Compositions and methods for gene delivery to the respiratory tract and / or lung

Abstract

To provide a new therapeutic strategy for dealing with fundamental genetic / molecular defects of hereditary pulmonary disease patients.SOLUTION: Provided are recombinant nucleic acids comprising one or more polynucleotides encoding a polypeptide (e.g., an inhaled therapeutic polypeptide, such as a human alpha-1-antitrypsin polypeptide); viruses comprising the recombinant nucleic acids; compositions and formulations comprising the recombinant nucleic acids and / or viruses; methods of use thereof (e.g., to deliver a polypeptide to one or more cells of the respiratory tract and / or for the treatment of a disease affecting the lungs, such as alpha-1-antitrypsin deficiency); and articles of manufacture or kits thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 951,523, filed December 20, 2019, the entire contents of which are incorporated herein by reference.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 761342001340SeqList.txt, Recorded: December 18, 2020, Size: 366KB).

[0003] Technical Field The present disclosure relates, in part, to recombinant nucleic acids comprising one or more polynucleotides encoding a polypeptide (e.g., an inhaled therapeutic polypeptide such as a human alpha-1-antitrypsin polypeptide); viruses comprising the recombinant nucleic acids; pharmaceutical compositions and formulations thereof; and methods of use thereof (e.g., for delivering the polypeptide to one or more cells of the respiratory tract and / or for treating diseases affecting the lungs, such as alpha-1-antitrypsin deficiency). [Background technology]

[0004] background Inherited lung diseases result in significant lifetime morbidity and mortality. Approximately 22% of all pediatric hospital admissions are for respiratory disorders, and congenital causes of respiratory disease are often fatal. Despite significant advances in clinical care and a better understanding of pathogenic mechanisms, definitive treatment options for these patients are lacking, and therapeutic approaches are often limited to supportive and compassionate care or lung transplantation. Therefore, novel therapeutic strategies are needed to address patients' underlying genetic / molecular defects.

[0005] All references cited herein, including patent applications, patent publications, non-patent literature, and NCBI / UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] overview To meet these and other needs, provided herein are recombinant nucleic acids (e.g., recombinant herpesvirus genomes) encoding one or more polypeptides (e.g., one or more inhaled therapeutic polypeptides) for use in viruses (e.g., herpesviruses), pharmaceutical compositions and formulations, medicaments, and / or methods useful for delivering one or more polypeptides to one or more cells of the respiratory tract (e.g., airway epithelial cells) and / or for treating one or more diseases affecting the airways and / or lungs in a subject in need thereof.

[0007] Accordingly, certain aspects of the present disclosure relate to recombinant herpesvirus genomes comprising one or more polynucleotides encoding inhaled therapeutic polypeptides. In some embodiments, the recombinant herpesvirus genome comprises two or more polynucleotides encoding inhaled therapeutic polypeptides. In some embodiments, the recombinant herpesvirus genome is replication-competent. In some embodiments, the recombinant herpesvirus genome is replication-deficient. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding inhaled therapeutic polypeptides within one or more viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is selected from a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, an Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof.

[0008] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

[0009] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome is engineered to reduce or eliminate expression of one or more virulent herpes simplex virus genes. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation. In some embodiments, the inactivating mutation is present in a herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from infected cell protein (ICP)0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and UL55. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the tk gene.In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the joint region. In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the joint region.

[0010] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in one or more viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in one or both ICP4 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in the ICP22 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in the UL41 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in one or both ICP0 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide in the ICP27 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide within the ICP47 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide within the tk viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding an inhaled therapeutic polypeptide within the UL55 viral locus.

[0011] In some embodiments that may be combined with any of the preceding embodiments, the inhaled therapeutic polypeptide is an alpha-1-antitrypsin polypeptide, a sodium-dependent phosphate transport protein 2B polypeptide, a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, a dynein intermediate chain 2 axonemal polypeptide, a radial spokehead 1 homolog polypeptide, a coiled-coil main-containing protein 114 polypeptide, a radial spokehead protein 4 homolog A polypeptide, a dynein assembly factor 1 axonemal polypeptide, a dynein assembly factor 2 axonemal polypeptide, a leucine-rich repeat-containing protein 6 polypeptide, a Peptide, pulmonary surfactant-associated protein B polypeptide, pulmonary surfactant-associated protein C polypeptide, homeobox protein Nkx-2.1 polypeptide, ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide, granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, bone morphogenetic protein receptor type 2 polypeptide, sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, serine / threonine protein kinase receptor R3 polypeptide, endoglin polypeptide, mother's against decapentaplegic homolog 9 polypeptide, caveolin-1 polypeptide, potassium channel subfamily K member 3 polypeptide, eIF-2-alpha kinase GCN2 polypeptide, pulmonary surfactant-associated protein A2 polypeptide, telomerase reverse transcriptase polypeptide, dyskerin polypeptide, regulator of telomere elongation helicase 1 polypeptide, poly(A)-specific ribonuclease PARN polypeptide, TERF1-interacting nuclear factor 2 polypeptide,In some embodiments that may be combined with any of the preceding embodiments, the inhaled therapeutic polypeptide is a human polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3-46. In some embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5-21. In some embodiments, the inhaled therapeutic polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.The inhaled therapeutic polypeptide comprises a sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 22-27. In some embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 28-35. In some embodiments, the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 23, 25, and 36-46.

[0012] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome has reduced cytotoxicity when introduced into a target cell compared to a corresponding wild-type herpesvirus genome. In some embodiments, the target cell is a human cell. In some embodiments, the target cell is a respiratory cell. In some embodiments, the target cell is an airway epithelial cell.

[0013] Another aspect of the present disclosure relates to a herpesvirus comprising any of the recombinant herpesvirus genomes described herein. In some embodiments, the herpesvirus is replication-competent. In some embodiments, the herpesvirus is replication-deficient. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus has reduced cytotoxicity compared to a corresponding wild-type herpesvirus. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is selected from herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is a herpes simplex virus. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is HSV-1.

[0014] Another aspect of the present disclosure relates to a pharmaceutical composition comprising any of the recombinant herpesvirus genomes and / or any of the herpesviruses described herein and a pharmaceutically acceptable carrier. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local, or epicutaneous administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for oral, intranasal, intratracheal, or inhalation administration. In some embodiments, the pharmaceutical composition is suitable for intranasal or inhalation administration. In some embodiments, the pharmaceutical composition is suitable for inhalation administration. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for use in a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, an electrohydrodynamic aerosol device, or any combination thereof. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is suitable for use in a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a phosphate buffer. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises glycerol. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a lipid carrier. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition comprises a nanoparticle carrier.

[0015] Another aspect of the present disclosure relates to the use of any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes), recombinant viruses (e.g., recombinant herpesviruses), and / or pharmaceutical compositions described herein as pharmaceutical agents.

[0016] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes), recombinant viruses (e.g., recombinant herpesviruses), and / or pharmaceutical compositions described herein in therapeutic methods.

[0017] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes), recombinant viruses (e.g., recombinant herpesviruses), and / or pharmaceutical compositions described herein in the preparation of a medicament for treating one or more lung diseases (e.g., genetic lung diseases).

[0018] Another aspect of the present disclosure relates to a method for expressing, enhancing, increasing, augmenting, and / or supplementing the level of an inhaled therapeutic polypeptide in one or more respiratory cells, airway epithelial cells, and / or lung cells in a subject, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the subject is afflicted with a chronic lung disease. In some embodiments that may be combined with any of the preceding embodiments, the subject is human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered orally, intranasally, intratracheally, or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered intranasally or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0019] Another aspect of the present disclosure relates to a method of reducing or inhibiting progressive lung destruction in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the subject has a chronic lung disease. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered orally, intranasally, intratracheally, or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered intranasally or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0020] Another aspect of the present disclosure relates to a method for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of a disease affecting the airways and / or lungs in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered orally, intranasally, intratracheally, or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered intranasally or via inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0021] Another aspect of the present disclosure relates to a method for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of alpha-1-antitrypsin deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SERPINA1 gene. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding an alpha-1-antitrypsin polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0022] Another aspect of the present disclosure relates to a method for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of alveolar microlithiasis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses and / or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SLC34A2 gene. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding a sodium-dependent phosphate transport protein 2B polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0023] Another aspect of the present disclosure relates to a method of providing prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of primary ciliary dyskinesia in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes selected from DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1, DNAAF2, and LRRC6. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm-associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, a dynein intermediate chain 2 axonemal polypeptide, a radial spokehead 1 homolog polypeptide, a coiled-coil domain-containing protein 114 polypeptide, a radial spokehead protein 4 homolog A polypeptide, a dynein assembly factor 1 axonemal polypeptide, a dynein assembly factor 2 axonemal polypeptide, and a leucine-rich repeat-containing protein 6 polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation.In some embodiments, the herpesvirus or pharmaceutical composition is administered to a subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to a subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0024] Another aspect of the present disclosure relates to a method for providing prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of congenital pulmonary alveolar proteinosis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from SFTPB, SFTPC, NKX2-1, ABCA3, CSF2RB, and CSF2RA. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from a pulmonary surfactant-associated protein B polypeptide, a pulmonary surfactant-associated protein C polypeptide, a homeobox protein Nkx-2.1 polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a cytokine receptor common subunit beta polypeptide, and a granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide. In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0025] Another aspect of the present disclosure relates to methods for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary arterial hypertension in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from BMPR2, ATP2A2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, and EIF2AK4. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from a bone morphogenetic protein receptor type 2 polypeptide, a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, a serine / threonine protein kinase receptor R3 polypeptide, an endoglin polypeptide, a mother's against decapentaplegic homolog 9 polypeptide, a caveolin-1 polypeptide, a potassium channel subfamily K member 3 polypeptide, and an eIF-2-alpha kinase GCN2 polypeptide. In some embodiments that can be combined with any of the preceding embodiments, the subject is a human. In some embodiments that can be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0026] Another aspect of the present disclosure relates to a method of providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary fibrosis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant herpesviruses or pharmaceutical compositions described herein. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes selected from SFTPC, ABCA3, SFTPA2, TERT, TERC, DKC1, RTEL, PARN, TINF2, NAF1, MUC5B, DSP, STN1, and DPP9. In some embodiments, the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from a pulmonary surfactant-associated protein C polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a pulmonary surfactant-associated protein A2 polypeptide, a telomerase reverse transcriptase polypeptide, a dyskerin polypeptide, a regulator of telomere elongation helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, an H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin-5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, and a dipeptidyl peptidase 9 polypeptide. In some embodiments that can be combined with any of the preceding embodiments, the subject is a human. In some embodiments that can be combined with any of the preceding embodiments, the herpesvirus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject intranasally or via inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered to the subject via inhalation, hi some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device.In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0027] Another aspect of the present disclosure relates to a method for delivering a polypeptide to one or more cells of the respiratory tract of a subject, the method comprising administering to the subject a pharmaceutical composition comprising: (a) a herpesvirus comprising a recombinant herpesvirus genome; and (b) a pharmaceutically acceptable carrier, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding the polypeptide. In some embodiments, the subject is afflicted with an inherited lung disease. In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, the disease is selected from alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis.

[0028] In some embodiments, the herpesvirus is replication-competent. In some embodiments, the herpesvirus is replication-deficient. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus has reduced cytotoxicity compared to a corresponding wild-type herpesvirus. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is selected from herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus is a herpes simplex virus. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is HSV-1.

[0029] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is selected from a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, an Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

[0030] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome is engineered to reduce or eliminate expression of one or more virulent herpes simplex virus genes. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation. In some embodiments, the inactivating mutation is present in a herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from infected cell protein (ICP)0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and UL55. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the tk gene.In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the joint region. In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the joint region.

[0031] In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in one or more viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in one or both ICP4 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in an ICP22 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in a UL41 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in one or both ICP0 viral loci. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in an ICP27 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide in an ICP47 viral locus. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide within the tk viral locus.In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding a polypeptide within the UL55 viral locus.

[0032] In some embodiments that may be combined with any of the preceding embodiments, the subject is a human. In some embodiments that may be combined with any of the preceding embodiments, the pharmaceutical composition is administered orally, intranasally, intratracheally, or by inhalation to the subject. In some embodiments, the pharmaceutical composition is administered intranasally or by inhalation to the subject. In some embodiments, the pharmaceutical composition is administered by inhalation to the subject. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0033] Other aspects of the present disclosure relate to articles of manufacture or kits that include any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein, and instructions for their administration. [The present invention 1001] A recombinant herpesvirus genome comprising one or more polynucleotides encoding an inhaled therapeutic polypeptide. [The present invention 1002] 1001. A replication-competent recombinant herpesvirus genome of the present invention. [The present invention 1003] 1001. A recombinant herpesvirus genome of the present invention that is replication-deficient. [The present invention 1004] A recombinant herpesvirus genome of any of claims 1001 to 1003 of the present invention, selected from the group consisting of a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivative thereof. [The present invention 1005] The recombinant herpesvirus genome of any one of 1001 to 1004 of the present invention, which is a recombinant herpes simplex virus genome. [The present invention 1006] 1005. The recombinant herpesvirus genome of the present invention, wherein said recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. [The present invention 1007] The recombinant herpesvirus genome of invention 1005 or invention 1006, wherein the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome. [The present invention 1008] 8. The recombinant herpes simplex virus genome of any one of claims 1004 to 1007, wherein the recombinant herpes simplex virus genome has been engineered to reduce or eliminate expression of one or more virulent herpes simplex virus genes. [The present invention 1009] 9. The recombinant herpesvirus genome of any one of claims 1004 to 1008, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation. [The present invention 1010] 1009. The recombinant herpesvirus genome of the present invention, wherein the inactivating mutation is present in a herpes simplex virus gene. [The present invention 1011] 10. The recombinant herpesvirus genome of the present invention, wherein the inactivating mutation is a deletion in the coding sequence of the herpes simplex virus gene. [The present invention 1012] The recombinant herpesvirus genome of the present invention 1010 or 1011, wherein the herpes simplex virus gene is selected from the group consisting of infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41 and UL55. [The present invention 1013] 1012. The recombinant herpes simplex virus genome of the present invention, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of said ICP4 gene. [The present invention 1014] The recombinant herpesvirus genome of invention 1012 or invention 1013, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. [The present invention 1015] The recombinant herpesvirus genome of any one of claims 1012 to 1014, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. [The present invention 1016] 10. The recombinant herpes simplex virus genome of any one of claims 1012 to 1015, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of the ICP0 gene. [The present invention 1017] The recombinant herpesvirus genome of any one of 1012 to 1016, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. [The present invention 1018] The recombinant herpesvirus genome of any one of claims 1012 to 1017, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. [The present invention 1019] The recombinant herpesvirus genome of any one of claims 1012 to 1018, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. [The present invention 1020] The recombinant herpes simplex virus genome of any of claims 1004 to 1019, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding the inhaled therapeutic polypeptide within one or both of the ICP4 viral loci. [The present invention 1021] The recombinant herpesvirus genome of any one of claims 1004 to 1020, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the inhaled therapeutic polypeptide within the ICP22 viral locus. [The present invention 1022] The recombinant herpesvirus genome of any one of claims 1004 to 1021, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the inhalation therapeutic polypeptide within the UL41 viral locus. [The present invention 1023] The recombinant herpes simplex virus genome of any of claims 1004 to 1022, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding the inhaled therapeutic polypeptide within one or both of the ICP0 viral loci. [The present invention 1024] The recombinant herpesvirus genome of any one of claims 1004 to 1023, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the inhaled therapeutic polypeptide within the ICP27 viral locus. [The present invention 1025] The recombinant herpesvirus genome of any one of claims 1004 to 1024, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the inhaled therapeutic polypeptide within the ICP47 viral locus. [The present invention 1026] The recombinant herpes simplex virus genome of any one of claims 1004 to 1025, wherein the recombinant herpes simplex virus genome comprises the one or more polynucleotides encoding the inhalation therapeutic polypeptide within the UL55 viral locus. [The present invention 1027] The inhaled therapeutic polypeptide may be an alpha-1-antitrypsin polypeptide, a sodium-dependent phosphate transport protein 2B polypeptide, a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm-associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, a dynein intermediate chain 2 axonemal polypeptide, a radial spoke head 1 homolog polypeptide, a coiled-coil domain-containing protein 114 polypeptide, a radial spoke head protein 4 homolog A polypeptide, a dynein assembly factor 1 axonemal polypeptide, a dynein assembly factor 2 axonemal polypeptide, a leucine-rich repeat-containing protein 6 polypeptide, a pulmonary surfactant-associated protein B polypeptide, a pulmonary surfactant-associated protein C polypeptide, or a homeobox protein Nkx-2.1 polypeptide, ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide, granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, bone morphogenetic protein receptor type 2 polypeptide, sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, serine / threonine protein kinase receptor R3 polypeptide, endoglin polypeptide, mother's against decapentaplegic homolog 9 polypeptide, caveolin-1 polypeptide, potassium channel subfamily K member 3 polypeptide, eIF-2-alpha kinase GCN2 polypeptide The recombinant herpesvirus genome of any of claims 1001 to 1026, wherein the recombinant herpesvirus genome is selected from the group consisting of a pulmonary surfactant-associated protein A2 polypeptide, a telomerase reverse transcriptase polypeptide, a dyskerin polypeptide, a regulator of telomere elongation helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, an H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin-5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, and a dipeptidyl peptidase 9 polypeptide. [The present invention 1028] The recombinant herpesvirus genome of any one of claims 1001 to 1027, wherein the polypeptide for inhalation therapy is a human polypeptide. [The present invention 1029] A recombinant herpesvirus genome of any of the present inventions 1001 to 1028, wherein the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 46. [The present invention 1030] The recombinant herpesvirus genome of any one of claims 1001 to 1029, wherein the polypeptide for inhalation therapy is an alpha-1-antitrypsin polypeptide. [The present invention 1031] The recombinant herpesvirus genome of any one of claims 1001 to 1030, wherein the polypeptide for inhalation therapy is a human alpha-1-antitrypsin polypeptide. [The present invention 1032] A recombinant herpesvirus genome of any of claims 1001 to 1031, wherein the inhaled therapeutic polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. [The present invention 1033] A recombinant herpesvirus genome according to any one of claims 1001 to 1032, which has reduced cytotoxicity when introduced into a target cell, as compared with a corresponding wild-type herpesvirus genome. [The present invention 1034] The recombinant herpesvirus genome of the present invention, wherein the target cell is a human cell. [This invention 1035] The recombinant herpesvirus genome of claim 1033 or 1034, wherein the target cell is a respiratory cell. [The present invention 1036] The recombinant herpesvirus genome of any one of claims 1033 to 1035, wherein the target cell is an airway epithelial cell. [This invention 1037] A herpesvirus comprising the recombinant herpesvirus genome of any one of 1001 to 1036 of the present invention. [The present invention 1038] 1037. A herpesvirus of the present invention having replication ability. [This invention 1039] 1037. The herpesvirus of the present invention, which is replication-deficient. [The present invention 1040] The herpesvirus of any one of 1037 to 1039 of the present invention, which has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. [The present invention 1041] Any of the herpesviruses of the present invention 1037 to 1040, which is selected from the group consisting of herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus. [The present invention 1042] The herpesvirus of any one of claims 1037 to 1041, which is a herpes simplex virus. [This invention 1043] The herpesvirus of invention 1041 or 1042, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof. [This invention 1044] The herpesvirus of any one of claims 1041 to 1043, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1). [This invention 1045] A recombinant herpesvirus genome according to any one of the present inventions 1001 to 1036 or a herpesvirus according to any one of the present inventions 1037 to 1044, and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising: [The present invention 1046] A pharmaceutical composition of the present invention 1045 suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, local, or epicutaneous administration. [This invention 1047] A pharmaceutical composition of invention 1045 or invention 1046, suitable for oral, intranasal, intratracheal, or inhalation administration. [This invention 1048] 8. The pharmaceutical composition of any one of claims 1045 to 1047, which is suitable for intranasal or inhalation administration. [This invention 1049] 9. The pharmaceutical composition of any one of claims 1045 to 1048, which is suitable for inhalation administration. [The present invention 1050] 1049. The pharmaceutical composition of any of claims 1045 to 1049, which is suitable for use in a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, an electrohydrodynamic aerosol device, or any combination thereof. [This invention 1051] The pharmaceutical composition of any one of claims 1045 to 1050, which is suitable for use in a nebulizer. [This invention 1052] The pharmaceutical composition of the present invention 1051, wherein the nebulizer is a vibrating mesh nebulizer. [This invention 1053] The pharmaceutical composition of any one of claims 1045 to 1052, which comprises a phosphate buffer solution. [This invention 1054] The pharmaceutical composition of any one of claims 1045 to 1053, which contains glycerol. [This invention 1055] The pharmaceutical composition of any one of claims 1045 to 1054, comprising a lipid carrier. [The present invention 1056] A pharmaceutical composition according to any one of claims 1045 to 1055, comprising a nanoparticle carrier. [This invention 1057] A herpesvirus according to any one of claims 1037 to 1044 or a pharmaceutical composition according to any one of claims 1045 to 1056 for use as a medical drug. [This invention 1058] A herpesvirus according to any one of claims 1037 to 1044 or a pharmaceutical composition according to any one of claims 1045 to 1056 for use in therapy. [This invention 1059] Use of any of the herpesviruses of the present inventions 1037 to 1044 or any of the pharmaceutical compositions of the present inventions 1045 to 1056 in the manufacture of a medicament for treating one or more inherited lung diseases. [The present invention 1060] 1. A method of expressing, enhancing, increasing, augmenting, and / or supplementing levels of an inhaled therapeutic polypeptide in one or more respiratory cells, airway epithelial cells, and / or lung cells in a subject, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1061] 1. A method of reducing or inhibiting progressive lung destruction in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1062] The method of claim 1060 or claim 1061, wherein the subject is suffering from a chronic lung disease. [This invention 1063] 1. A method for providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of a disease affecting the airways and / or lungs in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1064] 1. A method for providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of alpha-1-antitrypsin deficiency in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1065] The method of claim 1064, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SERPINA1 gene. [The present invention 1066] 106. The method of claim 1064 or claim 1065, wherein said recombinant herpesvirus genome comprises one or more polynucleotides encoding an alpha-1-antitrypsin polypeptide. [This invention 1067] 1. A method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of alveolar microlithiasis in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [The present invention 1068] 1067. The method of claim 1067, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SLC34A2 gene. [This invention 1069] 1069. The method of claim 1067 or claim 1068, wherein said recombinant herpesvirus genome comprises one or more polynucleotides encoding a sodium-dependent phosphate transport protein 2B polypeptide. [The present invention 1070] 1. A method for providing preventative, palliative, or therapeutic alleviation of one or more signs or symptoms of primary ciliary dyskinesia in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1071] 1070. The method of claim 1070, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from the group consisting of DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1, DNAAF2, and LRRC6. [This invention 1072] 1072. The method of claim 1070 or 1071, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from the group consisting of a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, a dynein intermediate chain 2 axonemal polypeptide, a radial spoke head 1 homolog polypeptide, a coiled-coil domain-containing protein 114 polypeptide, a radial spoke head protein 4 homolog A polypeptide, a dynein assembly factor 1 axonemal polypeptide, a dynein assembly factor 2 axonemal polypeptide, and a leucine-rich repeat-containing protein 6 polypeptide. [This invention 1073] 1. A method for providing preventative, palliative, or therapeutic alleviation of one or more signs or symptoms of congenital pulmonary alveolar proteinosis in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1074] 1073. The method of claim 1073, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from the group consisting of SFTPB, SFTPC, NKX2-1, ABCA3, CSF2RB, and CSF2RA. [This invention 1075] The method of claim 1073 or claim 1074, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from the group consisting of a pulmonary surfactant-associated protein B polypeptide, a pulmonary surfactant-associated protein C polypeptide, a homeobox protein Nkx-2.1 polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a cytokine receptor common subunit beta polypeptide, and a granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide. [This invention 1076] 1. A method for providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary arterial hypertension in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [This invention 1077] 1076. The method of claim 1076, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from the group consisting of BMPR2, ATP2A2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, and EIF2AK4. [This invention 1078] The method of claim 1076 or claim 1077, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from the group consisting of a bone morphogenetic protein receptor type 2 polypeptide, a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, a serine / threonine protein kinase receptor R3 polypeptide, an endoglin polypeptide, a mothers-against-decapentaplegic homolog 9 polypeptide, a caveolin-1 polypeptide, a potassium channel subfamily K member 3 polypeptide, and an eIF-2-alpha kinase GCN2 polypeptide. [This invention 1079] 1. A method for providing preventative, palliative, or therapeutic alleviation of one or more signs or symptoms of pulmonary fibrosis in a subject in need thereof, comprising: The method, which comprises administering to the subject an effective amount of any of the herpesviruses of the present inventions Nos. 1037 to 1044 or any of the pharmaceutical compositions of the present inventions Nos. 1045 to 1056. [The present invention 1080] 1079. The method of claim 1079, wherein the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in one or more genes selected from the group consisting of SFTPC, ABCA3, SFTPA2, TERT, TERC, DKC1, RTEL, PARN, TINF2, NAF1, MUC5B, DSP, STN1, and DPP9. [This invention 1081] The method of claim 1079 or 1080, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding a polypeptide selected from the group consisting of a pulmonary surfactant-associated protein C polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a pulmonary surfactant-associated protein A2 polypeptide, a telomerase reverse transcriptase polypeptide, a dyskerin polypeptide, a regulator of telomere elongation helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, an H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin-5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, and a dipeptidyl peptidase 9 polypeptide. [This invention 1082] The method of any one of claims 1060 to 1081, wherein the subject is a human. [This invention 1083] The method of any of claims 1060 to 1082, wherein said herpesvirus or pharmaceutical composition is administered to said subject orally, intranasally, intratracheally, or via inhalation. [This invention 1084] The method of any of claims 1060 to 1083, wherein said herpesvirus or pharmaceutical composition is administered to said subject intranasally or via inhalation. [This invention 1085] The method of any of claims 1060 to 1084, wherein said herpesvirus or pharmaceutical composition is administered to said subject via inhalation. [The present invention 1086] 1086. The method of any of claims 1060 to 1085, wherein the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. [This invention 1087] The method of any one of claims 1060 to 1086, wherein the herpesvirus or pharmaceutical composition is administered using a nebulizer. [This invention 1088] 1087. The method of claim 1087, wherein the nebulizer is a vibrating mesh nebulizer. [This invention 1089] 1. A method for delivering a polypeptide to one or more cells of the respiratory tract of a subject, comprising: The subject: (a) a herpesvirus comprising a recombinant herpesvirus genome, wherein the recombinant herpesvirus genome comprises one or more polynucleotides encoding the polypeptide; (b) a pharmaceutically acceptable carrier; The method comprises administering a pharmaceutical composition comprising: [The present invention 1090] The method of claim 1089, wherein said subject is suffering from a disease affecting the airways and / or lungs. [This invention 1091] 1090. The method of claim 1090, wherein said disease is selected from the group consisting of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. [This invention 1092] 1092. The method of any one of claims 1089 to 1091, wherein the herpesvirus has replication ability. [This invention 1093] 1092. The method of any one of claims 1089 to 1091, wherein said herpesvirus is replication-deficient. [This invention 1094] The method of any one of claims 1089 to 1093, wherein the herpes virus is a herpes simplex virus. [This invention 1095] The method of any one of claims 1089 to 1094, wherein the recombinant herpesvirus genome is a recombinant herpes simplex virus genome. [This invention 1096] 1095. The method of claim 1095, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation. [This invention 1097] 1096. The method of claim 1096, wherein said inactivating mutation is present in a herpes simplex virus gene. [This invention 1098] 1097. The method of claim 1097, wherein said inactivating mutation is a deletion in the coding sequence of said herpes simplex virus gene. [This invention 1099] 1097. The method of claim 1098, wherein the herpes simplex virus gene is selected from the group consisting of infected cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55. [The present invention 1100] 1099. The method of claim 1099, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of said ICP4 gene. [The present invention 1101] The method of claim 1099 or claim 1100, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in said ICP22 gene. [The present invention 1102] 1102. The method of any one of claims 1099 to 1101, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in said UL41 gene. [The present invention 1103] 13. The method of any of claims 1099 to 1102, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of said ICP0 gene. [The present invention 1104] 1104. The method of any one of claims 1099 to 1103, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in said ICP27 gene. [This invention 1105] 15. The method of any one of claims 1099 to 1104, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. [The present invention 1106] 16. The method of any one of claims 1099 to 1105, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. [This invention 1107] The method of any of claims 1089 to 1106, wherein said pharmaceutical composition is administered to said subject orally, intranasally, intratracheally, or via inhalation. [This invention 1108] 8. The method of any one of claims 1089 to 1107, wherein said pharmaceutical composition is administered to said subject intranasally or via inhalation. [This invention 1109] 1108. The method of any one of claims 1089 to 1108, wherein said pharmaceutical composition is administered to said subject via inhalation. [The present invention 1110] 1109. The method of any of claims 1089 to 1109, wherein said pharmaceutical composition is administered to said subject via inhalation. [The present invention 1111] The method of any of claims 1089 to 1110, wherein said pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. [The present invention 1112] The method of any one of claims 1089 to 1111, wherein the pharmaceutical composition is administered using a nebulizer. [The present invention 1113] 1112. The method of claim 1112, wherein said nebulizer is a vibrating mesh nebulizer. [Brief explanation of the drawings]

[0034] [Figure 1-1]Figures 1A-1I show schematic diagrams of wild-type and modified herpes simplex virus genomes. Figure 1A shows a wild-type herpes simplex virus genome. Figure 1B shows a schematic diagram of wild-type and modified herpes simplex virus genomes. A modified herpes simplex virus genome containing a deletion of the coding sequence for ICP4 (both copies) and having an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into each of the ICP4 loci. Figure 1C shows a schematic diagram of wild-type and modified herpes simplex virus genomes. A modified herpes simplex virus genome containing a deletion of the coding sequence for ICP4 (both copies) and UL41 and having an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into each of the ICP4 loci. Figure 1D shows a schematic diagram of wild-type and modified herpes simplex virus genomes. A modified herpes simplex virus genome containing a deletion of the coding sequence for ICP4 (both copies) and UL41 and having an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into the UL41 locus. Figure 1E shows a schematic diagram of wild-type and modified herpes simplex virus genomes. This shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies) and ICP22, with an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into each of the ICP4 loci. Figure 1F shows a schematic diagram of wild-type and modified herpes simplex virus genomes. This shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies) and ICP22, with an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into each of the ICP22 loci. Figure 1G shows a schematic diagram of wild-type and modified herpes simplex virus genomes. This shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, with an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into each of the ICP4 loci. Figure 1H shows a schematic diagram of wild-type and modified herpes simplex virus genomes.Figure 1I shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, and having an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into the UL41 locus. Figure 1I shows a schematic diagram of wild-type and modified herpes simplex virus genomes. Figure 1I shows a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, and having an expression cassette containing a nucleic acid encoding an inhaled therapeutic polypeptide integrated into the ICP22 locus. [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 1-4] See description of Figure 1-1. [Figure 1-5] See description of Figure 1-1. [Figure 2] Figure 2 shows vector transduction and transgene expression in the airways of wild-type and CFTR-deficient mice after nebulization of a modified herpes simplex virus encoding a human CFTR transgene ("HSV-CFTR") or a negative control (vehicle). Figure 2A shows the levels of human CFTR DNA present in biopsies taken from the indicated airway tissues of mice 48 hours after nebulization of HSV-CFTR or the vehicle control, as assessed by qPCR analysis. Figure 2B shows the levels of human CFTR transcript present in biopsies taken from the indicated airway tissues of mice 48 hours after nebulization of HSV-CFTR or the vehicle control, as assessed by qRT-PCR analysis. Data are presented as the mean of two tissue samples (two replicates / tissue) ± standard error of the mean (SEM). [Figure 3] FIG. 1 shows representative hematoxylin and eosin (H&E)-stained airway tissue samples taken from wild-type and CFTR-deficient mice after nebulization with a modified herpes simplex virus encoding a human CFTR transgene ("HSV-CFTR") or a negative control (vehicle). [Figure 4]Figure 1 shows cellular infiltrates in bronchoalveolar lavage fluid (BALF) collected from the lungs of wild-type and CFTR-deficient mice after nebulization with a modified herpes simplex virus encoding a human CFTR transgene ("HSV-CFTR") or a negative control (vehicle). Data are presented as the mean of duplicate samples ± SEM. Statistics were calculated using a two-tailed Student's T-test. [Figure 5] FIG. 1 shows a schematic of the study design for multiple-dose nebulization of modified herpes simplex virus encoding a human CFTR transgene ("HSV-CFTR") in non-human primates. [Figure 6] Figure 6 shows vector transduction and transgene expression in selected tissues of non-human primates after aerosolization of a modified herpes simplex virus encoding a human CFTR transgene ("HSV-CFTR"). Figure 6A shows the levels of human CFTR DNA present in biopsies taken from the indicated tissues 48 hours after aerosolization of a high dose of HSV-CFTR, as assessed by qPCR analysis. Figure 6B shows the levels of human CFTR transcript present in biopsies taken from the indicated tissues 48 hours after aerosolization of a high dose of HSV-CFTR, as assessed by qRT-PCR analysis. Data are presented as the mean ± SEM of two replicates per tissue. nd: not detected. [Figure 7] Figure 1 shows Western blot detection of intracellular human alpha-1-antitrypsin (A1AT) in uninfected control cells (mock) or cells infected with a modified herpes simplex virus encoding a human SERPINA1 transgene at a multiplicity of infection (MOI) of 1 or 2. Recombinant human A1AT (rA1AT) was used as a positive control. [Figure 8]Western blot detection of secreted human alpha-1-antitrypsin (A1AT) in cell culture supernatants of uninfected control cells (mock) or cells infected with a modified herpes simplex virus encoding the human SERPINA1 transgene at a multiplicity of infection (MOI) of 1 or 2. Recombinant human A1AT (rA1AT) was used as a positive control. Blank wells were left between each infected cell supernatant sample when loading the gel (lanes 5, 7, and 9). DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description The following description sets forth example methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0036] I. General techniques The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and may be adapted from conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988), Oligonucleotide Synthesis (M.J.Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J.E.Cellis, ed., 1998) Academic Press, Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994) and the widely used methodology described in Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0037] II. Definition Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0038] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0039] As used herein, the term "and / or" may include any and all combinations of one or more of the associated listed items. For example, the term "a and / or b" can refer to "a only," "b only," "a or b," or "a and b," and the term "a, b and / or c" can refer to "a only," "b only," "c only," "a or b," "a or c," "b or c," "a, b or c," "a and b," "a and c," "b and c," or "a, b and c," etc.

[0040] As used herein, the term "about" refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0041] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0042] As used herein, the terms "polynucleotide," "nucleic acid sequence," "nucleic acid," and variations thereof are intended to refer collectively to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, such as substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications.

[0043] As used herein, a nucleic acid is "operatively linked" or "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operatively linked" or "operably linked" means that the DNA or RNA sequences being linked are contiguous.

[0044] As used herein, the term "vector" refers to an individual element used to introduce heterologous nucleic acids into cells for either their expression or replication. Expression vectors include vectors capable of expressing nucleic acids operably linked to regulatory sequences, such as promoter regions, which can effect expression of such nucleic acids. Thus, an expression vector can refer to a DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, when introduced into an appropriate host cell, results in expression of a nucleic acid. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and those that remain episomal or integrate into the host cell genome.

[0045] As used herein, "open reading frame" or "ORF" refers to a continuous stretch of nucleic acid, either DNA or RNA, that encodes a protein or polypeptide. Typically, the nucleic acid includes a translation initiation signal or start codon, such as ATG or AUG, and a stop codon.

[0046] As used herein, "untranslated region" or "UTR" refers to untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. The inclusion of one or more UTRs in a polynucleotide may affect post-transcriptional regulation, mRNA stability, and / or translation of the polynucleotide.

[0047] As used herein, the term "transgene" refers to a polynucleotide that can be introduced into a cell and then transcribed into RNA, translated, and / or expressed under appropriate conditions. In some aspects, a transgene confers a desired characteristic on the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result.

[0048] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0049] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic animals, and livestock, as well as zoo, sport, or pet animals, such as dogs, horses, cats, cows, and animals used in research, such as mice, rats, hamsters, rabbits, and non-human primates. In some embodiments, the mammal is a human.

[0050] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient(s) is effective and which does not contain additional components that have unacceptable toxicity to the subject receiving the composition or formulation. A "pharmaceutically acceptable" excipient (e.g., vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient(s) used.

[0051] As used herein, an "effective amount" is at least the minimum amount necessary to bring about a measurable improvement or prevention of one or more symptoms of a particular disorder. An "effective amount" may vary depending on factors such as the patient's condition, age, sex, and weight. An effective amount is also one in which the therapeutic beneficial effects outweigh any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the development of the disease, its complications, and intermediate pathological phenotypes manifested during disease development. For therapeutic use, beneficial or desired results include clinical results such as alleviation of one or more symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other medications used to treat the symptoms of the disease, delaying disease progression, and / or extending survival. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective amount of a recombinant nucleic acid, virus, and / or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0052] As used herein, "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of a clinical condition. Desirable effects of treatment include slowing the rate of progression of the disease / disorder / condition, improving or alleviating the disease / disorder / condition, and achieving remission or improving prognosis. For example, an individual is successfully "treated" if one or more signs or symptoms associated with alpha-1-antitrypsin deficiency are reduced or eliminated.

[0053] As used herein, the term "delaying the progression of" a disease / disorder / disorder refers to postponing, preventing, slowing, inhibiting, stabilizing, and / or delaying the onset of the disease / disorder / disorder. This delay may be of different length or duration depending on the history of the disease / disorder / disorder and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0054] III. Recombinant Nucleic Acids Certain aspects of the present disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) comprising one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) polynucleotides encoding polypeptides (e.g., inhalation therapeutic polypeptides). In some embodiments, the recombinant nucleic acid comprises one polynucleotide encoding an inhalation therapeutic polypeptide. In some embodiments, the recombinant nucleic acid comprises two polynucleotides encoding inhalation therapeutic polypeptides. In some embodiments, the recombinant nucleic acid comprises three polynucleotides encoding inhalation therapeutic polypeptides. In some embodiments, the recombinant nucleic acid comprises one or more polynucleotides encoding two or more inhalation therapeutic polypeptides. In some embodiments, the two or more inhalation therapeutic polypeptides are the same. In some embodiments, the two or more inhalation therapeutic polypeptides are different.

[0055] In some embodiments, the recombinant nucleic acid is a vector. In some embodiments, the recombinant nucleic acid is a viral vector. In some embodiments, the recombinant nucleic acid is a herpes virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is a recombinant herpes virus genome. In some embodiments, the recombinant herpes virus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome.

[0056] Polynucleotides encoding inhalation therapeutic polypeptides In some embodiments, the present disclosure relates to recombinant nucleic acids comprising one or more polynucleotides encoding a polypeptide (e.g., an inhaled therapeutic polypeptide). In some embodiments, the inhaled therapeutic polypeptide is a wild-type and / or functional variant of a polypeptide correlated with, causing, or contributing to one or more diseases affecting the airways and / or lungs (e.g., a mutant and / or truncated polypeptide correlated with, causing, or contributing to one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and / or pulmonary fibrosis).

[0057] In some embodiments, the recombinant nucleic acids of the present disclosure comprise one or more polynucleotides comprising the coding sequence of a wild-type and / or functional form of a gene that has been identified as containing a pathogenic variant and / or loss-of-function mutation that correlates with, causes, or contributes to one or more diseases affecting the airways and / or lungs (e.g., a pathogenic variant and / or loss-of-function mutation in a gene identified in patients suffering from one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, pulmonary fibrosis, etc.). Genes harboring pathogenic variants and / or loss-of-function mutations that correlate with, cause, or contribute to one or more diseases or conditions affecting the airways and / or lungs (e.g., alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, pulmonary fibrosis) include, for example, SERPINA1, SLC34A2, DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, and ZMYND. 10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1, DNAAF2, LRRC6, SFTPB, SFTPC, NKX2-1, ABCA3, CSF2RB, CSF2RA, BMPR2, ATP2A2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, EIF2AK4, SFTPA2, TERT, TERC, DKC1, RTEL, PARN, TINF2, NAF1, MUC5B, DSP, STN1, and DPP9. In some embodiments, a polynucleotide of the present disclosure comprises a wild-type coding sequence of any of the genes described herein (such as any isoform thereof). An exemplary polynucleotide comprising the wild-type coding sequence of the human SERPINA1 gene is provided as SEQ ID NO: 1. In some embodiments, a polynucleotide of the present disclosure comprises a codon-optimized variant of the wild-type coding sequence of any of the genes described herein. An exemplary polynucleotide comprising a codon-optimized variant of the wild-type coding sequence of the human SERPINA1 gene is provided as SEQ ID NO:2.In some embodiments, the use of a codon-optimized variant of a gene's coding sequence increases the stability and / or yield of heterologous expression of the encoded polypeptide (RNA and / or protein) in a target cell compared to the stability and / or yield of heterologous expression of the corresponding, non-codon-optimized wild-type sequence. Any suitable method known in the art for codon-optimizing a sequence for expression in one or more target cells (e.g., one or more human cells) can be used, for example, by the method described by Fath et al. (PLoS One. 2011 Mar3;6(3):e17596).

[0058] Any suitable polypeptide known in the art can be encoded by the polynucleotides of the present disclosure, for example, alpha-1-antitrypsin polypeptide, sodium-dependent phosphate transport protein 2B polypeptide, dynein heavy chain 5 axonemal polypeptide, dynein heavy chain 11 axonemal polypeptide, coiled-coil domain-containing protein 39 polypeptide, dynein intermediate chain 1 axonemal polypeptide, coiled-coil domain-containing protein 40 polypeptide, coiled-coil domain-containing protein 103 polypeptide, sperm-associated antigen 1 polypeptide, zinc finger MYND domain-containing protein 10 polypeptide, armadillo repeat-containing protein 4 polypeptide, coiled-coil domain-containing protein 151 polypeptide, dynein intermediate chain 2 axonemal polypeptide, radial spokehead 1 homolog polypeptide, coiled-coil domain-containing protein 114 polypeptide, radial spokehead protein 4 homolog A polypeptide, dynein assembly factor 1 axonemal polypeptide, dynein assembly factor 2 axonemal polypeptide, leucine-rich repeat-containing protein 6 polypeptide, pulmonary surfactant-associated protein B polypeptide, pulmonary surfactant-associated protein C polypeptide, and homeobox protein Nkx-2.Examples of polypeptides include ATP-binding cassette subfamily A member 3 polypeptide, cytokine receptor common subunit beta polypeptide, granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide, bone morphogenetic protein receptor type 2 polypeptide, sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, serine / threonine protein kinase receptor R3 polypeptide, endoglin polypeptide, mother's against decapentaplegic homolog 9 polypeptide, caveolin-1 polypeptide, potassium channel subfamily K member 3 polypeptide, eIF-2-alpha kinase GCN2 polypeptide, pulmonary surfactant-associated protein A2 polypeptide, telomerase reverse transcriptase polypeptide, dyskerin polypeptide, regulator of telomere elongation helicase 1 polypeptide, poly(A)-specific ribonuclease PARN polypeptide, TERF1-interacting nuclear factor 2 polypeptide, H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, mucin-5B polypeptide, desmoplakin polypeptide, CST complex subunit STN1 polypeptide, and dipeptidyl peptidase 9 polypeptide. In some embodiments, an inhaled therapeutic polypeptide of the present disclosure comprises a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any of the polypeptides described herein.

[0059] In some embodiments, a polynucleotide of the present disclosure encodes an alpha-1-antitrypsin polypeptide. In some embodiments, the alpha-1-antitrypsin polypeptide is a human alpha-1-antitrypsin polypeptide (see, e.g., UniProt Accession No. P01009). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type SERPINA1 gene (see, e.g., NCBI Gene ID: 5265) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the alpha-1-antitrypsin polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:3. In some embodiments, the polynucleotide encoding the alpha-1-antitrypsin polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3.

[0060] In some embodiments, the polynucleotide encoding the alpha-1-antitrypsin polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 3. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 418, consecutive amino acids of SEQ ID NO:3.

[0061] In some embodiments, a polynucleotide of the present disclosure encodes a sodium-dependent phosphate transport protein 2B polypeptide. In some embodiments, the sodium-dependent phosphate transport protein 2B polypeptide is a human sodium-dependent phosphate transport protein 2B polypeptide (see, e.g., UniProt Accession No.: O95436). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type SLC34A2 gene (see, e.g., NCBI Gene ID: 10568) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the sodium-dependent phosphate transport protein 2B polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide encoding the sodium-dependent phosphate transport protein 2B polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4.

[0062] In some embodiments, the polynucleotide encoding the sodium-dependent phosphate transport protein 2B polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 4. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 690, consecutive amino acids of SEQ ID NO:4.

[0063] In some embodiments, a polynucleotide of the present disclosure encodes a dynein heavy chain 5 axoneme polypeptide. In some embodiments, the dynein heavy chain 5 axoneme polypeptide is a human dynein heavy chain 5 axoneme polypeptide (see, e.g., UniProt Accession No. Q8TE73). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAH5 gene (see, e.g., NCBI Gene ID: 1767) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the dynein heavy chain 5 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:5. In some embodiments, the polynucleotide encoding the dynein heavy chain 5 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0064] In some embodiments, the polynucleotide encoding the dynein heavy chain 5 axoneme polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 5. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, but fewer than 4624 consecutive amino acids of SEQ ID NO: 5.

[0065] In some embodiments, a polynucleotide of the present disclosure encodes a dynein heavy chain 11 axonemal polypeptide. In some embodiments, the dynein heavy chain 11 axonemal polypeptide is a human dynein heavy chain 11 axonemal polypeptide (see, e.g., UniProt Accession No. Q96DT5). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAH11 gene (see, e.g., NCBI Gene ID: 8701) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the dynein heavy chain 11 axonemal polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:6. In some embodiments, the polynucleotide encoding the dynein heavy chain 11 axonemal polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6.

[0066] In some embodiments, the polynucleotide encoding the dynein heavy chain 11 axonemal polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 6. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, but fewer than 4516 consecutive amino acids of SEQ ID NO: 6.

[0067] In some embodiments, a polynucleotide of the present disclosure encodes a coiled-coil domain-containing protein 39 polypeptide. In some embodiments, the coiled-coil domain-containing protein 39 polypeptide is a human coiled-coil domain-containing protein 39 polypeptide (see, e.g., UniProt Accession No. Q9UFE4). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CCDC39 gene (see, e.g., NCBI Gene ID 339829) or a codon-optimized variant thereof. In some embodiments, the coiled-coil domain-containing protein 39 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:7. In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 39 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

[0068] In some embodiments, a polynucleotide encoding a coiled-coil domain-containing protein 39 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 7. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, but fewer than 941 contiguous amino acids of SEQ ID NO: 7.

[0069] In some embodiments, a polynucleotide of the present disclosure encodes a dynein intermediate chain 1 axoneme polypeptide. In some embodiments, the dynein intermediate chain 1 axoneme polypeptide is a human dynein intermediate chain 1 axoneme polypeptide (see, e.g., UniProt Accession No. Q9UI46). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAI1 gene (see, e.g., NCBI Gene ID: 27019) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding a dynein intermediate chain 1 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:8. In some embodiments, the polynucleotide encoding the dynein intermediate chain 1 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

[0070] In some embodiments, the polynucleotide encoding the dynein intermediate chain 1 axoneme polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 8. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 699, consecutive amino acids of SEQ ID NO:8.

[0071] In some embodiments, a polynucleotide of the present disclosure encodes a coiled-coil domain-containing protein 40 polypeptide. In some embodiments, the coiled-coil domain-containing protein 40 polypeptide is a human coiled-coil domain-containing protein 40 polypeptide (see, e.g., UniProt Accession No. Q4G0X9). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CCDC40 gene (see, e.g., NCBI Gene ID: 55036) or a codon-optimized variant thereof. In some embodiments, the coiled-coil domain-containing protein 40 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:9. In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 40 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0072] In some embodiments, a polynucleotide encoding a coiled-coil domain-containing protein 40 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 9. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1142, consecutive amino acids of SEQ ID NO: 9.

[0073] In some embodiments, a polynucleotide of the present disclosure encodes a coiled-coil domain-containing protein 103 polypeptide. In some embodiments, the coiled-coil domain-containing protein 103 polypeptide is a human coiled-coil domain-containing protein 103 polypeptide (see, e.g., UniProt Accession No. Q8IW40). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CCDC103 gene (see, e.g., NCBI Gene ID 388389) or a codon-optimized variant thereof. In some embodiments, the coiled-coil domain-containing protein 103 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 10. In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 103 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:10.

[0074] In some embodiments, a polynucleotide encoding a coiled-coil domain-containing protein 103 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 10. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 242 contiguous amino acids of SEQ ID NO: 10.

[0075] In some embodiments, the polynucleotide of the present disclosure encodes a sperm associated antigen 1 polypeptide. In some embodiments, the sperm associated antigen 1 polypeptide is a human sperm associated antigen 1 polypeptide (see, e.g., UniProt Accession No. Q07617). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type SPAG1 gene (see, e.g., NCBI Gene ID: 6674) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the sperm associated antigen 1 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide encoding the sperm associated antigen 1 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:11.

[0076] In some embodiments, the polynucleotide encoding the sperm associated antigen 1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 11. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, but fewer than 926, consecutive amino acids of SEQ ID NO: 11.

[0077] In some embodiments, a polynucleotide of the present disclosure encodes a zinc finger MYND domain-containing protein 10 polypeptide. In some embodiments, the zinc finger MYND domain-containing protein 10 polypeptide is a human zinc finger MYND domain-containing protein 10 polypeptide (see, e.g., UniProt Accession No.: O75800). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ZMYND10 gene (see, e.g., NCBI Gene ID: 51364) or a codon-optimized variant thereof. In some embodiments, the zinc finger MYND domain-containing protein 10 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide encoding the zinc finger MYND domain-containing protein 10 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:12.

[0078] In some embodiments, a polynucleotide encoding a zinc finger MYND domain-containing protein 10 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 12. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 440 contiguous amino acids of SEQ ID NO: 12.

[0079] In some embodiments, a polynucleotide of the present disclosure encodes an armadillo repeat-containing protein 4 polypeptide. In some embodiments, the armadillo repeat-containing protein 4 polypeptide is a human armadillo repeat-containing protein 4 polypeptide (see, e.g., UniProt Accession No. Q5T2S8). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ARMC4 gene (see, e.g., NCBI Gene ID: 55130), or a codon-optimized variant thereof. In some embodiments, the armadillo repeat-containing protein 4 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide encoding the armadillo repeat-containing protein 4 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:13.

[0080] In some embodiments, a polynucleotide encoding an armadillo repeat-containing protein 4 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 13. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1044, consecutive amino acids of SEQ ID NO: 13.

[0081] In some embodiments, a polynucleotide of the present disclosure encodes a coiled-coil domain-containing protein 151 polypeptide. In some embodiments, the coiled-coil domain-containing protein 151 polypeptide is a human coiled-coil domain-containing protein 151 polypeptide (see, e.g., UniProt Accession No. A5D8V7). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CCDC151 gene (see, e.g., NCBI Gene ID: 115948) or a codon-optimized variant thereof. In some embodiments, the coiled-coil domain-containing protein 151 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 151 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:14.

[0082] In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 151 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 14. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, but fewer than 595, consecutive amino acids of SEQ ID NO: 14.

[0083] In some embodiments, a polynucleotide of the present disclosure encodes a dynein intermediate chain 2 axoneme polypeptide. In some embodiments, the dynein intermediate chain 2 axoneme polypeptide is a human dynein intermediate chain 2 axoneme polypeptide (see, e.g., UniProt Accession No. Q9GZS0). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAI2 gene (see, e.g., NCBI Gene ID: 64446) or a codon-optimized variant thereof. In some embodiments, a polynucleotide encoding a dynein intermediate chain 2 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide encoding the dynein intermediate chain 2 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0084] In some embodiments, the polynucleotide encoding the dynein intermediate chain 2 axoneme polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 15. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 605, consecutive amino acids of SEQ ID NO: 15.

[0085] In some embodiments, a polynucleotide of the present disclosure encodes a radial spoke head 1 homolog polypeptide. In some embodiments, the radial spoke head 1 homolog polypeptide is a human radial spoke head 1 homolog polypeptide (see, e.g., UniProt Accession No. Q8WYR4). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type RSPH1 gene (see, e.g., NCBI Gene ID: 89765) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the radial spoke head 1 homolog polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 16. In some embodiments, the polynucleotide encoding the radial spokehead 1 homolog polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:16.

[0086] In some embodiments, the polynucleotide encoding the radial spokehead 1 homolog polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 16. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 309, consecutive amino acids of SEQ ID NO: 16.

[0087] In some embodiments, a polynucleotide of the present disclosure encodes a coiled-coil domain-containing protein 114 polypeptide. In some embodiments, the coiled-coil domain-containing protein 114 polypeptide is a human coiled-coil domain-containing protein 114 polypeptide (see, e.g., UniProt Accession No. Q96M63). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CCDC114 gene (see, e.g., NCBI Gene ID 93233) or a codon-optimized variant thereof. In some embodiments, the coiled-coil domain-containing protein 114 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide encoding the coiled-coil domain-containing protein 114 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:17.

[0088] In some embodiments, a polynucleotide encoding a coiled-coil domain-containing protein 114 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 17. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 670, consecutive amino acids of SEQ ID NO: 17.

[0089] In some embodiments, a polynucleotide of the present disclosure encodes a radial spokehead protein 4 homolog A polypeptide. In some embodiments, the radial spokehead protein 4 homolog A polypeptide is a human radial spokehead protein 4 homolog A polypeptide (see, e.g., UniProt Accession No. Q5TD94). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type RSPH4A gene (see, e.g., NCBI Gene ID 345895) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the radial spokehead protein 4 homolog A polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encoding the radial spokehead protein 4 homolog A polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:18.

[0090] In some embodiments, the polynucleotide encoding the radial spokehead protein 4 homolog A polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 18. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, but fewer than 716, consecutive amino acids of SEQ ID NO: 18.

[0091] In some embodiments, a polynucleotide of the present disclosure encodes a dynein assembly factor 1 axonemal polypeptide. In some embodiments, the dynein assembly factor 1 axonemal polypeptide is a human dynein assembly factor 1 axonemal polypeptide (see, e.g., UniProt Accession No. Q8NEP3). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAAF1 gene (see, e.g., NCBI Gene ID: 123872) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the dynein assembly factor 1 axonemal polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the dynein assembly factor 1 axonemal polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:19.

[0092] In some embodiments, the polynucleotide encoding the dynein assembly factor 1 axonemal polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 19. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, but fewer than 725 consecutive amino acids of SEQ ID NO: 19.

[0093] In some embodiments, a polynucleotide of the present disclosure encodes a dynein assembly factor 2 axonemal polypeptide. In some embodiments, the dynein assembly factor 2 axonemal polypeptide is a human dynein assembly factor 2 axonemal polypeptide (see, e.g., UniProt Accession No. Q9NVR5). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DNAAF2 gene (see, e.g., NCBI Gene ID: 55172) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding a dynein assembly factor 2 axonemal polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:20. In some embodiments, the polynucleotide encoding the dynein assembly factor 2 axoneme polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:20.

[0094] In some embodiments, the polynucleotide encoding the dynein assembly factor 2 axoneme polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 20. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, but fewer than 837, consecutive amino acids of SEQ ID NO: 20.

[0095] In some embodiments, a polynucleotide of the present disclosure encodes a leucine-rich repeat-containing protein 6 polypeptide. In some embodiments, the leucine-rich repeat-containing protein 6 polypeptide is a human leucine-rich repeat-containing protein 6 polypeptide (see, e.g., UniProt Accession No. Q86X45). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type LRRC6 gene (see, e.g., NCBI Gene ID: 23639) or a codon-optimized variant thereof. In some embodiments, the leucine-rich repeat-containing protein 6 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 21. In some embodiments, the polynucleotide encoding a leucine-rich repeat-containing protein 6 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:21.

[0096] In some embodiments, the polynucleotide encoding the leucine-rich repeat-containing protein 6 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 21. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 466, consecutive amino acids of SEQ ID NO: 21.

[0097] In some embodiments, the polynucleotide of the present disclosure encodes a pulmonary surfactant-associated protein B polypeptide. In some embodiments, the pulmonary surfactant-associated protein B polypeptide is a human pulmonary surfactant-associated protein B polypeptide (see, e.g., UniProt Accession No. P07988). In some embodiments, the polynucleotide comprises a coding sequence of a wild-type SFTPB gene (see, e.g., NCBI Gene ID: 6439) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein B polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 22. In some embodiments, the polynucleotide encoding a pulmonary surfactant-associated protein B polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:22.

[0098] In some embodiments, the polynucleotide encoding the pulmonary surfactant associated protein B polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 22. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 381 consecutive amino acids of SEQ ID NO: 22.

[0099] In some embodiments, the polynucleotide of the present disclosure encodes a pulmonary surfactant-associated protein C polypeptide. In some embodiments, the pulmonary surfactant-associated protein C polypeptide is a human pulmonary surfactant-associated protein C polypeptide (see, e.g., UniProt Accession No. P11686). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type SFTPC gene (see, e.g., NCBI Gene ID: 6440) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein C polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide encoding a pulmonary surfactant-associated protein C polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:23.

[0100] In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein C polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 23. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 197, consecutive amino acids of SEQ ID NO: 23.

[0101] In some embodiments, the polynucleotide of the present disclosure encodes a homeobox protein Nkx-2.1 polypeptide. In some embodiments, the homeobox protein Nkx-2.1 polypeptide is a human homeobox protein Nkx-2.1 polypeptide (see, e.g., UniProt Accession No. P43699). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type NKX2-1 gene (see, e.g., NCBI Gene ID: 7080) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the homeobox protein Nkx-2.1 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 24. In some embodiments, the polynucleotide encoding the homeobox protein Nkx-2.1 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:24.

[0102] In some embodiments, the polynucleotide encoding the homeobox protein Nkx-2.1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 24. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 371 consecutive amino acids of SEQ ID NO: 24.

[0103] In some embodiments, a polynucleotide of the present disclosure encodes an ATP-binding cassette subfamily A member 3 polypeptide. In some embodiments, the ATP-binding cassette subfamily A member 3 polypeptide is a human ATP-binding cassette subfamily A member 3 polypeptide (see, e.g., UniProt Accession No. Q99758). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ABCA3 gene (see, e.g., NCBI Gene ID: 21) or a codon-optimized variant thereof. In some embodiments, the ATP-binding cassette subfamily A member 3 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide encoding the ATP-binding cassette subfamily A member 3 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:25.

[0104] In some embodiments, the polynucleotide encoding the ATP-binding cassette subfamily A member 3 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 25. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, but fewer than 1704, consecutive amino acids of SEQ ID NO: 25.

[0105] In some embodiments, a polynucleotide of the present disclosure encodes a cytokine receptor common subunit beta polypeptide. In some embodiments, the cytokine receptor common subunit beta polypeptide is a human cytokine receptor common subunit beta polypeptide (see, e.g., UniProt Accession No. P32927). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CSF2RB gene (see, e.g., NCBI Gene ID: 1439), or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the cytokine receptor common subunit beta polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:26. In some embodiments, the polynucleotide encoding the cytokine receptor common subunit beta polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:26.

[0106] In some embodiments, the polynucleotide encoding the cytokine receptor common subunit beta polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 26. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, but fewer than 897, consecutive amino acids of SEQ ID NO:26.

[0107] In some embodiments, the polynucleotide of the present disclosure encodes a granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide. In some embodiments, the granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide is a human granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide (see, e.g., UniProt Accession No. P15509). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CSF2RA gene (see, e.g., NCBI Gene ID: 1438) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 27. In some embodiments, the polynucleotide encoding the granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

[0108] In some embodiments, the polynucleotide encoding the granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 27. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 400 consecutive amino acids of SEQ ID NO: 27.

[0109] In some embodiments, the polynucleotide of the present disclosure encodes a bone morphogenetic protein receptor type 2 polypeptide. In some embodiments, the bone morphogenetic protein receptor type 2 polypeptide is a human bone morphogenetic protein receptor type 2 polypeptide (see, e.g., UniProt Accession No.: Q13873). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type BMPR2 gene (see, e.g., NCBI Gene ID: 659) or a codon-optimized variant thereof. In some embodiments, the bone morphogenetic protein receptor type 2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 28. In some embodiments, the polynucleotide encoding the bone morphogenetic protein receptor type 2 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:28.

[0110] In some embodiments, the polynucleotide encoding the bone morphogenetic protein receptor type 2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 28. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1038, consecutive amino acids of SEQ ID NO: 28.

[0111] In some embodiments, the polynucleotide of the present disclosure encodes a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide. In some embodiments, the sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide is a human sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide (see, e.g., UniProt Accession No. P16615). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ATP2A2 gene (see, e.g., NCBI Gene ID: 488) or a codon-optimized variant thereof. In some embodiments, a polynucleotide encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 29. In some embodiments, a polynucleotide encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 29.

[0112] In some embodiments, a polynucleotide encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 29. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1042, consecutive amino acids of SEQ ID NO: 29.

[0113] In some embodiments, the polynucleotide of the present disclosure encodes a serine / threonine protein kinase receptor R3 polypeptide. In some embodiments, the serine / threonine protein kinase receptor R3 polypeptide is a human serine / threonine protein kinase receptor R3 polypeptide (see, e.g., UniProt Accession No. P37023). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ACVRL1 gene (see, e.g., NCBI Gene ID: 94) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the serine / threonine protein kinase receptor R3 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 30. In some embodiments, the polynucleotide encoding the serine / threonine protein kinase receptor R3 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:30.

[0114] In some embodiments, the polynucleotide encoding the serine / threonine protein kinase receptor R3 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 30. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, but fewer than 503, consecutive amino acids of SEQ ID NO: 30.

[0115] In some embodiments, a polynucleotide of the present disclosure encodes an endoglin polypeptide. In some embodiments, the endoglin polypeptide is a human endoglin polypeptide (see, e.g., UniProt Accession No. P17813). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type ENG gene (see, e.g., NCBI Gene ID: 2022) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the endoglin polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide encoding the endoglin polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 31.

[0116] In some embodiments, a polynucleotide encoding an endoglin polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 31. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 658, consecutive amino acids of SEQ ID NO: 31.

[0117] In some embodiments, a polynucleotide of the present disclosure encodes a Mothers Against Decapentaplegic Homolog 9 polypeptide. In some embodiments, the Mothers Against Decapentaplegic Homolog 9 polypeptide is a human Mothers Against Decapentaplegic Homolog 9 polypeptide (see, e.g., UniProt Accession No.: O15198). In some embodiments, the polynucleotide comprises a coding sequence for a wild-type SMAD9 gene (see, e.g., NCBI Gene ID: 4093) or a codon-optimized variant thereof. In some embodiments, a polynucleotide encoding a Mothers Against Decapentaplegic Homologue 9 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 32. In some embodiments, a polynucleotide encoding a Mothers Against Decapentaplegic Homologue 9 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 32.

[0118] In some embodiments, a polynucleotide encoding a Mothers Against Decapentaplegic Homologue 9 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 32. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 467 consecutive amino acids of SEQ ID NO: 32.

[0119] In some embodiments, a polynucleotide of the present disclosure encodes a caveolin-1 polypeptide. In some embodiments, the caveolin-1 polypeptide is a human caveolin-1 polypeptide (see, e.g., UniProt Accession No. Q03135). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type CAV1 gene (see, e.g., NCBI Gene ID: 857) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the caveolin-1 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide encoding the caveolin-1 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:33.

[0120] In some embodiments, the polynucleotide encoding the caveolin-1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 33. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, but fewer than 178 consecutive amino acids of SEQ ID NO: 33.

[0121] In some embodiments, a polynucleotide of the present disclosure encodes a potassium channel subfamily K member 3 polypeptide. In some embodiments, the potassium channel subfamily K member 3 polypeptide is a human potassium channel subfamily K member 3 polypeptide (see, e.g., UniProt Accession No.: O14649). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type KCNK3 gene (see, e.g., NCBI Gene ID: 3777) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the potassium channel subfamily K member 3 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 34. In some embodiments, the polynucleotide encoding the potassium channel subfamily K member 3 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:34.

[0122] In some embodiments, the polynucleotide encoding the potassium channel subfamily K member 3 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 34. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 394, consecutive amino acids of SEQ ID NO: 34.

[0123] In some embodiments, the polynucleotide of the present disclosure encodes an eIF-2-alpha kinase GCN2 polypeptide. In some embodiments, the eIF-2-alpha kinase GCN2 polypeptide is a human eIF-2-alpha kinase GCN2 polypeptide (see, e.g., UniProt Accession No. Q9P2K8). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type EIF2AK4 gene (see, e.g., NCBI Gene ID: 440275) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the eIF-2-alpha kinase GCN2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 35. In some embodiments, the polynucleotide encoding the eIF-2-alpha kinase GCN2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:35.

[0124] In some embodiments, the polynucleotide encoding the eIF-2-alpha kinase GCN2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 35. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, but fewer than 1649 contiguous amino acids of SEQ ID NO: 35.

[0125] In some embodiments, the polynucleotide of the present disclosure encodes a pulmonary surfactant-associated protein A2 polypeptide. In some embodiments, the pulmonary surfactant-associated protein A2 polypeptide is a human pulmonary surfactant-associated protein A2 polypeptide (see, e.g., UniProt Accession No. Q8IWL1). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type SFTPA2 gene (see, e.g., NCBI Gene ID: 729238) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein A2 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 36. In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein A2 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:36.

[0126] In some embodiments, the polynucleotide encoding the pulmonary surfactant-associated protein A2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 36. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, but fewer than 248 consecutive amino acids of SEQ ID NO: 36.

[0127] In some embodiments, the polynucleotide of the present disclosure encodes a telomerase reverse transcriptase polypeptide. In some embodiments, the telomerase reverse transcriptase polypeptide is a human telomerase reverse transcriptase polypeptide (see, e.g., UniProt Accession No. O14746). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type TERT gene (see, e.g., NCBI Gene ID: 7015) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the telomerase reverse transcriptase polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 37. In some embodiments, the polynucleotide encoding the telomerase reverse transcriptase polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:37.

[0128] In some embodiments, the polynucleotide encoding the telomerase reverse transcriptase polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 37. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1132, consecutive amino acids of SEQ ID NO: 37.

[0129] In some embodiments, a polynucleotide of the present disclosure encodes a dyskerin polypeptide. In some embodiments, the dyskerin polypeptide is a human dyskerin polypeptide (see, e.g., UniProt Accession No. 060832). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DKC1 gene (see, e.g., NCBI Gene ID 1736) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the dyskerin polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 38. In some embodiments, the polynucleotide encoding the dyskerin polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 38.

[0130] In some embodiments, the polynucleotide encoding the dyskerin polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 38. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, but fewer than 514, consecutive amino acids of SEQ ID NO: 38.

[0131] In some embodiments, a polynucleotide of the disclosure encodes a modulator of a telomere elongation helicase 1 polypeptide. In some embodiments, the modulator of a telomere elongation helicase 1 polypeptide is a human modulator of a telomere elongation helicase 1 polypeptide (see, e.g., UniProt Accession No.: Q9NZ71). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type RTEL gene (see, e.g., NCBI Gene ID: 51750), or a codon-optimized variant thereof. In some embodiments, a polynucleotide encoding a modulator of a telomere elongation helicase 1 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 39. In some embodiments, a polynucleotide encoding a modulator of a telomere elongation helicase 1 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 39.

[0132] In some embodiments, the polynucleotide encoding a modulator of a telomere elongation helicase 1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 39. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, but fewer than 1219, contiguous amino acids of SEQ ID NO: 39.

[0133] In some embodiments, a polynucleotide of the present disclosure encodes a poly(A)-specific ribonuclease PARN polypeptide. In some embodiments, the poly(A)-specific ribonuclease PARN polypeptide is a human poly(A)-specific ribonuclease PARN polypeptide (see, e.g., UniProt Accession No.: O95453). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type PARN gene (see, e.g., NCBI Gene ID: 5073) or a codon-optimized variant thereof. In some embodiments, a polynucleotide encoding a poly(A)-specific ribonuclease PARN polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 40. In some embodiments, a polynucleotide encoding a poly(A)-specific ribonuclease PARN polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

[0134] In some embodiments, the polynucleotide encoding the poly(A)-specific ribonuclease PARN polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 40. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, but fewer than 639, consecutive amino acids of SEQ ID NO:40.

[0135] In some embodiments, a polynucleotide of the present disclosure encodes a TERF1-interacting nuclear factor 2 polypeptide. In some embodiments, the TERF1-interacting nuclear factor 2 polypeptide is a human TERF1-interacting nuclear factor 2 polypeptide (see, e.g., UniProt Accession No. Q9BSI4). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type TINF2 gene (see, e.g., NCBI Gene ID: 26277) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding a TERF1-interacting nuclear factor 2 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:41. In some embodiments, the polynucleotide encoding a TERF1-interacting nuclear factor 2 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:41.

[0136] In some embodiments, the polynucleotide encoding the TERF1-interacting nuclear factor 2 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 41. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 451 contiguous amino acids of SEQ ID NO:41.

[0137] In some embodiments, the polynucleotide of the present disclosure encodes an H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide. In some embodiments, the H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide is a human H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide (see, e.g., UniProt Accession No.: Q96HR8). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type NAF1 gene (see, e.g., NCBI Gene ID: 92345) or a codon-optimized variant thereof. In some embodiments, the H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide is a polynucleotide that encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 42. In some embodiments, the polynucleotide that encodes the H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 42.

[0138] In some embodiments, the H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 42. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, but fewer than 494, consecutive amino acids of SEQ ID NO: 42.

[0139] In some embodiments, a polynucleotide of the present disclosure encodes a mucin-5B polypeptide. In some embodiments, the mucin-5B polypeptide is a human mucin-5B polypeptide (see, e.g., UniProt Accession No. Q9HC84). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type MUC5B gene (see, e.g., NCBI Gene ID: 727897) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the mucin-5B polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO:43. In some embodiments, the polynucleotide encoding the mucin-5B polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:43.

[0140] In some embodiments, the polynucleotide encoding the mucin-5B polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO:43. N-terminal truncations, C-terminal truncations, or fragments may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, at least 4750, at least 5000, at least 5250, at least 5500, at least 5750 but fewer than 5762 consecutive amino acids of SEQ ID NO:43.

[0141] In some embodiments, the polynucleotide of the present disclosure encodes a desmoplakin polypeptide. In some embodiments, the desmoplakin polypeptide is a human desmoplakin polypeptide (see, e.g., UniProt Accession No. P15924). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DSP gene (see, e.g., NCBI Gene ID: 1832) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the desmoplakin polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 44. In some embodiments, the polynucleotide encoding the desmoplakin polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 44.

[0142] In some embodiments, the polynucleotide encoding the Desmoplakin polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 44. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, but fewer than 2871 consecutive amino acids of SEQ ID NO: 44.

[0143] In some embodiments, the polynucleotide of the present disclosure encodes a CST complex subunit STN1 polypeptide. In some embodiments, the CST complex subunit STN1 polypeptide is a human CST complex subunit STN1 polypeptide (see, e.g., UniProt Accession No. Q9H668). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type STN1 gene (see, e.g., NCBI Gene ID: 79991) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the CST complex subunit STN1 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 45. In some embodiments, the polynucleotide encoding the CST complex subunit STN1 polypeptide is a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:45.

[0144] In some embodiments, the polynucleotide encoding the CST complex subunit STN1 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 45. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, but fewer than 368, consecutive amino acids of SEQ ID NO: 45.

[0145] In some embodiments, a polynucleotide of the present disclosure encodes a dipeptidyl peptidase 9 polypeptide. In some embodiments, the dipeptidyl peptidase 9 polypeptide is a human dipeptidyl peptidase 9 polypeptide (see, e.g., UniProt Accession No. Q86TI2). In some embodiments, the polynucleotide comprises the coding sequence of a wild-type DPP9 gene (see, e.g., NCBI Gene ID: 91039) or a codon-optimized variant thereof. In some embodiments, the polynucleotide encoding the dipeptidyl peptidase 9 polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 46. In some embodiments, the polynucleotide encoding the dipeptidyl peptidase 9 polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:46.

[0146] In some embodiments, the polynucleotide encoding the dipeptidyl peptidase 9 polypeptide is a polynucleotide encoding an N-terminal truncation, a C-terminal truncation, or a fragment of the amino acid sequence of SEQ ID NO: 46. The N-terminal truncation, C-terminal truncation, or fragment may comprise at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, but fewer than 892 consecutive amino acids of SEQ ID NO:46.

[0147] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 3-46. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 3-46.

[0148] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 5-21. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 5-21.

[0149] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 22-27. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 22-27.

[0150] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 28-35. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 28-35.

[0151] In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 23, 25, and 36-46. In some embodiments, a polynucleotide of the disclosure encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 23, 25, and 36-46.

[0152] Polynucleotides of the present disclosure that encode a polypeptide (e.g., an inhalation therapeutic polypeptide) may further encode additional coding and non-coding sequences. Examples of additional coding and non-coding sequences may include, but are not limited to, sequences encoding additional polypeptide tags (e.g., polypeptide tags encoded in-frame with a polypeptide to produce a fusion protein), introns (e.g., natural introns, modified introns, or heterologous introns), 5'UTRs and / or 3'UTRs (e.g., natural 5'UTRs and / or 3'UTRs, modified 5'UTRs and / or 3'UTRs, or heterologous 5'UTRs and / or 3'UTRs), etc. Examples of suitable polypeptide tags may include, but are not limited to, purification tags (e.g., his-tags, flag-tags, maltose-binding protein, and glutathione-S-transferase tags), detection tags such as tags that can be detected photometrically (e.g., green fluorescent protein or red fluorescent protein), and tags with detectable enzymatic activity (e.g., alkaline phosphatase), tags containing secretory sequences, signal sequences, leader sequences, and / or stabilizing sequences, any combination of protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.). In some embodiments, the 5' UTR and / or 3' UTR improves the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5' UTR and / or 3' UTR improves the level and / or duration of protein expression. In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites) that can prevent or reduce off-target expression (e.g., inhibit expression in particular cell types (e.g., neuronal cells), at particular times in the cell cycle, at particular developmental stages, etc.). In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites) that can enhance expression of the encoded polypeptide in particular cell types.

[0153] In some embodiments, a polynucleotide of the present disclosure encoding a polypeptide (e.g., an inhalation therapeutic polypeptide) is operably linked to one or more (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) regulatory sequences. The term "regulatory sequence" can include enhancers, insulators, promoters, and other expression control elements (e.g., polyadenylation signals). Any suitable enhancer(s) known in the art can be used, including, for example, enhancer sequences derived from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), enhancer sequences derived from eukaryotic viruses (e.g., the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, etc.), and any combination thereof. Any suitable insulator(s) known in the art can be used, including, for example, the HSV chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2, the chicken hypersensitive site 4 insulator (cHS4), the human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), the scaffold / matrix attachment region (S / MAR) from the human interferon beta gene (IFNB1), and any combination thereof.Any suitable promoter known in the art (e.g., a promoter suitable for transcription in a mammalian host cell) can be used, including, for example, promoters obtained from the genomes of viruses (e.g., polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), promoters derived from heterologous mammalian genes (e.g., from an actin promoter (e.g., a β-actin promoter), a ubiquitin promoter (e.g., a ubiquitin C (UbC) promoter), a phosphoglycerate kinase (PGK) promoter, an immunoglobulin promoter, a heat shock promoter, etc.), promoters derived from homologous mammalian genes, synthetic promoters (e.g., a CAG promoter), and any combination thereof (provided such promoters are compatible with the host cell). Regulatory sequences can include those that direct constitutive expression of the nucleic acid, as well as tissue-specific regulatory sequences and / or inducible or repressible sequences.

[0154] In some embodiments, the polynucleotides of the present disclosure are operably linked to one or more heterologous promoters. In some embodiments, the one or more heterologous promoters are one or more of a constitutive promoter, a tissue-specific promoter, a temporal promoter, a spatial promoter, an inducible promoter, and a repressible promoter. In some embodiments, the one or more heterologous promoters are one or more of a human cytomegalovirus (HCMV) immediate early promoter, a human elongation factor-1 (EF1) promoter, a human β-actin promoter, a human UbC promoter, a human PGK promoter, a synthetic CAGG promoter, and any combination thereof. In some embodiments, the polynucleotides of the present disclosure encoding a polypeptide (e.g., an inhaled therapeutic polypeptide) are operably linked to an HCMV promoter.

[0155] In some embodiments, a polynucleotide of the present disclosure encoding a polypeptide (e.g., an inhaled therapeutic polypeptide such as alpha-1-antitrypsin) expresses the polypeptide when the polynucleotide is delivered to one or more target cells of a subject (e.g., one or more cells of the subject's respiratory tract, airway, lungs, etc.). In some embodiments, expression of the polypeptide (e.g., an inhaled therapeutic polypeptide such as alpha-1-antitrypsin) enhances, increases, augments, and / or supplements the level, function, and / or activity of the polypeptide in one or more target cells of the subject (e.g., compared to the level of endogenous polypeptide expressed in the cell, compared to before expression of the polypeptide, etc.). In some embodiments, expression of a polypeptide (e.g., an inhaled therapeutic polypeptide such as alpha-1-antitrypsin) provides prophylactic, palliative, or therapeutic relief in a subject (e.g., compared to before expression of the polypeptide) of one or more signs or symptoms of a disease affecting the airways and / or lungs (e.g., alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, pulmonary fibrosis, etc.).

[0156] In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence for (e.g., a transgene encoding) a collagen alpha-1 (VII) chain polypeptide (COL7). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence for (e.g., a transgene encoding) a lysyl hydroxylase 3 polypeptide (LH3). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence for (e.g., a transgene encoding) a keratin type I cytoskeletal 17 polypeptide (KRT17). In some embodiments, a polynucleotide of the present disclosure does not include a coding sequence for (e.g., a transgene encoding) a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide such as a human TGM1 polypeptide and / or a human TGM5 polypeptide). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a coding sequence of a transgene encoding) a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a coding sequence of a transgene encoding) an antibody (e.g., a full-length antibody, an antibody fragment, etc.). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a coding sequence of a transgene encoding) a serine protease inhibitor Kazal-type (SPINK) polypeptide (e.g., a human SPINK polypeptide such as a SPINK5 polypeptide). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence (e.g., a coding sequence of a transgene encoding) a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide). In some embodiments, the polynucleotides of the disclosure comprise a coding sequence for a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide (e.g., a human CFTR polypeptide) (e.g.,In some embodiments, the polynucleotides of the present disclosure do not include the coding sequence of a transgene encoding the same. In some embodiments, the polynucleotides of the present disclosure do not include an ichthyosis-associated polypeptide (e.g., an ATP-binding cassette subfamily A member 12 polypeptide, an 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 polypeptide, an aldehyde dehydrogenase family 3 member A2 polypeptide, an arachidonate 12-lipoxygenase type 12R polypeptide, a hydroperoxide isomerase ALOXE3 polypeptide, an AP-1 complex subunit sigma-1A polypeptide, an arylsulfatase E polypeptide, a caspase-14 polypeptide, a corneodesmosin polypeptide, a ceramide synthase 3 polypeptide, a carbohydrate sulfotransferase 8 polypeptide, a claudin-1 polypeptide, a cystatin-A polypeptide, a cytochrome P450 4F22 polypeptide, 3-beta-hydroxysteroid-delta(8), delta(7)-isomerase polypeptide, elongation of very long chain fatty acid protein 4 polypeptide, filaggrin polypeptide, filaggrin 2 polypeptide, gap junction beta-2 polypeptide, gap junction beta-3 polypeptide, gap junction beta-4 polypeptide, gap junction beta-6 polypeptide, 3-ketodihydrosphingosine reductase polypeptide, keratin, type II cytoskeleton 1 polypeptide, keratin, type II cytoskeleton 2 epidermal polypeptide, keratin, type I cytoskeleton 9 polypeptide, keratin, type I cytoskeleton 1 polypeptide 0 polypeptide, lipase member N polypeptide, loricrin polypeptide, membrane-bound transcription factor site 2 protease polypeptide, magnesium transporter NIPA4 polypeptide, sterol-4-alpha-carboxylate 3-dehydrogenase decarboxylating polypeptide, peroxisomal targeting signal 2 receptor polypeptide, D-3-phosphoglycerate dehydrogenase polypeptide, phytanoyl-CoA dioxygenase, peroxisomal polypeptide, patatin-like phospholipase domain-containing protein 1 polypeptide, proteasome maturation protein polypeptide, phosphoserine aminotransferase polypeptide,In some embodiments, the polynucleotides of the present disclosure do not include coding sequences (e.g., coding sequences of transgenes encoding) short-chain dehydrogenase / reductase family 9C member 7 polypeptides, serpin B8 polypeptides, long-chain fatty acid transport protein 4 polypeptides, synaptosomal-associated protein 29 polypeptides, tumorigenesis inhibitor 14 protein polypeptides, sterylsulfatase polypeptides, vacuum protein sorting-associated protein 33B polypeptides, and CAAX prenyl protease 1 homolog polypeptides. In some embodiments, the polynucleotides of the present disclosure do not include coding sequences (e.g., coding sequences of transgenes encoding) collagen alpha-1(VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, and / or any chimeric polypeptides thereof. In some embodiments, the polynucleotides of the present disclosure do not include coding sequences for collagen alpha-1(VII) chain polypeptides, lysyl hydroxylase 3 polypeptides, keratin type I cytoskeleton 17 polypeptides, transglutaminase (TGM) polypeptides, filaggrin polypeptides, cosmetic proteins, antibodies, SPINK polypeptides, CFTR polypeptides, ichthyosis-associated polypeptides, and / or any chimeric polypeptides thereof (e.g., transgenes encoding the same).

[0157] Recombinant nucleic acids In some embodiments, the present disclosure relates to a recombinant nucleic acid comprising any one or more of the polynucleotides described herein. In some embodiments, the recombinant nucleic acid is a vector (e.g., an expression vector, a display vector, etc.). In some embodiments, the vector is a DNA vector or an RNA vector. Generally, any vector suitable for maintaining, propagating, and / or expressing a polynucleotide to produce one or more polypeptides in a subject can be used. Examples of suitable vectors can include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, Sindbis viral vectors, measles vectors, herpes viral vectors, lentiviral vectors, retroviral vectors, etc.). In some embodiments, the vector is a herpes viral vector. In some embodiments, the vector is capable of autonomous replication in a host cell. In some embodiments, the vector is not capable of autonomous replication in a host cell. In some embodiments, the vector is capable of integration into host DNA. In some embodiments, the vector is not capable of integration into host DNA (e.g., is episomal). Methods for producing vectors containing one or more polynucleotides of interest are well known to those of skill in the art and include, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid (e.g., by genetic engineering techniques).

[0158] In some embodiments, the recombinant nucleic acid of the present disclosure is a herpes simplex virus (HSV) amplicon. Herpes virus amplicons, including their structural characteristics and methods of production, are generally known to those of skill in the art (see, e.g., de Silva S. and Bowers W., "Herpes Virus Amplicon Vectors," Viruses 2009, 1, 594-629). In some embodiments, the herpes simplex virus amplicon is an HSV-1 amplicon. In some embodiments, the herpes simplex virus amplicon is an HSV-1 hybrid amplicon. Examples of HSV-1 hybrid amplicons include, but are not limited to, HSV / AAV hybrid amplicons, HSV / EBV hybrid amplicons, HSV / EBV / RV hybrid amplicons, and / or HSV / Sleeping Beauty hybrid amplicons. In some embodiments, the amplicon is an HSV / AAV hybrid amplicon. In some embodiments, the amplicon is an HSV / Sleeping Beauty hybrid amplicon.

[0159] In some embodiments, the recombinant nucleic acid of the present disclosure is a recombinant herpesvirus genome. The recombinant herpesvirus genome can be a recombinant genome derived from any member of the DNA virus family Herpesviridae known in the art, including, for example, a recombinant herpes simplex virus genome, a recombinant varicella-zoster virus genome, a recombinant human cytomegalovirus genome, a recombinant herpesvirus 6A genome, a recombinant herpesvirus 6B genome, a recombinant herpesvirus 7 genome, a recombinant Epstein-Barr virus genome, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. In some embodiments, the recombinant herpesvirus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. As used herein, an "inactivating mutation" may refer to any mutation that results in a gene or regulon product (RNA or protein) that is reduced in amount and / or function, undetectable, or eliminated (e.g., compared to the corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations may include, but are not limited to, deletions, insertions, point mutations, and rearrangements in transcriptional control sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method for measuring the amount of a gene product or regulon product known in the art can be used, including, for example, qPCR, Northern blot, RNA-seq, Western blot, ELISA, etc. In some embodiments, one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpesvirus genes. In some embodiments, the recombinant herpesvirus genome is attenuated (e.g., compared to a corresponding wild-type herpesvirus genome). In some embodiments, the recombinant herpesvirus genome is replication-competent. In some embodiments, the recombinant herpesvirus genome is replication-deficient.

[0160] In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) genome. In some embodiments, the recombinant herpes simplex virus genome comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) inactivating mutations. In some embodiments, the one or more inactivating mutations are present in one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, etc.) herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome is attenuated (e.g., compared to the corresponding wild-type herpes simplex virus genome). In some embodiments, the recombinant herpes simplex virus genome is replication-competent. In some embodiments, the recombinant herpes simplex virus genome is replication-deficient.

[0161] In some embodiments, the recombinant herpesvirus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant HSV-1 genome is selected from the group consisting of strains such as, for example, strain 17, Ty25, R62, S25, Ku86, S23, R11, Ty148, Ku47, H166syn, 1319-2005, F-13, M-12, 90237, F-17, KOS, 3083-2008, F12g, L2, CD38, H193, M-15, India 2011, 0116209, F-11I, 66-207, 2762, 369-2007, 3355, MacIntyre, McKrae, 7862, 7-h se, HF10, 1394,2005, 270-2007, OD4, SC16, M-19, 4J1037, 5J1060, J1060, KOS79, 132- The recombinant HSV-1 genome may be derived from any HSV-1 strain known in the art, such as 1988, 160-1982, H166, 2158-2007, RE, 78326, F18g, F11, 172-2010, H129, F, E4, CJ994, F14g, E03, E22, E10, E06, E11, E25, E23, E35, E15, E07, E12, E14, E08, E19, E13, ATCC 2011, etc. (See, e.g., Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is derived from the KOS strain. In some embodiments, the recombinant HSV-1 genome is not derived from the McKrae strain. In some embodiments, the recombinant HSV-1 genome is attenuated (e.g., compared to a corresponding wild-type HSV-1 genome), in some embodiments, the recombinant HSV-1 genome is replication-competent, in some embodiments, the recombinant HSV-1 genome is replication-deficient.

[0162] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the infected cell protein (or infected cell polypeptide) (ICP)0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and / or UL55 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 herpes simplex virus gene (one or both copies) and / or the ICP47 herpes simplex virus gene (e.g., to avoid production of immunostimulatory virus). In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 herpes simplex virus gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP34.5 herpes simplex virus gene (one or both copies) and the ICP47 herpes simplex virus gene, hi some embodiments, the recombinant herpes simplex virus genome is not oncolytic.

[0163] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and further comprises an inactivating mutation in the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, the ICP47 gene, the UL41 gene, and / or the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene.In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, and / or the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP27 gene, the ICP47 gene, and / or the UL55 gene.

[0164] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP22 gene, the ICP27 gene, the ICP47 gene, the UL41 gene, and / or the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies), an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP4 gene (one or both copies), the ICP22 gene, and / or the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP27 gene, the ICP47 gene, and / or the UL55 gene.

[0165] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP27 gene, the ICP47 gene, the UL41 gene, and / or the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP22 gene and / or the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP27 gene, the ICP47 gene, and / or the UL55 gene.

[0166] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP47 gene, the UL41 gene, and / or the UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP27 gene.

[0167] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, the UL41 gene, and / or the UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP47 gene.

[0168] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, the ICP47 gene, and / or the UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL41 gene.

[0169] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene and further comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, the ICP47 gene, and / or the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL55 gene.

[0170] In some embodiments, the recombinant herpes simplex virus genome comprises an internal repeat long (IR repeat). L ) region and internal repeat short (IR SThe virus contains an inactivating mutation (e.g., deletion) in the internal repeat (joint) region, including the ICP4 gene and the ICP0 gene. In some embodiments, the inactivation (e.g., deletion) of the joint region removes one copy each of the ICP4 gene and the ICP0 gene. In some embodiments, the inactivation (e.g., deletion) of the joint region further inactivates (e.g., deletes) the promoters of the ICP22 and ICP47 genes. If necessary, expression of one or both of these genes can be restored by inserting an immediate-early promoter into the recombinant herpes simplex virus genome (see, e.g., Hill et al. (1995). Nature 375(6530):411-415; Goldsmith et al. (1998). J Exp Med 187(3):341-348). Without wishing to be bound by theory, it is believed that inactivating (e.g., deleting) the joint region may contribute to the stability of the recombinant herpes simplex virus genome and / or allow the recombinant herpes simplex virus genome to accommodate more and / or larger transgenes.

[0171] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 gene (one or both copies), the ICP22 gene, and the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 gene (one or both copies), the ICP27 gene, and the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, the ICP47 gene, and the UL55 gene. In some embodiments, the inactivating mutations in the ICP4 gene (one or both copies), the ICP27 gene, and / or the UL55 gene are deletions of the coding sequence of the ICP4 gene (one or both copies), the ICP27 gene, and / or the UL55 gene. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes (e.g., the coding sequences of ICP22 and ICP47 are intact but not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome comprises deletions in the coding sequences of the ICP4 gene (one or both copies), the ICP27 gene, and the UL55 gene, and deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 gene (one or both copies) and / or the UL41 gene.

[0172] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and the ICP4 gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), and the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, and the ICP27 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, and the UL55 gene. In some embodiments, the inactivating mutation in the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, and / or the UL55 gene comprises a deletion of the coding sequence of the ICP0 gene (one or both copies), the ICP4 gene (one or both copies), the ICP22 gene, the ICP27 gene, and / or the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP47 gene and / or the UL41 gene.

[0173] In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one, two, three, four, five, six, or more viral loci. Examples of suitable viral loci may include, but are not limited to, the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, tk, UL41, and UL55 herpes simplex virus loci. In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both ICP4 viral loci (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within one or both ICP4 loci). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the ICP22 viral locus (e.g., a recombinant virus comprising a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP22 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the UL41 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the ICP27 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP27 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the ICP47 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP47 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the UL55 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the UL55 locus).In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the tk viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the tk locus).

[0174] In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the ICP4 viral loci and one or more polynucleotides of this disclosure within the ICP22 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within one or both of the ICP4 loci and a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP22 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the ICP4 viral loci and one or more polynucleotides of this disclosure within the UL41 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within one or both of the ICP4 loci and a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within the ICP22 viral locus and one or more polynucleotides of this disclosure within the UL41 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP22 locus and a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the ICP4 viral loci, one or more polynucleotides of this disclosure within the viral ICP22 locus, and one or more polynucleotides of this disclosure within the UL41 viral locus (e.g., the recombinant virus comprises a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within one or both of the ICP4 loci, a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the ICP22 locus, and a polynucleotide encoding a polypeptide (such as an inhaled therapeutic polypeptide) within the UL41 locus).In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one or both of the ICP4 viral loci, one or more polynucleotides of the present disclosure in the ICP22 viral locus, one or more polynucleotides of the present disclosure in the UL41 viral locus, one or more polynucleotides of the present disclosure in the ICP27 viral locus, one or more polynucleotides of the present disclosure in the ICP47 viral locus, one or more polynucleotides of the present disclosure in the tk viral locus, and / or one or more polynucleotides of the present disclosure in the UL55 viral locus.

[0175] In some embodiments, the recombinant herpesvirus genome (e.g., a recombinant herpes simplex virus genome) has been engineered to reduce or eliminate expression of one or more herpesvirus genes (e.g., one or more virulent herpesvirus genes), such as both copies of the HSV ICP0 gene, one or both copies of the HSV ICP4 gene, the HSV ICP22 gene, the HSV UL41 gene, the HSV ICP27 gene, the HSV ICP47 gene, the HSV tk gene, the HSV UL55 gene, etc. In some embodiments, the recombinant herpesvirus genome (e.g., a recombinant herpes simplex virus genome) has been engineered to reduce the cytotoxicity of the recombinant genome (e.g., when introduced into a target cell) compared to a corresponding wild-type herpesvirus genome (e.g., a wild-type herpes simplex virus genome). In some embodiments, the target cell is a human cell (a primary cell or a cell line derived therefrom). In some embodiments, the target cell is a respiratory cell (a primary cell or a cell line derived therefrom). In some embodiments, the target cells are airway epithelial cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are lung cells (primary cells or cell lines derived therefrom).In some embodiments, the cytotoxicity (e.g., in target cells) of the recombinant genome (e.g., recombinant herpes simplex virus genome) is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to the corresponding wild-type herpes simplex virus genome (e.g., measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) herpes simplex virus genome versus a wild-type herpes simplex virus genome in target cells; measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome versus a wild-type herpes simplex virus genome in target cells, etc.). In some embodiments, the cytotoxicity (e.g., in a target cell) of the recombinant herpes genome (e.g., a recombinant herpes simplex virus genome) is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, at least about 150 ... fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more (e.g., measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) herpes simplex virus genome versus a wild-type herpes simplex virus genome in a target cell; measuring the relative cytotoxicity of a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome versus a wild-type herpes simplex virus genome in a target cell, etc.).Methods for measuring cytotoxicity are known to those of skill in the art and include, for example, the use of vital dyes (formazan dyes), protease biomarkers, MTT assays (or assays using related tetrazolium salts such as XTT, MTS, water-soluble tetrazolium salts), measuring ATP content, etc.

[0176] In some embodiments, the recombinant genome (e.g., recombinant herpes simplex virus genome) is engineered to have a reduced effect on target cell proliferation following exposure of the target cell to the recombinant genome compared to the corresponding wild-type genome (e.g., wild-type herpes simplex virus genome). In some embodiments, the target cell is a human cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a respiratory cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is an airway epithelial cell (primary cell or a cell line derived therefrom). In some embodiments, the target cell is a pulmonary cell (primary cell or a cell line derived therefrom). In some embodiments, target cell growth following exposure to the recombinant genome is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% faster than target cell growth following exposure to the corresponding wild-type genome (e.g., measuring the relative cell growth following exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome in a target cell relative to the cell growth following exposure to a wild-type herpes simplex virus genome; measuring the relative cell growth following exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome in a target cell relative to the cell growth following exposure to a wild-type herpes simplex virus genome, etc.).In some embodiments, target cell proliferation after exposure to the recombinant genome is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold faster than target cell proliferation after exposure to the corresponding wild-type genome (e.g., measuring the relative cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome in a target cell relative to the cell proliferation after exposure to a wild-type herpes simplex virus genome; measuring the relative cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome in a target cell relative to the cell proliferation after exposure to a wild-type herpes simplex virus genome, etc.). Methods for measuring cell proliferation are known to those of skill in the art and include, for example, by using the Ki67 cell proliferation assay, the BrdU cell proliferation assay, and the like.

[0177] A vector (e.g., a herpes virus vector) can contain one or more polynucleotides of the present disclosure in a form suitable for expression of the polynucleotide in a host cell. The vector can include one or more regulatory sequences operably linked (e.g., as described above) to the polynucleotide to be expressed.

[0178] In some embodiments, the present disclosure relates to one or more heterologous polynucleotides (e.g., bacterial artificial chromosomes (BACs)) comprising any of the recombinant nucleic acids described herein.

[0179] In some embodiments, a recombinant nucleic acid (e.g., a recombinant herpes simplex virus genome) of the present disclosure comprises one or more of the polynucleotides described herein inserted into the recombinant nucleic acid in any orientation. When a recombinant nucleic acid comprises two or more polynucleotides described herein (e.g., two or more, three or more, etc.), the polynucleotides can be inserted in the same or opposite orientation to each other. Without wishing to be bound by theory, incorporating two polynucleotides (e.g., two transgenes) into a recombinant nucleic acid (e.g., a vector) in an antisense orientation can be useful to avoid read-through and ensure proper expression of each polynucleotide.

[0180] In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a collagen alpha-1 (VII) chain polypeptide (COL7). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a lysyl hydroxylase 3 polypeptide (LH3). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a keratin type I cytoskeletal 17 polypeptide (KRT17). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide such as a human TGM1 polypeptide and / or a human TGM5 polypeptide). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.). In some embodiments, a recombinant nucleic acid of the present disclosure does not include a polynucleotide encoding an antibody (e.g., a full-length antibody, an antibody fragment, etc.). In some embodiments, a recombinant nucleic acid of the present disclosure does not comprise a polynucleotide encoding a serine protease inhibitor Kazal-type (SPINK) polypeptide (e.g., a human SPINK polypeptide such as a SPINK5 polypeptide). In some embodiments, a recombinant nucleic acid of the present disclosure does not comprise a polynucleotide encoding a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide). In some embodiments, a recombinant nucleic acid of the present disclosure does not comprise a polynucleotide encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide (e.g., a human CFTR polypeptide). In some embodiments, a recombinant nucleic acid of the present disclosure does not comprise a polynucleotide encoding an ichthyosis-associated polypeptide (e.g., an ATP-binding cassette subfamily A member 12 polypeptide, a 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 polypeptide, an aldehyde dehydrogenase family 3 member A2 polypeptide,Arachidonate 12-lipoxygenase 12R type polypeptide, hydroperoxide isomerase ALOXE3 polypeptide, AP-1 complex subunit sigma-1A polypeptide, arylsulfatase E polypeptide, caspase-14 polypeptide, corneodesmosin polypeptide, ceramide synthase 3 polypeptide, carbohydrate sulfotransferase 8 polypeptide, claudin-1 polypeptide, cystatin-A polypeptide, cytochrome P450 4F22 polypeptide, 3-beta-hydroxysteroid-delta(8), delta(7)-isomerase polypeptide, elongation of very long chain fatty acid protein 4 polypeptide, filaggrin polypeptide, filaggrin 2 polypeptide, gap junction beta-2 polypeptide, gap junction beta-3 polypeptide, gap junction beta-4 polypeptide, gap junction beta-6 polypeptide, 3-ketodihydrosphingosine reductase polypeptide, keratin, type II cytoskeleton 1 polypeptide, keratin, type II cytoskeleton 2 epidermal polypeptide, keratin, type I cytoskeleton 9 polypeptide, keratin, type I cytoskeleton 10 polypeptide, lipase member N polypeptide, loricrin polypeptide, membrane-bound transcription factor site 2 protease polypeptide, magnesium transporter NIPA4 polypeptide polypeptide, sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating polypeptide, peroxisomal targeting signal 2 receptor polypeptide, D-3-phosphoglycerate dehydrogenase polypeptide, phytanoyl-CoA dioxygenase, peroxisomal polypeptide, patatin-like phospholipase domain-containing protein 1 polypeptide, proteasome maturation protein polypeptide, phosphoserine aminotransferase polypeptide, short-chain dehydrogenase / reductase family 9C member 7 polypeptide, serpin B8 polypeptide, long-chain fatty acid transport protein 4 polypeptide, synaptosomal-associated protein 29 polypeptide, tumorigenesis inhibitor 14 protein polypeptide, sterylsulfatase polypeptide, vacuum protein sorting-associated protein 33B polypeptide,and CAAX prenyl protease 1 homolog polypeptide). In some embodiments, the recombinant nucleic acid of the disclosure does not comprise a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, and / or any chimeric polypeptide thereof. In some embodiments, the recombinant nucleic acid of the disclosure does not comprise a polynucleotide encoding a collagen alpha-1(VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeletal 17 polypeptide, a transglutaminase (TGM) polypeptide, a filaggrin polypeptide, a cosmetic protein, an antibody, a SPINK polypeptide, a CFTR polypeptide, an ichthyosis-associated polypeptide, and / or any chimeric polypeptide thereof.

[0181] IV. Viruses Certain aspects of the present disclosure relate to viruses comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the viruses are capable of infecting one or more target cells of a subject (e.g., a human). In some embodiments, the viruses are suitable for delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human subject). In some embodiments, the one or more target cells are human cells. In some embodiments, the one or more target cells are one or more cells that contain a genetic defect (e.g., a pathogenic variant and / or a loss-of-function mutation) in an endogenous gene that encodes a mutant variant of a polypeptide encoded by the polynucleotide and / or recombinant genome (e.g., expression of the polypeptide from the polynucleotide and / or recombinant genome molecularly corrects the underlying protein defect). In some embodiments, the one or more target cells are one or more airway epithelial cells. In some embodiments, the one or more target cells are one or more cells of the respiratory tract (e.g., airway epithelial cells (such as goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, tumor cells, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (such as type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); salivary gland cells in the bronchi (serous cells, mucous cells, and / or duct cells); etc.). In some embodiments, the one or more target cells are one or more cells of the lung.

[0182] Any suitable virus known in the art can be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, papillomavirus, herpesvirus (e.g., herpes simplex virus), vaccinia virus, and / or any hybrid or derivative thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replication-competent. In some embodiments, the virus is replication-deficient. In some embodiments, the virus has been modified to alter its tissue tropism compared to the tissue tropism of the corresponding unmodified wild-type virus. In some embodiments, the virus has reduced cytotoxicity (e.g., in target cells) compared to the corresponding wild-type virus. Methods for producing viruses containing recombinant nucleic acids are well known to those of skill in the art.

[0183] In some embodiments, the virus is a member of the DNA virus family Herpesviridae, which includes, for example, herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus. In some embodiments, the herpesvirus is attenuated. In some embodiments, the herpesvirus is replication-deficient. In some embodiments, the herpesvirus is replication-competent. In some embodiments, the herpesvirus has been engineered to reduce or eliminate expression of one or more herpesvirus genes (e.g., one or more toxic herpesvirus genes). In some embodiments, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments, the herpesvirus is not oncolytic.

[0184] In some embodiments, the virus is a herpes simplex virus. The herpes simplex virus comprising the recombinant nucleic acid may be produced by the processes disclosed in, for example, WO2015 / 009952, WO2017 / 176336, WO2019 / 200163, and / or WO2019 / 210219. In some embodiments, the herpes simplex virus is attenuated. In some embodiments, the herpes simplex virus is replication-deficient. In some embodiments, the herpes simplex virus is replication-competent. In some embodiments, the herpes simplex virus has been engineered to reduce or eliminate expression of one or more herpes simplex virus genes (e.g., one or more toxic herpes simplex virus genes). In some embodiments, the herpes simplex virus has reduced cytotoxicity compared to the corresponding wild-type herpes simplex virus. In some embodiments, the herpes simplex virus is not oncolytic. In some embodiments, the herpes simplex virus is HSV-1, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is an HSV-1 virus. In some embodiments, the HSV-1 is attenuated. In some embodiments, the HSV-1 is replication-deficient. In some embodiments, the HSV-1 is replication-competent. In some embodiments, the HSV-1 has been engineered to reduce or eliminate expression of one or more HSV-1 genes (e.g., one or more virulent HSV-1 genes). In some embodiments, the HSV-1 has reduced cytotoxicity compared to corresponding wild-type HSV-1. In some embodiments, the HSV-1 is not oncolytic.

[0185] In some embodiments, the herpes simplex virus is modified to alter its tissue tropism compared to the tissue tropism of the unmodified wild-type herpes simplex virus. In some embodiments, the herpes simplex virus comprises a modified envelope. In some embodiments, the modified envelope comprises one or more (e.g., one or more, two or more, three or more, four or more, etc.) mutated herpes simplex virus glycoproteins. Examples of herpes simplex virus glycoproteins can include, but are not limited to, glycoproteins gB, gC, gD, gH, and gL. In some embodiments, the modified envelope alters the tissue tropism of the herpes simplex virus compared to the wild-type herpes simplex virus.

[0186] In some embodiments, the transduction efficiency (in vitro and / or in vivo) of a virus of the present disclosure (e.g., a herpes virus, such as herpes simplex virus) of one or more target cells (e.g., one or more cells of the respiratory tract) is at least about 25%. For example, the transduction efficiency of the virus of one or more target cells can be at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.5% or more. In some embodiments, the virus is a herpes simplex virus, and the transduction efficiency of the virus of one or more target cells (e.g., one or more cells of the respiratory tract) is between about 85% and about 100%. In some embodiments, the virus is a herpes simplex virus, and the viral transduction efficiency of one or more target cells (e.g., one or more cells of the respiratory tract) is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. Methods for measuring viral transduction efficiency in vitro or in vivo are well known to those skilled in the art and include, for example, qPCR analysis, deep sequencing, Western blotting, fluorescence analysis (e.g., fluorescent in situ hybridization (FISH), fluorescent reporter gene expression, immunofluorescence, FACS), etc.

[0187] V. Pharmaceutical Compositions and Formulations Certain aspects of the present disclosure relate to pharmaceutical compositions or formulations comprising any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes) and / or viruses (e.g., herpesviruses comprising a recombinant genome) described herein (such as a herpes simplex virus comprising a recombinant herpes simplex virus genome), and a pharmaceutically acceptable excipient or carrier.

[0188] In some embodiments, the pharmaceutical composition or formulation comprises any one or more of the viruses (e.g., herpes viruses) described herein. In some embodiments, the pharmaceutical composition or formulation comprises about 10 4 ~about 10 12 For example, the pharmaceutical composition or formulation may contain about 10 plaque-forming units (PFU) / mL of virus. 4 ~about 10 12 , about 10 5 ~about 10 12 , about 10 6 ~about 10 12 , about 10 7 ~about 10 12 , about 10 8 ~about 10 12 , about 10 9 ~about 10 12 , about 10 10 ~about 10 12 , about 10 11 ~about 10 12 , about 10 4 ~about 10 11 , about 10 5 ~about 10 11 , about 10 6 ~about 10 11 , about 10 7 ~about 10 11 , about 10 8 ~about 10 11 , about 10 9 ~about 10 11 , about 10 10 ~about 10 11 , about 10 4 ~about 10 10 , about 10 5 ~about 10 10 , about 10 6 ~about 10 10 , about 10 7 ~about 10 10, about 10 8 ~about 10 10 , about 10 9 ~about 10 10 , about 10 4 ~about 10 9 , about 10 5 ~about 10 9 , about 10 6 ~about 10 9 , about 10 7 ~about 10 9 , about 10 8 ~about 10 9 , about 10 4 ~about 10 8 , about 10 5 ~about 10 8 , about 10 6 ~approx. 108, approx. 10 7 ~about 10 8 , about 10 4 ~about 10 7 , about 10 5 ~about 10 7 , about 10 6 ~about 10 7 , about 10 4 ~about 10 6 , about 10 5 ~about 10 6 or about 10 4 ~about 10 5 In some embodiments, the pharmaceutical composition or formulation may contain about 10 PFU / mL of virus. 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 or about 10 12 containing PFU / mL of virus.

[0189] Pharmaceutical compositions and formulations can be prepared by mixing the active ingredient(s) (such as recombinant nucleic acids and / or viruses) having the desired degree of purity with one or more pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers (e.g., phosphate, citrate, acetate, and other organic acids); antioxidants (e.g., ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); low molecular weight (less than about 10 residues) polypeptides; proteins. The carrier may comprise a substance (e.g., serum albumin, gelatin, or immunoglobulin); a polyol (such as glycerol, e.g., formulations containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., of glycerol); a hydrophilic polymer (e.g., polyvinylpyrrolidone); monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); a chelating agent (e.g., EDTA); a sugar (e.g., sucrose, mannitol, trehalose, or sorbitol); a salt-forming counterion (e.g., sodium); a metal complex (e.g., Zn-protein complex); and / or a non-ionic surfactant (e.g., polyethylene glycol (PEG)). A complete discussion of pharmaceutically acceptable carriers is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0190] In some embodiments, the pharmaceutical composition or formulation comprises one or more lipid (e.g., cationic lipid) carriers. In some embodiments, the pharmaceutical composition or formulation comprises one or more nanoparticle carriers. Nanoparticles are submicron (less than about 1000 nm) sized drug delivery vehicles that can carry encapsulated drugs (such as synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid-based biologicals) for immediate or controlled release. Various molecules (e.g., proteins, peptides, recombinant nucleic acids, etc.) can be efficiently encapsulated in nanoparticles using processes well known in the art. In some embodiments, a molecule "encapsulated" in a nanoparticle can refer to a molecule (such as a virus) that is contained within the nanoparticle, or attached and / or associated with the surface of the nanoparticle, or any combination thereof. Nanoparticles for use in the compositions or formulations described herein can be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles comprising poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combination thereof (see, e.g., Vauthier et al. Adv Drug Del Rev. (2003) 55:519-48, US2007 / 0148074, US2007 / 0092575, US2006 / 0246139, US5753234, US7081483, and WO2006 / 052285).

[0191] In some embodiments, the pharmaceutically acceptable carrier or excipient may be compatible or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intracerebroventricular, and / or intranasal administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is compatible or suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is compatible or suitable for intranasal and / or inhalation administration. In some embodiments, the pharmaceutically acceptable carrier or excipient is compatible or suitable for inhalation administration.

[0192] In some embodiments, the pharmaceutical composition or formulation may be adapted or suitable for any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, dermal, oral, intranasal, intratracheal, sublingual, buccal, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreal, subretinal, transmucosal, intraarticular, implant, inhalation, intrathecal, intracerebroventricular, or intranasal administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for intranasal and / or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for inhalation administration.

[0193] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional ingredients. Examples of additional ingredients include binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, vinegar, etc.). The polynucleotide may include, but is not limited to, an agent capable of stabilizing or preventing degradation of the polynucleotide, such as, but not limited to, an ester or a mixture of esters, such as, but not limited to, an ester of cellulose, a cellulose acetate, a cellulose acetate copolymer ... In some embodiments, the pharmaceutical composition or formulation comprises glycerol (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer and glycerol.

[0194] Pharmaceutical compositions and formulations to be used for in vivo administration are generally sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0195] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to deliver one or more polynucleotides encoding a polypeptide (e.g., an inhaled therapeutic polypeptide such as an alpha-1-antitrypsin polypeptide) to one or more cells of a subject (e.g., one or more cells of the subject's respiratory tract). In some embodiments, the subject is suffering from a disease affecting the airways and / or lungs. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat a disease or condition (e.g., one affecting the airways and / or lungs, such as alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and / or pulmonary fibrosis) that would benefit from expression of the encoded polypeptide. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to prevent or treat progressive lung destruction. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat acute or chronic lung diseases. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat alpha-1-antitrypsin deficiency. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat pulmonary alveolar microlithiasis. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat primary ciliary dyskinesia. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat pulmonary alveolar proteinosis. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used to treat pulmonary arterial hypertension.In some embodiments, any of the recombinant nucleic acids, viruses and / or pharmaceutical compositions or formulations described herein can be used to treat pulmonary fibrosis.

[0196] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in preparing a medicament useful for delivering one or more polynucleotides encoding a polypeptide (e.g., an inhaled therapeutic polypeptide, such as an alpha-1-antitrypsin polypeptide) to one or more cells of a subject (e.g., one or more cells of the subject's respiratory tract). In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in preparing a medicament useful for treating a disease or condition (e.g., one affecting the airways and / or lungs, such as alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and / or pulmonary fibrosis) that would benefit from expression of the encoded polypeptide. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in preparing a medicament useful for preventing or treating progressive lung destruction. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating acute or chronic pulmonary disease. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating alpha-1-antitrypsin deficiency. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating alveolar microlithiasis. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating primary ciliary dyskinesia. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating pulmonary alveolar proteinosis.In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating pulmonary arterial hypertension. In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein can be used in the preparation of a medicament useful for treating pulmonary fibrosis.

[0197] VI. Method Certain aspects of the present disclosure relate to methods of delivering a polypeptide to one or more cells of a subject's respiratory tract (e.g., airway epithelial cells (goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells), serous cells, brush cells, tumor cells, non-ciliated columnar cells, and / or metaplastic cells); alveolar cells (type 1 pneumocytes, type 2 pneumocytes, and / or cuboidal non-ciliated cells); bronchial salivary gland cells (such as serous gland cells, mucous cells, and / or duct cells); etc.), the method comprising: (a) administering to the subject a pharmaceutical composition comprising any of the viruses described herein (e.g., herpes simplex virus, e.g., HSV-1), including any of the recombinant nucleic acids described herein (e.g., recombinant herpes simplex virus genomes, e.g., recombinant HSV-1 genomes), comprising one or more polynucleotides encoding the polypeptide; and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or by inhalation. In some embodiments, the pharmaceutical composition is administered to the subject intranasally or by inhalation. In some embodiments, the pharmaceutical composition is administered to the subject by inhalation. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the herpesvirus or pharmaceutical composition is administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0198] In some embodiments, the herpes virus (e.g., herpes simplex virus) is replication-competent. In some embodiments, the herpes virus (e.g., herpes simplex virus) is replication-deficient. In some embodiments, the subject is human. In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, the subject is afflicted with one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject does not have cystic fibrosis and / or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject is afflicted with alpha-1-antitrypsin deficiency. In some embodiments, the subject is afflicted with alveolar microlithiasis. In some embodiments, the subject is afflicted with primary ciliary dyskinesia. In some embodiments, the subject is afflicted with pulmonary alveolar proteinosis. In some embodiments, the subject has pulmonary arterial hypertension. In some embodiments, the subject has pulmonary fibrosis. In some embodiments, the polypeptide is any of the polypeptides described herein. In some embodiments, the herpesvirus is any of the herpesviruses described herein. In some embodiments, the recombinant herpesvirus genome is any of the recombinant nucleic acids described herein.

[0199] Other aspects of the present disclosure relate to expressing, enhancing, increasing, augmenting, and / or supplementing levels of a polypeptide (e.g., an inhaled therapeutic polypeptide) in one or more cells of a subject, the method comprising administering to the subject any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, the subject is afflicted with acute and / or chronic pulmonary disease. In some embodiments, the subject is afflicted with one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject does not have cystic fibrosis and / or chronic obstructive pulmonary disease (COPD).

[0200] In some embodiments, administration of the recombinant nucleic acid, virus, medicinal agent, and / or pharmaceutical composition or formulation to a subject increases polypeptide (e.g., inhaled therapeutic polypeptide) levels (transcript or protein levels) by at least about two-fold in one or more contacted or treated cells of the subject compared to the endogenous level of the polypeptide in one or more corresponding untreated cells in the subject. For example, administration of a recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation can increase polypeptide (e.g., inhaled therapeutic polypeptide) levels (transcript or protein levels) in one or more contacted or treated cells of a subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, at least about 1000-fold, or more, compared to the endogenous level of the polypeptide in one or more corresponding untreated cells in the subject. In some embodiments, the one or more contacted or treated cells are one or more cells of the respiratory tract (e.g., one or more cells of the airway epithelium). Methods for measuring transcript or protein levels from a sample are well known to those of skill in the art, such as, for example, qPCR, Western blot, mass spectrometry, etc.

[0201] Another aspect of the present disclosure relates to a method of improving at least one respiratory volume measurement in a subject in need thereof, the method comprising administering to the subject any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, the subject is afflicted with acute and / or chronic pulmonary disease. In some embodiments, the subject is afflicted with one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject does not have cystic fibrosis and / or chronic obstructive pulmonary disease (COPD).

[0202] In some embodiments, administering the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation to a subject improves at least one respiratory volume measurement by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more, compared to at least one reference respiratory volume measured in the subject before treatment. Examples of suitable respiratory volumes that can be measured include, for example, total lung capacity (TLC), the volume in the lungs at maximum inflation; tidal volume (TV), the volume of air entering and leaving the lungs during quiet breathing; residual volume (RV), the volume of air remaining in the lungs after a maximum expiration; expiratory reserve volume (ERV), the maximum volume of air (in excess of the tidal volume) that can be exhaled during a forced expiration; inspiratory reserve volume (ERV), the maximum volume of air that can be inhaled from the end of inspiration; inspiratory volume (IC), the sum of IRV and TV; inspiratory vital capacity (IVC), the maximum volume of air inhaled from the point of maximum expiration; vital capacity (VC), the volume of air exhaled after the deepest inspiration; functional residual capacity (FRC), the volume of the lungs at the end of expiration; forced vital capacity (FVC), a measure of the vital capacity from a maximum forced expiration; forced expiratory volume (time) (FEV t ), the volume of air exhaled under exertion in the first t seconds; forced inspiratory flow (FIF), a specific measurement of the forced inspiratory curve; forced expiratory flow (PEF), the maximal forced expiratory flow measured with a peak flow meter; maximal voluntary ventilation (MVV), the volume of air exhaled in a specific period during repeated maximal efforts. Methods are generally known to those skilled in the art.

[0203] Another aspect of the present disclosure relates to a method of reducing, preventing, or treating chronic inflammation in the lungs in a subject in need thereof, the method comprising administering to the subject any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject is afflicted with a disease affecting the airways and / or lungs. In some embodiments, the subject is afflicted with acute and / or chronic pulmonary disease. In some embodiments, the subject is afflicted with one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject does not have cystic fibrosis and / or chronic obstructive pulmonary disease (COPD). Methods of measuring pulmonary inflammation, including improvements thereto, are well known to those skilled in the art, such as by measuring exhaled nitric oxide, determining the percentage of eosinophils in sputum and / or blood, etc.

[0204] Another aspect of the present disclosure relates to a method of reducing, inhibiting, or treating progressive lung destruction in a subject in need thereof, the method comprising administering to the subject any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject is suffering from a disease affecting the airways and / or lungs. In some embodiments, the subject is suffering from acute and / or chronic lung disease. In some embodiments, the subject is suffering from one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject is free of cystic fibrosis and / or chronic obstructive pulmonary disease (COPD). Methods for measuring lung destruction are well known to those skilled in the art, for example, by the method described in Saetta et al. (Am Rev Respir Dis. 1985 May;131(5):764-9).

[0205] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of a disease affecting the airways and / or lungs in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject is afflicted with acute and / or chronic pulmonary disease. In some embodiments, the subject is afflicted with one or more of alpha-1-antitrypsin deficiency, alveolar microlithiasis, primary ciliary dyskinesia, congenital pulmonary alveolar proteinosis, pulmonary arterial hypertension, and pulmonary fibrosis. In some embodiments, the subject is not afflicted with cystic fibrosis and / or chronic obstructive pulmonary disease (COPD).

[0206] Another aspect of the present disclosure relates to methods for providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of alpha-1-antitrypsin deficiency in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SERPINA1 gene (one or both copies). In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding an alpha-1-antitrypsin polypeptide. In some embodiments, administration of the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of alpha-1-antitrypsin deficiency.

[0207] Signs and symptoms of alpha-1-antitrypsin deficiency may include, but are not limited to, shortness of breath (especially during exercise); wheezing and / or whistling sounds while breathing; increased susceptibility to lung infections; fatigue; rapid heart rate upon standing; weight loss; chronic cough (often containing blood); emphysema (commonly panacinar); and increased sputum production.

[0208] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of alveolar microlithiasis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicinal agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the SLC34A2 gene (one or both copies). In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding a sodium-dependent phosphate transport protein 2B polypeptide. In some embodiments, administration of the recombinant nucleic acid, virus, medicinal agent, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of alveolar microlithiasis.

[0209] Signs and symptoms of alveolar microlithiasis may include, but are not limited to, shortness of breath, dry cough (which may contain blood); chest pain; asthenia; and pneumothorax.

[0210] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of primary ciliary dyskinesia in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes (one or both copies) selected from DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1, DNAAF2, and LRRC6. In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding a dynein heavy chain 5 axonemal polypeptide, a dynein heavy chain 11 axonemal polypeptide, a coiled-coil domain-containing protein 39 polypeptide, a dynein intermediate chain 1 axonemal polypeptide, a coiled-coil domain-containing protein 40 polypeptide, a coiled-coil domain-containing protein 103 polypeptide, a sperm-associated antigen 1 polypeptide, a zinc finger MYND domain-containing protein 10 polypeptide, an armadillo repeat-containing protein 4 polypeptide, a coiled-coil domain-containing protein 151 polypeptide, a dynein intermediate chain 2 axonemal polypeptide, a radial spoke head 1 homolog polypeptide, a coiled-coil domain-containing protein 114 polypeptide, a radial spoke head protein 4 homolog polypeptide, a dynein assembly factor 1 axonemal polypeptide, a dynein assembly factor 2 axonemal polypeptide, and / or a leucine-rich repeat-containing protein 6 polypeptide.In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes associated with primary ciliary dyskinesia, the subject is treated with a recombinant nucleic acid comprising one or more polynucleotides encoding wild-type and / or functional variants of the corresponding polypeptides (e.g., the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in the DNAH5 gene (one or both copies)), and the subject is administered a virus, medicament, and / or pharmaceutical composition or formulation (including a recombinant nucleic acid comprising one or more polynucleotides encoding, for example, a dynein heavy chain 5 axoneme polypeptide). In some embodiments, administration of the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of primary ciliary dyskinesia.

[0211] Signs and symptoms of primary ciliary dyskinesia may include, but are not limited to, chronic infections of the sinuses, ears, and / or lungs; chronic nasal congestion; runny nose with mucus and pus discharge; hearing loss; difficulty breathing (especially in newborns); chronic cough; recurrent pneumonia; chronic sinusitis; bronchiectasis; and partial or total collapse of the lung.

[0212] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary alveolar proteinosis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes (one or both copies) selected from SFTPB, SFTPC, NKX2-1, ABCA3, CSF2RB, and / or CSF2RA. In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding a pulmonary surfactant-associated protein B polypeptide, a pulmonary surfactant-associated protein C polypeptide, a homeobox protein Nkx-2.1 polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a cytokine receptor common subunit beta polypeptide, and a granulocyte-macrophage colony-stimulating factor receptor subunit alpha polypeptide. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes associated with pulmonary alveolar proteinosis, the subject is treated with a recombinant nucleic acid comprising one or more polynucleotides encoding wild-type and / or functional variants of the corresponding polypeptides (e.g., the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in an SFTPB gene (one or both copies)), and the subject is administered a virus, medicament, and / or pharmaceutical composition or formulation (including a recombinant nucleic acid comprising one or more polynucleotides encoding, for example, a pulmonary surfactant-associated protein B polypeptide). In some embodiments, administration of the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of pulmonary alveolar proteinosis.

[0213] Signs and symptoms of pulmonary alveolar proteinosis may include, but are not limited to, difficulty breathing; coughing occasionally with mucus or blood; bluish complexion; general fatigue; low-grade fever; weight loss; chest pain or tension; low levels of blood oxygen; and clubbing.

[0214] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary arterial hypertension in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes (one or both copies) selected from BMPR2, ATP2A2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, and / or EIF2AK4. In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding a bone morphogenetic protein receptor type 2 polypeptide, a sarcoplasmic / endoplasmic reticulum calcium ATPase 2 polypeptide, a serine / threonine protein kinase receptor R3 polypeptide, an endoglin polypeptide, a mothers-against-decapentaplegic homolog 9 polypeptide, a caveolin-1 polypeptide, a potassium channel subfamily K member 3 polypeptide, and / or an eIF-2-alpha kinase GCN2 polypeptide. In some embodiments, the subject's genome comprises a pathogenic mutation and / or a loss-of-function mutation in one or more genes associated with pulmonary arterial hypertension, the subject is treated with a recombinant nucleic acid comprising one or more polynucleotides encoding wild-type and / or functional variants of the corresponding polypeptides (e.g., the subject's genome comprises a pathogenic variant and / or a loss-of-function mutation in the BMPR2 gene (one or both copies)), and the subject is administered a virus, medicament, and / or pharmaceutical composition or formulation (e.g., comprising a recombinant nucleic acid comprising one or more polynucleotides encoding a bone morphogenetic protein receptor type 2 polypeptide, etc.). In some embodiments, administration of the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of pulmonary arterial hypertension.

[0215] Signs and symptoms of pulmonary arterial hypertension may include, but are not limited to, shortness of breath, initially during exertion and eventually at rest; fatigue; dizziness or fainting spells; chest tightness or pain; swelling of the ankles, legs, and / or abdomen; bluish lips and skin; and a racing or pounding pulse.

[0216] Another aspect of the present disclosure relates to a method of providing preventative, palliative, or therapeutic relief of one or more signs or symptoms of pulmonary fibrosis in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes (one or both copies) selected from SFTPC, ABCA3, SFTPA2, TERT, TERC, DKC1, RTEL, PARN, TINF2, NAF1, MUC5B, DSP, STN1, and / or DPP9. In some embodiments, the recombinant nucleic acid (e.g., a recombinant herpesvirus genome) comprises one or more polynucleotides encoding a pulmonary surfactant-associated protein C polypeptide, an ATP-binding cassette subfamily A member 3 polypeptide, a pulmonary surfactant-associated protein A2 polypeptide, a telomerase reverse transcriptase polypeptide, a dyskerin polypeptide, a regulator of telomere elongation helicase 1 polypeptide, a poly(A)-specific ribonuclease PARN polypeptide, a TERF1-interacting nuclear factor 2 polypeptide, an H / ACA ribonucleoprotein complex non-core subunit NAF1 polypeptide, a mucin-5B polypeptide, a desmoplakin polypeptide, a CST complex subunit STN1 polypeptide, and / or a dipeptidyl peptidase 9 polypeptide. In some embodiments, the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in one or more genes associated with pulmonary fibrosis, the subject is treated with a recombinant nucleic acid comprising one or more polynucleotides encoding wild-type and / or functional variants of the corresponding polypeptides (e.g., the subject's genome comprises a pathogenic variant and / or loss-of-function mutation in an SFTPC gene (one or both copies)), and the subject is administered a virus, medicinal agent, and / or pharmaceutical composition or formulation (e.g., one comprising a recombinant nucleic acid comprising one or more polynucleotides encoding a pulmonary surfactant-associated protein C polypeptide, etc.).In some embodiments, administration of the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation affects a measurable improvement or prevention of one or more signs or symptoms of pulmonary fibrosis.

[0217] Signs and symptoms of pulmonary fibrosis may include, but are not limited to, shortness of breath, especially during exercise; a dry, frequent cough; rapid, shallow breathing; gradual, involuntary weight loss; fatigue; joint and muscle pain; swelling of the legs; and clubbing of the fingertips or toes.

[0218] The recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein can be administered by any suitable method or route known in the art, including, but not limited to, orally, intranasally, intratracheally, sublingually, bucally, topically, rectally, by inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, topically, epicutaneously, or any combination thereof. Accordingly, the present disclosure encompasses methods of delivering any of the recombinant nucleic acids, viruses, medicaments, or pharmaceutical compositions or formulations described herein to an individual (e.g., an individual having or at risk of developing a disease affecting the respiratory tract and / or lungs). In some embodiments, the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein are administered orally, intranasally, intratracheally, and / or via inhalation. In some embodiments, the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein are administered intranasally or via inhalation. In some embodiments, the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein are administered via inhalation. In some embodiments, the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein are administered using a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein are administered using a nebulizer. In some embodiments, the nebulizer is a vibrating mesh nebulizer.

[0219] Methods for delivering drugs to the respiratory tract and / or lungs via oral, intranasal, intratracheal, and / or inhalation routes of administration are generally known to those skilled in the art (e.g., Gardenhire et al. A Guide to Aerosol Delivery Devices for Respiratory Therapists, 4th Edition, American Association for Respiratory care, 2017; Patil et al. Pulmonary Drug Delivery Strategies: A Concise, Systematic Review, Lung India. 2012.29(1):44-9; Marx et al. Intranasal Drug Administration-An Attractive Delivery Route for Some Drugs, 2015).

[0220] In some embodiments, the recombinant nucleic acid, virus, medicinal agent, and / or composition or formulation is delivered to the lungs by inhalation of an aerosolized formulation. Inhalation can occur through the subject's nose and / or mouth. Exemplary devices for delivering the recombinant nucleic acid, virus, medicinal agent, and / or pharmaceutical composition or formulation to the lungs can include, but are not limited to, dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers), impingement jets, extruded jets, surface wave microfluidic nebulization, capillary aerosol generation, electrohydrodynamic aerosol devices, and the like (see, e.g., Carvalho and McConville. The function and performance of aqueous devices for inhalation therapy. (2016) Journal of Pharmacy and Pharmacology).

[0221] Liquid formulations can be administered to a subject's lungs using, for example, a pressurized metered-dose inhaler (pMDI). pMDIs generally include at least two components: a canister in which the liquid formulation is held under pressure in combination with one or more propellants, and a container used to hold and actuate the canister. The canister may contain a single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, through which the contents of the canister can be expelled. Aerosolized medication is administered from a pMDI by applying force to the canister, forcing it into a receptacle, thereby opening the valve and transporting medication particles from the valve through the receptacle outlet. Upon expulsion from the canister, the liquid formulation is atomized, forming an aerosol. pMDIs typically use one or more propellants to pressurize the contents of the canister and propel the liquid formulation through the receptacle outlet, forming an aerosol. Any suitable propellant can be utilized and can take a variety of forms, such as a compressed gas or a liquefied gas.

[0222] Liquid formulations can be administered to a subject's lungs using, for example, a nebulizer. A nebulizer is a liquid aerosol generator that converts a liquid formulation into a mist or cloud of droplets, often with a median aerodynamic diameter of less than about 5 microns, which can be inhaled into the lower respiratory tract. When the aerosol cloud is inhaled, the droplets carry the active agent(s) to the nose, upper respiratory tract, and / or deep lung. The formulation can be administered to a patient using any type of nebulizer known in the art, including, but not limited to, pneumatic (jet) nebulizers, electromechanical nebulizers (e.g., ultrasonic nebulizers, vibrating mesh nebulizers), etc. Pneumatic (jet) nebulizers use a pressurized gas supply as the driving force for atomizing the liquid formulation. The compressed gas is delivered through a nozzle or jet, creating a low-pressure field that entrains the surrounding liquid formulation, shearing it into a thin film or filaments. The thin film or filaments are unstable and break into small droplets, which are carried into the inhaled air by the compressed gas flow. A baffle inserted into the droplet plume sifts out large droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers nebulize liquid formulations using electrically generated mechanical force. The electromechanical driving force can be applied, for example, by vibrating the liquid formulation at ultrasonic frequencies or by forcing the bulk liquid through small holes in a thin film. This force generates a thin liquid film or filament stream, which breaks into droplets and forms a slow-moving aerosol stream that can be entrained in the inhaled airflow. In some embodiments, the nebulizer is a vibrating mesh nebulizer. Examples of vibrating mesh nebulizers include, for example, Phillips InnoSpire, Aerogen Solo, and PARI eFlow.

[0223] Liquid formulations can be administered to the subject's lungs, for example, using electrohydrodynamic (EHD) aerosol devices, which use electrical energy to aerosolize liquid drug solutions or suspensions.

[0224] Dry powder formulations can be administered to a subject's lungs using, for example, a dry powder inhaler (DPI). DPIs typically use a mechanism such as a burst of gas to create a cloud of dry powder in a container, which can be inhaled by the subject. In DPIs, the administered dose is stored in the form of a non-pressurized dry powder, and upon actuation of the inhaler, the powder particles are inhaled by the subject. In some cases, compressed gas can be used to administer the powder, similar to a pMDI. In some cases, DPIs can be breath-activated (aerosols are generated precisely in response to inspiration). Typically, dry powder inhalers administer doses of less than tens of milligrams per inhalation to avoid inducing coughing. Examples of DPIs include, for example, the Turbohaler® inhaler (AstraZeneca), the Clickhaler® inhaler (Innovata), the Diskus® inhaler (Glaxo), the EasyHaler® (Orion), and the Exubera® inhaler (Pfizer).

[0225] In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to the subject once, hi some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to the subject at least twice (e.g., at least twice, at least three times, at least four times, at least five times, at least ten times, etc.). In some embodiments, at least about 1 hour (e.g., at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 120 days, etc.) elapses between administrations (e.g., between the first and second administrations, between the second and third administrations, etc.). In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to a subject once, twice, three times, four times, or five or more times daily. In some embodiments, the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation is administered to a subject once, twice, three times, four times, five or more times per month.

[0226] VII.Host cells Certain aspects of the present disclosure relate to one or more host cells comprising any of the recombinant nucleic acids described herein. Any suitable host cell (prokaryotic or eukaryotic) known in the art can be used, including, for example, eubacteria, such as gram-negative or gram-positive microorganisms, for example, Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), and the like. Prokaryotic cells, including Enterobacteriaceae such as S. yphimurium, Serratia (e.g., S. marcescans), and Shigella, and bacilli such as B. subtilis and B. licheniformis; fungal cells (e.g., S. cerevisiae); insect cells (e.g., S2 cells); and SV40-transformed monkey kidney strain CV1 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma line (Hep G2), DHFR These include Chinese hamster ovary (CHO) cells, including CHO cells, and mammalian cells, including myeloma cell lines such as NS0 and Sp2 / 0. In some embodiments, the host cells are human or non-human primate cells.In some embodiments, host cell is a cell derived from a cell line.Examples of suitable host cell or cell line can include, but are not limited to, 293 cell, HeLa cell, SH-Sy5y cell, Hep G2 cell, CACO-2 cell, A549 cell, L929 cell, 3T3 cell, K562 cell, CHO-K1 cell, MDCK cell, HUVEC cell, Vero cell, N20 cell, COS-7 cell, PSN1 cell, VCaP cell, CHO cell, etc.

[0227] In some embodiments, the recombinant nucleic acid is a herpes simplex virus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is an HSV-1 amplicon or an HSV-1 hybrid amplicon. In some embodiments, a virus comprising one or more recombinant nucleic acids described herein is produced by contacting a host cell containing a helper virus with an HSV-1 amplicon or an HSV-1 hybrid amplicon described herein. In some embodiments, the virus is recovered from the supernatant of the contacted host cells. Methods for producing a virus by contacting a host cell containing a helper virus with an HSV-1 amplicon or an HSV-1 hybrid amplicon are known in the art.

[0228] In some embodiments, the host cell is a complementing host cell. In some embodiments, the complementing host cell expresses one or more genes inactivated in any of the viral vectors described herein. In some embodiments, the complementing host cell is contacted with a recombinant herpesvirus genome described herein (e.g., a recombinant herpes simplex virus genome). In some embodiments, contacting the complementing host cell with the recombinant herpesvirus genome produces a herpesvirus comprising one or more recombinant nucleic acids described herein. In some embodiments, the virus is recovered from the supernatant of the contacted host cell. Methods of producing virus by contacting a complementing host cell with a recombinant herpes simplex virus are generally described in WO2015 / 009952, WO2017 / 176336, WO2019 / 200163, and / or WO2019 / 210219.

[0229] VIII. Products or Kits Certain aspects of the present disclosure relate to articles of manufacture or kits that include any of the recombinant nucleic acids, viruses, medicaments, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the articles of manufacture or kits include a package insert that includes instructions for administering the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation.

[0230] Suitable containers for the recombinant nucleic acid, virus, medicament, and / or pharmaceutical composition or formulation may include, for example, bottles, vials, bags, tubes, and syringes. Containers may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or alloys (such as stainless steel or Hastelloy). In some embodiments, the container comprises a label on or associated with the container, the label indicating directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, inhalers, nebulizers, intranasal administration devices, package inserts, etc.

[0231] The specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. Various modifications of the disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [Example]

[0232] The present disclosure will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0233] Example 1: Modified herpes simplex virus vectors encoding inhaled therapeutic polypeptides To generate a modified herpes simplex virus genome vector capable of expressing an inhaled therapeutic polypeptide in target mammalian cells (such as cells of the respiratory tract), the herpes simplex virus genome (FIG. 1A) is first modified to inactivate one or more herpes simplex virus genes. Such modifications can reduce the toxicity of the genome in mammalian cells. Variants of these modified / attenuated recombinant virus constructs are then generated so that they carry one or more polynucleotides encoding the desired inhaled therapeutic polypeptide.These variants include: 1) a recombinant ΔICP4-modified HSV-1 genome comprising an expression cassette containing a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1B); 2) a recombinant ΔICP4 / ΔUL41-modified HSV-1 genome comprising an expression cassette containing a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1C); 3) a recombinant ΔICP4 / ΔUL41-modified HSV-1 genome comprising an expression cassette containing a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each UL41 locus (Figure 1D); 4) a recombinant ΔICP4 / ΔICP22-modified HSV-1 genome comprising an expression cassette containing a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1E); 5) a recombinant ΔICP4 / ΔICP22-modified HSV-1 genome comprising an expression cassette containing a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1E). 1) a recombinant ΔICP4 / ΔICP22 modified HSV-1 genome containing an expression cassette comprising a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into the 22 locus (Figure 1F); 6) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome containing an expression cassette comprising a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1G); 7) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome containing an expression cassette comprising a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each UL41 locus (Figure 1H); and 8) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome containing an expression cassette comprising a coding sequence for an inhaled therapeutic polypeptide (e.g., SEQ ID NO: 3) under the control of a heterologous promoter integrated into each ICP22 locus (Figure 1I).

[0234] These modified herpes simplex virus genome vectors are transfected into engineered cells that are modified to express one or more herpes simplex virus genes.These engineered cells secrete the replication-defective herpes simplex virus that has the modified genome packaged in it into the supernatant of cell culture.Then, the supernatant is collected, concentrated, and filtered through a 5 μm filter for sterilization.

[0235] Example 2: In vivo administration of modified herpes simplex virus vectors to the respiratory tract of wild-type and mutant animals Eight C57BL / 6 mice ("wild type") and four intestine-corrected CFTR-deficient Cftr tm1Unc Tg(FABPCFTR)1 Jaw / J mice (CFTR - / - In vivo experiments were performed in four wild-type and four CFTR mice to determine whether a modified herpes simplex virus vector ("HSV-CFTR," an engineered HSV-1 encoding human CFTR) was amenable to non-invasive inhalation administration and capable of transducing tissue throughout the airways of treated animals. - / - Mice were administered HSV-CFTR in parallel using a clinically approved nebulizer. Using this same inhalation device, four wild-type mice were separately administered a vehicle control. Cage-side and clinical observations performed throughout the experiment confirmed no differences between HSV-CFTR and vehicle-treated animals. No differences in body weight were observed between groups. Forty-eight hours after nebulization, animals were euthanized, and the following tissue samples were collected separately from the entire airway of each animal: trachea (superior and inferior), bronchi (left and right), right lung (anterior, middle, posterior, and accessory lobes), and left lung. Tissues for qPCR / qRT-PCR analysis (2 animals per group) were flash-frozen in liquid nitrogen. Tissues for histology (2 animals per group) were fixed in 10% neutral-buffered formalin and embedded in paraffin. No gross observations were observed in any treated animals during necropsy.

[0236] Biopsies taken from the airways of animals exposed to HSV-CFTR revealed detectable levels of human CFTR DNA by qPCR analysis, with the majority of vector disseminated relatively evenly to the right and left lungs (Figure 2A). Surprisingly, CFTR-deficient animals demonstrated improved transduction efficiency in all lung tissues tested, with 31.9-fold, 7.1-fold, 3.2-fold, 6.4-fold, and 3.4-fold increases in vector genomes detected in the right accessory lobe, right posterior lobe, right anterior lobe, right middle lobe, and left lobe, respectively, compared to wild-type counterparts. Even ignoring the right accessory lobe lung as an outlier, a 5.1-fold increase in HSV-CFTR transduction was observed in the CFTR-deficient animals. - / - Human CFTR RNA was detected in the lungs of treated animals, and a similar trend was observed at this transcript level, with higher transgene expression detected in CFTR-deficient versus wild-type animals in most tissues tested (Figure 2B). Little or no human CFTR DNA or RNA was observed in vehicle-treated animals, suggesting the specificity of the assay for the human transgene-encoded HSV-CFTR.

[0237] Histological examination of tissues collected throughout the airways from each treatment group was performed to determine whether infection with the engineered vector caused acute inflammation, necrosis, or other gross physiological changes that could indicate potential safety concerns for HSV-CFTR inhalation therapy in vivo. Compared to vehicle-treated mice, no overt signs of immune cell infiltration, fibrosis, or necrosis were detected in either HSV-CFTR-treated tissues from wild-type or CFTR-deficient immunocompetent animals (Figure 3). Bronchoalveolar lavage (BAL) fluid collected from these animals 48 hours post-infection showed no significant differences in cellular infiltration into the lungs between groups, further demonstrating the safety of this vector in both wild-type and CFTR-deficient animals after inhalation (Figure 4).

[0238] Collectively, this data demonstrates that this modified herpes simplex virus vector is suitable for noninvasive inhalation administration using a clinically approved nebulizer and that the vector can be effectively delivered in the context of CFTR-deficient lung epithelium, revealing potent transduction and subsequent expression of the encoded human CFTR transgene in target airway tissue.

[0239] Example 3: In vivo tolerability and biodistribution of repeated-dose modified herpes simplex virus vectors in non-human primates Part of the purpose of this study was to evaluate the delivery of modified herpes simplex virus vectors in nonhuman primates (NHPs) after nebulization. This study was conducted, in part, to ensure that repeat delivery of modified herpes simplex virus vectors is feasible for future inhalation studies. A single male cynomolgus monkey received a total of three exposures (vehicle (day 1), low-dose HSV-CFTR (day 5), and high-dose HSV-CFTR (day 17)) before being euthanized and tissue collection performed (Figure 5). Tissues collected included the brain, spleen, kidney, liver, lungs (three unique locations), heart, and lymph nodes (axillary and inguinal). Blood was also collected pre- and post-dose to determine systemic exposure to the drug product after inhalation application. All procedures performed complied with applicable animal welfare laws and were approved by the respective national Institutional Animal Care and Use Committees (IACUCs).

[0240] No abnormal cage side or clinical observations were observed in the animals throughout the study. Furthermore, no changes in food consumption or body weight were observed during the dosing period, indicating that repeated dosing with the modified herpes simplex virus vector was well tolerated.

[0241] On day 19, 48 hours after administration of nebulized high-dose HSV-CFTR, blood and tissue samples were collected to analyze biodistribution and effector expression via qPCR and qRT-PCR, respectively. Significant vector accumulation was observed in all three lung tissues tested, while little or no vector was detected in the remaining tissues (Figure 6A). All blood samples were below the detection limit of the qPCR assay, suggesting that the vector was restricted to the respiratory tract without significant spread to the circulatory system. Human CFTR RNA was detected primarily in the animals' lungs, with little, if any, effector expression in the other tested tissues (Figure 6B). Interestingly, transgene expression levels in the lungs were 1–2 orders of magnitude higher (on a tissue copy / gram basis) than the vector genome in the tested tissues, suggesting that the modified herpes simplex virus vector robustly expresses the encoded human transgene upon infection of the respiratory epithelium after nebulization.

[0242] In summary, without wishing to be bound by theory, the preclinical data provided herein demonstrate...

Claims

[Claim 1] The invention described in the specification of this application.

Images