ENGINEERING THE RRM2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE TO RESIST DEGRADATION

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.

Claims

1. An isolated nucleic acid molecule encoding an Rrm2 polypeptide that, together with Rrm1 polypeptide comprises ribonucleotide reductase activity, the encoded Rrm2 polypeptide comprising a mutation that increases the intracellular level of the polypeptide as compared to wild-type Rrm2 polypeptide.

2. The isolated nucleic acid of claim 1, wherein the mutation increases the intracellular stability of the Rrm2 polypeptide relative to wild-type.

3. The isolated nucleic acid molecule of claim 1, wherein the mutation is in a ubiquitin-binding degron.

4. The isolated nucleic acid molecule of claim 3, wherein the mutation in the ubiquitin-binding degron prevents or inhibits ubiquitin-mediated degradation of its gene product.

5. The isolated nucleic acid molecule of claim 1, wherein the Rrm2 gene sequence encodes an Rrm2 polypeptide having 2 to 10 amino acid changes as compared to wild-type Rrm2 polypeptide sequence.

6. The isolated nucleic acid molecule of claim 3, wherein the ubiquitin-binding degron is encoded at nucleotides 88-96, 97-99, and/or 145-153 of SEQ ID NO: 3 or SEQ ID NO: 5.

7. The isolated nucleic acid molecule of claim 1, wherein the mutation is a mutation of a nucleotide selected from the group consisting of: 88-96, 97-99, and/or 145-153 positions of SEQ ID NO: 3 or SEQ ID NO: 5.

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

9. The isolated nucleic acid molecule of claim 1, wherein the mutation 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.

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

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

12. The vector of claim 11, wherein the vector is a viral vector.

13. The vector of claim 12, 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.

14. A polypeptide encoded by the isolated nucleic acid of claim 1.

15. The polypeptide of claim 14, wherein the polypeptide sequence is selected from SEQ ID NO: 4, 21-25.

16. A protein complex comprising the polypeptide of claim 14 and an Rrm1 polypeptide.

17. A composition comprising an isolated nucleic acid molecule of claim 1, a mutant Rrm2 polypeptide encoded by the isolated nucleic acid molecule, or a vector comprising a nucleic acid molecule comprising the sequence of the isolated nucleic acid molecule.

18. A pharmaceutical composition comprising the composition of claim 17 and a pharmaceutically acceptable carrier.

19. A method of treating a cardiac disease or disorder, the method comprising administering to a subject in need thereof a composition of claim 17.

20. The method of claim 19, wherein the cardiac disease or disorder is myocardial infarction or myocardial ischemia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] FIG. 1A-FIG. 1E are a series of graphs and schematics showing that dATP increases contractility in cardiac myosin. FIG. 1A is a line graph showing that isometric force of demembranated cardiac trabeculae increases with 2% dATP, 98% ATP vs. 100% ATP. FIG. 1A is adapted from Korte et al., J Mol Cell Cardiol. 2011 December; 51(6):894-901. FIG. 1B-FIG. 1E are a series of graphs and schematics showing that AAV6-mediated cardiac-specific overexpression of ribonucleotide reductase enhances myocardial contractility. FIG. 1B is a schematic showing an AAV6 vector expressing human RRM1 cDNA and human RRM2 cDNA. FIG. 1C is a series of bar graphs showing that cardiac function was increased with RRM1 and RRM2 overexpression. Cardiac function was assessed in isolated perfused mouse hearts 4 weeks post-rAAV6 injection (grey hatched) or control (white). Left ventricular (LV) developed pressure denotes the difference of LV systolic and LV diastolic pressures. Positive rate of pressure change calculated by the first derivative of the ascending LV pressure wave (+dP/dt) is used as an index of the rate of pressure development. Negative rate of pressure change calculated by the first derivative of the descending LV pressure wave (dP/dt) is used as an index of the rate of ventricular relaxation. FIG. 1D shows Western blot analysis of Rrm1 and Rrm2 protein levels in mouse liver, heart, and skeletal muscle 4 weeks postsystemic rAAV6 delivery. Significant overexpression of both Rrm1 and Rrm2 was detected in ventricles but not in the livers of treated mice, and essentially no expression of either endogenous subunit was detected in control heart or liver. FIG. 1E is a line graph showing that injection of rAAV6-rat(R1R2)cTnT455 improves in vivo cardiac function in rats subjected to myocardial infarction (MI). Direct injection of rAAV6-rat(R1R2) into the noninfarcted myocardium was made 5 days after ligation of the left anterior descending artery (MIR1R2) or sham surgery. Serial echocardiography measures were performed at 2, 4, and 8 weeks postinfarction. FIG. 1B-FIG. 1E are adapted from Kolwicz et al., Mol Ther. 2016 February; 24(2):240-250.

[0070] FIG. 2 is a series of line graphs showing quantification of deoxyribonucleotide triphosphates (dNTPs) in muscle tissues by high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). On the left are ion chromatograms showing relative abundance over time for dGTP, ATP, dATP, labeled C-13 dATP, dTTP, and dCTP upon injection of 62.5 fmol dNTPs, 1,250 fmol ATP, and 25,000 fmol of labeled C-13 dATP.

[0071] FIG. 3 is a series of dot plots showing that overexpression of RNR increases dATP content in cardiomyocytes. The term hiPSC-CMs denotes human induced pluripotent stem cell derived cardiomyocytes.

[0072] FIG. 4 shows a Western blot analysis of Rrm1 and Rrm2 proteins. Note that AAV-induced overexpression of Rrm2 can be highly variable.

[0073] FIG. 5A-FIG. 5B is series and graphs showing that Rrm2 is degraded in cardiomyocytes by ubiquitin-proteasome activity. FIG. 5A shows a Western blot analysis of Rrm1 and Rrm2 proteins in neonatal rat cardiomyocytes transduced for three days in vitro with AV-RNR, with or without a 3-hour incubation in MG-132, a potent proteasome inhibitor. FIG. 5B is a bar graph showing dATP levels. Note that Rrm2 protein levels and dATP levels are highest with MG132 treatment.

[0074] FIG. 6A-FIG. 6C is series and graphs showing that engineered mutations in Rrm2 increase RNR levels in hiPSC-CMs. Exemplary Rrm2 mutants include but are not limited to 49ARA, 30AAA/49ARA double mutant, 30AAN, 30AAA, 33T/A (see e.g., FIG. 9). FIG. 6A shows a Western blot analysis of Rrm1 and Rrm2 proteins in WT or Rrm2 mutant cells. FIG. 6B is a dot plot showing the ratio of the Rrm2 protein levels for each Rrm mutant compared to the protein level for wild-type (WT) Rrm2. FIG. 6C is a bar graph showing the ratio of dATP levels to ATP levels for each Rrm mutant or WT Rrm2.

[0075] FIG. 7 is a bar graph showing that engineered Rrm2 elevates [dATP] in neonatal rat cardiomyocytes in vitro compared to WT Rrm2. DM designates the Rrm2 30AAA/49ARA double mutant.

[0076] FIG. 8 is a series of schematics showing degron locations and engineered mutations in the Rrm2 nucleic acid sequence. Rrm2 is shown on top with five exemplary Rrm2 mutants aligned at the bottom: 30AAA, 30AAN, 33A, 49ARA, and 30AAA/49ARA double mutant. Note that the nucleic acid sequences shown are the non-coding sequence of each, i.e., reverse complement of the coding sequence. For coding sequences of each mutant see e.g., SEQ ID NOs: 16-20.

DETAILED DESCRIPTION

Ribonucleotide Reductase Complex

[0077] Ribonucleotide reductase (RNR), also known as ribonucleotide diphosphate reductase (rNDP), is an enzyme that catalyzes the reaction of ribonucleotides to deoxyribonucleotides, which are essential components in the synthesis of DNA. RNR is conserved in all living organisms. The RNR enzyme catalyzes the de novo synthesis of dNDPs. Catalysis of ribonucleoside 5-diphosphates (NDPs) involves a reduction at the 2-carbon of ribose 5-phosphate to form the 2-deoxy derivative-reduced 2-deoxyribonucleoside 5-diphosphates (dNDPs). This reduction is initiated with the generation of a free radical. Following a single reduction, RNR requires electrons donated from the dithiol groups of the protein thioredoxin. Regeneration of thioredoxin occurs when nicotinamide adenine dinucleotide phosphate (NADPH) provides two hydrogen atoms that are used to reduce the disulfide groups of thioredoxin.

[0078] Three classes of RNR have similar mechanisms for the reduction of NDPs. All classes use free-radical chemistry. Class I reductases use an iron center with ferrous to ferric conversion to generate a tyrosyl free radical. Reduction of NDP substrates occurs under aerobic conditions. Class I reductases are divided into IA and IB due to differences in regulation. Class IA reductases are distributed in eukaryotes, eubacteria, bacteriophages, and viruses. Class IB reductases are found in eubacteria. Class IB reductases can also use a radical generated with the stabilization of a binuclear manganese center. Class II reductases generate the free radical 5-deoxyadenosyl radical from cobalamin (coenzyme B12) and have a simpler structure than class I and class III reductases. Reduction of NDPs or ribonucleotide 5-triphosphates (NTPs) occurs under either aerobic or anaerobic conditions. Class II reductases are distributed in archaebacteria, eubacteria, and bacteriophages. Class III reductases use a glycine radical generated with the help of an S-adenosyl methionine and an iron sulphur center. Reduction of NTPs is limited to anaerobic conditions. Class III reductases are distributed in archaebacteria, eubacteria, and bacteriophages. Organisms are not limited to having one class of enzymes. For example, E. coli have both class I and class III RNR.

[0079] The RNR complex consists of two subunitsRrm1 and Rrm2. The larger Rrm1 subunit contains the catalytic site and 2 allosteric sites that can bind dATP, whereas the smaller Rrm2 subunit contains the free radical generator. The RNR complex is tightly allosterically regulated, with <5% of the ATP pool present as dATP. Each RNR1 monomer consists of three domains: (1) one mainly helical domain comprising the 220 N-terminal residues; (2) a second large ten-stranded / structure comprising 480 residues; and (3) a third small five-stranded / structure comprising 70 residues.

[0080] As used herein, Rrm1 or ribonucleotide reductase catalytic subunit M1 refers to the large, catalytic site containing, subunit of the RNR complex. Sequences for Rrm1 are known for a number of species, e.g., human Rrm1 (NCBI Gene ID: 6240) mRNA (NCBI Ref Seq: NM_001033.5) and polypeptide (NCBI Ref Seq: NP_001024.1). In some embodiments of any of the aspects, the Rrm1 nucleic acid or polypeptide can be an isoform, ortholog, variant, and/or allele of SEQ ID NO: 1 or 2, respectively.

[0081] As used herein, Rrm2 or ribonucleotide reductase catalytic subunit M2 refers to the small subunit of the RNR complex. Sequences for Rrm2 are known for a number of species, e.g., human Rrm2 (NCBI Gene ID: 6241) mRNA (NCBI Ref Seq: NM_001034.4) and polypeptide (NCBI Ref Seq: NP_001025.1). In some embodiments of any of the aspects, the Rrm2 nucleic acid or polypeptide can be an isoform, ortholog, variant, and/or allele of SEQ ID NO: 3 or 4, respectively.

[0082] SEQ ID NO: 1 is the nucleotide sequence encoding Rrm1, isoform 1.

TABLE-US-00001 (SEQIDNO:1) atgcatgtgatcaagcgagatggccg 241 ccaagaacgagtcatgtttgacaaaattacatctcgaatccagaagctttgttatggact 301 caatatggattttgttgatcctgctcagatcaccatgaaagtaatccaaggcttgtacag 361 tggggtcaccacagtggaactagatactttggctgctgaaacagctgcaaccttgactac 421 taagcaccctgactatgctatcctggcagccaggatcgctgtctctaacttgcacaaaga 481 aacaaagaaagtgttcagtgatgtgatggaagacctctataactacataaatccacataa 541 tggcaaacactctcccatggtggccaagtcaacattggatattgttctggccaataaaga 601 tcgcctgaattctgctattatctatgaccgagatttctcttacaattacttcggctttaa 661 gacgctagagcggtcttatttgttgaagatcaatggaaaagtggctgaaagaccacaaca 721 tatgttgatgagagtatctgttgggatccacaaagaagacattgatgcagcaattgaaac 781 atataatcttctttctgagaggtggtttactcatgcttcgcccactctcttcaatgctgg 841 taccaaccgcccacaactttctagctgttttcttctgagtatgaaagatgacagcattga 901 aggcatttatgacactctaaagcaatgtgcattgatttctaagtctgctggaggaattgg 961 tgttgctgtgagttgtattcgggctactggcagctacattgctgggactaatggcaattc 1021 caatggccttgtaccgatgctgagagtatataacaacacagctcgatatgtggatcaagg 1081 tgggaacaagcgtcctggggcatttgctatttacctggagccttggcatttagacatctt 1141 tgaattccttgatttaaagaagaacacaggaaaggaagagcagcgtgccagagatctttt 1201 ctttgctctttggattccggatctcttcatgaaacgagtggagactaatcaggactggtc 1261 tttgatgtgtccaaatgagtgtcctggtctggatgaggtttggggagaggaatttgagaa 1321 actatatgcaagttatgagaaacaaggtcgtgtccgcaaagttgtaaaagctcagcagct 1381 ttggtatgccatcattgagtctcagacggaaacaggcaccccgtatatgctctacaaaga 1441 ttcctgtaatcgaaagagcaaccagcagaacctgggaaccatcaaatgcagcaacctgtg 1501 cacagaaatagtggagtacaccagcaaagatgaggttgctgtttgtaatttggcttccct 1561 ggccctgaatatgtatgtcacatcagaacacacatacgactttaagaagttggctgaagt 1621 cactaaagtcgttgtccgaaacttgaataaaattattgatataaactactatcctgtacc 1681 agaggcatgcctatcaaataaacgccatcgccccattggaattggggtacaaggtctggc 1741 agatgcttttatcctgatgagatacccttttgagagtgcagaagcccagttactgaataa 1801 gcagatctttgaaactatttattatggtgctctggaagccagctgtgaccttgccaagga 1861 gcagggcccatacgaaacctatgagggctctccagttagcaaaggaattcttcagtatga 1921 tatgtggaatgttactcctacagacctatgggactggaaggttctcaaggagaagattgc 1981 aaagtatggtataagaaacagtttacttattgccccgatgcctacagcttccactgctca 2041 gatcctggggaataatgagtccattgaaccttacaccagcaacatctatactcgcagagt 2101 cttgtcaggagaatttcagattgtaaatcctcacttattgaaagatcttaccgagcgggg 2161 cctatggcatgaagagatgaaaaaccagattattgcatgcaatggctctattcagagcat 2221 accagaaattcctgatgacctgaagcaactttataaaactgtgtgggaaatctctcagaa 2281 aactgttctcaagatggcagctgagagaggtgctttcattgatcaaagccaatctttgaa 2341 catccacattgctgagcctaactatggcaaactcactagtatgcacttctacggctggaa 2401 gcagggtttgaagactgggatgtattatttaaggacaagaccagcggctaatccaatcca 2461 gttcactctaaataaggagaagctaaaagataaagaaaaggtatcaaaagaggaagaaga 2521 gaaggagaggaacacagcagccatggtgtgctctttggagaatagagatgaatgtctgat 2581 gtgtggatcctga SEQIDNO:2istheaminoacidsequenceforRrm1,isoform1. (SEQIDNO:2) MHVIKRDGRQERVMFDKITSRIQKLCYGLNMDFVDPAQITMKVI QGLYSGVTTVELDTLAAETAATLTTKHPDYAILAARIAVSNLHKETKKVFSDVMEDLY NYINPHNGKHSPMVAKSTLDIVLANKDRLNSAITYDRDFSYNYFGEKTLERSYLLKIN GKVAERPQHMLMRVSVGIHKEDIDAAIETYNLLSERWFTHASPTLFNAGTNRPQLSSC FLLSMKDDSIEGIYDTLKQCALISKSAGGIGVAVSCIRATGSYIAGTNGNSNGLVPML RVYNNTARYVDQGGNKRPGAFAIYLEPWHLDIFEFLDLKKNTGKEEQRARDLFFALWI PDLFMKRVETNQDWSLMCPNECPGLDEVWGEEFEKLYASYEKQGRVRKVVKAQQLWYA IIESQTETGTPYMLYKDSCNRKSNQQNLGTIKCSNLCTEIVEYTSKDEVAVCNLASLA LNMYVTSEHTYDFKKLAEVTKVVVRNLNKIIDINYYPVPEACLSNKRHRPIGIGVQGL ADAFILMRYPFESAEAQLLNKQIFETIYYGALEASCDLAKEQGPYETYEGSPVSKGIL QYDMWNVTPTDLWDWKVLKEKIAKYGIRNSLLIAPMPTASTAQILGNNESIEPYTSNI YTRRVLSGEFQIVNPHLLKDLTERGLWHEEMKNQIIACNGSIQSIPEIPDDLKQLYKT VWEISQKTVLKMAAERGAFIDQSQSLNIHIAEPNYGKLTSMHFYGWKQGLKTGMYYLR TRPAANPIQFTLNKEKLKDKEKVSKEEEEKERNTAAMVCSLENRDECLMCGS SEQIDNO:3isthenucleotidesequenceencodingRrm2,isoform2. (SEQIDNO:3) ATGCTCTCCCTCCGTGTCCCGCTCGCGCCCATCACGGACCCGCAGCAGCTGCAGCTCTCGCCGCTGAAGGGGC TCAGCTTGGTCGACAAGGAGAACACGCCGCCGGCCCTGAGCGGGACCCGCGTCCTGGCCAGCAAGACCGCGAG GAGGATCTTCCAGGAGCCCACGGAGCCGAAAACTAAAGCAGCTGCCCCCGGCGTGGAGGATGAGCCGCTGCTG AGAGAAAACCCCCGCCGCTTTGTCATCTTCCCCATCGAGTACCATGATATCTGGCAGATGTATAAGAAGGCAG AGGCTTCCTTTTGGACCGCCGAGGAGGTGGACCTCTCCAAGGACATTCAGCACTGGGAATCCCTGAAACCCGA GGAGAGATATTTTATATCCCATGTTCTGGCTTTCTTTGCAGCAAGCGATGGCATAGTAAATGAAAACTTGGTG GAGCGATTTAGCCAAGAAGTTCAGATTACAGAAGCCCGCTGTTTCTATGGCTTCCAAATTGCCATGGAAAACA TACATTCTGAAATGTATAGTCTTCTTATTGACACTTACATAAAAGATCCCAAAGAAAGGGAATTTCTCTTCAA TGCCATTGAAACGATGCCTTGTGTCAAGAAGAAGGCAGACTGGGCCTTGCGCTGGATTGGGGACAAAGAGGCT ACCTATGGTGAACGTGTTGTAGCCTTTGCTGCAGTGGAAGGCATTTTCTTTTCCGGTTCTTTTGCGTCGATAT TCTGGCTCAAGAAACGAGGACTGATGCCTGGCCTCACATTTTCTAATGAACTTATTAGCAGAGATGAGGGTTT ACACTGTGATTTTGCTTGCCTGATGTTCAAACACCTGGTACACAAACCATCGGAGGAGAGAGTAAGAGAAATA ATTATCAATGCTGTTCGGATAGAACAGGAGTTCCTCACTGAGGCCTTGCCTGTGAAGCTCATTGGGATGAATT GCACTCTAATGAAGCAATACATTGAGTTTGTGGCAGACAGACTTATGCTGGAACTGGGTTTTAGCAAGGTTTT CAGAGTAGAGAACCCATTTGACTTTATGGAGAATATTTCACTGGAAGGAAAGACTAACTTCTTTGAGAAGAGA GTAGGCGAGTATCAGAGGATGGGAGTGATGTCAAGTCCAACAGAGAATTCTTTTACCTTGGATGCTGACTTCT AA SEQIDNO:4istheaminoacidsequenceforRrm2,isoform2. (SEQIDNO:4) MLSLRVPLAPITDPQQLQLSPLKGLSLVDKENTPPALSGTRVLASKTARRIFQEPTEPKTKAAAPGVEDEPLL RENPRRFVIFPIEYHDIWQMYKKAEASFWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLV ERFSQEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETMPCVKKKADWALRWIGDKEA TYGERVVAFAAVEGIFESGSFASIFWLKKRGLMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREI IINAVRIEQEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGESKVERVENPFDEMENISLEGKTNFFEKR VGEYQRMGVMSSPTENSFTLDADF

[0083] One aspect described herein is an RNR complex comprising, consisting of, or consisting essentially of, a wild type Rrm1 protein and a Rrm2 protein encoded by any of the isolated nucleic acids described herein. As used herein, RNR complex refers to Rrm1 and Rrm2 in physical association with each other in the form that provides RNR activity. One skilled in the art can assess whether the RNR complex is formed, for example, by sucrose gradient analysis or co-immunoprecipitation under non-denaturing conditions.

[0084] In one embodiment, the RNR complex provided herein comprises the same enzymatic function of a wild-type RNR complex, for example, catalyzing the formation of deoxyribonucleotides from ribonucleotides. Assays for assessing the enzymatic function of a complex provided herein include, but are not limited to nucleotide binding assays, for example, as described in Chimploy, K., and Mathews, C K. J of Biol Chem, 2001; Hendricks, S P, and Mathews CK. J of Biol Chem, 1997; and Hendricks, S P, and Mathews CK. J of Biol Chem, 1998; the content of which are incorporated herein by reference.

[0085] In one embodiment, the nucleic acid (or vector comprising a nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system. In one embodiment, the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism). In one embodiment, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In one embodiment, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In one embodiment, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In one embodiment, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In one embodiment, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell. In one embodiment, the Rrm2 (and/or Rrm1) nucleic acid sequence is codon-optimized (see e.g., SEQ ID NO: 5).

TABLE-US-00002 SEQIDNO:5isanexemplarycodon-optimizedRrm2 nucleicacidsequence. ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCT GCAGCTGAGCCCACTGAAAGGCCTGAGTCTGGTCGATAAAGAGAACACAC CACCTGCACTGAGTGGCACTCGGGTGCTGGCATCAAAGACCGCCCGGAGA ATTTTCCAGGAGCCAACCGAACCCAAAACAAAGGCCGCTGCACCTGGGGT CGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGATTTTTC CTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGT TTCTGGACAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGA ATCCCTGAAGCCAGAGGAAAGGTACTTCATTTCTCATGTGCTGGCATTCT TTGCCGCTAGTGACGGGATCGTGAACGAGAATCTGGTCGAACGCTTTAGC CAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATTTCAGATTGC TATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATA TCAAAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATG CCATGTGTGAAGAAAAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAA GGAGGCTACTTACGGGGAAAGAGTGGTCGCATTCGCAGCCGTGGAGGGAA TTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCTGAAAAAGCGAGGC CTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAAGG CCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGC CCTCTGAGGAACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAG CAGGAGTTCCTGACAGAAGCCCTGCCTGTCAAACTGATTGGCATGAATTG CACTCTGATGAAGCAGTACATCGAGTTTGTGGCCGACAGGCTGATGCTGG AACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATTTTATG GAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGG GGAATATCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCA CCCTGGACGCCGATTTTTGA

[0086] In one embodiment, the Rrm2 and Rrm1 mRNAs are bicistronic, i.e., on the same vector and/or under the control of the same promoter. In one embodiment, the Rrm2 nucleic acid is linked to Rrm1, e.g., through a P2A sequence. P2A is a non-limiting example of a 2A self-cleaving peptide, which can induce the cleaving of the recombinant protein in cell. In one embodiment, the Rrm2 nucleic acid is linked to Rrm1, e.g., through a 2A self-cleaving peptide sequence. Non-limiting examples of 2A self-cleaving peptides include T2A, P2A, E2A, and F2A. Any self-cleaving peptide sequence known in the art can be used to link Rrm2 to Rrm2.

TABLE-US-00003 SEQIDNO:6isanexemplarynucleicacidsequencecomprisingaKozak sequence,Rrm1,P2A,andRrm2. GCTAGCGAATTCGCCACCATGCACGTCATCAAGAGAGACGGGAGGCAGGAAAGAGTCATGT TCGATAAAATCACTTCAAGAATCCAGAAACTGTGTTACGGGCTGAACATGGACTTCGTCGAT CCTGCCCAGATTACCATGAAAGTGATCCAGGGACTGTACTCTGGCGTCACCACAGTGGAGCT GGACACACTGGCCGCTGAAACCGCAGCCACACTGACTACCAAACACCCAGATTATGCAATT CTGGCTGCACGGATCGCCGTGAGTAATCTGCATAAGGAGACAAAGAAAGTCTTCTCAGACG TGATGGAGGACCTGTACAATTATATCAACCCTCACAATGGGAAACATTCACCAATGGTCGCT AAGAGCACTCTGGACATTGTGCTGGCCAACAAAGATCGGCTGAACAGCGCTATCATCTACG ACCGGGATTTCAGTTACAACTACTTCGGCTTTAAGACACTGGAGAGATCATATCTGCTGAAA ATCAATGGGAAGGTGGCCGAACGGCCTCAGCACATGCTGATGAGAGTCAGCGTGGGCATTC ATAAGGAGGACATTGATGCCGCTATCGAAACTTACAACCTGCTGAGCGAGCGCTGGTTCAC CCACGCTTCCCCTACACTGTTTAACGCAGGAACCAATCGACCACAGCTGAGCAGCTGCTTCC TGCTGAGCATGAAGGACGATTCCATCGAGGGCATCTACGACACCCTGAAACAGTGCGCACT GATTTCTAAGAGTGCCGGCGGGATCGGAGTCGCTGTGAGTTGTATTCGGGCAACCGGCTCAT ATATCGCCGGCACAAACGGCAACAGCAACGGGCTGGTCCCCATGCTGAGGGTGTACAACAA TACAGCCCGCTATGTGGATCAGGGAGGCAACAAGAGACCAGGAGCATTTGCCATCTACCTG GAACCCTGGCACCTGGACATTTTCGAGTTTCTGGATCTGAAGAAAAATACTGGCAAAGAGG AACAGAGGGCTCGCGACCTGTTCTTTGCACTGTGGATTCCCGACCTGTTCATGAAGAGGGTG GAGACCAACCAGGACTGGAGCCTGATGTGCCCCAATGAGTGTCCTGGGCTGGATGAAGTGT GGGGAGAGGAATTTGAAAAACTGTACGCCAGTTATGAGAAGCAGGGCCGAGTGCGGAAAG TGGTCAAGGCCCAGCAGCTGTGGTACGCTATCATTGAGAGCCAGACAGAAACTGGCACCCC CTACATGCTGTATAAAGACTCTTGCAACCGCAAGAGTAACCAGCAGAATCTGGGGACCATC AAATGCAGCAATCTGTGTACAGAGATTGTGGAATATACTTCCAAGGATGAGGTCGCCGTGT GTAACCTGGCATCACTGGCCCTGAATATGTACGTCACAAGCGAGCACACTTATGACTTCAAG AAACTGGCTGAAGTGACCAAAGTGGTCGTGAGGAATCTGAACAAGATCATTGACATCAACT ACTATCCCGTGCCTGAGGCCTGCCTGAGCAATAAGAGACATAGGCCCATCGGGATTGGAGT GCAGGGCCTGGCTGACGCATTCATCCTGATGCGCTACCCTTTTGAGTCCGCCGAAGCTCAGC TGCTGAACAAGCAGATTTTTGAAACAATCTACTACGGGGCTCTGGAGGCATCTTGTGACCTG GCCAAAGAACAGGGACCCTACGAGACTTATGAAGGCTCCCCTGTGTCTAAGGGCATCCTGC AGTACGATATGTGGAACGTCACACCAACTGACCTGTGGGATTGGAAAGTGCTGAAGGAGAA AATTGCAAAGTATGGCATCCGGAACAGCCTGCTGATCGCCCCAATGCCCACTGCCTCTACCG CTCAGATTCTGGGCAACAATGAGTCCATCGAACCATACACTTCTAACATCTACACCCGGAGA GTCCTGAGCGGGGAGTTCCAGATCGTGAATCCCCACCTGCTGAAAGACCTGACCGAACGGG GACTGTGGCATGAGGAAATGAAGAACCAGATCATTGCCTGCAATGGCAGTATCCAGTCAAT TCCTGAGATCCCAGACGATCTGAAACAGCTGTACAAGACAGTCTGGGAGATCAGCCAGAAA ACTGTGCTGAAGATGGCAGCCGAAAGAGGGGCTTTCATTGATCAGTCACAGAGCCTGAACA TCCACATTGCCGAGCCCAATTACGGAAAGCTGACCTCCATGCATTTTTATGGGTGGAAACAG GGACTGAAGACTGGCATGTACTATCTGCGCACCCGACCAGCTGCAAACCCCATCCAGTTTAC CCTGAATAAGGAGAAACTGAAGGACAAAGAAAAGGTGTCCAAAGAGGAAGAGGAAAAGG AGAGAAACACAGCCGCTATGGTGTGTTCTCTGGAGAATAGGGATGAATGCCTGATGTGTGG CAGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC CCTGGACCTCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCT GAGCCCACTGAAAGGCCTGAGTCTGGTCGATAAAGAGAACACACCACCTGCACTGAGTGGC ACTCGGGTGCTGGCATCAAAGACCGCCCGGAGAATTTTCCAGGAGCCAACCGAACCCAAAA CAAAGGCCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTT CGTGATTTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTT TCTGGACAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCC AGAGGAAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGA ACGAGAATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTA TGGATTTCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCT ATATCAAAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTG AAGAAAAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAA AGAGTGGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTT TGGCTGAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGA CGAAGGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTG AGGAACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGA AGCCCTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTG TGGCCGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTC GATTTTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGG AATATCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGA TTTTTGAGCTAGC SEQIDNO:7isanexemplaryKozaksequence(asfoundinSEQIDNO:6). GCCACC SEQIDNO:8isanexemplaryRrm1sequence(asfoundinSEQIDNO:6). ATGCACGTCATCAAGAGAGACGGGAGGCAGGAAAGAGTCATGTTCGATAAAATCACTTCAA GAATCCAGAAACTGTGTTACGGGCTGAACATGGACTTCGTCGATCCTGCCCAGATTACCATG AAAGTGATCCAGGGACTGTACTCTGGCGTCACCACAGTGGAGCTGGACACACTGGCCGCTG AAACCGCAGCCACACTGACTACCAAACACCCAGATTATGCAATTCTGGCTGCACGGATCGC CGTGAGTAATCTGCATAAGGAGACAAAGAAAGTCTTCTCAGACGTGATGGAGGACCTGTAC AATTATATCAACCCTCACAATGGGAAACATTCACCAATGGTCGCTAAGAGCACTCTGGACAT TGTGCTGGCCAACAAAGATCGGCTGAACAGCGCTATCATCTACGACCGGGATTTCAGTTACA ACTACTTCGGCTTTAAGACACTGGAGAGATCATATCTGCTGAAAATCAATGGGAAGGTGGC CGAACGGCCTCAGCACATGCTGATGAGAGTCAGCGTGGGCATTCATAAGGAGGACATTGAT GCCGCTATCGAAACTTACAACCTGCTGAGCGAGCGCTGGTTCACCCACGCTTCCCCTACACT GTTTAACGCAGGAACCAATCGACCACAGCTGAGCAGCTGCTTCCTGCTGAGCATGAAGGAC GATTCCATCGAGGGCATCTACGACACCCTGAAACAGTGCGCACTGATTTCTAAGAGTGCCG GCGGGATCGGAGTCGCTGTGAGTTGTATTCGGGCAACCGGCTCATATATCGCCGGCACAAA CGGCAACAGCAACGGGCTGGTCCCCATGCTGAGGGTGTACAACAATACAGCCCGCTATGTG GATCAGGGAGGCAACAAGAGACCAGGAGCATTTGCCATCTACCTGGAACCCTGGCACCTGG ACATTTTCGAGTTTCTGGATCTGAAGAAAAATACTGGCAAAGAGGAACAGAGGGCTCGCGA CCTGTTCTTTGCACTGTGGATTCCCGACCTGTTCATGAAGAGGGTGGAGACCAACCAGGACT GGAGCCTGATGTGCCCCAATGAGTGTCCTGGGCTGGATGAAGTGTGGGGAGAGGAATTTGA AAAACTGTACGCCAGTTATGAGAAGCAGGGCCGAGTGCGGAAAGTGGTCAAGGCCCAGCA GCTGTGGTACGCTATCATTGAGAGCCAGACAGAAACTGGCACCCCCTACATGCTGTATAAA GACTCTTGCAACCGCAAGAGTAACCAGCAGAATCTGGGGACCATCAAATGCAGCAATCTGT GTACAGAGATTGTGGAATATACTTCCAAGGATGAGGTCGCCGTGTGTAACCTGGCATCACTG GCCCTGAATATGTACGTCACAAGCGAGCACACTTATGACTTCAAGAAACTGGCTGAAGTGA CCAAAGTGGTCGTGAGGAATCTGAACAAGATCATTGACATCAACTACTATCCCGTGCCTGA GGCCTGCCTGAGCAATAAGAGACATAGGCCCATCGGGATTGGAGTGCAGGGCCTGGCTGAC GCATTCATCCTGATGCGCTACCCTTTTGAGTCCGCCGAAGCTCAGCTGCTGAACAAGCAGAT TTTTGAAACAATCTACTACGGGGCTCTGGAGGCATCTTGTGACCTGGCCAAAGAACAGGGA CCCTACGAGACTTATGAAGGCTCCCCTGTGTCTAAGGGCATCCTGCAGTACGATATGTGGAA CGTCACACCAACTGACCTGTGGGATTGGAAAGTGCTGAAGGAGAAAATTGCAAAGTATGGC ATCCGGAACAGCCTGCTGATCGCCCCAATGCCCACTGCCTCTACCGCTCAGATTCTGGGCAA CAATGAGTCCATCGAACCATACACTTCTAACATCTACACCCGGAGAGTCCTGAGCGGGGAG TTCCAGATCGTGAATCCCCACCTGCTGAAAGACCTGACCGAACGGGGACTGTGGCATGAGG AAATGAAGAACCAGATCATTGCCTGCAATGGCAGTATCCAGTCAATTCCTGAGATCCCAGA CGATCTGAAACAGCTGTACAAGACAGTCTGGGAGATCAGCCAGAAAACTGTGCTGAAGATG GCAGCCGAAAGAGGGGCTTTCATTGATCAGTCACAGAGCCTGAACATCCACATTGCCGAGC CCAATTACGGAAAGCTGACCTCCATGCATTTTTATGGGTGGAAACAGGGACTGAAGACTGG CATGTACTATCTGCGCACCCGACCAGCTGCAAACCCCATCCAGTTTACCCTGAATAAGGAGA AACTGAAGGACAAAGAAAAGGTGTCCAAAGAGGAAGAGGAAAAGGAGAGAAACACAGCC GCTATGGTGTGTTCTCTGGAGAATAGGGATGAATGCCTGATGTGTGGCAGT SEQIDNO:9isanexemplaryP2Asequence(asfoundinSEQIDNO:6). GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

Rrm2 Mutant

[0087] Mutations found within the ubiquitin binding domain (i.e., the site of ubiquitin addition or ubiquitination) of Rrm2 are shown herein to decrease ubiquitination of Rrm2, increase Rrm2 stability (e.g., half-life of Rrm2), and result in increased dATP in the cell. Accordingly, provided herein is an isolated nucleic acid molecule encoding an Rrm2 polypeptide that, together with Rrm1 polypeptide comprises ribonucleotide reductase activity, the encoded Rrm2 polypeptide comprising a mutation that increases the intracellular level of the polypeptide as compared to wild-type Rrm2 polypeptide. In one embodiment, the mutation is in a ubiquitin binding degron of Rrm2. In one embodiment, the ubiquitin binding degrons of Rrm2 are found at nucleotides 88-96 (which encode amino acids that can associate with the APC/FZR1 proteasome) and nucleotides 97-99 and 145-153 (which can associate with the SCF/CyclinF proteasome) of wild-type Rrm2 (SEQ ID NO: 3). In one embodiment, the ubiquitin binding degrons of Rrm2 are found at amino acids 30-32 (which can associate with the APC/FZR1 proteasome) and amino acids 33 and 49-51 (which can associate with the SCF/CyclinF proteasome) of wild-type Rrm2 (SEQ ID NO: 4).

[0088] In one embodiment, the ubiquitin binding degrons of Rrm2 have the nucleic acid sequence of SEQ ID NOs: 10-12 or the amino acid sequence of SEQ ID NOs: 13-15. SEQ ID NO: 10 AAGGAGAAC; SEQ ID NO: 11 ACG; SEQ ID NO: 12 AGGAGGATC; SEQ ID NO: 13 KEN; SEQ ID NO: 14 T; SEQ ID NO: 15 RRI.

[0089] In one embodiment, the mutant Rrm2 having mutations within the ubiquitin binding degrons of Rrm2 have one mutation, at least one mutation, at least 2 mutations, at least 3 mutations, at least 4 mutations, at least 5 mutations, at least 6 mutations, at least 7 mutations, at least 8 mutations, at least 9 mutations, or 10 mutations, or more. Alternatively, the mutant Rrm2 can include 1 mutation, two or fewer mutations, three or fewer mutations, four or fewer mutations, five or fewer mutations, six or fewer mutations, seven or fewer mutations, eight or fewer mutations, nine or fewer mutations or 10 or fewer mutations, relative to wild-type Rrm2 polypeptide. Mutations found within a ubiquitin binding degron can be in succession, e.g., two or three amino acids in a row can be mutated. Alternatively, mutations found within a ubiquitin binding degron can be have wild-type amino acids in between. A mutation described herein can be an amino acid substitution, deletion, or insertion. It is contemplated herein that a mutation can be any amino acid change within the ubiquitin binding domain that results in at least decreased ubiquitination of Rrm2, increased stability of Rrm2, and/or increased dATP levels in the cell. Considerations for mutating a ubiquitination site while maintaining Rrm2 activity in terms of complex formation and ribonucleotide reductase activity with Rrm1 are discussed herein above. In some embodiments, the mutation is found near a ubiquitin binding degron, e.g., within 1-10 nucleotides of a ubiquitin binding degron, i.e., nucleotides not encoding a ubiquitin binding degron. In some embodiments, the mutation is found near a ubiquitin binding degron, e.g., within 1-10 amino acids of a ubiquitin binding degron, i.e., amino acids not encoding a ubiquitin binding degron.

[0090] In one embodiment, the mutation is selected from Table 1.

TABLE-US-00004 TABLE 1 Rrm2 mutations (amino acid and its corresponding number in SEQ ID NO: 3 shown) Positon 30 31 32 33 49 51 Wild-type K E N T R I Mutant A A A A A A

[0091] In one embodiment, the isolated nucleic acid has a sequence of SEQ ID NOs: 16-20. In one embodiment, the isolated nucleic acid consists of or consists essentially of the sequence of SEQ ID NO: 16-20.

TABLE-US-00005 SEQIDNO:16isanexemplarynucleicacidsequenceforRrm2mutant30AAA ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCTGAGCCC ACTGAAAGGCCTGAGTCTGGTCGATGCAGCGGCCACACCACCTGCACTGAGTGGCACTCGG GTGCTGGCATCAAAGACCGCCCGGAGAATTTTCCAGGAGCCAACCGAACCCAAAACAAAGG CCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGAT TTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTTTCTGGA CAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCCAGAGG AAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGAACGAG AATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATT TCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATATCA AAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTGAAGAA AAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAAAGAGT GGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCT GAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAA GGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTGAGGA ACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGAAGCC CTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTGTGGC CGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATT TTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGGAATA TCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGATTTTT GA SEQIDNO:17isanexemplarynucleicacidsequenceforRrm2mutant30AAN ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCTGAGCCC ACTGAAAGGCCTGAGTCTGGTCGATGCAGCGAACACACCACCTGCACTGAGTGGCACTCGG GTGCTGGCATCAAAGACCGCCCGGAGAATTTTCCAGGAGCCAACCGAACCCAAAACAAAGG CCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGAT TTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTTTCTGGA CAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCCAGAGG AAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGAACGAG AATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATT TCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATATCA AAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTGAAGAA AAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAAAGAGT GGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCT GAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAA GGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTGAGGA ACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGAAGCC CTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTGTGGC CGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATT TTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGGAATA TCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGATTTTT GA SEQIDNO:18isanexemplarynucleicacidsequenceforRrm2mutant33A ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCTGAGCCC ACTGAAAGGCCTGAGTCTGGTCGATAAAGAGAACGCACCACCTGCACTGAGTGGCACTCGG GTGCTGGCATCAAAGACCGCCCGGAGAATTTTCCAGGAGCCAACCGAACCCAAAACAAAGG CCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGAT TTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTTTCTGGA CAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCCAGAGG AAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGAACGAG AATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATT TCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATATCA AAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTGAAGAA AAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAAAGAGT GGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCT GAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAA GGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTGAGGA ACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGAAGCC CTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTGTGGC CGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATT TTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGGAATA TCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGATTTTT GA SEQIDNO:19isanexemplarynucleicacidsequenceforRrm2mutant49ARA ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCTGAGCCC ACTGAAAGGCCTGAGTCTGGTCGATAAAGAGAACACACCACCTGCACTGAGTGGCACTCGG GTGCTGGCATCAAAGACCGCCGCGAGAGCTTTCCAGGAGCCAACCGAACCCAAAACAAAGG CCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGAT TTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTTTCTGGA CAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCCAGAGG AAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGAACGAG AATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATT TCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATATCA AAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTGAAGAA AAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAAAGAGT GGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCT GAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAA GGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTGAGGA ACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGAAGCC CTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTGTGGC CGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATT TTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGGAATA TCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGATTTTT GA SEQIDNO:20isanexemplarynucleicacidsequenceforRrm2mutant30AAA/49ARA ATGCTGAGTCTGAGGGTCCCACTGGCACCTATCACCGATCCACAGCAGCTGCAGCTGAGCCC ACTGAAAGGCCTGAGTCTGGTCGATGCAGCGGCCACACCACCTGCACTGAGTGGCACTCGG GTGCTGGCATCAAAGACCGCCGCGAGAGCTTTCCAGGAGCCAACCGAACCCAAAACAAAGG CCGCTGCACCTGGGGTCGAGGACGAACCACTGCTGAGAGAGAATCCCAGGCGCTTCGTGAT TTTTCCTATCGAATACCACGATATTTGGCAGATGTATAAGAAAGCTGAGGCAAGTTTCTGGA CAGCTGAGGAAGTGGACCTGAGCAAAGACATCCAGCACTGGGAATCCCTGAAGCCAGAGG AAAGGTACTTCATTTCTCATGTGCTGGCATTCTTTGCCGCTAGTGACGGGATCGTGAACGAG AATCTGGTCGAACGCTTTAGCCAGGAGGTGCAGATCACTGAAGCCCGATGCTTCTATGGATT TCAGATTGCTATGGAGAACATCCATTCAGAAATGTACAGCCTGCTGATTGACACCTATATCA AAGATCCTAAGGAGCGCGAGTTCCTGTTTAATGCCATTGAGACAATGCCATGTGTGAAGAA AAAGGCAGACTGGGCTCTGCGATGGATCGGCGATAAGGAGGCTACTTACGGGGAAAGAGT GGTCGCATTCGCAGCCGTGGAGGGAATTTTCTTTTCTGGCAGTTTCGCTTCCATCTTTTGGCT GAAAAAGCGAGGCCTGATGCCTGGGCTGACCTTTTCCAACGAGCTGATTTCTCGCGACGAA GGCCTGCACTGCGATTTCGCCTGTCTGATGTTTAAACACCTGGTGCATAAGCCCTCTGAGGA ACGAGTCCGGGAGATCATTATCAACGCAGTGAGGATCGAGCAGGAGTTCCTGACAGAAGCC CTGCCTGTCAAACTGATTGGCATGAATTGCACTCTGATGAAGCAGTACATCGAGTTTGTGGC CGACAGGCTGATGCTGGAACTGGGATTCTCAAAGGTGTTTCGCGTCGAGAACCCATTCGATT TTATGGAGAATATCAGCCTGGAAGGCAAAACAAACTTCTTTGAGAAGAGAGTCGGGGAATA TCAGAGGATGGGCGTGATGAGCAGCCCCACTGAGAATAGCTTCACCCTGGACGCCGATTTTT GA

[0092] In one embodiment, an isolated nucleic acid comprises, consists of, or consists essentially of a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to one of SEQ ID NO: 16-20, and retains at least 50% of the function of one of SEQ ID NO: 16-20, e.g., complex formation with Rrm1 and increasing the cellular level of dATP.

[0093] In one embodiment, the isolated polypeptide has a sequence of SEQ ID NOs: 21-25. In one embodiment, the isolated polypeptide consists of or consists essentially of a polypeptide having the sequence of SEQ ID NO: 21-25.

TABLE-US-00006 SEQIDNO:21isanexemplaryaminoacidsequence forRrm2mutant30AAA MLSLRVPLAPITDPQQLQLSPLKGLSLVDAAATPPALSGTRVLASKTARR IFQEPTEPKTKAAAPGVEDEPLLRENPRRFVIFPIEYHDIWQMYKKAEAS FWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLVERFS QEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETM PCVKKKADWALRWIGDKEATYGERVVAFAAVEGIFFSGSFASIFWLKKRG LMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGFSKVFRVENPFDFM ENISLEGKTNFFEKRVGEYQRMGVMSSPTENSFTLDADF SEQIDNO:22isanexemplaryaminoacidsequence forRrm2mutant30AAN MLSLRVPLAPITDPQQLQLSPLKGLSLVDAANTPPALSGTRVLASKTARR IFQEPTEPKTKAAAPGVEDEPLLRENPRRFVIFPIEYHDIWQMYKKAEAS FWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLVERFS QEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETM PCVKKKADWALRWIGDKEATYGERVVAFAAVEGIFFSGSFASIFWLKKRG LMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGFSKVFRVENPFDFM ENISLEGKTNFFEKRVGEYQRMGVMSSPTENSFTLDADF SEQIDNO:23isanexemplaryaminoacidsequence forRrm2mutant33A MLSLRVPLAPITDPQQLQLSPLKGLSLVDKENAPPALSGTRVLASKTARR IFQEPTEPKTKAAAPGVEDEPLLRENPRRFVIFPIEYHDIWQMYKKAEAS FWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLVERFS QEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETM PCVKKKADWALRWIGDKEATYGERVVAFAAVEGIFFSGSFASIFWLKKRG LMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGFSKVFRVENPFDFM ENISLEGKTNFFEKRVGEYQRMGVMSSPTENSFTLDADF SEQIDNO:24isanexemplaryaminoacidsequence forRrm2mutant49ARA MLSLRVPLAPITDPQQLQLSPLKGLSLVDKENTPPALSGTRVLASKTAAR AFQEPTEPKTKAAAPGVEDEPLLRENPRRFVIFPIEYHDIWQMYKKAEAS FWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLVERFS QEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETM PCVKKKADWALRWIGDKEATYGERVVAFAAVEGIFFSGSFASIFWLKKRG LMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGFSKVFRVENPFDFM ENISLEGKTNFFEKRVGEYQRMGVMSSPTENSFTLDADF SEQIDNO:25isanexemplaryaminoacidsequence forRrm2mutant30AAA/49ARA MLSLRVPLAPITDPQQLQLSPLKGLSLVDAAATPPALSGTRVLASKTAAR AFQEPTEPKTKAAAPGVEDEPLLRENPRRFVIFPIEYHDIWQMYKKAEAS FWTAEEVDLSKDIQHWESLKPEERYFISHVLAFFAASDGIVNENLVERFS QEVQITEARCFYGFQIAMENIHSEMYSLLIDTYIKDPKEREFLFNAIETM PCVKKKADWALRWIGDKEATYGERVVAFAAVEGIFFSGSFASIFWLKKRG LMPGLTFSNELISRDEGLHCDFACLMFKHLVHKPSEERVREIIINAVRIE QEFLTEALPVKLIGMNCTLMKQYIEFVADRLMLELGFSKVFRVENPFDFM ENISLEGKTNFFEKRVGEYQRMGVMSSPTENSFTLDADF

[0094] In one embodiment, an isolated polypeptide comprises, consists of, or consists essentially of polypeptide having a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to one of SEQ ID NO: 21-25, and retains as least 50% of the function of one of SEQ ID NO: 21-25, e.g., complex formation with Rrm1 and increasing the cellular level of dATP.

Ubiquitin-Mediated Degradation

[0095] The ubiquitin-mediated degradation pathway promotes selective, controlled degradation of intracellular proteins in eukaryotic cells. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from, e.g., a few minutes to days or more, and can vary considerably depending on the type of protein, e.g., structural protein vs enzyme or regulatory protein, cell type, nutritional and environmental conditions, as well as the stage of the cell-cycle.

[0096] Proteins that undergo selective degradation, presumably through the actions of a ubiquitin-dependent proteasome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.

[0097] The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin is then transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases.

[0098] Depending on the ubiquitin-conjugating enzyme and the nature of the substrate, specific lysine residues of ubiquitin are used as acceptor sites for further ubiquitinations. This can lead to either a linear multiubiquitin chain (when a single lysine residue of ubiquitin is used) or multi-ubiquitin trees (when more than one lysine reside of ubiquitin is used). Although the attachment of a single ubiquitin moiety to a substrate can be sufficient for targeted degradation, multi-ubiquitination appears to be required in most cases.

[0099] Amino acids that are likely to be ubiquitinated (i.e., a ubiquitination site) can be identified, e.g., using ubiquitination site prediction software. Exemplary software includes UbPred: predictor of protein ubiquitination sites (found on the world wide web at www.ubpred.org), UbiSite (found on the world wide web at http://csb.cse.yzu.edu.tw/UbiSite/), BDM Pub (e.g., found on the world wide web at http://bdmpub.biocuckoo.org/). Methods for identifying ubiquitination sites are further reviewed in, e.g., He F., et al. BMC Systems Biol. 2018, 12(Supp 6): 109; Radivojac, P., et al. Proteins. 2010 Feb. 1; 78(2): 365-380; and Yadav, S., et al. Advances in Fuzzy Systems. Volume 2018, Article ID 5125103, 10 pages; the contents of which are incorporated herein by reference in their entireties.

[0100] In one embodiment, mutations placed in the ubiquitin binding degron prevents, inhibits, or decreases conjugation of ubiquitin to Rrm2. In one embodiment, the mutations in the Rrm2 ubiquitin binding degron nucleotide sequence results in decreased ubiquitination levels of the encoded Rrm2 protein. In one embodiment, the level of ubiquitin of the encoded Rrm2 protein is decreased at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more as compared to ubiquitin level of a wild-type Rrm2 protein at that site. Assays for detecting the level of ubiquitination of a protein are known in the art, and include, for example, co-immunoprecipitation of the protein of interest under denaturing conditions and SDS-PAGE gel and western blotting techniques with an anti-ubiquitin antibody to visualize the level of ubiquitin on the protein of interest. Additional assays for determining the level of ubiquitination of a protein include Ubi-Detect protein ubiquitination detection kit (e.g., available from R&D Systems).

[0101] Mutations in the Rrm2 ubiquitin binding degron that decrease the level of ubiquitination result in an increased stability of the Rrm2 protein, as it is no longer targeted by the ubiquitin mediated degradation system. In one embodiment, the mutations in the Rrm2 ubiquitin binding degron nucleotide sequence results in increased stability of the of the encoded Rrm2 protein (i.e., an increased half-life of the protein). In one embodiment, the level of stability of the encoded Rrm2 protein is increased at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more as compared to the stability level of a wild-type Rrm2 protein. Assays for detecting the stability of a protein are known in the art, and include, treating a cell lysate or an in vitro system having the protein of interest and components of the ubiquitin mediated degradation system with cyclohexamide to halt protein translation and measuring the level of the protein of interest over time (e.g., in a time course) via Western blotting. Alternatively, protein stability can be measured using a standard pulse-chase experiment.

2-deoxy-ATP (2-deoxyadenosine; dATP)

[0102] Deoxyadenosine is a derivative of the nucleoside adenosine. It is composed of adenine covalently attached to a deoxyribose moiety via an N9-glycosidic bond. Deoxyribose differs from ribose by the absence of oxygen in the 3 position of its ribose ring. Deoxyadenosine is a critical component of

[0103] DNA; deoxyadenosine is the DNA nucleoside A, which pairs with deoxythymidine (T) in double-stranded DNA.

[0104] In one embodiment, cellular expression of an isolated rrm2 nucleic acid or an Rrm2 polypeptide encoded by the nucleic acid, or a composition thereof, increases dATP in the cell. For example, cellular expression of an isolated rrm2 nucleic acid or an Rrm2 polypeptide encoded by it, or a composition thereof increases intracellular dATP levels at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold or more as compared to expression of wild-type Rrm2. Deoxyadenosine levels in a cell, such as a cell expressing an Rrm2 mutant polypeptide as described herein can be measured via e.g., HPLC or mass spectrometry using standard protocols.

[0105] In one embodiment, expression of an Rrm2 mutant as described herein can, on its own, provide an increase in dATP level; that is, increasing the stability of the normally unstable Rrm2 subunit of the Rrm1/Rrm2 RNR complex can provide an increase in RNR activity. Alternatively, expression of both Rrm1 and an Rrm2 mutant can be provided, either from two separate vectors or from a single vector encoding both factors. When both Rrm1 and a mutant Rrm2 as described herein are used, the additional expression of the Rrm1 can permit enhancement in RNR activity beyond that achievable by providing the stabilized Rrm2 mutant alone.

Vectors

[0106] Provided herein are vectors comprising any of the isolated nucleic acids described herein that encode Rrm2 mutants with increased intracellular half-life. The term vector, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term vector encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0107] As used herein, the term expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression products include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term gene means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5 untranslated (5UTR) or leader sequences and 3 UTR or trailer sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0108] In one embodiment, a vector is a viral vector. As used herein, the term viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Exemplary viral vectors for delivery of a gene or gene product to a subject, include, but are not limited to an adeno associated virus (AAV), a lentivirus (LV), a herpes simplex virus (HSV), an adeno virus (AV), or a pox virus (PV). Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found, e.g., in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals (e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics. John Wiley and Sons, Inc.: 1997). Further, production of AAV vectors is further described, e.g., in U.S. Pat. No. 9,441,206, the contents of which is incorporated herein by reference in its entirety.

[0109] By recombinant vector is meant a vector that includes a heterologous nucleic acid sequence, or transgene that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments of any of the aspects, be combined with other suitable compositions and therapies. In some embodiments of any of the aspects, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0110] While it is generally considered that therapeutic approaches using Rrm2 mutant polypeptides as described herein will express the Rrm2 polypeptide in a cell, e.g., a cardiac cell, it is contemplated that the Rrm2 polypeptide mutants described herein can alternatively be used in purified protein form. Thus, in some embodiments, a vector drives polypeptide (i.e., protein) expression in insect cells using baculovirus expression vectors, which together provide very efficient expression of recombinant proteins, which can be isolated for direct use. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0111] In one embodiment, an isolated nucleic acid as described herein is operatively linked to a promotor for expression of the isolated nucleic acid. Exemplary promoters that can be used in the present technology include, but are not limited to, constitutive promoters, repressible promoters, and/or inducible promoters, some non-limiting examples of which include viral promoters (e.g., CMV, SV40), tissue specific promoters (e.g., muscle MCK), heart (e.g., NSE), eye (e.g., MSK) and synthetic promoters (SP1 elements) and the. chicken beta actin promoter (CB or CBA).

[0112] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), and are further described in, e.g., U.S. Pat. Nos. 8,187,836; 8,455,219; 8,980,626; 7,384,776; and 6,451,539; the contents of which are incorporated herein by reference in their entireties. When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Muller, D., et al. (2006) Microbial Cell Factories.

[0113] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the synthetic nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid in, for example, a hematopoietic cell). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable cardiac tissue-specific promoters include the NCX1 promoter (e.g., as described in Nicholas S B., et al. Am J Physiol. 1998), the MLC-2v (e.g., as described Griscelli, F., et al. C R Acad Sci III. 1997 February; 320(2):103-12); cardiac troponin-I proximal promoter (TNNI3) (e.g., as described in Gallo, P., et al. Gene Therapy. 15, pages 161-170 (2008); and the aMHC promoter. All citations provided herein are incorporated herein by reference in their entireties.

[0114] In one embodiment, the synthetic nucleic acid molecules described herein are introduced to a cell via a non-viral method. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam and Lipofectin). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). In one embodiment, human IPS-derived cardiomyocytes are modified in vitro to express an Rrm2 mutant as described herein before being introduced to or transplanted into cardiac tissue in vivo.

[0115] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Compositions

[0116] Various aspects described herein provide a composition comprising, consisting of, or consisting essentially of any of the isolated nucleic acids, vectors, polypeptides, or RNR complexes described herein.

[0117] One aspect provided herein is a pharmaceutical composition comprising, consisting of, or consisting essentially of any of the isolated nucleic acids, vectors, polypeptides, or RNR complexes described herein. As used herein, the term pharmaceutical composition refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, 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. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found to occur in nature.

[0118] Pharmaceutically acceptable or physiologically tolerable materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid, including, but not limited to, e.g., lyophilized, forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present technology can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used with the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

Method of Treating Cardiac Disease or Disorder

[0119] One aspect provided herein is a method of administering a composition or pharmaceutical composition described herein to a subject suffering from a cardiac disease or disorder, e.g., a disease or disorder characterized by insufficient cardiac function or defects in the heart structure. Exemplary cardiac diseases or disorders include congestive heart failure, cardiomyopathy, myocardial infarction, cardiac ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, cardiomyopathy, dysfunctional conduction systems, dysfunctional coronary arteries, and pulmonary hypertension. In some embodiments, the disease is selected from the group consisting of congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle degeneration, infective myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, an autoimmune endocarditis and congenital heart disease. In one embodiment, the compositions and methods as disclosed herein are useful for treatment of a disease or disorder which is myocardial infarction or myocardial ischemia.

[0120] Other aspects relate to the use of a therapeutic agent as described herein (e.g., an isolated nucleic acid encoding an Rrm2 polypeptide mutant, a vector encoding an Rrm2 polypeptide mutant, a vector encoding Rrm1 and an Rrm2 polypeptide mutant, a cell comprising sequence encoding any of these, an Rrm2 polypeptide mutant, or a protein complex consisting of Rrm1 and an Rrm2 polypeptide mutant) in a method for the treatment of a cardiovascular disease or disorder in a subject. Other aspects of the present technology relate to a composition or pharmaceutical composition comprising said therapeutic agent, for use in the treatment of a cardiac disease or disorder. In alternative embodiments, a population of cardiac cells, e.g., a population of cardiomyocytes, or cardiomyocyte precursor cells (e.g., cardiac stem cells) can be contacted with a therapeutic agent or a composition thereof in vitro, in vivo or ex vivo, and in some embodiments, where the contact occurs in vitro or ex vivo, the population of cardiac cells, e.g., cardiomyocytes can be transplanted into a subject for the treatment and/or prevention of cardiac diseases, or for the treatment of existing cardiac muscle which is damaged by disease or injury, e.g., induced by a myocardial infarction.

[0121] In alternative embodiments, the present technology provides for methods to treat a cardiovascular disease or disorder in a subject comprising administering to the subject a composition comprising a population of cardiac cells, e.g., population of cardiac stem cells, ventricular cardiac stem cells or cardiac cells differentiated from human stem cells, e.g., IPS cells, where the population of cells comprises sequences encoding an Rrm2 mutant polypeptide as described herein, alone or together with an Rrm1 polypeptide. In some embodiments, the iPS cell is an autologous iPS cell e.g., an iPS cell which was reprogrammed from a somatic cell obtained from a subject to which the pharmaceutical composition is being administered. In some embodiments, a cardiac cell is a population of primordial or cardiac cell precursors, or their progeny, as disclosed in International Patent Applications, WO/2008/054819, WO2010/144678, WO2010/042856, and U.S. Patent Applications 2010/0166714, US2011/0033430, US2010/021713 and US2011/0003327, which are incorporated herein in their entirety by reference. In some embodiments, a cell population is a Isl1+primordial cell, or progeny thereof as disclosed in WO2010/144678.

[0122] With reference to the treatment of, for example, a cardiovascular condition or disease in a subject, the term therapeutically effective amount refers to the amount of a therapeutic agent that is safe and sufficient to treat, prevent or delay the development of a cardiovascular disease or disorder. The amount can thus cure or cause the cardiovascular disease or disorder to go into remission, slow the course of cardiovascular disease progression, reduce or inhibit a symptom of a cardiovascular disease or disorder, slow or inhibit the establishment of secondary symptoms of a cardiovascular disease or disorder or inhibit the development of a secondary symptom of a cardiovascular disease or disorder. The effective amount for the treatment of the cardiovascular disease or disorder depends on the type of cardiovascular disease to be treated, the severity of the symptoms, the subject being treated, the age and general condition of the subject, the mode of administration and so forth. Thus, it is not possible to specify any exact effective amount. However, for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

[0123] Administering

[0124] As used herein, the terms administering, introducing and transplanting are used interchangeably and refer to the placement of a pharmaceutical composition comprising a therapeutic agent described herein (e.g., an isolated nucleic acid encoding Rrm2 polypeptide mutant, an Rrm2 polypeptide mutant, or a protein complex consisting of Rrm1 and an Rrm2 polypeptide mutant), or a composition comprising a population of cells and therapeutic agent, or a composition comprising a population cells that have previously been contacted with a therapeutic agent as described herein, into a subject by a method or route which results in at least partial localization of any of the compositions, at a desired site or tissue location, e.g., cardiac tissue. In various embodiment, the terms administering, introducing and transplanting are used interchangeably and refer to the placement of a pharmaceutical composition comprising a therapeutic agent, or a composition comprising a population of cardiac cells and a therapeutic agent, or a composition comprising a population of cardiomyocytes that have been previously contacted by a therapeutic agent as described herein, into a subject by a method or route which results in at least partial localization of the pharmaceutical composition, at a desired cardiac site or cardiac tissue location. In some embodiments, the pharmaceutical composition comprising a therapeutic agent can be administered by any appropriate route which results in effective treatment in the subject, i.e. administration results in delivery to a desired location or tissue in the subject where at least a portion of the therapeutic agent is located at a desired target tissue, e.g., cardiac tissue, or target cell (e.g., cardiac cell, such as a cardiomyocyte) location.

[0125] The phrases parenteral administration and administered parenterally as used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The phrases systemic administration, administered systemically, peripheral administration and administered peripherally as used herein mean the administration of pharmaceutical compositions comprising a therapeutic agent described herein other than directly into a target tissue or organ, such that it enters the animal's system and, thus, is subject to metabolism and other like processes.

[0126] These pharmaceutical preparations are a further object of the technology. Usually the amount of active ingredient (polypeptide, nucleic acid encoding polypeptide, vector, etc.) is between 0.1-95% by weight of the preparation, preferably between 0.2-20% by weight in preparations for parenteral use.

[0127] Combination Therapies

[0128] While it is contemplated herein that a pharmaceutical composition comprising a therapeutic agent described herein (e.g., an isolated nucleic acid encoding an Rrm2 polypeptide mutant, an Rrm2 polypeptide mutant, or a protein complex consisting of Rrm1 and an Rrm2 polypeptide mutant), or a composition comprising a population of cells and therapeutic agent, or a composition comprising a population of cells that have previously been contacted with a therapeutic agent as described herein can be administered as a monotherapy to a subject, combination therapy can be used to treat a cardiac disease or disorder in a subject. In various embodiments, the subject is further administered any other approved therapy for a cardiac disease or disorder. Such therapies include, but are not limited to ACE Inhibitors; Angiotension II Receptor Blockers; Antiarrhythmics; Antiplatelet Drugs; Aspirin Therapy; Beta-Blocker Therapy; Calcium Channel Blocker Drugs; Clot Buster Drugs (e.g., thrombolytic therapy); Digoxin; Diuretics; Nitrates; or Blood Thinners (e.g., Warfarin). Therapies can further include lifestyle changes (e.g., weight loss, increased exercise; change in diet (e.g., reduction in red meat); surgical procedures (e.g., coronary angioplasty, artificial heart valve surgery, atheretomy, cardiomyoplasty; limited access coronary artery surgery; transmyocardial revascularization; or coronary artery bypass graft surgery (CABG)); or placement of a therapeutic device (e.g., a stent).

[0129] Administered in combination, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with a cardiac disorder or disease and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as simultaneous or concurrent delivery. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The therapeutic agents described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the therapeutic agent described herein can be administered first, and the additional therapy can be administered second, or the order of administration can be reversed. The therapeutic agent and/or other therapy, e.g., procedures or modalities, can be administered during periods of active disorder, or during a period of remission or less active disease. The therapeutic agent can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder. It is understood that the therapeutic agent and an additional therapy are to be administered using appropriate means known in the art, and that these means can be the same or different. For example, a composition comprising cardiomyocytes that have previously been contacted by a therapeutic agent described herein will be administered locally to the cardiac tissue, and Warfarin will be administered orally for systemic administration.

[0130] Dosage

[0131] In some embodiments, a composition as described herein is administered in unit dosage form. Unit dosage form as the term is used herein refers to a dosage suitable for one administration. By way of example a unit dosage form can be an amount of therapeutic disposed in a delivery device, e.g., a syringe, catheter, e.g., a cardiac catheter, or intravenous drip bag. In one embodiment, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously.

[0132] The dosage of the therapeutic agent as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytotoxic effects. The dosage can also be adjusted by the individual physician in the event of any complication.

[0133] The dosage range depends upon the potency, and includes amounts large enough to produce the desired effect, e.g., an improvement in cardiac function. Generally, the dosage will vary with the type of therapeutic agent given (e.g., an isolated nucleic acid encoding an Rrm2 polypeptide mutant, an Rrm2 polypeptide mutant, or a protein complex consisting of Rrm1 and an Rrm2 polypeptide mutant; or a composition comprising a population of cells and therapeutic agent; or a composition comprising a population cells that have previously been contacted with a therapeutic agent as described herein) and with the age, sex, and condition of the patient. Dosages for nucleic acids and vectors can be in the nanograms to micrograms range (e.g., 10 ng, 100 ng, 1 microgram, 10 micrograms, 100 micrograms, 1 milligram or more), particularly when administered directly, e.g., to cardiac tissue. Dosages for cells can be in the range of 1000 cells to 10.sup.4 cells, 510.sup.4 cells, 10.sup.5 cells, 510.sup.5 cells, 10.sup.6 cells, 510.sup.6 cells, 10.sup.7 cells, 510.sup.7 cells, 10.sup.8 cells, 510.sup.8 cells, 10.sup.9 cells, 510.sup.9 cells, 10.sup.10 cells or more, including ranges between these dosages (e.g., 1000-5,000 cells, 1000-10,000 cells, 1000-100,000 cells, 1000-10.sup.6 cells, 1000-10.sup.7 cells, 1000-10.sup.8 cells, 1000-10.sup.9 cells, 1000-10.sup.10 cells, 10.sup.6 to 10.sup.10 cells, 10.sup.6-10.sup.9 cells, 10.sup.6-10.sup.8 cells, 10.sup.6-10.sup.7 cells and all integer values within those ranges. If necessary, cell compositions can also be administered multiple times at these dosages. The cells can be administered by using injection or infusion techniques that are commonly known in the field (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). For injection or implantation, cells can be suspended in a biocompatible gel or polymer, or impregnated within a matrix or scaffold, e.g., to slow the rate of diffusion or wash-out of the cells from the site of administration.

[0134] Efficacy

[0135] The efficacy of treatment can be judged by an ordinarily skilled practitioner. For example, efficacy can be assessed by determining whether a composition or formulation leads to a decrease of at least one symptom of the disease or disorder as disclosed herein, e.g., cardiovascular disease. For example, any of increased heart ejection fraction, decreased rate of heart failure, decreased infarct size, decreased associated morbidity (pulmonary edema, renal failure, arrhythmias) improved exercise tolerance or other quality of life measures, and decreased mortality indicates effective treatment. Increased or efficient engraftment of transplanted cells (i.e., greater than or equal to 2%, greater than or equal to 4%, greater than or equal to 5% or more) including, but not limited to autologous cells bearing a nucleic acid or vector as described herein also indicates effective treatment. In embodiments where the compositions are used for the treatment of a cardiovascular disease or disorder, the efficacy of the composition can be judged using an experimental animal model of cardiovascular disease, e.g., animal models of ischemia-reperfusion injury (Headrick J P, Am J Physiol Heart circ Physiol 285; H1797; 2003) and animal models acute myocardial infarction. (Yang Z, Am J Physiol Heart Circ. Physiol 282:H949:2002; Guo Y, J Mol Cell Cardiol 33; 825-830, 2001).

[0136] As used herein, the term treating when used in reference to a treatment of a cardiovascular disease or disorder is used to refer to the reduction of a symptom and/or a biochemical marker of a cardiovascular disease or disorder, for example a reduction in at least one biochemical marker of a cardiovascular disease by at least about 10% would be considered an effective treatment. Examples of such biochemical markers of cardiovascular disease include a reduction of, for example, creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) in the blood, and/or a decrease in a symptom of cardiovascular disease and/or an improvement in blood flow and cardiac function as determined by someone of ordinary skill in the art as measured by electrocardiogram (ECG or EKG), or echocardiogram (heart ultrasound), Doppler ultrasound and nuclear medicine imaging. A reduction in a symptom of a cardiovascular disease by at least about 10% would also be considered effective treatment by the methods as disclosed herein. As alternative examples, a reduction in a symptom of cardiovascular disease, for example a reduction of at least one of the following; dyspnea, chest pain, palpitations, dizziness, syncope, edema, cyanosis etc. by at least about 10% or a cessation of such systems, or a reduction in the size one such symptom of a cardiovascular disease by at least about 10% would also be considered as affective treatments by the methods as disclosed herein. In some embodiments, it is preferred, but not required that the therapeutic agent actually eliminate the cardiovascular disease or disorder, rather just reduce a symptom to a manageable extent.

[0137] Subjects in need of treatment by the methods as disclosed herein can be identified by any method to diagnose myocardial infarction (commonly referred to as a heart attack). Methods of diagnosing these conditions are well known by persons of ordinary skill in the art. By way of non-limiting example, myocardial infarction can be diagnosed by (i) blood tests to detect levels of creatine phosphokinase (CPK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and other enzymes released during myocardial infarction; (ii) electrocardiogram (ECG or EKG) which is a graphic recordation of cardiac activity, either on paper or a computer monitor. An ECG can be beneficial in detecting disease and/or damage; (iii) echocardiogram (heart ultrasound) used to investigate congenital heart disease and assessing abnormalities of the heart wall, including functional abnormalities of the heart wall, valves and blood vessels; (iv) Doppler ultrasound can be used to measure blood flow across a heart valve; (v) nuclear medicine imaging (also referred to as radionuclide scanning in the art) allows visualization of the anatomy and function of an organ, and can be used to detect coronary artery disease, myocardial infarction, valve disease, heart transplant rejection, check the effectiveness of bypass surgery, or to select patients for angioplasty or coronary bypass graft.

[0138] Methods described herein can be used to treat and/or prevent myocardial ischemia. As used herein, the term ischemia refers to any localized tissue ischemia due to reduction of the inflow of blood. The term myocardial ischemia refers to circulatory disturbances caused by coronary atherosclerosis and/or inadequate oxygen supply to the myocardium. For example, an acute myocardial infarction represents an irreversible ischemic insult to myocardial tissue. This insult results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation and produces an environment in which the myocardial metabolic demands exceed the supply of oxygen to the myocardial tissue.

[0139] The technology provided herein can further be described in the following numbered paragraphs: [0140] 1. An isolated nucleic acid molecule encoding an Rrm2 polypeptide that, together with Rrm1 polypeptide comprises ribonucleotide reductase activity, the encoded Rrm2 polypeptide comprising a mutation that increases the intracellular level of the polypeptide as compared to wild-type Rrm2 polypeptide. [0141] 2. The isolated nucleic acid of paragraph 1, wherein the mutation increases the intracellular stability of the Rrm2 polypeptide relative to wild-type. [0142] 3. The isolated nucleic acid molecule of paragraph 1, wherein the mutation is in a ubiquitin-binding degron. [0143] 4. The isolated nucleic acid molecule of paragraph 3, wherein the mutation in the ubiquitin-binding degron prevents or inhibits ubiquitin-mediated degradation of its gene product. [0144] 5. The isolated nucleic acid molecule of paragraph 1, wherein the Rrm2 gene sequence encodes an Rrm2 polypeptide having 2 to 10 amino acid changes as compared to wild-type Rrm2 polypeptide sequence. [0145] 6. The isolated nucleic acid molecule of paragraph 1, wherein the wild-type Rrm2 gene sequence is SEQ ID NO: 3 or 5. [0146] 7. The isolated nucleic acid molecule of paragraph 3, wherein the ubiquitin-binding degron is encoded at nucleotides 88-96, 97-99, and/or 145-153 of SEQ ID NO: 3 or SEQ ID NO: 5. [0147] 8. The isolated nucleic acid molecule of paragraph 1, wherein the mutation is an amino acid substitution, deletion, or insertion. [0148] 9. The isolated nucleic acid molecule of paragraph 1, wherein the mutation is a mutation of a nucleotide selected from the group consisting of: 88-96, 97-99, and/or 145-153 positions of SEQ ID NO: 3 or SEQ ID NO: 5. [0149] 10. The isolated nucleic acid molecule of any of paragraphs 1-9, wherein the sequence is selected from SEQ ID NO: 16-20. [0150] 11. The isolated nucleic acid molecule of paragraph 2, wherein stability of the encoded polypeptide is increased at least 50% as compared to wild-type Rrm2. [0151] 12. The isolated nucleic acid molecule of paragraph 1, wherein the mutation 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. [0152] 13. The isolated nucleic acid molecule of paragraph 12, wherein the level of cytosolic dATP is increased at least 50% as compared to wild-type Rrm2. [0153] 14. The isolated nucleic acid molecule of paragraph 1, operably linked to a sequence that permits expression of the encoded polypeptide in a cell. [0154] 15. The isolated nucleic acid molecule of paragraph 14, wherein the sequence that permits expression of the encoded polypeptide in a cell comprises a promoter. [0155] 16. The isolated nucleic acid molecule of paragraph 15, wherein the promoter is selected from the group consisting of: a constitutive promoter, a ubiquitous promoter, an inducible promoter, a viral promoter, a tissue specific promoter, and a synthetic promoter. [0156] 17. The isolated nucleic acid molecule of paragraph 16, wherein the tissue specific promoter is a cardiac tissue specific promoter. [0157] 18. A vector comprising the isolated nucleic acid molecule of any one of paragraphs 1-17. [0158] 19. The vector of paragraph 18, wherein the vector is a viral vector. [0159] 20. The vector of any one of paragraphs 18-19, wherein the vector is a DNA or RNA virus. [0160] 21. The vector of any one of paragraphs 18-20, 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. [0161] 22. A polypeptide encoded by the isolated nucleic acid of any one of paragraphs 1-17. [0162] 23. The polypeptide of paragraph 22, wherein the polypeptide sequence is selected from SEQ ID NO: 4, 21-25. [0163] 24. A protein complex comprising the polypeptide of paragraph 22 or 23 and at least one polypeptide of a different sequence. [0164] 25. The protein complex of paragraph 24, wherein the complex comprises an Rrm1 polypeptide. [0165] 26. A composition comprising an isolated nucleic acid molecule of any one of paragraphs 1-17, a vector of any one of paragraphs 20-23, or a polypeptide of any one of paragraphs 22-25. [0166] 27. The composition of paragraph 28, further comprising a pharmaceutically acceptable carrier. [0167] 28. A pharmaceutical composition comprising an isolated nucleic acid molecule of any one of paragraphs 1-17, a vector of any one of paragraphs 18-21, or a polypeptide of any one of paragraphs 22-25. [0168] 29. A method of treating a cardiac disease or disorder, the method comprising administering to a subject in need thereof a composition of any one of paragraphs 26-27, or the pharmaceutical composition of paragraph 28. [0169] 30. The method of paragraph 29, wherein the cardiac disease or disorder is myocardial infarction or myocardial ischemia. [0170] 31. The method of paragraph 29, wherein the composition is administered directly to a cardiac cell. [0171] 32. The method of paragraph 31, wherein the cardiac cell is a cardiomyocyte. [0172] 33. The method of paragraph 29, wherein administering increases dATP in a cardiac cell as compared to an appropriate control. [0173] 34. The method of paragraph 33, wherein dATP is increased at least 50% as compared to an appropriate control. [0174] 35. The method of paragraphs 33 and 34, wherein dATP levels are measured via HPLC and/or mass spectrometry. [0175] 36. The method of paragraph 29, wherein administering increases RNR enzymatic activity in a cell as compared to an appropriate control. [0176] 37. The method of paragraph 36, wherein RNR enzymatic activity is increased at least 50% as compared to an appropriate control. [0177] 38. The method of paragraph 29, further comprising, prior to administering, diagnosing a subject as having a cardiac disease or disorder. [0178] 39. The method of paragraph 29, further comprising, prior to administering, receiving the results of an assay that diagnoses a subject as having a cardiac disease or disorder. [0179] 40. The method of paragraph 29, further comprising administering at least a second therapeutic. [0180] 41. The method of paragraph 1, wherein the polypeptide comprises two mutations. [0181] 42. A method of treating myocardial infarction in a subject, comprising increasing the amount of dATP content in cardiomyocytes in the subject. [0182] 43. The method of paragraph 42, wherein the method comprises overexpression of ribonucleotide reductase (RNR) in the cardiomyocytes in the subject. [0183] 44. A method of increasing the amount of dATP content in cardiomyocytes in a subject, comprising administering to the subject an effective amount of a vector comprising mutants of Rrm2, wherein the mutants overexpress RNR in the cardiomyocytes. [0184] 45. The method of paragraph 44, wherein the mutants of Rrm2 contain one or more mutations in the ubiquitin-binding region of the Rrm2 amino acid sequence. [0185] 46. The method of any previous paragraph, wherein the increased amount of dATP is measured by HPLC. [0186] 47. The method of any previous paragraph, wherein the overexpression is of Rrm1 or Rrm2. [0187] 48. Other methods and compositions as described and paragraphed herein.

[0188] This technology is further illustrated by the following example which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures are incorporated herein by reference.

Example 1

[0189] As described herein, engineering mutations into the ubiquitin-binding motif of Rrm2 increases the amount of Rrm2 overexpression and results in greater cell levels of dATP than unmutated (wild type) Rrm2 in RNR complex. An improved RNR vector using these engineered Rrm2 construct can be developed that is resistant to degradation through the ubiquitin-proteasome pathway and therefore enable more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes in the heart. Without engineering Rrm2, RNR levels after AAV transduction are highly variable in mouse models of cardiomyopathy.

[0190] 2-deoxy-ATP (dATP) is a nucleotide used in DNA synthesis but present in extremely low levels in post-mitotic cells. Methods of increasing dATP levels in cardiomyocytes is a potential therapy for cardiomyopathies. As little as 1% dATP increases both the magnitude and rate of contraction (see e.g., FIG. 1A; Korte et al., J Mol Cell Cardiol. 2011 December; 51(6):894-901).

[0191] The concentration of dATP (i.e., [dATP]) can be elevated in cardiomyocytes by overexpression of ribonucleotide reductase (RNR). AAV-mediated overexpression of RNR in cardiomyocytes improved in vivo function in healthy mice and in rats subjected to myocardial infarction (see e.g., FIG. 1B-FIG. 1E; Kolwicz et al., Mol Ther. 2016 February; 24(2):240-250).

[0192] dNTPs can be quantified in muscle tissues by mass spectrometry. A fast and sensitive HPLC-MS/MS method has been developed for direct quantification of intracellular dNTPs from tissue (see e.g., FIG. 2). Exemplary dNTPs that can be detected by said HPLC-MS/MS method include but are not limited to deoxyguanosine triphosphate (dGTP), adenosine triphosphate (ATP), deoxyadenosine triphosphate dATP, deoxyadenosine triphosphate dATP containing carbon-13 isotype (dATP-C13), deoxythymidine triphosphate (dTTP), and deoxycytidine triphosphate (dCTP).

[0193] Overexpression of RNR increases dATP content in cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes, in vitro adenovirus-transduced neonatal rat cardiomyocytes, adult transgenic mouse cardiomyocytes, and in vivo AAV-transduced mouse cardiomyocytes all displayed elevated levels of dATP (see e.g., FIG. 3).

[0194] AAV-induced overexpression of Rrm2 is highly variable. AAV-induced transduction of RNR is consistent in wild-type mice (see e.g., Kolwicz et al., Mol Ther. 2016 February; 24(2):240-250), but not in disease models. A transgenic mouse model of dilated cardiomyopathy showed overexpression of Rrm1 but inconsistent overexpression of Rrm2 (see e.g., FIG. 4).

[0195] Rrm2 is degraded in cardiomyocytes by ubiquitin-proteasome activity. Neonatal rat cardiomyocytes transduced for three days in vitro with RNR have greater levels of Rrm2 after 3 hours of incubation in MG-132, a potent proteasome inhibitor (see e.g., FIG. 5A). MG132-treated cells also have higher [dATP] in preliminary measurements (see e.g., FIG. 5B).

[0196] Engineered mutations in Rrm2 increase RNR levels in hiPSC-CMs. Human induced-pluripotent stem cells were transfected in vitro with plasmids for RNR containing mutations in the ubiquitin-binding region of Rrm2 (see e.g., FIG. 6A-FIG. 6C). A version containing two mutations was chosen for adenovirus production based on protein levels. FIG. 8 shows nucleotide sequences for 5 exemplary Rrm2 mutants compared to WT Rrm2.

[0197] Engineered Rrm2 elevates [dATP] in neonatal rat cardiomyocytes in vitro. Cardiomyocytes transduced by AV with Rrm1 and double-mutant Rrm2 (AV-RNR-DM) have elevated [dATP] as measured by mass spectrometry (see e.g., FIG. 7).

[0198] Degradation of Rrm2 via the ubiquitin-proteasome complex can be prevented in vitro by the introduction of engineered mutations in the ubiquitin-binding region. Pharmacological inhibition of proteasome activity in transduced cells leads to increased levels of Rrm2 protein and dATP. Without engineering Rrm2, RNR levels after AAV transduction are highly variable in mouse models of cardiomyopathy. dATP levels can be measured in cultured and adult cardiomyocytes with high sensitivity by HPLC/MS/MS and can be elevated by overexpressing RNR.

[0199] The proteasome thus plays a key role in cardiac disease. An improved RNR vector can be developed that is be resistant to degradation through the ubiquitin-proteasome pathway, therefore enabling more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes. Double-mutant Rrm2 is one approach to titrate the amount of dATP produced in myocytes. An AAV can be created for muscle-specific transduction of RNR-DM in vivo.