(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/164762 Al 13 October 2016 (13.10.2016) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 14/705 (2006.01) C12N 15/11 (2006.01) kind of national protection available): AE, AG, AL, AM, C12N 15/09 (2006.01) C12N 15/12 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US20 16/026703 HN, HR, HU, ID, IL, ΓΝ , IR, IS, JP, KE, KG, KN, KP, KR, (22) International Filing Date: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, 8 April 2016 (08.04.2016) MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, (25) Filing Language: English SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (26) Publication Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 62/144,895 8 April 2015 (08.04.2015) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (71) Applicant: MODERNA THERAPEUTICS, INC. TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, [US/US]; 200 Technology Square, Cambridge, Massachu TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, setts 02139 (US). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors: ELLSWORTH, Jeff Lynn; 23 Meriam Street, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Lexington, Massachusetts 02420 (US). BOLEN, Joseph GW, KM, ML, MR, NE, SN, TD, TG). Beene; 1 Avery Street, 29B, Boston, Massachusetts 021 11 (US). GREGOIRE, Francine M.; 184 Austin Street SI, Published: Newton, Massachusetts 02465 (US). GUILD, Justin; 495 — with international search report (Art. 21(3)) Edgell Rd, Framingham, Massachusetts 01701 (US). — with sequence listing part of description (Rule 5.2(a)) (74) Agents: NANNENGA-COMBS, Bonnie et al; Sterne Kessler Goldstein & Fox P.L.L.C, 1100 New York Aven ue, N.W., Washington, District of Columbia 20005 (US). (54) Title: POLYNUCLEOTIDES ENCODING LOW DENSITY LIPOPROTEIN RECEPTOR EGF-A AND INTRACELLULAR DOMAIN MUTANTS AND METHODS OF USING THE SAME II O FIG. 2 0 (57) Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynuc leotide molecules encoding low density lipoprotein receptor comprising least one amino acid mutation in the EGF-A domain and at least one or a single amino acid mutation in the intracellular domain. POLYNUCLEOTIDES ENCODING LOW DENSITY LIPOPROTEIN RECEPTOR EGF-A AND INTRACELLULAR DOMAIN MUTANTS AND METHODS OF USING THE SAME REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB [0001] The content of the electronically submitted sequence listing (Name: 3529_021PC01_SeqListing_ST25.txt"; Size: 2,607,509 bytes; and date of creation: April 07, 2016) filed herewith the application is incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] High cholesterol is one of a number of risk factors for heart attack and stroke. Although poor diet and lack of excise are common causes of high cholesterol, genetic changes can also influence cholesterol levels. For example, familiar hypercholesterolemia (FH) is a genetic disorder characterized by high cholesterol levels in the blood, specifically very high levels of low-density lipoprotein (LDL). FH also leads to early cardiovascular disease. [0003] Cholesterol targeting drugs on the market include statins, fibrates, niacin, bile acid sequestrants (resins), phytosterols, or other compounds that prevent absorption of fats, reduce absorption of cholesterol, or target genes in the cholesterol trafficking pathway. Cholesterol lowering drugs approved for the treatment of homozygous FH include mipomersen, an antisense oligonucleotide inhibitor which targets ApoB-100, and lomitapide, an inhibitor of the microsomal triglyceride transfer protein (MTP or MTTP) which is necessary for very low-density lipoprotein (VLDL) assembly and secretion in the liver. [0004] Not all cholesterol targeting drugs can adequately lower LDL levels, particularly in FH patients. Liver related problems are commonly associated with cholesterol targeting drugs, particularly elevation in serum transaminases and accumulation of hepatic fat (or hepatic steatosis) have been reported. In addition, there are risks and contraindications associated with certain conditions or other medications. SUMMARY OF THE INVENTION [0005] Certain aspects of the invention are directed to a polynucleotide comprising an open reading frame of linked nucleosides encoding a human low density lipoprotein receptor (LDLR) or functional fragment thereof comprising at least one amino acid mutation in the EGF-A domain and at least one or a single amino acid mutation in the intracellular domain, wherein the at least one amino acid mutation in the EGF-A domain abrogates binding of PCSK9 to the LDLR or functional fragment thereof, and wherein the open reading frame is optionally codon-optimized. [0006] Another aspect of the invention is directed to a polynucleotide comprising an open reading frame of linked nucleosides encoding a human low density lipoprotein receptor (LDLR) or functional fragment thereof comprising at least one amino acid mutation in the EGF-A domain and at least one amino acid mutation in the intracellular domain, wherein the at least one amino acid mutation in the EGF-A domain abrogates binding of PCSK9 to the LDLR or functional fragment thereof and, optionally, at least one of the amino acids corresponding to K830 or C839 of human LDLR is intact, and wherein the open reading frame is optionally codon-optimized. [0007] In some embodiments, the at least one mutation in the EGF-A domain is at an amino acid residue corresponding to N316, L339, or the combination of N316 and L339 of human LDLR (SEQ ID NO:43). [0008] In some embodiments, the at least one mutation in the EGF-A domain comprises a deletion of the amino acid, an insertion of at least one amino acid, a conservative substitution, or a substitution. [0009] In some embodiments, the at least one mutation in the EGF-A domain is a substitution from the N (asparagine) corresponding to amino acid residue 316 of SEQ ID NO:43 to an amino acid selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine. [0010] In some embodiments, the at least one mutation in the EGF-A domain is a substitution from the L (leucine) corresponding to amino acid residue 339 of SEQ ID NO:43 to an amino acid selected from the group consisting of aspartate, glutamate, glutamine, and asparagine. [0011] In some embodiments, the single mutation or at least one mutation in the intracellular domain is at an amino acid residue corresponding to K816, K830, or C839 of human LDLR (SEQ ID NO:43). [0012] In some embodiments, the single mutation or at least one mutation in the intracellular domain comprises a deletion of the amino acid, an insertion of at least one amino acid, a conservative substitution, or a substitution. [0013] In some embodiments, the single mutation or at least one mutation in the intracellular domain is a substitution from the K (lysine) corresponding to an amino acid residue 816 or 830 of SEQ ID NO:43 to an amino acid selected from the group consisting of histidine or arginine. [0014] In some embodiments, the single mutation or at least one mutation in the intracellular domain is a substitution from the C (cysteine) corresponding to an amino acid residue 839 of SEQ ID NO:43 to an amino acid selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine. [0015] In some embodiments, the LDLR or functional fragment thereof comprises one mutation in the EGF-A domain and one mutation in the intracellular domain. [0016] In some embodiments, the one mutation in the EGF-A domain corresponds to N316A of human LDLR and the one mutation in the intracellular domain corresponds to K830R of human LDLR. [0017] In some embodiments, the polynucleotide comprises at least one chemically modified nucleoside. In some embodiments, the at least one chemically modified nucleoside is selected from any of those listed in Table 5. In some embodiments, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 5- methylcytosine, 5-methoxyuridine, and a combination thereof. In some embodiments, the nucleosides in the open reading frame are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 9 5% , at least 99%, or 100%. In some embodiments, the chemically modified nucleosides in the open reading frame are selected from the group consisting of uridine, adenine, cytosine, guanine, and any combination thereof. [0018] In some embodiments, the uridine nucleosides in the open reading frame are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. [0019] In some embodiments, the adenine nucleosides in the open reading frame are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.