US20220087935A1 - Composition and Methods for Treatment of Primary Ciliary Dyskinesia - Google Patents

Composition and Methods for Treatment of Primary Ciliary Dyskinesia Download PDF

Info

Publication number
US20220087935A1
US20220087935A1 US17/420,346 US202017420346A US2022087935A1 US 20220087935 A1 US20220087935 A1 US 20220087935A1 US 202017420346 A US202017420346 A US 202017420346A US 2022087935 A1 US2022087935 A1 US 2022087935A1
Authority
US
United States
Prior art keywords
seq
mrna
coding sequence
composition
dnah5
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US17/420,346
Other languages
English (en)
Inventor
Anusha Dias
Darshan Parekh
Jeffrey S. Dubins
Christian Cobaugh
Shrirang Karve
Zarna Patel
Sara J. Dunaj
Frank DeRosa
Michael Heartlein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Translate Bio Inc
Original Assignee
Translate Bio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Translate Bio Inc filed Critical Translate Bio Inc
Priority to US17/420,346 priority Critical patent/US20220087935A1/en
Publication of US20220087935A1 publication Critical patent/US20220087935A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • PCD Primary ciliary dyskinesia
  • cilia is an auto recessive disorder characterized by abnormal cilia and flagella that are found in the linings of the airway, the reproductive system, and other organs and tissues.
  • PCD occurs in approximately 1 in 16,000. Symptoms are present as early as at birth, with breathing problems, and the affected individuals develop frequent respiratory tract infections beginning in early childhood. People with PCD also have year-round nasal congestion and chronic cough. Chronic respiratory tract infections can result in condition called bronchiectasis, which damages the passages, called bronchi, and can cause life-threatening breathing problems.
  • Some individuals with PCD also have infertility, recurrent ear infections, abnormally placed organs within their chest and abdomen.
  • DNAH1 or DNAH5 genes account for about a third of all cases of primary ciliary dyskinesia.
  • the DNAH5 gene encodes dynein axonemal heavy chain 5, which forms the inner structure of cilia. With an absent or abnormal dynein axonemal heavy chain 5, defective cilia cannot produce the force and movement needed to eliminate fluid, bacteria, and particles from the lungs. The movement of cilia also helps establish the left-right axis during embryonic development and propel the sperm cells forward to the female egg cell.
  • the present invention provides, among other things, methods and compositions for use in the treatment of primary ciliary dyskinesia (PCD).
  • PCD primary ciliary dyskinesia
  • the present invention is based, in part, on the surprising discovery that DNAH5 mRNA, which is approximately 14 kb in length, can be successfully encapsulated in a liposome and effectively delivered to target tissues in vivo.
  • the present invention provides a method of delivery of a 10 kb or greater mRNA encoding for a protein or peptide in vivo comprising administering to a subject in need of delivery a 10 kb or greater mRNA encoding a protein or peptide.
  • the 10 kb or greater mRNA is encapsulated in a liposome.
  • the 10 kb or greater mRNA is 11 kb or greater in length.
  • the 10 kb or greater mRNA is 12 kb or greater in length.
  • the 10 kb or greater mRNA is 13 kb or greater in length.
  • the 10 kb or greater mRNA is 14 kb or greater in length.
  • the present invention provides a method of delivery of human axonemal dynein heavy chain 5 (DNAH5) in vivo comprising administering to a subject in need of delivery an mRNA encoding a human DNAH5 protein.
  • the DNAH5 mRNA is encapsulated in a liposome.
  • the present invention provides a method of treating primary ciliary dyskinesia (PCD) comprising administering to a subject in need of treatment an mRNA encoding human axonemal dynein heavy chain 5 (DNAH5) at an effective dose and an administration interval such that at least one symptom or feature of PCD is reduced in intensity, severity, or frequency or has delayed in onset.
  • PCD primary ciliary dyskinesia
  • the DNAH5 mRNA is encapsulated in a liposome.
  • the liposome comprises one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids.
  • the one or more cationic lipids are selected from the group consisting of cKK-E12, OF-02, C12-200, MC3, DLinDMA, DLinkC2DMA, ICE (Imidazol-based), HGT5000, HGT5001, HGT4003, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, DLinSSDMA, KLin-K-DMA, DLin-K-XTC2-DMA, 3-(4-(bis(2-hydroxydodecyl)amino)butyl)-6-(4-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)butyl)-1,4-dioxan
  • the cationic lipid is ICE.
  • the one or more non-cationic lipids are selected from DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) or combinations thereof.
  • the non-cationic lipid is DOPE.
  • the one or more PEG-modified lipids comprise a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
  • the cationic lipid constitutes about 30-60% of the liposome by molar ratio.
  • the cationic lipid constitutes about 30%, 40%, 50%, or 60% of the liposome by molar ratio.
  • the liposome comprises ICE, DOPE and DMG-PEG2K.
  • the liposome has a size of about 80 nm to 200 nm, optionally wherein the liposome has a size of about 100 nm or less than 100 nm.
  • the DNAH5 mRNA is codon optimized.
  • the DNAH5 mRNA comprises one or more modified nucleotides.
  • the one or more modified nucleotides are selected from pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.
  • the mRNA is unmodified.
  • the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3.
  • the mRNA comprises a 3′-untranslated region (3′-UTR) that has a sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31.
  • the mRNA comprises a coding sequence at least 95% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6 to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 6 to SEQ ID NO: 31.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 6.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 7.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 8.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 9.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 10.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 11.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 12.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 13.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 14.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 15.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 16.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 17.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 18.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 19.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 20. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 20. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 20. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 20. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 20.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 20.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 21. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 21. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 21. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 21.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 21.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 22. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 22. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 22. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 22. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 22.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 22.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 23. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 23. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 23. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 23. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 23.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 23.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 24. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 24. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 24. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 24. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 24.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 24.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 25. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 25. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 25. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 25.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 25. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 25.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 26. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 26. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 26. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 26. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 26.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 26. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 26.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 27. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 27. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 27. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 27.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 27. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 27.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 28. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 28. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 28. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 28.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 28. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 28.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 29. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 29. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 29. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 29.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 29. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 29.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 30. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 30. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 30. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 30.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 30. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 30.
  • the mRNA comprises a coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 70% identical to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 90% identical to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence at least 95% identical to SEQ ID NO: 31.
  • the mRNA comprises a coding sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 31. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 31.
  • administering the mRNA to the subject is performed by intratracheal, intranasal, intravenous, intramuscular or subcutaneous delivery.
  • administering the mRNA to the subject is performed by intratracheal delivery.
  • administering the mRNA to the subject is performed by intranasal delivery.
  • administering the mRNA to the subject is performed by aerosol delivery.
  • administering the mRNA to the subject is performed by nebulized delivery.
  • administering the mRNA to the subject is performed by dry powder inhalation.
  • the composition is administered once a week.
  • the composition is administered once every two weeks.
  • the composition is administered twice a month.
  • the composition is administered once a month.
  • the administering the mRNA results in DNAH5 protein expression detectable in one or more internal organs selected from lung, heart, liver, spleen, kidney, brain, stomach, intestines, ovary and testis.
  • the administering the mRNA results in DNAH5 protein expression detectable in the lung.
  • the administering the mRNA results in DNAH5 protein expression detectable in the lung epithelium.
  • the invention provides a composition for use in the treatment of primary ciliary dyskinesia (PCD), the composition comprising an mRNA encoding human axonemal dynein heavy chain 5 (DNAH5) encapsulated in a liposome, wherein the liposome comprises one or more cationic lipids, one or more non-cationic lipids and one or more PEG-modified lipids.
  • PCD primary ciliary dyskinesia
  • DNAH5 human axonemal dynein heavy chain 5
  • the mRNA comprises a DNAH5 coding sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to any one of SEQ ID NO: 6 to SEQ ID NO: 31.
  • mRNA comprises a coding sequence at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 6. In some embodiments, mRNA comprises a coding sequence at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 7.
  • the mRNA comprises a coding sequence set forth in SEQ ID NO: 6. In some embodiments, the mRNA comprises a coding sequence set forth in SEQ ID NO: 7.
  • the mRNA has a 5′-untranslated region (5′-UTR) that has a sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, and a 3′-untranslated region (3′-UTR) that has a sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • 5′-UTR 5′-untranslated region
  • 3′-UTR 3′-untranslated region
  • the mRNA has one or more modified nucleotides.
  • the modified one or more nucleotides is selected from pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine.
  • the mRNA is unmodified.
  • the liposome is 100 nm in diameter or less.
  • the invention provides a pharmaceutical composition comprising the composition described above and a suitable excipient.
  • the present invention provides a method of delivery of a mRNA encoding for a protein or peptide in vivo comprising administering to a subject in need of delivery a mRNA encoding a protein or peptide and having a 5′-untranslated region (5′-UTR) that has a sequence at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2 and that is not SEQ ID NO: 3.
  • the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 70% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3.
  • the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 75% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3. In some embodiments, the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 80% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3. In some embodiments, the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 85% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3.
  • the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 90% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3. In some embodiments, the mRNA comprises a 5′-untranslated region (5′-UTR) that has a sequence at least 95% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3. In some embodiments, the mRNA comprises a 5′-untranslated region (5′-UTR) that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2 that is not SEQ ID NO: 3.
  • the mRNA comprises a 5′-untranslated region (5′-UTR) set forth in SEQ ID NO: 2.
  • 5′-UTR 5′-untranslated region
  • FIG. 1A is a schematic diagram that shows the dissection and usage of various parts of mouse trachea and lungs for quantitative PCR analysis (qPCR) and immunohistochemistry (IHC) analysis, 24 hours after mRNA administration.
  • FIG. 1B (left) and (right), are graphs that show qPCR data for hDNA5 mRNA in the different regions of the respiratory system as indicated in the figure.
  • FIG. 1B (left) shows data from Group 1 mice who were administered MRT-1 hDNA5 mRNA;
  • FIG. 1B (right) shows data from Group 2 mice who were administered MRT-1 hDNA5-GFP mRNA.
  • FIG. 2A and FIG. 2B show series of photomicrographs depicting results from IHC analysis for hDNA protein expression in the respiratory airways.
  • FIG. 2A depicts representative IHC data for hDNA-5 protein staining in MRT-1 hDNA5 mRNA treated mice compared to saline-treated control (Group 1, left); and IHC data for GFP protein staining in MRT-1 hDNA5-GFP mRNA treated mice compared to saline-treated control (Group 2, right).
  • FIG. 2B shows detailed localization of the respective hDNA5 mRNA derived protein in epithelial tissue of the airways in Group 1 (upper panel) and Group 2 (lower panel) mice.
  • alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 15 carbon atoms (“C1-15 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). Examples of C1-3 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), and isopropyl (C3). In some embodiments, an alkyl group has 8 to 12 carbon atoms (“C8-12 alkyl”).
  • C8-12 alkyl groups include, without limitation, n-octyl (C8), n-nonyl (C9), n-decyl (C10), n-undecyl (C11), n-dodecyl (C12) and the like.
  • n- normal refers to unbranched alkyl groups.
  • n-C8 alkyl refers to (CH2)7CH3
  • n-C10 alkyl refers to (CH2)9CH3, etc.
  • amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • an amino acid has the general structure H2N—C(H)(R)—COOH.
  • an amino acid is a naturally occurring amino acid.
  • an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid.
  • Standard amino acid refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond.
  • Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).
  • chemical entities e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.
  • amino acid is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • Codon-optimized As used herein, the term describes a nucleic acid in which one or more of the nucleotides present in a naturally occurring nucleic acid sequence (also referred to as ‘wild-type’ sequence) has been substituted with an alternative nucleotide to optimize protein expression without changing the amino acid sequence of the polypeptide encoded by the naturally occurring nucleic acid sequence.
  • the codon AAA may be altered to become AAG without changing the identity of the encoded amino acid (lysine).
  • the nucleic acids of the invention are codon optimized to increase protein expression of the protein encoded by the nucleic acid.
  • nucleobase thymidine (T) and uracil (U) are used interchangeably in narration of mRNA sequences.
  • delivery encompasses both local and systemic delivery.
  • delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient's circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).
  • patient's circulation system e.g., serum
  • Dosing interval in the context of a method for treating a disease is the frequency of administering a therapeutic composition in a subject (mammal) in need thereof, for example an mRNA composition, at an effective dose of the mRNA, such that one or more symptoms associated with the disease is reduced; or one or more biomarkers associated with the disease is reduced, at least for the period of the dosing interval.
  • Dosing frequency and dosing interval may be used interchangeably in the current disclosure.
  • expression refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme).
  • intact protein e.g., enzyme
  • post-translational modification e.g., enzyme
  • an effective dose is a dose of the mRNA in the pharmaceutical composition which when administered to the subject in need thereof, hereby a mammalian subject, according to the methods of the invention, is effective to bring about an expected outcome in the subject, for example reduce a symptom associated with the disease.
  • a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Half-life is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • the terms “improve,” “increase” or “reduce,” or grammatical equivalents indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).
  • Local distribution or delivery As used herein, the terms “local distribution,” “local delivery,” or grammatical equivalent, refer to tissue specific delivery or distribution. Typically, local distribution or delivery requires a protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system.
  • a protein e.g., enzyme
  • messenger RNA As used herein, the term “messenger RNA (mRNA)” refers to a polyribonucleotide that encodes at least one polypeptide. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, in vitro transcribed, chemically synthesized, etc. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. Typically, the mRNA of the present invention is synthesized from adenosine, guanosine, cytidine and uridine nucleotides that bear no modifications.
  • mRNA with unmodified nucleotides is referred to herein as mRNA with unmodified nucleotides or ‘unmodified mRNA’ for short.
  • the mRNA of the present invention does not comprise any of the following nucleoside analogs: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxo
  • An mRNA suitable for practising the claimed invention commonly does not comprise nucleosides comprising chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
  • nucleosides comprising chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • a patient refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.
  • pharmaceutically acceptable refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4 alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.
  • Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
  • systemic distribution or delivery As used herein, the terms “systemic distribution,” “systemic delivery,” or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream. Compared to the definition of “local distribution or delivery.”
  • subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Target tissues refers to any tissue that is affected by a disease to be treated.
  • target tissues include those tissues that display disease-associated pathology, symptom, or feature.
  • therapeutically effective amount of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
  • PCD Primary Ciliary Dyskinesia
  • PCD Primary ciliary dyskinesia
  • DNAH5 which encodes the dynein axonemal heavy chain 5 protein that forms the inner structure of cilia, cause PCD.
  • Over 80 different mutations in the DNAH5 gene have been identified in patients with PCD.
  • Mutations in the DNAH5 gene result in an absent or abnormal dynein axonemal heavy chain 5, which is required for the proper functioning of cilia. Without a normal version of dynein axonemal heavy chain 5, defective cilia cannot produce the force and movement needed to eliminate fluid, bacteria, and particles from the lungs, to establish the left-right axis during embryonic development, and to propel the sperm cells. PCD can lead to chronic respiratory tract infections, bronchiectasis, year-round nasal congestion, abnormally placed organs within their chest and abdomen, and infertility.
  • Polyribonucleotides of the disclosure can be used, for example, to treat a subject having or at risk of having primary ciliary dyskinesia or any other condition associated with a defect or malfunction of a gene whose function is linked to cilia maintenance and function.
  • Non limiting examples of genes that have been associated with primary ciliary dyskinesia include: armadillo repeat containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain containing 103 (CCDC103), coiled-coil domain containing 114 (CCDC114), coiled-coil domain containing 39 (CCDC39), coiled-coil domain containing 40 (CCDC40), coiled-coil domain containing 65 (CCDC65), cyclin O (CCNO), dynein (axonemal) assembly factor 1 (DNAAF1), dynein (axonemal) assembly factor 2 (DNAAF2), dynein (axonemal) assembly factor 3 (DNAAF3), dynein (axonemal) assembly factor 5 (DNAAF5), dynein axonemal heavy chain 11 (DNAH11), dynein axonemal heavy chain 5 (DNAH5), dyn
  • the present invention provides methods and compositions for delivering mRNA encoding to a subject for the treatment of PCD.
  • a suitable DNAH5 mRNA encodes any full length, fragment or portion of a DNAH5 protein which can be substituted for naturally-occurring DNAH5 protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with PCD.
  • a suitable mRNA sequence is an mRNA sequence encoding a human DNAH5 protein.
  • the naturally-occurring human DNAH5 mRNA coding sequence and the corresponding amino acid sequence are shown in Table 1:
  • a suitable mRNA is a wild-type human DNAH5 mRNA of sequence.
  • a suitable therapeutic candidate mRNA is a codon-optimized hDNAH5 sequence that can encodes a DNAH5 amino acid sequence shown in Table 1 as SEQ ID NO: 1 or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • an mRNA according to the present invention encodes a DNAH5 protein with an amino acid sequence that is identical to SEQ ID NO: 1.
  • mRNAs contain numerous layers of information that overlap the amino acid code.
  • codon optimization has been used to remove rare codons which were thought to be rate-limiting for protein expression. While fast growing bacteria and yeast both exhibit strong codon bias in highly expressed genes, higher eukaryotes exhibit much less codon bias, making it more difficult to discern codons that may be rate-limiting.
  • codon bias per se does not necessarily yield high expression but requires other features.
  • codon optimization can interfere with this fine-tuning mechanism, resulting in less efficient protein translation or an increased amount of incorrectly folded proteins.
  • codon optimization may disrupt the normal patterns of cognate and wobble tRNA usage, thereby affecting protein structure and function because wobble-dependent slowing of elongation may likewise have been selected as a mechanism for achieving correct protein folding.
  • the inventors have arrived at a codon-optimized hDNAH5 sequence that improves expression of the DNAH5 protein at least threefold over the coding sequence of the wild type gene.
  • the increase in expression is not limited to cell cultures of mammalian cells but was also observed in vivo in a mouse model. It is expected that the observed improvement in expression of the codon-optimised DNAH5 coding sequence will result in an improved, more cost-effective mRNA replacement therapy for patients suffering from PCD, because it does not require the use of modified nucleotides for the preparation of the mRNA and allows treatment with a reduced dose and/or at extended dosing intervals.
  • sequences that follow recite select, exemplary codon-optimized DNAH5 mRNA sequences.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 6.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 7.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 8.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 9.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 10.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 11.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 12.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 13.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 14.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 15.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 16.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 17.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 18.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 19.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 20.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 21.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 22.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 23.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 24.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 25.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 26.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 27.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 28.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 29.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 30.
  • a suitable mRNA may be a codon-optimized sequence, as shown in SEQ ID NO: 31.
  • a suitable mRNA sequence may be an mRNA sequence a homolog or an analog of human DNAH5 protein.
  • a homolog or an analog of human DNAH5 protein may be a modified human DNAH5 protein containing one or more amino acid substitutions, deletions, and/or insertions as compared to a wild-type or naturally-occurring human DNAH5 protein while retaining substantial DNAH5 protein activity.
  • an mRNA suitable for the present invention encodes an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1.
  • an mRNA suitable for the present invention encodes a protein substantially identical to human DNAH5 protein. In some embodiments, an mRNA suitable for the present invention encodes an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1. Typically, an mRNA according to the present invention encodes a DNAH5 protein with an amino acid sequence that is identical to SEQ ID NO: 1.
  • an mRNA suitable for the present invention encodes a fragment or a portion of human DNAH5 protein. In some embodiments, an mRNA suitable for the present invention encodes a fragment or a portion of human DNAH5 protein, wherein the fragment or portion of the protein still maintains DNAH5 activity similar to that of the wild-type protein.
  • a suitable mRNA encodes a fusion protein comprising a full length, fragment or portion of a DNAH5 protein fused to another protein (e.g., an N or C terminal fusion).
  • the protein fused to the mRNA encoding a full length, fragment or portion of a DNAH5 protein encodes a signal or a cellular targeting sequence.
  • an mRNA suitable for the present invention comprises a nucleotide sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.
  • an mRNA in accordance with the present invention comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 6.
  • an mRNA according to the present invention comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 7.
  • an mRNA according to the present invention comprises the nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
  • RNAs according to the present invention may be synthesized according to any of a variety of known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.
  • the mRNA according to the present invention is synthesized as unmodified mRNA.
  • the mRNAs of the invention are synthesized from naturally occurring nucleotides including purines (adenine (A), guanine (G)) or pyrimidines (cytosine (C), uracil (U)).
  • mRNA synthesis includes the addition of a “cap” on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end.
  • the presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells.
  • the presence of a “tail” serves to protect the mRNA from exonuclease degradation.
  • mRNAs include a 5′ cap structure.
  • a 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.
  • mRNAs include a 3′ poly(A) tail structure.
  • a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 800 adenosine nucleotides (e.g., about 300 to 500 adenosine nucleotides, about 300 to 800 adenosine nucleotides, about 10 to 500 adenosine nucleotides, about 10 to 300 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • a poly-A tail in an mRNA in accordance with the invention is about 300 to about 800 adenosine nucleotides long. More commonly, the poly-A tail is about 300 adenosine nucleotides long. In some embodiments, the poly(A) tail structure comprises at least 85%, 90%, 95% or 100% adenosine.
  • mRNAs include a 3′ poly(C) tail structure.
  • a suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the mRNA further comprises a 5′ untranslated region (5′ UTR) comprising a nucleotide sequence and positioned between the 5′ cap structure and coding sequence, and/or a 3′ untranslated region (3′ UTR) comprising a nucleotide sequence and positioned between the coding sequence and the poly(A) tail structure.
  • a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
  • a 5′ untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer.
  • mRNAs according to the present invention are typically synthesized as unmodified mRNAs.
  • mRNAs are modified to enhance their stability or reduce their immunogenic properties, in particular when administered to a subject as naked mRNAs or in complexed form. Therefore, providing an mRNA encoding a codon-optimized DNAH5 coding sequence of the present invention may have synergistic effects, resulting in sustained in vivo function that exceeds that observed with unmodified mRNAs.
  • Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA.
  • a modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications.
  • mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • mRNAs of the present invention may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5′-O-(1-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
  • mRNAs of the present invention may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine 5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate), 2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine 5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide (2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate), 2′-C-alkyloli
  • mRNAs of the present invention may contain modifications of the bases of the nucleotides (base modifications).
  • a modified nucleotide which contains a base modification is also called a base-modified nucleotide.
  • base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate, 5-methyluridine
  • mRNAs include a 5′ cap structure.
  • a 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.
  • Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5′)ppp(5′)N, where N is any nucleoside.
  • the cap is added enzymatically.
  • the cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase.
  • the addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription.
  • the terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.
  • a common cap for mRNA produced by in vitro transcription is m7G(5′)ppp(5′)G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5′-termini.
  • the prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m7G(5′)ppp(5′)G (“m7GpppG”) as an initiator of transcription.
  • ARCA Anti-Reverse Cap Analog
  • modified ARCA which is generally a modified cap analog in which the 2′ or 3′ OH group is replaced with —OCH3.
  • Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).
  • a suitable cap is a 7-methyl guanylate (“m7G”) linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in m7G(5′)ppp(5′)N, where N is any nucleoside.
  • m7G 7-methyl guanylate
  • a preferred embodiment of a m7G cap utilized in embodiments of the invention is m7G(5′)ppp(5′)G.
  • the cap is a Cap0 structure.
  • Cap0 structures lack a 2′-O-methyl residue of the ribose attached to bases 1 and 2.
  • the cap is a Cap1 structure.
  • Cap1 structures have a 2′-O-methyl residue at base 2.
  • the cap is a Cap2 structure.
  • Cap2 structures have a 2′-O-methyl residue attached to both bases 2 and 3.
  • m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as the ARCA 3′-OCH3 and 2′-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E.
  • a tail serves to protect the mRNA from exonuclease degradation.
  • the poly-A tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly-A tail can be added to an mRNA molecule thus rendering the RNA more stable.
  • Poly-A tails can be added using a variety of art-recognized techniques. For example, long poly-A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly-A tails. In addition, poly-A tails can be added by transcription directly from PCR products.
  • Poly-A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).
  • mRNAs include a 3′ poly(A) tail structure.
  • the length of the poly-A tail can be at least about 10, 50, 100, 200, 300, 400 or 500 nucleotides in length.
  • a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 800 adenosine nucleotides (e.g., about 300 to 500 adenosine nucleotides, about 300 to 800 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • a poly-A tail in an mRNA in accordance with the invention is about 300 to about 800 adenosine nucleotides long. More commonly
  • mRNAs include a 3′ poly(C) tail structure.
  • a suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein.
  • the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.
  • mRNAs include a 5′ untranslated region (UTR). In some embodiments, mRNAs include a 3′ untranslated region. In some embodiments, mRNAs include both a 5′ untranslated region and a 3′ untranslated region. In some embodiments, a 5′ untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5′ untranslated region may be between about 50 and 500 nucleotides in length.
  • a 3′ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3′ untranslated region may be between 50 and 500 nucleotides in length or longer.
  • Exemplary 3′ and 5′ untranslated region sequences can be derived from mRNA molecules which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule.
  • a 5′ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improve the nuclease resistance and/or improve the half-life of the polynucleotide.
  • IE1 immediate-early 1
  • hGH human growth hormone
  • modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to their unmodified counterparts, and include, for example modifications made to improve such polynucleotides' resistance to in vivo nuclease digestion.
  • the codon-optimized DNAH5 mRNA includes a coding region having a codon-optimized coding region flanked by 5′ and 3′ untranslated regions as represented as X and Y, respectively (vide infra)
  • X (5′ UTR Sequence) is [SEQ ID NO.: 2] AGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG or a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2, or (SEQ ID NO: 3) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAG ACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGC GGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG or a sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
  • a codon-optimized human dynein axonemal heavy chain 5 messenger RNA (DNAH5 mRNA) is synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which is followed by the addition of a 5′ cap structure (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail of approximately 100, 200, 250, 300, 400, 500 or 800 nucleotides in length as determined by gel electrophoresis.
  • DNAH5 mRNA codon-optimized human dynein axonemal heavy chain 5 messenger RNA
  • mRNA encoding a DNAH5 protein may be delivered as naked RNA (unpackaged) or via delivery vehicles.
  • delivery vehicle delivery vehicle
  • transfer vehicle nanoparticle or grammatical equivalent
  • mRNAs encoding a DNAH5 protein may be delivered via a single delivery vehicle.
  • mRNAs encoding a DNAH5 protein may be delivered via one or more delivery vehicles each of a different composition.
  • suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinylene
  • a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein.
  • liposomal delivery vehicles as used herein, also encompass polymer containing nanoparticles.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI).
  • PEI polyethylenimine
  • a suitable delivery vehicle is a liposome.
  • liposomes are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998).
  • Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • liposome typically serves to transport a desired mRNA to a target cell or tissue.
  • a typical liposome in accordance with the invention comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.
  • cationic lipids refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH.
  • Suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2010/144740, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate, having a compound structure of:
  • compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO 2013/149140, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of one of the following formulas:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, an optionally substituted, variably saturated or unsaturated C 1 -C 20 alkyl and an optionally substituted, variably saturated or unsaturated C 6 -C 20 acyl; wherein L 1 and L 2 are each independently selected from the group consisting of hydrogen, an optionally substituted C 1 -C 30 alkyl, an optionally substituted variably unsaturated C 1 -C 30 alkenyl, and an optionally substituted C 1 -C 30 alkynyl; wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., where m is three); and wherein n is zero or any positive integer (e.g., where n is one).
  • compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-15,18-dien-1-amine (“HGT5000”), having a compound structure of:
  • compositions and methods of the present invention include the cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-4,15,18-trien-1-amine (“HGT5001”), having a compound structure of:
  • compositions and methods of the present invention include the cationic lipid and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-5,15,18-trien-1-amine (“HGT5002”), having a compound structure of:
  • compositions and methods of the invention include cationic lipids described as aminoalcohol lipidoids in International Patent Publication WO 2010/053572, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/118725, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/118724, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • Suitable cationic lipids for use in the compositions and methods of the invention include a cationic lipid having the formula of 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2013/063468 and WO 2016/205691, each of which are incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of the following formula:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015/184256, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of the following formula:
  • the compositions and methods are independently hydrogen, optionally substituted C1-50 al
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/004202, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • the cationic lipids of the compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015/199952, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/004143, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/075531, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of the following formula:
  • L 1 or L 2 is —O(C ⁇ O)—, —(C ⁇ O)O—, —C( ⁇ O)—, —O—, —S(O) x , —S—S—, —C( ⁇ O)S—, —SC( ⁇ O)—, —NR a C( ⁇ O)—, —C( ⁇ O)NR a —, NR a C( ⁇ O)NR a —, —OC( ⁇ O)NR a —, or —NR a C( ⁇ O)O—; and the other of L 1 or L 2 is —O(C ⁇ O)—, —(C ⁇ O)O—, —C( ⁇ O)—, —O—, —S(O) x , —S—S—, —C( ⁇ O)S—, SC( ⁇ O)—, —NR a C( ⁇ O)—, —C( ⁇ O)NR
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/117528, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • compositions and methods of the present invention include a cationic lipid having the compound structure:
  • Suitable cationic lipids for use in the compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/049245, which is incorporated herein by reference.
  • the cationic lipids of the compositions and methods of the present invention include a compound of one of the following formulas:
  • R 4 is independently selected from —(CH 2 ) n Q and —(CH 2 ) n CHQR;
  • Q is selected from the group consisting of —OR, —OH, —O(CH 2 ) n N(R) 2 , —OC(O)R, —CX 3 , —CN, —N(R)C(O)R, —N(H)C(O)R, —N(R)S(O) 2 R, —N(H)S(O) 2 R, —N(H)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(H)C(O)N(R) 2 , —N(H)C(O)N(H)(R), —N(R)C(S)N(R) 2 , —N(H)C(S)N(R) 2 , —N(H)C(S)N(H)(R), and a
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/173054 and WO 2015/095340, each of which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid having a compound structure of:
  • compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO 2012/170889, which is incorporated herein by reference.
  • compositions and methods of the present invention include a cationic lipid of the following formula:
  • R 1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino (e.g., an alkyl amino such as dimethylamino) and pyridyl; wherein R 2 is selected from the group consisting of one of the following two formulas:
  • compositions and methods of the present invention include a cationic lipid, “HGT4001”, having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid, “HGT4002”, having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid, “HGT4003”, having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid, “HGT4004”, having a compound structure of:
  • compositions and methods of the present invention include a cationic lipid “HGT4005”, having a compound structure of:
  • compositions and methods of the present invention include cleavable cationic lipids as described in U.S. Provisional Application No. 62/672,194, filed May 16, 2018, and incorporated herein by reference.
  • the compositions and methods of the present invention include a cationic lipid that is any of general formulas or any of structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in U.S. Provisional Application No. 62/672,194.
  • the compositions and methods of the present invention include a cationic lipid that has a structure according to Formula (I′),
  • compositions and methods of the present invention include the cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”).
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • cationic lipids suitable for the compositions and methods of the present invention include, for example, 5-carboxyspermylglycinedioctadecylamide (“DOGS”); 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (“DOSPA”) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), U.S. Pat. Nos. 5,171,678; 5,334,761); 1,2-Dioleoyl-3-Dimethylammonium-Propane (“DODAP”); 1,2-Dioleoyl-3-Trimethylammonium-Propane (“DOTAP”).
  • DOGS 5-carboxyspermylglycinedioctadecylamide
  • DOSPA 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethy
  • Additional exemplary cationic lipids suitable for the compositions and methods of the present invention also include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (“DSDMA”); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”); N-dioleyl-N,N-dimethylammonium chloride (“DODAC”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”); 3-dimethylamin
  • one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • one or more cationic lipids suitable for the compositions and methods of the present invention include 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (“XTC”); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (“ALNY-100”) and/or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide (“NC98-5”).
  • XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
  • the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the composition, e.g., a lipid nanoparticle.
  • the compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the composition, e.g., a lipid nanoparticle.
  • the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the composition, e.g., a lipid nanoparticle.
  • the compositions of the present invention include one or more cationic lipids that constitute about 30-70% (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the composition, e.g., a lipid nanoparticle
  • sterol-based cationic lipids may be use instead or in addition to cationic lipids described herein.
  • Suitable sterol-based cationic lipids are dialkylamino-, imidazole-, and guanidinium-containing sterol-based cationic lipids.
  • certain embodiments are directed to a composition comprising one or more sterol-based cationic lipids comprising an imidazole, for example, the imidazole cholesterol ester or “ICE” lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, as represented by structure (I) below.
  • imidazole cholesterol ester or “ICE” lipid 3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-
  • a lipid nanoparticle for delivery of RNA (e.g., mRNA) encoding a functional protein may comprise one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or “ICE” lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by the following structure:
  • the percentage of cationic lipid in a liposome may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.
  • cationic lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by weight.
  • the cationic lipid e.g., ICE lipid
  • provided liposomes contain one or more non-cationic (“helper”) lipids.
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE
  • non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids.
  • the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10% to about 70% of the total lipid present in a liposome.
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • the percentage of non-cationic lipid in a liposome may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • provided liposomes comprise one or more cholesterol-based lipids.
  • suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.
  • the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the liposome may be greater than 5, %, 10%, greater than 20%, greater than 30%, or greater than 40%.
  • provided liposomes comprise one or more PEGylated lipids.
  • PEG polyethylene glycol
  • derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids together which comprise the liposome.
  • Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 2 kDa, up to 3 kDa, up to 4 kDa or 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
  • a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al.
  • a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K.
  • particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18).
  • particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18).
  • the PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 15%, about 0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposome.
  • PEG-modified phospholipid and derivatized lipids may constitute at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total lipids in a suitable lipid solution by weight or by molar.
  • PEGylated lipid lipid(s) constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the total lipids in a suitable lipid solution by weight or by molar.
  • the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the liposome, as well as the relative molar ratio of such lipids to each other is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.
  • a suitable liposome for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEGylated lipids and/or polymers described herein at various ratios.
  • a liposome in accordance with the present invention comprises a cationic lipid, a non-cationic lipid, a cholesterol lipid and a PEGylated lipid.
  • a suitable liposome formulation may include a combination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K or ICE, DOPE and DMG-PEG2K. Additional combinations of lipids are described in the art, e.g., U.S. Ser. No. 62/420,421 (filed on Nov. 10, 2016), U.S. Ser. No. 62/421,021 (filed on Nov. 11, 2016), U.S. Ser. No. 62/464,327 (filed on Feb. 27, 2017), and PCT Application entitled “Novel ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA,” filed on Nov. 10, 2017, the disclosures of which are included here in their full scope by reference.
  • cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome by molar ratio.
  • the percentage of cationic lipids (e.g., cKK-E12, C12-200, ICE, and/or HGT4003) is or greater than about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the liposome by molar ratio.
  • the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:30:25:5, respectively.
  • the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEGylated lipid(s) is approximately 50:25:20:5.
  • the liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques which are presently known in the art.
  • the liposomes for use in provided compositions can be prepared by various techniques which are presently known in the art.
  • multilamellar vesicles may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase then may be added to the vessel with a vortexing motion which results in the formation of MLVs.
  • Unilamellar vesicles can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles.
  • unilamellar vesicles can be formed by detergent removal techniques.
  • compositions comprise a liposome wherein the mRNA is associated on both the surface of the liposome and encapsulated within the same liposome.
  • cationic liposomes may associate with the mRNA through electrostatic interactions.
  • cationic liposomes may associate with the mRNA through electrostatic interactions.
  • the compositions and methods of the invention comprise mRNA encapsulated in a liposome.
  • the one or more mRNA species may be encapsulated in the same liposome.
  • the one or more mRNA species may be encapsulated in different liposomes.
  • the mRNA is encapsulated in one or more liposomes, which differ in their lipid composition, molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and/or combinations thereof.
  • the one or more liposome may have a different composition of cationic lipids, neutral lipid, PEG-modified lipid and/or combinations thereof.
  • the one or more liposomes may have a different molar ratio of cationic lipid, neutral lipid, cholesterol and PEG-modified lipid used to create the liposome.
  • the process of incorporation of a desired mRNA into a liposome is often referred to as “loading”. Exemplary methods are described in Lasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference.
  • the mRNA of the invention is encapsulated in a liposome using the methods described in WO 2018/089801 (the teachings of which are incorporated herein by reference in their entirety). Briefly, the mRNA is encapsulated by mixing of a solution comprising pre-formed liposomes with mRNA such that liposomes encapsulating mRNA are formed.
  • the liposome-incorporated nucleic acids are completely located in the interior space of the liposome within the bilayer membrane of the liposome, although as discussed above, some of the mRNA (e.g., no more than 10% of total mRNA in the liposome composition) may also be associated with the exterior surface of the liposome membrane.
  • the incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation”.
  • the purpose of incorporating an mRNA into a liposome is to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids.
  • a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA to the target cell or tissue.
  • Suitable liposomes in accordance with the present invention may be made in various sizes.
  • provided liposomes may be made smaller than previously known mRNA encapsulating liposomes.
  • decreased size of liposomes is associated with more efficient delivery of mRNA. Selection of an appropriate liposome size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made.
  • an appropriate size of liposome is selected to facilitate systemic distribution of antibody encoded by the mRNA.
  • a liposome may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; in such cases the liposome could readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a liposome may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • a liposome may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomes to hepatocytes.
  • the size of a liposome is determined by the length of the largest diameter of the liposome particle.
  • a suitable liposome has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm).
  • a suitable liposome has a size ranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm).
  • a suitable liposome has a size ranging from about 100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm, 100-150 nm). Liposomes with a size of 80-200 nm are particularly suitable for some application. In some embodiments, a suitable liposome has a size ranging from about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm). In a particular embodiment, a suitable liposome has a size less than about 100 nm.
  • the size of the liposomes may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • QELS quasi-electric light scattering
  • This section provides exemplary liposome formulations for effective delivery and expression of DNAH5 mRNA in vivo.
  • the formulations described herein include a multi-component lipid mixture of varying ratios employing one or more cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol-based lipids) and PEGylated lipids designed to encapsulate mRNA encoding DNAH5 protein.
  • helper lipids e.g., non-cationic lipids and/or cholesterol-based lipids
  • PEGylated lipids designed to encapsulate mRNA encoding DNAH5 protein.
  • Cationic lipids can include (but not exclusively) DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids” J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.
  • Helper lipids can include (but not exclusively) DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.
  • the PEGylated lipids can include (but not exclusively) a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a
  • Clinical or therapeutic candidate mRNA formulations are selected from the exemplary codon-optimized mRNA sequences having a 5′-cap and a 3′-poly A tail, which is formulated in a suitable lipid combination as described above.
  • Clinically relevant mRNA candidates are characterized by efficient delivery and uptake by in vivo tissue, high level of expression and sustained protein production, without detectable adverse effects in the subject to whom the therapeutic is administered, either caused by the pharmacologically active ingredient or by the lipids in the liposome, or by any excipients used in the formulation. In general, high efficiency with low dose administration is favorable for the selection process of a relevant candidate therapeutic.
  • the present invention provides compositions for use in the treatment of primary ciliary dyskinesia (PCD).
  • the compositions of the present invention are for use in the manufacture of a medicament for the treatment of primary ciliary dyskinesia (PCD).
  • delivery vehicles such as liposomes can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients.
  • Provided liposomally-encapsulated or associated mRNAs, and compositions containing the same, may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.
  • the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject, the mammal, (e.g., treating, modulating, curing, preventing and/or ameliorating PCD).
  • a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and/or prophylactic effect.
  • the amount of a therapeutic agent e.g., mRNA encoding aDNAH5 protein
  • the amount of a therapeutic agent administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject.
  • a therapeutic agent e.g., mRNA encoding aDNAH5 protein
  • administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject.
  • characteristics include the condition, disease severity, general health, age, sex and body weight of the subject.
  • objective and subjective assays may optionally be employed to identify optimal dosage ranges.
  • an effective therapeutic dose of the pharmaceutical composition comprising an mRNA encoding dynein axonemal heavy chain 5 protein is administered to the mammal at a dosing interval sufficient to reduce for the period of the dosing interval or longer the level of at least one symptom or biomarker associated with PCD in the mammal relative to its state prior to the treatment.
  • the mammal is a human.
  • a suitable therapeutic dose that may be applicable for a human being can be derived based on animal studies.
  • a basic guideline for deriving a human equivalent dose from studies performed in animals can be obtained from the U.S. Food and Drug Administration (FDA) website at https://www.fda.gov/downloads/drugs/guidances/ucm078932.pdf, entitled, “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers.”
  • FDA Food and Drug Administration
  • a suitable dose of, for example, 0.6 mg/kg in a mouse would relate to a human equivalent dose of 0.0048 mg/kg.
  • a projected human therapeutic dose can be derived based on studies in other animals.
  • the dosing interval is once every 15 days or longer, or once every 20 days or longer, or once every 21 days, or once every 22 days, or once every 23 days, or once every 24 days, or once every 25 days, once every 26 days, or once every 27 days, or once every 28 days, or once every 29 days or longer, or once every 30 days or longer, or once every 31 days or longer.
  • the dosing interval is once every 40, 45 or 50 days or 60 days, or any number of days in between.
  • the dosing interval is once every 80, 90 or 120 days or 150 days, or any number of days in between.
  • the therapeutic low dose is administered at a dosing interval of once every 2 weeks or longer, which is sufficient to reduce the level of at least one symptom or biomarker associated with PCD in the mammal relative to the state prior to the treatment. In some embodiments, the therapeutic low dose is administered at a dosing interval of once every 3 weeks or longer, which is sufficient to reduce the level of at least one symptom or biomarker associated with PCD in the mammal relative to the state prior to the treatment. In some embodiments, the dosing interval is once every 4 weeks or longer. In some embodiments, the dosing interval is once every 5 weeks or longer. In some embodiments, the dosing interval is once every 6 weeks or longer. In some embodiments, the dosing interval is once every 8 weeks or longer. In some embodiments, the dosing interval is once every 12 or 15 or 18 weeks or longer.
  • the dosing interval is once a month. In some embodiments, the dosing interval is once in every two months. In some embodiments, the dosing interval is once every three months, or once every four months or once every five months or once every six months or anywhere in between.
  • administering the provided composition results in an increased dynein axonemal heavy chain 5 mRNA expression level in a biological sample from a subject as compared to a baseline expression level before treatment.
  • the baseline level is measured immediately before treatment.
  • Biological samples include, for example, whole blood, serum, plasma, urine and tissue samples (e.g., muscle, liver, skin fibroblasts).
  • administering the provided composition results in an increased DNAH5 mRNA expression level by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to the baseline level immediately before treatment.
  • administering the provided composition results in an increased DNAH5 mRNA expression level as compared to a DNAH5 mRNA expression level in subjects who are not treated
  • a therapeutically effective dose of the provided composition when administered regularly, results in an increased DNAH5 protein expression or activity level in a subject as compared to a baseline DNAH5 protein expression or activity level before treatment.
  • the DNAH5 protein expression or activity level is measured in a biological sample obtained from the subject such as blood, plasma or serum, urine, or solid tissue extracts.
  • the administering of a composition of the invention results in DNAH5 expression detectable in the liver.
  • administering the provided composition results in an increased DNAH5 protein expression or activity level in a biological sample (e.g., plasma/serum or urine) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment.
  • a biological sample e.g., plasma/serum or urine
  • administering the provided composition results in an increased DNAH5 protein expression or activity level in a biological sample (e.g., plasma/serum or urine) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment for at least 24 hours, at least 48 hours, at least 72 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days.
  • a biological sample e.g., plasma/serum or urine
  • administering the provided composition results in an increased DNAH5 protein expression or activity level in a biological sample (e.g., plasma/serum or urine) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment for at least 24 hours, at
  • the therapeutic dose is sufficient to achieve at least some stabilization, improvement or elimination of symptoms and other indicators, such as biomarkers, are selected as appropriate measures of disease progress, disease regression or improvement by those of skill in the art.
  • Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal.
  • the therapeutically effective dose comprising the mRNA encoding dynein axonemal heavy chain protein is administered to the subject by intramuscular administration.
  • the therapeutically effective dose comprising the mRNA encoding dynein axonemal heavy chain protein is administered to the subject by subcutaneous administration.
  • the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments the administration results in delivery of the mRNA to a muscle cell. In some embodiments the administration results in delivery of the mRNA to a hepatocyte (i.e., liver cell). In a particular embodiment, the intramuscular administration results in delivery of the mRNA to a muscle cell.
  • the therapeutically effective dose comprising the mRNA encoding dynein axonemal heavy chain protein is administered to the subject by intravenous administration.
  • liposomally encapsulated mRNAs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • Formulations containing provided compositions complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.
  • DNAH5 encoding mRNA is administered intravenously, wherein intravenous administration is associated with delivery of the mRNA to hepatocytes.
  • the therapeutically effective dose comprising the mRNA encoding dynein axonemal heavy chain protein is administered for suitable delivery to the mammal's liver. In some embodiments, the therapeutically effective dose comprising the mRNA encoding dynein axonemal heavy chain protein is administered for suitable expression in hepatocytes of the administered mammal.
  • Therapeutic agents e.g., mRNA encoding a DNAH5 protein
  • Therapeutic agents can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition (e.g., PCD).
  • a therapeutically effective amount of the therapeutic agents (e.g., mRNA encoding a DNAH5 protein) of the present invention may be administered intrathecally periodically at regular intervals (e.g., once every year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily or continuously).
  • regular intervals e.g., once every year, once every six months, once every five months, once every three months, bimonthly (once every two months), monthly (once every month), biweekly (once every two weeks), twice a month, once every 30 days, once every 28 days, once every 14 days, once every 10 days, once every 7 days, weekly, twice a week, daily or continuously).
  • provided liposomes and/or compositions are formulated such that they are suitable for extended-release of the mRNA contained therein.
  • extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • the compositions of the present invention are administered to a subject twice a day, daily or every other day.
  • compositions of the present invention are administered to a subject twice a week, once a week, once every 7 days, once every 10 days, once every 14 days, once every 28 days, once every 30 days, once every two weeks, once every three weeks, once every four weeks, once a month, twice a month, once every six weeks, once every eight weeks, once every other month, once every three months, once every four months, once every six months, once every eight months, once every nine months or annually.
  • compositions of the present invention are administered to a subject once a week, once every two weeks or once a month. In a more preferred embodiment, the compositions of the present invention are administered to a subject once every two weeks or once every month. In the most preferred embodiment, the compositions of the present invention are administered to a subject once every month.
  • the mRNA is administered concurrently with an additional therapy.
  • compositions and liposomes which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release an mRNA over extended periods of time.
  • depot administration e.g., intramuscularly, subcutaneously, intravitreally
  • the extended-release means employed are combined with modifications made to the mRNA to enhance stability.
  • a therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses.
  • a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents.
  • the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; and like factors as is well known in the medical arts.
  • a therapeutically effective dose of the provided composition when administered regularly, results in at least one symptom or feature of PCD is reduced in intensity, severity, or frequency or has delayed onset.
  • lyophilized pharmaceutical compositions comprising one or more of the liposomes disclosed herein and related methods for the use of such compositions as disclosed for example, in International Patent Application PCT/US12/41663, filed Jun. 8, 2012, the teachings of which are incorporated herein by reference in their entirety.
  • lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo.
  • a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.
  • the pharmaceutical composition comprises a lyophilized liposomal delivery vehicle that comprises a cationic lipid, a non-cationic lipid, a PEG-modified lipid and cholesterol.
  • the pharmaceutical composition has a Dv50 of less than 500 nm, 300 nm, 200 nm, 150 nm, 125 nm, 120 nm, 100 nm, 75 nm, 50 nm, 25 nm or smaller upon reconstitution.
  • the pharmaceutical composition has a Dv90 of less than 750 nm, 700 nm, 500 nm, 300 nm, 200 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or smaller upon reconstitution.
  • the pharmaceutical composition has a polydispersity index value of less than 1, 0.95, 0.9, 0.8, 0.75, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, 0.1, 0.05 or less upon reconstitution.
  • the pharmaceutical composition has an average particle size of less than 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or upon reconstitution.
  • the lyophilized pharmaceutical composition further comprises one or more lyoprotectants, such as sucrose, trehalose, dextran or inulin. Typically, the lyoprotectant is sucrose.
  • the pharmaceutical composition is stable for at least 1 month or at least 6 months upon storage at 4° C., or for at least 6 months upon storage at 25° C.
  • the biologic activity of the mRNA of the reconstituted lyophilized pharmaceutical composition exceeds 75% of the biological activity observed prior to lyophilization of the composition.
  • Provided liposomes and compositions may be administered to any desired tissue.
  • the DNAH5 mRNA delivered by provided liposomes or compositions is expressed in the tissue in which the liposomes and/or compositions were administered.
  • the mRNA delivered is expressed in a tissue different from the tissue in which the liposomes and/or compositions were administered.
  • Exemplary tissues in which delivered mRNA may be delivered and/or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid.
  • the timing of expression of delivered mRNAs can be tuned to suit a particular medical need.
  • the expression of the protein encoded by delivered mRNA is detectable 1, 2, 3, 6, 12, 24, 48, 72, 96 hours, 1 week, 2 weeks, or 1 month after administration of provided liposomes and/or compositions.
  • a therapeutically effective dose of the provided composition when administered regularly, results in a reduced methylmalonic acid level in a subject as compared to a baseline methylmalonic acid level before treatment.
  • administering the provided composition results in an increased level of DNAH5 protein in a liver cell (e.g., a hepatocyte) of a subject as compared to a baseline level before treatment. Typically, the baseline level is measured immediately before treatment. In some embodiments, administering the provided composition results in an increased DNAH5 protein level in the liver cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment. In some embodiments, administering the provided composition results in an increased DNAH5 protein level in a liver cell as compared to the DNAH5 protein level a liver cell of subjects who are not treated.
  • a liver cell e.g., a hepatocyte
  • administering the provided composition results in an increased DNAH5 protein level in plasma or serum of subject as compared to a baseline level before treatment. Typically, the baseline level is measured immediately before treatment. In some embodiments, administering the provided composition results in an increased DNAH5 protein level in plasma or serum by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level before treatment. In some embodiments, administering the provided composition results in an increased DNAH5 protein level in plasma or serum as compared to a DNAH5 protein level in plasma or serum of subjects who are not treated.
  • administering the provided composition results in increased DNAH5 enzyme activity in a biological sample from a subject as compared to the baseline level before treatment.
  • the baseline level is measured immediately before treatment.
  • Biological samples include, for example, whole blood, serum, plasma, urine and tissue samples (e.g., liver).
  • administering the provided composition results in an increased DNAH5 enzyme activity by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared to a baseline level immediately before treatment.
  • administering the provided composition results in an increased DNAH5 enzyme activity as compared to DNAH5 enzyme activity in subjects who are not treated.
  • the subject is a mammal. In some embodiments, the mammal is an adult. In some embodiments the mammal is an adolescent. In some embodiments the mammal is an infant or a young mammal. In some embodiments, the mammal is a primate. In some embodiments the mammal is a human. In some embodiments the subject is 6 years to 80 years old.
  • Example 1 Exemplary Liposome Formulations for DNAH5 mRNA Delivery and Expression
  • This example provides exemplary liposome formulations for effective delivery and expression of hDNAH5 mRNA in vivo.
  • the formulations described in the following Examples contain a multi-component lipid mixture of varying ratios employing one or more cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol lipids) and PEGylated lipids designed to encapsulate human dynein axonemal heavy chain 5 (hDNAH5) mRNA.
  • the multi-component lipid mixture used in the following Examples were ethanolic solutions of an imidazole cholesterol ester (“ICE”) cationic lipid, a non-cationic lipid such as DOPE, and a PEGylated lipid such as DMG-PEG2K.
  • ICE imidazole cholesterol ester
  • Codon-optimized hDNAH5 messenger RNA was synthesized by in vitro transcription from a plasmid DNA template encoding the gene. Following in vitro transcription, a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA cap structures by viral and cellular proteins” J. Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail were added. The poly(A) tail was approximately 135 nucleotides in length on average.
  • the 5′ and 3′ untranslated regions present in each mRNA product are represented as X and Y, respectively, and defined as stated (vide infra).
  • the MRT-1 codon-optimized hDNAH5 messenger RNA coding region comprised the sequence of SEQ ID NO. 6 or SEQ ID NO. 7.
  • a 3′-GFP-tagged version of MRT-1 codon-optimized hDNA5 was likewise prepared, MRT-hDNA5-GFP using molecular cloning techniques well known in the art.
  • hDNAH5 mRNA was encapsulated in multi-component liposomes as described in WO 2018/089790, published May 17, 2018 (incorporated herein by reference), at an N/P ratio of approximately 10.
  • This example illustrates exemplary methods of administering hDNAH5 mRNA-loaded liposome nanoparticles and methods for analyzing delivered mRNA and subsequently expressed hDNAH5 protein in lung epithelium in vivo.
  • the test article for Group 1 was 10 ⁇ g/animal of MRT-1 hDNAH5 mRNA prepared as described in Example 1.
  • the test article for Group 2 was 10 ⁇ g/animal (unless otherwise specified) of hDNAH5-GFP mRNA (i.e., a sequence including both MRT1 hDNAH5 mRNA and green fluorescent protein (GFP) mRNA) prepared as described in Example 1.
  • the control included either saline administered at the same volume or an irrelevant mRNA in the same delivery vehicle as the test articles. Mice were euthanized at 24 hours ( ⁇ 5%) post dose administration.
  • FIG. 1A depicts the dissection scheme of the lung.
  • the left section of the entire airway was fixed in buffer for subsequent immunohistochemical and histological analysis.
  • the right section of the entire airway was snap-frozen and stored at ⁇ 70° C. for subsequent qPCR analysis of the trachea (1), superior lobe (2), middle lobe (3), inferior lobe (4), and post-caval lobe (5).
  • the liver also was snap-frozen and stored at ⁇ 70° C.
  • RNA is reverse transcribed (RT) into cDNA using random primers.
  • RT reverse transcribed
  • qPCR taqman fluorophore probe
  • the hDNAH5 and GFP protein in the trachea and lungs was characterized by IHC staining. Briefly, the harvested tissues were fixed in formalin and embedded in paraffin blocks. Sections (5 micron thick) along the length of the tissues were mounted on glass slides for staining. Antigen retrieval was performed using EDTA based buffer, followed by blocking with hydrogen peroxide and goat serum. Primary antibodies against hDNAH5 (Ab122390) and GFP (Ab290) were incubated with respective samples overnight at 4° C. Enzyme-conjugated secondary antibodies were used for detection of the bound primary antibodies. The images of the stained slides were captured at 20 ⁇ magnification. Results of the IHC analysis are shown in FIG. 2 .
  • FIG. 1B provides qPCR data showing successful hDNAH5 mRNA deposition in cells in each of the trachea (1), superior lobe (2), middle lobe (3), inferior lobe (4), and post-caval lobe (5) of the lung for each mouse in Groups 1 and 2.
  • FIG. 1B provides qPCR data showing successful hDNAH5 mRNA deposition in cells in each of the trachea (1), superior lobe (2), middle lobe (3), inferior lobe (4), and post-caval lobe (5) of the lung for each mouse in Groups 1 and 2.
  • FIG. 2A provides exemplary IHC images showing positive staining for hDNAH5 protein expressed from the hDNAH5 mRNA lung tissue from mice in each of Groups 1 and 2.
  • FIG. 2B shows IHC images with positive staining for hDNAH5 protein, from mice in Groups 1 and 2, in tissue from the trachea as well as tissue across the entire lung, from top to bottom (left to right in FIG. 2B ).
  • Exemplary codon-optimized mRNA sequences are shown in SEQ ID NO: 6-31.
  • U and T are used interchangeably.
US17/420,346 2019-01-07 2020-01-07 Composition and Methods for Treatment of Primary Ciliary Dyskinesia Abandoned US20220087935A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/420,346 US20220087935A1 (en) 2019-01-07 2020-01-07 Composition and Methods for Treatment of Primary Ciliary Dyskinesia

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201962789414P 2019-01-07 2019-01-07
US17/420,346 US20220087935A1 (en) 2019-01-07 2020-01-07 Composition and Methods for Treatment of Primary Ciliary Dyskinesia
PCT/US2020/012529 WO2020146344A1 (en) 2019-01-07 2020-01-07 Composition and methods for treatment of primary ciliary dyskinesia

Publications (1)

Publication Number Publication Date
US20220087935A1 true US20220087935A1 (en) 2022-03-24

Family

ID=69423424

Family Applications (3)

Application Number Title Priority Date Filing Date
US16/736,417 Active 2040-03-14 US11559561B2 (en) 2019-01-07 2020-01-07 Composition and methods for treatment of primary ciliary dyskinesia
US17/420,346 Abandoned US20220087935A1 (en) 2019-01-07 2020-01-07 Composition and Methods for Treatment of Primary Ciliary Dyskinesia
US18/062,435 Pending US20230226147A1 (en) 2019-01-07 2022-12-06 Composition and methods for treatment of primary ciliary dyskinesia

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US16/736,417 Active 2040-03-14 US11559561B2 (en) 2019-01-07 2020-01-07 Composition and methods for treatment of primary ciliary dyskinesia

Family Applications After (1)

Application Number Title Priority Date Filing Date
US18/062,435 Pending US20230226147A1 (en) 2019-01-07 2022-12-06 Composition and methods for treatment of primary ciliary dyskinesia

Country Status (8)

Country Link
US (3) US11559561B2 (de)
EP (1) EP3908597A1 (de)
JP (2) JP2022516356A (de)
CN (1) CN113412273A (de)
AU (1) AU2020206109A1 (de)
CA (1) CA3125588A1 (de)
IL (1) IL284480A (de)
WO (1) WO2020146344A1 (de)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK3463483T3 (da) 2016-05-27 2024-03-04 Transcriptx Inc Behandling af primær ciliedyskinesi med syntetisk messenger-RNA
US11559561B2 (en) * 2019-01-07 2023-01-24 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia
EP4308703A1 (de) * 2021-03-19 2024-01-24 Recode Therapeutics, Inc. Polynukleotidzusammensetzungen, zugehörige formulierungen und verfahren zur verwendung davon
WO2023086893A1 (en) * 2021-11-10 2023-05-19 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia

Family Cites Families (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4401796A (en) 1981-04-30 1983-08-30 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4737323A (en) 1986-02-13 1988-04-12 Liposome Technology, Inc. Liposome extrusion method
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
FR2645866B1 (fr) 1989-04-17 1991-07-05 Centre Nat Rech Scient Nouvelles lipopolyamines, leur preparation et leur emploi
US5334761A (en) 1992-08-28 1994-08-02 Life Technologies, Inc. Cationic lipids
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US5744335A (en) 1995-09-19 1998-04-28 Mirus Corporation Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein
ATE536418T1 (de) 2004-06-07 2011-12-15 Protiva Biotherapeutics Inc Lipidverkapselte interferenz-rna
JP2009544754A (ja) 2006-07-28 2009-12-17 アプライド バイオシステムズ， エルエルシー ジヌクレオチドｍｒｎａキャップアナログ
EP2094849B1 (de) * 2006-11-09 2014-01-08 Dynavax Technologies Corporation Langfristige modifikation einer erkrankung mithilfe immunstimulatorischer oligonukleotide
WO2009058911A2 (en) 2007-10-31 2009-05-07 Applied Biosystems Inc. Preparation and isolation of 5' capped mrna
AU2009303345B2 (en) 2008-10-09 2015-08-20 Arbutus Biopharma Corporation Improved amino lipids and methods for the delivery of nucleic acids
JP6087504B2 (ja) 2008-11-07 2017-03-01 マサチューセッツ インスティテュート オブ テクノロジー アミノアルコールリピドイドおよびその使用
DK2440183T3 (en) 2009-06-10 2018-10-01 Arbutus Biopharma Corp Improved lipid formulation
CN103748078B (zh) 2011-06-08 2016-11-09 夏尔人类遗传性治疗公司 可裂解脂质
AU2012328570B2 (en) 2011-10-27 2017-08-31 Massachusetts Institute Of Technology Amino acid derivatives functionalized on the n-terminus capable of forming drug encapsulating microspheres and uses thereof
CN104334161A (zh) 2012-03-29 2015-02-04 夏尔人类遗传性治疗公司 可电离的阳离子脂质
PT3083556T (pt) 2013-12-19 2020-03-05 Novartis Ag Lípidos e composições lipídicas para a entrega de agentes ativos
EP3699274A1 (de) * 2014-03-24 2020-08-26 Translate Bio, Inc. Mrna-therapie zur behandlung von augenerkrankungen
MA48050A (fr) 2014-05-30 2020-02-12 Translate Bio Inc Lipides biodégradables pour l'administration d'acides nucléiques
SI3766916T1 (sl) 2014-06-25 2023-01-31 Acuitas Therapeutics Inc. Formulacije novih lipidov in lipidnih nanodelcev za dostavo nukleinskih kislin
WO2016004202A1 (en) 2014-07-02 2016-01-07 Massachusetts Institute Of Technology Polyamine-fatty acid derived lipidoids and uses thereof
US20180000953A1 (en) 2015-01-21 2018-01-04 Moderna Therapeutics, Inc. Lipid nanoparticle compositions
EP3247398A4 (de) 2015-01-23 2018-09-26 Moderna Therapeutics, Inc. Lipidnanopartikelzusammensetzungen
AU2016233135B2 (en) * 2015-03-19 2021-07-08 Translate Bio, Inc. mRNA therapy for pompe disease
US10201618B2 (en) 2015-06-19 2019-02-12 Massachusetts Institute Of Technology Alkenyl substituted 2,5-piperazinediones, compositions, and uses thereof
WO2017004143A1 (en) 2015-06-29 2017-01-05 Acuitas Therapeutics Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
AU2016324310B2 (en) 2015-09-17 2021-04-08 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
CN113636947A (zh) 2015-10-28 2021-11-12 爱康泰生治疗公司 用于递送核酸的新型脂质和脂质纳米颗粒制剂
WO2017117528A1 (en) 2015-12-30 2017-07-06 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
BR112018069795A2 (pt) 2016-03-30 2019-01-29 Intellia Therapeutics Inc formulações de nanopartículas lipídicas para componentes de crispr/cas
DK3463483T3 (da) 2016-05-27 2024-03-04 Transcriptx Inc Behandling af primær ciliedyskinesi med syntetisk messenger-RNA
EP3538073A1 (de) 2016-11-10 2019-09-18 Translate Bio, Inc. Verbessertes verfahren zur herstellung von mrna-beladenen lipidnanopartikeln
CA3043033A1 (en) 2016-11-10 2018-05-17 Translate Bio, Inc. Improved ice-based lipid nanoparticle formulation for delivery of mrna
EP3592728A1 (de) * 2017-03-07 2020-01-15 Translate Bio, Inc. Polyanionische verabreichung von nukleinsäuren
EP4311541A3 (de) 2018-04-25 2024-02-07 Ethris GmbH Kryoprotektive mittel für teilchenförmige formulierungen
US11559561B2 (en) * 2019-01-07 2023-01-24 Translate Bio, Inc. Composition and methods for treatment of primary ciliary dyskinesia

Also Published As

Publication number Publication date
US11559561B2 (en) 2023-01-24
US20200215157A1 (en) 2020-07-09
WO2020146344A1 (en) 2020-07-16
JP2022516356A (ja) 2022-02-25
US20230226147A1 (en) 2023-07-20
CN113412273A (zh) 2021-09-17
EP3908597A1 (de) 2021-11-17
JP2024028651A (ja) 2024-03-04
IL284480A (en) 2021-08-31
CA3125588A1 (en) 2020-07-16
AU2020206109A1 (en) 2021-07-29

Similar Documents

Publication Publication Date Title
US20230050301A1 (en) Mrna therapy for phenylketonuria
US20220218803A1 (en) Mrna therapy for argininosuccinate synthetase deficiency
US11104652B2 (en) Stereochemically enriched compositions for delivery of nucleic acids
US11712463B2 (en) MRNA therapy for pompe disease
US20210205421A1 (en) Messenger rna therapy for the treatment of ornithine transcarbamylase deficiency
US20220072152A1 (en) Composition and Methods for Treatment of Ornithine Transcarbamylase Deficiency
US11559561B2 (en) Composition and methods for treatment of primary ciliary dyskinesia
US20230145188A1 (en) Composition and methods for treatment of methylmalonic acidemia
AU2014340083A1 (en) mRNA therapy for phenylketonuria
WO2023086893A1 (en) Composition and methods for treatment of primary ciliary dyskinesia

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION UNDERGOING PREEXAM PROCESSING

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STCB Information on status: application discontinuation

Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION