Engineering the RRM2 subunit of ribonucleotide reductase to resist degradation

Patent metadata

Field	Value
Publication number	US-12208133-B2
Application number	US-201916457441-A
Country	US
Kind code	B2
Filing date	Jun 28, 2019
Priority date	Jun 29, 2018
Publication date	Jan 28, 2025
Grant date	Jan 28, 2025

Abstract

Provided herein are isolated nucleic acids that encode a stable form of Rrm2 for the use of increasing the intracellular Rrm2 protein levels and cytosolic 2-deoxy-ATP (dATP) levels. Further provided herein are methods for treating a cardiac disease or disorder, e.g., myocardial infarction or myocardial ischemia, by administering the isolated nucleic acids, a polypeptide encoded by the isolated nucleic acids, or composition comprising the isolated nucleic acids to a subject in need thereof.

First claim

The invention claimed is:

1. An isolated nucleic acid molecule encoding a mutant Rrm2 polypeptide, wherein the isolated nucleic acid molecule comprises a mutated version of SEQ ID NO: 3 or SEQ ID NO: 5, the version having at least a mutation of a nucleotide selected from the group consisting of: positions 88-96, 97-99, and 145-153 of SEQ ID NO: 3 or SEQ ID NO: 5,
wherein the encoded mutant Rrm2 polypeptide comprises a mutation in a ubiquitin-binding degron relative to a wild-type Rrm2 polypeptide, wherein the ubiquitin-binding degron is encoded by wild-type nucleotides 88-96, 97-99, and/or 145-153 of SEQ ID NO: 3 or SEQ ID NO: 5, and
wherein the mutation in the ubiquitin-binding degron increases the intracellular stability of the mutant Rrm2 polypeptide compared to the wild-type.

2. The isolated nucleic acid molecule of claim 1 , wherein the mutation in the ubiquitin-binding degron prevents or inhibits ubiquitin-mediated degradation of its gene product.

3. The isolated nucleic acid molecule of claim 1 , wherein the encoded mutant Rrm2 polypeptide comprises 2 to 10 amino acid changes as compared to wild-type Rrm2 polypeptide sequence.

4. The isolated nucleic acid molecule of claim 1 , wherein the isolated nucleic acid molecule has a nucleic acid sequence selected from SEQ ID NO: 16-20.

5. The isolated nucleic acid molecule of claim 1 , wherein the mutation in the ubiquitin-binding degron increases the level of cytosolic 2-deoxy-ATP (dATP) in a cell when the isolated nucleic acid molecule is expressed in a cell as compared to expression of wild-type Rrm2.

6. The isolated nucleic acid molecule of claim 1 , wherein the isolated nucleic acid molecule is operably linked to a sequence that permits expression of the encoded polypeptide in a cell.

7. A vector comprising the isolated nucleic acid molecule of claim 1 .

8. The vector of claim 7 , wherein the vector is a viral vector.

9. The vector of claim 8 , wherein the viral vector is an adeno associated virus (AAV), a lentivirus (LV), a herpes simplex virus (HSV), an adeno virus (AV), or a pox virus (PV) vector.

10. A composition comprising:
(i) the isolated nucleic acid molecule of claim 1 ; or
(ii) a vector comprising a nucleic acid molecule comprising the sequence of the isolated nucleic acid molecule of claim 1 .

Assignees

• Univ Washington

Inventors

• Regnier Michael
• Murry Charles E
• Guan Xuan

Classifications

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• A61K48/0066

  Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid · CPC title

• C12Y117/04001

  Ribonucleoside-diphosphate reductase (1.17.4.1) · CPC title

• C12N15/86

  Viral vectors · CPC title

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title