WO2017027371A1 - Production of adamts13 using mrna - Google Patents

Abstract

The disclosure provides polypeptides (e.g., mRNAs) capable of modulating expression of an ADAMTS13 protem, fragment, or variant that are useful for, among other things, cleaving vWF and, thus, treating diseases or disorders with unregulated vWF functions.

Description

PRODUCTION OF ADAMTS 13 USING MRNA

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on August 4, 2016, is named 0357 SEQ Listing___ST25.TXT and is 23.9kb in size.

BACKGROUND

ADAMTS 13 (a dismtegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is a zmc-metalloprotease belonging to ADAMTS family that specifically cleaves von Willebrand factor (vWF), which is an important factor of platelet aggregation. VWF released through stimulation or circulating in blood is important in forming platelet thrombus because it plays a role in collaboration with collagen on platelet adhesion and agglutination in the subendothelial tissue of a damaged vascular wall.

Thrombotic thrombocytopenic purpura (TTP) is a disorder of the blood characterized by low platel ets, low red blood cell count (caused by premature breakdown of th e cells), and neurological abnormalities. The sharp drop in the number of red blood cells and platelets is associated with severe problems affecting the kidneys and brain, as well as fever and bleeding. The neurological symptoms associated with this disease include headaches, confusion, speech changes, and alterations in consciousness, which vary from lethargy to coma. Other symptoms include development of kidney abnormalities. In addition,

ADAMTS .13 levels in plasma of patients with thrombotic disease, including disseminated intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), TTP, pulmonary embolism, cerebral infarction and systemic lupus erythematosus (SLE), are significantly reduced compared to healthy adult. Moreover, with respect to DIC associated with septicemia, it was shown that patients having ADAMTS 13 blood level of less than 20% are significantly likely to develop nephropathy as compared to patients having ADAMTS 13 blood level of 20% or more.

Gene therapy may be useful for treating a disease or disorder related to unregulated vWF functions. However, current viral vectors for gene therapy have serious safety

concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. SUMMARY

The disclosure relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture and formulation of nucleic acid molecules, especially polynucleotides (e.g. , messenger RNA and modified messenger RNA).

In one embodiment, the disclosure is directed to a composition that provides for the expression of an ADAMTS13 protein or a biologically active fragment or variant thereof, in a target cell, wherein said composition comprises a messenger RN A (mRNA) molecule comprising at least one modification. In a particular embodiment, the modification increases the stability of the mRNA molecule in vivo. In a particular embodiment, the mRNA is formulated in a pharmaceutically acceptable carrier, e.g., a lipid nanoparticle. In a particular embodiment, the mRNA molecule comprises a first region comprising at least one open reading frame encoding the ADAMTS13 protein or biologically active fragment or variant thereof. In a particular embodiment, the compositions described herein can further comprise a) a flanking region located at the 5' terminus of the first regi on, comprising: i) a

physiological 5' UTR of the ADAMTS 13 protein or a biologically active fragment or variant thereof; and ii) at least one 5' terminal cap; b) a flanking region located at the 3' terminus of the first region, comprising: i) a physiological 3' UTR of the ADAMTS 13 protein or a biologi cally active fragment or variant thereof; and ii) a 3' tail sequence; or c) both a) and b). In a particular embodiment, the at least one 5' terminal cap is selected from the group consisting of: a 5' triphosphate cap (5'-ppp), a guanosine-triphosphate cap (5' Gppp), a 5' N7- methylguanosine-triphosphate cap (5' N7~MeGppp, 7mGppp), a m7GpppG cap, a 5' adenylated cap (rApp), Cap 0, Cap I, ARCA, inosine, Nl-methyl-guanosine, 2' fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine. In a particular embodiment, the 3' tail sequence is selected from the group consisting of a poiy-A tail and a poly_A-G quartet. In a particular embodiment, the first region further encodes a signal peptide and/or a leader sequence at the 5' terminus of the ADAMTS13 protein or a biologically active fragment or variant thereof. In a particular embodiment, the first region further encodes a detectable label, e.g. , a fluorescent label, a luminescent label, a heavy metal label, a radioactive label or an enzymatic label, linked to the ADAMTS 13 protein or a biologically active fragment or variant thereof. In a particular embodiment, the at least one mRNA molecule comprises at least one modification of at least one nucleoside in the flanking region located at the 5' terminus of the first region. In a particular embodiment, the at least one mRNA molecule comprises at least one modification of at least one nucleoside in the flanking region located at the 3' terminus of the first region. In a particular embodiment, the at least one mi NA molecule comprises at least one

modification of at least one nucleoside in the first region. In a particular embodiment, the at least one modification is located in a nucleoside base and/or sugar portion. In a particular embodiment, the compositions described herein can further comprise an agent for facilitating transfer of the at least one RNA molecule to an intracellular compartment of a target ceil. In a particular embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof is a fusion protein. In a particular embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof comprise an ammo acid sequence as set forth in SEQ ID NO: 4. In a particular embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof comprises an polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NOS:5 or 6. In a particular embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof further comprise at least one amino acid substitution, e.g. , a substitution to histidine or alanine. In a particular

embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof comprising the at least one amino acid substitution has an increased stability or in vivo half- life when compared to the unsubstituted ADAMTS 13 protein or biologically active fragment or variant thereof. In a particular embodiment, the ADAMTS13 protein or a biologically active fragment or variant thereof binds to vWF, e.g. , human vWF and/or at least one vWF from a non-human mammal.

In one embodiment, the disclosure is directed to a method for expressing in a target cell an ADAMTS13 protein or biologically active fragment or variant thereof comprising maintaining a composition as described herein, under conditions and for a time sufficient to allow expression by the target ceil of the ADAMTS 13 protein or biologically active fragment or variant thereof.

In one embodiment, the disclosure is directed to a therapeutic kit comprising: (i) a composition described herein: and (ii) means for deliver}' of the at least one RNA molecule to a subject.

In one embodiment, the disclosure is directed to an article of manufacture comprising: a) a container comprising a label: and b) a composition described herein, wherein the label indicates that the composition is to be administered to a human having, suspected of having or at risk for developing, a condition related to unregulated vWF expression or activity. In a particular embodiment, the article of manufacture further comprises one or more additional active therapeutic agents for use in treating such human. In one embodiment, the disclosure is directed to a method for treating a patient having, suspected of having or at risk for developing a condition related to misregulated or unregulated vWF expression or activity, the method comprising administering to said subject a composition described herein in an amount effective to treat the condition. In a particular embodiment, the condition is a thrombotic disease such as, for example, disseminated intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), thrombocytopenic purpura (TTP), pulmonary embolism, cerebral infarction or systemic lupus erythematosus (SLE). TTP can be a congenital TTP or an acquired TTP. In a particular embodiment, the composition is administered by a route selected from the group consisting of: intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary,

intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrastemal injection and infusion. In a particular embodiment, the composition is administered in combination with a second therapeutic agent.

In one embodiment, the disclosure is directed to a composition for modulating the expression of an ADAMTS 13 protein or a biologically active fragment or variant thereof. In some embodiments, the composition comprises at least one polynucleotide (e.g. , mRNA) molecule and an optional transfer vehicle. In some embodiments, the mRNA comprises at least one modification that confers stability to the mRNA. In some embodiments, the composition includes an agent for modul ating the expression of an ADAMTS 13 protein or a biologically active fragment or variant thereof in a target cell.

The composition(s) described herein, e.g. , mRNA composition(s), can comprise, for example, at least one open reading frame encoding an ADAMTS 13 protein or a biologically active fragment or variant thereof. The composition can further or optionally comprise at least one amino acid substitution in any one of its domains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of Western blot graphs that depict the expression of FLAG-tagged ADAMTS 13 in HeLa (panel A) and human primary hepatocyte (panel B). In panel A, lane 1 : molecular weight markers; lane 2: HeLa cell lysate control (no mRNA transfection); lane 3: FLAG-tag positive control; lane 4: HeLa cell lysate (transfected with FLAG-AD AMTS 13 mRNA). In panel B, lane 1 : molecular weight markers; lane 2: hepatocyte cell lysate (transfected with FLAG- ADAMTS 13 mRNA); lane 3: hepatocyte cell lysate control (no mRNA transfection). The arrows point to the expressed FLAG-ADAMTS 13 protein in both panels. The asterisks represent endogenous GAPDH protein as a protein loading control.

FIG. 2 is a series of graphs showing the activity readings of the expressed

FLAG-tagged ADAMTS13. Each box represents one well of a 96- well plate containing different reactions (run in duplicate). Box 1 contains blank control. Box 2 contains positive control. Box 3 contains negative control. Box 14 contains culture media of hepatocytes iransfected with wild-type non-tagged ADAMTS 13. Box 16 contains culture media of HeLa ceils iransfected with FLAG-tagged ADAMTS 13. Box 18 contains culture media of HeLa ceils iransfected with FLAG-tagged AD AMTS 13.

FIG. 3 is a bar graph showing the activity of the expressed ADAMTSI3 proteins after a 60 minute reaction. Lane 1 : blank control; lane 2: recombinant ADAMTS 13 as a positive control; lane 3: recombinant ADAMTS13 plus enzyme inhibitor; lane 4: hepatocyte cell lysate (iransfected with FLAG-AD AMTS 13 mRNA); lane 5: hepatocyte cell lysate

(iransfected with FLAG- AD AMTS 13 mRNA) plus enzyme inhibitor; lane 6: hepatocyte cell lysate (transfected with non-tagged AD AMTS 13 mRNA); lane 7: hepatocyte cell lysate

(iransfected with non-tagged ADAMTS 13 mRNA) plus enzyme inhibitor; lane 8: HeLa cell lysate (iransfected with FLAG-AD AMTS 13 mRNA); lane 9: HeLa cell lysate (transfected with FLAG- AD AMTS 13 mRNA) plus enzyme inhibitor; lane 10: HeLa cell lysate

(transfected with non-tagged ADAMTS13 mRNA); lane 11 : HeLa ceil lysate control (iransfected with non-tagged ADAMTS13 mRNA) plus enzyme inhibitor; lane 12:

hepatocyte cell (transfected with FLAG- AD AMTS 13 mRNA) culture media; lane 13:

hepatocyte cell (transfected with FLAG-ADAMTS13 mRNA) culture media plus enzyme inhibitor; lane 14: hepatocyte ceil (iransfected with non-tagged AD AMTS 13 mRNA) culture media; lane 15: hepatocyte cell (transfected with non-tagged ADAMTSI3 mRNA) culture media plus enzyme inhibitor; lane 16: HeLa cell (transfected with FLAG- AD AMTS 13 mRNA) culture media; lane 17: HeLa cell (transfected with FLAG-ADAMTS13 mRNA) culture media plus enzyme inhibitor; lane 18: HeLa cell (transfected with non-tagged ADAMTS13 mRNA) culture media; lane 19: HeLa cell (transfected with non-tagged ADAMTS13 mRNA) culture media plus enzyme inhibitor.

Figure 4 is a graph showing the activity of the expressed ADAMTS 13 proteins after a

150 minute reaction. Lanes 1 through 19 represent the same loading sequence as in FIG. 3.

Figure 5 is a graph showing the activity of the expressed ADAMTS 13 proteins after a 210 minute reaction. Lanes 1 to 19 represent the same loading sequence as in FIG. 3. Figure 6 is a graph showing the activity of the expressed ADAMTS13 proteins after a one day reaction. Lanes 1 to 19 represent the same loading sequence as in FIG. 3.

Figure 7 is a Western blot showing the expressed non-tagged wild-type ADAMTS13 protein in liver tissue homogenates of C57bl/6 mice. MW: molecular weight markers;

A1-A5: five mice in Group A with PBS injections; B6-B10: five mice in Group B with vehicle injections; G31-G35: five mice in Group G administered with mRNAs encoding the wild-type ADAMTS13 for 24 hours; 071-075: five mice in Group O administered with mRNAs encoding the wild-type ADAMTS13 for 48 hours.

FIGS. 8A and 8B are graphs comparing ADAMTS 13 in vivo expression levels (FIG. 8A) using the data from FIG. 7. Endogenous GAPDH expression levels were used as a protein loading control (FIG. 8B). The "24 hrs" and "48 hrs" data points represent animals administered with mRNAs encoding wild-type ADAMTS13 for 24 hours (Group G) or for 48 hours (Group O). The asterisk represents statistically significant difference in expression of hADAMTS13 in mice as compared to the V ehicle (encapsulating Luciferase) negative control in liver lysate.

FIG. 9 is a series of images showing the expressed FLAG-tagged ADAMTS13 protein in C57bl/6 mice hepatocytes (24 hours post injection) by FLAG-IHC (40*). A2, B6, H36 and H39 are representative samples from Groups A, B and H, respectively.

DETAILED DESCRIPTION

Described herein are compositions of nucleic acids capable of providing protein expression and or regulating (modulating) protein expression of ADAMTS 13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; also known as von Willebrand factor-cleaving protease (vWFCP) or a biologically active fragment or variant in a target cell. Methods and processes of preparing and delivering such nucleic acids to a target cell are also provided. Furthermore, kits and devices for the design, preparation, manufacture and formulation of such nucleic acids are also described.

In particular, the compositions provided herein are useful for treating diseases or disorders with unregulated expression and/or activity of von Willebrand Factor (v WF). Nucleic acids include, for example, polynucleotides, which further include, for example, ribonucleic acids (RN As) including mRNAs (including those comprising a modified or unmodified backbone and/or one or more modified ribobases); deoxyribonucleic acids (DNAs); threose nucleic acids (TNAs; Yu, H. ei al. , Nat. Chem. , 4: 183-7 , 2012), glycol nucleic acids (GNAs; Ueda, N. et a!., J. Heterocyclic Chem. , 8:827-9, 1971; Zhang, L. et al , J. Am, Chem. Soc, 127:4174-5, 2005), peptide nucleic acids (PNAs; Nielsen, P. et al , Science, 254: 1497-500, 1991), locked nucleic acids (LNAs; Koshkm, A. et al, Tetrahedron, 54:3607-30, 1998), and other polynucleotides.

The nucleic acid molecule can be, for example, a messenger RNA (mRNA). In some embodiments, the mRNA encodes an ADAMTS13 protein or its biologically active fragments or valiants. In one embodiment, the mRNA is delivered into a target cell to express such ADAMTS 13 protein or biologically active fragment or variant in vivo, in situ or ex vivo. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and that is capable of being translated to produce the encoded polypeptide of interest.

As used herein, "fragment" refers to a portion of a molecule, e.g. , a gene, expression product of a gene, coding sequence, mRNA or protein, that retains a desired length or function. A "biologically active" fragment retains a desired activity of the molecule. An ADAMTS13 fragment, for example, can be a fragment of the full length ADAMTS13 protein that binds vWF. In general, a fragment of an enzyme or signaling molecule can be, for example, that portion(s) of the molecule that retains its signaling or enzymatic activity. A fragment of a gene or coding sequence, for example, can be that portion of the gene or coding sequence that produces an expression product fragment. As used herein, "gene" is a term used to describe a genetic element that gives rise to expression products (e.g. , pre-mRNA, mRNA, and polypeptides). A fragment does not necessarily have to be defined functionally, as it can also refer to a portion of a molecule that is not the whole molecule, but has some desired characteristic or length (e.g. , restriction fragments, amplification fragments, etc. ).

In another embodiment, the mRNA is delivered to a subject, e.g. , a mammal, to express such ADAMTS 1 3 protein or biologically active fragment or variant. The terms "individual," "subject," "host" and "patient" are used to refer to any subject for whom diagnosis, treatment or therapy is desired, particularly humans. Other subject include, but are not limited to, cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses and the like.

The mRNA is capable of treating or alleviating a thrombotic disease or disorder, or other vWF-reiated disease or disorder in the subject. Examples of such vWF -related diseases or disorders include, but are not limited to, disseminated intravascular coagulation (DIC), hemolytic uremic syndrome (HUS), deep vein thrombosis (DVT), thrombocytopenic purpura (TTP)- both congenital and acquired TTPs, pulmonary embolism, cerebral infarction and systemic lupus erythematosus (SLE). RNA structure

The RNA molecule(s) described herein, for example, can increase or promote expression of an ADAMTS 13 protein, fragment or variant. In some embodiments, the composition comprises an artificially synthesized or isolated natural RNA molecule with or without a transfer vehicle. Such RNA molecule can comprise, for example, an mRNA encoding an ADAMTS13 protein, fragment or variant. In some embodiments, an encoding RNA molecule (such as an mRNA) comprises a nucleic acid sequence structure as follows:

A B C D E

Domain C in the above structure can include, but is not limited to, the nucleic acid sequence encoding an ADAMTS 13 protein or its fragments or variants. Domain C can comprise at its 5' terminus one or more signal sequences. Domain B represents an optional flanking region comprising one or more complete or incomplete 5' UTR (Untranslated Region) sequences. Domain A represents an optional 5' terminal cap to the mRNA sequence. Domain D represents an optional flanking region comprising one or more complete or incomplete 3' UTR sequences. Domain E represents an optional flanking region comprising a 3' tail , e.g. , a poly-A tail or a poly-A-G quartet. Bridging the 5' terminus of Domain C and the flanking Domain B is an optional first operational region. Traditionally this first operational region comprises a start codon. The operational region can alternatively comprise any translation initiation sequence or signal including a start codon. Bridging the 3' terminus of Domain C and the flanking region Domain D is an optional second operational region. Traditionally this second operational region comprises a stop codon. The operational region can alternatively comprise any translation initiation sequence or signal including a stop codon. Multiple serial stop codons can be used.

An mRNA molecule can be, for example, cyclized or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5 -end binding proteins. Such cyclization or concatemerization can be formed through various mechanisms including, for example, chemical, enzymatic and/or ribozyme-catalyzed mechanisms. The newly formed 573' linkage can be intramolecular or intermolecular. In some embodiments, the 5'-end and/or the 3'-end of the mRNA comprises one or more chemically reactive group(s) that, when close together, form a new covalent linkage between the 5'-end and the 3 '-end of the mRNA. The 5'-end can optionally comprise an NHS-ester reactive group and the 3'-end can comprise a 3'-amino terminated nucleotide such that in an organic solvent the 3'-amino terminated nucleotide on the 3'-end of a synthetic mRNA molecule undergoes nucleophihc attack at the 5'-NHS-ester moiety forming a new 5'/3'-amide bond. In some embodiments, T4 RNA ligase is used to enzymatically link a

5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. Alternatively, either the 5'- or 3'-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5 -end of a nucleic acid molecule to the 3'-end of a nucleic acid molecule. The ligase ribozyme can be derived from, for example, the Group I intron, Group II intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment).

RNA sequence

mRNA sequences that encode an ADAMTS 13 protein, biologically active fragment or variant are described herein. The mRNA molecules are useful for, among other things, inhibiting vWF function and, thus, treating disorders with unregulated vWF functions (e.g. , unregulated vWF protein levels or protein functions).

ADAMTS13

The ADAMTS 13 protein encoded by a polynucleotide (e.g., mRNA) described herein includes fragments, e.g., biologically active fragments, and variants. Such ADAMTS13 protein or its fragments or variants can specifically bind to and cleave vWF at, e.g. ,

Tyr 1605 -Met 1606, which corresponds to 842-843 after cleavage of a preprosequence of vWF. The ADAMTS13 protein, for example, ca comprise a mammalian ADAMTS13 protein. In some embodiment, the encoded ADAMTS13 protein is a non-human animal, e.g. , a mammalian ADAMTS 13. The term "mammal" (and correspondingly "mammalian") refers to (a source of) a human or a non-human mammal, such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, a camelid or a non-human primate. The ADAMTS 13 protein can comprise, for example, a labeled or tagged ADAMTS 13. Such labels or tags can include, but are not limited to, FLAG (DYKDDDDK; SEQ ID NO: l), polyhistidine (6xHis; HHHHHH; SEQ ID NO: 2), Streptag, hemagglutinin (HA; YPYDVPDYA; SEQ ID NO:3), giutathione-S -transferase (GST), maltose-binding protein (MBP), luciferase, a fluorescent protein (e.g. , green fluorescent protein (GFP)), and chloramphenicol acetyl transferase (CAT). A wild-type human ADAMTS 13 protein sequence is given below as SEQ ID NO:4 (GenBank Accession number: AAL1 1095). mhqrhprarc pplc agila cgfllgcwgp shfqqsciqa lepqavs syl spgaplkgrp pspgfqrqrq rqrraaggil hlellvavgp dvfqahqedt eryvltnlni gaellrdpsl gaqfrvhlvk mviltepega pnitanltss llsvcgwsqt inpeddtdpg hadlvlyitr fdlelpdgnr qvrgvtqlgg acsptwscli tedtgfdlgv tiaheighsf glehdgapgs gcgpsghvma sdgaapragl awspcsrrcti Isllsagrar c wdpprpqp gsaghppdaq pglyysaneq crvafgpka actfarehld mcqalschtd pldqsscsrl

Ivplldgtec gvekwcskgr crslveltpi aavhgrwssw gprspesrse gggvvtrrrq cnnprpafgg racvgadlqa emcntqacek tqlefrtisqqc artdgqplrs spggasfyhw gaavphsqgd alcrhmcrai gesfimkrgd sfldgtrcmp sgpredgtls lcvsgscrtf gcdgrmdsqq vwdrcqvegg dnstcsprkg sftagra ey vtfltvtpnl tsvyianhrp Ifthlavrig gryvvagkms ispnttypsl ledgrveyrv alted Ί prl

edadiqvyrr ygeeygnltr pditftyfqp kprqawvwaa vrgpcs scg aglrwvnysc ldqarkelve tvqcqgsqqp pawpeacvle peppywavgd fgpcsascgg glrerpv cv eaqgsllktl pparcragaq qpavaletcn pqpcparwev sepssctsag gaglalenet c pgadglea pvtegpgsvd eklpapepcv gmscppgwgh Idatsageka pspwgsirtg aqaahvwtpa agscs scgr glmelrf1cm dsalrvpvqe elcglaskpg srrevcqavp cparwqykla acsvscgrgv vrrilycara hgeddgeeil Idtqcqglpr pepqeacsle pcpprwkvms lgpcsascgl gtarrsvacv qldqgqdvev deaacaalvr peasvpclia dctyrwhvgt wmecsvscgd giqr rdtel gpqaqapvpa dfcqhlpkp tvrgcwagpc vgqgtpslvp heeaaapgrt tatpagasle wsqargllfs papqpr rllp gpqensvqs s acgrqhlept gtidmrgpgq adcavaigrp lgevv lrvl esslncsagd mlllwgrltw rkmcrklldm tfssktntlv vrqrcgrpgg gvllrygsql apetfy ecd mqlfgpwgei vspsispats nagger Ifin vaphariaih alatnmgagt eganasyili rdthslrtta fhgqqvlywe sessqaemef segflkaqas

Irgqywtlqs wvpemqdpqs wkgkegt iSEQ !D NO:4) An exemplary DNA sequence for producing an niRNA described herein encoding a non-tagged ADAMTS13 is given below as SEQ ID NO:5,

atgcaccagc gtcacccccg ggcaagatgc cctcccctct gtgtggccgg aatccttgcc tgtggctttc tcctgggctg ctggggaccc tcccatttcc agcagagttg tcttcaggct ttggagccac aggccgtgtc ttcttacttg agccctggtg ctcccttaaa aggccgccct ccttcccctg gcttccagag gcagaggcag aggcagaggc gggctgcagg cggcatccta cacctggagc tgctggtggc cgtgggcccc gatgtcttcc aggctcacca ggaggacaca gagcgctatg tgctcaccaa cctcaacatc ggggcagaac tgcttcggga cccgtccctg ggggctcagt ttcgggtgca cctggtgaag atggtcattc tgacagagcc gagggtgct ccaaatatca cagccaacct cacctcgtcc ctgctgagcg tctgtgggtg gagccagacc atcaacccta aggacgacac ggatcctggc catgctgacc tggtcctcta tatcactagg tttgacctgg agttgcctga tggtaaccgg caggtgcggg gcgtcaccca gctgggcggt gcctgctccc caacctggag ctgcctcatt accgaggaca ctggcttcga cctgggagtc accattgccc atgagattgg gcacagcttc ggcctggagc acgacggcgc gcccggcagc ggctgcggcc ccagcggaca cgtgatggct tcggacggcg ccgcgccccg cgccggcctc gcctggtccc cctgcagccg ccggcagctg ctgagcctgc tcagcgcagg acgggcgcgc tgcgtgtggg acccgccgcg gcctcaaccc gggtccgcgg ggcacccgcc ggatgcgcag cctggcctct actacagcgc caacgagcag tgccgcgtgg ccttcggccc caaggctgtc gcctgcacct tcgccaggga gcacctggat atgtgccagg ccctctcctg ccacacagac ccgctggacc aaagcagctg cagccgcctc ctcgttcctc tcctggatgg gacagaatgt ggcgtggaga agtggtgctc caagggtcgc tgccgctccc tggtggagct gacccccata gcagcagtgc atgggcgctg gtctagctgg ggtccccgaa gtccttgctc ccgctcctgc ggaggaggtg tggtcaccag gaggcggcag tgcaacaacc ccagacctgc ctttgggggg cgtgcatgtg ttggtgctga cctccaggcc gagatgtgca acactcaggc ctgcgagaag acccagctgg agttcatgtc gcaacagtgc gccaggaccg acggccagcc gctgcgctcc tcccctggcg gcgcctcctt ctaccactgg ggtgctgctg taccacacag ccaaggggat gctctgtgca gacacatgtg ccgggccatt ggcgagagct tcatcatgaa gcgtggagac agcttcctcg atgggacccg gtgtatgcca agtggccccc gggaggacgg gaccctgagc ctgtgtgtgt cgggcagctg caggacattt ggctgtgatg gtaggatgga ctcccagcag gtatgggaca ggtgccaggt gtgtggtggg gacaacagca cgtgcagccc acggaagggc tctttcacag ctggcagagc gagagaatat gtcacgtttc tgacagttac ccccaacctg accagtgtct acattgccaa ccacaggcct ctcttcacac acttggcggt gaggatcgga li gggcgctatg cgtggctgg gaagatgagc acaccaccta ctggaggatg gtcgtgtcga gtacagagtg gccctcaccg aggaccggc gccccgcctg gaggagatcc gcatctgggg acccctccag gaagatgctg acatccaggt ttacaggcgg tatggcgagg agtatggcaa cctcacccgc ccagacatca ccttcaccta cttccagcct aagccacggc aggcctgggt gtgggccgct gtgcgtgggc cctgctcggt gagctgtggg gcagggctgc gctgggtaaa ctacagctgc ctggaccagg ccaggaagga gttggtggag actgtccagt gccaagggag ccagcagcca ccagcgtggc cagaggcctg cgtgctcgaa ccctgccctc cctactgggc ggtgggagac ttcggcccat gcagcgcctc ctgtgggggt ggcctgcggg agcggccagt gcgctgcgtg gaggcccagg gcagcctcct gaagacattg cccccagccc ggtgcagagc aggggcccag cagccagctg tggcgctgga aacctgcaac ccccagccct gccctgccag gtgggaggtg tcagagccca gctcatgcac atcagctggt ggagcaggcc tggcct tgga gaacgagacc tgtgtgccag gggcagatgg cctggaggct ccagtgactg aggggcctgg ctccgtagat gagaagctgc ctgcccctga gccctgtgtc gggatgtcat gtcctccagg ctggggccat ctggatgcca cctctgcagg ggagaaggct ccctccccat ggggcagcat caggacgggg gctcaagctg cacacgtgtg gacccctgcg gcagggtcgt gctccgtctc ctgcgggcga ggtctgatgg agctgcgttt cctgtgcatg gactctgccc t cagggtgcc tgtccaggaa gagctgtgtg gcctggcaag caagcctggg agccggcggg aggtctgcca ggctgtcccg tgccctgctc ggtggcagta caagctggcg gcctgcagcg tgagctgtgg gagaggggtc gtgcggagga t cctgtattg tgcccgggcc catggggagg acgatggtga ggagatcctg ttggacaccc agtgccaggg gctgcctcgc ccggaacccc aggaggcctg cagcctggag ccctgccca c ctaggtggaa agtcatgtcc cttggcccat gttcggccag ctgtggcctt ggcactgcta gacgctcggt ggcctgtgtg cagctcgacc aaggccagga cgtggaggtg gacgaggcgg cctgtgcggc gctggtgcgg cccgaggcca gtgtcccctg tctcattgcc gactgcacct a ccgctggca tgttggcacc tggatggagt gctctgtttc ctgtggggat ggcatccagc gccggcgtga cacctgcctc ggaccccagg cccaggcgcc tgtgccagct gatttctgcc agcacttgcc caagccggtg actgtgcgtg gctgctgggc tgggccctgt gtgggacagg gtacgcccag cctggtgccc cacgaagaag ccgctgctcc aggacggacc acagccaccc ctgctggtgc ctccctggag tggtcccagg cccggggcct gctcttctcc ccggctcccc agcctcggcg gctcctgccc gggccccagg aaaactcagt gcagtccagt gcctgtggca ggcagcacct tgagccaaca ggaaccattg acatgcgagg cccagggcag gcagactgtg cag ggccat tgggcggccc ctcggggagg tggtgaccct ccgcgtcctt gagagttctc tcaactgcag gcgggggac atgttgctgc tttggggccg gctcacctgg aggaagatgt gcaggaagct gttggacatg actttcagct ccaagaccaa cacgctggtg gtgaggcagc gctgcgggcg gccaggaggt ggggtgctgc tgcggtatgg gagccagctt gctcctgaaa ccttctacag agaatgtgac atgcagctct ttgggccctg gggtgaaatc gtgagcccct cgctgagtcc agccacgagt aatgcagggg gctgccggct cttcattaat gtggctccgc acgcacggat tgccatccat gccctggcca ccaacatggg cgctgggacc gagggagcca atgccagcta catcttgatc cgggacaccc acagcttgag gaccacagcg ttccatgggc agcaggtgct ctactgggag tcagagagca gccaggctga gatggagttc agcgagggct tcctgaaggc tcaggccagc ctgcggggcc agtactggac cctccaatca tgggtaccgg agatgcagga ccctcagtcc tggaagggaa aggaaggaac ctaatagtga (SEQ D N0:5)

An exemplar}' DNA sequence for producing an mRNA encoding a FLAG-tagged ADAMTS13 is given below as SEQ ID NO:6.

atgcaccagc gtcacccccg ggcaagatgc cctcccctct gtgtggccgg aatccttgcc tgtggctttc tcctgggctg ctggggaccc tcccatttcc agcagagttg tcttcaggct ttggagccac aggccgtgtc ttcttacttg agccctggtg ctcccttaaa aggccgccct ccttcccctg gcttccagag gcagaggcag aggcagaggc gggctgcagg cggcatccta cacctggagc tgctggtggc cgtgggcccc gatgtcttcc aggctcacca ggaggacaca gagcgctatg tgctcaccaa cctcaacatc ggggcagaac tgcttcggga cccgtccctg ggggctcagt ttcgggtgca cctggtgaag atggtcattc tgacagagcc tgagggtgct ccaaatatca cagccaacct cacctcgtcc ctgctgagcg tctgtgggtg gagccagacc atcaaccctg aggacgacac ggatcctggc catgctgacc tggtcctcta tatcactagg tttgacctgg agttgcctga tggtaaccgg caggtgcggg gcgtcaccca gctgggcggt gcctgctccc caacctggag ctgcctcatt accgaggaca ctggcttcga cctgggagtc accattgccc atgagattgg gcacagcttc ggcctggagc acgacggcgc gcccggcagc ggctgcggcc ccagcggaca cgtgatggct tcggacggcg ccgcgccccg cgccggcctc gcctggtccc cctgcagccg ccggcagctg ctgagcctgc tcagcgcagg acgggcgcgc tgcgtgtggg acccgccgcg gcctcaaccc gggtccgcgg ggcacccgcc ggatgcgcag cctggcctct actacagcgc caacgagcag tgccgcgtgg ccttcggccc caaggctgtc gcctgcacct tcgccaggga gcacctggat atgtgccagg ccctctcctg ccacacagac ccgctggacc aaagcagctg cagccgcctc ctcgttcctc tcctggatgg gacagaatgt ggcgtggaga agtggtgctc caagggtcgc tgccgctccc tggtggagct gacccccata gcagcagtgc atgggcgctg gtctagctgg ggtccccgaa gtccttgct c ccgctcctgc ggaggaggtg tggtcaccag gaggcggcag tgcaacaacc ccagacctgc ctttgggggg cgtgcatgtg t tggtgctga cctccaggcc gagatgtgca acactcaggc ctgcgagaag acccagctgg agttcatgt c gcaacagtgc gccaggaccg acggccagcc gctgcgctcc tcccctggcg gcgcctcctt ctaccactgg ggtgctgctg taccacacag ccaaggggat getctgtgca gacacatgtg ccgggccatt ggcgagagct tcatcatgaa gcgtggagac agcttcctcg atgggacccg gtgtatgcca agtggccccc gggaggaegg gaccctgagc ctgtgtgtgt cgggcagctg caggacattt ggctgtgatg gtaggatgga ctcccagcag gtatgggaca ggtgccaggt gtgtggtggg gacaacagca cgtgcagccc acggaagggc tctttcacag ctggcagagc gagagaatat gtcacgtttc tgacagttac ccccaacctg accagtgtct acattgccaa ccacaggcct ctcttcacac acttggcggt gaggategga gggcgctatg tcgtggctgg gaagatgagc atctccccta acaccaccta cccctccctc ctggaggatg gtcgtgtcga gtacagagtg gccctcaccg aggaccggct gccccgcctg gaggagatcc gcatctgggg acccctccag gaagatgctg acatccaggt ttacaggcgg tatggcgagg agtatggcaa cctcacccgc ccagacatca ccttcaccta cttccagcct aagccacggc aggcctgggt gtgggccgct gtgcgtgggc cctgctcggt gagctgtggg gcagggctgc gctgggtaaa ctacagctgc ctggaccagg ccaggaagga gttggtggag actgtccagt gccaagggag ccagcagcca ccagcgtggc cagaggcctg cgtgctcgaa ccctgccctc cctactgggc ggtgggagac ttcggcccat gcagcgcctc ctgtgggggt ggcctgcggg ageggecagt gcgctgcgtg gaggcccagg gcagcctcct gaagacattg cccccagccc ggtgcagagc aggggcccag cagccagctg tggcgctgga aacctgcaac ccccagccct gccctgccag gtgggaggtg tcagagccca getcatgeae atcagctgg ggagcaggcc tggccttgga gaacgagacc tgtgtgccag gggcagatgg cctggaggct ccagtgactg aggggcctgg ctccgtagat gagaagctgc ctgcccctga gccctgtgtc gggatgtcat gtcctccagg ctggggccat ctggatgcca cctctgcagg ggagaaggct ccctccccat ggggcagcat caggacgggg gctcaagctg cacacgtgtg gacccctgcg gcagggtcgt gc ccgtctc ctgcgggcga ggtctgatgg agctgcgttt cctgtgcatg gactctgccc tcagggtgcc tgtccaggaa gagctgtgtg gcctggcaag caagcctggg agccggcggg aggtctgcca ggctgtcccg tgccctgctc ggtggcagta caagctggcg gcctgcagcg tgagctgtgg gagaggggtc gtgcggagga tcctgtattg tgcccgggcc catggggagg acgatggtga ggagatcctg ttggacaccc agtgccaggg gctgcctcgc ccggaacccc aggaggcctg cagcctggag ccctgcccac ctaggtggaa agtcatgtcc cttggcccat gttcggccag ctgtggcctt ggcactgcta gacgctcggt ggcctgtgtg cagctcgacc aaggccagga cgtggaggtg gacgaggcgg cctgtgcggc gctggtgcgg cccgaggcca gtgtcccctg tctcattgcc gactgcacct accgctggca tgttggcacc tggatggagt gctctgtttc ctgtggggat ggcatccagc gccggcgtga cacctgcctc ggaccccagg cccaggcgcc tgtgccagct gatttctgcc agcacttgcc caagccggtg actgtgcgtg gctgctgggc tgggccctgt gtgggacagg gtacgcccag cctggtgccc cacgaagaag ccgctgctcc aggacggacc acagccaccc ctgctggtgc ctccctggag tggtcccagg cccggggcct gctcttctcc ccggctcccc agcctcggcg gctcctgccc gggccccagg aaaactcagt gcagtccagt gcctgtggca ggcagcacct tgagccaaca ggaaccattg acatgcgagg cccagggcag gcagactgtg cagtggccat tgggcggccc ctcggggagg tggtgaccct ccgcgtcctt gagagttctc tcaactgcag tgcgggggac atgttgctgc tttggggccg gctcacctgg aggaagatgt gcaggaagct gttggacatg actttcagct ccaagaccaa ca cgctggtg gtgaggcagc gctgcgggcg gccaggaggt ggggtgctgc tgcggtatgg gagccagctt gctcctgaaa ccttctacag agaatgtgac atgcagctct ttgggccctg gggtgaaatc gtgagcccct cgctgagtcc agccacgagt aatgcagggg gctgccggct cttcattaat gtggctccgc acgcacggat tgccatccat gccctggcca ccaacatggg cgctgggacc gagggagcca atgccagcta catcttgatc cgggacaccc acagcttgag gaccacagcg ttccatgggc agcaggtgct ctactgggag tcagagagca gccaggctga gatggagttc agcgagggct tcctgaaggc tcaggccagc ctgcggggcc agtactggac cctccaatca tgggtaccgg agatgcagga ccctcagtcc tggaagggaa aggaaggaac cgactacaaa gacga gacg acaagtaata gtga (SEQ ID NO:6) The ADAMTS 13 protein encoded by the mRNA described herein includes a biologically active fragment or variant of the wild-type ADAMTS13 protein. As described herein, "biologically active" refers to the ability to cleave vWF, or substantially cleave vWF, e.g. , retaining at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100% or more vWF cleavage activity relative to wild-type ADAMTS 13. ADAMTS 13 variants, such as, for example, those described in U.S. Patent Nos, 8,906,661, 8,394,373, and 7,572,59 ! or described by Jian, C. et al. {Blood, 1 19:3836-43, 2012), the entire contents of each of which are incorporated herein by reference. Such variants can, for example, increase the target-binding ability, affinity, efficacy and/or stability of ADAMTS13. Some substitutions facilitate the preparation and/or delivery of the coding polynucleotide of the ADAMTS13 protein, fragments or variants encoded by the mRNA. Other substitutions improve the expression and/or therapeutic function of the ADAMTS 13 protein, fragments, or variants encoded by the mRNA. Some substitutions, for example, can decrease host immunogenicity and/or increase stability and/or in vivo hal f-life of the ADAMTS 13 protein, fragments or variants encoded by the mRNA.

The disclosure provides for the use of sequences that at least about 71%), about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%», about 96°,_:.. about 97%, about 98%, about 99%», or about 100% identity to desired reference sequence. As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used. The terms "homology" or "identity " or "similarity" refer to sequence relationships between two nucleic acid molecules and can be determined by comparing a nucleotide position in each sequence when aligned for purposes of comparison. The term "homology" refers to the relatedness of two nucleic acid or protein sequences. The term "identity" refers to the degree to which nucleic acids are the same between two sequences. The term "similarity" refers to the degree to which nucleic acids are the same, but includes neutral degenerate nucleotides that can be substituted within a codon without changing the amino acid identity of the codon, as is well known, in the art. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequences that alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant," Such variants can be useful, for example, to alter the physical properties of the peptide, e.g. , to increase stability or efficacy of the peptide.

Conservative substitution tables providing functionally similar amino acids are known to those of ordinary skill in the art. Such conservatively substituted variants are in addition to and do not exclude polymorphic variants, interspecies homoiogs (orthologs) and alternate alleles. The following groups provide non-limiting examples of ammo acids that can be conservatively substituted for one another: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V): 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

In some embodiments, the ADAMTS 13 protein, fragments, or variants encoded by the mRNA described herein is a fusion protein. For example, such encoded ADAMTS13 protein can comprise ADAMTS13 connected to another moiety with or without a linker. The linker (e.g. , a linker peptide) can either connect the N-terminus of the ADAMTS 13 portion, or vice versa, resulting in two different 5'-to-3' orientations for the encoded ADAMTS 13 protein. Such linker peptide may be any one known in the art, including, e.g., those peptides usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect to the N-terminus or C-terminus of the ADAMTS 13.

RNA formation and Modifications

Provided herein are SEQ ID OS:5 and 6, which represent exemplary nucleic acid sequences used to produce mRNA sequences encoding ADAMTS13 proteins. Any sequence variation (which, e.g. , contains at least one different three-base codon but encoding the same amino acid sequence) may also be used to prepare mRNA molecules. Variations to the standard codons are known in the art and are included herein.

Codon usage bias refers to differences in the frequency of occurrence of synonymous codons in coding DNA. A codon is a seri es of three nucleotides (triplets) that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation (stop codons). There are 64 different codons (61 codons encoding for amino acids plus 3 stop codons) for only 20 different translated amino acids. The overabundance in the number of codons allows many amino acids to be encoded by more tha one codon. The genetic code is thus "degenerate" because of such redundancy. Different organisms often exhibit preferences for one of the several codons that encode the same amino acid. Such codon preferences are argued to reflect a balance between mutational biases and natural selection for translational optimization. Optimal codons in fast-growing microorganisms, like Escherichia coli or Saccharomyces cerevisiae, reflect the composition of their respective genomic tRNA pool. Optimal codons may help to achieve faster translation rates and high accuracy. As a result of these factors, translational selection is expected to be stronger in highly expressed genes.

In organisms that do not have high growth rates or that present small genomes, codon usage optimization is normally absent, and codon preferences are determined by the characteristic mutational biases seen in that particular genome. Examples of this are Homo sapiens and Helicobacter pylori . Organisms that show an intermediate level of codon usage optimization include at least Drosophila melanogaster, Caenorhabditis elegans,

Strongylocentrotus purpuratus and Arahidopsis th.ali.ana. (Hershberg, R. & Petrov, D., Annu. Rev. Genet. , 42:287-99, 2008; Eyre-Walker, A„ J. Mot Evol, 33:442-9, 1991; the entire contents of each of which are herein incorporated by reference).

The mRNA molecule(s) described herein can comprise at least one codon substituted to the corresponding biased codon in the target cell {e.g. , a desired cell in which the

ADAMTS13 protein, fragment or variant is to be expressed). One exemplar)' and non-limiting rationale for this substitution is to decrease host immunogenicity and/or to facilitate protein translation in a subject (e.g. , since the biased codon is a better "fit" for the endogenous translation machinery). In other embodiments, an mRNA molecule may comprise at least one codon substituted to a non-preferred codon in the target ceil. One exemplary and non-limiting rationale for this substitution is to increase differentiation of the expressed ADAMTS13 protein, fragment, or variant from endogenous ADAMTS 13 and/or to add some preferred properties to the expressed ADAMTSl 3 protein, fragments, or variant.

The mRNAs described herein can be natural or recombinant in nature and isolated or chemically synthesized. Isolated mRNAs can be isolated, for example, from cell cultures expressing an exogenous expression vector that allows for transcription of the mRNA.

Additionally, mRNAs can be isolated from organisms such as plants or animals that comprise an exogenous expression vector that allows for the production of the desired mRNA.

Variant, fragment or codon-optimized mRNA sequences can be, for example, determined in silico prior to synthesis.

Described herein are compositions and methods for the manufacture and optimization of mRNA molecules, e.g. , by modifying the architecture of mRNA molecules. In some embodiments, increased production of an ADAMTS13 protein, fragment or variant, encoded by the mRNA molecules is achieved by altering the terminal regions of such mRNA molecules. Terminal regions can include, for example, the 5'-untranslated region (UTR) and 3'-UTR. A 5' cap and poly-A tail are also optionally contained in the terminal regions.

The mRNAs described herein can comprise one or morenucieosides comprising a modification relati ve to the naturally occurring nucleoside (a "modified nucleoside" or modified nucleotide"). In some embodiments, such modification, for example, reduce the innate immune response of a cell and/or surrounding tissue into which the mRNA molecule is introduced, impro v e the stability of the mRNA moiecule, improve the efficiency of protein (e.g. , an ADAMTS13 protein, fragment, or variant, encoded by the mRNA of the instant invention) production, improve intracellular retention and/or the half-life of the mRNA molecules, improve viability of contacted cells; and/or reduce immunogenicity. Exemplary modification methods and compositions can be seen in, for example, PCT publication Nos: WO2014081507 and WO2013151664, the entire contents of each of which are herein incorporated by reference.

Two or more linked nucleotides can be inserted, deleted, duplicated, inverted or randomized in the mRNA molecule without significant chemical modification to the mRNA. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, stmctural modifications are indeed chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the

polynucleotide.

In some embodiments, the chemical modifications can be located on the sugar moiety of the mRNA molecule. In other embodiments, the chemical modifications can be located on the phosphate backbone of the mRNA.

In certain embodiments it is desirable to intracelluiarly degrade a modified mRNA molecule introduced into the cell, for example, if precise timing of protein production is desired. Included herein, therefore are modified mRNA molecules containing a degradation domain that is capable of being acted on in a directed manner within a cell.

In some embodiments, more than one mRNA molecules are linked together using a ftmctionaiized linker molecule. A functionaiized saccharide molecule, for example, can be chemically modified to contain multiple chemical reactive groups (SH-, NH₂-, N₃, etc.) to react with the cognate moiety on a 3'-fuiictionalized mRNA molecule (e.g. , a 3'-maleimide ester, 3'-NHS-ester, alkynvl, etc.). The number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conj gated nucleic acid or mRNA.

To further enhance protein production, the mRNA can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. , acridines), cross-linkers (e.g. , psoralene, mitomycin C), poiphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphate, amino acids, PEG (e.g. , PEG-40K), MPEG, [MPEG]₂, radiolabeled markers, enzymes, haptens (e.g. , biotin), transport/absorption facilitators (e.g. , aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins (e.g. , glycoproteins), peptides (e.g. , molecules having a specific affinity for a co-iigand), antibodies (e.g. , an antibody that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell), hormones and hormone receptors, non-peptidic species (such as lipids, lectins, carbohydrates, vitamins and cofactors), or a drug.

Conj ugation can result in increased stability and/or half-life and can be particularly useful in targeting the mRNA molecule to specific sites in the cell, tissue or organism.

The mRN A molecule(s) described herein can be, for example, hi functional or multi-functional, which means the mRNA molecule has or is capable of two functions or multiple functions. The multiple functionalities, structural or chemical, can be encoded by the mRNA (e.g. , the function may not manifest until the encoded product is translated) or can be a property of the mRNA itself. Similarly, Afunctional mRNA molecules can comprise a function that is covalently or electrostatically associated with the mRNA. Further, the two functions can be provided in the context of a complex of a modified RNA and another molecule.

Provided herein is a modified mRNA molecule containing a translatable region and one, two, or more than two different nucleoside modifications. Basic components of an mRNA molecule can include, for example, a 5' cap, a 5'-UTR, a coding region, a 3'-UTR and a poly- A tail. The mRNA moiecule(s) described herein, for example, can undergo capping and/or tailing reactions. A capping reaction can be performed, for example, by methods known in the art to add a 5' cap to the 5' end of the mRNA. Methods for capping include, but are not limited to, using a Vaccinia capping enzyme (New England Biolabs, Ipswich, MA). A poly- A tailing reaction can be performed by methods known in the ait, including, but not limited to, using 2'-0-methyltransferase. Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5'-UTR begins at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3 -UTR begins immediately following the stop codon and continues to the transcription termination signal. Regulatory features of a UTR ca be incorporated into the mRNA molecule(s) described herein. Specific features can also be incorporated to ensure controlled modulation (e.g. , down-regulation nor up-regulation), for example, regulator}^" elements that down-regulate the mRNA are desirable in situations where the mRNA is misdirected to undesired cells, tissues or organs.

Some 5'-UTRs help translation initiation, for example, naturally occurring or endogenous 5'-UTRs. They harbor signatures like Kozak sequences, which facilitate translation initiation by the ribosome for many genes. Kozak sequences havea consensus sequence comprising a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another G. 5'-UTRs also can form secondary structures that are involved in elongation factor binding.

Other non-naturally occurring or exogenous UTR sequences can be incorporated into the 5^!- or 3'-UTR of the mRN A. For example, introns or portions of introns sequences can be incorporated into the flanking regions of the mRNA. Incorporation of intronic sequences can, for example, increase protein production as well as mRNA levels.

3'-UTRs are rich in adenosines and uridines. These AU-nch signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes: Class I AREs (such as those in c-Myc and MyoD) contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of ARE include GM-CSF and TNFa. Class III ARES are less well defined. These U-rich regions do not contain an AUUUA motif. c-Jun and myogenin are two examples of this class. Most proteins binding to the AREs destabilize the messenger, whereas members of the ELAV family, most notably HuR, increase the stability of mRNA. HuR binds to AREs of all the three classes.

Engineering the HuR-speeific binding sites into the 3'-UTR of the mRNA leads to HuR binding and thus, stabilization of the mRNA in vivo.

Introduction, removal or modification of 3'-UTR AREs can modulate the stability of mRNA. When engineering specific mRNA, one or more copies of an ARE can be introduced to make such mRNA less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or altered to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments, for example, can be conducted in relevant cell lines with the mRNA. The produced ADAMTS 13 protein, fragment or variant can be assayed at various time points post-transfection. For example, cells can be transfected with different

ARE-engineering mRNAs and an ELISA kit can be used to detect the expressed ADAMTS 13 protein, fragment, or variant at, for example, 6 hours, 12 hours, 24 hours, 48 hours and/or 7 days post-transfection.

The 5 '-cap structure of an mRNA is involved in nuclear export and mRNA stability in the cell. The cap binds to Cap Binding Protein (CBP), which is responsible for in vivo mRNA stability and translation competency through the interaction of CBP with poly-A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns during mRNA splicing.

The mRNA molecules described herein can be capped to generate, for example, a 5'~ppp~5'~triphosphate linkage. The linkage site is between a terminal guanosine cap residue and the 5'-temiinal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can be methylated to generate an N7 -methyl -guanylate residue. The ribose sugars of the terminal and/or ante-terminal transcribed nucleotides of the S'-end of the mRNA can optionally also be 2'-0-methyiated. 5 -decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRN A molecule, for degradation.

Modifications to the mRNA molecule can generate a non-hydrolyzable cap structure preventing decappmg and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides can be used during the capping reaction. A Vaccinia capping enzyme from New England Biolabs (Ipswich, MA), for example, can be used with a-thio-guanosine nucleotides to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides can be used such as, for example, a-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the mRNA on the

2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from naturally occuring (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure. while still retaining cap function. In some embodiments, cap analogs are chemically or enzymatically synthesized and/or linked to the mRNA. For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3^!-0-dimethyl- guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which may equivalently be designated as 3' 0-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the mRNA, The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped mRNA. Another exemplar}⁷ cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e. , N7, 2'-0- dimethyl-guanosine-5'-triphosphate-5 '-guanosine, m7Gm-ppp-G). Cap structures include, but are not limited to, 5' Triphosphate cap (5'-ppp), Guanosine-triphosphate Cap (5'-Gppp), 5' N7-methylguanosine-triphosphate Cap (5' N7-MeGppp, 7mGppp), 5' Adenylated cap (rApp), 7mG(5')ppp(5')N, pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1 ), and 7mG(5')- ppp(5')NlmpN2mp (cap 2). For a detailed discussion of possibl e 5'-terminal cap structure, see onarska, M. et al. (Cell, 38:731-6, 1984), the entire contents of which are herein incorporated by reference. In certain embodiments, a separate capping enzyme is used to append the 5'-cap, either endogenous or analog. In other embodiments, an mRNA that preferentially incorporates a cap analog during transcription initiation is used to produce a capped mRNA.

5'-terminal caps can include endogenous caps or cap analogs. A 5'-terminal cap can comprise, for example, a guanine analog (e.g. , inosine, Nl-methyi-guanosine, 2 -fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine.

Additional viral sequences such as, for example, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus (PCT Pub. No. WO2012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the 3'-UTR of the mRNA and can stimulate the translation of the mRNA,

mRNA described herein can contain an internal ribosome entry site (IRES), which play s a role in initiating protein synthesis in absence of a 5'-cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more tha one functional ribosome binding site can encode several peptides or polypeptides that are translated independently by the ribosomes

("multicistronic nucleic acid molecules"). When the mRNA is provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used include, without limitation, those from picornaviruses (e.g. , FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) and cricket paralysis viruses (CrPV).

During RNA processing, a long chain of adenine nucleotides (poiy-A tail) can be added to the mRNA molecule, for example, to increase mRNA stability. After transcription, the 3'-end of the transcript can be cleaved to form a free 3'-hydroxyl. A poly-A polymerase, for example, then adds a chain of adenine nucleotides to the mRNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, about 100 and about 250 residues long. In some embodiments, unique poly-A tail lengths provide certain advantages to the mRNA. Generally, the length of a poly-A tail for the mRNA is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g. , at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90,

100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1, 100, 1 ,200, 1,300, 1,400, 1,500, 1,600, 1 ,700, 1 ,800, 1,900, 2,000, 2,500 or 3,000 nucleotides). In some embodiments, the mRNA can comprise a poly-A tail of a length from about 30 to about 3,000 nucleotides (e.g. , from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1 ,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1 ,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1 ,000 to 1 ,500, from 1 ,000 to 2,000, from 1 ,000 to 2,500, from 1 ,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500 or from 2,500 to 3,000.

In some embodiments, the poly-A tail is designed relative to the length of the overall mRNA. This design can be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product (e.g. , an ADAMTS 13 protein, fragment, or variant) expressed from the mRNA.

The poly-A tail can also be designed as a fraction of such mRNA. In this context, the length of the poly -A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% or more of the total length of the mRNA or the total length of the mRNA minus the poly- A tail. Further, engineered binding sites and conjugation of the mRNA for Poly- A Binding Protein (PABP) may enhance expression. mRNA described herein can be linked together to the PABP, for example, through the 3'-end using modified nucleotides at the 3'-terminus of the poly-A tail. mRNA described herein can be designed to include a poly-A-G quartet. The

G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G~rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant mRNA is assayed for its stability, protein production and other parameters, including half-life at various time points. The poly-A-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.

Other RNA modification methods include a combination of nucleotide modifications abrogating mRNA interaction with Toll-like receptor 3 (TLR3), TLR7, TLR8 and retinoid-inducible gene 1 (RIG-1), resulting in low immunogenicity and higher stability in mice ( ormann, M. et al. , Nat. Biotechnol. , 29: 154-7, 2011; the contents of which are incorporated by reference herein in their entirety).

mRNA molecules can be purified after isolating from a cell, a tissue, an organism or chemical synthesis reaction mixture. The purification process can include clean-up, quality assurance, and quality control. The clean-up can be performed, for example, by methods known in the aits such as AGENCOURT¹*' beads (Beckman Coulter Genomics, Danvers,

MA), poly-T beads, LNA™ oligo-T capture probes (EXIQON^® Inc, Vedbaek, Denmark) or HPLC-based purification methods such as, for example, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in relation to a polynucleotide such as a "purifi ed mRN A" refers to one that is separated from at least one contaminant. As used herein, a ^"'contaminant" is any substance that makes another unfit, impure or inferior. In certain embodiments where an altered mRNA is being purified (e.g., a modified mRNA, a capped mRNA, a truncated or conjugated mRNA, etc. ), the unaltered form of the mRNA can be considered a contaminant. Thus, a purified polynucleotide (e.g. , mRNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

A quality assurance and/or quality control check can be conducted using methods such as, for example, gel electrophoresis, UV absorbance or analytical HPLC. In another embodiment, the mR A molecule of the instant invention may be sequenced by methods including, but not limited to, reverse-transcriptase-PCR.

in one embodiment, the mRNA molecule can be quantified using methods such as, for example, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANQDROP¹* spectrometer (ThermoFisher, Waltham, MA). The mRNA molecule can be analyzed, for example, to determine if the mRNA is of a desired or proper size or if any degradation has occurred. Degradation of th e mRNA can be checked by methods such as, for example, agarose gel electrophoresis, HPLC -based purification methods (e.g. , strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC

(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)), liquid chromatography mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis

(CGE).

mRNA described herein can be quantified and/or delivered, for example, in exosomes, e.g. , derived from one or more bodily fluid(s), e.g. , peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveoiar lavage fluid, semen, prostatic fluid, cowper s fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes can be retrieved from an organ such as, for example, lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver and/or placenta.

In certain quantification method(s), a sample of mRNA solution is obtained and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfiuidic separation or combinations thereof. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease

biomarker. The assay can be performed using constract-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, while the exosomes can be isolated using immunohistochemical methods, such as enzyme-linked immunosorbent assay (ELISA) methods. Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfiuidic separation, or combinations thereof.

These methods afford an investigator the ability to monitor, in real time, the level of mRNA remaining or delivered. To further improve the sensitivity of such monitoring, analysis, or quantification of the mRNA in a host, mRNA can be engineered to include one or more structural or chemical modification(s) to distinguish it from endogenous mRNAs.

Modifed nucleosides that can be incorporated into the mRNAs described ehrien include, for example, a modification to a uridine (U), a cytidine (C), an adenine (A) or guanine (G). The modified nucleoside can be for example, m⁵C (5-methyl cy tidine), m⁶A (N6-methyladenosine), s U (2-thiouridien), ψ (pseudouri dine), or Urn (2'-0-methyluridine).

Some exemplary chemical modifications of nucleosides that can be used to modify the mRNA described herein include, but are not limited to, pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyIuridine, 5 -carboxymethyl -uridine, 1 -carboxymethyi- pseudouridine, 5-propynyl-uridine, 1 -propynyl -pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5 -taurinomethyl-2-thio- ridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l -methyl-pseudouridine, 2-thio- l -methyl - pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio-l -methyl-l -deaza-pseudouridine, dihydrotiridme, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy uridine, 2-methoxy-4-thio-uri dine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidme, 5- formylcytidine, N4-methyl cytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl -1 -deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy -pseudoisocytidine, 4-methoxy-l -methyl- pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyl adenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2 -methyl thio-N6-(cis~ hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N⁶- threonylcarbamoyiadenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N^b,N°- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosme, 1- methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methyl guanosine, N²- methylguanosine, N ,N²-dimethyl guanosine, 8-oxo-guanosine, 7-methyi-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N²-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In another embodiment, the modifications are independently selected from the group consisting of 5 -methyl cytosine, pseudouridine and l -methylpseudouridine.

In some embodiments, the modified nucleobase in the mRNA molecule is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include, for example, pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5 -hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridme (e.g., 5-iodo- uridine or 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5- oxy acetic acid (cmo³U), undine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl- uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm'U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcnrU), 5-methoxycarbonylmethyl-2-thio-uridine (mcnrs2U), 5-aminomethyl -2-thio- uridine (nm⁵s2U), 5-methylaminomethyl -uridine (mnm⁵U), 5-meihylaminomeihyl-2-thio- uridine (mnm⁵s2U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se U), 5- carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5- carboxymethylaminomethyl -2-thio-uridine (cmnm⁵s2U), 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyl-uridine (xcm⁵U), 1 -taurinomethyl -pseudouridine, 5- taurinomethyi-2-thio-uridine (rm⁵s2U), l-tam"inomethyl-4-thio-pseudouridine, 5-methyl- uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1 -methyl -pseudouridine (m ), 5- methyl-2-thio-uridine (m^,s2U), l-methyl-4-thio-pseudouridine (^ηι¹3⁴ψ), 4-thio- 1-methylpseudouridine, 3 -methyl-pseud ouri dine (^ητ'ψ), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1- deaza-pseudouridine, 2-thio-l -methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (rn⁵D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp³U), 1 -methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp^), 5-(isopentenylaminomethyl)uridine (inn U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), . alpha. -thio-uri dine, 2'-0-niethyi-uridme (Um), 5,2'-0-dimethyl-uridine (ra⁵Um), 2'-0-methyl-pseudouridine (fin), 2-thio-2'-0- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcm⁵Um), 5- carbamo}⁷lmethyl-2'-0-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2'-0- methyl-uridine (cmnm⁵Um), 3,2'-0-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)- 2'-0-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F- uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxy vinyl) uridine, and 5-[3-(l-E- propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include, for example, 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5- formyl-cytidine (f ), N4-methyl-cytidine (rn⁴C), 5-methyl-cytidine (m^C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyi-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidiiie, 4-thi o-pseudoisocytidine, 4-thio- 1 -methyl-pseudoi socyti dine, 4-thi o- 1 -methyl - 1 -deaza- pseudoisocytidine, 1 -methyl-] -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyi-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy -pseudoisocytidine, 4-methoxy-l -methyl- pseudoi socyti dine, lysidine (k C), . alpha, -thio-cyti dine, 2'-0-rnethyl-cytidine (Cm), 5,2 -0- dimethyl-cytidine (m⁵Cm), N4-acetyl-2'~0-methyl-cytidine (ac⁴Cm), N4,2'-0-dimethyl- cytidme (m⁴Cm), 5-formyl-2'-0-methyl-cytidine (f'Cm), N4,N4,2'-0-trimethyl-cytidine (m⁴ ₂Cm), 1 -thio-cyti dine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include, for example, 2-amino- purine, 2,6-diaminopunne, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza~8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza~8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyi-adenosiiie (m' A), 2-methyl- adenine (n A), N6-methyl-adenosine (m^uA), 2-methylthio-N6-methyl-adenosine (ms2m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis- hydroxyisopentenyl)adenosine (io°A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io^bA), N6-glycinylcarbamoyl-adenosine (g.sup.6A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl -adenosine (m°t⁶A), 2-methylthio-N6- threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A), N6- hydroxynorvalylcarbamoyl-adenosine (hn^bA), 2-methylthio-N6-hydroxynorvalylcarbamoyl- adenosine (ms2hn°A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, . alpha. -thio-adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl- adenosine (m⁶Am), N6,N6,2'-0-trimethyl -adenosine (m⁶ ₂Am), l,2'-0-dimethyl-adenosine (n^Am), 2'-0-ribosyl adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio- adenosine, 8-azido~adenosine, 2'-F-ara-adenosine, 2 -F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucieobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include, for example, inosine (I), 1- methyi-mosine (m¹!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyWy), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (oQ), galactosyl -queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQ , archaeosine (G⁺), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl -guanosine (m⁷G), 6-thi o-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (m^lG), N2-methyl-guanosine (m^'G), N2,N2- dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²''G), N2, N2,7-dimethyl-guanosine (m^{2 7}G), 8-oxo-guatiosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6- thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, alpha, -thio-guanosine, 2 -O-methyl- guanosine (Gm), N2-methyl-2'-0-methyl-guanosine (m²Gm), N2,N2-dimethyl-2'-0-methyl- guanosine (m²?.Gm), l-methyl-2'-0-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl- guanosine (m²'⁷Gm), 2'-0-methyl -inosine (Im), l,2'-0-dimethyl-mosine (m^m), 2^!~0~ ribosyiguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methyl -guanosine, 2'-F-ara- guanosine, and 2'-F-guanosine.

The nucieobase of the nucleotide, for example, can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. The nucieobase, for example, can be independently selected from adenine, cytosine, guanine, uracil or hypoxanthine. In another embodiment, the nucieobase can also include, for example, naturally occurring and synthetic derivatives of a base, including, for example, pyrazolo[3,4-d]pyrimidines, 5 -methyl cytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyi and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haIo (e.g., 8-bromo), 8-amino, 8-thioi, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3- deazaadenine, pyrazolo[3,4-d]pyrimidme, imidazo[1 ,5-a] 1 ,3,5 triazinones, 9-deaza urines, irrudazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1 ,2,4-triazine, pyndazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and/or derivatives thereof, e.g. , A includes adenine or adenine analogs.

Other modifications include those described in U. S. Patent No. 8,835,108, U.S. Patent Application Publication No. 20130156849; Tavernier, G. et al. , J. Control. Release,

150:238-47, 201 1 ; Anderson, B, et al , Nucleic Acids Res., 39:9329-38, 201 1 ; Pascolo, S,, Methods Mol. Med, 127:23-40, 2006; Kariko, K. et al , Mol Ther., 16: 1833-40, 2008;

Kariko, K. et al , Immunity, 23: 165-75, 2005; and Warren, L. et al., Cell Stem Cell, 7:618-30, 2010. Every possibility exemplified in the instant disclosure and/or cited in the references represents a separate embodiment of modification of the mRNA described herein. Compositions

mRNA described herein can be delivered into a host, such as a mammal {e.g. , a human), to express a protein of interest (e.g. , an ADAMTS 13 protein, fragment, or variant).

The mRNA can comprise at least one ex on of the protein of interest for in vivo expression.

Optionally, the mRNA can have at least one of the introns of the protein of interest or another protein to facilitate gene expression.

Delivery

Systemic delivery of chemically modified messenger RNA (mRNA) as an alternative to plasmid DNA (pDNA) is described herein. Modified mRNA evades recognition by the innate immune system and is less immunostimuiatory than dsDNA or unmodified mRN A. Moreover, the cytoplasmic delivery of mRN A circumvents the nuclear envelope, which results in a higher gene expression level. When formulated in the nanoparticle formulation liposome-protamine-RNA (LPR), modified mRNA shows increased nuclease tolerance and is more effectively taken up by tumor cells after systemic administration (Wang, Y. et al. , Mol. Ther. , 21 : 358-367, 2013, the contents of which are incorporated by reference herein in their entirety).

mRNA described herein can be delivered by multiple methods to the host organism. For disclosures on such delivery systems, please see PCT publication Nos: WO2013185069, WO2012075040, and WO2011068810, the content of which are incorporated by reference herein in their entirety. In some embodiments, the mRNA is delivered to at least one human tissue or organ (such as, liver, muscle, lung, etc.).

The use of lipid carrier vehicles to facilitate the deliver}_' of nucl eic acids to target cells, as discussed in PCT publication No: WO2013185069, is described herein. Lipid carrier vehicles (e.g., liposomes and lipid-derived nanoparticles) are generally useful in a variety of applications in research, industry, and medicine, particularly for their use as transfer vehicles of diagnostic or therapeutic compounds in vivo (Lasic, D., Trends Biotechnol., 16:307-21, 1998; Drummond, D. et al., Pharmacol. Rev. , 51 :691-743, 1999) and are usually

characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains.

A lipid carrier vehicle can transport mRNA to a target cell (e.g. , a liver cell, e.g. , an hepatocyte) or tissue or organ (e.g., liver). Administration of mRNA, which is encapsulated within a lipid carrier vehicle, results in delivery of mRNA and/or the protein to desired ceil(s0 or tissues. The liposomal transfer vehicles can be prepared, for example, to contain a desired nucleic acid for a protein of interest (e.g., ADAMTS 13 protein, fragment or variant thereof). The process of incorporation of a desired entity (e.g. , a nucleic acid) into a liposome is referred to as "loading" (Lasic. D. et al. , FEBS Lett., 312:255-8, 1992). The liposome-incorporated nucleic acids can be completely or be partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is referred to herein as "encapsulation," wherein the nucleic acid is entirely contained within the interior space of the liposome. The purpose of incorporating an mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment that may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, the selected transfer vehicle is capable of enhancing the stability of the mRNA contained therein. The liposome can allow the encapsulated mRNA to reach the target cell and'Or can allow the encapsulated mRNA to reach a desired target cell, tissue or organ.

In some embodiments, the compositions provided herein are capable of modulating the expression of an AD AMI^'S 13 protein, fragment, or variant by increasing the level/amount of the mRNA encoding the ADAMTS13 protein, fragment, or variant in a target cell or tissue. mRNA deliver}⁷ into a mammalian organism for protein production is a technique known in the art. In some embodiments, the mRNA described herein is more stable {e.g., has limited or reduced nuclease susceptibility) in the composition described herein compared to a wild-type and/or endogenous version of the mRNA. In some embodiments, the mRNA comprises one or more modifications and/or amino acid substitutions that confer m vivo stability (e.g. , by increasing the half-life) to the mRNA. In other embodiments, the mRNA comprises one or more modifications and/or amino acid substitutions that correct a defect implicated in an associated aberrant expression of the endogenous ADAMTS13.

As used herein, the term "target cell" refers to a cell or tissue to which a composition is to be directed or targeted. In some embodiments, the target cells are deficient in a protein or enzyme of interest. For example, where it is desired to deliver a nucleic acid to a

hepatocyte, the hepatocyte represents the target cell. In some embodiments, the methods, nucleic acids and compositions described herein transfect the target cells in a specific and selective manner (i.e. , do not transfect non-target cells, or, only on a limited or reduced basis). mRNA, therefore, can be produced and formulaterd to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells,

hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stern cells, mesenchymal cells, neural cells (e.g. , meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g. , rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining ceils, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor ceils.

The compositions comprising the mRNA can be administered and dosed in accordance with reasonable medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The "effective amount" for the purposes herein may be determined by such relevant

considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient expression of the ADAMTS 13 protein, fragment, or variant in the target cell.

The route of delivery used in the methods described herein allows for noninvasive, self-administration of the therapeutic compositions of the mRNA. The method(s) involve, for example, intratracheal or pulmonary administration by aerosolization, nebulization, or instillation of compositions comprising the mRNA in a suitable transfection or lipid earner vehicles as described herein.

Although the local cells and tissues of the liver represent a potential target capable of funciioning as a biological depot or reservoir for production and secretion of the ADAMTS 13 protein, fragment or variant, encoded by the mRNA described herein, other administration of the composition comprising the mRNA to a desired tissue or organ, for example, via aerosolization, nebulization, or instillation results in the distribution of the expressed

ADAMTS 13 protein, fragment or variant. Without wishing to be bound by any particular theory, it is contemplated that nanoparticle compositions of the mRNA can pass through the lung airway-blood barrier and result in translation of the intact nanoparticle to cells and tissues, such as, for example, the heart, the liver, the spleen, where it results in the production of the encoded ADAMTS 13 protein, fragment or variant in these non-lung tissues.

The compositions and methods described herein are useful, for example, for the management and treatment of a large number of diseases, and in particular diseases associated with or caused by unregulated vWF. In certain embodiments, the compositions comprising the mRNA are distributed in encapsulated nanoparticles and produce the encoded ADAMTS 13 protein, fragment, or variant in the liver, spleen, heart, and/or other cells, tissues or organs. For example, administration of the compositions, e.g. , a nanoparticle comprising the mRNA, by aerosolization, nebulization, or instillation to the lung results in the composition itself and its protein product (e.g. , the encoded ADAMTS 13 protein, fragment, or variant) being detectable in both the local ceils and tissues and elsewhere, e.g., lung, depending on the route of administration. If desired, peripheral target cells, tissues and organs can express the ADAMTS 13 protein, fragment or variant as well.

Following administration of the composition to the subject, the ADAMTS 13 protein, fragment or variant encoded by the mRNA is detectable in the target tissue(s) for at least about one to seven days or longer following administration of the composition to the subject. The amount of expressed ADAMTS 13 protein, fragment, or variant necessary to achieve a therapeutic effect vanes depending on the condition being treated and the condition of the patient. For example, the expressed ADAMTS 13 protein, fragment, or variant may be detectable in the target tissues at a concentration (e.g. , a therapeutic concentration) of at least 0.025-1.5 pg/mL (e.g., at least 0.050 pg/mL, at least 0.075 pg/mL, at least 0.1 ug/mL, at least 0.2 pg/mL, at least 0.3 pg/mL, at least 0.4 pg/mL, at least 0.5 pg/mL, at least 0.6 pg/mL, at least 0.7 pg/mL, at least 0.8 pg/mL, at least 0.9 pg/mL, at least 1.0 pg/niL, at least

1.1 pg/mL, at least 1.2 pg/mL, at least 1.3 pg/mL, at least 1.4 pg/mL, or at least 1.5 pg/mL), for at least about ! . 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or 45 days or longer following administration of the composition to the subject.

In certain embodiments, the compositions described herein can be formulated such that they are delivered as a particulate liquid or solid prior to or upon administration to a subject. Such compositions can be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, for example, an aerosolized aqueous solution or suspension) to generate particles that are easily mhaiable by the subject. In other embodiments, compositions are formulated to allow for intravenous or subcutaneous dosing of the mRNA. In some embodiments, the compositions described herein are administered to a subject such that a concentration of at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 3.0 mg/kg, at least 4.0 mg/kg, at least 5.0 mg/kg, at least 6.0 mg/kg, at least 7.0 mg/kg, at least 8.0 mg/kg, at least 9.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 rag/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, or at least 100 mg/kg body weight is administered in a single dose. In some embodiments, the compositions described herein are administered to a subject such that a total amount of the dose is at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 nig, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 5 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg.

Pharmaceutical Compositions and Formulations

The compositions of mRNA described can be formulated as a pharmaceutical solution, e.g. , for administration to a subj ect for the treatment or prevention of a disease or disorder with misregulated or unregulated v WF functions. The pharmaceutical compositions can include a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g. , an acid addition salt or a base addition salt (Berge, S. et al. , J. Pharm. Sci., 66: 1 -19, 1977).

Compositions can be formulated according to standard methods. Pharmaceutical formulation is an established art (Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20^lh Edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999) "Pharmaceutical Dosage Forms and Drug Deliver}' Systems," 7^l!l Edition, Lippincott Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000) "Handbook of Pharmaceutical Excipients American Pharmaceutical Association," 3^fd Edition (ISBN:

091733096X)). In some embodiments, a composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage at 2-8C (e.g. , 4C). In some embodiments, a composition can be formulated for storage at a temperature below 0C (e.g. , -20C or -80C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g. , one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, 1 ½ years, or 2 years) at 2-8C (e.g., 4C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8C (e.g., 4C).

Compositions can be formulated for administration by a parenteral mode (e.g. , intravenous, subcutaneous, intraperitoneal, or intramuscular injection). "Parenteral administration," "administered parenteral ly" and other grammatically equivalent phrases, as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

Compositions can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.

Compositions can also be formulated in immunoliposome compositions. Such formulations can be prepared, for example, by methods known in the art (Eppstein, D. et al. , Proa Natl. Acad. Set USA, 82:3688-92, 1985; Hwang, K. et al, Proa Natl. Acad. Set USA, 77:4030-4, 1980; and U.S. Patent Nos. 4,485,045 and 4,544,545; the entire contents of each of which are herein incorporated by reference). Liposomes with enhanced circulation time are disclosed, for example, in U.S. Patent No. 5,013,556, the entire contents of which are herein incorporated by reference.

in certain embodiments, compositions can be formulated with a carrier that protects the mRNA against rapid release, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyiactic acid (J.R. Robinson (1978) "Sustained and Controlled Release Drug Deliver}' Systems," Marcel Dekker, Inc., New York).

In some embodiments, compositions can be formulated in a composition suitable for intrapulmonary administration (e.g. , for administration via an inhaler or nebulizer) to a mammal such as a human (U.S. Patent Application Publication No. 20080202513; U.S. Patent Nos. 7,112,341 and 6,019,968; and PCX Publication Nos. WO 00/061178 and WO 06/122257, the disclosures of each of which are incorporated herein by reference in their entirety).

In some embodiments, compositions can be administered locally, for example, by way of topical application or intravitreal injection. For example, in some embodiments, the compositions can be formulated for administration by way of an eye drop. The therapeutic preparation for treating the eye ca contain one or more active agents in a concentration from about 0.01 to about 1% by weight, preferably from about 0.05 to about 0.5% in a

pharmaceutically acceptable solution, suspension or ointment. Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like. Suitable buffers include, e.g., boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, and sodium biphosphate, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, and sodium chloride.

Suitable antioxidants and stabilizers for use in compositions and formulations described herein include, for example, sodium bisulfite, sodium metabi sulfite, sodium thiosulfite, and thiourea. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydiOxyethyiceliulose,

hvdroxymethylpropylceiluiose, lanolin, raethylcellulose, petroiatura, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethylcellulose.

As described above, relatively high concentration compositions can be made. For example, the compositions can be formulated at an mRNA concentration between about 10 mg/mL to 100 mg/mL (e.g. , between about 9 mg/mL and 90 mg/mL; between about 9 mg/mL and 50 mg/mL; between about 10 mg/mL and 50 mg/mL; between about 15 mg/mL and 50 mg/mL; between about 15 mg/mL and 110 mg/mL; between about 15 mg/mL and 100 mg/mL; between about 20 mg/mL and 100 mg/mL; between about 20 mg/mL and 80 mg/mL; between about 25 mg/mL and 100 mg/mL; between about 25 mg/mL and 85 mg/mL; between about 20 mg/mL and 50 mg/mL; between about 25 mg/mL and

50 mg/mL; between about 30 mg/mL and 100 mg/mL; between about 30 mg/mL and 50 mg/mL; between about 40 mg mL and 100 mg mL; or between about 50 mg/mL and 100 mg/mL). In some embodiments, compositions can be formulated at a concentration of greater than 5 mg/mL and less than 50 mg/mL. Methods for formulating a protein in an aqueous solution are known in the art and are described in, for example, U.S. Patent No. 7,390,786; McNally and Hastedt (2007), "Protein Formulation and Delivery," Second Edition, Drugs and the Pharmaceutical Sciences, Volume 175, CRC Press; and Banga (2005), "Therapeutic peptides and proteins: formulation, processing, and delivery systems, Second Edition" CRC Press.

In some embodiments, the aqueous solution has a neutral pH, e.g. , a pH between, e.g. , 6.5 and 8 (e.g. , between and inclusive of 7 and 8). In some embodiments, the aqueous solution has a pH of about 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4. 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. In some embodiments, the aqueous solution has a pH of greater than (or equal to) 6 (e.g. , greater than or equal to 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7,9), but less than pH 8.

In some embodiments, compositions can be formulated with one or more additional therapeutic agents. When compositions are to be used in combination with a second active agent, the compositions can be co-formulated with the second agent or the compositions can be formulated separately from the second agent formulation. For example, the respective pharmaceutical compositions can be mixed, e.g. , just prior to administration, and

administered together or can be administered separately, e.g. , at the same or different times.

Methods for Treatment

The compositions described herein can be administered to a subject, e.g. , a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, or intramuscular injection (TM).

Administration ca be achieved, for example, by local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous or gelatinous material, including membranes, such as siaiastic membranes or fibers. The implant can be configured for sustained or periodic release of the composition to the subject (U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795;

EP488401 ; and EP 430539, the disclosures of each of which are incorporated herein by- reference in their entirety). A composition described herein can be delivered to the subject by way of an implantable device based on, e.g. , diffusive, erodible, or convective systems, e.g. , osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems or electromechanical systems.

In some embodiments, a composition is delivered to a subject by way of local administration. As used herein, 'local administration" or 'local delivery" refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system. For example, the composition can be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. Following local administration in the vicinity of a target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site. In some embodiments, the compositions are distributed in unit dosage form, which can be particularly suitable for self-administration. A formulated product described herein can be included within a container, typically, for example, a vial, cartridge, prefiiled syringe or disposable pen. A doser such as the doser device described in U.S. Patent No. 6,302,855 can also be used, for example, with an injection system. An injection system can employ a delivery pen as described in U.S. Patent No. 5,308,341. Such devices can comprise at least one injection needle (e.g. , a 31 gauge needle of about 5 to 8 mm in length), are typically pre-filied with one or more therapeutic unit doses of a therapeutic solution comprising an mRNA described herein, and are useful for rapidly delivering the solution to a subject with as little pain as possible.

The present disclosure also includes controlled-release or extended-release formulations suitable for chronic and/or self-administration of a composition comprising an mRNA described herein. The various formulations can be administered to a patient in need of treatment as a bolus or by continuous infusion over a period of time.

In some embodiments, a high concentration composition comprising an mRNA described herein is formulated for sustained-release, extended-release, timed-release, controlled-release or continuous-release administration. In some embodiments, depot formulations are used to administer the composition to the subject in need thereof. In this method, the composition is formulated with one or more carriers providing a gradual release of active agent over a period of a number of hours or days. Such formulations are often based upon a degrading matrix that gradually disperses in the body to release the active agent.

A pharmaceutical solution can include a therapeutically effective amount of a composition comprising an mRNA described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. based, in part, on the effect of the administered composition, or the combinatorial effect of the composi tion and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of a composition described herein can also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition (and one or more additional active agents) to elicit a desired response in the individual, e.g., amelioration of at least one condition parameter, e.g. , amelioration of at least one symptom of the disease or disorder with misregulated or unregulated vWF functions. For example, a therapeutically effective amount of a composition described herein ca inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a particular disorder, and/or any one of the symptoms of the particular disorder known in the art or described herein. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. Suitable human doses of any of the compositions described herein can further be evaluated by methods known in the art (van Gurp, E, et αί , Αιη. J. Transplant, , 8: 171 1-8, 2008; Hanauske, A, et al, Clin. Cancer Res. , 13:523-31, 2007; and Hetherington, S. e\ ' al , Antimicrob. Agents Chemother. , 50:3499-500, 2006).

The terms "therapeutically effective amount" or "therapeutically effective dose," or similar terms used herein are intended to mea an amount of an agent (e.g. , mRNA) that elicits a desired biological or medical response (e.g. , an improvement in one or more symptoms of a disease or disorder with misregulated or unregulated vWF functions). In some embodiments, a pharmaceutical solution described herein contains a therapeutically effective amount of at least one of said compositions. In some embodiments, the solutions contain one or more compositions and one or more (e.g. , two, three, four, five, six, seven, eight, nine, 10 or 1 1 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the composition described herein lies generally within a range of circulating conce trations of the compositions that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a composition described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5₀ (/. «· . the concentration of the ADAMTS13 protein, fragment or variant that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. In some embodiments, cell culture or animal modeling can be used to determine a dose required to achieve a therapeutically effective concentration.

As used herein, a subject "in need of prevention," "in need of treatment," or "in need thereof," refers to one, who by the judgment of an appropriate medical practitioner (e.g. , a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment.

The term "preventing" is art-recognized, and when used in relation to a condition, is well understood in the art, and includes administration of a composition that reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject that does not receive a composition described herein. Thus, prevention of a disease or disorder with misregulated or unregulated vWF functions such as, for example, TTP includes, for example, reducing the extent or frequency of coughing, wheezing, or chest pain in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the occurrence of coughing or wheezing in a treated population versus an untreated control population, e.g. , by a statistically and/or clinically significant amount.

As described herein, the compositions of mRNA can be used to treat a variety of diseases or disorders related to misregulated or unregulated v WF functions, such as, for example, thrombotic thrombocytopenic purpura (TTP) or other thrombotic diseases, including disseminated intravascular coagulation (DIC), hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), pulmonary embolism, cerebral infarction and systemic lupus eiythematosus (SLE). In some embodiments, TTP may be a congenital TTP or an acquired TTP. In some embodiments, a composition described herein is administered to a subject to treat, prevent or ameliorate at least one symptom of a disease or disorder related to misregulated or unregulated vWF functions in a subject.

Monitoring a subject (e.g. , a human patient) for an improvement in a disorder as described herein includes evaluating the subject for a change in a disease parameter, e.g. , an improvement in one or more symptoms of a given disorder. In some embodiments, the evaluation is performed, for example, at least I, 2, 4, 6, 8, 12, 24 or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration of a composition described herein. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g. , evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g. , adding or dropping any of the treatments for a disorder described herein.

All references cited in the present disclosure are incorporated by reference herein in their entirety. The following examples are meant to illustrate, not to limit, the disclosure. EXAMPLES

EXAMPLE 1. In vitro Expression of AD AMTS 13

mRNA expression of AD AMTS 13 was tested in HeLa cells and primary hepatocytes

(grown in each well of 24-well plates). HeLa cells were transfected with 1.25 μ§ mRNA (encoding FLAG- AD AMTS 13 as set forth in SEQ ID NO: l encoded at the C-terminus of

SEQ ID NO: 4) using a 1 :2 ratio of Lipofectamine*' 2000 (Life Technologies, Carlsbad, CA).

For HeLa transfections, 150 LIL Opti-MEM (Life Technologies) medium (pre-warmed to

37C) were mixed with 7.5 \iL Lipofectamine 2000. In a different tube, 37.5 μ.Ι_, of 100 ng^L

ADAMTS13 mRN A were mixed with 187.5 μί Opti-MEM and incubated for 5 minutes. The mRNA mixture was mixed with the Lipofectamine 2000 mixture and incubated at room temperature for >15 minutes to form complex. 125 μΐ, of the mixture were then added to 3 wells of cells and incubated for 24 hours. The same was repeated with the

FLAG-ADAMTS13 mRNA. IV AL primary hepatocytes were transfected with 2.5 μg mRNA (same as above) using a 1 :6 ratio of mRNA-IN™ (Amsbio, Cambridge, MA). For hepatocytes two hours before transfection, the culture medium was exchanged with 500 uL HIM (hepatocyte induction medium, pre-warmed in water bath). For transfections, 144 μΐ. Opti-MEM^ (Life Technologies) medium (pre-warmed to 37C) was mixed with 6 μί, mRNA-IN™. In a different tube, 25 μΐ. of 100 ng/μΐ, FLAG- AD AMTS 13 mRNA were mixed with 125 JJL Opti-MEM and incubated for 5 minutes. The mRNA mixture was mixed with the mRNA-IN™ mixture and incubated at room temperature for >15 minutes to form complex. Three hundred microliters of the mixture were then added to each well of cells and incubated for 24 hours.

Ceils were then lysed in RIPA buffer (protease tablet added). First, HeLa ceils were washed with PBS and collected via trypsination. The three treatment wells were pooled and the washed cell pellet was lysed in 100 μΐ. RIPA. For hepatocytes, the medium was removed and saved, each well was washed gently with 1 mL of PBS, then 100 μΐ, RIPA was added to each well, followed by scrapping the ceils using routine methods. The ceil lysates were concentrated using a Pierce concentrator at 15,000 rpm for 10 minutes (4C). Protein concentration was calculated, and 30 μg of total protein were loaded for Western blot.

Ceil lysate and ceil culture media were used for Western blots to test if FLAG-tagged

ADAMTS 13 was expressed. Twenty microliters of 30 ug total protein (for control, only 5 μg of total protein was added from the lysates of cells expressing the FLAG-tag) for cell lysate and 20 ₍uL of 20 ng total protein for cell culture media were individually mixed with 20 μΐ, of 2x Laemmli buffer and heated to 95C for 10 minutes. The 40 μΐ. of sample mixture was loaded onto a 4-12% Bis Tris PAGE gel and run at 200V for 40 minutes. The gel was transferred to a nitrocellulose membrane using iBlot2 for 7 minutes. The membrane was blocked for 1 hour at room temperature with Li-Cor blocking buffer. Primary anti-FLAG antibody (at a 1 :500 dilution) was added to the membrane in blocking buffer (with 0.1% Tween 20) and left overnight shaking at 4C. The blots were then placed in Snap ID and washed 4x with 30 mL of TBS + 0.1% Tween 20. Rabbit GAPDH (a housekeeping protein in cell lysates used as a loading control) antibody (diluted 1 : 1666) was added and incubated for 10 minutes. After washing the membrane 4* with 30 mL TBS (with 0.1% Tween 20), mouse and rabbit secondary antibodies ( 1 : 1400) were added and incubated for 10 minutes. The membrane was further washed 4x with 30 mL TBS (with 0. 1% Tween 20) and one wash of 30 mL TBS The membrane was air dried and visualized using a Li-Cor Odyssey ^~' imager.

As shown in FIG. 1 , FLAG-ADAMTS 13 was expressed (the boxed band) in both HeLa (FIG. 1 A) and human primary hepatocyte (FIG. IB). Consistent with references, ADAMTS13 (with a predicted molecular weight of 153 kDa) ran at about 190 kDa on SDS-PAGE, as a result of glycosylation.

EXAMPLE 2. In vitro Activity of ADAMTS13

Both FLAG-AD AMTS 13 (SEQ ID NO: ! added to the C-termmus of SEQ ID NO:4) and non-tagged wild-type AD AMTS 13 (SEQ ID NO: 4) were expressed in HeLa cells and primary human hepatocytes. The expressed proteins were tested for activity with a

Sensolyte* 520 activity assay kit (AnaSpec, Fremont, CA). Samples were run according to the manufacturer's protocol B for biological samples with some minor modifications.

Specifically, 20 uL of cell lysate was mixed with 30 μΐ, of reaction buffer, while 40 uL of ceil culture media was mixed with 10 μί, of reaction buffer as control. The resulting 50 iL of mixture was incubated for 10 minutes and then further mixed with 50 μί, of vWF73 FRET peptide substrate. Both positive (recombinant rhADAMTS13) and negative (rhADAMTS plus its inhibitor, 1,10-Phenanthroline, IC5₀ = 206 uM) were used. Upon substrate addition, ADAMTS13 cleaved the vWF73 FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence, while Ex/Em = 490/520 nm. Each plate was read in the kinetic mode at 60, 150 and 210 minute time points and read again in next day. A plot of each reading is provided in FIG. 2. The absolute reading results (in RFU) were further compared in FIGS. 3-6. HeLa cells clearly secrete active AD AMTS 13 (either tagged or non-tagged) into the culture media (lanes 16 and 18 in FIGS. 3-6), indicating high levels of expression from the mRNA constructs. Primary hepatocytes secreted the expressed wild-type ADAMTS .13, which shows activity over time (lane 14 in FIGS. 3-6). However, the

FLAG-ADAMTS13 only expressed in low levels in hepatocytes, possibly due to a negative effect of the FLAG-tag, but yet to be determined. ADAMTS13 activity was observed in HeLa ceil lysates with increased activity over time (lanes 8 and 10 in FIGS. 3-6).

EXAMPLE 3. In vivo Expression of ADAMTS13

Both FL AG- AD AMTS 13 (SEQ ID NO: l added to the C-terminus of SEQ ID NO:4) and non-tagged wild type ADAMTS13 (SEQ ID NO: 4) were tested for expression in C57bl/6 mice. Sixteen groups of animals (fi ve in each group) were used as outlined in Table 1.

Table 1 Study design for AD AMTS 13 expression

0 ADAMTS 13-WT 1.5

P ADAMTS 13-FLAG 1.5

Results for Groups A, B, G, H, O and P were analyzed. Specifically, at the corresponding terminal time points, 100 mg of liver tissue was lysed in RIP A buffer with protease inhibitor. After centrifugation at 12,000 rcf for 10 rain at 4C, the corresponding supernatant was aspirated into a clean tube. Protein concentration was measured using

DirectDetect Spectrometer (EMD Millipore, Billerica, MA). PAGE and Western blot were performed with 30 ,ug protein loaded with ADAMTS 13 specific antibody (#AP14()85 from Acns Antibodies, San Diego, CA).

As shown in FIG. 7, ADAMTS 13 expression in liver tissue iy sates (the right side of the graph showing the result for Group G and Group O mice) was significantly increased compared to vehicle groups (the left side of the graph showing the result for Group A and Group B) (P<0.5). FIG. 8 illustrates the quantitative result of FIG. 7, showing increased expression of ADAMTS 13 at least at 24 hours post injection (panel A).

FLAG- AD AMTS 13 imniunihistoeheinistry (IHC) was performed. Mouse liver tissues were fixed in 10% neutral buffered formalin (NBF) for 48 hours. After tissue processing, liver tissues were embedded in paraffin blocks. Slides were cut into 5 μηι thickness. Anti-FLAG antibody (#14793 from Cell Signaling Technology, Danvers, MA) was used at 1 :250 dilution. Irnmunohistochemistry was performed on Leica Bond Rx. Briefly, after de-paraffinization and blocking with 10% normal horse serum (NHS), primary antibody was applied for 30 minutes and processed with polymer and D AB detection. Slides were viewed under 40 magnification on the Olympus CX41 microscope using bright field. As shown in FIG. 9, ADAMTS 13 expression was detected by FLAG IHC and observed in hepatocytes 24 hours post injection, especially around liver blood vessels. Increased AD AMTS 13 expression was also observed in liver sinusoid.

While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particul ar situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

Claims

CLAIMS What is claimed is:

1. A composition that provides for the expression of an ADAMTS13 protein or a

biologically active fragment or variant thereof, in a target cell wherein said composition comprises a messenger RNA (mRN A) molecule comprising at least one modification.

2. The composition of Claim 1, wherein the modification increases the stability of the mRNA molecule in vivo.

3. The composition of Claim 1, wherein the mRNA is formulated in a pharmaceutically acceptable carrier.

4. The Composition of Claim 3, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle.

5. The composition of Claim 1, wherein the mRNA molecule comprises a first region comprising at least one open reading frame encoding the ADAMTS 13 protein or biologically active fragment or variant thereof.

6. The composition of Claim 5, further comprising:

a) a flanking region located at the 5' terminus of the first region, comprising: i) a physiological 5' UTR of the ADAMTS 13 protein or a biologically active fragment or variant thereof; and

ii) at least one 5' terminal cap;

b) a flanking region located at the 3' terminus of the first region, comprising: i) a physiological 3' UTR of the ADAMTS 13 protein or a biologically active fragment or varia t thereof; and

ii) a tail sequence; or

c) both a) and b).

7. The composition of Claim 6, wherein the at least one 5' terminal cap is selected from the group consisting of: a 5' triphosphate cap (5'-ppp), a guanosine-triphosphate cap (5^! Gppp), a 5' N7-methyl guanosine-triphosphate cap (5' N7-MeGppp, 7mGppp), a m7GpppG cap, a 5' adenylated cap (rApp), Cap 0, Cap 1, ARCA, inosine, Nl-methyi- guanosine, 2' fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.

8. The composition of Claim 6, wherein the 3' tail sequence is selected from the group consisting of a poly-A tail and a polyA-G quartet.

9. The composition of Claim 6, wherein the first region further encodes a signal peptide and/or a leader sequence at the 5' terminus of the ADAMTS13 protein or a biologically active fragment or variant thereof.

10. The composition of Claim 6, wherein the first region further encodes a detectable label linked to the ADAMTS 13 protein or a biologically active fragment or variant thereof.

11. The composition of Claim 10, wherein the detectable label is a fluorescent label, a luminescent label, a heavy metal label, a radioactive label or an enzymatic label.

12. The composition of Claim 6, wherein the at least one mRNA molecule comprises at least one modification of at least one nucleoside in the flanking region located at the 5' terminus of the first region.

13. The composition of Claim 6, wherein the at least one mRNA molecule comprises at least one modification of at least one nucleoside in the flanking region located at the 3' terminus of the first region.

14. The composition of Claim 5, wherein the at least one mRNA molecule comprises at least one modification of at least one nucleoside in the first region.

15. The composition of any one of the previous claims, wherein the at least one

modification is located in a nucleoside base and/or sugar portion.

16. The composition of Claim 1, further comprising an agent for facilitating transfer of the at least one RNA molecule to an intracellular compartment of a target cell.

17. The composition of Claim 1 , wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof is a fusion protein.

18. The composition of Claim 1, wherein the ADAMTS13 protein or a biologically active fragment or variant thereof comprise an amino acid sequence as set forth in SEQ ID NO:4.

19. The composition of Claim 1 , wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof comprises an polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NOS:5 or 6.

20. The composition of any one of Claims 1-19, wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof further comprise at least one amino acid substitution.

21. The composition of any one of Claims 1-19, wherein the at least one amino acid substitution is a substitution to histidine or alanine.

22. The composition of Claim 1, wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof comprising the at least one amino acid substitution has an increased stability or in vivo half-life when compared to the unsubstituted

ADAMTS 13 protein or biologically active fragment or variant thereof.

23. The composition of any one of Claims 1-22, wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof binds to vWF.

24. The composition of Claim 23, wherein the ADAMTS 13 protein or a biologically active fragment or variant thereof binds to human vWF and at least one vWF from a non-human mammal.

25. A method expressing in a target ceil an ADAMTS 13 protein or biologically active fragment or variant thereof comprising maintaining a composition of any one of Claims 1 -24 under conditions and for a time sufficient to allow expression by the target cell of the ADAMTS 13 protein or biologically active fragment or variant thereof.

26. A therapeutic kit comprising: (i) the composition of any one of claims 1-23; and (li) means for deliver ' of the at least one RNA molecule to a subject.

27, An article of manufacture comprising: a) a container comprising a label; and

b) the composition of any one of Claims 1-23, wherein the label indicates that the composition is to be administered to a human having, suspected of having or at risk for developing, a condition related to unregulated vWF expression or activity.

The article of manufacture of Claim 27, further comprising one or more additional active therapeutic agents for use in treating such human.

A method for treating a patient having, suspected of having or at risk for developing a condition related to unregulated vWF expression or activity, the method comprising administering to said subject the composition of any one of Claims 1-24 in an amount effective to treat the condition.

The method of Claim 29, wherein the condition is a thrombotic disease.

The method of claim 30, wherein the thrombotic disease comprises is selected from the group consisting of: disseminated intravascular coagulation (DIC),

hemolytic-uremic syndrome (HUS), deep vein thrombosis (DVT), thrombocytopenic purpura (TTP), pulmonary embolism, cerebral infarction and systemic lupus erythematosus (SLE).

The method of Claim 31 , wherein the thrombotic disease is TTP,

The method of Claim 32, wherein the TTP is a congenital TTP or an acquired TTP.

The method of any one of Claims 29-33, wherein the composition is administered by a route selected from the group consisting of: intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

The method of any one of Claims 29-34, wherein the composition is administered in combination with a second therapeutic agent.