Compositions and methods for delivering CFTR polypeptides

Abstract

To provide a kit comprising a composition and a neburizer for the treatment of a chronic lung disease, such as cystic fibrosis.SOLUTION: A pharmaceutical composition comprises (a) herpes simplex virus type 1(HSV-1) comprising the recombinant HSV-1 genome, where the recombinant HSV-1 genome comprises one or more polynucleotides encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide; and (b) a pharmaceutically acceptable excipient. The pharmaceutical composition is formulated with a neburizer.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 / 802,871, filed February 8, 2019, the entirety of which is 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: Computer Readable Form (CRF) of the Sequence Listing (Filename: 7613420001140SEQLIST.txt, Recorded on: January 17, 2020, Size: 44KB).

[0003] FIELD OF THEINVENTION The present disclosure relates, in part, to recombinant nucleic acids comprising one or more polynucleotides encoding a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) polypeptide, viruses comprising same, compositions and formulations thereof, and methods of their use (e.g., for the prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of chronic pulmonary diseases, such as cystic fibrosis). [Background technology]

[0004] background The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride and bicarbonate channel that is important for lung homeostasis. Reduced or lost CFTR channel function often leads to a chronic inflammatory response that promotes mucus congestion, chronic bacterial infections, and associated progressive lung damage. Reduced CFTR expression has been suggested to be a component of the lung pathology observed in patients with chronic obstructive pulmonary disease (COPD), and loss-of-function mutations in the CFTR gene lead to the dire consequences associated with cystic fibrosis (CF). Over 2,000 unique mutations in the CFTR gene have been described.

[0005] CF is a genetic disease characterized by the accumulation of copious amounts of sticky mucus that can damage many of the body's organs, but the most severe pathological consequences are lung-related. CF patients present with dehydrated mucus in the lungs that leads to airway obstruction, chronic bacterial infections (and associated inflammatory responses), bronchiectasis, and ultimately respiratory failure. Currently, more than 70,000 people are living with cystic fibrosis worldwide. Historically, children born with CF died as infants, and until 1980, the median survival time was less than 20 years. While medical advances in the past 30 years have significantly improved both the quality of life and life expectancy of CF patients (40.6 years in the United States as of 2013), there is a clear need for novel therapeutic options that target the molecular correction of the CFTR deficiency observed in CF patients, as well as patients suffering from other chronic lung diseases such as COPD.

[0006] All references cited herein, including patent applications, patent publications, non-patent literature, and NCBI / UniProtKB / Swiss-Prot accession numbers, are hereby 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

[0007] Quick Overview To meet these and other needs, provided herein are recombinant nucleic acids (e.g., recombinant herpesvirus genomes) encoding one or more CFTR polypeptides for use in viruses (e.g., herpesviruses), pharmaceutical compositions and formulations, medicaments, and / or methods useful for treating a CFTR deficiency in a subject in need of such treatment, and / or for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of a chronic lung disease, such as cystic fibrosis.

[0008] The inventors have shown that the recombinant viruses described herein can effectively transduce airway epithelial cells derived from CF patients and successfully express their encoded exogenous human CFTR (see, e.g., Example 2). In addition, the inventors have shown that the recombinant viruses described herein express full-length functional human CFTR that is properly transported to the plasma membrane (see, e.g., Example 2). Furthermore, the inventors have shown that the recombinant viruses described herein rescue disease phenotypes in clinically relevant 3D organotypic cultures prepared from biopsies taken from multiple CF patients carrying various underlying CFTR mutations (see, e.g., Example 3). Furthermore, the inventors have shown that recombinant HSV vectors can be administered to the lungs of immunocompetent animals via multiple routes, and further, that non-invasive inhalation administration routes induce lower cell entry in the lungs while expressing similar levels of the encoded transgene in the lungs (see, e.g., Example 4). Without wishing to be bound by theory, it is believed that increasing, enhancing, and / or replenishing CFTR polypeptide levels in one or more cells of a subject in need thereof by administering one or more of the recombinant nucleic acids, viruses, agents, and / or compositions described herein will 1) reduce or prevent mucus accumulation in one or more organs (e.g., lungs) of the individual, 2) reduce or prevent airway obstruction in the individual, 3) reduce or prevent chronic bacterial infection and / or associated chronic inflammation in the lungs of the individual, 4) reduce or prevent bronchiectasis in the individual, 5) reduce, inhibit, or treat progressive lung damage in the individual, and / or 6) provide preventative, palliative, or therapeutic relief of one or more signs or symptoms of a chronic lung disease (e.g., cystic fibrosis, COPD, etc.).

[0009] Thus, certain aspects of the present disclosure relate to a recombinant herpesvirus genome comprising one or more polynucleotides encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide. 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 a CFTR polypeptide 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, a recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof.

[0010] In some embodiments that may be combined with any of the above-mentioned embodiments, the CFTR polypeptide is a human CFTR polypeptide.In some embodiments that may be combined with any of the above-mentioned embodiments, the CFTR 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 with the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.In some embodiments that may be combined with any of the above-mentioned embodiments, the CFTR 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 with the amino acid sequence of SEQ ID NO:5.In some embodiments, the CFTR polypeptide comprises the amino acid sequence of SEQ ID NO:5.

[0011] In some embodiments that may be combined with any of the previous 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, 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.

[0012] 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 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 junction region. In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the junction region.

[0013] 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 CFTR 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 CFTR polypeptide in one or both of the 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 CFTR 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 a CFTR 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 a CFTR polypeptide in one or both of the 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 CFTR 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 a CFTR 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 a CFTR polypeptide within the UL55 viral locus.

[0014] 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 the corresponding wild-type herpesvirus genome. In some embodiments, the target cell is a human cell. In some embodiments that may be combined with any of the preceding embodiments, the target cell is a respiratory cell. In some embodiments that may be combined with any of the preceding embodiments, the target cell is an airway epithelial cell or a submucosal gland cell.

[0015] Other aspects of the present disclosure relate 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 defective. In some embodiments that may be combined with any of the preceding embodiments, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus when introduced into a target cell. In some embodiments, the target cell is a human cell. In some embodiments that may be combined with any of the preceding embodiments, the target cell is a respiratory cell. In some embodiments that may be combined with any of the preceding embodiments, the target cell is an airway epithelial cell or a submucosal gland cell. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus is selected from herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpes virus 6A, herpes virus 6B, herpes virus 7, Kaposi's sarcoma-associated herpes virus, and any combination or derivative thereof. In some embodiments that may be combined with any of the preceding embodiments, the herpes virus is 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.

[0016] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the recombinant herpesvirus genomes described herein and / or any of the herpesviruses described herein and a pharma- ceutically acceptable excipient. In some 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, and / or epicutaneous administration. In some embodiments, the pharmaceutical composition is suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharmaceutical composition is suitable for inhalation administration. In some embodiments, the pharmaceutical composition is suitable for non-invasive inhalation administration. In some 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, the pharmaceutical composition is suitable for nebulization (using 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.

[0017] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein as a medicament.

[0018] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein in therapy.

[0019] Other aspects of the present disclosure relate to the use of any of the recombinant nucleic acids, herpes viruses, and / or pharmaceutical compositions described herein in the production or manufacture of a medicament for treating one or more signs or symptoms of CFTR deficiency and / or chronic pulmonary disease (e.g., cystic fibrosis, COPD, etc.).

[0020] Another aspect of the present disclosure is a method of enhancing, increasing, enhancing, and / or supplementing CFTR polypeptide levels in one or more cells of a subject, comprising administering to the subject an effective amount of any of the recombinant herpesvirus genomes described herein, any of the herpesviruses described herein, and / or any of the pharmaceutical compositions described herein. In some embodiments, the one or more cells are one or more cells of the respiratory tract. In some embodiments, the one or more cells are one or more airway epithelial cells and / or one or more cells of a submucosal gland. In some embodiments that may be combined with any of the preceding embodiments, the subject suffers from a chronic lung disease. In some embodiments, the chronic lung disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). 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 subject's genome comprises a loss-of-function mutation in the CFTR gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, sublingually, bucally, topically, rectally, via inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, locally, and / or epicutaneously. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via non-invasive inhalation.In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / 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 recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered via a nebulizer (e.g., a vibrating mesh nebulizer).

[0021] Another aspect of the present disclosure relates to a method of reducing or inhibiting progressive lung damage in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant herpesvirus genomes described herein, any of the herpesviruses described herein, and / or any of the pharmaceutical compositions described herein. In some embodiments, the subject suffers from a chronic lung disease. In some embodiments, the chronic lung disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). 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 subject's genome comprises a loss-of-function mutation in the CFTR gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, sublingually, bucally, topically, rectally, via inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, locally, and / or epicutaneously. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via non-invasive inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / 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 recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered via a nebulizer (e.g., a vibrating mesh nebulizer).

[0022] 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 cystic fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant herpesvirus genomes described herein, any of the herpesviruses described herein, and / or any of the pharmaceutical compositions described herein. In some embodiments, the one or more signs or symptoms of cystic fibrosis are selected from persistent coughing that produces a lot of mucus, a lot of sticky mucus that accumulates in the airways, wheezing, difficulty breathing, sinusitis, recurrent lung infections, inflamed nasal passages, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof. 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 subject's genome comprises a loss-of-function mutation in the CFTR gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, sublingually, bucally, topically, rectally, via inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, locally, and / or epicutaneously. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via non-invasive inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / 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 recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered via a nebulizer (eg, a vibrating mesh nebulizer).

[0023] 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 COPD in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant herpesvirus genomes described herein, any of the herpesviruses described herein, and / or any of the pharmaceutical compositions described herein. In some embodiments, the one or more signs or symptoms of COPD are selected from shortness of breath, wheezing, chest tightness, excess mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof. 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 subject's genome comprises a loss-of-function mutation in the CFTR gene. In some embodiments that may be combined with any of the preceding embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, sublingually, bucally, topically, rectally, via inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, locally, and / or epicutaneously. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition is administered to the subject via non-invasive inhalation. In some embodiments, the recombinant herpesvirus genome, herpesvirus, and / 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 recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered via a nebulizer (eg, a vibrating mesh nebulizer).

[0024] Other aspects of the disclosure relate to an article of manufacture or kit that includes any of the recombinant herpesvirus genomes, herpesviruses, medicaments, and / or pharmaceutical compositions described herein and instructions for administering the recombinant herpesvirus genomes, herpesviruses, medicaments, or pharmaceutical compositions. In some embodiments, the article of manufacture or kit further includes a device for aerosolizing the recombinant herpesvirus genomes, herpesviruses, medicaments, and / or pharmaceutical compositions. In some embodiments, the device is a dry powder inhaler, a pressurized metered dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol device. In some embodiments, the device is a nebulizer (e.g., a vibrating mesh nebulizer). [The present invention 1001] A recombinant herpesvirus genome comprising one or more polynucleotides encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide. [The present invention 1002] 1001. A recombinant herpesvirus genome of the present invention that is capable of replication. [The present invention 1003] 1001. A recombinant herpesvirus genome of the present invention which is replication-deficient. [The present invention 1004] 1004. The recombinant herpesvirus genome of any one of claims 1001 to 1003, comprising said one or more polynucleotides encoding said CFTR polypeptide within one or more viral loci. [The present invention 1005] The recombinant herpesvirus genome of any one of the present inventions 1001 to 1004, selected from the group consisting of recombinant herpes simplex virus genome, recombinant varicella-zoster virus genome, recombinant human cytomegalovirus genome, recombinant herpesvirus 6A genome, recombinant herpesvirus 6B genome, recombinant herpesvirus 7 genome, recombinant Kaposi's sarcoma-associated herpesvirus genome, and any derivatives thereof. [The present invention 1006] The recombinant herpesvirus genome according to any one of claims 1001 to 1005, wherein the CFTR polypeptide is a human CFTR polypeptide. [The present invention 1007] The recombinant herpesvirus genome of any one of claims 1001 to 1006, wherein the CFTR 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:5 or SEQ ID NO:6. [The present invention 1008] The recombinant herpesvirus genome of any one of claims 1001 to 1007, wherein the CFTR 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:5. [The present invention 1009] The recombinant herpesvirus genome according to any one of claims 1001 to 1008, which is a recombinant herpes simplex virus genome. [The present invention 1010] The recombinant herpesvirus genome of the present invention 1009, 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 1011] The recombinant herpesvirus genome of the present invention 1009 or 1010, wherein the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. [The present invention 1012] 12. The recombinant herpes simplex virus genome of any one of claims 1009 to 1011, wherein said recombinant herpes simplex virus genome is engineered to reduce or eliminate expression of one or more toxic herpes simplex virus genes. [The present invention 1013] 13. The recombinant herpesvirus genome of any one of claims 1009 to 1012, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation. [The present invention 1014] The recombinant herpesvirus genome of the present invention, wherein the inactivating mutation is present in a herpes simplex virus gene. [The present invention 1015] 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 1016] The recombinant herpesvirus genome of the present invention 1014 or 1015, 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 1017] 1016. 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 1018] The recombinant herpesvirus genome of claim 1016 or 1017, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP22 gene. [The present invention 1019] The recombinant herpesvirus genome according to any one of claims 1016 to 1018, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL41 gene. [The present invention 1020] 1020. The recombinant herpes simplex virus genome of any one of claims 1016 to 1019, wherein said recombinant herpes simplex virus genome comprises an inactivating mutation in one or both copies of said ICP0 gene. [The present invention 1021] The recombinant herpesvirus genome according to any one of claims 1016 to 1020, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP27 gene. [The present invention 1022] The recombinant herpesvirus genome according to any one of claims 1016 to 1021, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP47 gene. [The present invention 1023] The recombinant herpesvirus genome according to any one of claims 1016 to 1022, wherein the recombinant herpes simplex virus genome comprises an inactivating mutation in the UL55 gene. [The present invention 1024] 13. The recombinant herpes simplex virus genome of any one of claims 1009 to 1023, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within one or both of the ICP4 viral loci. [The present invention 1025] 15. The recombinant herpes simplex virus genome of any one of claims 1009 to 1024, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within an ICP22 viral locus. [The present invention 1026] 6. The recombinant herpes simplex virus genome of any one of claims 1009 to 1025, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within a UL41 viral locus. [The present invention 1027] 1027. The recombinant herpes simplex virus genome of any one of claims 1009 to 1026, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within one or both of the ICP0 viral loci. [The present invention 1028] 10. The recombinant herpes simplex virus genome of any one of claims 1009 to 1027, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within an ICP27 viral locus. [The present invention 1029] 9. The recombinant herpes simplex virus genome of any one of claims 1009 to 1028, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within an ICP47 viral locus. [The present invention 1030] 1029. The recombinant herpes simplex virus genome of any one of claims 1009 to 1029, wherein said recombinant herpes simplex virus genome comprises said one or more polynucleotides encoding said CFTR polypeptide within the UL55 viral locus. [The present invention 1031] 1030. The recombinant herpesvirus genome of any one of claims 1001 to 1030, which when introduced into a target cell has reduced cytotoxicity compared to the corresponding wild-type herpesvirus genome. [The present invention 1032] The recombinant herpesvirus genome of the present invention, wherein the target cell is a human cell. [The present invention 1033] The recombinant herpesvirus genome of the present invention 1031 or 1032, wherein the target cell is an airway epithelial cell. [The present invention 1034] The recombinant herpesvirus genome of claim 1031 or 1032, wherein the target cell is a respiratory cell. [The present invention 1035] A herpesvirus comprising the recombinant herpesvirus genome of any one of claims 1001 to 1034. [The present invention 1036] 1035 herpesvirus of the present invention, which is replication-competent. [The present invention 1037] 1035. A herpesvirus of the invention which is replication-deficient. [The present invention 1038] The herpesvirus according to any one of claims 1035 to 1037, which has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. [The present invention 1039] The herpesvirus according to any one of claims 1035 to 1038, which is selected from the group consisting of herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, and Kaposi's sarcoma-associated herpesvirus. [The present invention 1040] The herpesvirus according to any one of claims 1035 to 1039, which is a herpes simplex virus. [The present invention 1041] The herpesvirus of the present invention 1039 or 1040, wherein said herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof. [The present invention 1042] The herpesvirus according to any one of claims 1039 to 1041, wherein the herpes simplex virus is HSV-1. [The present invention 1043] A pharmaceutical composition comprising the recombinant herpesvirus genome of any one of claims 1001 to 1034 or the herpesvirus of any one of claims 1035 to 1042, and a pharma- ceutically acceptable excipient. [The present invention 1044] A pharmaceutical composition of the present invention 1043 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. [The present invention 1045] A pharmaceutical composition of invention 1043 or invention 1044 which is suitable for oral, intranasal, intratracheal, or inhalation administration. [The present invention 1046] 6. The pharmaceutical composition of any one of claims 1043 to 1045, which is suitable for inhalation administration. [The present invention 1047] 1047. The pharmaceutical composition of any one of claims 1043 to 1046, which is suitable for non-invasive inhalation administration. [The present invention 1048] 8. The pharmaceutical composition of any one of claims 1043 to 1047, 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. [The present invention 1049] 1048. The pharmaceutical composition of any one of claims 1043 to 1048, which is suitable for use in a nebulizer. [The present invention 1050] 1049. The pharmaceutical composition of claim 10, wherein said nebulizer is a vibrating mesh nebulizer. [The present invention 1051] The pharmaceutical composition of any one of claims 1043 to 1050, comprising a phosphate buffer. [The present invention 1052] 2. The pharmaceutical composition of any one of claims 1043 to 1051, comprising glycerol. [The present invention 1053] 3. The pharmaceutical composition of any one of claims 1043 to 1052, comprising a lipid carrier. [The present invention 1054] 1053. The pharmaceutical composition of any one of claims 1043 to 1053, comprising a nanoparticle carrier. [The present invention 1055] A method for enhancing, increasing, enhancing and / or replenishing CFTR polypeptide levels in one or more cells of a subject, the method comprising administering to the subject an effective amount of a herpesvirus of any one of claims 1035 to 1042 or a pharmaceutical composition of any one of claims 1043 to 1054. [The present invention 1056] The method of any one of claims 10 to 55, wherein said one or more cells are one or more cells of the respiratory tract. [The present invention 1057] The method of claim 1055 or claim 1056, wherein said one or more cells are one or more airway epithelial cells or one or more cells of a submucosal gland. [The present invention 1058] A method for reducing or inhibiting progressive lung damage in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus according to any one of claims 1035 to 1042 or a pharmaceutical composition according to any one of claims 1043 to 1054. [The present invention 1059] 9. The method according to any one of claims 1055 to 1058, wherein said subject is suffering from a chronic lung disease. [The present invention 1060] The method of claim 1059, wherein said chronic lung disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). [The present invention 1061] A method for providing prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of cystic fibrosis in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus of any one of claims 1035 to 1042 or a pharmaceutical composition of any one of claims 1043 to 1054. [The present invention 1062] The method of claim 1061, wherein the one or more signs or symptoms of cystic fibrosis are selected from the group consisting of persistent coughing that produces a lot of mucus, a lot of sticky mucus accumulating in the airways, wheezing, difficulty breathing, sinusitis, recurrent lung infections, inflamed nasal passages, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof. [The present invention 1063] A method for providing prophylactic, palliative or therapeutic relief of one or more signs or symptoms of COPD in a subject in need thereof, comprising administering to the subject an effective amount of a herpesvirus of any one of claims 1035 to 1042 or a pharmaceutical composition of any one of claims 1043 to 1054. [The present invention 1064] The method of claim 1063, wherein the one or more signs or symptoms of COPD are selected from the group consisting of shortness of breath, wheezing, chest tightness, excess mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof. [The present invention 1065] The method according to any one of claims 1055 to 1064, wherein the subject is a human. [The present invention 1066] 1066. The method of any one of claims 1055 to 1065, wherein the subject's genome comprises a loss-of-function mutation in the CFTR gene. [The present invention 1067] The method of any one of claims 1055 to 1066, wherein said herpesvirus or pharmaceutical composition is administered to said subject orally, intranasally, intratracheally or via inhalation. [The present invention 1068] The method of any one of claims 1055 to 1067, wherein said herpesvirus or pharmaceutical composition is administered to said subject via inhalation. [The present invention 1069] The method according to any one of claims 1055 to 1068, wherein said herpesvirus or pharmaceutical composition is administered via non-invasive inhalation administration. [The present invention 1070] The method of any one of claims 1055 to 1069, wherein the herpes virus 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. [The present invention 1071] The method of any one of claims 1055 to 1070, wherein said herpesvirus or pharmaceutical composition is administered using a nebulizer. [The present invention 1072] 1071. The method of claim 1071, wherein the nebulizer is a vibrating mesh nebulizer. [Brief description of the drawings]

[0025] [Figure 1-1]Figure 1A shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A wild-type herpes simplex virus genome is shown. Figure 1B shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing a deletion of the coding sequence of ICP4 (both copies) in which an expression cassette containing a nucleic acid encoding a human CFTR polypeptide is integrated into each of the ICP4 loci. [Figure 1-2] Figure 1C shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing a deletion of the coding sequence of ICP4 (both copies) and UL41, in which an expression cassette containing a nucleic acid encoding a human CFTR polypeptide is integrated into each of the ICP4 loci. Figure 1D shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing a deletion of the coding sequence of ICP4 (both copies) and UL41, in which an expression cassette containing a nucleic acid encoding a CFTR polypeptide is integrated into the UL41 locus. [Figure 1-3] Figure 1E shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing a deletion of the coding sequence of ICP4 (both copies) and ICP22, in which an expression cassette containing a nucleic acid encoding a human CFTR polypeptide is integrated into each of the ICP4 loci. Figure 1F shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing a deletion of the coding sequence of ICP4 (both copies) and ICP22, in which an expression cassette containing a nucleic acid encoding a CFTR polypeptide is integrated into the ICP22 locus. [Figure 1-4]Figure 1G shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing deletions of the coding sequences of ICP4 (both copies), UL41, and ICP22, in which an expression cassette containing a nucleic acid encoding a human CFTR polypeptide is integrated into each of the ICP4 loci. Figure 1H shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome. A modified herpes simplex virus genome containing deletions of the coding sequences of ICP4 (both copies), UL41, and ICP22, in which an expression cassette containing a nucleic acid encoding a CFTR polypeptide is integrated into the UL41 locus. [Figure 1-5] Figure 1I shows a schematic diagram of a wild-type herpes simplex virus genome and a modified herpes simplex virus genome containing deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, in which an expression cassette containing a nucleic acid encoding a CFTR polypeptide has been integrated into the ICP22 locus. [Diagram 2] Figure 1 shows the expression of human CFTR in primary airway epithelial cells (SAECs) from cystic fibrosis (CF) patients infected with HSV-CFTR vector at the indicated multiplicity of infection (MOI), as assessed by qRT-PCR analysis. Mock-infected CF SAECs were used as a negative control. Data are presented as the mean ± standard error of two replicates. [Diagram 3] Figure 1 shows human CFTR protein expression in primary SAECs from CF patients infected with HSV-CFTR vector at the indicated MOI, as assessed by Western blot analysis. Mock-infected CF SAECs were used as a negative control. GAPDH was used as a loading control. [Figure 4]Representative immunofluorescence images of human CFTR protein expression in mock-infected or HSV-CFTR-infected primary CF patient SAECs are shown. A shows the dose-dependent increase in human CFTR protein expression upon infection of primary CF SAECs with increasing MOIs of HSV-CFTR. B shows the relative cellular localization of human CFTR protein in HSV-CFTR-infected (MOI 3) or mock-infected (MOI 0) primary CF SAECs. DAPI staining was used to visualize nuclei. [Diagram 5] Figure 1 shows human CFTR protein functionality in primary SAECs from CF patients infected with HSV-CFTR vectors at the indicated MOI, as assessed by fluorescent dye uptake assay. Mock-infected CF SAECs were used as negative controls. Data are presented as mean ± SEM. [Figure 6A] Analysis of G542X / G542X cystic fibrosis patient derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI is shown. Vehicle alone or mCherry encoding HSV vector (mCherry) was used as negative control and G418 was used as positive control. Representative brightfield images of G542X / G542X PDO after 24 hours of vehicle treatment or transduction with either HSV-CFTR or HSV-mCherry at an MOI of 10 are shown. Vehicle treated PDO isolated from a healthy individual (wild type) was included and imaged as a comparison. [Figure 6B] Analysis of G542X / G542X cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control, and G418 was used as positive control. Representative images of calcein-stained organoids before addition of forskolin (Frsk) (t=0) and quantification of average organoid size are shown. [Figure 6C]Analysis of G542X / G542X cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control, and G418 was used as positive control. Representative images of calcein-stained organoids and quantification of mean organoid size 60 min after addition of 2 μM Frsk (t=60) are shown. ***p<0.001, ****p<0.0001. [Figure 7A] Analysis of F508del / F508del cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control, and Orkambi® was used as positive control. Representative images of calcein-stained organoids before addition of forskolin (Frsk) (t=0) and quantification of mean organoid size are shown. [Figure 7B] Analysis of F508del / F508del cystic fibrosis patient derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry encoding HSV vector (mCherry) was used as negative control and Orkambi® was used as positive control. Representative images of calcein stained organoids and quantification of mean organoid size 60 min after addition of 2 μM Frsk (t=60) are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8A] Analysis of W1282X / W1282X cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control. Representative images of calcein-stained organoids before addition of forskolin (Frsk) (t=0) and quantification of mean organoid size are shown. [Figure 8B]Analysis of W1282X / W1282X cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control. Representative images of calcein-stained organoids and quantification of mean organoid size 60 min (t=60) after addition of 2 μM Frsk are shown. *p<0.05, ***p<0.001. [Figure 9A] Analysis of F508del / F508del cystic fibrosis patient-derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry-encoding HSV vector (mCherry) was used as negative control, and Orkambi® was used as positive control. Representative images of calcein-stained organoids before addition of forskolin (Frsk) (t=0) and quantification of mean organoid size are shown. [Figure 9B] Analysis of F508del / F508del cystic fibrosis patient derived intestinal organoids (PDO) infected with HSV-CFTR at the indicated MOI. Vehicle alone or mCherry encoding HSV vector (mCherry) was used as negative control and Orkambi® was used as positive control. Representative images of calcein stained organoids and quantification of mean organoid size 60 min after addition of 2 μM Frsk (t=60) are shown. ****p<0.0001. [Figure 10A] mCherry nucleic acid and protein analysis in lung and tracheal biopsies taken 48 hours after intranasal or intratracheal administration of mCherry-encoding HSV vector (HSV-mCherry) or vehicle control (mock). mCherry transcript levels in lung and tracheal biopsies as assessed by qRT-PCR analysis are shown. Data are presented as mean ± standard error of six replicates for HSV-mCherry, and data are presented as mean ± standard error of four replicates for vehicle control. [Figure 10B]mCherry nucleic acid and protein analysis in lung and tracheal biopsies taken 48 hours after intranasal or intratracheal administration of mCherry-encoding HSV vector (HSV-mCherry) or vehicle control (mock). Representative immunofluorescence images of mCherry protein expression in lung biopsies after intranasal administration of HSV-mCherry or vehicle control are shown. DAPI staining was used to visualize nuclei and cytokeratin staining was used to visualize epithelial cells. [Figure 10C] mCherry nucleic acid and protein analysis in lung and tracheal biopsies taken 48 hours after intranasal or intratracheal administration of mCherry-encoding HSV vector (HSV-mCherry) or vehicle control (mock). Representative immunofluorescence images of mCherry protein expression in lung biopsies after intratracheal administration of HSV-mCherry or vehicle control are shown. DAPI staining was used to visualize nuclei and cytokeratin staining was used to visualize epithelial cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 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.

[0027] I. General Techniques Additionally, the specifications for the specifications are given in Sambrook et al.,Molecular Cloning:A Laboratory Manual 3d edition(2001)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Current Protocols in Molecular Biology (FMAusubel, et al.eds., (2003)); Lane,eds.(1988)、Oligonucleotide Synthesis(MJGait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(JECellis,ed.,1998)Academic Press、Animal Cell Culture(RIFreshney),ed.,1987)、Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.These methods are generally well understood and commonly employed by those skilled in the art using conventional methodologies, such as those widely used methodologies described in Short Protocols in Molecular Biology (Wiley and Sons, 1999), and in Quantitative Biology (Wiley and Sons, 1999).

[0028] 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.

[0029] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0030] 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" may refer to "a only," "b only," "a or b," or "a and b," while the term "a, b, and / or c" may 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.

[0031] As used herein, the term "about" refers to the normal range of error for the respective value, which is readily known to 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.

[0032] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0033] As used herein, the terms "polynucleotide", "nucleic acid sequence", "nucleic acid" and variations thereof are intended to be inclusive of 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 for 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 analogs of naturally occurring nucleotides, and internucleotide modifications.

[0034] As used herein, a nucleic acid is "operably linked" or "operably linked" when it is placed in 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 coding sequence, and a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. In general, "operably linked" or "operably linked" means that the DNA or RNA sequences being linked are in close proximity.

[0035] As used herein, the term "vector" refers to a separate element used to introduce a heterologous nucleic acid into a cell for either expression or replication thereof. Expression vectors include vectors capable of expressing a nucleic acid operably linked to a regulatory sequence, such as a promoter region, capable of effecting expression of such a nucleic acid. 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 a suitable 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.

[0036] As used herein, "open reading frame" or "ORF" refers to a continuous stretch of 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.

[0037] As used herein, "untranslated region" or "UTR" refers to untranslated nucleic acid at the 5' and / or 3' end of an open reading frame. Inclusion of one or more UTRs in a polynucleotide can affect post-transcriptional regulation, mRNA stability, and / or translation of the polynucleotide.

[0038] As used herein, the term "transgene" refers to a polynucleotide that, after being introduced into a cell, can be transcribed into RNA and translated and / or expressed under appropriate conditions, which in some embodiments confers a desired property to the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome.

[0039] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably and may refer to a polymer of two or more amino acids.

[0040] As used herein, "subject," "host," or "individual" refers to any animal classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, 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.

[0041] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition or formulation is administered. A "pharmaceutical acceptable" excipient (e.g., vehicle, additive) is one that may be reasonably administered to a target mammal to provide an effective dosage of the active ingredient(s) employed.

[0042] As used herein, an "effective amount" is at least the minimum amount necessary to affect 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 disease state, age, sex, and weight of the patient. An effective amount is also an amount in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other drugs used to treat the symptoms of the disease, delaying the progression of the disease, and / or increasing survival time. An effective amount may 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 will be 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 administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in conjunction with one or more other agents.

[0043] As used herein, "treatment" refers to a clinical intervention designed to change the natural course of the treated individual or cell in the course of clinical pathology. The desired effects of treatment include slowing the rate of progression of a disease / disorder / deficiency, improving or alleviating the disease / disorder / deficiency condition, and remission or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a chronic lung disease (e.g., cystic fibrosis or COPD) are reduced or eliminated.

[0044] As used herein, the term "delaying progression" of a disease / disorder / deficiency refers to prolonging, preventing, delaying, retarding, stabilizing, and / or postponing the onset of a disease / disorder / deficiency (e.g., cystic fibrosis or COPD). This delay can be of varying length or time, depending on the disease / disorder / deficiency and / or the medical history of the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop the disease.

[0045] III. Recombinant Nucleic Acids Certain aspects of the disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) that include 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 a CFTR polypeptide (e.g., a human CFTR polypeptide). In some embodiments, a recombinant nucleic acid includes one polynucleotide that encodes a CFTR polypeptide. In some embodiments, a recombinant nucleic acid includes two polynucleotides that encode a CFTR polypeptide. In some embodiments, a recombinant nucleic acid includes three polynucleotides that encode a CFTR polypeptide.

[0046] 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 nucleic acid is a recombinant herpes simplex virus genome. In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus type 1 (HSV-1) genome.

[0047] Polynucleotides encoding cystic fibrosis transmembrane conductance regulator (CFTR) polypeptides In some embodiments, the disclosure relates to a recombinant nucleic acid comprising one or more polynucleotides comprising a coding sequence of a CFTR gene (e.g., a human CFTR gene), or any portion thereof. Any suitable CFTR gene known in the art (including any isoforms thereof) can be encoded by a polynucleotide of the present disclosure, including, for example, the human CFTR gene (see, e.g., NCBI Gene ID: 1080; SEQ ID NO: 1 or SEQ ID NO: 3), chimpanzee CFTR gene (see, e.g., NCBI Gene ID: 463674), mouse CFTR gene (see, e.g., NCBI Gene ID: 12638), rat CFTR gene (see, e.g., NCBI Gene ID: 24255), dog CFTR gene (see, e.g., NCBI Gene ID: 492302), rabbit CFTR gene (see, e.g., NCBI Gene ID: 100009471), bovine CFTR gene (see, e.g., NCBI Gene ID: 281067), rhesus monkey CFTR gene (see, e.g., NCBI Gene ID: 574346), and the like. In some embodiments, the polynucleotides of the present disclosure comprise 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 any of the sequences of the CFTR gene (and / or its coding sequence) described herein or known in the art. Methods for identifying CFTR gene homologs / orthologs from additional species are known to those of skill in the art, including, for example, using nucleic acid sequence alignment programs such as the BLAST® blastn suite.

[0048] In some embodiments, the polynucleotides of the present disclosure comprise a codon-optimized variant of any of the CFTR genes described herein or known in the art. In some embodiments, the polynucleotides of the present disclosure comprise a codon-optimized variant of any of the coding sequences of the CFTR genes described herein or known in the art. In some embodiments, the use of a codon-optimized variant of the CFTR gene increases the stability and / or yield of heterologous expression (RNA and / or protein) of the encoded CFTR polypeptide in a target cell (e.g., a target human cell such as a human airway epithelial 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 cells of the lung) can be used, including, for example, the method described by Fath et al. (PLoS One. 2011 Mar 3;6(3):e17596).

[0049] In some embodiments, the one or more polynucleotides of the disclosure comprise a coding sequence of the human CFTR gene.

[0050] In some embodiments, a polynucleotide of the 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 sequence of SEQ ID NO: 1. In some embodiments, a polynucleotide of the disclosure comprises the sequence of SEQ ID NO:1.

[0051] In some embodiments, the polynucleotides of the disclosure comprise a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 1. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 1 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, 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, but fewer than 4443 contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the polynucleotides of the disclosure comprise 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 sequence of nucleic acids 1-4440 of SEQ ID NO: 1. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-4440 of SEQ ID NO: 1.

[0052] In some embodiments, a polynucleotide of the 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 sequence of SEQ ID NO: 3. In some embodiments, a polynucleotide of the disclosure comprises the sequence of SEQ ID NO: 3.

[0053] In some embodiments, the polynucleotides of the disclosure comprise a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 3. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 3 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, 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, but fewer than 4260 contiguous nucleotides of SEQ ID NO: 3. In some embodiments, the polynucleotides of the disclosure comprise 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 sequence of nucleic acids 1-4257 of SEQ ID NO: 3. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-4257 of SEQ ID NO: 3.

[0054] In some embodiments, a polynucleotide of the disclosure comprises a coding sequence for a codon-optimized variant of the human CFTR gene.

[0055] In some embodiments, a polynucleotide of the 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 sequence of SEQ ID NO: 2. In some embodiments, a polynucleotide of the disclosure comprises the sequence of SEQ ID NO: 2.

[0056] In some embodiments, the polynucleotides of the disclosure comprise a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 2. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 2 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, 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, but fewer than 4443 contiguous nucleotides of SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise 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 sequence of nucleic acids 1-4440 of SEQ ID NO: 2. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-4440 of SEQ ID NO: 2.

[0057] In some embodiments, a polynucleotide of the 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 sequence of SEQ ID NO: 4. In some embodiments, a polynucleotide of the disclosure comprises the sequence of SEQ ID NO: 4.

[0058] In some embodiments, the polynucleotides of the disclosure comprise a 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 4. In some embodiments, the 5' truncation, 3' truncation, or fragment of the sequence of SEQ ID NO: 4 is a polynucleotide having at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, or at least 350, at least 400, at least 450, 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, but fewer than 4260 contiguous nucleotides of SEQ ID NO: 4. In some embodiments, the polynucleotides of the disclosure comprise 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 sequence of nucleic acids 1-4257 of SEQ ID NO: 4. In some embodiments, the polynucleotides of the disclosure comprise the sequence of nucleic acids 1-4257 of SEQ ID NO: 4.

[0059] In some embodiments, the polynucleotides of the disclosure comprise 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 a nucleic acid sequence selected from SEQ ID NO:1-4. In some embodiments, the polynucleotides of the disclosure comprise 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 a nucleic acid sequence selected from SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotides of the disclosure comprise a sequence selected from SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, a polynucleotide of the disclosure 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 a nucleic acid sequence selected from SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, a polynucleotide of the disclosure comprises a sequence selected from SEQ ID NO:3 or SEQ ID NO:4.

[0060] A polynucleotide of the disclosure (e.g., encoding a human CFTR polypeptide) may further encode additional coding and non-coding sequences, including, but not limited to, sequences encoding additional polypeptide tags (e.g., encoded in frame with the CFTR protein to produce a fusion protein), introns (e.g., natural, modified, or heterologous introns), 5'UTRs and / or 3'UTRs (e.g., natural, modified, or heterologous 5'UTRs and / or 3'UTRs), and the like. Examples of suitable polypeptide tags include, but are not limited to, purification tags, such as his-tags, flag tags, maltose binding protein and glutathione-S-transferase tags, detection tags, such as tags that can be photometrically detected (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), any combination of tags including secretion sequences, signal sequences, leader sequences, and / or stabilization sequences, protease cleavage sites (e.g., furan cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.), etc. In some embodiments, the 5'UTR and / or 3'UTR increase the stability, localization, and / or translation efficiency of the polynucleotide. In some embodiments, the 5'UTR and / or 3'UTR improve protein expression levels and / or duration. In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites, etc.) that can block or reduce off-target expression (e.g., inhibit expression in a particular cell type (e.g., neuronal cell) at a particular time in the cell cycle, at a particular developmental stage). In some embodiments, the 5'UTR and / or 3'UTR contain elements (e.g., one or more miRNA binding sites, etc.) that can enhance CFTR protein expression in a particular cell type.

[0061] In some embodiments, a polynucleotide of the present disclosure (e.g., encoding a human CFTR 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 from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, insulin, etc.), enhancer sequences from eukaryotic cell viruses (e.g., SV40 enhancer on the late side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancer, polyoma enhancer on the late side of the replication origin, adenovirus enhancer, etc.), and any combination thereof. Any suitable insulator(s) known in the art may be used, including, for example, the herpes simplex virus (HSV) chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2 from the human interferon beta gene (IFNB1), chicken hypersensitive site 4 insulator (cHS4), human HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE), scaffold / matrix attachment region (S / MAR), and any combination thereof.Any suitable promoter known in the art (e.g., a promoter suitable for transcription in a mammalian host cell) may be used, including, for example, promoters obtained from the genome of a virus (e.g., polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), etc.), promoters from heterologous mammalian genes (e.g., actin promoters (e.g., β-actin promoter), ubiquitin promoters (e.g., ubiquitin C (UbC) promoter), phosphoglycerate kinase (PGK) promoter, immunoglobulin promoters, heat shock protein promoters, etc.), promoters from natural and / or homologous mammalian genes (e.g., human CFTR gene promoter), synthetic promoters (such as the CAGG promoter), and any combination thereof, provided that such promoter is compatible with the host cell. Regulatory sequences may include sequences directing constitutive expression of the nucleic acid, as well as tissue-specific regulatory sequences and / or inducible or repressible sequences.

[0062] In some embodiments, the polynucleotide of the present disclosure is 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 polynucleotide of the present disclosure (e.g., encoding a human CFTR polypeptide) is operably linked to a HCMV promoter.

[0063] In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a collagen alpha-1 (VII) chain polypeptide (COL7) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a lysyl hydroxylase 3 polypeptide (LH3) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a keratin type I cytoskeletal 17 polypeptide (KRT17) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a transglutaminase (TGM) polypeptide (e.g., a human transglutaminase polypeptide, such as a human TGM1 polypeptide and / or a human TGM5 polypeptide) (e.g., a transgene encoding the same). In some embodiments, the polynucleotides of the present disclosure do not include a coding sequence for a cosmetic protein (e.g., a collagen protein, fibronectin, elastin, lumican, vitronectin / vitronectin receptor, laminin, neuromodulators, fibrillin, additional skin extracellular matrix proteins, etc.) (e.g., a transgene encoding the same). In some embodiments, a polynucleotide of the disclosure does not include a coding sequence for an antibody (e.g., a full-length antibody, an antibody fragment, etc.) (e.g., a transgene encoding the same). In some embodiments, a polynucleotide of the disclosure does not include a coding sequence for a serine protease inhibitor Kazal type (SPINK) polypeptide (e.g., a human SPINK polypeptide, such as a human SPINK5 polypeptide) (e.g., a transgene encoding the same). In some embodiments, a polynucleotide of the disclosure does not include a coding sequence for a filaggrin or filaggrin 2 polypeptide (e.g., a human filaggrin or filaggrin 2 polypeptide) (e.g., a transgene encoding the same). In some embodiments, a polynucleotide of the disclosure does not include a coding sequence for a collagen alpha-1 (VII) chain polypeptide, a lysyl hydroxylase 3 polypeptide, a keratin type I cytoskeleton 17 polypeptide, and / or any chimeric polypeptides thereof (e.g., a transgene encoding the same).In some embodiments, the polynucleotides of the present disclosure do not comprise a coding sequence for 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, and / or any chimeric polypeptide thereof (e.g., a transgene encoding the same).

[0064] Cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide In some embodiments, the present disclosure relates to one or more polynucleotides encoding CFTR polypeptides (e.g., human CFTR polypeptides) or any part thereof. Any suitable CFTR polypeptide known in the art can be encoded by the polynucleotides of the present disclosure, including, for example, human CFTR polypeptides (e.g., see UniProt Accession No. P13569; SEQ ID NO: 5 or SEQ ID NO: 6), chimpanzee CFTR polypeptides (e.g., see UniProt Accession No. Q2QLE5), mouse CFTR polypeptides (e.g., see UniProt Accession No. P26361), rat CFTR polypeptides (e.g., see UniProt Accession No. P34158), rabbit CFTR polypeptides (e.g., see UniProt Accession No. Q00554), rhesus CFTR polypeptides (e.g., see UniProt Accession No. Q00553), etc. In some embodiments, the CFTR polypeptides of the present disclosure include sequences 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 CFTR polypeptides described herein or known in the art. Methods for identifying CFTR polypeptide homologs / orthologs from additional species are known to those of skill in the art, including, for example, the use of amino acid sequence alignment programs such as the BLAST® blastp suite or OrthoDB.

[0065] In some embodiments, a CFTR polypeptide of the disclosure is a human CFTR polypeptide.

[0066] In some embodiments, a polynucleotide encoding a human CFTR 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: 5. In some embodiments, a polynucleotide encoding a human CFTR polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0067] In some embodiments, the polynucleotide encoding a human CFTR polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or 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 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, but fewer than 1480 consecutive amino acids of SEQ ID NO:5.

[0068] In some embodiments, a polynucleotide encoding a human CFTR 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: 6. In some embodiments, a polynucleotide encoding a human CFTR polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6.

[0069] In some embodiments, the polynucleotide encoding a human CFTR polypeptide is a polynucleotide encoding an N-terminal truncation, C-terminal truncation, or fragment of the amino acid sequence of SEQ ID NO: 6. The N-terminal truncation, C-terminal truncation, or fragment may include 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 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, but fewer than 1419 consecutive amino acids of SEQ ID NO: 6.

[0070] In some embodiments, a polynucleotide of the disclosure that encodes a CFTR polypeptide is a polynucleotide that encodes a polypeptide comprising 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:5 or SEQ ID NO:6. In some embodiments, a polynucleotide of the disclosure that encodes a CFTR polypeptide is a polynucleotide that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0071] In some embodiments, a polynucleotide of the disclosure encoding a CFTR polypeptide (e.g., a human CFTR polypeptide) expresses the CFTR polypeptide when the polynucleotide is delivered to one or more target cells of the subject (e.g., one or more cells of the airway and / or lungs of the subject). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) enhances, increases, enhances, and / or replenishes the level, function, and / or activity of the CFTR polypeptide in one or more target cells of the subject (e.g., compared to before expression of the CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces mucus secretion by one or more cells of the subject and / or in one or more organs (e.g., lungs) of the subject (e.g., compared to before expression of the CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces and / or inhibits mucus accumulation in one or more organs (e.g., lungs) of the subject (e.g., compared to before expression of the CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces, prevents, or treats airway obstruction in a subject (e.g., compared to before expression of a CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces, prevents, or treats chronic bacterial infection and / or associated chronic inflammation in the lungs of a subject (e.g., compared to before expression of a CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces, inhibits, prevents, or treats bronchiectasis in a subject (e.g., compared to before expression of a CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) reduces, inhibits, prevents, or treats progressive lung injury in a subject (e.g., compared to before expression of a CFTR polypeptide).In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) provides preventative, palliative, or therapeutic alleviation of a chronic lung disease (e.g., cystic fibrosis, chronic obstructive pulmonary disorder) in a subject (e.g., compared to before expression of a CFTR polypeptide). In some embodiments, expression of a CFTR polypeptide (e.g., a human CFTR polypeptide) provides preventative, palliative, or therapeutic alleviation of one or more signs or symptoms of cystic fibrosis in a subject (e.g., compared to before expression of a CFTR polypeptide).

[0072] Recombinant Nucleic Acids In some embodiments, the 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. In general, a vector suitable for maintaining, propagating, and / or expressing a polynucleotide to produce one or more polypeptides in a subject may be used. Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, Sindbis virus vectors, measles vectors, herpes virus vectors, lentivirus vectors, retrovirus vectors, etc.). In some embodiments, the vector is a herpes virus 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 constructing vectors containing one or more polynucleotides of interest, such as by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid (eg, by genetic engineering techniques), are well known to those of skill in the art.

[0073] In some embodiments, the recombinant nucleic acid of the present disclosure is a herpes simplex virus (HSV) amplicon. Herpes virus amplicons, including structural features, and methods of making same are generally known to those of skill in the art (see, for example, 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 may 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.

[0074] In some embodiments, the recombinant nucleic acid of the present disclosure is a recombinant herpesvirus genome. For example, the recombinant herpesvirus genome can be a recombinant genome from any member of the Herpesviridae family of DNA viruses known in the art, including recombinant herpes simplex virus genome, recombinant varicella zoster virus genome, recombinant human cytomegalovirus genome, recombinant herpesvirus 6A genome, recombinant herpesvirus 6B genome, recombinant herpesvirus 7 genome, recombinant Kaposi's sarcoma-associated herpesvirus genome, and any combination or derivative thereof. As used herein, an "inactivating mutation" can refer to any mutation that results in a gene or regulon product (RNA or protein) with reduced, undetectable, or eliminated amount and / or function (e.g., compared to a corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations can include, but are not limited to, deletions, insertions, point mutations, and rearrangements of the transcriptional control sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. Any suitable method of measuring the amount of a gene or regulon product known in the art may be used, including, for example, qPCR, Northern blot, RNAseq, Western blot, ELISA, etc. 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. 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.) 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.

[0075] In some embodiments, the recombinant nucleic acid is a recombinant herpes simplex virus (HSV) 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 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 a corresponding wild-type herpes simplex virus genome). In some embodiments, the recombinant herpes simplex virus genome is replicable. In some embodiments, the recombinant herpes simplex virus genome is replication-deficient.

[0076] In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome. In some embodiments, the recombinant HSV-1 genome is, 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 from any HSV-1 strain known in the art, including 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, and the like (see, e.g., Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is from a KOS strain. In some embodiments, the recombinant HSV-1 genome is not from a McKrae strain. In some embodiments, the recombinant HSV-1 genome is attenuated. In some embodiments, the recombinant HSV-1 genome is replication competent. In some embodiments, the recombinant HSV-1 genome is replication defective. In some embodiments, the recombinant HSV-1 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.) HSV-1 genes.

[0077] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation 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 (ICP)0 (one or both copies), ICP4 (one or both copies), 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 (e.g., is capable of expressing) an inactivating mutation in the ICP0 (one or both copies) herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise (e.g., is capable of expressing) an inactivating mutation in the ICP27 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise (e.g., is capable of expressing) an inactivating mutation in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not comprise (e.g., is capable of expressing) an inactivating mutation in the ICP0 (one or both copies), ICP27, and / or ICP47 herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the junction region. In some embodiments, the recombinant herpes simplex virus genome does not comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene 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 comprise an inactivating mutation in the ICP34.5 (one or both copies) herpes simplex virus gene and the ICP47 herpes simplex virus gene.In some embodiments, the recombinant herpes simplex virus genome is not oncolytic.

[0078] 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 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 gene. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP0 gene (one or both copies) and 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 ICP0 gene (one or both copies) and comprises 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 comprises 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 in the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and / or UL41 genes.In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP27, ICP47, and / or UL55 gene.

[0079] 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 (one or both copies), ICP22, ICP27, ICP47, UL41, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 gene (one or both copies) and comprises 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 comprises 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), comprises an inactivating mutation in the ICP22 gene, and comprises an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP4 (one or both copies), ICP22, and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0080] 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 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, UL41, and / or UL55 genes. 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 UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP22 and / or UL41 genes. In some embodiments, the recombinant herpes simplex virus genome further comprises an inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP27, ICP47, and / or UL55 genes.

[0081] 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 an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP47, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP27 gene.

[0082] 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 an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, UL41, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP47 gene.

[0083] 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 an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL55 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL41 gene.

[0084] 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 an ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the UL55 gene.

[0085] In some embodiments, the recombinant herpes simplex virus genome comprises an internal repeat long (IR L ) region and internal repeat short (IR SIn some embodiments, the inactivation (e.g., deletion) of the joint region eliminates one copy each of the ICP4 and ICP0 genes. 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 desired, expression of one or both of these genes can be restored by insertion of an immediate early promoter into a 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 inactivation (e.g., deletion) of 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.

[0086] In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome comprises an inactivating mutation in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutation in the ICP4 (one or both copies), ICP27, and / or UL55 genes is a deletion of the coding sequence of the ICP4 (one or both copies), ICP27, and / or UL55 gene. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions of the promoter regions of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome comprises a deletion of the coding sequences of the ICP4 (one or both copies), ICP27, and UL55 genes, and a deletion of 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.

[0087] In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies) and ICP4 (one or both copies) genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), and ICP22 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome comprises inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and UL55 genes. In some embodiments, the inactivating mutation in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or UL55 gene comprises a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or 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.

[0088] In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one, two, three, four, five, six, seven, or more viral loci. Examples of suitable viral loci may include, but are not limited to, ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, tk, UL41, and / or UL55 herpes simplex virus loci. In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in one or both viral ICP4 loci (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide in one or both ICP4 loci). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure in the viral ICP22 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide in the ICP22 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral UL41 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at the UL41 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within one or both of the viral ICP0 loci (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at one or both of the ICP0 loci). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP27 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at the ICP27 locus). In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of the present disclosure within the viral ICP47 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at the ICP47 locus).In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the viral ICP4 loci and comprises one or more polynucleotides of this disclosure within the viral ICP22 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at one or both of the ICP4 loci and a polynucleotide encoding a human CFTR polypeptide at the ICP22 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the viral ICP4 loci and comprises one or more polynucleotides of this disclosure within the viral UL41 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at one or both of the ICP4 loci and a polynucleotide encoding a human CFTR polypeptide at the UL41 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the disclosure within the viral ICP22 locus and one or more polynucleotides of the disclosure within the viral UL41 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at the ICP22 locus and a polynucleotide encoding a human CFTR polypeptide at the UL41 locus). In some embodiments, a recombinant herpes simplex virus genome comprises one or more polynucleotides of the disclosure within one or both of the viral ICP4 loci, comprises one or more polynucleotides of the disclosure within the viral ICP22 locus, and comprises one or more polynucleotides of the disclosure within the viral UL41 locus (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide at one or both of the ICP4 loci, a polynucleotide encoding a human CFTR polypeptide at the ICP22 locus, and a polynucleotide encoding a human CFTR polypeptide at the UL41 locus, etc.).In some embodiments, the recombinant herpes simplex virus genome comprises one or more polynucleotides of this disclosure within one or both of the viral ICP4 loci, one or more polynucleotides of this disclosure within the viral ICP22 locus, one or more polynucleotides of this disclosure within the viral UL41 locus, one or more polynucleotides of this disclosure within one or both of the viral ICP0 locus, one or more polynucleotides of this disclosure within the viral ICP27 locus, and / or one or more polynucleotides of this disclosure within the ICP47 locus.

[0089] In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce or eliminate expression of one or more herpesvirus genes (e.g., one or more toxic herpesvirus genes), such as one or 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, etc. In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce the cytotoxicity of the recombinant genome compared to the corresponding wild-type herpesvirus genome (e.g., when introduced into a target cell). 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 mucosal cell. 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 cells are lung cells (primary cells or cell lines derived therefrom). In some embodiments, the cytotoxicity of the recombinant herpesvirus 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 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.).In some embodiments, the cytotoxicity of the recombinant herpesvirus genome is reduced by 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, at least about 1000-fold, or more, 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 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 of measuring cytotoxicity are known to those of skill in the art, including, for example, the use of vital dyes (formazan dyes), protease biomarkers, the MTT assay (or assays using related tetrazolium salts such as XTT, MTS, water soluble tetrazolium salts), measuring ATP content, and the like.

[0090] In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce its effect on target cell proliferation following exposure of the target cell to the recombinant genome compared to the corresponding wild-type herpesvirus genome. In some embodiments, the target cell is a human cell (primary cell or cell line derived therefrom). In some embodiments, the target cell is a mucosal cell. In some embodiments, the target cell is a respiratory cell (primary cell or cell line derived therefrom). In some embodiments, the target cell is an airway epithelial cell (primary cell or cell line derived therefrom). In some embodiments, the target cell is a pulmonary cell (primary cell or cell line derived therefrom). In some embodiments, target cell proliferation 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 proliferation following exposure to a corresponding wild-type herpes simplex virus genome (e.g., measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome relative to the cell proliferation following exposure to a wild-type herpes simplex virus genome in a target cell, measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome relative to the wild-type herpes simplex virus genome in a target cell, etc.).In some embodiments, target cell proliferation following exposure to the recombinant genome is at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 750 times, or at least about 1000 times faster than target cell proliferation following exposure to a corresponding wild-type herpes simplex virus genome (e.g., measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome relative to the cell proliferation following exposure to a wild-type herpes simplex virus genome in a target cell, measuring the relative cell proliferation following exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome relative to the wild-type herpes simplex virus genome in a target cell, etc.). Methods for measuring cell proliferation are known to those of skill in the art, including, for example, the use of the Ki67 cell proliferation assay, the BrdU cell proliferation assay, and the like.

[0091] A vector (e.g., a herpes virus vector) can include 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 to the polynucleotide to be expressed (e.g., as described above).

[0092] 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 (e.g., two or more, three or more, etc.) polynucleotides described herein, these polynucleotides can be inserted in the same orientation or in 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 antisense orientation can help avoid read-through and ensure proper expression of each polynucleotide.

[0093] IV. Viruses Certain aspects of the present disclosure relate to a virus comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the virus is capable of infecting one or more target cells of a subject (e.g., a human). In some embodiments, the virus is suitable for delivering the polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., a human). 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 having a CFTR deficiency (e.g., one or more cells comprising a genomic mutation in a native CFTR gene). In some embodiments, the one or more target cells are one or more mucosal cells. 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 (e.g., goblet cells, ciliated cells, Clara cells, neuroendocrine cells, basal cells, intermediate or parabasal cells, serous cells, brush cells, oncocytes, 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 (such as 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.

[0094] Any suitable virus known in the art may be used, including, for example, adenovirus, adeno-associated virus, retrovirus, lentivirus, Sendai virus, herpes virus, vaccinia virus, and / or any hybrid or derivative virus thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replication competent. In some embodiments, the virus is replication defective. In some embodiments, the virus is 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.

[0095] In some embodiments, the virus is a member of the Herpesviridae family of DNA viruses, including, for example, herpes simplex virus, varicella zoster virus, human cytomegalovirus, herpes virus 6A, herpes virus 6B, herpes virus 7, and Kaposi's sarcoma-associated herpes virus. In some embodiments, the herpes virus is attenuated. In some embodiments, the herpes virus is replication-deficient. In some embodiments, the herpes virus is replication-competent. In some embodiments, the herpes virus is engineered to reduce or eliminate expression of one or more herpes virus genes (e.g., one or more toxic herpes virus genes). In some embodiments, the herpes virus has reduced cytotoxicity compared to the corresponding wild-type herpes virus. In some embodiments, the herpes virus is not oncolytic.

[0096] In some embodiments, the herpes 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, WO2019 / 210219, and / or WO2020 / 006486. 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 is 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 virus, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is HSV-1 virus. In some embodiments, the herpes simplex virus is HSV-1. 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 is engineered to reduce or eliminate expression of one or more HSV-1 genes (e.g., one or more toxic HSV-1 genes). In some embodiments, the HSV-1 has reduced cytotoxicity compared to the corresponding wild-type HSV-1. In some embodiments, the HSV-1 is not oncolytic.

[0097] 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.) mutant herpes simplex virus glycoproteins. Examples of herpes simplex virus glycoproteins may include, but are not limited to, glycoproteins gB, gC, gD, gH, and gL. In some embodiments, the modified envelope alters herpes simplex virus tissue tropism compared to the wild-type herpes simplex virus.

[0098] In some embodiments, the transduction efficiency (in vitro and / or in vivo) of a virus of the present disclosure (e.g., a herpes virus) on 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 on 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%, at least about 99.5%, or more. In some embodiments, the virus is a herpes simplex virus and the transduction efficiency of the virus on one or more target cells (e.g., one or more cells of the respiratory tract) is about 85% to about 100%. In some embodiments, the virus is a herpes simplex virus, and the viral transduction efficiency for 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, including, for example, qPCR analysis, deep sequencing, Western blotting, fluorescent quantitative analysis (fluorescent in situ hybridization (FISH), fluorescent reporter gene expression, immunofluorescence, FACS, etc.), etc.

[0099] V. Pharmaceutical Compositions and Formulations Certain aspects of the present disclosure relate to pharmaceutical compositions and formulations comprising any of the recombinant nucleic acids (e.g., recombinant herpesvirus genomes) and / or viruses (e.g., herpes viruses comprising a recombinant genome) described herein (such as a herpes simplex virus comprising a recombinant herpes simplex virus genome) and a pharma- ceutical acceptable excipient or carrier.

[0100] 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 1010 , 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 Contains PFU / mL of virus.

[0101] Pharmaceutical compositions and formulations can be prepared by mixing the active ingredient(s) (such as recombinant nucleic acid and / or virus) having a desired degree of purity with one or more pharma- ceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally non-toxic to recipients at the dosages and concentrations employed and may include buffers (such as phosphate, citric acid, acetic acid, and other organic acids), antioxidants (such as ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), and / or other suitable amines. The carrier may include a hydrophilic polymer (such as polyvinylpyrrolidone), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (such as Zn-protein complexes), and / or non-ionic surfactants (such as polyethylene glycol (PEG)). A thorough discussion of pharma- ceutically acceptable carriers is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991).

[0102] 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 biological therapeutics) for immediate or controlled release. A variety of 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 in the nanoparticle, attached to 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).

[0103] In some embodiments, the pharma- ceutically acceptable carrier or excipient may be adapted for 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, by implant, by inhalation, intrathecal, intraventricular, and / or intranasal administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted for or suitable for oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, the pharma- ceutically acceptable carrier or excipient is adapted for or suitable for inhalation administration. In some embodiments, the pharma- ceutical acceptable carrier or excipient is adapted for or suitable for non-invasive inhalation administration. In some embodiments, the pharma- ceutical acceptable carrier or excipient is adapted for or suitable for nebulization (using a vibrating mesh nebulizer).

[0104] 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, by implant, by inhalation, intrathecal, intraventricular, or intranasal administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for oral, intranasal, intratracheal, or inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for non-invasive inhalation administration. In some embodiments, the pharmaceutical composition or formulation is adapted or suitable for nebulization (using a vibrating mesh nebulizer).

[0105] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional ingredients. Examples of additional ingredients include binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, etc.). , sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), wetting agents (e.g., sodium lauryl sulfate, etc.), salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, sweeteners, flavorings, fragrances, colorants, moisturizers, sunscreens, antibacterial agents, agents capable of stabilizing polynucleotides or preventing their degradation, etc. In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer. 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 15%, etc.). In some embodiments, the pharmaceutical composition or formulation comprises a phosphate buffer and glycerol. In some embodiments, the pharmaceutical composition or formulation comprises less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% glycerol. In some embodiments, the pharmaceutical composition or formulation does not comprise glycerol.

[0106] Pharmaceutical compositions and formulations used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0107] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to deliver one or more polynucleotides encoding a CFTR polypeptide to one or more cells of a subject (e.g., one or more CFTR-deficient cells, one or more cells with a CFTR genetic mutation, one or more cells of the respiratory tract, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to treat a disease or condition that would benefit from expression of a CFTR polypeptide (e.g., a disease associated with CFTR deficiency and / or a disease associated with a CFTR genetic mutation). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prevent or treat a chronic lung disease (e.g., cystic fibrosis, COPD, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prevent or treat cystic fibrosis.

[0108] In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prepare a medicament useful for delivering one or more polynucleotides encoding a CFTR polypeptide to one or more cells of a subject (e.g., one or more CFTR-deficient cells, one or more cells with a CFTR gene mutation, one or more cells of the respiratory tract, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prepare a medicament useful for preventing or treating a disease or condition that would benefit from expression of a CFTR polypeptide (e.g., a disease associated with CFTR deficiency and / or a disease associated with a CFTR gene mutation). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prepare a medicament useful for preventing or treating a chronic lung disease (e.g., cystic fibrosis, COPD, etc.). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prepare a medicament useful for preventing or treating cystic fibrosis.

[0109] VI. Method Certain aspects of the present disclosure relate to enhancing, increasing, enhancing, and / or supplementing CFTR polypeptide levels in one or more cells of a subject, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, such as cystic fibrosis, COPD, etc. In some embodiments, the subject suffers from cystic fibrosis.

[0110] In some embodiments, administration of a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to a subject increases the CFTR level (transcript or protein level) in one or more contacted or treated cells of the subject by at least about 2-fold, as compared to the endogenous CFTR level in one or more corresponding untreated cells in the subject. For example, administration of a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation may increase the CFTR level (transcript or protein level) in one or more contacted or treated cells of the 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, as compared to the endogenous CFTR level 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 and / or one or more cells of a submucosal gland). Methods for measuring transcript or protein levels from a sample are well known to those of skill in the art, including, for example, qPCR, Western blot, mass spectrometry, and the like.

[0111] Another aspect of the present disclosure relates to a method of reducing cellular sodium levels in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, e.g., cystic fibrosis, COPD, etc. In some embodiments, the subject suffers from cystic fibrosis.

[0112] In some embodiments, administration of a recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to a subject reduces intracellular sodium levels in one or more contacted or treated cells by 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 intracellular sodium levels in one or more corresponding untreated cells in the subject. Methods for measuring intracellular sodium levels are generally known to those of skill in the art.

[0113] Another aspect of the present disclosure is a method of improving at least one respiratory volume measurement in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, such as cystic fibrosis, COPD, etc.

[0114] In some embodiments, administration of the recombinant nucleic acid, virus, agent, 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 prior to treatment. Examples of suitable respiratory volumes that may be measured include, for example, total lung capacity (TLC), the volume in the lungs at maximum expansion; tidal volume (TV), the amount of air moved in and out of the lungs during quiet breathing; residual volume (RV), the amount of air remaining in the lungs after maximum expiration; expiratory reserve volume (ERV), the maximum amount (more than the tidal volume) that can be exhaled during a forced breath; inspiratory reserve volume (ERV), the maximum amount of air that can be inhaled from the end-inspiration position; inspiratory capacity (IC), the sum of IRV and TV; inspiratory vital capacity (IVC), the maximum amount of air inhaled from the point of maximum expiration; vital capacity (VC), the amount of air exhaled after the deepest inspiration; functional residual capacity (FRC), the volume in the lungs at the end-expiration position; forced vital capacity (FVC), the determination of vital capacity from a maximum forced expiratory effort; forced expiratory volume (time) (FEV t ), the volume expired under effortful conditions in the first t seconds; forced inspiratory flow (FIF), a specific measurement of the forced inspiratory curve; peak expiratory flow (PEF), maximal forced expiratory flow measured with a peak flow meter; maximum ventilatory volume (MVV), the volume of air expired in a specific period during repeated maximal efforts, etc. Methods for measuring respiratory volume are generally known to those skilled in the art.

[0115] Another aspect of the present disclosure relates to a method of reducing or preventing chronic bacterial infection in the lungs in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, e.g., cystic fibrosis, COPD, etc. In some embodiments, the subject suffers from cystic fibrosis. Methods of monitoring bacterial infection in the lungs, including improvements thereto, are known to those skilled in the art, including, for example, by performing blood tests or cultures, oximetry, arterial blood gas measurements, bronchoscopy, transtracheal mucus cultures, lung biopsies, thoracentesis, computed tomography scans, etc.

[0116] Another aspect of the present disclosure is a method of reducing, preventing, or treating chronic bacterial infections of the lungs in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, e.g., cystic fibrosis, COPD, etc. In some embodiments, the subject suffers from cystic fibrosis. Methods of measuring lung inflammation, including improvements therein, are well known to those skilled in the art, including, for example, by measuring exhaled nitric oxide, determining the percentage of eosinophils in sputum and blood, etc.

[0117] Another aspect of the present disclosure is a method of reducing, inhibiting, or treating progressive lung damage in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies). In some embodiments, the subject suffers from a chronic lung disease, such as cystic fibrosis, COPD, etc. In some embodiments, the subject suffers from cystic fibrosis. Methods of measuring lung damage are well known to those skilled in the art, including, for example, by the method described by Saetta et al. (Am Rev Respir Dis. 1985 May; 131(5): 764-9).

[0118] 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 cystic fibrosis in a subject in need thereof, comprising administering to the subject any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject's genome comprises a mutation (e.g., a loss-of-function mutation) in the endogenous CFTR gene (one or both copies).

[0119] Signs and symptoms of cystic fibrosis may include, but are not limited to, persistent coughing that produces a lot of mucus, excessive sticky mucus accumulating in the airways, wheezing, difficulty breathing, sinusitis, recurrent lung infections, inflamed nasal passages, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof.

[0120] 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 COPD in a subject in need thereof, comprising administering to the subject an effective amount of any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a smoker or ex-smoker.

[0121] Signs and symptoms of COPD can include, but are not limited to, shortness of breath, wheezing, chest tightness, excess mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof.

[0122] The recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein may be administered by any suitable method or route known in the art, including, but not limited to, orally, intranasally, intratracheally, sublingually, buccally, topically, rectally, via inhalation, transdermally, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardially, intraosseous, intraperitoneally, transmucosally, intravaginally, intravitreally, intraorbitally, subretinally, intraarticularly, periarticularly, topically, epicutaneously, or any combination thereof. Thus, the present disclosure encompasses methods of delivering any of the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein to an individual (e.g., an individual having or at risk of developing a chronic lung disease such as cystic fibrosis).

[0123] In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation described herein is administered orally, intranasally, intratracheally, or via inhalation. Methods for delivering drugs to the lungs via oral, intranasal, intratracheal, and / or inhalation routes of administration are generally known to those of skill in the art (see, e.g., Gardenhire et al. A Guide to Aerosol Delivery Devices for Respiratory Therapists, 4). thEdition, 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).

[0124] In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is delivered to the lung by inhalation of an aerosolized formulation. Inhalation can occur through the subject's nose and / or mouth. Exemplary devices for delivering recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to the lung 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).

[0125] Liquid formulations can be administered to the lungs of a subject 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 receptacle used to hold and actuate the canister. The canister can contain a single or multiple dose formulation. The canister can include a valve, typically a metering valve, through which the contents of the canister can be expelled. Aerosolized drug is dispensed from a pMDI by applying force to the canister to force it into the receptacle, thereby opening the valve and causing drug particles to be transported out of the valve through the receptacle outlet. Upon expulsion from the canister, the liquid formulation is atomized to form an aerosol. pMDIs typically use one or more propellants to pressurize the contents of the canister, propelling the liquid formulation out of the receptacle outlet and forming an aerosol. Any suitable propellant can be utilized and can take a variety of forms, including, for example, compressed gas or liquefied gas.

[0126] The liquid formulation may be administered to the lungs of a subject, for example, using a nebulizer. A nebulizer is a liquid aerosol generator that converts the liquid formulation into a mist or cloud of small droplets, often with an aerodynamic mass median particle diameter of less than about 5 microns in diameter, that can be inhaled into the lower respiratory tract. The droplets carry the active agent(s) to the nose, upper airways, and / or deep lungs when the aerosol cloud is inhaled. 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.), etc., may be used to administer the formulation to a patient. Pneumatic (jet) nebulizers use a pressurized gas supply as the driving force for nebulization of the liquid formulation. The compressed gas is delivered through a nozzle or jet to create a low pressure region that entraps the surrounding liquid formulation and shears it into a thin film or filament. The film or filament is unstable and breaks up into small droplets that are carried by the compressed gas flowing in the inhaled breath. A baffle inserted into the droplet plume screens out larger droplets and returns them to a bulk reservoir. Electromechanical nebulizers use electrically generated mechanical force to nebulize liquid formulations. 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 membrane. The force creates a thin liquid film or filament stream that breaks into small droplets to form a slow-moving aerosol stream that can be trapped in the inhalation air stream. In some embodiments, the nebulizer is a vibrating mesh nebulizer. Examples of vibrating mesh nebulizers include, for example, Phillips InnoSpire, Aerogen Solo, PARI eFlow, etc.

[0127] Liquid formulations can be administered to the lungs of a subject, for example, using electrohydrodynamic (EHD) aerosol devices, which use electrical energy to aerosolize a liquid drug solution or suspension.

[0128] Dry powder formulations can be administered to the lungs of a subject, for example, using a dry powder inhaler (DPI). DPIs typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which can then be inhaled by the subject. In DPIs, the dose to be administered 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 dispense the powder, similar to pMDIs. In some cases, DPIs can be breath-actuated (aerosols are created exactly 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, Turbohaler® inhaler (AstraZeneca), Clickhaler® inhaler (Innovata), Diskus® inhaler (Glaxo), EasyHaler® (Orion), Exubera® inhaler (Pfizer), and the like.

[0129] In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to the subject once, hi some embodiments, the recombinant nucleic acid, virus, agent, 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 one 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.) passes between administrations (e.g., between a first administration and a second administration, between a second administration and a third administration, etc.). In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to a subject once, twice, three times, four times, five times, or more per day. In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to a subject 1, 2, 3, 4, 5, or more times per month. In some embodiments, the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation is administered to a subject 1, 2, 3, 4, 5, or more times per year.

[0130] VII.Host cells Certain aspects of the present disclosure pertain to one or more host cells containing any of the recombinant nucleic acids described herein. For example, prokaryotic cells including eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), 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, etc.), and mammalian cells (e.g., monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney lines (293 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 Any suitable host cell (prokaryotic or eukaryotic) known in the art may be used, including human ovarian cancer cells (Humanoid spp., ATCC CCL 131, ATCC CCL 132, ATCC CCL 133), human ovarian cancer cells (Humanoid spp., ATCC CCL 134, ATCC CCL 135), human ovarian cancer cells (Humanoid spp., ATCC CCL 136, ATCC CCL 137), human ovarian cancer cells (Humanoid spp., ATCC CCL 138, ATCC CCL 139), human ovarian cancer cells (Humanoid spp., ATCC CCL 139), human ovarian cancer cells (Humanoid spp., ATCC CCL 137), human ovarian cancer cells (Humanoid spp., ATCC CCL 138 ...9), human ovarian cancer cells (Humanoid spp., ATCC CCL 139), human ovarian cancer cells (Humanoid spp., ATCC CCL 139), human ovarian cancer cells (Humanoid spp., ATCC CCL 139), human ovarian cancer cells (Humanoid spp., In some embodiments, the host cell is a cell from a cell line.Examples of suitable host cells or cell lines may include, but are not limited to, 293, HeLa, SH-Sy5y, Hep G2, CACO-2, A549, L929, 3T3, K562, CHO-K1, MDCK, HUVEC, Vero, N20, COS-7, PSN1, VCaP, CHO cells, and the like.

[0131] 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 host cell containing a helper virus is contacted with an HSV-1 amplicon or an HSV-1 hybrid amplicon described herein, resulting in the production of a virus containing one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. 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.

[0132] 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 (e.g., a recombinant herpes simplex virus genome) described herein. In some embodiments, contacting the complementing host cell with the recombinant herpesvirus genome results in the production of a herpesvirus comprising one or more recombinant nucleic acids described herein. In some embodiments, the virus is collected from the supernatant of the contacted host cell. Methods of producing a virus by contacting a complementing host cell with a recombinant herpes simplex virus are generally described in WO2015 / 009952, WO2017 / 176336, WO2019 / 200163, WO2019 / 210219, and / or WO2020 / 006486.

[0133] VIII. Products or Kits Certain aspects of the present disclosure relate to an article of manufacture or kit that includes any of the recombinant nucleic acids, viruses, agents, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the article of manufacture or kit includes a package insert that includes instructions for administering the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation to treat CFTR deficiency (e.g., in subjects with homozygous CFTR loss-of-function mutations) and / or to provide preventive, palliative, or therapeutic relief of one or more signs or symptoms of chronic lung disease (such as cystic fibrosis or COPD). In some embodiments, the article of manufacture or kit further includes a device for administering the recombinant nucleic acid, virus, agent, and / or pharmaceutical composition or formulation. In some embodiments, the device is a nebulizer (e.g., a vibrating mesh nebulizer).

[0134] Suitable containers for the recombinant nucleic acid, virus, drug, and / or pharmaceutical compositions or formulations may include, for example, bottles, vials, bags, tubes, and syringes. The containers may be formed from a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container comprises a label on or associated with the container, the label indicating instructions for use. The article 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, and the like.

[0135] 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. EXAMPLES

[0136] The present disclosure will be more fully understood by referring to the following examples. However, it should not be interpreted as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light thereof, various modifications or changes will be suggested to those skilled in the art and will fall within the spirit and scope of the present application and the scope of the appended claims.

[0137] Example 1: Modified herpes simplex virus vector encoding the human CFTR protein To generate a modified herpes simplex virus vector capable of expressing a CFTR polypeptide in a target mammalian cell (such as a lung cell), 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 such that they carry one or more polynucleotides encoding the desired CFTR polypeptide.These variants include: 1) a recombinant ΔICP4 modified HSV-1 genome comprising an expression cassette comprising a coding sequence (e.g., SEQ ID NO:2) of a human CFTR polypeptide (e.g., SEQ ID NO:5) 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 comprising a coding sequence of a human CFTR polypeptide 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 comprising a coding sequence of a human CFTR polypeptide under the control of a heterologous promoter integrated into the UL41 locus (Figure 1D); and 4) a recombinant ΔICP4 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence of a human CFTR polypeptide 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 comprising a coding sequence for a human CFTR polypeptide under the control of a heterologous promoter integrated into the ICP22 locus (FIG. 1F); 6) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for a human CFTR polypeptide under the control of a heterologous promoter integrated into each ICP4 locus (FIG. 1G); 7) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for a human CFTR polypeptide under the control of a heterologous promoter integrated into the UL41 locus (FIG. 1H); and 8) a recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome comprising an expression cassette comprising a coding sequence for a human CFTR polypeptide under the control of a heterologous promoter integrated into the ICP22 locus (FIG. 1I).

[0138] These modified herpes simplex virus genome vectors are transfected into engineered cells that are modified to express one or more herpes virus genes. These engineered cells secrete the replication-defective herpes simplex virus with the modified genome packaged inside into the supernatant of the cell culture. The supernatant is then collected, concentrated, and sterile-filtered through a 5 μm filter.

[0139] Example 2: Construction and in vitro characterization of HSV-1 vectors encoding human CFTR in 2D cultures Following the discovery of the genetic defect that causes cystic fibrosis, the earliest pulmonary gene therapy clinical trials were conducted in the early 1990s. Recombinant adenovirus was one of the early vectors tested for CFTR delivery, but adeno-based viruses failed in these trials, primarily due to a paucity of viral receptors on the apical lung surface and the severity of the host immune response to repeated viral delivery. Other viral gene therapy vectors administered to CF patients were based on adeno-associated viruses (a number of AAV serotypes have been tested in CF clinical settings). Large-scale repeated administration studies of AAV-based gene therapy vectors have provided disappointing results in improving CF lung function in medicated patients. Much like adenovirus, recombinant AAV vectors do not efficiently infect the apical lung surface, and due to physical limitations in the size of the encoded cargo, AAV vectors do not efficiently deliver full-length human CFTR. Despite more than 20 years of intensive efforts, viral-based gene therapy has yet to help patients with CF (or any other obstructive pulmonary disease).

[0140] Currently, according to the US Cystic Fibrosis Foundation, there are no ongoing clinical trials of viral gene therapy in CF, and only two viral-based gene therapy vectors are in preclinical development (both of which are based on AAV, a vector that, as mentioned above, has already failed multiple clinical trials in CF patients). Instead, focus has shifted from viral-based vectors to non-viral methods of CFTR delivery (e.g., DNA plasmids or mRNA complexed with liposomes). Unfortunately, these non-viral vectors have only met with limited success, due at least in part to significant hurdles faced by product instability and / or inefficient delivery / transfection of liposomal formulations. More than 25 clinical trials involving over 470 patients testing viral and non-viral gene vectors have failed to show clinical benefit, primarily due to inefficient gene transfer into target cells and host immune-mediated clearance after repeated exposure.

[0141] To this end, a recombinant herpes simplex virus type 1 (HSV-1) vector encoding full-length human CFTR (HSV-CFTR) has been developed as a novel gene therapy for the treatment of CF patients. Without wishing to be bound by theory, it is believed that the HSV-based approach overcomes many of the hurdles experienced by other gene therapy vectors for CF, including the ability to encode full-length human CFTR, high efficiency of target cell transduction (HSV preferentially infects the apical membrane of polarized epithelial cells), viral stability, and established clinical safety of repeated administration of products using the same viral backbone as HSV-CFTR in the context of the highly inflammatory environment of wounded skin (ClinicalTrials.gov Identifier: NCT03536143). The following example describes an experiment showing that this novel HSV-based gene therapy vector was able to express functional full-length human CFTR in a dose-dependent manner in small airway epithelial cells (SAEC) derived from cystic fibrosis patients.

[0142] HSV-CFTR was constructed as described in Example 1 above. Primary CF patient SAECs grown in 2D cultures were left uninfected (mock) or infected with HSV-CFTR at a multiplicity of infection (MOI) of 0.3, 1, or 3. Human CFTR expression was assessed 48 hours after infection in harvested cells by quantitative reverse transcription PCR (qRT-PCR). Codon-optimized CFTR transcripts were detected in primary CF SAECs infected at MOIs as low as 0.3 and appeared to show a dose-dependent increase in transgene expression up to MOIs of 3.0 (Figure 2). Little exogenous CFTR RNA was observed in mock-infected control samples, demonstrating the specificity of the assay for the HSV-encoded human transgene.

[0143] CFTR protein expression in HSV-CFTR-infected primary CF SAEC was assessed via Western blot analysis. GAPDH was used as a control to ensure consistent loading of samples. CF patient SAEC overexpressed human CFTR when infected with HSV-CFTR compared to mock-infected control cells (Figure 3). Interestingly, endogenous CFTR protein in mock-infected cells was resolved as a single band slightly larger than 150 kDa (the predicted size of full-length human CFTR is 168 kDa), while exogenous CFTR protein expressed in HSV-CFTR-transduced cells appeared as a doublet of significantly larger size. Human CFTR is known to exist in three different forms depending on the glycosylation state: (1) nonglycosylated, (2) core glycosylated, and (3) fully mature, complex glycosylated (Scanlin, 2001, Respir Res, 2(5), pp. 276-9). The appearance of a single low molecular weight band in mock-infected CF patient cells suggested that the endogenous (mutant) protein was present only in a non-glycosylated form, indicative of an immature protein variant that is not properly transported through the endoplasmic reticulum (ER) to the cell surface. In stark contrast, the appearance of two larger forms of CFTR in HSV-CFTR-infected cells revealed extensive post-translational modifications of the human transgene, likely representing core- and complex-glycosylated variants of CFTR, suggesting proper maturation and transport of the exogenous protein through the ER.

[0144] Next, CFTR protein expression and relative localization were examined by immunofluorescence. Primary CF patient SAECs were transduced with HSV-CFTR at the indicated MOI for 48 hours and immunofluorescence staining of human CFTR was performed. Mock-infected control samples were added to show the baseline levels and cellular localization of endogenous mutant CFTR protein in these diseased cells. When analyzed in the context of control cells, immunofluorescence data demonstrated that transduced SAECs showed an HSV-CFTR dose-dependent increase in CFTR protein expression (Figure 4A). When comparing the relative cellular localization of CFTR expressed in mock-infected versus HSV-CFTR-infected CF patient SAECs (Figure 4B), CFTR expressed in uninfected cells appeared to be translocated to the perinuclear region (suggesting capture and turnover in the ER), whereas CFTR was found throughout the cytoplasm and on the cell surface of HSV-CFTR-transduced cells (indicating proper maturation and trafficking in the ER). This data was consistent with the Western blot data suggesting that wild-type, HSV-CFTR-expressed CFTR was fully glycosylated, whereas endogenous mutant CFTR was aglycosylated (Fig. 3 ).

[0145] Finally, functionality of HSV-CFTR-expressed human CFTR in infected CF patient SAECs was confirmed using a dihydrorhodamine 6G (dR6G) fluorescent dye uptake assay previously validated as a functional endpoint of virus-mediated CFTR restoration in 2D CF patient epithelial cell cultures (Wersto, 1996, Proc Natl Acad Sci USA, 93(3), pp. 1167-72). Briefly, HSV-CFTR or mock-infected primary CF patient SAECs were incubated with dR6G-containing cell culture medium for 15 min, washed four times with PBS, lysed in RIPA buffer, and 526 nm excitation / 555 nm emission fluorescence was read for each sample on a plate reader. dR6G itself is non-fluorescent, but upon cellular uptake and exposure to intracellular dehydrogenases, is converted to the fluorescent compound rhodamine 6G, a process that depends on the presence of functional CFTR (Wersto, 1996, Proc Natl Acad Sci USA, 93(3), pp. 1167-72). BCA assays were performed on each cell lysate to quantify total protein content, and relative fluorescence per μg of total protein was calculated for each sample (Figure 5). HSV-CFTR infection of primary CF patient SAEC caused a modest dose-dependent increase in dR6G uptake compared to mock-infected controls, indicating that HSV-CFTR can restore CFTR function in these diseased primary epithelial cells.

[0146] Example 3: In vitro HSV-CFTR dose range and pharmacology in 3D organotypic cultures using CF patient-derived organoids Mutations in the CFTR gene are classified into one of six classes depending on the primary mechanism that results in CFTR dysfunction. Mutations that affect synthesis and processing result in more severe disease because little protein reaches the cell surface and do not impede luminal transport, whereas mutations that reduce CFTR-mediated anion efflux often result in less severe symptoms due to the retention of some residual CFTR function at the apical membrane (Foundation, 2019, 2018 Annual Data Report, Bethesda: Cystic Fibrosis Foundation). Because CFTR mutations affect distinct steps of protein synthesis and function, recent drug development efforts have focused on small molecule modulator therapies that target the specific source of the protein defect. For example, ivacaftor, subclassified as a CFTR protein "potentiator," enhances chloride secretion of membrane CFTR (providing clinical benefit to individuals with certain class III and IV CFTR gating and conductance mutations), while elexacaftor, subclassified as a CFTR protein "corrector," acts by promoting proper folding and cellular processing of CFTR that would otherwise be degraded by the endoplasmic reticulum quality control pathway (providing clinical benefit to individuals with certain class II CFTR trafficking mutations) (Clancy, 2019, Am J Respir Crit Care Med, 186(7), pp. 593-7). While the recent FDA approval of four of these modulator therapies has been a boon for CF patients with specific mutations that respond to these drugs, these modulators only treat a subset of the CF population. Particularly important in need of effective drug intervention are patients with Class I mutations (which account for approximately 10% of CF cases worldwide), which encompass frameshift, splicing, and nonsense mutations that result in severely reduced or absent CFTR expression; these patients suffer from the most severe and most lethal forms of CF (Wilschanski, 2012, Front Pharmacol, 20(3), pp.1-3).

[0147] Due to the lack of suitable CF animal models, efficacy studies in air-liquid interface differentiated bronchial epithelial cells derived from CF patient lung explant material have been used for some drug development efforts after proof-of-concept experiments in heterologous 2D cell systems (Neuberger, 2011, Methods Mol Biol, 741(1), pp. 39-54) (Randell, 2011, Methods Mol Biol, 742(1), pp. 285-310). However, the limited availability of lung explant tissue and the invasive procedures required to obtain bronchial cells from CF patients without end-stage disease have led to the development of 3D organotypic systems derived from "easy access" tissues taken from CFTR mutant patients to test novel therapeutics for treating CF. One such technique, using the forskolin-induced swelling (FIS) assay, has been used to study CFTR protein function, either alone or in response to pharmaceutical intervention, using CF patient-derived intestinal organoids (PDOs) (Dekkers, 2013, Nat Med, 19(7), pp. 939-45), and has proven to be a breakthrough in CF drug development. Upon exposure to forskolin, organoids rapidly increase their cyclic AMP content, resulting in the opening of CFTR channels. Organoids derived from biopsies taken from healthy individuals swell as a result of CFTR-mediated ion and water transport into the organoid lumen, whereas organoids derived from CFTR mutant patient biopsies (or wild-type organoids exposed to specific pharmacological inhibition of CFTR protein function) have reduced or completely inhibited swelling capacity (Boj, 2017, J Vis Exp, 120(1), p. e55159). The use of CF PDO allows for quantitative measurement of CFTR protein function (via detection of organoid swelling) upon treatment with novel therapeutic agents, and positive results from this 3D organotypic system have been shown to directly correlate with clinical benefit, including both changes in pulmonary response and sweat chloride concentrations in treated CF patients (Berkers, 2019, Cell Rep, 26(7), pp.1701-1708).

[0148] The following example describes experiments demonstrating that the recombinant HSV-1 vector HSV-CFTR, characterized in Example 2 above, is able to rescue the cystic phenotype of CF PDO, regardless of the underlying CFTR mutation.

[0149] The ability of HSV-CFTR to restore functional CFTR expression was tested in clinically relevant 3D organotypic cultures using intestinal organoids derived from four different CF patients: (1) a female patient homozygous for the F508del CFTR mutation (class II mutation), (2) a male patient also homozygous for the F508del mutation, (3) a female patient homozygous for the G542X nonsense CFTR mutation (class I mutation), and (4) a female patient homozygous for the W1282X nonsense CFTR mutation (class I mutation). To assess CFTR activity in transduced organoids, organoid morphology and size were evaluated 24 or 48 hours after infection and FIS assays were performed as previously described (Boj, 2017, J Vis Exp, 120(1), p.e55159). For efficient infection of CF organoids, organoids were sheared into small pieces, incubated with HSV-CFTR in solution at the indicated MOI for 1 h, and seeded into 96-well clear-bottom plates for analysis. FIS assays were performed 24 or 48 h after seeding, as described in more detail below.

[0150] First, G542X / G542X PDO was infected at MOIs of 10, 20, and 40 to assess the vector's impact on both organoid swelling and cell viability. Intestinal organoids derived from healthy patients were plated in parallel as a comparison. Strikingly, HSV-CFTR-transduced organoids showed lumen formation and distinct cystic morphology mimicking wild-type PDO 24 hours after infection, suggesting complete functional correction of the disease phenotype by the engineered vector prior to the addition of forskolin (Figure 6A). An mCherry-expressing HSV vector was used as a negative control to demonstrate that the alterations in PDO morphology observed in HSV-CFTR-treated samples were not due to a nonspecific response to viral infection. Next, FIS assays were performed 48 hours after infection. At t=0, prior to the addition of forskolin and subsequent activation of CFTR, HSV-CFTR-transduced organoids already had a significantly enlarged luminal area compared to vehicle-treated or mCherry-infected organoids, consistent with the observations at 24 h post-infection (Figure 6B). Interestingly, only a moderate increase in organoid swelling was observed 60 min after addition of forskolin (t=60) in HSV-CFTR-transduced organoids, likely due to these organoids approaching their maximum swelling potential prior to forskolin exposure (Figure 6C). The G542X / G542X mutation can be corrected (at least in part) by exposure to the aminoglycoside geneticin (G418), which allows translational readout of the nonsense mutation, and G418 was included in this assay as a positive control. While G542X / G542X PDO swelled in the presence of G418 at t=60, the average organoid size in these positive control samples was significantly smaller than that of HSV-CFTR-exposed PDO (Figures 6B and 6C). Mild to moderate toxicity of the vector in G542X / G542X PDO was observed 48 hours after infection when HSV-CFTR was used at an MOI of 20 or 40, with toxicity at MOIs of 20 and above likely responsible for the reduced capacity for swelling observed in these organoids compared to samples infected at an MOI of 10.However, despite the observed cytotoxic effects at high MOI, treated organoids still outperformed the positive small molecule control.

[0151] Because HSV-CFTR corrected diseased organoids to wild-type morphology (large cystic lumen) at all tested MOIs within 24 hours and higher HSV-CFTR doses appeared to adversely affect organoids in the swelling assay, the three remaining cystic fibrosis PDOs were tested at lower HSV-CFTR doses (MOIs of 1, 5, and 10) and analyzed via FIS assay 24 hours after infection. First, HSV-CFTR was tested in PDOs derived from patients homozygous for the F508del mutation of CFTR. F508del is the most common mutation in cystic fibrosis patients, with at least one copy of this allele found in approximately 85% of CF patients worldwide, and F508del accounts for approximately 70% of CFTR loss-of-function mutations (Maiuri, 2015, Ann Transl Med, 3(Supple 1), p.S24). The majority of F508del organoid cultures tested showed cystic (wild-type) morphology 24 hours after infection with HSV-CFTR, even at the lowest dose tested (MOI of 1). The average size of F508del organoids treated with HSV-CFTR was significantly increased compared to vehicle control or mCherry-infected organoids before forskolin addition (Figure 7A). No significant change in average organoid size was detected after forskolin addition in HSV-CFTR-transduced samples, and these organoids are considered to be already at or close to their maximum expansion capacity, i.e., "pre-expansion" (Figure 7B). Importantly, the functional correction of CFTR defects in F508del organoids appeared to be similar between HSV-CFTR-treated organoids before forskolin treatment and positive control Orkambi®-exposed organoids 60 minutes after forskolin treatment (Figure 7A vs. Figure 7B). Orkambi® is a lumacaftor / ivacaftor combination therapy that is FDA approved for the treatment of CF patients 2 years of age and older who are homozygous for the F508del mutation. No overt vector-attributable cytotoxicity was observed at any of the MOIs tested.

[0152] Next, organoids derived from a patient homozygous for the second nonsense CFTR mutation (W1282X) were infected with HSV-CFTR and organoid size was quantified before and after addition of forskolin. Consistent with the data presented in Figure 6 above, HSV-CFTR efficiently restored the wild-type cystic phenotype and increased the average organoid size 24 hours after infection in W1282X / W1282X nonsense CFTR PDO before addition of forskolin (Figure 8A). Also, HSV-CFTR at an MOI as low as 1 appeared to correct both disease forms before and after addition of forskolin (Figures 8A and 8B). G418 was also included in these experiments, but a positive control could not be included in these experiments because W1282X / W1282X PDO appeared to be unresponsive to this readout aminoglycoside (since there is currently no effective therapy against all nonsense CFTR mutations). This data suggested that HSV-CFTR was able to restore CFTR function in both G418-responsive and G418-unresponsive CFTR-null patient samples.

[0153] Finally, organoids from a second F508del homozygous patient were examined. HSV-CFTR-infected PDOs had a slightly increased mean size compared to vehicle-treated organoids, but this difference was not statistically significant (Figures 9A and 9B).

[0154] Data from these studies revealed that transduction of intestinal CF organoids with HSV-CFTR resulted in a marked modification of organoid morphology within 24 h of infection at MOIs ranging from 1 to 40, from a compact budding CF phenotype to a cystic organoid phenotype containing a well-defined lumen exhibiting wild-type characteristics. This "pre-swollen" wild-type phenotype was quantitatively demonstrated by measuring total organoid size prior to addition of forskolin and the resulting activation of CFTR, in comparison to multiple negative controls. Due to the "pre-swollen" nature of HSV-CFTR-transduced organoids, the ability of forskolin to stimulate further swelling was limited. The observation of corrected cystic morphology in CF organoids exposed to low doses of HSV-CFTR suggested that high levels of exogenous wild-type CFTR expressed in a small number of cells was sufficient to establish disease correction, demonstrating the "dominant" effect of this therapeutic modality. Although one F508del organoid showed a slightly less efficient restoration of the wild-type phenotype compared to the other examined CF organoid cultures, cystic morphology was observed in all CF organoids infected with HSV-CFTR at an MOI of 5 or higher. The differences observed between the various CF intestinal organoid cultures are most likely due to subtle changes in their proliferation or differentiation state upon infection, and therefore it is unlikely that the CFTR genotype itself contributed meaningfully to the efficiency of HSV-CFTR transduction or functional CFTR expression. In other words, HSV-CFTR corrected the CF disease phenotype regardless of the underlying CFTR mutation in this clinically translatable 3D organotypic system.

[0155] Taken together, the data provided in these Examples show that HSV-CFTR successfully infected relevant airway epithelia, efficiently produced functional human CFTR, and molecularly corrected multiple CFTR defects without significant toxicity. Without wishing to be bound by theory, it is believed that these studies represent the first case of experimental validation of an attenuated HSV-based gene therapy vector to deliver full-length functional human CFTR, and support the application of HSV-CFTR as a novel and broadly applicable gene therapy for the treatment of CF.

[0156] Example 4: Proof-of-concept in vivo administration of inhaled HSV-based vectors The following examples describe proof-of-concept in vivo studies examining the feasibility of administering HSV-based vectors to the trachea and / or lungs of immunocompetent animals following intranasal or intratracheal administration of the virus.

[0157] All procedures performed in this example were in compliance with applicable animal welfare laws and approved by the local Institutional Animal Care and Use Committee (IACUC). Ten 5-6 week old C57BL / 6 mice were used in the study, five of which received either HSV-mCherry (as described above) or vehicle control by intratracheal administration, and five of which received HSV-mCherry or vehicle control by intranasal administration. Prior to experimental procedures, animals were sedated with an intraperitoneal injection of a mixture of Telazol / Dexdomitol, and ophthalmic ointment was applied to the eyes to prevent corneal drying.

[0158] For intratracheal administration, the neck of each mouse was shaved using an electric razor and depilatory cream was applied to remove all remaining fur. The surgical area was then washed twice with 70% ethanol soaked swabs and the anesthetized mouse was placed on an angled restraining stand. A small incision in the neck was made using surgical scissors and the thymus, platysma and anterior trachealis muscles were moved out of the way to visualize and access the tracheal rings. 4.9375×10 8Three animals received a 25 μL intratracheal injection of plaque forming units (PFU) of HSV-mCherry, while two animals received a 25 μL intratracheal injection of vehicle control, and each mouse was held in a suspended position until its breathing gradually returned to normal. The incision sites were closed with simple individually tied sutures.

[0159] For intranasal administration, mice were anesthetized as described above and placed on an angled restraining stand. Three mice were administered 4.9375x10 formulated in 25 μL (12.5 μL per nostril). 8 PFU of virus was inoculated intranasally, respectively. The rate of formulation release was adjusted to allow the mice to inhale the inoculum without forming air bubbles during the inspiratory phase of breathing. Two mice were administered 25 μL of vehicle control using the same procedure. After administration, the animals were held in a hanging position until breathing returned to normal.

[0160] All animals were allowed to recover from anesthesia and were given water and food ad libitum until the time of sacrifice. 48 hours after dosing, mice were euthanized and bronchoalveolar lavage (BAL) was performed on the left and right lungs using sterile saline. BAL fluid was collected, centrifuged, and cell pellets were collected. The upper part of the trachea was then harvested and snap frozen in liquid nitrogen for nucleic acid quantification. Lungs (left, right upper, right middle, and right lower lobes and retrocaval lobes) were individually harvested and snap frozen in liquid nitrogen for nucleic acid analysis or perfused in 4% neutral buffered formalin and embedded in paraffin for immunofluorescence analysis.

[0161] For immunofluorescence staining of paraffin-embedded lung tissue, an Alexa Fluor® 488-conjugated pan-cytokeratin antibody was used to detect epithelial cells (Invitrogen Cat. No. 53-9003-82), and a rabbit anti-mCherry primary antibody (Abcam Cat. No. ab213511) and an Alexa Fluor® 594-conjugated secondary antibody (Abcam Cat. No. ab150080) were used to detect infected cells. Tissue samples were mounted in mounting medium containing DAPI to visualize nuclei.

[0162] Intranasal versus intratracheal administration of HSV-mCherry resulted in similar levels of mCherry transcripts being detected in lung tissue of transduced animals (Figure 10A). Interestingly, while little transgene transcript was identified in the trachea of ​​intranasally exposed mice, robust mCherry transcription was detected in the trachea of ​​intratracheally exposed mice, and no statistically significant differences in transgene expression were observed between the lungs and trachea of ​​these invasively treated animals. In addition, a greater mean total cell count per mL of BAL fluid was observed in intratracheally administered animals (646,667 cells / mL and 393,333 cells / mL for intratracheal and intranasal administration, respectively), suggesting a greater influx of inflammatory cells into the lungs following intratracheal administration of HSV-based vectors. Transgene protein expression in lung epithelial tissue was observed in both intranasally (FIG. 10B) and intratracheally (FIG. 10C) challenged animals dosed with HSV-mCherry, but not in the corresponding vehicle controls.

[0163] Taken together, this data indicates that engineered HSV vectors can be administered to the lungs of immunocompetent animals via multiple routes of administration, and further indicates that the non-invasive inhalation route of administration allows similar levels of transgene expression in the lungs as more direct invasive routes of administration, while at the same time inducing less (inflammatory) cellular invasion.

[0164] Example 5: Nebulization of HSV-CFTR The following examples describe studies examining a non-invasive, nebulizer-based delivery route for HSV-CFTR to the airways of wild-type and CFTR-deficient immunocompetent mice.

[0165] Sixteen mice are used in the study: 12 immunocompetent C57BL / 6 animals, and 4 immunocompetent gut-corrected CFTR-deficient animals. Table 1 provides a summary of the study. Four wild-type animals receive HSV-CFTR via intranasal instillation, while the remaining animals receive HSV-CFTR (or vehicle control) via nebulization (e.g., using a vibrating mesh nebulizer). 48 hours after dosing, animals are euthanized, BAL fluid is collected, and tissue samples along the respiratory tract and lungs are taken, i.e., upper and lower trachea, left and right bronchi, left lung, and right lung (upper, middle, lower, and retrocaval lobes, respectively). Tissues from two animals per group are snap frozen in liquid nitrogen and processed for nucleic acid analysis. Vector genomes / 50ng total DNA will be quantified in each tissue via qPCR analysis, and human CFTR transcripts / 50ng total RNA will be quantified in each tissue via qRT-PCR analysis. Tissues from the remaining two animals per group will be perfused and embedded in paraffin for immunofluorescence / immunohistochemistry. BAL fluid will be processed to examine immune cell infiltration into the lungs. (Table 1) Study design TIFF2025013873000002.tif28146

[0166] Sequence information SEQUENCE LISTING <110> Krystal Biotech, Inc. <120> COMPOSITIONS AND METHODS FOR DELIVERING CFTR POLYPEPTIDES <150> US 62 / 802,871 <151> 2019-02-08 <160> 6 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 4443 <212> DNA <213> Homo sapiens <400> 1 atgcagaggt cgcctctgga aaaggccagc gttgtctcca aacttttttt cagctggacc 60 agaccaattt tgaggaaagg atacagacag cgcctggaat tgtcagacat ataccaaatc 120 ccttctgttg attctgctga caatctatct gaaaaattgg aaagagaatg ggatagagag 180 ctggcttcaa agaaaaatcc taaactcatt aatgcccttc ggcgatgttt tttctggaga 240 tttatgttct atggaatctt tttatattta ggggaagtca ccaaagcagt acagcctctc 300 ttactgggaa gaatcatagc ttcctatgac ccggataaca aggaggaacg ctctatcgcg 360 atttatctag gcataggctt atgccttctc tttattgtga ggacactgct cctacaccca 420 gccatttttg gccttcatca cattggaatg cagatgagaa tagctatgtt tagtttgatt 480 tataagaaga ctttaaagct gtcaagccgt gttctagata aaataagtat tggacaactt 540 gttagtctcc tttccaacaa cctgaacaaa tttgatgaag gacttgcatt ggcacatttc 600 gtgtggatcg ctcctttgca agtggcactc ctcatggggc taatctggga gttgttacag 660 gcgtctgcct tctgtggact tggtttcctg atagtccttg ccctttttca ggctgggcta 720 gggagaatga tgatgaagta cagagatcag agagctggga agatcagtga aagacttgtg 780 attacctcag aaatgattga aaattccaa tctgttaagg catactgctg ggaagaagca 840 atggaaaaaa tgattgaaaa cttaagacaa acagaactga aactgactcg gaaggcagcc 900 tatgtgagat acttcaatag ctcagccttc ttcttctcag ggttctttgt ggtgttttta 960 tctgtgcttc cctatgcact aatcaaagga atcatcctcc ggaaaatatt caccaccatc 1020 tcattctgca ttgttctgcg catggcggtc actcggcaat ttccctgggc tgtacaaaca 1080 tggtatgact ctcttggagc aataaacaaa atacaggatt tcttacaaaa gcaagaatat 1140 aagacattgg aatataactt aacgactaca gaagtagtga tggagaatgt aacagccttc 1200 tgggaggagg gatttgggga attatttgag aaagcaaaac aaaacaataa caatagaaaa 1260 acttctaatg gtgatgacag cctctctttc agtaatttct cacttcttgg tactcctgtc 1320 ctgaaagata ttaatttcaa gatagaaaga ggacagttgt tggcggttgc tggatccact 1380 ggagcaggca agacttcact tctaatggtg attatgggag aactggagcc ttcagagggt 1440 aaaattaagc acagtggaag aatttcattc tgttctcagt tttcctgt tgcctggc 1500 accattaaag aaatatcat ctttggtgtt tcctatgatg atatagata cagaagcgtc 1560 atcaagcat gccaactaga agaggacatc tccaagttg cagagaaaga caatagtt 1620 cttggagaag gtggaatcac actgagtgga ggtcaacgag cagaatttc tttagcaga 1680 gcagtataca aagatgctga tttgtattta ttagactctc cttttggata cctagatgtt 1740 ttaacagaaaagaatatt tgaaagctgt gtctgtaaac tgatggctaa caaactagg 1800 atttggtca cttctaaaat ggaacattta aagaaagctg acaaatatt aattttgcat 1860 gaaggtagca gctattttta tgggacattt tcagaactcc aaaatctaca gccagacttt 1920 agctcaaac tcatgggatg tgattctttc gaccaattta gtgcagaaag aagaattca 1980 atcctaactg agaccttaca ccgttctca ttagaggag atgctcctgt ctcctggaca 2040 gaaaaaaaaaacaattttt aacagact gggagtttg gggaaaaag gagaattct 2100 attctcaatc caatcaactc tatacgaaa ttttccattg tgcaaagac tcccttacaa 2160 atgaatggca tcgaagagga ttctgatgag cctttagaga gaaggctgtc cttagtacca 2220 gattctgagc agggaggc gattgcct cgcatcagcg tgatcagcac tggccccacg 2280 cttcaggcac gaaggaggca gtctgtcctg aacctgatga cacactcagt taaccaggt 2340 cagaacattc accgaagac aacagcatcc acacgaaag tgtcactggc ccctcaggca 2400 aacttgactg aactgatat atttcaaga aggttatctc aagaactgg cttggaata 2460 agtgaagaaa ttaacgaaga agacttaag gagtgcttttt ttgatgatat ggagagcata 2520 ccagcagtga ctacatggaa cacatacctt cgatatatta ctgtccacaa gagctttatt 2580 ttgtgctaa ttgtgctt agtaatttt ctggcagagg tggctgcttc ttggttgtg 2640 ctgtggctcc ttggaacac tcctctcaa gataaggga atagtactca tagtagaat 2700 aacagctatg cagtgattat caccagcacc agttcgtatt atgtgtttta catttacgtg 2760 ggagtagccg acacttgct tgctatggga ttctcagag gtctaccact ggtgcatact 2820 ctaatcacag tgtcgaaaat ttcaccac aaaatgttac attctgttct tcaagcacct 2880 atgtcaaccc tcaacacgtt gaaagcaggt gggattctta atagattctc caaagatata 2940 gcaatttg atgaccttct gcctcttacc atattgact tcatccagtt gttattaatt 3000 gtgattggag ctatagcagt tgtcgcagtt ttacaaccct acatctttgt tgcaacagtg 3060 ccagtgatag tggcttttat tatgttgaga gcatatttcc tccaaacctc acagcaactc 3120 aaacaactgg aatctgaagg caggagtcca atttcactc atcttgttac aagcttaaaa 3180 ggactatgga cacttcgtgc cttcggacgg cagccttact ttgaaactct gttccacaaa 3240 gctctgaatt tacatactgc caactggttc ttgtacctgt caacactgcg ctggttccaa 3300 atgagaatag aaatgattt tgtcatcttc ttcattgctg ttaccttcat ttccatttta 3360 acaacaggag aaggagaagg aagagttggt attatcctga ctttagccat gaatatcatg 3420 agtacattgc agtgggctgt aaactccagc atagatgtgg atagcttgat gcgatctgtg 3480 agccgagtct ttaagttcat tgacatgcca acagaaggta aacctaccaa gtcaaccaaa 3540 ccatacaaga atggccaact ctcgaaagtt atgattattg agaattcaca cgtgaagaaa 3600 gatgacatct ggccctcagg gggccaaatg actgtcaaag atctcacagc aaaatacaca 3660 gaaggtggaa atgccatatt agagaacatt tccttctcaa taagtcctgg ccagagggtg 3720 ggcctcttgg gaagaactgg atcagggaag agtactttgt tatcagcttt tttgagacta 3780 ctgaacactg aaggaaat ccagatcgat ggtgtgtctt gggattcaat aactttgcaa 3840 cagtggagga aagcctttgg agtgatacca cagaagtt ttatttttc tggaacattt 3900 3960 gaggttgggc tcagatctgt gatagaacag tttcctggga agcttgactt tgtccttgtg 4020 gatggggct gtgtcctaag ccatggccac aagcagttga tgtgcttggc tagatctgtt 4080 ctcagtaagg cgaagatctt gctgcttgat gaacccagtg ctcatttgga tccagtaaca 4140 taccaataa ttaagaagaac tctaaaacaa gcatttgctg attgcacagt aattctctgt 4200 4260 gtgcggcagt acgattccat ccagaaactg ctgaacgaga ggagcctctt ccggcaagcc 4320 atcagcccct ccgacagggt gaagctcttt ccccaccgga actcaagcaa gtgcaagtct 4380 aagccccaga ttgctgctct gaaagaggag acagaagaag aggtgcaaga tacaaggctt 4440 tag 4443 <210> 2 <211> 4443 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 atgcagagaa gccctctgga aaaggccagc gtggtgtcca agctgttctt cagctggacc 60 cggcctatcc tgcggaaggg ctatagacag agactggaac tgagcgacat ctatcagatc 120 cccagcgtgg acagcgccga caacctgtct gagaagctgg aaagagagtg ggacagagag 180 ctggcctcca agaagaaccc caagctgatc aacgccctgc ggcggtgctt cttctggcgg 240 tttatgttct acggcatctt cctgtacctg ggcgaagtga ccaaagccgt gcagcctctg 300 ctgctgggca gaatcattgc cagctacgac cccgacaaca aagaggaacg gtctatcgcc 360 atctacctcg gcatcggcct gtgcctgctg tttatcgtca gaaccctgct gctgcacccc 420 gccatctttg gactgcacca catcggcatg cagatgcgga tcgccatgtt cagcctgatc 480 tacaagaaaa ccctgaagct gtccagcaga gtgctggaca agatcagcat cggacagctg 540 gtgtccctgc tgagcaacaa cctgaacaag ttcgacgaag gcctggctct ggcccacttc 600 gtgtggattg ctcctctgca agtggccctg ctgatgggcc tgatttggga actgctgcag 660 gccagcgcct tttgcggact gggatttctg attgtgctgg ccctgttcca ggccggactg 720 gggagaatga tgatgaagta ccgggaccag agagccggca agatctccga gagactggtc 780 atcaccagcg agatgatcga gaacatccag agcgtgaagg cctactgctg ggaagaggcc 840 atggaaaaga tgattgagaa tctgcggcag accgagctga agctgacaag aaaggccgcc 900 tacgtgcgct acttcaacag cagcgccttc ttcttctccg gcttcttcgt ggtgttcctg 960 agcgtgctgc cctacgctct gatcaagggc atcatcctga gaaagatttt caccaccatt 1020 tctttctgca tcgtgctgcg gatggccgtg accagacagt ttccttgggc tgtgcagact 1080 tggtacgata gcctgggcgc catcaacaag atccaggact tcctgcagaa gcaagagtac 1140 aagaccctcg agtacaacct gaccaccacc gaggtggtca tggaaaacgt gaccgccttc 1200 tgggaggaag gcttcggcga gctgtttgag aaggccaagc agaacaacaa caaccgcaag 1260 accagcaacg gcgacgacag cctgttctttc tccaacttct ccctgctggg gacccctgtg 1320 ctgaaggaca tcaacttcaa gatcgagcgg ggacagctgc tggccgtggc tggatctaca 1380 ggcgccggaa aaaccagcct gctcatggtc atcatgggcg agctggaacc tagcgagggc 1440 aagatcaagc acagcggcag gatcagcttc tgtagccagt tctcctggat catgcccggc 1500 accatcaaag agaacatcat cttcggcgtg tcctacgacg agtacagata ccgcagcgtg 1560 atcaaggcct gccagctgga agaggacatc agcaagttcg ccgagaagga caacatcgtg 1620 ctcggcgaag gcggcatcac actgtctggc ggacagaggg ccagaatctc tctggctaga 1680 gccgtgtaca aggacgccga tctgtacctg ctggatagcc cctcggcta cctggatgtg 1740 ctgaccgaga aagagatctt cgagagctgc gtgtgcaagc tgatggccaa caagaccaga 1800 atcctggtca cctccaagat ggaacacctg aagaaggccg acaagatcct gattctgcac 1860 gagggcagca gctactttta cggcaccttc agcgagctcg agaacctgca gcctgacttc 1920 agcagcaaac tgatgggctc cgactccttc gaccagttca gcgccgagcg gagaaacagc 1980 atcctgacag agacactgca ccggttctcc ctggaaggcg acgctcctgt gtcttggacc 2040 gagacaaaga agcagagctt caaccagacc ggcgagtttg gcgagaagcg gaagaactcc 2100 attctgaacc ccatcaactc catccggaag ttcagcatcg tccagaaaac ccctctgcag 2160 atgaacggca tcgaagga tagggacgag cccctggaaa gacggctgtc tctggtgcct 2220 gatagcgaac agggcgaagc catcctgcct cggatctccg tgattagcac aggccctaca 2280 ctgcaggctc ggagaaggca gagtgtgctg aacctgatga cccacagcgt gaaccaggga 2340 agaatatcc agaaagac caccgccagc aacgggaaag tgtcactggc ccctcaggcc 2400 aacctgactg agctggacat ctacagcaga cggctgagcc aagagacagg cctggaaatc 2460 2520 cccgccgtga caacctggaa tacctacctg cggtacatca ccgtgcacaa gtccctgatc 2580 ttcgtgctga tctggtgtct cgtgatcttc ctggccgaag tggccgcttc tctggtggtt 2640 ctgtggctgc tcggaaacac cccactgcag gacaagggca atagcaccca cagccggaac 2700 aacagctacg ccgtgatcat cacctccacc agctcctact acgtgttcta catctacgtg 2760 ggcgtcgccg acactctgct cgccatgggc ttttttagag gactgcccct ggtgcacacc 2820 ctgatcaccg tgtctaagat cctgcaccat aagatgctgc acagcgtcct gcaggcccct 2880 atgagcacac tgaacaccct gaagccggc ggaatcctga acagattcag caaggacatt 2940 gccatcctgg acgacctgct gcctctgacc atcttgact tcatccagct gctgctgatc 3000 gtgatcggcg ccattgctgt ggtggctgtg ctgcagcctt acatcttcgt ggccaccgtg 3060 cctgtgatcg tggccttcat tatgctgcgg gcctactttc tgcagacctc tcagcagctg 3120 aagcagctcg agtctgaggg cagaagcccc atctttaccc acctcgtgac cagcctgaaa 3180 ggcctgtgga ccctgagagc ctttggcaga cagccctact tcgagacact gttccacaag 3240 gccctgaacc tgcacaccgc caactggttt ctgtatctga gcaccctgcg gtggttccag 3300 atgaggatcg agatgatttt cgtcatcttc tttatcgccg tgaccttcat cagcatcctc 3360 accactggcg aaggcgaggg cagagtggga atcattctga ccctggccat gaacatcatg 3420 tccacactcc agtgggccgt gaacagcagc atcgatgtgg acagcctgat gcggagcgtg 3480 tcccgggtgt tcaagttcat cgacatgccc acagagggca agcccaccaa gagcaccaag 3540 ccttacaaga acggccagct gagcaaagtc atgatcatcg agaactccca cgtcaagaag 3600 gacgacattt ggcccagcgg aggccagatg accgtgaagg atctgaccgc caagtacacc 3660 gaaggcgaa acgccattct ggaaaacatc agctttagca tcagccctgg ccagcgcgtg 3720 ggactccttg gaagaaccgg aagcggcaag tctactctgc tgagcgcctt cctgagactg 3780 ctgaataccg agggcgagat ccagatcgat ggggtgtcct gggacagcat caccctgcaa 3840 caatggcgga aggcctttgg cgtgatccct cagaaggtgt tcattttcag cggcacgttc 3900 cggagaatc tggaccccta cgagcagtgg agcgaccaag agatttggaa ggtggccgat 3960 gaagtgggac tgagaagcgt gatcgagcag tttcccggca agctggattt cgtgctgggtg 4020 gatggcggct gtgtgctgtc tcacggacac aagcagctga tgtgcctggc cagatccgtg 4080 ctgtccaagg ccaagattct gctgctcgac gagcctagcg ctcacctcga tcctgtgacc 4140 taccagatca tccggcggac actgaagcag gcctttgccg attgcaccgt gatcctgtgc 4200 gagcacagaa ttgaggccat gctggaatgc cagcagtttc tggttatcga agaacaaa 4260 gtgcggcagt acgacagcat ccagaagctg ctgaacgagc ggagcctgtt cagacaggcc 4320 atctctccca gcgacagagt gaagctgttc cctcaccgga acagctccaa gtgcaagagc 4380 aagcctcaga tcgccgctct gaaagagaa accgaggaag aggtgcagga cacacggctg 4440 that 4443 <210> 3 <211> 4260 <212> DNA <213> Homo sapiens <400> 3 atgcagaggt cgcctctgga aaaggccagc gttgtctcca aacttttttt cagctgggacc 60 agaccaattt tgaggaaagg atacagacag cgcctggaat tgtcagacat ataccaaatc 120 180 ctggcttcaa agaaaaatcc taaactcatt aatgcccttc ggcgatgttt tttctggaga 240 tttatgttct atggaatctt tttattta ggggaagtca ccaaagcagt acagcctctc 300 ttactgggaa gaatcatagc ttcctatgac ccggataaca aggaggaacg ctctatcgcg 360 attatctag gcataggctt atgccttctc tttattgtga ggacactgct cctacaccca 420 gccattttg gccttcatca cattggaatg cagatgagaa tagctatgtt tagttgatt 480 tataagaaga ctttaaagct gtcaagccgt gttctagata aaataagtat tggacaactt 540 gttagtctcc tttccaacaa cctgaacaaa tttgatgaag gacttgcatt ggcacatttc 600 gtgtggatcg ctcctttgca agtggcactc ctcatggggc taatctggga gttgttacag 660 gcgtctgcct tctgtggact tggtttcctg atagtccttg ccctttttca ggctgggcta 720 gggagaatga tgatgaagta cagagatcag agagctggga agatcagtga aagacttgtg 780 attacctcag aaatgattga aaattccaa tctgttaagg catactgctg ggaagaagca 840 atggaaaaaa tgattgaaaa cttaagacaa acagaactga aactgactcg gaaggcagcc 900 tatgtgagat acttcaatag ctcagccttc ttcttctcag ggttctttgt ggtgttttta 960 tctgtgcttc cctatgcact aatcaaagga atcatcctcc ggaaaatatt caccaccatc 1020 tcattctgca ttgttctgcg catggcggtc actcggcaat ttccctgggc tgtacaaaca 1080 tggtatgact ctcttggagc aataaacaaa atacaggatt tcttacaaaa gcaagaatat 1140 aagacattgg aatataactt aacgactaca gaagtagtga tggagaatgt aacagccttc 1200 tgggaggaga cttcacttct aatggtgatt atgggagaac tggagccttc agagggtaaa 1260 attaagcaca gtggaagaat ttcattctgt tctcagtttt cctggattat gcctggcacc 1320 attaaagaaa atatcatctt tggtgtttcc tatgatgaat atagatacag aagcgtcatc 1380 aaagcatgcc aactagaaga ggacatctcc aagtttgcag agaaagacaa tatagttctt 1440 ggagaaggtg gaatcacact gagtggaggt caacgagcaa gaatttcttt agcaagagca 1500 gtatacaaag atgctgattt gtatttatta gactctcctt ttggatacct agatgtttta 1560 acagaaaaag aaatatttga aagctgtgtc tgtaaactga tggctaacaa aactaggatt 1620 1680 ggtagcagct atttttatgg gacattttca gaactccaaa atctacagcc agactttagc 1740 tcaaaactca tgggatgtga ttctttcgac caatttagtg cagaagaag aaattcaatc 1800 ctaactgaga ccttacaccg tttctcatta gaaggagatg ctcctgtctc ctggagagaa 1860 acaaaaaaaac aatcttttaa agagactgga gagtttgggg aaaaaaggaa gaattctatt 1920 ctcaatccaa tcaactctat acgaaaattt tccattgtgc aaaagactcc cttaacaaatg 1980 aatggcatcg aagaggattc tgatgagcct ttagagagaa ggctgtcctt agtaccagat 2040 tctgagcagg gagaggcgat actgcctcgc atcagcgtga tcagcactgg ccccacgctt 2100 caggcacgaa ggaggcagtc tgtcctgaac ctgatgacac actcagttaa ccaaggtcag 2160 aacattcacc gaagacaac agcatccaca cgaaaagtgt cactggcccc tcaggcaaac 2220 2280 gaaatta acgaaagaa cttaaaggag tgctttttg atgatatgga gagcatacca 2340 gcagtgacta catggaacac ataccttcga tatattactg tccacaagag cttaattttt 2400 gtgctaattt ggtgcttagt aatttttctg gcagaggtgg ctgcttcttt ggttgtgctg 2460 tggctccttg gaaacactcc tcttcaagac aaagggaata gtactcatag tagaaataac 2520 agctatgcag tgattatcac cagcaccagt tcgtattatg tgttttacat ttacgtggga 2580 gtagccgaca ctttgcttgc tatgggattc ttcagaggtc taccactggt gcataccta 2640 atcacagtgt cgaaaatttt acaccacaaa atgttacatt ctgttcttca agcacctatg 2700 tcaaccctca acacgttgaa agcaggtggg attcttaata gattctccaa agatatagca 2760 atttggatg accttctgcc tcttaccata tttgacttca tccagttgtt attaattgtg 2820 attggagcta tagcagttgt cgcagtttta caaccctaca tctttgttgc aacagtgcca 2880 gtgatagtgg cttttattat gttgagagca tatttcctcc aaacctcaca gcaactcaaa 2940 caactggaat ctgaaggcag gagtccaatt ttcactcatc ttgttacaag cttaaaagga 3000 ctatggacac ttcgtgcctt cggacggcag ccttactttg aaactctgtt ccacaaagct 3060 ctgaatttac atactgccaa ctggttcttg tacctgtcaa cactgcgctg gttccaaatg 3120 agaataaa tgatttttgt catcttcttc attgctgtta ccttcatttc cattttaaca 3180 aggagaag gagagaag agttggtatt atcctgactt tagccatgaa tatcatgagt 3240 acattgcagt gggctgtaaa ctccagcata gatgtggata gcttgatgcg atctgtgagc 3300 cgagtcttta agttcattga catgccaaca gaaggtaac ctaccaagtc aaccaaacca 3360 tacaagaatg gccaactctc gaaagttatg attattgaga attcacacgt gaaaagat 3420 gacatctggc cctcaggggg ccaaatgact gtcaaagatc tcacagcaaa atacacagaa 3480 ggtggaaatg ccatattaga gaacatttcc ttctcaataa gtcctggcca gagggtgggc 3540 ctcttgggaa gaactggatc agggaagagt actttgttat cagctttttt gagactactg 3600 3660 tgggaaag cctttggagt gataccacag aaagtttta ttttttctgg aacatttaga 3720 3780 gttgggctca gatctgtgat agaacagttt cctgggaagc ttgactttgt ccttgtggat 3840 ggggggctgtg tcctaagcca tggccacaag cagttgatgt gcttggctag atctgttctc 3900 agtaaggcga agatcttgct gcttgatgaa cccagtgctc atttggatcc agtaacatac 3960 caataaatta gaagaactct aaaacaagca tttgctgatt gcacagtaat tctctgtgaa 4020 cacaggatag aagcaatgct ggaatgccaa caatttttgg tcatagaaga gaacaaagtg 4080 cggcagtacg attccatcca gaaactgctg aacgagga gcctcttccg gcaagccatc 4140 4200 cccagaattg ctgctctgaa agaggagaca gaagagagg tgcaagatac aaggctttag 4260 <210> 4 <211> 4260 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 atgcagagaa gccctctgga aaaggccagc gtggtgtcca agctgttctt cagctgggacc 60 cggcctatcc tgcggaaggg ctagacag agatggaac tgagcgacat ctatcagatc 120 180 ctggcctcca agaagaaccc caagctgatc aacgccctgc ggcggtgctt cttctggcgg 240 tttatgttct acggcatctt cctgtacctg ggcgaagtga ccaaagccgt gcagcctctg 300 ctgctgggca gaatcattgc cagctacgac cccgacaaca aaggaacg gtctatcgcc 360 atctacctcg gcatcggcct gtgcctgctg tttatcgtca gaaccctgct gctgcacccc 420 gccatctttg gactgcacca catcggcatg cagatgcgga tcgccatgtt cagcctgatc 480 tacaagaaaa ccctgaagct gtccagcaga gtgctggaca agatcagcat cggacagctg 540 gtgtccctgc tgagcaacaa cctgaacaag ttcgacgaag gcctggctct ggcccacttc 600 gtgtggattg ctcctctgca agtggccctg ctgatgggcc tgatttggga actgctgcag 660 gccagcgcct tttgcggact gggatttctg attgtgctgg ccctgttcca ggccggactg 720 gggagaatga tgatgaagta ccgggaccag agagccggca agatctccga gagactggtc 780 atcaccagcg agatgatcga gaacatccag agcgtgaagg cctactgctg ggaagaggcc 840 atggaaaaga tgattgagaa tctgcggcag accgagctga agctgacaag aaaggccgcc 900 tacgtgcgct acttcaacag cagcgccttc ttcttctccg gcttcttcgt ggtgttcctg 960 agcgtgctgc cctacgctct gatcaagggc atcatcctga gaaagatttt caccaccatt 1020 tctttctgca tcgtgctgcg gatggccgtg accagacagt ttccttgggc tgtgcagact 1080 tggtacgata gcctgggcgc catcaacaag atccaggact tcctgcagaa gcaagagtac 1140 aagaccctcg agtacaacct gaccaccacc gaggtggtca tggaaaacgt gaccgccttc 1200 tgggaagaaa ccagcctgct catggtcatc atgggcgagc tggaacctag cgagggcaag 1260 atcaagcaca gcggcaggat cagcttctgt agccagttct cctggatcat gcccggcacc 1320 atcaaagaga acatcatctt cggcgtgtcc tacgacgagt acagataccg cagcgtgatc 1380 aaggcctgcc agctggaaga ggacatcagc aagttcgccg agaaggacaa catcgtgctc 1440 ggcgaaggcg gcatcacact gtctggcgga cagagggcca gaatctctct ggctagagcc 1500 gtgtacaagg acgccgatct gtacctgctg gatagcccct tcggctacct ggatgtgctg 1560 accgagaaag agatcttga gagctgcgtg tgcaagctga tggccaacaa gaccagaatc 1620 ctggtcacct ccaagatgga acacctgaag aaggccgaca agatcctgat tctgcaggag 1680 ggcagcagct acttttacgg caccttcagc gagctccaga acctgcagcc tgacttcagc 1740 agcaactga tgggctgga ctccttgca cagttcagcg ccgagcggag aaacagcatc 1800 ctgacagaga cactgcaccg gttctccctg gaaggcgacg ctcctgtgtc ttggaccgag 1860 1920 ctgaacccca tcaactccat ccggaagttc agcatcgtcc agaaaacccc tctgcagatg 1980 aacggcatcg aaggatag cgacgagccc ctggaaagac ggctgtctct ggtgcctgat 2040 agcgaacagg gcgaagccat cctgcctcgg atctccgtga ttagcacagg ccctacactg 2100 caggctcgga gaaggcagtc tgtgctgaac ctgatgaccc acagcgtgaa ccagggacag 2160 aatatccaca gaagaccac cgccagcaca cggaaagtgt cactggcccc tcaggccaac 2220 ctgactgagc tggacatcta cagcagacgg ctgagccaag agacaggcct ggaaatcagc 2280 gaggaaatca acgaagagga cctgaaagag tgctttttcg acgacatgga atctatcccc 2340 gccgtgacaa cctggaatac ctacctgcgg tacatcaccg tgcacaagtc cctgatcttc 2400 gtgctgatct ggtgtctcgt gatcttcctg gccgaagtgg ccgcttctct ggtggttctg 2460 tggctgctgg ggaatacccc actgcaggac aagggcaata gcacccacag ccggaacaac 2520 agctacgccg tgatcatcac ctccaccagc tcctactacg tgttctacat ctacgtgggc 2580 gtcgccgaca ctctgctcgc catgggcttt tttagaggac tgcccctggt gcacaccctg 2640 atcaccgtgt ctaagatcct gcaccataag atgctgcaca gcgtcctgca ggcccctatg 2700 agcacactga acaccctgaa agccggcgga atcctgaaca gattcagcaa ggacattgcc 2760 atcctggacg acctgctgcc tctgaccatc ttcgacttca tccagctgct gctgatcgtg 2820 atcggcgcca ttgctgtggt ggctgtgctg cagccttaca tcttcgtggc caccgtgcct 2880 gtgatcgtgg ccttcattat gctgcgggcc tactttctgc agaccagcca gcagctgaag 2940 cagctcgagt ctgagggcag aagccccatc tttacccacc tcgtgaccag cctgaaaggc 3000 ctgtggaccc tgagagcctt tggcagacag ccctacttcg agacactgtt ccacaaggcc 3060 ctgaacctgc acaccgccaa ctggtttctg tatctgagca ccctgcggtg gttccagatg 3120 aggatcgaga tgattttcgt catcttcttt atcgccgtga ccttcatcag catcctcacc 3180 actggcgaag gcgagggcag agtgggaatc attctgaccc tggccatgaa catcatgtcc 3240 acactccagt gggccgtgaa cagcagcatc gatgtggaca gcctgatgcg gagcgtgtcc 3300 cgggtgttca agttcatcga catgcccaca gagggcaagc ccaccaagag caccaagcct 3360 tacaagaacg gccagctgag caaagtcatg atcatcgaga actcccacgt caagaaggac 3420 gacatttggc ccagcggagg ccagatgacc gtgaaggatc tgaccgccaa gtacaccgaa 3480 ggcggaaacg ccattctgga aaacatcagc tttagcatca gccctggcca gcgcgtggga 3540 ctgcttggaa gaacaggatc tggcaagtct actctgctga gcgccttcct gagactgctg 3600 aataccgagg gcgagatcca gatcgatggg gtgtcctggg acagcatcac cctgcaacaa 3660 tggcggaagg cctttggcgt gatccctcag aaggtgttca ttttcagcgg cacgttccgg 3720 aagaatctgg accccctacga gcagtggagc gaccaagaga tttggaaggt ggccgatgaa 3780 gtgggactga gaagcgtgat cgagcagttt cccggcaagc tggatttcgt gctggtggat 3840 ggcggctgtg tgctgtctca cggacacaag cagctgatgt gcctggccag aagcgtgctg 3900 tctaaggcca agatcctcct gctggacgag ccctctgctc acctcgatcc tgtgacctac 3960 cagatcatcc ggcggacact gaagcaggcc tttgccgatt gcaccgtgat cctgtgcgag 4020 cacagaatcg aggccatgct ggaatgccag cagtttctgg ttatcgaaga gaacaaagtg 4080 cggcagtacg acagcattca gaagctgctg aacgagcgga gcctgttcag acaggccatc 4140 tctcccagcg acagagtgaa gctgttccct caccggaaca gctccaagtg caagagcaag 4200 cctcagatcg ccgctctgaa agaagaaacc gaggaagagg tgcaggacac acggctgtaa 4260 <210> 5 <211> 1480 <212> PRT <213> Homo sapiens <400> 5 Met Gln Arg Ser Pro Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe 1 5 10 15 Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gly Tyr Arg Gln Arg Leu 20 25 30 Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Val Asp Ser Ala Asp Asn 35 40 45 Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu Leu Ala Ser Lys 50 55 60 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg 65 70 75 80 Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gly Glu Val Thr Lys Ala 85 90 95 Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp 100 105 110 Asn Lys Glu Glu Arg Ser Ile Ala Ile Tyr Leu Gly Ile Gly Leu Cys 115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly 130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp 180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val 195 200 205 Ala Leu Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe 210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val 260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu 275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr 290 295 300 Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg 340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile 355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu 370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Glu Gly Phe Gly Glu Leu Phe Glu Lys Ala Lys Gln Asn Asn 405 410 415 Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Ser Leu Phe Phe Ser Asn 420 425 430 Phe Ser Leu Leu Gly Thr Pro Val Leu Lys Asp Ile Asn Phe Lys Ile 435 440 445 Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Ser Thr Gly Ala Gly Lys 450 455 460 Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro Ser Glu Gly 465 470 475 480 Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln Phe Ser Trp 485 490 495 Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Phe Gly Val Ser Tyr 500 505 510 Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln Leu Glu Glu 515 520 525 Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu Gly Glu Gly 530 535 540 Gly Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser Leu Ala Arg 545 550 555 560 Ala Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser Pro Phe Gly 565 570 575 Tyr Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser Cys Val Cys 580 585 590 Lys Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser Lys Met Glu 595 600 605 His Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu Gly Ser Ser 610 615 620 Tyr Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln Pro Asp Phe 625 630 635 640 Ser Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe Ser Ala Glu 645 650 655 Arg Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe Ser Leu Glu 660 665 670 Gly Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln Ser Phe Lys 675 680 685 Gln Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro 690 695 700 Ile Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr Pro Leu Gln 705 710 715 720 Met Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu Arg Arg Leu 725 730 735 Ser Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu Pro Arg Ile 740 745 750 Ser Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg Arg Gln Ser 755 760 765 Val Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln Asn Ile His 770 775 780 Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gln Ala 785 790 795 800 Asn Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gln Glu Thr 805 810 815 Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys 820 825 830 Phe Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr Trp Asn Thr 835 840 845 Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile 850 855 860 Trp Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser Leu Val Val 865 870 875 880 Leu Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly Asn Ser Thr 885 890 895 His Ser Arg Asn Asn Ser Tyr Ala Val Ile Ile Thr Ser Thr Ser Ser 900 905 910 Tyr Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr Leu Leu Ala 915 920 925 Met Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val 930 935 940 Ser Lys Ile Leu His His Lys Met Leu His Ser Val Leu Gln Ala Pro 945 950 955 960 Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe 965 970 975 Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe 980 985 990 Asp Phe Ile Gln Leu Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val 995 1000 1005 Ala Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile Val 1010 1015 1020 Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser Gln Gln Leu 1025 1030 1035 1040 Lys Gln Leu Glu Ser Glu Gly Arg Ser Pro Ile Phe Thr His Leu Val 1045 1050 1055 Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe Gly Arg Gln Pro 1060 1065 1070 Tyr Phe Glu Thr Leu Phe His Lys Ala Leu Asn Leu His Thr Ala Asn 1075 1080 1085 Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe Gln Met Arg Ile Glu 1090 1095 1100 Met Ile Phe Val Ile Phe Phe Ile Ala Val Thr Phe Ile Ser Ile Leu 1105 1110 1115 1120 Thr Thr Gly Glu Gly Glu Gly Arg Val Gly Ile Ile Leu Thr Leu Ala 1125 1130 1135 Met Asn Ile Met Ser Thr Leu Gln Trp Ala Val Asn Ser Ser Ile Asp 1140 1145 1150 Val Asp Ser Leu Met Arg Ser Val Ser Arg Val Phe Lys Phe Ile Asp 1155 1160 1165 Met Pro Thr Glu Gly Lys Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn 1170 1175 1180 Gly Gln Leu Ser Lys Val Met Ile Ile Glu Asn Ser His Val Lys Lys 1185 1190 1195 1200 Asp Asp Ile Trp Pro Ser Gly Gly Gln Met Thr Val Lys Asp Leu Thr 1205 1210 1215 Ala Lys Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe 1220 1225 1230 Ser Ile Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg Thr Gly Ser 1235 1240 1245 Gly Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr Glu 1250 1255 1260 Gly Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile Thr Leu Gln 1265 1270 1275 1280 Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val Phe Ile Phe 1285 1290 1295 Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu Gln Trp Ser Asp 1300 1305 1310 Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gly Leu Arg Ser Val Ile 1315 1320 1325 Glu Gln Phe Pro Gly Lys Leu Asp Phe Val Leu Val Asp Gly Gly Cys 1330 1335 1340 Val Leu Ser His Gly His Lys Gln Leu Met Cys Leu Ala Arg Ser Val 1345 1350 1355 1360 Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Glu Pro Ser Ala His Leu 1365 1370 1375 Asp Pro Val Thr Tyr Gln Ile Ile Arg Arg Thr Leu Lys Gln Ala Phe 1380 1385 1390 Ala Asp Cys Thr Val Ile Leu Cys Glu His Arg Ile Glu Ala Met Leu 1395 1400 1405 Glu Cys Gln Gln Phe Leu Val Ile Glu Glu Asn Lys Val Arg Gln Tyr 1410 1415 1420 Asp Ser Ile Gln Lys Leu Leu Asn Glu Arg Ser Leu Phe Arg Gln Ala 1425 1430 1435 1440 Ile Ser Pro Ser Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser 1445 1450 1455 Lys Cys Lys Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu Thr Glu 1460 1465 1470 Glu Glu Val Gln Asp Thr Arg Leu 1475 1480 <210> 6 <211> 1419 <212> PRT <213> Homo sapiens <400> 6 Met Gln Arg Ser Pro Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe 1 5 10 15 Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gly Tyr Arg Gln Arg Leu 20 25 30 Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Val Asp Ser Ala Asp Asn 35 40 45 Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu Leu Ala Ser Lys 50 55 60 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg 65 70 75 80 Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gly Glu Val Thr Lys Ala 85 90 95 Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp 100 105 110 Asn Lys Glu Glu Arg Ser Ile Ala Ile Tyr Leu Gly Ile Gly Leu Cys 115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly 130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp 180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val 195 200 205 Ala Leu Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe 210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val 260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu 275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr 290 295 300 Phe Asn Ser Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg 340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile 355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu 370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Glu Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro 405 410 415 Ser Glu Gly Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln 420 425 430 Phe Ser Trp Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Phe Gly 435 440 445 Val Ser Tyr Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln 450 455 460 Leu Glu Glu Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu 465 470 475 480 Gly Glu Gly Gly Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser 485 490 495 Leu Ala Arg Ala Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser 500 505 510 Pro Phe Gly Tyr Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser 515 520 525 Cys Val Cys Lys Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser 530 535 540 Lys Met Glu His Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu 545 550 555 560 Gly Ser Ser Tyr Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln 565 570 575 Pro Asp Phe Ser Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe 580 585 590 Ser Ala Glu Arg Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe 595 600 605 Ser Leu Glu Gly Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln 610 615 620 Ser Phe Lys Gln Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile 625 630 635 640 Leu Asn Pro Ile Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr 645 650 655 Pro Leu Gln Met Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu 660 665 670 Arg Arg Leu Ser Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu 675 680 685 Pro Arg Ile Ser Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg 690 695 700 Arg Gln Ser Val Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln 705 710 715 720 Asn Ile His Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala 725 730 735 Pro Gln Ala Asn Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser 740 745 750 Gln Glu Thr Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu 755 760 765 Lys Glu Cys Phe Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr 770 775 780 Trp Asn Thr Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe 785 790 795 800 Val Leu Ile Trp Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser 805 810 815 Leu Val Val Leu Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly 820 825 830 Asn Ser Thr His Ser Arg Asn Asn Ser Tyr Ala Val Ile Ile Thr Ser 835 840 845 Thr Ser Ser Tyr Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr 850 855 860 Leu Leu Ala Met Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu 865 870 875 880 Ile Thr Val Ser Lys Ile Leu His His Lys Met Leu His Ser Val Leu 885 890 895 Gln Ala Pro Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu 900 905 910 Asn Arg Phe Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu 915 920 925 Thr Ile Phe Asp Phe Ile Gln Leu Leu Leu Ile Val Ile Gly Ala Ile 930 935 940 Ala Val Val Ala Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro 945 950 955 960 Val Ile Val Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser 965 970 975 Gln Gln Leu Lys Gln Leu Glu Ser Glu Gly Arg Ser Pro Ile Phe Thr 980 985 990 His Leu Val Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe Gly 995 1000 1005 Arg Gln Pro Tyr Phe Glu Thr Leu Phe His Lys Ala Leu Asn Leu His 1010 1015 1020 Thr Ala Asn Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe Gln Met 1025 1030 1035 1040 Arg Ile Glu Met Ile Phe Val Ile Phe Phe Ile Ala Val Thr Phe Ile 1045 1050 1055 Ser Ile Leu Thr Thr Gly Glu Gly Glu Gly Arg Val Gly Ile Ile Leu 1060 1065 1070 Thr Leu Ala Met Asn Ile Met Ser Thr Leu Gln Trp Ala Val Asn Ser 1075 1080 1085 Ser Ile Asp Val Asp Ser Leu Met Arg Ser Val Ser Arg Val Phe Lys 1090 1095 1100 Phe Ile Asp Met Pro Thr Glu Gly Lys Pro Thr Lys Ser Thr Lys Pro 1105 1110 1115 1120 Tyr Lys Asn Gly Gln Leu Ser Lys Val Met Ile Ile Glu Asn Ser His 1125 1130 1135 Val Lys Lys Asp Asp Ile Trp Pro Ser Gly Gly Gln Met Thr Val Lys 1140 1145 1150 Asp Leu Thr Ala Lys Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn 1155 1160 1165 Ile Ser Phe Ser Ile Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg 1170 1175 1180 Thr Gly Ser Gly Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu 1185 1190 1195 1200 Asn Thr Glu Gly Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile 1205 1210 1215 Thr Leu Gln Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val 1220 1225 1230 Phe Ile Phe Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu Gln 1235 1240 1245 Trp Ser Asp Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gly Leu Arg 1250 1255 1260 Ser Val Ile Glu Gln Phe Pro Gly Lys Leu Asp Phe Val Leu Val Asp 1265 1270 1275 1280 Gly Gly Cys Val Leu Ser His Gly His Lys Gln Leu Met Cys Leu Ala 1285 1290 1295 Arg Ser Val Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Glu Pro Ser 1300 1305 1310 Ala His Leu Asp Pro Val Thr Tyr Gln Ile Ile Arg Arg Thr Leu Lys 1315 1320 1325 Gln Ala Phe Ala Asp Cys Thr Val Ile Leu Cys Glu His Arg Ile Glu 1330 1335 1340 Ala Met Leu Glu Cys Gln Gln Phe Leu Val Ile Glu Glu Asn Lys Val 1345 1350 1355 1360 Arg Gln Tyr Asp Ser Ile Gln Lys Leu Leu Asn Glu Arg Ser Leu Phe 1365 1370 1375 Arg Gln Ala Ile Ser Pro Ser Asp Arg Val Lys Leu Phe Pro His Arg 1380 1385 1390 Asn Ser Ser Lys Cys Lys Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu 1395 1400 1405 Glu Thr Glu Glu Glu Val Gln Asp Thr Arg Leu 1410 1415

Claims

1. (a) a recombinant herpes simplex virus type 1 (HSV-1) genome, the recombinant HSV-1 genome comprising one or more polynucleotides encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide; and (b) Pharmaceutically acceptable excipients Including, Formulated with a nebulizer Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the recombinant HSV-1 genome comprises the one or more polynucleotides encoding the CFTR polypeptide within one or more viral loci.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the CFTR polypeptide comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

6.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the recombinant HSV-1 genome comprises an inactivating mutation in an HSV-1 gene 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.

5. 5. The pharmaceutical composition of claim 4, wherein the recombinant HSV-1 genome contains an inactivating mutation in one or both copies of the ICP4 gene.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more polynucleotides encoding the CFTR polypeptide are operably linked to a promoter and expressed from a recombinant HSV-1 genome.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the HSV-1 is replication-deficient.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the HSV-1 is attenuated.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the HSV-1 is aerosolized via nebulization.

10. 10. The pharmaceutical composition of claim 9, wherein the aerosolized HSV-1 comprises droplets having an aerodynamic mass median particle diameter of less than about 5 microns in diameter.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the nebulizer is a jet nebulizer, an ultrasonic nebulizer, or a vibrating mesh nebulizer.

12. The pharmaceutical composition of claim 11 , which is aerosolized via the nebulizer.

13. 13. The pharmaceutical composition of claim 12, wherein the aerosolized pharmaceutical composition comprises droplets having an aerodynamic mass median particle diameter of less than about 5 microns in diameter.

14. 14. Use of a pharmaceutical composition according to any one of claims 1 to 13 in the manufacture of a medicament for enhancing, increasing, enhancing and / or replenishing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) polypeptide levels.

15. The pharmaceutical composition of any one of claims 1 to 13 for enhancing, increasing, enhancing and / or replenishing CFTR polypeptide levels.

16. Use of a pharmaceutical composition according to any one of claims 1 to 13 in the manufacture of a medicament for reducing or inhibiting progressive lung damage.

17. A pharmaceutical composition according to any one of claims 1 to 13 for reducing or inhibiting progressive lung damage.

18. 18. The pharmaceutical composition of claim 17, wherein the progressive lung injury is cystic fibrosis or chronic obstructive pulmonary disease (COPD).

19. 14. Use of a pharmaceutical composition according to any one of claims 1 to 13 in the manufacture of a medicament for providing prophylactic, palliative or therapeutic alleviation of one or more signs or symptoms of cystic fibrosis.

20. A pharmaceutical composition according to any one of claims 1 to 13 for providing prophylactic, palliative or therapeutic alleviation of one or more signs or symptoms of cystic fibrosis.

21. 21. The pharmaceutical composition of claim 20, wherein the one or more signs or symptoms of cystic fibrosis are selected from the group consisting of persistent coughing that produces a lot of mucus, thick sticky mucus accumulating in the airways, wheezing, difficulty breathing, sinusitis, recurrent lung infections, inflamed nasal passages, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof.

22. 14. Use of a pharmaceutical composition according to any one of claims 1 to 13 in the manufacture of a medicament for providing prophylactic, palliative or therapeutic relief of one or more signs or symptoms of COPD.

23. A pharmaceutical composition according to any one of claims 1 to 13 for providing preventive, palliative or therapeutic relief of one or more signs or symptoms of COPD.

24. 24. The pharmaceutical composition of claim 23, wherein the one or more signs or symptoms of COPD are selected from the group consisting of shortness of breath, wheezing, chest tightness, excess mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof.

25. 25. The pharmaceutical composition of any one of claims 15, 17, 18, 20, 21, 23, and 24, wherein the nebulizer is a jet nebulizer, an ultrasonic nebulizer, or a vibrating mesh nebulizer.

26. (a) HSV-1 comprising a recombinant HSV-1 genome, the recombinant HSV-1 genome comprising one or more polynucleotides encoding a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide; and (b) a pharma- ceutically acceptable excipient A pharmaceutical composition comprising: Nebulizer Including the kit.

27. 27. The kit of claim 26, wherein the nebulizer is a jet nebulizer, an ultrasonic nebulizer, or a vibrating mesh nebulizer.

28. 28. The kit of claim 26 or 27, further comprising instructions for administering the pharmaceutical composition using a nebulizer to the lungs of a subject suffering from cystic fibrosis or chronic obstructive pulmonary disease (COPD).

29. A nebulizer comprising the pharmaceutical composition of any one of claims 1 to 13, 15, 17, 18, 20, 21, and 23 to 25.

30. 30. The nebulizer of claim 29, wherein the pharmaceutical composition is a liquid formulation.

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