Engineered nucleic acids and methods of use thereof

• US 9,464,124 B2
• Filed: 11/05/2014
• Issued: 10/11/2016
• Est. Priority Date: 09/12/2011
• Status: Active Grant

• Current Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• Original Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• Inventors: Bancel, Stephane, Schrum, Jason P., Aristarkhov, Alexander
• Primary Examiner(s): Swartz, Rodney P
• Application Number:
  US14/533,264
• Publication Number:
  US 20150056253A1
• Time in Patent Office: 706 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/1729 Cationic antimicrobial peptides, e.g. defensins
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 48/0075 characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• CPC Class Codes: C07K 14/4723 Cationic antimicrobial peptides, e.g. defensins
• CPC Class Codes: C12Y 301/27 Endoribonucleases producing 3'-phosphomonoesters (3.1.27)

Abstract

Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to kill or reduce the growth of microorganisms. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent microbial infection, or to improve a subject'"'"'s heath or wellbeing.

Claims

1. A pharmaceutical composition comprising:

a lipid-based nanoparticle comprising a synthetic messenger ribonucleic acid (mRNA) encoding a defensin polypeptide in an amount effective to permit production of the defensin polypeptide in a cell, wherein the synthetic mRNA comprises a translatable region that contains at least one nucleoside modification, and wherein 75-100% of uridine nucleotides in the synthetic mRNA are modified.

2. The pharmaceutical composition of claim 1, wherein the at least one nucleoside modification is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

3. The pharmaceutical composition of claim 1, wherein the composition is formulated for administration via a route selected from the group consisting of:

systemic, local, intravenous, topical, oral, administration via a dressing, administration via a bandage, rectal, vaginal, intramuscular, transarterial, intraperitoneal, intranasal, subcutaneous, endoscopic, transdermal and intrathecal.

4. The pharmaceutical composition of claim 3, wherein the route is intravenous.

5. The pharmaceutical composition of claim 1, wherein the defensin polypeptide is selected from the group consisting of α

-defensins, and β

-defensins and θ

-defensins.

6. The pharmaceutical composition of claim 1, wherein the anti-microbial polypeptide is hBD-2 (SEQ ID NO:

191 or

     192).

8. The pharmaceutical composition of claim 5, wherein the defensin is an α

-defensin selected from the group consisting of neutrophil defensin 1, defensin alpha 1, neutrophil defensin 3, neutrophil defensin 4, defensin 5 and defensin 6.

9. The pharmaceutical composition of claim 5, wherein the defensin is a β

-defensin selected from the group consisting of beta-defensin 1, beta-defensin 2, beta-defensin 103, beta-defensin 107, beta-defensin 110 and beta-defensin 136.

10. The pharmaceutical composition of claim 5, wherein the at least one nucleoside modification is pseudouridine.

11. The pharmaceutical composition of claim 5, wherein 100% of uridine nucleotides in the synthetic mRNA are modified.

12. The pharmaceutical composition of claim 5, wherein the synthetic mRNA further comprises a 5′

untranslated region that contains at least one nucleoside modification.

13. The pharmaceutical composition of claim 5, wherein the synthetic mRNA further comprises a 5′

untranslated region that contains at least two nucleoside modifications.

14. The pharmaceutical composition of claim 5, wherein the synthetic mRNA further comprises a 3′

untranslated region that contains at least one nucleoside modification.

15. The pharmaceutical composition of claim 5, wherein the synthetic mRNA further comprises a 3′

untranslated region that contains at least two nucleoside modifications.

7. A kit comprising:

(a) a synthetic messenger ribonucleic acid (mRNA) encoding a defensin polypeptide packaged in a container, wherein the synthetic mRNA comprises a translatable region that contains at least one nucleoside modification, and wherein 75-100% of uridine nucleotides in the synthetic mRNA are modified; and

(b) a pharmaceutically acceptable carrier packaged in a container.

16. A lipid-based nanoparticle comprising a synthetic messenger ribonucleic acid (mRNA) encoding a defensin polypeptide in an amount effective to permit production of the defensin polypeptide in a cell,

wherein 75-100% of uridine nucleotides in the synthetic mRNA are modified, and

wherein the synthetic mRNA comprises a translatable region, a 5′

untranslated region and a 3′

untranslated region, each of which contains at least one nucleoside modification.

17. The lipid-based nanoparticle of claim 16, wherein the at least one nucleoside modification is pseudouridine.