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 of U.S. Provisional Application No. 62 / 802,871, filed on February 8, 2019, the entire content of which is incorporated herein by reference.

[0002] Submission of Sequence Listing in ASCII Text File The content of the following submission in ASCII text file is incorporated herein by reference in its entirety: Computer - Readable Format (CRF) of the Sequence Listing (file name: 7613420001140SEQLIST.txt, recording date: January 17, 2020, size: 44KB).

[0003] Field of the Invention The present disclosure relates, in part, to recombinant nucleic acids comprising one or more polynucleotides encoding cystic fibrosis transmembrane conductance regulator (CFTR) polypeptides, viruses comprising the same, their compositions and formulations, and methods of using them (e.g., for prophylactic, palliative, or therapeutic alleviation of one or more signs or symptoms of chronic lung diseases such as cystic fibrosis).

Background Art

[0004] Background Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP - activated chloride and bicarbonate channel that is important for lung homeostasis. Decreased or lost CFTR channel function often results in a chronic inflammatory response that promotes mucus stagnation, chronic bacterial infections, and associated progressive lung injury. Decreased 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 result in the tragic consequences associated with cystic fibrosis (CF). More than 2,000 unique mutations in the CFTR gene have been described.

[0005] Cystic fibrosis (CF) is a genetic disorder characterized by the accumulation of large amounts of sticky mucus, which can damage many organs in the body, but the most severe pathological outcomes are lung-related. CF patients present with dehydrated mucus in the lungs, leading to airway obstruction, chronic bacterial infections (and associated inflammatory responses), bronchiectasis, and ultimately respiratory failure. Currently, more than 70,000 people worldwide live with cystic fibrosis. Historically, children born with CF died as infants, and until 1980, the median survival time was less than 20 years. While medical advances over the past 30 years have significantly improved both the quality of life and life expectancy for CF patients (40.6 years in the US as of 2013), there is a clear need for novel therapeutic options targeting the molecular correction of CFTR deficiency observed in CF patients, as well as in patients with 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 acceptance numbers, are incorporated herein by reference in whole, as if each individual reference were explicitly and individually indicated to be incorporated by reference. [Overview of the project]

[0007] Brief Overview To satisfy these and other needs, 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, agents and / or methods useful for treating CFTR deficiency in subjects requiring treatment of CFTR deficiency, and / or for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of chronic lung diseases such as cystic fibrosis.

[0008] The inventors have shown that the recombinant viruses described herein were able to effectively transduce airway epithelial cells derived from CF patients and successfully express the encoded exogenous human CFTR (see, for example, Example 2). In addition, the inventors have shown that the recombinant viruses described herein expressed full-length functional human CFTR that was appropriately transported to the plasma membrane (see, for example, Example 2). Furthermore, the inventors have shown that the recombinant viruses described herein rescued disease phenotypes in clinically relevant 3D organoid cultures prepared from biopsies taken from multiple CF patients carrying various underlying CFTR mutations (see, for example, Example 3). Furthermore, the inventors have shown that recombinant HSV vectors can be administered to the lungs of immunocompetent animals via multiple routes, and that the non-invasive inhalation route induced lower cell entry in the lungs while still expressing a similar level of encoded transgene in the lungs (see, for example, Example 4). Without being bound by theory, it is conceivable that administering one or more of the recombinant nucleic acids, viruses, drugs, and / or compositions described herein to increase, enhance, and / or supplement CFTR polypeptide levels in one or more cells of a subject requiring such enhancement and / or supplementation would 1) reduce or prevent mucus buildup in one or more organs of an individual (e.g., lungs), 2) reduce or prevent airway obstruction in an individual, 3) reduce or prevent chronic bacterial infections and / or associated chronic inflammation in the lungs of an individual, 4) reduce or prevent bronchiectasis in an individual, 5) reduce, inhibit, or treat progressive lung injury in an individual, and / or 6) provide prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of chronic lung disease (e.g., cystic fibrosis, COPD, etc.).

[0009] Accordingly, certain aspects of this disclosure relate to recombinant herpesvirus genomes comprising one or more polynucleotides encoding cystic fibrosis membrane conductance regulator (CFTR) polypeptides. In some embodiments, the recombinant herpesvirus genome is replicable. In some embodiments, the recombinant herpesvirus genome is replication-deficient. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpesvirus genome comprises one or more polynucleotides encoding CFTR polypeptides within one or more viral loci. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpesvirus genome is selected from recombinant herpes simplex virus genomes, recombinant varicella-zoster virus genomes, recombinant human cytomegalovirus genomes, recombinant herpesvirus 6A genomes, recombinant herpesvirus 6B genomes, recombinant herpesvirus 7 genomes, recombinant Kaposi's sarcoma-associated herpesvirus genomes, and any combination or derivatives thereof.

[0010] In some embodiments that can be combined with any of the embodiments described above, the CFTR polypeptide is a human CFTR polypeptide. In some embodiments that can be combined with any of the embodiments described above, the CFTR polypeptide includes 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 can be combined with any of the embodiments described above, the CFTR polypeptide includes 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 includes the amino acid sequence of SEQ ID NO: 5.

[0011] In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpesvirus genome is a recombinant herpes simplex virus genome. In some embodiments, the recombinant herpes simplex virus genome is a recombinant herpes simplex virus type 1 (HSV-1) genome, a recombinant herpes simplex virus type 2 (HSV-2) genome, or any derivative thereof. In some embodiments, the recombinant herpes simplex virus genome is a recombinant HSV-1 genome.

[0012] In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation. In some embodiments, the inactivating mutation is present in the herpes simplex virus gene. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the herpes simplex virus gene. In some embodiments, the herpes simplex virus gene is selected from infecting 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, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP22 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL41 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP27 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the ICP47 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL55 gene. In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes an inactivating mutation in the linking region. In some embodiments, the recombinant herpes simplex virus genome includes a deletion in the linking region.

[0013] In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within one or more viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within one or both of the ICP4 viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within the ICP22 viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within the UL41 viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within one or both of the ICP0 viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding the CFTR polypeptide within the ICP27 viral loci. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding a CFTR polypeptide within the ICP47 viral locus. In some embodiments that can be combined with any of the embodiments described above, the recombinant herpes simplex virus genome includes one or more polynucleotides encoding a CFTR polypeptide within the UL55 viral locus.

[0014] In some embodiments, which may be combined with any of the embodiments described above, the recombinant herpesvirus genome, when introduced into target cells, exhibits reduced cytotoxicity compared to the corresponding wild-type herpesvirus genome. In some embodiments, the target cells are human cells. In some embodiments, which may be combined with any of the embodiments described above, the target cells are respiratory cells. In some embodiments, which may be combined with any of the embodiments described above, the target cells are airway epithelial cells or submucosal gland cells.

[0015] Other aspects of this disclosure relate to herpesviruses comprising any of the recombinant herpesvirus genomes described herein. In some embodiments, the herpesvirus is replicable. In some embodiments, the herpesvirus is replication-deficient. In some embodiments, which may be combined with any of the embodiments described above, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments, when introduced into target cells, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments, the target cells are human cells. In some embodiments, which may be combined with any of the embodiments described above, the target cells are respiratory cells. In some embodiments, which may be combined with any of the embodiments described above, the target cells are airway epithelial cells or submucosal gland cells. In some embodiments that can be combined with any of the embodiments described above, the herpesvirus is selected from herpes simplex virus, varicella-zoster virus, human cytomegalovirus, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, Kaposi's sarcoma-associated herpesvirus, and any combination or derivative thereof. In some embodiments that can be combined with any of the embodiments described above, the herpesvirus 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 this disclosure relate to pharmaceutical compositions comprising any of the recombinant herpesvirus genomes and / or herpesviruses described herein, and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, oral cavity, rectal, vaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreous, subretinal, intra-articular, periarticular, local, and / or cutaneous 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 dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, electrohydrodynamic aerosol devices, or any combination thereof. In some embodiments, the pharmaceutical composition is suitable for spraying (using a vibrating mesh nebulizer). In some embodiments, which may be combined with any of the embodiments described above, the pharmaceutical composition comprises a phosphate buffer. In some embodiments, which may be combined with any of the embodiments described above, the pharmaceutical composition comprises glycerol. In some embodiments, which may be combined with any of the embodiments described above, the pharmaceutical composition comprises a lipid carrier. In some embodiments, which may be combined with any of the embodiments described above, the pharmaceutical composition comprises a nanoparticle carrier.

[0017] Other aspects of this disclosure relate to the use of any of the recombinant nucleic acids, herpesviruses, and / or pharmaceutical compositions described herein as pharmaceutical agents.

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

[0019] Other aspects of this disclosure relate to the use of any of the recombinant nucleic acids, herpesviruses, and / or pharmaceutical compositions described herein in the production or manufacture of agents for treating one or more signs or symptoms of CFTR deficiency and / or chronic lung diseases (e.g., cystic fibrosis, COPD, etc.).

[0020] Other aspects of this disclosure relate to methods for 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 the submucosal gland. In some embodiments which may be combined with any of the embodiments described above, 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 which may be combined with any of the embodiments described above, the subject is human. In some embodiments which may be combined with any of the embodiments described above, the genome of the subject contains a loss-of-function mutation in the CFTR gene. In some embodiments, which may be combined with any of the embodiments described above, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, sublingually, oral cavity, topically, rectally, by inhalation, percutaneously, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardiacly, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreously, intraorbitally, subretinally, intraarticularly, periarticularly, topically, and / or onto the skin. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, or by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by non-invasive inhalation.In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered using dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, or electrohydrodynamic aerosol devices. In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered via nebulizers (e.g., vibrating mesh nebulizers).

[0021] Other aspects of this disclosure relate to methods for reducing or inhibiting progressive lung injury in subjects requiring such reduction or inhibition, 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 subjects suffer from a chronic lung disease. In some embodiments, the chronic lung disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). In some embodiments, which may be combined with any of the embodiments described above, the subjects are human. In some embodiments, which may be combined with any of the embodiments described above, the genome of the subjects contains a loss-of-function mutation in the CFTR gene. In some embodiments, which may be combined with any of the embodiments described above, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, sublingually, oral cavity, topically, rectally, by inhalation, percutaneously, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardiacly, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreously, intraorbitally, subretinally, intraarticularly, periarticularly, topically, and / or onto the skin. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, or by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by non-invasive inhalation. In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered using dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, or electrohydrodynamic aerosol devices. In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered via nebulizers (e.g., vibrating mesh nebulizers).

[0022] Other aspects of the present disclosure relate to methods for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cystic fibrosis in a subject requiring such relief, 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 cough producing large amounts of mucus, large amounts of sticky mucus accumulating in the airways, wheezing, dyspnea, sinusitis, recurrent lung infections, inflammatory rhinitis, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof. In some embodiments which may be combined with any of the embodiments described above, the subject is human. In some embodiments which may be combined with any of the embodiments described above, the genome of the subject contains a loss-of-function mutation in the CFTR gene. In some embodiments, which may be combined with any of the embodiments described above, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, sublingually, oral cavity, topically, rectally, by inhalation, percutaneously, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardiacly, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreously, intraorbitally, subretinally, intraarticularly, periarticularly, topically, and / or onto the skin. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, or by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by non-invasive inhalation. In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered using dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, or electrohydrodynamic aerosol devices.In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered via a nebulizer (e.g., a vibrating mesh nebulizer).

[0023] Other aspects of this disclosure relate to methods for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of COPD in a subject requiring such relief, 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, excessive mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof. In some embodiments which may be combined with any of the embodiments described above, the subject is human. In some embodiments which may be combined with any of the embodiments described above, the genome of the subject contains a loss-of-function mutation in the CFTR gene. In some embodiments, which may be combined with any of the embodiments described above, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, sublingually, oral cavity, topically, rectally, by inhalation, percutaneously, subcutaneously, intradermally, intravenously, intraarterially, intramuscularly, intracardiacly, intraosseously, intraperitoneally, transmucosally, intravaginally, intravitreously, intraorbitally, subretinally, intraarticularly, periarticularly, topically, and / or onto the skin. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject orally, intranasally, intratracheally, or by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by inhalation. In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered to a subject by non-invasive inhalation. In some embodiments, recombinant herpesvirus genomes, herpesviruses, and / or pharmaceutical compositions are administered using dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, or electrohydrodynamic aerosol devices.In some embodiments, recombinant herpesvirus genome, herpesvirus, and / or pharmaceutical composition are administered via a nebulizer (e.g., a vibrating mesh nebulizer).

[0024] Other aspects of this disclosure relate to a product or kit comprising any of the recombinant herpesvirus genomes, herpesviruses, drugs, and / or pharmaceutical compositions described herein, and instructions for administering the recombinant herpesvirus genomes, herpesviruses, drugs, or pharmaceutical compositions. In some embodiments, the product or kit further includes an apparatus for aerosolizing the recombinant herpesvirus genomes, herpesviruses, drugs, and / or pharmaceutical compositions. In some embodiments, the apparatus is a dry powder inhaler, a pressurized metered-dose inhaler, a soft mist inhaler, a nebulizer, or an electrohydrodynamic aerosol apparatus. In some embodiments, the apparatus is a nebulizer (e.g., a vibrating mesh nebulizer). [Invention 1001] A recombinant herpesvirus genome containing one or more polynucleotides encoding a cystic fibrosis membrane conductance regulator (CFTR) polypeptide. [Invention 1002] A replicable recombinant herpesvirus genome according to the present invention 1001. [Invention 1003] A recombinant herpesvirus genome of the present invention 1001, which is replication-deficient. [Invention 1004] A recombinant herpesvirus genome according to any one of claims 1001 to 1003 of the present invention, comprising one or more polynucleotides encoding the CFTR polypeptide within one or more viral loci. [Invention 1005] A recombinant herpesvirus genome according to any one of claims 1001 to 1004 of the present invention, 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 derivative thereof. [Invention 1006] A recombinant herpesvirus genome according to any one of claims 1001 to 1005 of the present invention, wherein the CFTR polypeptide is a human CFTR polypeptide. [Invention 1007] A recombinant herpesvirus genome according to any one of claims 1001 to 1006 of the present invention, 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 with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. [Invention 1008] A recombinant herpesvirus genome according to any one of claims 1001 to 1007 of the present invention, 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 with the amino acid sequence of SEQ ID NO: 5. [Invention 1009] A recombinant herpes simplex virus genome, which is a recombinant herpesvirus genome according to any one of claims 1001 to 1008 of the present invention. [Invention 1010] The recombinant herpesvirus genome of the present invention 1009, wherein the recombinant herpesvirus genome is a recombinant herpesvirus type 1 (HSV-1) genome, a recombinant herpesvirus type 2 (HSV-2) genome, or any derivative thereof. [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. [Invention 1012] A recombinant herpesvirus genome according to any one of claims 1009 to 1011 of the present invention, wherein the recombinant herpesvirus genome is manipulated to reduce or eliminate the expression of one or more virulent herpesvirus genes. [Invention 1013] The recombinant herpes simplex virus genome is a recombinant herpesvirus genome according to any one of claims 1009 to 1012 of the present invention, wherein the recombinant herpes simplex virus genome includes an inactivating mutation. [Invention 1014] The recombinant herpesvirus genome of the present invention 1013, wherein the aforementioned inactivating mutation is present in the herpes simplex virus gene. [Invention 1015] The recombinant herpesvirus genome of the present invention 1014, wherein the inactivating mutation is a deletion of the coding sequence of the herpes simplex virus gene. [Invention 1016] A recombinant herpesvirus genome according to Invention 1014 or Invention 1015, wherein the herpes simplex virus gene is selected from the group consisting of infection cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55. [Invention 1017] The recombinant herpes simplex virus genome of the present invention 1016, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP4 gene. [Invention 1018] The recombinant herpes simplex virus genome of the invention 1016 or the invention 1017, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP22 gene. [Invention 1019] The recombinant herpes simplex virus genome according to any one of claims 1016 to 1018 of the present invention, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the UL41 gene. [Invention 1020] The recombinant herpes simplex virus genome according to any one of claims 1016 to 1019 of the present invention, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in one or both copies of the ICP0 gene. [Invention 1021] The recombinant herpes simplex virus genome according to any one of claims 1016 to 1020 of the present invention, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP27 gene. [Invention 1022] The recombinant herpes simplex virus genome according to any one of claims 1016 to 1021 of the present invention, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP47 gene. [Invention 1023] The recombinant herpes simplex virus genome according to any one of claims 1016 to 1022 of the present invention, wherein the recombinant herpes simplex virus genome contains an inactivating mutation in the UL55 gene. [Invention 1024] A recombinant herpes simplex virus genome according to any one of claims 1009 to 1023 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within one or both of the ICP4 viral loci. [Invention 1025] A recombinant herpes simplex virus genome according to any one of claims 1009 to 1024 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within the ICP22 viral locus. [Invention 1026] A recombinant herpes simplex virus genome according to any one of claims 1009 to 1025 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within the UL41 viral locus. [Invention 1027] A recombinant herpes simplex virus genome according to any one of claims 1009 to 1026 of the present invention, wherein the recombinant herpes simplex virus genome comprises one or more polynucleotides encoding the CFTR polypeptide within one or both of the ICP0 viral loci. [Invention 1028] The recombinant herpes simplex virus genome according to any one of claims 1009 to 1027 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within the ICP27 viral locus. [Invention 1029] A recombinant herpes simplex virus genome according to any one of claims 1009 to 1028 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within the ICP47 viral locus. [Invention 1030] The recombinant herpes simplex virus genome according to any one of claims 1009 to 1029 of the present invention, wherein the recombinant herpes simplex virus genome contains one or more polynucleotides encoding the CFTR polypeptide within the UL55 viral locus. [Invention 1031] A recombinant herpesvirus genome according to any one of claims 1001 to 1030 of the present invention, which, when introduced into target cells, exhibits reduced cytotoxicity compared to the corresponding wild-type herpesvirus genome. [Invention 1032] The recombinant herpesvirus genome of the present invention 1031, wherein the target cell is a human cell. [Invention 1033] The recombinant herpesvirus genome of the present invention 1031 or 1032, wherein the target cells are airway epithelial cells. [Invention 1034] The recombinant herpesvirus genome of the present invention 1031 or 1032, wherein the target cell is a respiratory cell. [Invention 1035] A herpesvirus comprising a recombinant herpesvirus genome according to any one of claims 1001 to 1034 of the present invention. [Invention 1036] A reproducible herpesvirus according to the present invention 1035. [Invention 1037] A herpesvirus of the present invention 1035, which is replication-deficient. [Invention 1038] A herpesvirus according to any one of claims 1035 to 1037 of the present invention, having reduced cytotoxicity compared to the corresponding wild-type herpesvirus. [Invention 1039] A herpesvirus according to any one of claims 1035 to 1038 of the present invention, 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. [Invention 1040] A herpes simplex virus, which is a herpes virus according to any one of claims 1035 to 1039 of the present invention. [Invention 1041] The herpesvirus of Invention 1039 or Invention 1040, wherein the herpes simplex virus is herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), or any derivative thereof. [Invention 1042] The herpes virus according to any one of claims 1039 to 1041 of the present invention, wherein the herpes simplex virus is HSV-1. [Invention 1043] A pharmaceutical composition comprising a recombinant herpesvirus genome according to any one of claims 1001 to 1034 of the present invention or a herpesvirus according to any one of claims 1035 to 1042 of the present invention, and a pharmaceutically acceptable excipient. [Invention 1044] A pharmaceutical composition according to Invention 1043, suitable for topical, transdermal, subcutaneous, intradermal, oral, intranasal, intratracheal, sublingual, oral cavity, rectal, vaginal, inhalation, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreous, subretinal, intra-articular, peri-articular, local, or cutaneous administration. [Invention 1045] A pharmaceutical composition according to Invention 1043 or Invention 1044, suitable for oral, intranasal, intratracheal, or inhalation administration. [Invention 1046] A pharmaceutical composition according to any one of claims 1043 to 1045 of the present invention, which is suitable for inhalation administration. [Invention 1047] A pharmaceutical composition according to any one of claims 1043 to 1046 of the present invention, which is suitable for non-invasive inhalation administration. [Invention 1048] A pharmaceutical composition according to any one of claims 1043 to 1047 of the present invention, suitable for use in dry powder inhalers, pressurized metered-dose inhalers, soft mist inhalers, nebulizers, electrohydrodynamic aerosol devices, or any combination thereof. [Invention 1049] A pharmaceutical composition according to any one of claims 1043 to 1048 of the present invention, which is suitable for use in a nebulizer. [Invention 1050] The pharmaceutical composition of the present invention 1049, wherein the nebulizer is a vibrating mesh nebulizer. [Invention 1051] A pharmaceutical composition according to any one of claims 1043 to 1050 of the present invention, comprising a phosphate buffer. [Invention 1052] A pharmaceutical composition comprising glycerol according to any one of claims 1043 to 1051 of the present invention. [Invention 1053] A pharmaceutical composition comprising a lipid carrier according to any one of claims 1043 to 1052 of the present invention. [Invention 1054] A pharmaceutical composition according to any one of claims 1043 to 1053 of the present invention, comprising a nanoparticle carrier. [Invention 1055] A method for 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 a herpesvirus of any one of the inventions 1035-1042 or a pharmaceutical composition of any one of the inventions 1043-1054. [Invention 1056] The method of the present invention 1055, wherein the one or more cells are one or more cells of the respiratory tract. [Invention 1057] The method of the present invention 1055 or 1056, wherein the one or more cells are one or more airway epithelial cells or one or more cells of a submucosal gland. [Invention 1058] A method for reducing or inhibiting progressive lung injury in a subject requiring it, comprising administering to the subject an effective amount of a herpes virus according to any one of claims 1035 to 1042 of the present invention or a pharmaceutical composition according to any one of claims 1043 to 1054 of the present invention. [Invention 1059] The method according to any one of claims 1055 to 1058 of the present invention, wherein the subject is suffering from a chronic lung disease. [Invention 1060] The method of the present invention 1059, wherein the chronic lung disease is cystic fibrosis or chronic obstructive pulmonary disease (COPD). [Invention 1061] A method for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cystic fibrosis in a subject requiring such relief, comprising administering to the subject an effective amount of a herpes virus according to any one of claims 1035 to 1042 of the present invention or a pharmaceutical composition according to any one of claims 1043 to 1054 of the present invention. [Invention 1062] The method of the present invention 1061, wherein one or more of the signs or symptoms of cystic fibrosis is selected from the group consisting of persistent cough producing large amounts of mucus, large amounts of sticky mucus accumulating in the airways, wheezing, dyspnea, sinusitis, recurrent lung infections, inflammatory rhinitis, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof. [Invention 1063] A method for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of COPD in a subject requiring such relief, comprising administering to the subject an effective amount of a herpes virus according to any one of claims 1035 to 1042 of the present invention or a pharmaceutical composition according to any one of claims 1043 to 1054 of the present invention. [Invention 1064] The method of the present invention 1063, wherein one or more of the signs or symptoms of COPD are selected from the group consisting of shortness of breath, wheezing, chest tightness, excessive mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof. [Invention 1065] The method according to any one of claims 1055 to 1064 of the present invention, wherein the subject is a human. [Invention 1066] The method according to any one of claims 1055 to 1065 of the present invention, wherein the target genome contains a loss-of-function mutation in the CFTR gene. [Invention 1067] The method according to any one of claims 1055 to 1066 of the present invention, wherein the herpes virus or pharmaceutical composition is administered to the subject orally, intranasally, intratracheally, or by inhalation. [Invention 1068] A method according to any one of claims 1055 to 1067 of the present invention, wherein the herpes virus or pharmaceutical composition is administered to the subject by inhalation. [Invention 1069] The method according to any one of claims 1055 to 1068 of the present invention, wherein the herpesvirus or pharmaceutical composition is administered via non-invasive inhalation. [Invention 1070] The method according to any one of claims 1055 to 1069 of the present invention, 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. [Invention 1071] The method according to any one of claims 1055 to 1070 of the present invention, wherein the herpes virus or pharmaceutical composition is administered using a nebulizer. [Invention 1072] The method of the present invention 1071, wherein the nebulizer is a vibrating mesh nebulizer. [Brief explanation of the drawing]

[0025] [Figure 1-1]Figure 1A shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. The wild-type herpes simplex virus genome is shown. Figure 1B shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. The modified herpes simplex virus genome, which includes deletions of the coding sequence of ICP4 (both copies), has an expression cassette containing nucleic acid encoding human CFTR polypeptide incorporated into each ICP4 locus. [Figure 1-2] Figure 1C shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies) and UL41, with an expression cassette containing nucleic acid encoding human CFTR polypeptide integrated into each of the ICP4 loci. Figure 1D shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies) and UL41, with an expression cassette containing nucleic acid encoding CFTR polypeptide integrated into the UL41 locus. [Figure 1-3] Figure 1E shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies) and ICP22, with an expression cassette containing nucleic acid encoding human CFTR polypeptide integrated into each of the ICP4 loci. Figure 1F shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies) and ICP22, with an expression cassette containing nucleic acid encoding CFTR polypeptide integrated into the ICP22 locus. [Figure 1-4]Figure 1G shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, with an expression cassette containing nucleic acid encoding human CFTR polypeptides integrated into each of the ICP4 loci. Figure 1H shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. It shows the modified herpes simplex virus genome, which includes deletions of the coding sequences for ICP4 (both copies), UL41, and ICP22, with an expression cassette containing nucleic acid encoding CFTR polypeptides integrated into the UL41 locus. [Figure 1-5] Figure 1I shows schematic diagrams of the wild-type herpes simplex virus genome and the modified herpes simplex virus genome. The modified herpes simplex virus genome shows deletions of the coding sequences of ICP4 (both copies), UL41, and ICP22, with an expression cassette containing nucleic acids encoding the CFTR polypeptide integrated into the ICP22 locus. [Figure 2] This study shows the expression of human CFTR in primary airway epithelial cells (SAECs) from cystic fibrosis (CF) patients infected with the HSV-CFTR vector at indicated multiples of infection (MOI), as assessed by qRT-PCR analysis. Mock-infected CF SAECs were used as negative controls. Data are presented as the mean ± standard error of two replicas. [Figure 3] This shows the expression of human CFTR protein in primary SAEC cells derived from CF patients infected with an HSV-CFTR vector-infected MOI, as assessed by Western blot analysis. Mock-infected CF SAEC cells were used as a negative control. GAPDH was used as a loading control. [Figure 4]Representative immunofluorescence images of human CFTR protein expression in primary CF patient SAECs infected with mock or HSV-CFTR are shown. A shows a dose-dependent increase in human CFTR protein expression upon infection of primary CF SAECs with increasing MOI of HSV-CFTR. B shows the relative cellular localization of human CFTR protein in primary CF SAECs infected with HSV-CFTR (MOI 3) or mock (MOI 0). Nuclei were visualized using DAPI staining. [Figure 5] This study demonstrates the functionality of human CFTR protein in primary SAECs derived from CF patients infected with an HSV-CFTR vector-infected MOI, as assessed by a fluorescent dye uptake assay. Mock-infected CF SAECs were used as negative controls. Data are presented as mean ± standard error. [Figure 6A] This section presents the analysis of intestinal organoids (PDOs) from G542X / G542X cystic fibrosis patients infected with HSV-CFTR at the indicated MOIs. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and G418 was used as a positive control. Representative bright-field images of G542X / G542X PDOs are shown 24 hours after vehicle treatment, or after transduction using either HSV-CFTR or HSV-mCherry at 10 MOIs. Vehicle-treated PDOs isolated from healthy individuals (wild-type) were also imaged for comparison. [Figure 6B] This report presents the analysis of prostate organoids (PDOs) from G542X / G542X cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and G418 was used as a positive control. Representative images of calcein-stained organoids before forskolin (Frsk) addition (t=0) and quantification of mean organoid size are shown. [Figure 6C]This report presents the analysis of prostate organoids (PDOs) from G542X / G542X cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and G418 was used as a positive control. Representative images of calcein-stained organoids 60 minutes (t=60) after the addition of 2 μM Frsk and quantification of mean organoid size are shown. ***p<0.001, ****p<0.0001. [Figure 7A] This section presents the analysis of prostate organoids (PDOs) from F508del / F508del cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and Orkambi® was used as a positive control. Representative images of calcein-stained organoids before forskolin (Frsk) addition (t=0) and quantification of mean organoid size are shown. [Figure 7B] This report presents the analysis of prostate organoids (PDOs) from F508del / F508del cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and Orkambi® was used as a positive control. Representative images of calcein-stained organoids 60 minutes (t=60) after the addition of 2 μM Frsk and quantification of the mean organoid size are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8A] This report presents the analysis of prostate organoids (PDOs) from W1282X / W1282X cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control. Representative images of calcein-stained organoids before forskolin (Frsk) addition (t=0) and quantification of mean organoid size are shown. [Figure 8B]This report presents the analysis of prostate organoids (PDOs) from W1282X / W1282X cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control. Representative images of calcein-stained organoids 60 minutes (t=60) after the addition of 2 μM Frsk and quantification of mean organoid size are shown. *p<0.05, ***p<0.001. [Figure 9A] This section presents the analysis of prostate organoids (PDOs) from F508del / F508del cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and Orkambi® was used as a positive control. Representative images of calcein-stained organoids before forskolin (Frsk) addition (t=0) and quantification of mean organoid size are shown. [Figure 9B] This paper presents the analysis of prostate organoids (PDOs) from F508del / F508del cystic fibrosis patients infected with HSV-CFTR at the indicated MOI. Vehicle alone or the mCherry-coded HSV vector (mCherry) was used as a negative control, and Orkambi® was used as a positive control. Representative images of calcein-stained organoids 60 minutes (t=60) after the addition of 2 μM Frsk and quantification of the mean organoid size are shown. ****p<0.0001. [Figure 10A] This report presents mCherry nucleic acid and protein analysis in lung and tracheal biopsies collected 48 hours after intranasal or intratracheal administration of the mCherry-coded HSV vector (HSV-mCherry) or vehicle control (mock). mCherry transcript levels in lung and tracheal biopsies, assessed by qRT-PCR analysis, are shown. Data for HSV-mCherry are presented as the mean ± standard error of six replicas, and data for the vehicle control are presented as the mean ± standard error of four replicas. [Figure 10B]This shows mCherry nucleic acid and protein analysis in lung and tracheal biopsies collected 48 hours after intranasal or intratracheal administration of the mCherry-coded HSV vector (HSV-mCherry) or a vehicle control (mock). Representative immunofluorescence images of mCherry protein expression in lung biopsies after intranasal administration of HSV-mCherry or the vehicle control are shown. Nuclei were visualized using DAPI staining, and epithelial cells were visualized using cytokeratin staining. [Figure 10C] This shows mCherry nucleic acid and protein analysis in lung and tracheal biopsies collected 48 hours after intranasal or intratracheal administration of the mCherry-coded HSV vector (HSV-mCherry) or a vehicle control (mock). Representative immunofluorescence images of mCherry protein expression in lung biopsies after intratracheal administration of HSV-mCherry or the vehicle control are shown. Nuclei were visualized using DAPI staining, and epithelial cells were visualized using cytokeratin staining. [Modes for carrying out the invention]

[0026] Detailed explanation The following descriptions illustrate exemplary methods and parameters. However, it should be noted that such descriptions are not intended to limit the scope of this disclosure, but rather are provided as descriptions of exemplary 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、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.The methodology described in *Short Protocols in Molecular Biology* (Wiley and Sons, 1994) and other widely used methodologies by those skilled in the art are generally well understood and commonly used.

[0028] II. Definition Before describing this disclosure in detail, please understand that this disclosure is not limited to any particular composition or biological system, and that these may, needless to say, vary. Also, please understand that the terminology used herein is intended solely to describe and not to limit any particular embodiment.

[0029] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. For example, a reference to "a molecule" can optionally include any combination of two or more such molecules.

[0030] As used herein, the term "and / or" may include any combination of one or more of the related listed items. For example, the term "a and / or b" may refer to "a only," "b only," "a or b," or "a and b," and 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."

[0031] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. References to “about” values ​​or parameters herein include (and are described) embodiments relating to the value or parameter itself.

[0032] It is understood that the aspects and embodiments of this disclosure include, consist of, and essentially consist of the aspects and embodiments.

[0033] As used herein, the terms “polynucleotide,” “nucleic acid sequence,” “nucleic acid,” and their variations are 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 nucleic acid bases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Therefore, these terms include known types of nucleic acid sequence modifications, such as substitutions with one or more analogues of naturally occurring nucleotides, and internucleotide modifications.

[0034] As used herein, a nucleic acid is "operatally linked" or "operatably linked" if it is in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatably linked to a coding sequence if it affects the transcription of that sequence, and a ribosome binding site is operatably linked to a coding sequence if it is positioned to facilitate translation. In general, "operatably linked" or "operatably linked" means that the linked DNA or RNA sequences are in close proximity.

[0035] As used herein, the term “vector” refers to a distinct element used to introduce a heterologous nucleic acid into a cell for either expression or replication. Expression vectors include vectors capable of expressing nucleic acids operatively linked to regulatory sequences, such as promoter regions, which can result in the expression of such nucleic acids. Thus, expression vectors may refer to DNA or RNA constructs, such as plasmids, phages, recombinant viruses, or other vectors, that result in the expression of nucleic acids when introduced into a suitable host cell. 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 in the episome or are incorporated into the host cell genome.

[0036] As used herein, “open reading frame” or “ORF” refers to the continuous elongation of the diffusion of either DNA or RNA that encodes a protein or polypeptide. Typically, nucleic acids include a translation start signal or start and stop codons, such as ATG or AUG.

[0037] As used herein, “untranslated region” or “UTR” refers to untranslated nucleic acid at the 5' and / or 3' ends of an open reading frame. The 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. In some embodiments, this confers a desired characteristic to the introduced cell or otherwise results in a desired therapeutic or diagnostic outcome.

[0039] As used herein, the terms “polypeptide,” “protein,” and “peptide” are interchangeable and may refer to polymers of two or more amino acids.

[0040] As used herein, “subject,” “host,” or “individual” means any animal classified as a mammal, including humans, livestock and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, cats, and cattle, 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 terms “pharmaceutical preparation” or “pharmaceutical composition” refer to a preparation that enables the biological activity of the active ingredient(s) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition or preparation is administered. A “pharmaceutically acceptable” excipient (e.g., vehicle, additive) is one that can be administered in moderation to the subject mammal to provide an effective dose of the active ingredient(s) used.

[0042] As used herein, “effective dose” is the minimum amount necessary to have a measurable effect on the improvement or prevention of one or more symptoms of a particular disorder. “Effective dose” may vary depending on factors such as the patient’s disease state, age, sex, and weight. The effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired outcomes include the elimination or reduction of the risk of disease, its complications, and intermediate pathological phenotypes presented during the onset of the disease, reduction of their severity, or delay of their onset. In the case of therapeutic use, beneficial or desired outcomes include clinical outcomes such as the reduction of one or more symptoms caused by the disease, an improvement in the quality of life of the person suffering from the disease, a reduction in the dose of other drugs used to treat the symptoms of the disease, delay in disease progression, and / or extension of survival. The effective dose may be administered in one or more doses. For the purposes of this disclosure, the effective dose of recombinant nucleic acids, viruses, and / or pharmaceutical compositions is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in clinical contexts, the effective dose of recombinant nucleic acids, viruses, and / or pharmaceutical compositions may or may not be achieved in combination with other drugs, compounds, or pharmaceutical compositions. Therefore, “effective dose” may be considered in situations involving the administration of one or more therapeutic agents, and a single agent may be considered administered in an effective dose if the desired outcome can or has been achieved in combination with one or more other agents.

[0043] As used herein, “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated in the course of clinicopathology. Desired effects of treatment include a reduction in the rate of progression of a disease / disability / deficiency, improvement or mitigation of the disease / disability / deficiency condition, and remission or improved 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 the progression” of a disease / disorder / deficiency means delaying, preventing, postponing, stabilizing, and / or postponing the onset of a disease / disorder / deficiency (e.g., cystic fibrosis or COPD). This delay may vary in length or duration depending on the disease / disorder / deficiency and / or the medical history of the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may, in practice, encompass prevention in that the individual does not develop the disease.

[0045] III. Recombinant Nucleic Acids Certain embodiments of this disclosure relate to recombinant nucleic acids (e.g., isolated recombinant nucleic acids) comprising one or more polynucleotides (e.g., one or more, two or more, three or more, four or more, five or more, ten or more, etc.) encoding a CFTR polypeptide (e.g., human CFTR polypeptide). In some embodiments, the recombinant nucleic acid comprises one polynucleotide encoding a CFTR polypeptide. In some embodiments, the recombinant nucleic acid comprises two polynucleotides encoding a CFTR polypeptide. In some embodiments, the recombinant nucleic acid comprises three polynucleotides encoding 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 herpesvirus vector. In some embodiments, the recombinant nucleic acid is a herpes simplex virus amplicon. In some embodiments, the recombinant nucleic acid is a recombinant herpesvirus 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 membrane conductance regulator (CFTR) polypeptides In some embodiments, the disclosure relates to recombinant nucleic acids, or any portion thereof, comprising one or more polynucleotides containing the coding sequence of a CFTR gene (e.g., the human CFTR gene). For example, any suitable CFTR gene known in the art (including any isoform thereof), such as the human CFTR gene (e.g., NCBI gene ID: 1080; see SEQ ID NO: 1 or SEQ ID NO: 3), the chimpanzee CFTR gene (e.g., see NCBI gene ID: 463674), the mouse CFTR gene (e.g., see NCBI gene ID: 12638), the rat CFTR gene (e.g., see NCBI gene ID: 24255), the canine CFTR gene (e.g., see NCBI gene ID: 492302), the rabbit CFTR gene (e.g., see NCBI gene ID: 100009471), the bovine CFTR gene (e.g., see NCBI gene ID: 281067), the rhesus macaque CFTR gene (e.g., see NCBI gene ID: 574346), etc., can be encoded by the polynucleotides of this disclosure. In some embodiments, the polynucleotides of this 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 with any of the CFTR genes (and / or coding sequences) described herein or known in the art. Methods for identifying additional species-derived CFTR gene homologs / orthologues are known to those skilled in the art, for example, by using nucleic acid sequence alignment programs such as BLAST® blastn suite.

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

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

[0050] In some embodiments, the polynucleotides of the Disclosure include 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 with the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotides of the Disclosure include the sequence of SEQ ID NO: 1.

[0051] In some embodiments, the polynucleotides of the present disclosure include 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 1. In some embodiments, the 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 1 are polynucleotides 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, and at least 4250 consecutive nucleotides, but less than 4443. In some embodiments, the polynucleotides of the 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 with the sequences of nucleic acids 1 to 4440 of SEQ ID NO: 1.

[0052] In some embodiments, the polynucleotides of the Disclosure include 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 with the sequence of SEQ ID NO: 3.

[0053] In some embodiments, the polynucleotides of the present disclosure include 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 3. In some embodiments, the 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 3 are polynucleotides 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, and at least 4250 consecutive nucleotides, but less than 4260. In some embodiments, the polynucleotides of the 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 with the sequences of nucleic acids 1 to 4257 of SEQ ID NO: 3.

[0054] In some embodiments, the polynucleotides of this disclosure include the coding sequence of a codon-optimized variant of the human CFTR gene.

[0055] In some embodiments, the polynucleotides of the Disclosure include 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 with the sequence of SEQ ID NO: 2. In some embodiments, the polynucleotides of the Disclosure include the sequence of SEQ ID NO: 2.

[0056] In some embodiments, the polynucleotides of the present disclosure include 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 2. In some embodiments, the 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 2 are polynucleotides 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, and at least 4250 consecutive nucleotides but less than 4443. In some embodiments, the polynucleotides of the 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 with the sequences of nucleic acids 1 to 4440 of SEQ ID NO: 2.

[0057] In some embodiments, the polynucleotides of the Disclosure include 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 with the sequence of SEQ ID NO: 4. In some embodiments, the polynucleotides of the Disclosure include the sequence of SEQ ID NO: 4.

[0058] In some embodiments, the polynucleotides of the present disclosure include 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 4. In some embodiments, the 5' cuts, 3' cuts, or fragments of the sequence of SEQ ID NO: 4 are polynucleotides 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, and at least 4250 consecutive nucleotides, but less than 4260. In some embodiments, the polynucleotides of the 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 with the sequences of nucleic acids 1 to 4257 of SEQ ID NO: 4.

[0059] In some embodiments, the polynucleotides of the Disclosure include 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 a nucleic acid sequence selected from SEQ ID NOs: 1 to 4. In some embodiments, the polynucleotides of the Disclosure include 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 a nucleic acid sequence selected from SEQ ID NOs: 1 or SEQ ID NOs: 2. In some embodiments, the polynucleotides of the Disclosure include a sequence selected from SEQ ID NOs: 1 or SEQ ID NOs: 2. In some embodiments, the polynucleotides of the Disclosure include 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 a nucleic acid sequence selected from SEQ ID NO: 3 or SEQ ID NO: 4.

[0060] The polynucleotides of this disclosure (e.g., encoding a human CFTR polypeptide) may further encode additional coding and non-coding sequences. Examples of additional coding and non-coding sequences include, but are 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., native, modified, or heterologous introns), 5'UTR and / or 3'UTR (e.g., native, modified, or heterologous 5'UTR and / or 3'UTR), etc. Examples of suitable polypeptide tags include, but are not limited to, any combination of tags including purification tags, e.g., his tags, flag tags, maltose-binding protein and glutathione-S-transferase tags; detection tags, e.g., photometrically detectable tags (e.g., green fluorescent protein, red fluorescent protein, etc.) and tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.); secretion sequences, signal sequences, reader sequences and / or stabilization sequences; protease cleavage sites (e.g., furan cleavage sites, TEV cleavage sites, thrombin cleavage sites, etc.). In some embodiments, the 5'UTR and / or 3'UTR increase the stability, localization and / or translation efficiency of polynucleotides. 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 include elements (e.g., one or more miRNA binding sites) that can block or reduce off-target expression (e.g., inhibit expression in a particular cell type (e.g., nerve cells) at a particular time in the cell cycle or at a particular developmental stage). In some embodiments, the 5'UTR and / or 3'UTR include elements (e.g., one or more miRNA binding sites) that can enhance CFTR protein expression in a particular cell type.

[0061] In some embodiments, the polynucleotides of the Disclosure (e.g., encoding human CFTR polypeptides) are 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” may include enhancers, insulators, promoters, and other expression regulatory elements (e.g., polyadenylation signals). Any suitable enhancer(s) known in the art may be used, including, for example, enhancer sequences derived from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein, insulin, etc.), enhancer sequences derived from eukaryotic viruses (e.g., SV40 enhancers on the posterior end of the origin of replication (bp100-270), cytomegalovirus early promoter enhancers, polyoma enhancers on the posterior end of the origin of replication, adenovirus enhancers, etc.), and any combination thereof. For example, any suitable insulator(s) known in the art may be used, including the herpes simplex virus (HSV) chromatin boundary (CTRL / CTCF binding / insulator) elements CTRL1 and / or CTRL2 derived from the human interferon-beta gene (IFNB1), the chicken susceptibility site 4 insulator (cHS4), the human HNRPA2B1-CBX3 ubiquita chromatin opening element (UCOE), the scaffold / matrix binding region (S / MAR), and any combination thereof.For example, any suitable promoter known in the art (e.g., a promoter suitable for transcription in mammalian host cells), including promoters obtained from the genomes of viruses (e.g., polyomavirus, fowlpox virus, adenovirus (adenovirus 2, etc.), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, Simian virus 40 (SV40), etc.), promoters derived from heterologous mammalian genes (e.g., actin promoter (e.g., β-actin promoter), ubiquitin promoter (e.g., ubiquitin C (UbC) promoter), phosphoglycerate kinase (PGK) promoter, immunoglobulin promoter, heat shock protein promoter, etc.), promoters derived from natural and / or homologous mammalian genes (e.g., human CFTR gene promoter), synthetic promoters (e.g., CAGG promoter), and any combination thereof, may be used, provided that such promoters are suitable for transcription in mammalian host cells. Regulatory sequences may include nucleic acids and sequences that direct the constitutive expression of tissue-specific regulatory sequences and / or inducible or repressive sequences.

[0062] In some embodiments, the polynucleotides of the Disclosure are operably ligated to one or more heterologous promoters. In some embodiments, one or more heterologous promoters are one or more constitutive promoters, tissue-specific promoters, temporal promoters, spatial promoters, inductive promoters, and repressive promoters. In some embodiments, one or more heterologous promoters are one or more of the human cytomegalovirus (HCMV) pre-early promoter, human elongation factor-1 (EF1) promoter, human β-actin promoter, human UbC promoter, human PGK promoter, synthetic CAGG promoter, and any combination thereof. In some embodiments, the polynucleotides of the Disclosure (e.g., encoding a human CFTR polypeptide) are operably ligated to an HCMV promoter.

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

[0064] Cystic fibrosis membrane conductance regulator (CFTR) polypeptide In some embodiments, the Disclosure relates to one or more polynucleotides encoding a CFTR polypeptide (e.g., a human CFTR polypeptide) or any portion thereof. Any suitable CFTR polypeptide known in the art, including, for example, human CFTR polypeptide (e.g., UniProt acceptance number P13569; see SEQ ID NO. 5 or SEQ ID NO. 6), chimpanzee CFTR polypeptide (e.g., see UniProt acceptance number Q2QLE5), mouse CFTR polypeptide (e.g., see UniProt acceptance number P26361), rat CFTR polypeptide (e.g., see UniProt acceptance number P34158), rabbit CFTR polypeptide (e.g., see UniProt acceptance number Q00554), rhesus monkey CFTR polypeptide (e.g., see UniProt acceptance number Q00553), etc., can be encoded by the polynucleotides of the Disclosure. In some embodiments, the CFTR polypeptides of this 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 with any of the amino acid sequences of the CFTR polypeptides described herein or known in the art. Methods for identifying additional species-derived CFTR polypeptide homologs / orthologues are known to those skilled in the art, including, for example, the use of amino acid sequence alignment programs such as BLAST® blastp suite or OrthoDB.

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

[0066] In some embodiments, the polynucleotide encoding the human CFTR polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of Sequence ID No. 5.

[0067] In some embodiments, the polynucleotide encoding the human CFTR polypeptide is a polynucleotide encoding an N-terminal cleavage, C-terminal cleavage, or fragment of the amino acid sequence of SEQ ID NO: 5. The N-terminal cleavage, C-terminal cleavage, or fragment may contain 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, or at least 1400 consecutive amino acids, but less than 1480.

[0068] In some embodiments, the polynucleotide encoding the human CFTR polypeptide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 6.

[0069] In some embodiments, the polynucleotide encoding the human CFTR polypeptide is a polynucleotide encoding an N-terminal cleavage, C-terminal cleavage, or fragment of the amino acid sequence of SEQ ID NO: 6. The N-terminal cleavage, C-terminal cleavage, or fragment may contain 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, or at least 1400 consecutive amino acids, but less than 1419.

[0070] In some embodiments, the polynucleotides of the Disclosure encoding the CFTR polypeptide are polynucleotides encoding 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 with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

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

[0072] Recombinant nucleic acids In some embodiments, this disclosure relates to recombinant nucleic acids comprising one or more polynucleotides described herein. In some embodiments, the recombinant nucleic acid is a vector (e.g., an expression vector, a display vector, etc.). In some embodiments, the vector is a DNA vector or an RNA vector. Generally, a vector suitable for maintaining, amplifying, and / or expressing polynucleotides to produce one or more polypeptides in a subject may be used. Examples of suitable vectors include plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, Sindbisvirus vectors, measles vectors, herpesvirus vectors, lentivirus vectors, retrovirus vectors, etc.). In some embodiments, the vector is a herpesvirus vector. In some embodiments, the vector can self-replicate in host cells. In some embodiments, the vector cannot self-replicate in host cells. In some embodiments, the vector can be incorporated into host DNA. In some embodiments, the vector cannot be incorporated into host DNA (e.g., it is episomal). For example, methods for producing a vector containing one or more target polynucleotides by chemical synthesis or artificial manipulation of isolated nucleic acid segments (e.g., genetic engineering techniques) are well known to those skilled in the art.

[0073] In some embodiments, the recombinant nucleic acids of this disclosure are herpes simplex virus (HSV) amplicons. Herpes virus amplicons, including their structural features, and methods for producing them are generally known to those skilled 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 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 acids of this disclosure are recombinant herpesvirus genomes. Recombinant herpesvirus genomes, including, for example, recombinant herpes simplex virus genomes, recombinant varicella-zoster virus genomes, recombinant human cytomegalovirus genomes, recombinant herpesvirus 6A genomes, recombinant herpesvirus 6B genomes, recombinant herpesvirus 7 genomes, recombinant Kaposi's sarcoma-associated herpesvirus genomes, and any combination or derivatives thereof, may be recombinant genomes derived from any member of the herpesviridae family of DNA viruses known in the art. As used herein, “inactivating mutation” may mean any mutation resulting in a gene or regulon product (RNA or protein) having reduced, undetectable, or eliminated amounts and / or function (compared to, for example, a corresponding sequence lacking the inactivating mutation). Examples of inactivating mutations include, but are not limited to, deletions, insertions, point mutations, and rearrangements of transcriptional regulatory sequences (promoters, enhancers, insulators, etc.) and / or coding sequences of a given gene or regulon. For example, any preferred method known in the art for measuring the amount of a gene or regulon product, including qPCR, Northern blotting, RNA-seq, Western blotting, ELISA, etc., may be used. In some embodiments, the recombinant herpesvirus genome contains one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) inactivating mutations. In some embodiments, one or more inactivating mutations are present in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) herpesvirus genes. In some embodiments, the recombinant herpesvirus genome is attenuated (e.g., compared to the corresponding wild-type herpesvirus genome). In some embodiments, the recombinant herpesvirus genome is replicable. 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 contains one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) inactivating mutations. In some embodiments, one or more inactivating mutations are present in one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) herpes simplex virus genes. In some embodiments, the recombinant herpes simplex virus genome is attenuated (e.g., compared to the corresponding wild-type herpes simplex virus genome). In some embodiments, the recombinant herpes simplex virus genome is replicable. In some embodiments, the recombinant herpes simplex virus genome is a replication defect.

[0076] In some embodiments, the recombinant herpes simplex virus genome is the 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- It may be derived 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, etc. (see, for example, Bowen et al. J Virol. 2019 Apr 3;93(8)). In some embodiments, the recombinant HSV-1 genome is derived from the KOS strain. In some embodiments, the recombinant HSV-1 genome is not derived from the McKrae strain. In some embodiments, the recombinant HSV-1 genome is attenuated. In some embodiments, the recombinant HSV-1 genome is replicable. In some embodiments, the recombinant HSV-1 genome is a replication defect. In some embodiments, the recombinant HSV-1 genome contains 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, 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 contains inactivating mutations in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the following herpes simplex virus genes: ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL)41, and / or UL55. In some embodiments, the recombinant herpes simplex virus genome does not contain an inactivating mutation in the ICP0 (one or both copies) herpes simplex virus gene (e.g., it can be expressed). In some embodiments, the recombinant herpes simplex virus genome does not contain an inactivating mutation in the ICP27 herpes simplex virus gene (e.g., it can be expressed). In some embodiments, the recombinant herpes simplex virus genome does not contain an inactivating mutation in the ICP47 herpes simplex virus gene (e.g., it can be expressed). In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP0 (one or both copies), ICP27, and / or ICP47 herpes simplex virus genes (e.g., they can be expressed). In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the linkage region. In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP34.5 (one or both copies) herpes simplex virus gene and / or ICP47 herpes simplex virus gene (e.g., to avoid the production of immunostimulatory viruses). In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP34.5 herpes simplex virus gene (one or both copies). In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP47 herpes simplex virus gene. In some embodiments, the recombinant herpes simplex virus genome does not contain inactivating mutations in the ICP34.5 (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 includes an inactivating mutation in one or both copies of the ICP0 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene and further includes inactivating mutations in one or both copies of the ICP4 gene, ICP22, ICP27, ICP47, UL41, and / or UL55 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene and an inactivating mutation in one or both copies of the ICP4 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene, an inactivating mutation in one or both copies of the ICP4 gene, and an inactivating mutation in the ICP22 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene, an inactivating mutation in one or both copies of the ICP4 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene, an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in one or both copies of the ICP0 gene, an inactivating mutation in one or both copies of the ICP4 gene, an inactivating mutation in the ICP22 gene, and an inactivating mutation in the UL41 gene. In some embodiments, the inactivating mutation is a deletion of the coding sequence of the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, and / or UL41 gene.In some embodiments, the recombinant herpes simplex virus genome further comprises inactivating mutations in the ICP27, ICP47, and / or UL55 genes.

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

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

[0082] In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP47 gene. In some embodiments, the recombinant herpes simplex virus genome contains an inactivating mutation in the ICP47 gene and further contains inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, UL41, and / or UL55 genes. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the ICP47 gene.

[0083] In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL41 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL41 gene and further includes inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL55 genes. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the UL41 gene.

[0084] In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL55 gene. In some embodiments, the recombinant herpes simplex virus genome includes an inactivating mutation in the UL55 gene and further includes inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, ICP47, and / or UL41 genes. In some embodiments, the inactivating mutation is a deletion in the coding sequence of the UL55 gene.

[0085] In some embodiments, the recombinant herpes simplex virus genome has internal repeat longs (IR). L ) area and internal repeat short (IR SThe internal repeat (joint) region containing the ) region includes an inactivating mutation (e.g., deletion thereof). In some embodiments, inactivation (e.g., deletion) of the joint region eliminates one copy each of the ICP4 and ICP0 genes. In some embodiments, 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 a pre-early promoter into the recombinant herpes simplex virus genome (see, e.g., Hill et al. (1995). Nature 375(6530):411-415, Goldsmith et al. (1998). J Exp Med 187(3):341-348). Without being constrained by theory, it is conceivable that inactivation (e.g., deletion) of joint regions may contribute to the stability of recombinant herpes simplex virus genomes and / or allow recombinant herpes simplex virus genomes to accommodate more and / or larger transgenes.

[0086] In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or both copies), ICP22, and ICP27 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or both copies), ICP27, and UL55 genes. In some embodiments, the recombinant herpes simplex virus genome contains inactivating mutations in the ICP4 (one or both copies), ICP22, ICP27, ICP47, and UL55 genes. In some embodiments, the inactivating mutations in the ICP4 (one or both copies), ICP27, and / or UL55 genes are deletions of the coding sequences in the ICP4 (one or both copies), ICP27, and / or UL55 genes. In some embodiments, the inactivating mutations in the ICP22 and ICP47 genes are deletions in the promoter regions of the ICP22 and ICP47 genes (e.g., the ICP22 and ICP47 coding sequences are intact but not transcriptionally active). In some embodiments, the recombinant herpes simplex virus genome includes deletions in the coding sequences of the ICP4 (one or both copies), ICP27, and UL55 genes, as well as deletions in the promoter regions of the ICP22 and ICP47 genes. In some embodiments, the recombinant herpes simplex virus genome further includes inactivating mutations in the ICP0 (one or both copies) and / or UL41 gene.

[0087] In some embodiments, the recombinant herpes simplex virus genome includes 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 includes 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 includes 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 includes inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and UL55 genes. In some embodiments, inactivating mutations in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or UL55 genes include deletions of the coding sequences in the ICP0 (one or both copies), ICP4 (one or both copies), ICP22, ICP27, and / or UL55 genes. In some embodiments, the recombinant herpes simplex virus genome further includes inactivating mutations in the ICP47 gene and / or UL41 gene.

[0088] In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of the Disclosure within one, two, three, four, five, six, seven, or more viral loci. Examples of preferred viral loci include, but are not limited to, the 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 contains one or more polynucleotides of the Disclosure within 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 contains one or more polynucleotides of the Disclosure within 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 contains 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 UL41 locus). In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of the 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 contains one or more polynucleotides of the 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 contains one or more polynucleotides of the 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, the recombinant herpes simplex virus genome contains one or more polynucleotides of the Disclosure within one or both of the viral ICP4 loci and one or more polynucleotides of the Disclosure within the viral ICP22 loci (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide in one or both of the ICP4 loci and a polynucleotide encoding a human CFTR polypeptide in the ICP22 loci). In some embodiments, the recombinant herpes simplex virus genome contains one or more polynucleotides of the Disclosure within one or both of the viral ICP4 loci and one or more polynucleotides of the Disclosure within the viral UL41 loci (e.g., a recombinant virus carrying a polynucleotide encoding a human CFTR polypeptide in one or both of the ICP4 loci and a polynucleotide encoding a human CFTR polypeptide in the UL41 loci). In some embodiments, the recombinant herpes simplex virus genome contains 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, the recombinant herpes simplex virus genome contains one or more polynucleotides of the Disclosure within one or both of the viral ICP4 loci, 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 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).In some embodiments, the recombinant herpes simplex virus genome includes one or more polynucleotides of the Disclosure in one or both of the viral ICP4 loci, one or more polynucleotides of the Disclosure in the viral ICP22 loci, one or more polynucleotides of the Disclosure in the viral UL41 loci, one or more polynucleotides of the Disclosure in one or both of the viral ICP0 loci, one or more polynucleotides of the Disclosure in the viral ICP27 loci, and / or one or more polynucleotides of the Disclosure in the ICP47 loci.

[0089] In some embodiments, the recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) is engineered to reduce or eliminate the 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, 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 target cells). In some embodiments, the target cells are human cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are mucosal cells. In some embodiments, the target cells are respiratory cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are airway epithelial cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are lung cells (primary cells or cell lines derived therefrom). In some embodiments, the cytotoxicity of recombinant herpesvirus genomes 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 herpesvirus genome (e.g., measurement of relative cytotoxicity of recombinant ΔICP4 (one or both copies) herpesvirus genome to wild-type herpesvirus genome in target cells, measurement of relative cytotoxicity of recombinant ΔICP4 (one or both copies) / ΔICP22 herpesvirus genome to wild-type herpesvirus genome in target cells, etc.).In some embodiments, the cytotoxicity of recombinant herpesvirus genomes is reduced by 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 75 times, at least about 1000 times, at least about 250 times, at least about 500 times, at least about 750 times, at least about 1000 times or more compared to the corresponding wild-type herpesvirus genome (e.g., measurement of relative cytotoxicity of recombinant ΔICP4 (one or both copies) herpesvirus genome to wild-type herpesvirus genome in target cells, measurement of relative cytotoxicity of recombinant ΔICP4 (one or both copies) / ΔICP22 herpesvirus genome to wild-type herpesvirus genome in target cells, etc.). For example, methods for measuring cytotoxicity, including the use of biological dyes (formazan dyes), protease biomarkers, MTT assays (or assays using related tetrazolium salts such as XTT, MTS, and water-soluble tetrazolium salts), and measurement of ATP content, are known to those skilled in the art.

[0090] In some embodiments, recombinant herpesvirus genomes (e.g., recombinant herpes simplex virus genomes) are engineered to reduce their effect on target cell proliferation after exposure to the recombinant genome compared to the corresponding wild-type herpesvirus genome. In some embodiments, the target cells are human cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are mucosal cells. In some embodiments, the target cells are respiratory cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are airway epithelial cells (primary cells or cell lines derived therefrom). In some embodiments, the target cells are lung cells (primary cells or cell lines derived therefrom). In some embodiments, target cell proliferation after exposure to a 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 after exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to cell proliferation after exposure to a wild-type herpes simplex virus genome in target cells, or the relative cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to cell proliferation after exposure to a wild-type herpes simplex virus genome in target cells.In some embodiments, target cell proliferation after exposure to a 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 75 times, at least about 1000 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 after exposure to a recombinant ΔICP4 (one or both copies) herpes simplex virus genome compared to cell proliferation after exposure to a wild-type herpes simplex virus genome, or at least about 1000 times faster than target cell proliferation after exposure to a recombinant ΔICP4 (one or both copies) / ΔICP22 herpes simplex virus genome compared to cell proliferation after exposure to a wild-type herpes simplex virus genome. For example, methods for measuring cell proliferation, including the use of Ki67 cell proliferation assays and BrdU cell proliferation assays, are known to those skilled in the art.

[0091] A vector (e.g., a herpesvirus vector) may contain one or more polynucleotides of the Disclosure in a form suitable for expression of polynucleotides in host cells. The vector may contain one or more regulatory sequences operatively linked to the polynucleotide to be expressed (e.g., as described above).

[0092] In some embodiments, the recombinant nucleic acid of this disclosure (e.g., recombinant herpes simplex virus genome) comprises one or more polynucleotides described herein inserted into the recombinant nucleic acid in any orientation. If the recombinant nucleic acid comprises two or more polynucleotides described herein (e.g., two or more, three or more, etc.), these polynucleotides may be inserted in the same orientation or in opposite orientations to each other. Without wishing to be bound by theory, incorporating two polynucleotides (e.g., two transgenes) into the recombinant nucleic acid (e.g., a vector) in antisense orientation may help to avoid read-through and ensure proper expression of each polynucleotide.

[0093] IV. Viruses Certain aspects of this disclosure relate to viruses comprising any of the polynucleotides and / or recombinant nucleic acids described herein. In some embodiments, the virus can infect one or more target cells of a subject (e.g., human). In some embodiments, the virus is suitable for delivering polynucleotides and / or recombinant nucleic acids to one or more target cells of a subject (e.g., 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 CFTR deficiency (e.g., one or more cells containing genomic mutations in the 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, 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, mesocellular or parabasal cells, serous cells, brush cells, oncocytes, non-ciliated columnar cells, and / or metaplastic cells), alveolar cells (type I lung cells, type II lung cells, and / or cuboid non-ciliated cells, etc.), salivary gland cells in the bronchi (serous cells, mucosal cells, and / or ductal cells, etc.)). In some embodiments, one or more target cells are one or more cells of the lung.

[0094] Any suitable virus known in the art may be used, for example, including adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, Sendai viruses, herpesviruses, vaccinia viruses, and / or any hybrid or derivative viruses thereof. In some embodiments, the virus is attenuated. In some embodiments, the virus is replicable. In some embodiments, the virus is replication-deficient. 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 skilled 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, herpesvirus 6A, herpesvirus 6B, herpesvirus 7, and Kaposi's sarcoma-associated herpesvirus. In some embodiments, the herpesvirus is attenuated. In some embodiments, the herpesvirus is replication-deficient. In some embodiments, the herpesvirus is replicable. In some embodiments, the herpesvirus is replicable. In some embodiments, the herpesvirus is engineered to reduce or eliminate the expression of one or more herpesvirus genes (e.g., one or more virulent herpesvirus genes). In some embodiments, the herpesvirus has reduced cytotoxicity compared to the corresponding wild-type herpesvirus. In some embodiments, the herpesvirus is not oncolytic.

[0096] In some embodiments, the herpesvirus is herpes simplex virus. Herpes simplex viruses containing recombinant nucleic acids can be produced by processes disclosed, for example, in 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 replicable. In some embodiments, the herpes simplex virus is replicable. In some embodiments, the herpes simplex virus is engineered to reduce or eliminate the expression of one or more herpes simplex virus genes (e.g., one or more virulent 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 the HSV-1 virus, HSV-2, or any derivative thereof. In some embodiments, the herpes simplex virus is the HSV-1 virus. In some embodiments, the herpes simplex virus is HSV-1. In some embodiments, HSV-1 is attenuated. In some embodiments, HSV-1 is replication-deficient. In some embodiments, HSV-1 is replicable. In some embodiments, HSV-1 is replicateable. In some embodiments, HSV-1 is engineered to reduce or eliminate the expression of one or more HSV-1 genes (e.g., one or more toxic HSV-1 genes). In some embodiments, HSV-1 has reduced cytotoxicity compared to the corresponding wild-type HSV-1. In some embodiments, HSV-1 is not oncolytic.

[0097] In some embodiments, the herpes simplex virus is modified to alter its tissue tropism compared to that of unmodified wild-type herpes simplex virus. In some embodiments, the herpes simplex virus includes a modified envelope. In some embodiments, the modified envelope includes 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 include, but are not limited to, glycoproteins gB, gC, gD, gH, and gL. In some embodiments, the modified envelope alters the herpes simplex virus tissue tropism compared to wild-type herpes simplex virus.

[0098] In some embodiments, the transduction efficiency (in vitro and / or in vivo) of the virus of the Disclosure (e.g., herpesvirus) to 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 to one or more target cells may 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 higher. In some embodiments, the virus is herpes simplex virus, and the transduction efficiency of the virus to 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 transduction efficiency of the virus to 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 the transduction efficiency of the virus in vitro or in vivo, including, for example, qPCR analysis, deep sequencing, Western blotting, and fluorescence quantitative analysis (fluorescence insight hybridization (FISH), fluorescence reporter gene expression, immunofluorescence, FACS, etc.), are well known to those skilled in the art.

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

[0100] In some embodiments, the pharmaceutical composition or formulation comprises any one or more of the viruses (e.g., herpesviruses) described herein. In some embodiments, the pharmaceutical composition or formulation comprises from about 10 4 to about 10 12 plaque forming units (PFU) / mL of virus. For example, the pharmaceutical composition or formulation comprises from about 10 4 to about 10 12 , from about 10 5 to about 10 12 , from about 10 6 to about 10 12 , from about 10 7 to about 10 12 , from about 10 8 to about 10 12 , from about 10 9 to about 10 12 , from about 10 10 to about 10 12 , from about 10 11 to about 10 12 , from about 10 4 to about 10 11 , from about 10 5 to about 10 11 , from about 10 6 to about 10 11 , from about 10 7 to about 10 11 , from about 10 8 [[ID=STRIKE]] 11 11 , from about 10 9 to about 10 11 , from about 10 10 to about 10 11 , from about 10 4 to about 10 10 , from about 10 5 to about 1​​​​​​​10 , about 10 8 ~about 10 10 , about 10 9 ~about 10 10 , about 10 4 ~about 10 9 , about 10 5 ~about 10 9 , about 10 6 ~about 10 9 , about 10 7 ~about 10 9 , about 10 8 ~about 10 9 , about 10 4 ~about 10 8 , about 10 5 ~about 10 8 , about 10 6 ~approximately 108, approximately 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 It may contain virus at a concentration of PFU / mL. In some embodiments, the pharmaceutical composition or formulation may contain about 10 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 virus at PFU / mL.

[0101] Pharmaceutical compositions and formulations can be prepared by mixing one or more active ingredients (recombinant nucleic acids and / or viruses, etc.) of desired purity with one or more pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers or excipients are generally non-toxic to the recipient at the dosage and concentration used and include buffers (phosphoric acid, citrate, acetic acid, and other organic acids, etc.), antioxidants (ascorbic acid and methionine, etc.), preservatives (octadecyldimethylbenzylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkylparabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, etc.), amino acids (glycine, glutamine, asparagine, histidine, arginine, or glycine, glutamine, asparagine, histidine, arginine, or glycine, glutamine, asparagine, histidine, arginine, or glycine, glutamine, asparagine, histidine, arginine, or glycine, glutamine, asparagine, histidine, arginine, or glycine, glutamine, asparagine, arginine, or glycine, glutamine These may include dins, low molecular weight (less than approximately 10 residues) polypeptides, proteins (serum albumin, gelatin, or immunoglobulins, etc.), polyols (glycerol, etc., e.g., formulations containing 10% glycerol), hydrophilic polymers (polyvinylpyrrolidone, etc.), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin), chelating agents (EDTA, etc.), sugars (sucrose, mannitol, trehalose, or sorbitol, etc.), salt-forming counterions (sodium, etc.), metal complexes (Zn-protein complexes, etc.), and / or nonionic surfactants (polyethylene glycol (PEG), etc.). A detailed discussion of pharmaceutically 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 capable of carrying encapsulated drugs (e.g., synthetic small molecules, proteins, peptides, cells, viruses, and nucleic acid-based biotherapeutic agents) for rapid or controlled release. Various molecules (e.g., proteins, peptides, recombinant nucleic acids, etc.) can be efficiently encapsulated in nanoparticles using processes well known in the art. In some embodiments, a molecule "encapsulated" in a nanoparticle may refer to a molecule (e.g., a virus) that is contained within 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 may be any type of biocompatible nanoparticle known in the art, including, for example, nanoparticles containing poly(lactic acid), poly(glycolic acid), PLGA, PLA, PGA, and any combination thereof (see, for example, 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, a pharmaceutically acceptable carrier or excipient may be adapted to or preferred to any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, cutaneous, oral, intranasal, intratracheal, sublingual, oral cavity, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreous, subretinal, transmucosal, intra-articular, implantable, inhalation, intrathecal, intraventricular, and / or intranasal administration. In some embodiments, a pharmaceutically acceptable carrier or excipient may be adapted to or preferred to oral, intranasal, intratracheal, and / or inhalation administration. In some embodiments, a pharmaceutically acceptable carrier or excipient may be adapted to or preferred to inhalation administration. In some embodiments, a pharmaceutically acceptable carrier or excipient may be adapted to or preferred to non-invasive inhalation administration. In some embodiments, a pharmaceutically acceptable carrier or excipient may be adapted to or preferred to aerosol (using a vibrating mesh nebulizer).

[0104] In some embodiments, the pharmaceutical composition or formulation may be adapted to or preferred to any route of administration known in the art, including, for example, intravenous, intramuscular, subcutaneous, cutaneous, oral, intranasal, intratracheal, sublingual, oral cavity, topical, transdermal, intradermal, intraperitoneal, intraorbital, intravitreous, subretinal, transmucosal, intra-articular, implantable, inhalation, intrathecal, intraventricular, or intranasal administration. In some embodiments, the pharmaceutical composition or formulation may be adapted to or preferred to be administered orally, intranasally, intratracheally, or by inhalation. In some embodiments, the pharmaceutical composition or formulation may be adapted to or preferred to be administered by inhalation. In some embodiments, the pharmaceutical composition or formulation may be adapted to or preferred to be administered by non-invasive inhalation. In some embodiments, the pharmaceutical composition or formulation may be adapted to or preferred to be administered by spraying (using a vibrating mesh nebulizer).

[0105] In some embodiments, the pharmaceutical composition or formulation further comprises one or more additional components. Examples of additional components include binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate) Examples of such substances include, but are not limited to, sodium acetate, disintegrants (e.g., starch, sodium starch glycolate, etc.), humectants (e.g., sodium lauryl sulfate, etc.), salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, sweeteners, flavorings, fragrances, colorants, humectants, sunscreens, antibacterial agents, and agents that can stabilize polynucleotides or prevent their degradation. 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 contains 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 contain glycerol.

[0106] Pharmaceutical compositions and formulations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

[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 target cells (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 treat diseases or conditions that would benefit from the expression of CFTR polypeptide (e.g., diseases associated with CFTR deficiency, and / or diseases associated with CFTR gene mutations). 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 chronic lung diseases (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 agents useful for delivering one or more polynucleotides encoding CFTR polypeptide to one or more target cells (e.g., one or more CFTR-deficient cells, one or more cells with CFTR gene mutations, 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 agents useful for the prevention or treatment of diseases or conditions that would benefit from the expression of CFTR polypeptide (e.g., diseases associated with CFTR deficiency, and / or diseases associated with CFTR gene mutations). In some embodiments, any of the recombinant nucleic acids, viruses, and / or pharmaceutical compositions or formulations described herein may be used to prepare agents useful for the prevention or treatment of chronic lung diseases (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 agents useful for the prevention or treatment of cystic fibrosis.

[0109] VI. Method Certain embodiments of this disclosure relate to enhancing, increasing, boosting, 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, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome contains mutations (e.g., loss-of-function mutations) in one or both copies of the endogenous CFTR gene. 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 recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations 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 twofold compared to the endogenous CFTR level in one or more corresponding untreated cells of the subject. For example, administration of recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations can increase the CFTR level (transcript or protein level) in one or more contacted or treated cells of the subject by at least about twofold, at least about threefold, at least about fourfold, at least about fivefold, at least about sixfold, at least about sevenfold, at least about eightfold, at least about ninefold, at least about tenfold, at least about fifteenfold, at least about twentyfold, at least about twenty-fivefold, at least about fifteenfold, at least about fifteenfold, at least about seventeenfold, at least about fifteenfold, at least about seventeenfold, at least about five hundredfold, at least about seventeenfold, at least about seventeenfold, at least about tenteenfold, at least about fifteenfold, at least about twentyfold, at least about twenty-fivefold, at least about fifteenfold, at least about seventeenfold, at least about fifteenfold, at least about fifteenfold, at least about twentyfold, at least about twenty-fifteenfold, at least about fifteenfold, at least about fifteenfold, at least about seventeenfold, at least about fif In some embodiments, one or more contact cells 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 the submucosal gland). Methods for measuring transcript or protein levels from a sample, including, for example, qPCR, Western blotting, mass spectrometry, etc., are well known to those skilled in the art.

[0111] Other aspects of this disclosure relate to a method for reducing cellular sodium levels in a subject requiring a reduction in cellular sodium levels, comprising administering to the subject one of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome contains a mutation (e.g., loss-of-function mutation) in one or both copies of the endogenous CFTR gene. 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.

[0112] In some embodiments, administration of recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations to a subject reduces the intracellular sodium level 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 the intracellular sodium level in one or more corresponding untreated cells in the subject. Methods for measuring intracellular sodium levels are generally known to those skilled in the art.

[0113] Other aspects of this disclosure relate to a method for performing an improvement in the measurement of at least one respiratory volume in a subject requiring such improvement, comprising administering to the subject one of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome contains a mutation (e.g., loss-of-function mutation) in one or both copies of the endogenous CFTR gene. In some embodiments, the subject suffers from a chronic lung disease, such as cystic fibrosis, COPD, etc.

[0114] In some embodiments, administration to a subject of recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations improves at least one respiratory volume measurement by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or more compared to at least one reference respiratory volume measured in the subject before treatment. Examples of suitable respiratory volumes that can be measured include, for example, total vital capacity (TLC), the volume of the lungs at maximum expansion; tidal volume (TV), the amount of air that moves in and out of the lungs during quiet breathing; residual volume (RV), the amount of air remaining in the lungs after maximum exhalation; expiratory reserve volume (ERV), the maximum amount that can be exhaled (exceeding tidal volume) during forced breathing; inspiratory reserve volume (ERV), the maximum amount of air that can be inhaled from the end-spiratory 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 expiratory volume; vital capacity (VC), the amount of air exhaled after the deepest inspiration; functional residual capacity (FRC), the volume of the lungs at the end-expiratory position; forced vital capacity (FVC), the determination of vital capacity from maximal forced expiratory effort; forced expiratory volume (time) (FEV1). t Examples include the amount of air exhaled under forced conditions during the first t seconds; forced inspiratory flow (FIF), specific measurements of the forced inspiratory curve; peak expiratory flow (PEF), the maximum forced expiratory flow measured with a peak flow meter; and maximal tidal volume (MVV), the amount of air exhaled during a specific period of repeated maximal effort. Methods for measuring respiratory volume are generally known to those skilled in the art.

[0115] Other aspects of this disclosure relate to methods for reducing or preventing chronic bacterial infections in the lungs in subjects requiring such reduction or prevention, comprising administering to the subject any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome includes a mutation (e.g., loss-of-function mutation) in one or both copies of the endogenous CFTR gene. 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 for monitoring bacterial infections in the lungs, including, for example, by performing blood tests or cultures, oxygen measurements, arterial blood gas measurements, bronchoscopy, transtracheal mucus culture, lung biopsy, thoracentesis, computed tomography scans, etc., including improvements thereto, are known to those skilled in the art.

[0116] Other aspects of this disclosure relate to methods for reducing, preventing, or treating chronic bacterial infections of the lungs in subjects requiring such treatment, comprising administering to the subject any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome includes a mutation (e.g., loss-of-function mutation) in one or both copies of the endogenous CFTR gene. 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 for measuring pneumonia, including, for example, measuring exhaled nitric oxide, determining the percentage of eosinophils in sputum and blood, including improvements thereto, are well known to those skilled in the art.

[0117] Other aspects of this disclosure relate to methods for reducing, inhibiting, or treating progressive lung injury in subjects requiring such treatment, comprising administering to the subject any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome contains a mutation (e.g., loss-of-function mutation) in one or both copies of the endogenous CFTR gene. 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 for measuring lung injury, including, for example, by the method described by Saetta et al. (Am Rev Respir Dis. 1985 May; 131(5): 764-9), are well known to those skilled in the art.

[0118] Other aspects of this disclosure relate to methods for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cystic fibrosis in a subject, comprising administering to the subject any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the subject is human. In some embodiments, the subject's genome contains mutations (e.g., loss-of-function mutations) in one or both copies of the endogenous CFTR gene.

[0119] Signs and symptoms of cystic fibrosis may include, but are not limited to, persistent cough producing large amounts of mucus, large amounts of sticky mucus accumulating in the airways, wheezing, dyspnea, sinusitis, recurrent lung infections, inflammatory nasal sinuses, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof.

[0120] Other aspects of this disclosure relate to methods for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of COPD in a subject, comprising administering to the subject an effective amount of any recombinant nucleic acid, virus, drug, and / or pharmaceutical composition or formulation described herein. In some embodiments, the subject is human. In some embodiments, the subject is a smoker or former smoker.

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

[0122] The recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein may be administered by any preferred method or route known in the art, including, but not limited to, oral, intranasal, intratracheal, sublingual, oral cavity, topical, rectal, inhalation, percutaneous, subcutaneous, intradermal, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intraperitoneal, transmucosal, intravaginal, intravitreous, intraorbital, subretinal, intra-articular, periarticular, local, on the skin, or any combination thereof. Accordingly, this disclosure includes methods for delivering any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein to an individual (for example, an individual having or at risk of developing a chronic lung disease such as cystic fibrosis).

[0123] In some embodiments, the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein are administered orally, intranasally, intratracheally, or by inhalation. Methods of delivering drugs to the lungs via oral, intranasal, intratracheally, and / or inhalation routes are generally known to those skilled in the art (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, recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations are delivered to the lungs by inhalation of an aerosolized formulation. Inhalation may occur through the nose and / or mouth of the subject. Exemplary devices for delivering recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations to the lungs may 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), impact jets, extrusion jets, surface wave microfluidic atomizers, capillary aerosol generators, electrohydrodynamic aerosol devices, etc. (see, for example, 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 target lungs, for example, using a pressurized metered-dose inhaler (pMDI). A pMDI typically comprises 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 operate the canister. The canister may contain a single-dose or multi-dose formulation. The canister may include a valve, typically a metering valve, from which the contents of the canister can be discharged. The aerosolized drug is dispensed from the pMDI by applying force to the canister, pushing it into the receptacle, thereby opening the valve and allowing the drug particles to be transported from the valve through the receptacle outlet. Upon discharge from the canister, the liquid formulation is sprayed and forms an aerosol. A pMDI typically uses 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 may be used, and can take a variety of forms, for example, including compressed or liquefied gases.

[0126] Liquid formulations can be administered to the target lungs, for example, using a nebulizer. A nebulizer is a liquid aerosol generator that converts a liquid formulation into a cloud of mist or small droplets, often having an aerodynamic mass median particle diameter of less than approximately 5 microns, which can be inhaled into the lower respiratory tract. The droplets, once the aerosol cloud is inhaled, carry the active agent(s) to the nose, upper respiratory tract, and / or deep lungs. Any type of nebulizer known in the art, including but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers (e.g., ultrasonic nebulizers, vibrating mesh nebulizers, etc.), can be used to administer formulations to patients. Pneumatic (jet) nebulizers use a pressurized gas supply as the driving force for atomizing the liquid formulation. The compressed gas is delivered through a nozzle or jet, creating a low-pressure region that draws in the surrounding liquid formulation and shears it into a thin membrane or filament. The membrane or filament is unstable and breaks into small droplets that are carried by the compressed gas flowing into inspiratory respiration. A baffle inserted into the droplet plume filters out larger droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers use electrically generated mechanical force to atomize liquid formulations. Electromechanical driving force can be applied, for example, by vibrating the liquid formulation at an ultrasonic frequency, or by forcing the bulk liquid through small pores in a thin film. The force generates a thin liquid film or filamentous flow, which breaks into small droplets, forming a slow-moving aerosol flow that can be trapped in the inhalation flow. In some embodiments, the nebulizer is a vibrating mesh nebulizer. Examples of vibrating mesh nebulizers include, for example, the Phillips InnoSpire, Aerogen Solo, and PARI eFlow.

[0127] Liquid formulations can be administered to the target lungs, for example, using an electrohydrodynamic (EHD) aerosol device. An EHD aerosol device uses 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). A DPI typically uses a mechanism such as gas bursting to create a cloud of dry powder inside the container, which is then inhaled by the subject. In a DPI, the dose to be administered is stored in the form of unpressurized dry powder, and when the inhaler is activated, the powder particles are inhaled by the subject. In some cases, compressed gas may be used to distribute the powder, similar to a pMDI. In some cases, a DPI can be respiratory-activated (an aerosol is produced in precise response to inhalation). Typically, dry powder inhalers administer doses of less than tens of milligrams per inhalation to avoid inducing cough. Examples of DPIs include, for example, the Turbohaler® inhaler (AstraZeneca), the Clickhaler® inhaler (Innovata), the Diskus® inhaler (Glaxo), the EasyHaler® inhaler (Orion), and the Exubera® inhaler (Pfizer).

[0129] In some embodiments, recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations are administered to a subject once. In some embodiments, recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations are administered to a subject at least twice (e.g., at least two times, at least three times, at least four times, at least five times, at least ten times, etc.). In some embodiments, at least one hour (for example, at least about one hour, at least about six hours, at least about twelve hours, at least about eighteen hours, at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about fifteen days, at least about twenty days, at least about thirty days, at least about forty days, at least about fifty days, at least about sixty days, at least about seven days, at least about fifteen days, at least about twenty days, at least about thirty days, at least about forty days, at least about fifty days, at least about sixty days, at least about seventy days, at least about eighty days, at least about ninety days, at least about one hundred days, at least about one In some embodiments, recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations are administered to a subject once, twice, three, four, five, or more times per month. In some embodiments, recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations are administered to a subject once, twice, three, four, five, or more times per year.

[0130] VII.Host cells Certain aspects of this disclosure relate to one or more host cells containing any of the recombinant nucleic acids described herein. For example, prokaryotic cells including eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae including Escherichia (e.g., E. coli), Enterobacter, Erminia, Klebsiella, Proteus, Salmonella (e.g., S. typhimurium), Serratia (e.g., S. marcescans), and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, fungal cells (e.g., S. cerevisiae), insect cells (e.g., S2 cells), and mammalian cells (e.g., monkey kidney CV1 strain (COS-7, ATCC CRL 1651) transformed with SV40, human embryonic kidney strain (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 10) Any suitable host cells (prokaryotic or eukaryotic) known in the art may be used, including 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL 51), TRI cells, MRC5 cells, FS4 cells, human hepatoma (Hep G2), Chinese hamster ovary (CHO) cells (e.g., DHFR'CHO cells), and myeloma cell lines such as NS0 and Sp2 / 0. In some embodiments, the host cells are human or non-human primate cells. In some embodiments, the host cells are cells derived from a cell line.Suitable host cells or cell lines 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, and CHO cells.

[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 as described herein, resulting in the production of a virus containing one or more recombinant nucleic acids as 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 complementary host cell. In some embodiments, the complementary host cell expresses one or more genes that are inactivated in any of the viral vectors described herein. In some embodiments, the complementary host cell is in contact with a recombinant herpesvirus genome (e.g., recombinant herpes simplex virus genome) described herein. In some embodiments, contact of the complementary host cell with the recombinant herpesvirus genome results in the production of a herpesvirus 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 complementary host cell with 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 embodiments of this disclosure relate to a product or kit comprising any of the recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations described herein. In some embodiments, the product or kit includes a package insert containing instructions for administering the recombinant nucleic acid, virus, drug, and / or pharmaceutical composition or formulation for the treatment of CFTR deficiency (e.g., in subjects with homozygous CFTR loss-of-function mutations) and / or for the prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of a chronic lung disease (such as cystic fibrosis or COPD). In some embodiments, the product or kit further includes a device for administering the recombinant nucleic acid, virus, drug, and / or pharmaceutical composition or formulation. In some embodiments, the device is a nebulizer (e.g., a vibrating mesh nebulizer).

[0134] Suitable containers for recombinant nucleic acids, viruses, drugs, and / or pharmaceutical compositions or formulations may include, for example, bottles, vials, bags, tubes, and syringes. Containers may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container may include a label on the container or a label associated with the container, which indicates instructions for use. The product or kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, inhalers, nebulizers, intranasal administration devices, and accompanying documents.

[0135] This specification is considered sufficient to enable those skilled in the art to implement the disclosure. In addition to the modifications shown and described herein, various modifications to the disclosure will be apparent to those skilled in the art from the foregoing description and will fall within the scope of the appended claims. [Examples]

[0136] This disclosure will be better understood by referring to the following examples. However, this should not be construed as limiting the scope of this disclosure. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof are proposed to those skilled in the art and should be understood as being within the spirit and scope of this application and the appended claims.

[0137] Example 1: Modified herpes simplex virus vector encoding human CFTR protein To construct modified herpes simplex virus vectors capable of expressing CFTR polypeptides in target mammalian cells (such as lung cells), the herpes simplex virus genome (Figure 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. Next, variants of these modified / attenuated recombinant viral constructs are generated so that they carry one or more polynucleotides encoding the desired CFTR polypeptide.These variants include: 1) a recombinant ΔICP4-modified HSV-1 genome containing an expression cassette with the 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 containing an expression cassette with the coding sequence of a human CFTR polypeptide (e.g., SEQ ID NO: 2) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1C); 3) a recombinant ΔICP4 / ΔUL41-modified HSV-1 genome containing an expression cassette with the coding sequence of a human CFTR polypeptide (e.g., SEQ ID NO: 2) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1D); and 4) a recombinant ΔICP4 / ΔICP22-modified HSV-1 genome containing an expression cassette with the coding sequence of a human CFTR polypeptide (e.g., SEQ ID NO: 2) under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1E). 5) Recombinant ΔICP4 / ΔICP22 modified HSV-1 genome including an expression cassette containing the coding sequence of human CFTR polypeptide under the control of a heterologous promoter integrated into the ICP22 locus (Figure 1F), 6) Recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome including an expression cassette containing the coding sequence of human CFTR polypeptide under the control of a heterologous promoter integrated into each ICP4 locus (Figure 1G), 7) Recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome including an expression cassette containing the coding sequence of human CFTR polypeptide under the control of a heterologous promoter integrated into the UL41 locus (Figure 1H), and 8) Recombinant ΔICP4 / ΔUL41 / ΔICP22 modified HSV-1 genome including an expression cassette containing the coding sequence of human CFTR polypeptide under the control of a heterologous promoter integrated into the ICP22 locus (Figure 1I).

[0138] These modified herpes simplex virus genome vectors are transfected into engineered cells modified to express one or more herpesvirus genes. These engineered cells secrete replication-deficient herpes simplex viruses, which have the modified genomes packaged within them, into the supernatant of the cell culture. The supernatant is then collected, concentrated, and filtered through a 5 μm filter for sterile filtration.

[0139] Example 2: Construction and in vitro characterization of an HSV-1 vector encoding human CFTR in 2D cultures. Following the discovery of the genetic defect causing cystic fibrosis, early clinical trials of lung gene therapy were conducted in the early 1990s. Recombinant adenovirus was one of the early vectors tested for CFTR delivery, but adeno-based viruses failed these trials, mainly due to a lack 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 adeno-associated virus-based (numerous AAV serotypes have been tested in CF clinical settings). Large-scale repeated-dose studies of AAV-based gene therapy vectors have yielded disappointing results in improving CF lung function in treated patients. Similar to adenoviruses, recombinant AAV vectors do not efficiently infect the apical lung surface, and due to the physical limitations of the encoded cargo size, AAV vectors do not efficiently deliver full-length human CFTR. Despite more than 20 years of intensive effort, virus-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 virus-based gene therapy vectors are in preclinical development (both of which are based on AAV, a vector that has already failed in multiple clinical trials in CF patients, as mentioned above). Instead, the focus is shifting from virus-based vectors to non-viral methods of CFTR delivery (e.g., DNA plasmids or mRNA complexed with liposomes). Unfortunately, these non-viral vectors have achieved only limited success, at least in part, due 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 demonstrate clinical benefit, primarily due to inefficient gene delivery to target cells and host immune-mediated clearance after repeated exposure.

[0141] For this purpose, a recombinant herpes simplex virus type 1 (HSV-1) vector encoding full-length human CFTR (HSV-CFTR) was developed as a novel gene therapy for the treatment of CF patients. Without wishing to be constrained by theory, it is conceivable that the HSV-based approach overcomes many of the hurdles experienced with other gene therapy vectors for CF, including its 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 a product using the same viral scaffold as HSV-CFTR in the context of the highly inflammatory environment of wounded skin (ClinicalTrials.gov identifier: NCT03536143). The following examples describe experiments demonstrating that this novel HSV-based gene therapy vector was able to dose-dependently express functional full-length human CFTR in small airway epithelial cells (SAECs) derived from cystic fibrosis patients.

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

[0143] CFTR protein expression in primary CF SAECs infected with HSV-CFTR was evaluated via Western blotting. GAPDH was used as a control to ensure consistent loading of samples. CF patient SAECs overexpressed human CFTR upon HSV-CFTR infection compared to mock-infected control cells (Figure 3). Interestingly, endogenous CFTR protein in mock-infected cells was analyzed 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 significantly larger double line. Human CFTR is known to exist in three distinct forms depending on its glycosylation status: (1) non-glycosylated, (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 suggests that the endogenous (mutant) protein exists only in a non-glycosylated form, indicating an immature protein variant that is not properly transported to the cell surface via the endoplasmic reticulum (ER). 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 glycosylation and complex glycosylation variants of CFTR, suggesting proper maturation and transport of the exogenous protein via the ER.

[0144] Next, CFTR protein expression and relative localization were investigated by immunofluorescence. Primary CF patient SAECs were transduced with HSV-CFTR for 48 hours at a specified MOI, and immunofluorescence staining for 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 disease cells. When analyzed in the context of control cells, immunofluorescence data demonstrated that transduced SAECs showed a dose-dependent increase in CFTR protein expression in HSV-CFTR (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 transported to the perinuclear region (suggesting capture and turnover in the ER), while CFTR was found throughout the cytoplasm and on the cell surface of HSV-CFTR transduced cells (indicating proper maturation and transport in the ER). This data was consistent with Western blot data suggesting that CFTR expressed by wild-type HSV-CFTR was completely glycosylated, while endogenous mutant CFTR was not glycosylated (Figure 3).

[0145] Finally, the functionality of HSV-CFTR-expressing human CFTR in infected CF patient SAECs was confirmed using a dihydrorhodamine 6G (dR6G) fluorescence uptake assay, which had been previously validated as a functional endpoint for virus-mediated CFTR recovery in 2D CF patient epithelial cell cultures (Wersto, 1996, Proc Natl Acad Sci USA, 93(3), pp.1167-72). Briefly, primary CF patient SAECs infected with HSV-CFTR or mock-infected cells were incubated in dR6G-containing cell culture medium for 15 minutes, washed four times with PBS, lysed in RIPA buffer, and the 526 nm excitation / 555 nm emission fluorescence was read for each sample on a plate reader. dR6G itself is nonfluorescent, but it is converted to the fluorescent compound rhodamine 6G upon cellular uptake and exposure to intracellular dehydrogenases, a process dependent 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 the total protein content, and the relative fluorescence per μg of total protein was calculated for each sample (Figure 5). HSV-CFTR infection in primary CF patient SAECs resulted in a moderate dose-dependent increase in dR6G uptake compared to mock-infected controls, demonstrating 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 organoid cultures using CF patient-derived organoids Mutations in the CFTR gene are classified into one of six classes based on the primary mechanism that leads to CFTR dysfunction. Mutations affecting synthesis and processing result in more severe disease because little of the protein reaches the cell surface and do not interfere with luminal transport, while 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 characteristic stages of protein synthesis and function, recent drug development efforts have focused on small molecule modulator therapies that target specific sources of protein defects. For example, ivacaftor, classified as a CFTR protein "potensiator," enhances chloride secretion of membrane CFTR (providing clinical benefit to individuals with certain class III and IV CFTR gating and conductance mutations), while elexacaftor, classified as a CFTR protein "collector," acts by promoting proper folding and cellular processing of CFTR, which would otherwise be degraded by the endoplasmic reticulum quality control pathway (providing clinical benefit to individuals with certain class II CFTR transport mutations) (Clancy, 2019, Am J Respir Crit Care Med, 186(7), pp.593-7). While the recent FDA approvals of four of these modulator therapies have benefited CF patients with specific mutations that respond to these drugs, these modulators treat only a subset of the CF population. In particular, the need for effective drug intervention is heightened in patients with class I mutations (accounting for approximately 10% of CF cases worldwide), including nonsense mutations resulting in frameshift, splicing, and severely reduced or absent CFTR expression, and these patients suffer from the most severe and fatal forms of CF (Wilschanski, 2012, Front Pharmacol, 20(3), pp.1-3).

[0147] Due to the lack of suitable animal models for CF, efficacy studies of air-liquid interface differentiated bronchial epithelial cells derived from extrapulmonary transplant material from CF patients have been used for some drug development efforts following proof-of-concept experiments in xenogeneic 2D cell lines (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 extrapulmonary transplant tissue and the invasive procedures required to obtain bronchial cells from CF patients without terminal disease have led to the development of 3D organoid systems derived from “easily accessible” tissues taken from CFTR variant patients for testing novel therapeutic agents 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 pharmacological interventions, 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 transport of ions and water into the organoid lumen, while organoids derived from CFTR mutant patient biopsies (or wild-type organoids exposed to specific pharmacological inhibition of CFTR protein function) show reduced or complete inhibition of swelling volume (Boj, 2017, J Vis Exp, 120(1), p. e55159). The use of CF PDOs enables quantitative measurement of CFTR protein function during treatment with novel therapeutic agents (via detection of organoid swelling), and positive results from this 3D organoid system have been shown to directly correlate with clinical benefits, including changes in lung response and sweat chloride concentration, in treated CF patients (Berkers, 2019, Cell Rep, 26(7), pp. 1701-1708).

[0148] The following examples illustrate experiments demonstrating that the recombinant HSV-1 vector HSV-CFTR, characterized in Example 2 above, can 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 organoid 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 evaluate CFTR activity in transduced organoids, organoid morphology and size were assessed 24 or 48 hours after infection, and FIS assays were performed as described above (Boj, 2017, J Vis Exp, 120(1), p.e55159). For efficient infection of CF organoids, the organoids were sheared into small fragments, incubated with HSV-CFTR in solution for 1 hour at the indicated MOI, and seeded onto 96-well clear bottom plates for analysis. FIS assays were performed 24 or 48 hours after seeding, as described in more detail below.

[0150] First, G542X / G542X PDO was infected at MOIs of 10, 20, and 40 to evaluate the effects of the vector on both organoid swelling and cell viability. Intestinal organoids from healthy patients were plated in parallel as a control. Surprisingly, 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 vector manipulated before forskolin addition (Figure 6A). Using an mCherry-expressing HSV vector as a negative control, it was shown that the alteration of PDO morphology observed in HSV-CFTR treated samples was not due to a nonspecific response to viral infection. Next, the FIS assay was performed 48 hours after infection. At t=0, prior to forskolin addition and subsequent CFTR activation, HSV-CFTR transduced organoids already possessed a significantly enlarged luminal area compared to vehicle-treated or mCherry-infected organoids, consistent with observations at 24 hours post-infection (Figure 6B). Interestingly, only a moderate increase in organoid swelling was observed 60 minutes after forskolin addition (t=60) in HSV-CFTR transduced organoids, which is likely due to these organoids approaching their maximum swelling potential before forskolin exposure (Figure 6C). The G542X / G542X mutation could be corrected (at least partially) by exposure to aminoglycoside geneticin (G418), which allows for translational reading of nonsense mutations, and G418 was included in this assay as a positive control. G542X / G542X PDOs swelled at t=60 in the presence of G418, while the mean organoid size in these positive control samples was significantly smaller than that of HSV-CFTR-exposed PDOs (Figures 6B and 6C). Mild to moderate toxicity of the vector in G542X / G542X PDOs was observed 48 hours after infection when HSV-CFTR was used at 20 or 40 MOIs, and toxicity at MOIs greater than 20 likely accounts for the reduced ability for swelling observed in these organoids compared to samples infected at 10 MOIs.However, despite the observation of cytotoxic effects at high MOI, treated organoids still outperformed positive small molecule controls.

[0151] HSV-CFTR corrected disease organoids to wild-type morphology (large cystic lumen) within 24 hours at all tested MOIs, and higher HSV-CFTR doses appeared to adversely affect organoids in the swelling assay. Therefore, 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 in 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 the F508del organoid cultures tested exhibited cystic (wild-type) morphology 24 hours after HSV-CFTR infection, even at the lowest dose tested (MOI of 1). The mean 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 mean organoid size was detected after forskolin addition in HSV-CFTR transduced samples, suggesting that these organoids were already at or near their maximum expansion capacity, i.e., "pre-expansion" (Figure 7B). Importantly, functional correction of CFTR defect in F508del organoids appeared 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 an FDA-approved lumacaftor / ibacaftor combination therapy for the treatment of CF patients aged 2 years or older who are homozygous for the F508del mutation. No apparent vector-related cytotoxicity was observed in any of the MOIs tested.

[0152] Next, organoids from patients homozygous for the second nonsense CFTR mutation (W1282X) were infected with HSV-CFTR, and organoid size was quantified before and after forskolin addition. Consistent with the data presented in Figure 6 above, HSV-CFTR efficiently restored the wild-type cystic phenotype and increased the mean organoid size 24 hours after infection in W1282X / W1282X nonsense CFTR PDOs before forskolin addition (Figure 8A). Furthermore, HSV-CFTR with a low MOI of 1 appeared to correct disease morphology both before and after forskolin addition (Figures 8A and 8B). G418 was also included in these experiments, but a positive control could not be included in this experiment because W1282X / W1282X PDOs did not appear to respond to this read aminoglycoside (as there is currently no effective therapy for all nonsense CFTR mutations). These data suggest that HSV-CFTR can restore CFTR function in both G418-responsive and G418-nonresponsive CFTR-null patient samples.

[0153] Finally, organoids from a second F508del homozygous patient were examined. PDOs infected with HSV-CFTR 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 significant modification of organoid morphology from a compact budding CF phenotype to a cystic organoid phenotype with a well-defined lumen exhibiting wild-type characteristics, within 24 hours of infection in MOIs ranging from 1 to 40. This “pre-swelling” wild-type phenotype was quantitatively demonstrated by measuring total organoid size before forskolin addition and the resulting CFTR activation compared to multiple negative controls. Due to the “pre-swelling” nature of HSV-CFTR-transduced organoids, the ability of forskolin to stimulate further swelling was limited. Observation of corrected cystic morphology in CF organoids exposed to low doses of HSV-CFTR suggests that high levels of exogenous wild-type CFTR expressed in a small number of cells are sufficient to establish disease correction, demonstrating the “superior” effect of this therapeutic modality. One F508del organoid showed a slightly less efficient recovery of the wild-type phenotype compared to other CF organoid cultures examined, but cystic morphology was observed in all CF organoids infected with HSV-CFTR at MOIs greater than 5. The differences observed among various CF intestinal organoid cultures are most likely due to slight changes in their proliferation or differentiation state at the time of infection, and therefore, it is unlikely that the CFTR genotype itself significantly contributed 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 organoid system.

[0155] In summary, the data provided in these examples demonstrate that HSV-CFTR successfully infects the relevant airway epithelium, efficiently produces functional human CFTR, and molecularly corrects multiple CFTR defects without significant toxicity. Without wishing to be constrained by theory, these studies represent the first experimental validation of attenuated HSV-based gene therapy vectors for delivering full-length functional human CFTR, and may support the application of HSV-CFTR as a novel and widely applicable gene therapy for the treatment of CF.

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

[0157] All procedures performed in this example complied with the applicable animal welfare laws and were 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 these received either HSV-mCherry (described above) or a vehicle control via intratracheal administration, and five of these received either HSV-mCherry or a vehicle control via intranasal administration. Prior to the experimental procedure, the animals were sedated by intraperitoneal injection of a terazole / dexdomitol mixture, and ophthalmic ointment was applied to the eyes to prevent corneal dryness.

[0158] For intratracheal administration, the necks of each mouse were shaved using an electric razor, and all remaining fur was removed by applying depilatory cream. The surgical area was then washed twice with a 70% ethanol swab, and the anesthetized mice were placed on an angled restraint stand. A small incision was made in the neck using surgical scissors, and the thymus, platysma, and anterior tracheal muscles were moved out of the way to visualize and access the tracheal ring. 4.9375×10 8Three animals were administered 25 μL of the plaque-forming unit (PFU) HSV-mCherry intratracheally, while two animals received 25 μL of a vehicle control intratracheally. Each mouse was kept in a suspended position until its respiration gradually returned to normal. The incisions were closed with simple, individually tied sutures.

[0159] For intranasal administration, mice were anesthetized as described above and placed on an angled restraint stand. Three mice were given 25 μL (12.5 μL per nostril) of the formulation 4.9375x10 8 Each mouse was inoculated intranasally with the PFU virus. The rate of drug release was adjusted to ensure that the mice inhaled the inoculum without forming air bubbles during the inspiratory phase of their respiratory cycle. Two mice were administered 25 μL of vehicle control using the same procedure. After administration, the animals were held in a suspended position until their respiration returned to normal.

[0160] All animals were allowed to recover from anesthesia and given free access to water and food until euthanasia. Forty-eight hours after administration, the mice were euthanized, and bronchoalveolar lavage (BAL) was performed in both lungs using sterile saline. The BAL fluid was collected, centrifuged, and a cell pellet was assembled. Next, the upper trachea was collected and rapidly frozen in liquid nitrogen for nucleic acid quantification. The lungs (left lobe, right upper lobe, right middle lobe, and right lower lobe and posterior lobe of the vena cava) were collected individually and rapidly frozen in liquid nitrogen for nucleic acid analysis, or perfused in 4% neutral buffered formalin for immunofluorescence analysis and embedded in paraffin.

[0161] For immunofluorescence staining of paraffin-embedded lung tissue, epithelial cells were detected using Alexa Fluor® 488 conjugate pancytokeratin antibody (Invitrogen catalog number 53-9003-82), and infected cells were detected using rabbit anti-mCherry primary antibody (Abcam catalog number ab213511) and Alexa Fluor® 594 conjugate secondary antibody (Abcam catalog number ab150080). Tissue samples were mounted in mount medium containing DAPI, and the nuclei were visualized.

[0162] Intranasal versus intratracheal administration of HSV-mCherry resulted in similar levels of mCherry transcripts detected in the lung tissue of transduced animals (Figure 10A). Interestingly, transgene transcripts were barely identified in the trachea of ​​intranasal-exposed mice, while 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 tracheas of these invasively treated animals. In addition, a larger 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 after intratracheal administration of the HSV-based vector. Transgene protein expression in lung epithelial tissue was observed in both intranasal (Figure 10B) and intratracheal (Figure 10C) animals treated with HSV-mCherry, but not in the corresponding vehicle controls.

[0163] In summary, these data demonstrate that the manipulated HSV vector can be administered to the lungs of immunocompetent animals via multiple routes of administration, and further, that the non-invasive inhalation route allows for similar levels of transgene expression in the lungs as a more direct and invasive route of administration, while simultaneously inducing less (inflammatory) cellular invasion.

[0164] Example 5: Spraying of HSV-CFTR The following examples describe a study investigating a non-invasive, nebulizer-based delivery route for HSV-CFTR into the airways of immune-normal mice, both wild-type and CFTR-deficient.

[0165] The following 16 mice were used in the study: 12 immunocompetent C57BL / 6 animals and 4 immunocompetent intestinal corrected CFTR-deficient animals. Table 1 provides a summary of the study. Four wild-type animals were administered HSV-CFTR via intranasal drops, while the remaining animals were administered HSV-CFTR (or vehicle control) via spray (e.g., using a vibrating mesh nebulizer). Forty-eight hours after administration, the animals were euthanized, BAL fluid was collected, and tissue samples were taken from the respiratory tract and along the lungs, i.e., the upper and lower trachea, left and right bronchi, left lung, and right lung (individually, upper, middle, lower, and posterior lobe of the vena cava). Tissues from two animals per group were rapidly frozen in liquid nitrogen and processed for nucleic acid analysis. Vector genome / 50ng of total DNA is quantified in each tissue via qPCR analysis, and human CFTR transcript / 50ng of total RNA is quantified in each tissue via qRT-PCR analysis. Tissues from the remaining two animals per group are perfused and embedded in paraffin for immunofluorescence / immunohistochemistry. BAL fluid is treated to examine immune cell infiltration into the lungs. (Table 1) Research Design TIFF0007883557000001.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) Attenuated HSV-1 comprising recombinant herpes simplex virus type 1 (HSV-1) genome, wherein the recombinant HSV-1 genome comprises one or more polynucleotides encoding cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptide, and (b) Pharmacologically acceptable excipients Includes, One or more polynucleotides encoding the CFTR polypeptide are expressed from the recombinant HSV-1 genome to reduce or inhibit progressive lung injury, and It is administered to the subject using a nebulizer. A pharmaceutical composition for reducing or inhibiting progressive lung injury in a subject.

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

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

6.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the recombinant HSV-1 genome contains an inactivating mutation in an HSV-1 gene selected from the group consisting of infecting cell protein (ICP) 0, ICP4, ICP22, ICP27, ICP47, thymidine kinase (tk), long unique region (UL) 41, and UL55.

5. The pharmaceutical composition according to 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 according to any one of claims 1 to 5, wherein one or more polynucleotides encoding the CFTR polypeptide are operably linked to a promoter.

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

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the nebulizer is a jet nebulizer, an ultrasonic nebulizer, or a vibrating mesh nebulizer.

9. The pharmaceutical composition according to claim 8, which is aerosolized using the nebulizer.

10. The pharmaceutical composition according to claim 9, wherein the aerosolized pharmaceutical composition comprises droplets having an aerodynamic mass median particle diameter of less than 5 microns.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the progressive lung injury is cystic fibrosis or chronic obstructive pulmonary disease (COPD).

12. The use of attenuated HSV-1 containing recombinant herpes simplex virus type 1 (HSV-1) genome in the manufacture of a pharmaceutical product for reducing or inhibiting progressive lung injury, The aforementioned pharmaceutical is for administration using a nebulizer, The recombinant HSV-1 genome comprises one or more polynucleotides encoding cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptides, and One or more polynucleotides encoding the CFTR polypeptide are expressed from the recombinant HSV-1 genome. The aforementioned use.

13. The use of attenuated HSV-1, comprising recombinant herpes simplex virus type 1 (HSV-1) genome, in the manufacture of a pharmaceutical product for enhancing, increasing, enhancing, and / or supplementing cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptide levels, The aforementioned pharmaceutical is for administration using a nebulizer, The recombinant HSV-1 genome comprises one or more polynucleotides encoding cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptides, and One or more polynucleotides encoding the CFTR polypeptide are expressed from the recombinant HSV-1 genome. The aforementioned use.

14. A pharmaceutical composition according to any one of claims 1 to 11 for strengthening, increasing, enhancing, and / or supplementing CFTR polypeptide levels.

15. The use of attenuated HSV-1 containing recombinant herpes simplex virus type 1 (HSV-1) genome in the manufacture of a pharmaceutical product for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cystic fibrosis, The aforementioned pharmaceutical is for administration using a nebulizer, The recombinant HSV-1 genome comprises one or more polynucleotides encoding cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptides, and One or more polynucleotides encoding the CFTR polypeptide are expressed from the recombinant HSV-1 genome. The aforementioned use.

16. A pharmaceutical composition according to any one of claims 1 to 11, for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of cystic fibrosis.

17. The pharmaceutical composition according to claim 16, wherein one or more of the signs or symptoms of cystic fibrosis are selected from the group consisting of persistent cough producing large amounts of mucus, large amounts of sticky mucus accumulating in the airways, wheezing, dyspnea, sinusitis, recurrent lung infections, inflammatory rhinitis, bronchiectasis, nasal polyps, hemoptysis, pneumothorax, pancreatitis, recurrent pneumonia, respiratory failure, and any combination thereof.

18. The use of attenuated HSV-1, comprising a recombinant herpes simplex virus type 1 (HSV-1) genome, in the manufacture of a pharmaceutical product for providing prophylactic, palliative, or therapeutic relief of one or more signs or symptoms of COPD, The aforementioned pharmaceutical is for administration using a nebulizer, The recombinant HSV-1 genome comprises one or more polynucleotides encoding cystic fibrosis membrane conductance regulatory factor (CFTR) polypeptides, and One or more polynucleotides encoding the CFTR polypeptide are expressed from the recombinant HSV-1 genome. The aforementioned use.

19. A pharmaceutical composition according to any one of claims 1 to 11, for providing preventive, palliative, or therapeutic relief of one or more signs or symptoms of COPD.

20. The pharmaceutical composition according to claim 19, wherein one or more of the signs or symptoms of COPD are selected from the group consisting of shortness of breath, wheezing, chest tightness, excessive mucus in the lungs, chronic cough, cyanosis, frequent respiratory infections, and any combination thereof.

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