAKp was used in the in vivo experiments, which offers a good starting place for determining the ideal dose in humans or other mammals. In some embodiments, the effective amount of the pharmaceutical composition is an amount that produces from about 0. 1 to about 20 mg/kg body weight of FAK, as needed to normalize or maintain lung function.
[0081] In some embodiments, the therapeutic embodiments are carried out by administering the kits to a subject, e.g., a patient suffering from vascular and/or capillary tissue injury. The term “administering” means applying as a remedy, such as by the placement of a drug in a manner in which such drug would be received, e.g., intranasally or intratracheally, and be effective in carrying out its intended purpose. Alternatively, the drug may be administered systemically (e g., parenteral administration).
[0082] A “subject” or “patient” in whom administration of the therapeutic compound is an effective therapeutic regimen for a disease or disorder is preferably a human, but can be any animal, including a laboratory animal in the context of a trial or screening or activity experiment. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods, compounds and compositions are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, humans, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, non-human primates (e.g. cynomolgus monkeys), etc., avian species, such as chickens, turkeys, songbirds, etc., e.g., for veterinary' medical use. Preferably, the “subject” is a human patient afflicted with a disease to be treated, e.g., AL1 or ARDS.
[0083] In some embodiments, the delivery' tool delivers a therapeutically effective amount of the active agent to the subject. The term “therapeutically effective amount” as used herein refers to the amount of an active agent that is non-toxic but sufficient to provide the desired therapeutic effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like as known to those skilled in the art.
[0084] Treatment of a subject with the pharmaceutical composition or the kit disclosed above and herein may be monitored using methods known in the art. See, e.g., Schmidt et al., Trials, 18:116, 2017 (PMID: 28274276). The efficacy of treatment using the combination is preferably evaluated by examining the subject’s symptoms in a quantitative way, e.g., by noting a decrease in the frequency of adverse symptoms, behaviors, or attacks, or an increase in the time for sustained worsening of symptoms. In a successful treatment, the subject’s status w ill have improved (i.e., frequency of relapses will have decreased or the time to sustained progression will have increased).
[0085] The pharmaceutical composition or the kit disclosed above and herein are used to treat various pathological conditions that are associated with capillary cell injury, e.g., to prevent or repair an injured vascular and/or capillary tissue, to prevent or treat ALI or ARDS, to treat lung vascular leak in a COVID- 19 patient. The term “treating” is used herein generally includes the administration of the compound or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition (e.g., ALI or ARDS) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject’s condition (e.g., repair damaged lung epithelial tissue in the alveoli).
[0086] In some embodiments, the pharmaceutical composition may be administered with other pharmaceutically active materials for combination therapy, e.g., anti-inflammatory agents (e.g., dexamethasone, budesonide, formoterol, aspirin, acetaminophen and ibuprofen, or a combination thereof), or anti-infectives (e.g., paxlovid, molnupiravir, remdesivir, (l-lactam antibiotics, fluoroquinoline antibiotics, tetracyclines, aminoglycosides, or a combination thereof). [0087] It is to be understood that while the disclosure has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.
111. Examples
[0088] The following examples are illustrative in nature and are in no way intended to be limiting.
EXAMPLE 1
Alveolar Epithelial Type II (AT2) Cell Targeting with AAV6.2FF
[0089] To determine cell-type specificity of AAV6.2FF, intranasal (i.n.) administration was utilized to transfect C57BL/6 mice with AAV6.2FF capsids encoding genes that express luciferase, mCherry, GFP, or Cre recombinase. In particular, Albino C57BL/6 mouse received 1 x io11 viral genomes (vg) by i.n. delivery of AAV6.2FF capsids. Luciferin was administered via intraperitoneal (z.p.) injection to mice 1 hour prior to imaging. Luciferase expression in lung field by whole body IVIS imaging 1 day post transduction is shown in FIG. 1A. The expression was quantified at Day 0, and Days 1, 3, 7, 14, 21, 28, and 56 post transduction and plotted on the X-Y graph with X representing days post transduction and Y average radiance. Luciferin was administered via i.p. injection to mice 1 hour prior to imaging at each time point. The results of the quantitative longitudinal analysis of AAV6.2FF-luciferase expression in live mice following a single administration of virus are shown in FIG. IB.
[0090] FACS analysis of wild-type AT2 cells (EpCAM+/MCHII+) and AT2 cells 7 days post- intratracheal administration of AAV6.2FF-GFP was also done. The results show high degree of targeting (72.9%) (see, FIG. 1C).
[0091] Next, immunostaining of wild-type lung sections 2 weeks post intranasal delivery of AAV6.2FF-mCherry virus was done and the results are shown in FIG. ID. It was observed that there was a lack of mCherry expression in TUBA1 A1+ or CCSP+ bronchiolar epithelial cells, but there was robust expression in the epithelium of terminal bronchioles and alveoli. [0092] Further, FACS analysis of wild-type lung cells 2 weeks post mtranasal delivery of AAV6.2FF-mCherry virus revealed a high proportion of mCberry + targeting to AT2 cells (81%, proSPC+/NKX2. H7H0PX-), and minimal targeting to ATI (HOPX+) and immune (CD45+) cells (see, FIG. IE). [0093] Overall, about 60-80% of AT2 cells were transfected and led to expression of luciferase, mCherry, or GFP 37‘39. It was shown that AAV-driven transgene expression was detectable in AT2 cells within 24 hrs and remained high out to a minimum of 56 days following a single administration. Furthermore, only -20% of CD45+ immune cells were transduced. In addition, <0.5% of mCherry+ cells expressed cell-specific markers of alveolar type I (ATI) cells.
[0094] Immunofluorescent staining of GFP (green) and the AT2-specific marker pro-SPC (red) in AAV6.2FF-GFP transduced control lungs as well as in influenza A virus (I AV) infected/AAV6.2FF-GFP transduced lungs were further analyzed. In IAV infected/ AAV6.2FF- GFP group, mice were infected with IAV on Day 0 followed by AAV6.2FF- GFP on Day 3. Both groups were harvested on Day 6 post-AAV6.2FF-GFP transduction. The results show abundance of GFP/pro-SPC double positive AT2 cells in AAV6.2-GFP transduced lung (FIG. 2A) and persistence of AAV6.2FF-GFP signal in pro-SPC positive cells of IAV+AAV6.2FF- GFP lung (FIG. 2B). That is, GFP expression, driven by AAV6.2FF-GFP transduction, persisted in AT2 cells following lAV-induced acute lung injury in mice.
[0095] These studies show that intranasal (/.«.) or intratracheal (i.t.) delivery of AAV6.2FF encapsulated transgenes led to selective epithelial cell targeting in the alveoli with potent and durable expression of the transgene in AT2 cells, which suggests that AAV6.2FF targets AT2 cells with high specificity and efficiency.
EXAMPLE 2
Construction of AAV6.2FF-PTK2 Vector
[0096] AAV6.2FF virus encoding aN-terminal FLAG-tagged version of human FAK cDNA (NCBI reference sequence NM_153831.4; SEQ ID NO: 1) is to be amplified from human AT2 cells via PCR, sub-cloned into the pGEM®-T Easy shuttle vector (Promega), and sequenced. The sequence is to be confirmed afterwards. Diagram of a FAK-FLAG construct for AAV- driven expression in AT2 cells is shown in FIG. 3A. In this construct, human FAK cDNA is tagged with FLAG in frame atN-terminus. Known phosphorylation sites and associated interacting proteins are also shown in FIG. 3A.
[0097] FIG. 3B demonstrates the ability of AAV6.2FF-PTK2 to transfect AT2 cells and produce the FAK protein, wherein expression of FAK-FLAG protein driven from the pCASI AAV vector in transiently transfected HEK293 cells is shown. FAK-transfected cells stained with anti-FLAG antibody are shown as pointed by the arrows, non-transfected cells are designated with asterisks (*). Enrichment of FAK-FLAG subtending the plasma membranes is noted in this transformed cell line that lacks bona fide focal adhesion complexes. [0098] No identifiable information about the primary human AT2 (hAT2) cells from deidentified organ donors, whose lungs are not suitable for transplantation, is accessible. Human AT2 cells are to be isolated by elastase digestion and panning on IgG plates as previously described (Wang J, Edeen K, Manzer R, et al. Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro. American Journal of Respiratory Cell and Molecular Biology. 2007;36(6):661-668. doi: 10.1165/rcmb.2006-041 Ooc; Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010; 120(5): 1736-1748. doi: 10. 1172/jci38061). Epitope tagging of FAK must be performed at the amino terminus of the protein as C-termmal tags interfere with FAK localization and activity (Hildebrand JD, Schaller MD, Parsons JT. Identification of sequences required for the efficient localization of the focal adhesion kinase, ppl25FAK, to cellular focal adhesions. J Cell Biol. Nov 1993;123(4):993-1005. doi: 10.1083/jcb. 123.4.993; Williams AS, Trefts E, Lantier L, et al. Integrm-Lmked Kinase Is Necessary for the Development of Diet-Induced Hepatic Insulin Resistance. Diabetes. Feb 2017;66(2):325-334. doi: 10.2337/dbl 6-0484). Following sequence confirmation, FAK-FLAG is to be subcloned into the pCASI_MCS_WPRE transfer plasmid to generate AAV preparations. AAV6.2FF-PTK2 virus is to be generated by co-transfection of the pCASI FAK-FLAG WPRE plasmid with the AAV6.2FF packaging plasmid into HEK293 cells as previously described (Rindler TN, Brown KM, Stockman CA, et al. Efficient Transduction of Alveolar Type 2 Cells with Adeno- associated Virus for the Study of Lung Regeneration. American Journal of Respiratory Cell and Molecular Biology. 2021;65(l):l 18-121. doi:10.1165/rcmb.2021-00491e). AAV6.2FF- PTK2 particles are to be purified, and titers are to be determined via qPCR.
EXAMPLE 3
Confirmation of AT2 Targeting and Longitudinal Analysis of AAV6.2FF-PTK2 Expression [0099] A single dose of AAV6.2FF-PTK2 or a control AAV that expresses an mCherry fluorescent reporter, AAV6.2FF-mCherry (1 x 1012 viral genomes (vg)) is to be administered to wild-type C57BL/6J mice (Jackson Labs, 8-10 weeks old; n = 5 mice/sex/group) via mtranasal (i n.) administration and lung tissue is to be harvested post-administration at 1, 3, 12, and 24 weeks. To ensure scientific rigor and responsibility, all experiments are to be conducted with male and female mice to assess the possibility of sex being a relevant biological variable.
[0100] Experimental readouts to be performed at each time point include: 1) FACS analysis of enzymatically dispersed lung cells w ith identification of AT2 cells (EpCAM/CD326+ (CD326 (EpCAM) Antibody, APC (17-5791-82). htps://www.thermofisher.com/antibody/product/CD326-EpCAM-Antibody-clone-G8-8- Monoclonal/17-5791-82) MHCII+ (MHC Class II (I-A/I-E) Antibody, Alexa Fluor® 700 (56- 5321-82). htps://www.thermofisher.com/antibody/product/MHC-Class-II-I-A-I-E-Antibody- clone-M5-114-15-2-Monoclonal/56-5321-82)), hematopoietic cells (CD45 (CD45 Antibody , APC (17-0451-82). htps://www.thermofisher.com/antibody/product/CD45-Antibody-clone- 30-F11 -Monocl onal/17-0451-82)), endothelial cells (CD31 (APC Rat Anti-Mouse CD31. htps://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research- reagents/single-color-antibodies-ruo/apc-rat-anti-mouse-cd31.561814)), fibroblasts (PDGFRa) and AAV+ cells from control lungs (mCherry) or FAK+ cells or AAV6.2FF-PTK2 lungs (human specific FAK (FAK Antibody (MA5- 15644). htps://www.thermofisher.com/antibody/product/FAK-Antibody-clone-10H7E9- Monoclonal/MA5-15644)); and 2) immunofluorescent staining of fixed lung tissue for colocalization of AAV-driven FAK (FLAG staining, M2 clone (Monoclonal ANTI -FLAG® M2 antibody produced in mouse clone M2, purified immunoglobulin (Punfied IgGl subclass), buffered aqueous solution (10 rnM sodium phosphate, 150 mMNaCl, pH 7.4, containing 0.02% sodium azide) | Sigma-Aldrich, htp://www.sigmaaldrich.com/)) with antibody labeling of AT2 epithelial cells (proSFTPC (Anti-Prosurfactant Protein C (proSP-C) Antibody | AB3786. htps://www.merckmillipore.com/INTL/en/product/Anti-Prosurfactant-Protein-C- proSP-C-Antibody,MM_NF-AB3786)), airway epithelial cells including Club and ciliated cells (CCSP (Acetyl-alpha Tubulin (Lys40) Antibody (32-2700). htps://www.thermofisher.com/antibody/product/Acetyl-alpha-Tubulin-Lys40-Antibody-clone- 6-llB-l-Monoclonal/32-2700; Anti-CCSP - Rabbit. htps://www.sevenhillsbioreagents.com/products/anti-ccsp-rabbit)), hematopoietic cells and neutrophils (CD45 (Anti-CD45 antibody (abl0558) | Abeam, htps://www.abcam.com/cd45- antibody-abl0558.html), Ly6G (Recombinant Anti-Ly6g antibody [EPR22909-135] (ab238132) Abeam, htps://www.abcam.com/ly6g-antibody-epr22909-135-ab238132.html)), endothelial cells (endomucin (Mouse Endomucin Antibody. htps://www.mdsystems.com/products/mouse-endomucin-antibody_af4666)), fibroblasts (PDGFRa) and myofibroblasts (aSMA (Monoclonal Anti -Actin, a-Smooth Muscle clone 1A4, ascites fluid | Sigma-Aldrich, http://www.sigmaaldrich.com/)). Enzymatic lung cell dispersion, multiparameter flow cytometry, and co-localization immunostaining are routine procedures and can be performed from whole mouse lungs by utilizing the left lung for immunostaining and the right lung for FACS analysis (Rindler TN, Brown KM, Stockman CA, et al. Efficient Transduction of Alveolar Type 2 Cells with Adeno-associated Virus for the Study of Lung Regeneration. American Journal of Respiratory Cell and Molecular Biology. 2021;65(l):118- 121. doi: 10.1165/rcmb.2021-00491e; Redente EF, Chakraborty S, Sajuthi S, et al. Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. JCI Insight. Dec 8 2020;6(l)doi: 10.1172/jci.insight. l41618). 160 mice are included in this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 4 timepoints x 2 experimental replicates.
EXAMPLE 4
Longitudinal Analysis of Lung Function and Structure Post- Administration of AAV6 2FF- PTK2
[0101] To assess potential long-term consequences of AAV6.2FF-PTK2 expression in AT2 cells, a total of 40 wild-type C57BL/6J (8-10 weeks old, n = 10 mice/sex/group) are to receive i.n. administration of AAV6.2FF-PTK2 or AAV6.2FF-mCherry control virus and undergo monthly microCT scans for 6 months (Bruker Skyscanl276). Changes in parenchymal lung structure are to be assessed and any development of fibrotic or hyperplastic lesions are to be monitored. Images are to be acquired at 35 pm resolution using a 0.5 mm Aluminum filter and a 0.7 rotation step. Scans are to be reconstructed using NRecon software (Bruker. vl. 17.7) and analysis be performed using the CTAn software (Bruker vl.17.7.2). Readouts for the microCT cohort include measurement of aerated lung volume, tissue volume, Hounsfield Units and, if necessary', hyperplastic lesion counting (Redente EF, Black BP, Backos DS, et al. Persistent, Progressive Pulmonary' Fibrosis and Epithelial Remodeling in Mice. Am J Respir Cell Mol Biol. Jan 6 2021;doi:10.1165/rcmb.2020-0542MA). 40 mice are included in this study: 10 mice/sex x 2 sexes x 2 treatments/group x 1 harvest timepoint x 1 experimental replicate.
EXAMPLE 5
AAV6,2FF-Mediated Targeting of AT2 Cells to Repair Lung Epithelial Tissue [0102] AAV6.2FF is to be instilled into mouse lungs to selectively target expression of FAK to alveolar type 11 (AT2) cells. Importantly, FAK is endogenously expressed in AT2 cells so AAV-delivered FAK is likely to have the appropriate post-translational modifications. The viral vector includes the FAK cDNA encoding FAK protein. This pharmacologically active protein strengthens cell-cell junctions and enhances cell survival and is therefore expected to enhance epithelial repair in the context of lung injury. The overall goal is to develop a genedelivery treatment to repair damaged lung epithelial tissue in the alveoli. Treatment administration includes two delivery methods: intranasal (i.n.) instillation in liquid suspension, which is an established method, compared to a dry nanoparticulate aerosol, which would be particularly suitable for efficient deposition in the deep lung. The specific goal of this study is to develop an AAV6.2FF-PTK2 vector, test its efficacy in preclinical animal models of acute lung infection (ALI) and manufacture large-scale dry powder formulations of the vector that can ultimately be delivered in non-cold chain conditions for clinical use in the field.
[0103] Two distinct models of ALI are to be tested with the AAV6.2FF-PTK2 vector. In this study, AAV6.2FF is to be used to deliver FAK to AT2 cells in mice before and after lung injury using two preclinical mouse models: 1) an influenza A virus (I AV) infection model that mimics severe viral infection in humans; and 2) an acid instillation model that mimics gastric acid aspiration in humans. The IAV infectious model results in ALI/ ARDS 6-7 days postinfection characterized by decreased body weight (see, FIG. 4A), oxygen saturation levels and static lung compliance, increased inflammation (see, FIG. 4B) and widespread alveolar epithelial cell injury and death (see, FIGS. 5A-5C).
[0104] To establish an acute IAV infection mouse model, wild-type C57BL/6 mice were administered 1 x 102, 1 x 103, or 1 x 104 plaque forming units (PFU) of influenza A virus (PR/8/34) via intratracheal (i.t.) delivery. Body weight measurements over time following IAV administration demonstrate a dose-dependent reduction in body weights and partial recovery 14 days post-administration (FIG. 4A). All mice in the 1 x 104 PFU group were sacrificed due to extreme moribundity. Total bronchoalveolar (BAL) cells were counted 7 days post IAV administration. The results demonstrate a dose dependent increase in pulmonary inflammation (FIG. 4B)
[0105] Additionally, increased tissue density was observed in lAV-infected lungs (FIGS. 5A- 5C). Images of air-inflated, perfusion-fixed lungs in naive animals or after IAV infection by microCT (9 mm images) demonstrate increased tissue opacity in transverse sections (FIG. 5A), widespread tissue thickening and loss of alveolar structure in 3D-reconstructions (FIG. 5B), and increased lung tissue volume (top; FIG. 5C) and the increased percentage of total lung tissue/total lung volume (bottom; FIG. 5C).
[0106] To establish a non-infectious ALI model, wild-type C57BL/6 mice were administered 2 ml/kg (0.1 N) of HC1 via intratracheal (i.t.) delivery and harvested after 1 week.: Permeability of the alveolar epitheli al/endothelial barrier was assessed by albumin levels in the bronchoalveolar lavage (BAL) fluid. The results show that it was significantly increased in HC1 treated mice compared to naive mice (see, FIG. 6A). Quantification of differential cell counts from the BAL indicated a significant increase in inflammatory cells after HC1 treatment (see, FIG. 6B). Static lung compliance in HCl-treated mice as measured by flexiVent was decreased compared to naive mice (see, FIG. 6C). Representative Tri chrome staining of lung sections from naive vs. HCl-treated mice demonstrated a significant accumulation of inflammatory' cells in the alveolar spaces (see, FIG. 6D). [0107] Overall, the non-infectious ALI model established through HC1 delivery to the lungs to induce rapid and robust epithelial cell injury showed an increased lung permeability, decreased static lung compliance, and a robust inflammatory response dominated by macrophage and neutrophil infiltration, which transitioned to resolution after 3 weeks. Lung repair in this model is accompanied by a mild and resolving fibrotic response (Patel BV, Wilson MR, Takata M. Resolution of acute lung injury and inflammation: a translational mouse model. Eur Respir J. 2012;39(5): 1162-1170. doi: 10. 1183/09031936.00093911).
EXAMPLE 6
Determination of Association of FAK with Focal Adhesion Complexes in AT2 Cells [0108] One of the functions of FAK is the association with paxillin and vinculin in focal adhesion complexes (Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM. Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility. J Cell Biol. 2004;165(3):371-381. doi: 10.1083/jcb.200308011). First, the activation status of AAV6.2FF-PTK2 is to be confirmed by transducing wild-type C57BL/6J mice (8-10 weeks old; n = 3 mice/sex/group) with AAV6.2FF-PTK2 or AAV6.2FF-mCherry control vector and lung tissue is to be harvested 2 weeks post-transduction. AT2 cells are to be isolated using a dispase-based enzymatic lung digestion protocol that is well established (Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010;120(5): 1736-1748. doi: 10.1172/jci38061). FAK-FLAG is to be immunoprecipitated from AT2 cell lysates with anti-FLAG antibody and immunoblotted for active FAK (phospho-FAK Y397, Cell Signaling (Phospho-FAK (Tyr397) Antibody. https://www.cellsignal.com/products/primary-antibodies/phospho-fak-tyr397-antibody/3283)). Then, it is to be determined if AAV-driven FAK associates with focal adhesion complex proteins (e.g., paxillin and vinculin) in AT2 cells similar to its well described role in endothelial cells. To determine association of AAV-driven FAK with paxillin and vinculin in isolated AT2 cells, immunoprecipitation of phospho-FAK from AAV6.2FF-PTK2 or AAV6.2FF-mCherry control transduced mice is done, followed by immunoblotting for paxillin (BD Biosciences (Purified Mouse Anti-Paxillin. https://www.bdbiosciences.com/en- us/products/reagents/microscopy-imaging-reagents/immunofluorescence-reagents/purified- mouse-anti-paxillin.610051)) and vinculin (SigmaAldrich (Anti -Vinculin antibody, Mouse monoclonal clone VIN-11-5, purified from hybridoma cell culture | Sigma- Aldrich. http://www.sigmaaldrich.com/)). FAK/paxillin/vinculin association will be further confirmed via confocal microscopy of immune-stained lung sections from AAV6.2FF-PTK2 treated mice at designated time points. Lungs from n = 3 mice per group will be analyzed for the coimmunoprecipitation and immunofluorescence experiments. Both experiments will be replicated twice to ensure reproducibility. 72 mice are included in this study: 3 mice/sex x 2 sexes x 2 treatments/group x 3 readouts (IP phospho-FAK, co-IP and immunostaining) x 2 experimental replicates.
EXAMPLE 7 Determination of Functional Effect of AAV-FAK in Cell Injury Models
[0109] The functional effect of FAK expression is to be determined in mock treated and AAV6.2FF-PTK2 infected primary mouse AT2 (mAT2) cell cultures by 1) measuring trans- epithelial resistance (TER) in cell monolayers, at baseline and following monolayer perturbation, 2) determining their ability to repair a monolayer after a scratch wound, and 3) measuring the susceptibility to bleomycin-induced apoptosis. For TER studies, primary mAT2 cells are to be isolated and immediately transduced with AAV6.2FF-PTK2 or AAV6.2FF- mCherry control (1000 vg/cell). The transduced cells will then be seeded on Matngel/collagen coated transwell filters (Millicell-PCF) as previously described (Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010;120(5): 1736-1748. doi: 10. 1172/j ci38061 ). mAT2 cells will be grown to confluence and the TER values will be measured using a Millicell ERS-2 epithelial volt-ohm meter (Merck Millipore) (Ma Y, Semba S, Khan MRI, et al. Focal adhesion kinase regulates intestinal epithelial barrier function via redistribution of tight junction. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2013;l 832(1): 151-159. doi: 10.1016/j.bbadis.2012.10.006). Differences in monolayer baseline resistance between the mock treated and AAV6.2FF-PTK2 transduced groups will be measured.
[0110] To determine if overexpression of FAK prevents or attenuates decreased TER levels by tight junction disassembly, AT2 cell monolayers will be subjected to a calcium switch assay (Zheng B, Cantley LC. Regulation of epithelial tight junction assembly and disassembly by AMP-activated protein kinase. Proceedings of the National Academy of Sciences. 2007;104(3):819-822. doi: 10. 1073/pnas.0610157104; Playford M, Vadali K, Cai X, Burridge K, Schaller M. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho. Exp Cell Res. 2008;314(17):3187-3197. doi: 10.1016/j.yexcr.2008.08.010). Switching of confluent epithelial monolayers from normal calcium medium (Spinner MEM, Sigma, containing 1.8 mM CaC12 and 5% dialyzed FBS) to no calcium medium (Spinner MEM (Purified Mouse Anti-Paxillin. https://www.bdbiosciences.com/en-us/products/reagents/microscopy-imaging- reagents/immunofluorescence-reagents/purified-mouse-anti-paxillin.610051) containing 5% dialyzed FBS) causes rapid loss of cell-cell tight junctions, while re-addition of calcium to the media induces junction assembly and re-establishment of trans-epithelial resistance. mAT2 cell monolayers will be grown in normal calcium medium then switched to no calcium-free media for 120 mm and TER measurements will be acquired every 15 mm as described (Zheng B, Cantley LC. Regulation of epithelial tight junction assembly and disassembly by AMP- activated protein kinase. Proceedings of the National Academy of Sciences. 2007 ; 104(3): 819- 822. doi: 10. 1073/pnas.0610157104; Playford M, Vadali K, Cai X, Burridge K, Schaller M. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho. Exp Cell Res. 2008;314(17):3187-3197. doi: 10.1016/j.yexcr.2008.08.010). TER values will be normalized to baseline prior to switching to low calcium medium. Parallel cultures in normal calcium medium and no calcium medium 1 -hour post-switch will be fixed with paraformaldehyde for immunostaining with ZO-1 (ThermoFisher (Anti-Vinculin antibody, Mouse monoclonal clone VIN-11-5, purified from hybndoma cell culture | Sigma-Aldrich. http://wvvw.sigmaaldrich.com/)) and occludin (ThermoFisher (Occludin Antibody (33-1500). https://www.thermofisher.corn/antibody/product/Occludin-Antibody-clone-OC-3F10- Monoclonal/33-1500)) to confirm disruption of tight junction disassembly.
[0111] For scratch-wound assays, transduced mAT2 cells will be grown to confluence on Matrigel/collagen-coated transwells and will undergo scratch wounding with a pipette tip on the apical surface. Cells will be imaged immediately after wound induction and 24 hours postwound. Wound width will be calculated using ImageJ software (NIH) and wound repair will be expressed as the percentage of initial wound remaining after 24 hours, as previously described (McClendon J, Jansing NL, Redente EF, et al. Hypoxia-Inducible Factor la Signaling Promotes Repair of the Alveolar Epithelium after Acute Lung Injury. The American Journal of Pathology. 2017;187(8):1772-1786. doi: 10. 1016/j.ajpath.2017.04.012).
[0112] To determine how the overexpression of FAK affects the apoptotic potential of the AT2 cultures, AAV6.2FF-PTK2 and AAV6.2FF-mCherry control transduced cells will be treated with bleomycin sulfate, an apoptotic mimetic drug (100 pg/ml for 48 hours) (Tounekti O, Pron G, J. B, Mir LM. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Journal of Cancer Research. 1993;88(22):5462-5469.; Karam H, Hurbain-Kosmath I, Housset B. Direct toxic effect of bleomycin on alveolar type 2 cells. Toxicol Lett. 1995;76(2): 155-163. doi: 10.1016/0378- 4274(94)03207-n). Apoptosis will be determined by caspase 3/7 and 8 activation (Caspase- Glo®, Promega (Caspase-Gio® 3/7 Assay System | Caspase 3 Activity Assay | Apoptosis Detection, https://worldwide.promega.com/products/cell-health-assays/apoptosis- assays/caspase_glo-3_7-assay-systems/?catNum=G8090)) as previously described (Bamberg A, Redente EF, Groshong SD, et al. Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis. Am J Respir Crit CareMed. 2018;198(7):914-927. doi: 10.1164/rccm.201707-1497oc).
[0113] Primary human AT2 (hAT2) cells will be isolated from lungs of deidentified organ donors whose lungs are not suitable for transplantation. The cells may be frozen down for subsequent thawing and culturing. After thawing, hAT2 cells will be transduced with AAV6.2FF-PTK2 or AAV6.2FF-mCherry control (1000 vg/cell) and cultured on Matrigel/collagen-coated inserts for two days for adherence and then six days under air/liquid (ALI) interface conditions in the presence of FGF7 and dexamethasone (Wang J, Edeen K, Manzer R, et al. Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro. American Journal of Respiratory Cell and Molecular Biology.
2007;36(6): 661-668. doi: 10.1165/rcmb.2006-0410oc; Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM. LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J Clin Invest. 2010;120(5):1736-1748. doi: 10. 1172/jci38061). All cell culture experiments will be repeated at least three times to ensure reproducibility. Experiments with human AT2 cells will utilize cells from at least five individual donor lungs. Differences between conditions will be analyzed with parametric (t-test and ANOVA with Newman-Keuls post hoc analysis) or nonparametric (Wilcoxon Signed Rank) statistical analyses as appropriate. All data will be presented as the mean ± SD. A p- value of <0.05 will be considered significant. 108 mice are needed to isolate primary AT2 cells: 3 mice/sex x 2 sexes x 2 treatments/group x 3 readouts (TER, scratch wound and apoptosis) x 3 experimental replicates.
EXAMPLE 8 Determination of Composition of AAV6.2FF-PTK2 Drug Substance
[0114] AAV6.2FF capsids containing the FAK transgene will be produced in 200 mL shake flasks by co-transfection of HEK293 cells with same plasmids used to generate AAV in adherent cells, i.e., the pCASI FAK-FLAG WPRE plasmid with the AAV6.2FF packaging plasmid. HEK293 cells from Mass Biologies will be expanded in Hy clone protein-free and animal-derived component-free (ADCF) cell culture media. pH, temperature, and stir rate will be altered in order to determine the optimal conditions for FAK transgene packaging into the viral capsid.
[0115] Shake flask conditions designed to generate 1014 viral capsids per flask will be repeated 20 times and purified from HEK293 using cell lysis, filtration, affinity chromatography, and anion exchange chromatography. The later two column chromatographic steps will reduce empty capsids, nucleic acid contaminants, host cell protein, host cell DNA and media components.
[0116] The purified AAV6.2FF-PTK2 viral capsids will be characterized by droplet digital PCR (ddPCR), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), Western bloting, and analytical ultracentrifugation. These analytical methods will assess transgene vector production levels, viral capsid quality, and the percent of empty capsids, respectively. As needed, dynamic light scatering (DLS) or asymmetrical flow field-flow fractionation (AF4) will also be used, equipped with MALS detection, to measure the size distribution of the viral capsids before and after reconstitution of lyophilized powders (Zorato S, Weiss VU, Friedbacher G, et al. Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy. ACS Omega. 2021 ;6(25): 16428- 16437. doi:10.1021/acsomega.lc01443). Denaturing and non-denaturmg agarose gels will be used to determine any variability in the transgene DNA.
EXAMPLE 9
Formulation Development of AAV6.2FF-PTK2 Lyophilized Powder
[0117] Formulations will be prepared according to methods known in the art (e.g., Zhang et al., International Journal of Pharmaceutics, 2021(606): 120912) and evaluated for their stability as a function of pH and buffer composition. Samples will be evaluated at time zero (tO) for content, osmolality, and pH. The purities of each composition will be measured using asymmetric-Flow Field Flow Fractionation monitored with a multi-angle light scatering detector (AF4-MALS) and anion exchange chromatography (AEX-HPLC). Samples will also be tested after one week at 40°C, two weeks at 25°C, and four weeks at 5°C.
[0118] Once the optimal buffer and pH are determined, a second round of screening will be conducted. 8-12 formulations will be screened to evaluate the impact of other excipients on liquid stability, with a focus on selecting materials that would be used in a lyophilized product. Storage conditions and testing procedures will be the same as those used in the first round of screening.
[0119] 6-8 potential lyophilized formulations will be identified for testing. Each of these will contain 1012 vg/mL and will include pharmaceutically acceptable excipients that are capable of forming completely amorphous matrices (using sucrose and/or trehalose). In addition, the effects of the following will be checked: 1) crystalline bulking agent (e.g., mannitol), 2) buffer (e.g., TRIS), 3) tonicity modifier (e.g., glycine, or sodium chloride) and 4) plasticizing agent (e.g., glycerol). Each selected formulation will be lyophilized using conditions that allow all preparations to fully dry and will be evaluated for moisture content, glass transition temperature (Tg), crystallinity, and reconstitution time at time zero (tO).
[0120] Storage stability samples will be characterized at tO, following two weeks at 40°C storage and after four weeks at 25°C. Stability will be judged based on changes in visual appearance and reconstitution times. Stability of the viral capsids will be monitored following reconstitution using AEX-HPLC and AF4-MALS. Formulations demonstrating the best behavior will be remade to demonstrate reproducibility and to support testing in the cellular and animal models.
EXAMPLE 10
Determination of Effect of AAV6.2FF-PTK2 in Murine Models of Acute Lung Injury [0121] Male and female wild-type C57BL/6J mice will be used in both models. For both models, AAV6.2FF-PTK2 or AAAV6.2FF-mCherry as a control will be delivered using two strategies: (1) by intranasal (/.«.) delivery, and (2) by aerosolization (1 x 1012 viral genomes/mouse). Mice will be sacrificed at the indicated endpoints and assessed for established ALI outcomes. Experimental readouts include: 1) assessment of clinical parameters in live animals, and 2) assessment of injury and inflammation markers in post-mortem samples. A total of 5 mice/sex will be used with two experimental replicates, to provide a >80% power to detect an effect size (between experimental groups and sex of mouse) of 1.6 with a Gaussian distribution (2 -sided t-test, p<0.05). Differences will be analyzed between conditions with parametric (t-test and ANOVA with Newman-Keuls post hoc analysis) or nonparametric (Wilcoxon Signed Rank) statistical analyses as appropriate. To determine an effect of sex, a 2 way-ANOVA will be used. All data will be presented as the mean ± SEM. A p-value of <0.05 will be considered significant. All of the proposed post-mortem readouts can be performed on the same animal by utilizing separate lobes for each assessment, due to the fact that the administration route results in uniform lung injury (Redente EF, Chakraborty S, Sajuthi S, et al. Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. JCI Insight. Dec 8 2020;6(l)doi: 10.1172/jci.insight. l41618; Hartzler GO, Holmes DR, Osbom MJ. Patient-activated transvenous cardiac stimulation for the treatment of supraventricular and ventricular tachycardia. The American Journal of Cardiology. 1981;47(4):903-909. doi: 10. 1016/0002-9149(81)90192-2). The left lung can be tied off and inflated for histology/immunostaining, while the right lung will undergo BAL. Post BAL, the upper right lobe will be processed for protein, and the lower right lobes will be digested for FACS and cell sorting. Utilizing the lungs in this manner will aid in reducing animal use and allow for a comprehensive, multi-parameter study of each animal.
[0122] To quantitate the effect of AAV6.2FF-PTK2, the following four measurement will be performed:!) daily body weight measurements as described in Example 5 above; 2) daily oxygen saturation measurements via pulse oximetry on conscious animals (MouseOX Plus, STARR Life Sciences); 3) microCT (Skyscan 1276, Bruker Inc.) assessments for aerated lung volume at the study termination as described in Example 5 above; and 4) lung function measurements via //e.wVent (Scireq Technologies) to quantitate static pulmonary compliance at the study termination as described in Example 5 above.
[0123] The following assays will be performed: 1) quantitation of bronchoalveolar lavage (BAL) cell counts and differentials; 2) quantitation of pro-inflammatory cytokine levels in BAL fluid (IL-6, TNFa, active TGFP); 3) lung permeability assays (IgM (IgM Mouse Uncoated ELISA Kit with Plates - Invitrogen. https://www.thermofisher.com/elisa/product/IgM-Mouse-Uncoated-ELISA-Kit-with-Plates/88- 50470-22), albumin (Mouse Albumin ELISA Kit (Colorimetric). https://www.novusbio.com/products/albumin-elisa-kit_nbp2-60484) in BALF) (Redente EF, Keith RC, Janssen W, et al. Tumor Necrosis Factor-a Accelerates the Resolution of Established Pulmonary Fibrosis in Mice by Targeting Profibrotic Lung Macrophages. American Journal of Respiratory Cell and Molecular Biology . 2014;50(4):825-837. doi: 10.1165/rcmb.2013-0386oc; Redente EF, Jacobsen KM, Solomon JJ, et al. Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis.
American journal of physiology Lung cellular and molecular physiology. 2011/10// 2011;301(4):L510-8. doi: 10.1152/ajplung.00122.2011); 4) histology (H&E, trichrome, mean linear intercept values (MLI)); 5) analysis of single cell populations by multi-parameter flow cytometry 60; 6) Co-immunofluorescence for cell linages: (AT2, ATI, fibroblasts/myofibroblasts, immune cells, and endothelial cells (as described in Example 5 above), proliferation markers (Ki67 (Anti-Ki67 antibody KO Tested (ab!5580) | Abeam. https://www.abcam.com/ki67-antibody-abl5580.html) and/or EdU incorporation (Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye.)) and FAK (total (FAK Antibody.) and FAK-phsophoY397 (Phospho-FAK (Tyr397) Antibody. https://www.cellsignal.com/products/primary-antibodies/phospho-fak-tyr397-antibody/3283)) on fixed frozen and fixed paraffin-embedded lung sections; 7) determination of apoptosis via cleaved caspase 3 or TUNEL immunostaining (Redente EF, Chakraborty S, Sajuthi S, et al. Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. JCI Insight. Dec 8 2020;6(l)doi: 10.1172/jci.insight. l41618; Wynes MW, Edelman BL, Kostyk AG, et al. Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis. The Journal of Immunology. 201 l;187(l):527-537. doi:10.4049/jimmunol.1100447); 8) spatial-distance mapping (SDM) between AT2 cells (proSFTPC+) and fibroblasts (PDGFRa+), as previously described (Liarski VM, Kaverina N, Chang A, et al. Cell Distance Mapping Identifies Functional T Follicular Helper Cells in Inflamed Human Renal Tissue. Science Translational Medicine. 2014;6(230):230ra46-230ra46. doi:10.1126/scitranslmed.3008146; Zepp JA, Zacharias WJ, Frank DB, et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell. 2017;170(6):l 134-1148. elO. doi: 10.1016/j.cell.2017.07.034; and 9) Western blot analysis of total FAK and FAK- phosphoY397 from sorted AT2 cells (EpCAM+/MHCII+) Hasegawa K, Sato A, Tanimura K, et al. Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation. Respiratory Research. 2017;18(l)doi: 10. 1186/sl2931-017-0635- 5. These studies will be conducted in accordance with ATS guidelines for histological analysis (Hsia CCW, Hyde DM, Ochs M, Weibel ER. An Official Research Policy Statement of the American Thoracic Society /European Respiratory Society: Standards for Quantitative Assessment of Lung Structure. Am J Respir Crit Care Med. 2010;181(4):394-418. doi: 10.1164/rccm.200809-1522st) and also as previously reported (Redente EF, Black BP, Backos DS, et al. Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice. Am J Respir Cell Mol Biol. Jan 6 2021;doi:10.1165/rcmb.2020-0542MA; Redente EF, Keith RC, Janssen W, et al. Tumor Necrosis Factor-a Accelerates the Resolution of Established Pulmonary Fibrosis in Mice by Targeting Profibrotic Lung Macrophages.
American Journal of Respiratory Cell and Molecular Biology. 2014;50(4):825-837. doi: 10.1165/rcmb.2013-0386oc; Redente EF, Jacobsen KM, Solomon JJ, et al. Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis. American journal of physiology Lung cellular and molecular physiology. 2011/10// 2011 ;301(4):L510-8. doi: 10.1152/ajplung.00122.2011).
[0124] In order to quantitate the targeting of AAV6.2FF-PTK2 to specific lung cell populations and to assess the level of cell loss after injury and the extent of protection with AAV6.2FF-PTK2, procedures have been optimized to generate single cell suspensions from enzymatically dispersed lung tissues (Rindler TN, Brown KM, Stockman CA, et al. Efficient Transduction of Alveolar Type 2 Cells with Adeno-associated Virus for the Study of Lung Regeneration. American Journal of Respiratory Cell and Molecular Biology. 2021 ;65(1): 118- 121. doi: 10.1165/rcmb.2021-00491e; Redente EF, Chakraborty S, Sajuthi S, et al. Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis. JCI Insight. Dec 8 2020;6(l)doi: 10. 1172/j ci. insight. 141618; Redente EF, Keith RC, Janssen W, et al. Tumor Necrosis Factor-a Accelerates the Resolution of Established Pulmonary Fibrosis in Mice by Targeting Profibrotic Lung Macrophages. American Journal of Respiratory Cell and Molecular Biology . 2014;50(4):825-837. doi: 10.1165/rcmb.2013-0386oc; Riemondy KA, Jansing NL, Jiang P, et al. Single cell RNA sequencing identifies TGFbeta as a key regenerative cue following LPS-induced lung injury. JCI Insight. Mar 26 2019;5doi: 10.1172/jci. insight. 123637) and multi-parameter flow cytometry protocols have been established using cell surface markers analyzed on a BD Fortessa™ instrument. The flow strategy involves initial identification immune (CD45 and Ly6G) and endothelial (CD31) cells, AT2 cells (EpCAM+MHCII+) and fibroblasts (PDGFRa) along with mCherry or FLAG detection of the AAV6.2FF-PTK2.
EXAMPLE 11
Determination of Role of FAK in Injury Prevention in Murine Injury Models [0125] To determine if AAV6.2FF-PTK2 expression in AT2 cells prior to infection protects against ALI, mice will be administered AAV6.2FF-PTK2 or AAV -mCherry by the i.n. route (1 x 1012 viral genomes/mouse) Iweek prior to disease initiation. To initiate disease, wild-type C57BL/6J mice (8-10 weeks; n = 5/sex/group) will be administered H1N1 IAV (mouse adapted H1N1 PR8 strain, A/Puerto Rico/8-34-9VMC3/1934, BEI Resources NR-29028) by intratracheal (i t.) instillation. Two independent doses of IAV will model moderate (1 x 102 PFU) and lethal (1 x 104 PFU) ALI as described in Example 5 above. Mice will be euthanized 1-week or 3-week post-IAV administration for the moderate dose and at prior to moribundity for the lethal dose. Tissues will be harvested for experimental readouts detailed above. These experiments will be repeated via dry aerosolization exposure based on the time and dose determined from the i.n. delivery' experiments that provides the most protection.
[0126] 80 mice will be included for i.n. administration (moderate dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV doses x 2 endpoints x 2 expenmental replicates. 40 mice will be included for i.n. administration (lethal dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoints x 2 experimental replicates.
[0127] 40 mice will be included for aerosol administration model: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoints x 2 experimental replicates. [0128] To determine if AAV6.2FF-PTK2 expression in AT2 cells prior to sterile lung injury, mice will first be administered AAV-FAK or AAV-mCherry by the i.n. route (1 x 1012 viral genomes/mouse). One week later, mice will be administered HC1 (2 ml/kg, 0. IN) in saline by i.t. instillation. Mice will be euthanized 1-week and 3-week post-HCl administration and tissues will be harvested for experimental readouts detailed above. 80 mice will be included in this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF- mCherry) x 1 acid dose x 2 endpoints x 2 experimental replicates.
EXAMPLE 12
Determination of Role of FAK in Injury Reversal in Murine Injury Models [0129] Injury can occur prior to therapeutic intervention, thus it is desirable to determine the effectiveness of AAV6.2FF-PTK2 expression in AT2 cells after established ALL Mice will first be administered H1N1 IAV (mouse adapted H1N1 PR8 strain, A/Puerto Rico/8-34- 9VMC3/1934, BEI Resources NR-29028) by i.t. instillation. Three days after receiving IAV, AAV6.2FF-PTK2 or AAV-mCherry will be given by the i.n. route (1 x 1012 viral genomes/mouse). Two independent doses of IAV will model moderate (1 x 102 PFU) and lethal (1 x 104 PFU) ALI injury as described in Example 5 above. Mice will be euthanized 1- week and 3-week post-IAV administration and tissues will be harvested for experimental readouts. These experiments will be repeated with the final formation and development from the large scale AAV6.2FF-PTK2 formulation via dry aerosolization exposure based on the time and dose determined from the i.n. delivery experiments that provides the most protection. Additionally, it will be determined if overexpression of FAK initiates de novo development of cancer or pulmonary fibrosis in the context of ALI. A separate cohort of mice (n = 5 mice/sex/group) will receive a moderate dose of IAV followed by AAV6.2FF-PTK2 or AAV- mCherry control and will undergo monthly CT scans over 6 months.
[0130] 80 mice will be used for i.n. administration (moderate dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 2 endpoints x 2 experimental replicates. 40 mice will be used for i.n. administration (lethal dose): 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCheny) x 1 IAV dose x 1 endpoint x 2 experimental replicates. 40 mice will be used for aerosol administration model: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 IAV dose x 1 endpoint x 2 experimental replicates. 20 mice will be used for moderate dose longitudinal CT analysis: 5 mice/sex x 2 sexes x 2 treatments/group.
[0131] To determine if AAV6.2FF-PTK2 expression in AT2 cells after the initial of sterile lung injury, mice will first be administered HC1 at 2 ml/kg (0.1N) in saline by i.t. instillation. Three days after receiving HC1, AAV6.2FF-PTK2 or AAV-mCherry will be given by the i.n. route (1 x 1012 viral genomes/mouse). Mice will be euthanized 1-week and 3-week post-HCl administration and tissues will be harvested for experimental readouts. 80 mice will be included for this study: 5 mice/sex x 2 sexes x 2 treatments/group (AAV6.2FF-PTK2 or AAV6.2FF-mCherry) x 1 acid dose x 2 endpoints x 2 experimental replicates.
EXAMPLE 13
AAV6 2FF-Driven FAK Expression In vitro and In vivo
[0132] Western blot analysis of FAK and pFAK in AAV-FAK transduced AT2 cells in vivo - C57B16 WT mice (10 weeks of age) were transduced with AAV6.2FF-FAK-FLAG (2xl012 vg, intranasally) or administered HBSS vehicle control (non-transduced). 14 days posttransduction, primary AT2 cells were isolated by dispase digestion of the lung followed by negative selection over a magnetic column with anti-CD45, -CD16/32, -CD31, -Teri 19, -CD40 antibodies. AT2 cells were lysed in modified RIPA buffer and FAK-FLAG was immunoprecipitated form the lysates with anti-FLAG agarose beads. Protein was eluted off of the beads with 2X laemmli buffer. Lysates were run on a 10-20% tn cine polyacrylamide gel, transferred to PVDF membrane, and immunoblotted with primary antibodies against phospho- FAK (Y397) followed by incubated with an HRP-conjugated secondary antibody. The blot was then incubated with HRP substrate and imaged. Following imaging, the blot was stripped with stripping buffer, blocked in milk, and incubated with primary antibodies against total FAK followed by incubation with an HRP-conjugated secondary antibody. Blot was then incubated with HRP substrate and imaged. Each lane represents AT2 cells from a single animal.
[0133] The results are shown in FIG. 9A, wherein FLAG immunoprecipitation followed by Western blot of FAK was detected by anti-phospho FAK (Y397) and total FAK antibodies in whole AT2 cell lysates of AAV6.2FF-FAK-FLAG mouse (transduced) vs. no signal was detected in non-transduced mouse.
[0134] Immunofluorescence of FAK-FLAG expression in AAV-FAK transduced AT2 cells in vivo - C57B16 WT mice (10 weeks of age) were transduced with AAV6.2FF-FAK-FLAG (2x1012 vg, intranasally) or administered HBSS vehicle control (non-transduced). 14 days posttransduction, lungs were perfused with PBS and inflation fixed with 4% PFA at 25 cm of pressure. Lung tissue was dehydrated, embedded in paraffin and 7 pm sections were cut. Sections were rehydrated, blocked, and incubated with primary antibodies against the FLAG epitope (FLAG), pro-surfactant protein C (pro-SPC) and nuclei w ere counterstained with DAPI. Sections were then incubated with fluorophore-conjugated secondary antibodies and mounted in imaging media. Images were obtained on an EVOS7000 fluorescent microscope. Images are representative of one section from a single animal. The results are shown in FIG. 9B, with the arrows pointing to FAK-FLAG in pro-SPC+ AT2 cells from a non-transduced mouse (top panel) and a transduced mouse (bottom panel).
[0135] Flow cytometry of FAK-FLAG expression in AAV-FAK transduced AT2 cells in vivo - C57B16 WT mice (10 weeks of age) were transduced with AAV 6.2FF -FAK-FLAG (2xl012 vg, intranasally) or administered HBSS vehicle control (non-transduced). 14 days posttransduction, primary AT2 cells were isolated by dispase digestion of the lung followed by negative selection over a magnetic column with anti-CD45, -CD16/32, -CD31, -Teri 19, -CD40 antibodies. Non-permeabilized cells were stained with anti-EpCAM and anti-MHCII antibodies conjugated to fluorophores, then permeabilized with saponin, blocked and stained with anti-FLAG antibody. Cells were washed and immediately analyzed by FACS analysis. Gates were set based on no stain and single-stained controls; 100,000 events were collected and analyzed for the triple-stained experimental samples. Data shown are representative plots from one sample per group. The results are shown in FIG. 9C. Therein, the dashed boxes represent lack of FLAG expression in non-EpCAM+ AT2 cells.
EXAMPLE 14
BAL Immune Cell Analysis in Mice Transduced with AAV-FAK and Challenged with LPS [0136] C57B16 WT mice (10 weeks of age, n = 5-6 mice per group) were transduced with AAV6.2FF-GFP control or AAV6.2FF-FAK-FLAG (2xl012 vg, intranasally). 14 days posttransduction, mice were challenged with LPS (50 pg, intratracheal) and harvested on Day 17. Bronchoalveolar lavage (BAL) was performed on mice to recover inflammatory cells from the airspaces. Total cell counts were performed on each sample via manual hemocytometer counting under a light microscope. Counts were performed by two separate lab personnel and the data were averaged. The results are shown in FIG. 10A. Similar immune response was observed in both groups.
[0137] For differential cell analysis, cytospins were prepared for each sample, fixed, and stained with the DiffQuik kit. Samples were analyzed under a light microscope and immune cells were manually differentiated by size and morphology, and were stained by two lab personnel and data were averaged. A total of 250-300 cells were counted per slide, with 1 slide analyzed per mouse. The results are shown in FIGS. 10B-10D (neutrophils, macrophages, and lymphocytes counts, respectively). Similar immune cell profiles were observed in both groups, dominated primarily by a neutrophilic response. Similar immune response was observed in both groups. EXAMPLE 15
Analysis of AAV-Driven Transgene Expression in Lung Cells Post-LPS Challenge [0138] C57B16 WT mice (10 weeks of age, n = 6 mice total) were divided into two groups: control and experimental groups (LPS challenged). The experimental group was challenged with LPS (50 pg intratracheally) and transduced with AAV6.2FF-GFP (2xl012 vg, intranasally) 3 days post-LPS. The control group did not receive LPS and were also transduced with AAV6.2FF-GFP. Ten days post LPS, mice were harvested, and lung cells were isolated via antibody and magnetic bead isolation. Non-permeabilized cells were stained with anti- CD45, anti-CD31, anti-PDGFRa, anti-EpCAM, and anti-MHCII antibodies. Cells were washed and immediately analyzed by FACS analysis. Gates were set based on no stain and singlestained controls; 100,000 events were collected and analyzed for the triple-stained experimental samples. Each data point represents AT2 cells from a single animal analyzed by enzyme-based dispersion of the lung, follow ed by antibody staining and FACS analysis. [0139] The results are shown in FTG. 11 A-l IB, wherein the data illustrated in FIG. 11 A are representative FACS histogram plots from one sample per group, and the data in FIG. 11B are quantitative data for GFP expression in EpCAM+/MHCII+ AT2 cells with each data point representing cells from a single animal. The data in FIG. 11A show that similar GFP expression level in AT2 cells with respect to cell number (counts) and GFP intensity (GFP fluorescence) was observed in naive and LPS-inflamed lungs, and minimal targeting of AAV- GFP in immune cell (CD45+), fibroblasts (PDGFRa+), and endothelial cells (CD31+) was observed in the LPS inflamed lung. The data in FIG. 11B show that no difference in AT2 targeting of AAV was observed in naive or LPS treated mice.
[0140] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

Claims

LAIMED: A construct, comprising: a nucleic acid sequence encoding Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof. The construct of claim 1 , wherein the construct is selected from the group consisting of an engineered mRNA encoding the FAK protein, fragment, derivative or variant, and an expression vector encoding the FAK protein, fragment, derivative, or variant. The construct of claim 2, wherein the expression vector is a viral vector. The construct of claim 3, wherein the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno-associated viral (AAV) vector. The construct of claim 4, wherein the viral vector is an AAV vector. The construct of claim 5, wherein the viral vector is an AAV2 vector, an AAV6 vector, or a combination thereof. The construct of any one of claims 1-6, wherein the nucleic acid sequence is at least 75% identical to SEQ ID NO: 1. The construct of any one of claims 1-7, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO: 1. The construct of any one of claims 1-8, wherein the nucleic acid sequence is 100% identical to SEQ ID NO: 1. The construct of any one of claims 1-9, wherein the construct comprises a nucleic acid sequence at least 75% identical to SEQ ID NO: 2. The construct of any one of claims 1-10, wherein the construct comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 2. The construct of any one of claims 1-11, wherein the construct comprises the nucleic acid sequence as set forth in SEQ ID NO: 2. A pharmaceutical composition, comprising: the construct of any one of claims 1-12. The pharmaceutical composition of claim 13, further comprising: a reagent capable of assisting delivery of the construct to a recipient cell. The pharmaceutical composition of claim 14, wherein the reagent is polyethylenimine. The pharmaceutical composition of any one of claims 13-15, further comprising: a stabilizing excipient. The pharmaceutical composition of claim 16, wherein the stabilizing excipient is selected from the group consisting of citrate, glycine, phosphate, tris, histidine leucine, raffinose, trehalose, mannitol, lactose and trileucine. The pharmaceutical composition of any one of claims 13-17, wherein the composition is in a form of a dry powder or a liquid. The pharmaceutical composition of claim 18, wherein the dry powder is produced by lyophilization or spray -drying. A kit, comprising: the construct of any one of claims 1-12 or the pharmaceutical composition of any one of claims 13-19 and instructions for use. A method of preventing or repairing an injured vascular and/or capillary tissue in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical composition of any one of claims 13-19 or using the kit of claim 20. The method of claim 21, wherein the subject suffers from an injury that involves endothelial cells and/or epithelial cells. The method of claim 22, wherein the injury involves endothelial cells found in the capillary tissue. The method of claim 22, wherein the injury involves epithelial cells that make up alveolar cells in the deep lung. The method of any one of claims 21-24, wherein the injury is a lung injury, a hemorrhagic stroke, an acute or chronic kidney disease, or a capillary leak syndrome. The method of claim 25, wherein the lung injury' is acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), emphysema, idiopathic pulmonary fibrosis, or asthma. The method of claim 26, wherein the lung injury' is caused by SARS-CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury'. The method of claim 27, wherein the lung pathogen(s) include pseudomonas aeruginosa. The method of claim 27, wherein the internal organ injury includes acute pancreatitis. The method of any one of claims 21-29 wherein the pharmaceutical composition is administered intranasally, intratracheally, or parenterally. The method of claim 30, wherein the pharmaceutical composition is administered via intratracheal or intranasal instillation of a liquid, nebuhzation of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol. The method of any one of claims 19-31, wherein the effective amount is an amount that produces from about 0.1 to about 20 mg/kg body weight of FAK. A method of preventing or treating acute lung injury (ALI)Zacute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical composition of any one of claims 13-19 or using the kit of claim 20. The method of claim 33, wherein the lung injury' is caused by SARS-CoV-2, lung pathogen(s), inhalation of excess smoke or water, and/or internal organ injury'. The method of claim 34, wherein the lung pathogen(s) include pseudomonas aeruginosa. The method of claim 34, wherein the internal organ injury includes acute pancreatitis. The method of any one of claims 33-36, wherein the pharmaceutical composition is administered intranasally, intratracheally, or parenterally. The method of claim 37, wherein the pharmaceutical composition is administered via intratracheal or intranasal instillation of a liquid, nebulization of a liquid aerosol, or using a dry powder inhaler (DPI) of a dry aerosol. The method of any one of claims 33-38, wherein the effective amount is an amount that produces from about 0.1 to about 20 mg/kg body weight of FAK. A method of treating lung vascular leak in a COVID- 19 patient, the method comprising: expressing an intracellular, barrier-enhancing therapeutic agent in the lung endothelium of the patient through a gene delivery' tool. The method of claim 40, wherein the therapeutic agent is Focal Adhesion Kinase (FAK) protein or a biologically active fragment, derivative, or variant thereof. The method of claim 40 or claim 41, wherein the gene delivery tool is selected from the group consisting of an engineered mRNA encoding the FAK protein, fragment, derivative, or variant, and an expression vector encoding the FAK protein, fragment, derivative, or variant The method of claim 42, wherein the expression vector is a viral vector. The method of claim 43, wherein the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, a herpes viral vector, a vaccinia viral vector, and an adeno-associated viral (AAV) vector. The method of claim 44, wherein the viral vector is an AAV vector The method of claim 45, wherein the viral vector is an AAV2 vector, an AAV6 vector, or a combination thereof. The method of any one of claims 44-46, wherein the AAV vector comprises a nucleic acid sequence encoding FAK protein or a biologically active fragment, derivative, or variant thereof. The method of claim 47, wherein the nucleic acid sequence is at least 75% identical to SEQ ID NO: 1. The method of claim 48, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO: 1. The method of any one of claims 47-49, wherein the nucleic acid sequence is 100% identical to SEQ ID NO: 1. The method of any one of claims 44-50, wherein the AAV vector comprises a nucleic acid sequence at least 75% identical to SEQ ID NO: 2. The method of any one of claims 44-51, wherein the AAV vector comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 2. The method of any one of claims 44-52, wherein the AAV vector comprises the nucleic acid sequence as set forth in SEQ ID NO: 2.
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