Modified polynucleotides for the production of secreted proteins

Patent metadata

Field	Value
Publication number	US-9828416-B2
Application number	US-201514750004-A
Country	US
Kind code	B2
Filing date	Jun 25, 2015
Priority date	Apr 2, 2012
Publication date	Nov 28, 2017
Grant date	Nov 28, 2017

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

First claim

We claim:

1. An isolated mRNA comprising;
(a) a first region of linked nucleosides, said first region having at least 80% identity to SEQ ID NO: 5972 encoding a polypeptide of interest, said polypeptide of interest having the sequence of SEQ ID NO: 2630;
(b) a first flanking region located at the 5′ terminus of said first region comprising;
(i) a sequence of linked nucleosides having the sequence of the native 5′ UTR of the nucleic acid that encodes SEQ ID NO: 2630, or having the sequence of SEQ ID NOs: 1-4; and
(ii) at least one 5′ terminal cap;

(c) a second flanking region located at the 3′ terminus of said first region comprising;
(i′) a sequence of linked nucleosides having the sequence of the native 3′ UTR of the nucleic acid that encodes SEQ ID NO: 2630, or having the sequence of SEQ ID NOs: 5-21; and
(ii′) a 3′ tailing sequence of linked nucleosides.

2. The isolated mRNA of claim 1 wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid having the sequence of SEQ ID NO: 5972.

3. The isolated mRNA of claim 1 , wherein the 3′ tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of approximately 160 nucleotides and a polyA-G quartet.

4. The isolated mRNA of claim 1 which is purified.

5. The isolated mRNA of claim 1 , wherein the at least one 5′ terminal cap is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

6. The isolated mRNA of claim 1 , wherein at least one of said linked nucleosides comprises at least one modification as compared to the chemical structure of an A, G, U or C ribonucleotide.

7. The isolated mRNA of claim 6 , wherein at least one said modification is located in a nucleoside base and/or sugar portion.

8. The isolated mRNA of claim 1 , wherein said first region comprises n number of linked nucleosides having Formula (Ia):

or a pharmaceutically acceptable salt or stereoisomer thereof,
wherein
U is O, S, N(R U ) nu , or C(R U ) nu , wherein nu is an integer from 0 to 2 and each R U is, independently, H, halo, or optionally substituted alkyl;
is a single or double bond;
- - - is a single bond or absent;
each of R 1′ , R 2′ , R 1″ , R 2″ , R 3 , R 4 , and R 5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, or absent; wherein the combination of R 3 with one or more of R 1′ , R 1″ , R 2′ , R 2″ , or R 5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl; wherein the combination of R 5 with one or more of R 1′ , R 1″ , R 2′ , or R 2″ can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl; and wherein the combination of R 4 and one or more of R 1′ , R 1″ , R 2′ , R 2″ , R 3 , or R 5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl;
each of Y 1 , Y 2 , and Y 3 , is, independently, O, S, —NR N1 —, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
each Y 4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y 5 is, independently, O, S, optionally substituted alkylene, or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and
B is a nucleobase, wherein the combination of B and R 1′ , the combination of B and R 2′ , the combination of B and R 1″ , or the combination of B and R 2″ can, taken together with the carbons to which they are attached, optionally form a bicyclic group or wherein the combination of B, R 1″ , and R 3 or the combination of B, R 2″ , and R 3 can optionally form a tricyclic or tetracyclic group.

9. The isolated mRNA of claim 8 , wherein B is not pseudouridine (ψ) or 5-methyl-cytidine (m 5 C).

10. The isolated mRNA of claim 8 , wherein
U is O or C(R U ) nu , wherein nu is an integer from 1 to 2 and each R U is, independently, H, halo, or optionally substituted alkyl;
each of R 1 , R 1′ , R 1″ , R 2 , R 2′ , and R 2″ , if present, is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;
each of R 3 and R 4 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxyalkoxy;
each of Y 1 , Y 2 , and Y 3 , is, independently, O, S, —NR N1 —, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

each Y 4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;
each Y 5 is, independently, O or optionally substituted alkylene; and
n is an integer from 10 to 10,000.

11. The isolated mRNA of claim 10 , wherein each of R 1 , R 1′ , and R 1″ , if present, is H.

12. The isolated mRNA of claim 11 , wherein each of R 2 , R 2′ , and R 2″ , if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

13. The isolated mRNA of claim 10 , wherein each of R 2 , R 2′ , and R 2″ , if present, is H.

14. The isolated mRNA of claim 13 , wherein each of R 1 , R 1′ , and R 1″ , if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

15. The isolated mRNA of claim 8 , wherein said first region comprises n number of linked nucleotides having Formula (IIa):

or a pharmaceutically acceptable salt or stereoisomer thereof.

16. The isolated mRNA of claim 8 , wherein said first region comprises n number of linked nucleosides having Formula (IIk):

or a pharmaceutically acceptab

Assignees

• Modernatx Inc

Inventors

• Bancel Stephane
• Chakraborty Tirtha
• De Fougerolles Antonin
• Elbashir Sayda M
• John Matthias
• Roy Atanu
• Whoriskey Susan
• Wood Kristy M
• Hatala Paul
• Schrum Jason P
• Ejebe Kenechi
• Ellsworth Jeff Lynn
• Guild Justin

Classifications

• C12N15/85

  for animal cells · CPC title

• C07K14/745

  Blood coagulation or fibrinolysis factors · CPC title

• A61K38/193

  Colony stimulating factors [CSF] · CPC title

• A61K9/1271

  Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) · CPC title

• A61K9/1277

  Preparation processes; Proliposomes · CPC title