(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`I
`
`(43) International Publication Date
`2 0 June 2013 (20.06.2013)
`
`P O P C T
`
`(10) International Publication Number
`WO 2013/090648 Al
`
`(51)
`
`International Patent Classification:
`C07H 21/02 (2006.01)
`A61K 48/00 (2006.01)
`
`(21)
`
`International Application Number:
`
`(71) Applicant: MODERNA THERAPEUTICS
`[US/US];
`161 First Street, First Floor, Cambridge, Massachusetts
`02142 (US).
`
`PCT/US20 12/0696 10
`
`(72)
`
`14 December 2012 (14. 12.2012)
`
`English
`
`English
`
`(22)
`
`International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`16 December 201 1 (16. 12.201 1)
`61/576,705
`2 April 2012 (02.04.2012)
`61/618,957
`17 May 2012 (17.05.2012)
`61/648,244
`10 August 2012 (10.08.2012)
`61/681,712
`4 September 2012 (04.09.2012)
`61/696,381
`3 October 2012 (03. 10.2012)
`61/709,303
`PCT/US2012/0585 19
`3 October 2012 (03. 10.2012)
`11 October 2012 ( 11. 10.2012)
`
`61/712,490
`
`Inventors: DE FOUGEROLLES, Antonin; 66 Summit
`02446 (US). WOOD,
`Avenue, Brookline, Massachusetts
`Kristy M.; 501 Huron Ave, Unit 2, Cambridge, Massachu
`setts 02138 (US). ELBASHIR,
`Sayda M.; 149 Grove
`Street, Cambridge, Massachusetts 021 38 (US). AFEYAN,
`Noubar B.; 1 Memorial Drive, 7th Floor, Cambridge, M as
`VALENCIA,
`Pedro;
`sachusetts
`02142
`(US).
`60
`Wadsworth
`Street, Apt. 5G, Cambridge, Massachusetts
`02142 (US). SCHRUM, Jason P.; 4601 Flat Rock Road,
`U S
`us
`Apt. 417, Philadelphia, Pennsylvania 19127 (US).
`us (74) Agent: WARD, Donna T.; DT Ward, P.C.
`us
`Street, Groton, Massachusetts 01450 (US).
`us
`us (81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`us
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`us
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`
`142A Main
`
`(54) Title: MODIFIED NUCLEOSIDE, NUCLEOTIDE, AND NUCLEIC ACID COMPOSITIONS
`
`[Continued on nextpage]
`
`p
`
`.
`
`;6 8
`
`(57) Abstract: The present disclosure
`inter alia, formulation com
`provides,
`positions comprising modified nucleic
`acid molecules which may encode
`a
`protein, a protein precursor, or a par
`tially or
`fully processed form of the
`protein or a protein precursor. The for
`mulation composition may further
`in
`clude a modified nucleic acid molecule
`and a delivery agent. The present
`in
`vention further provides nucleic acids
`useful
`for encoding polypeptides
`cap
`able of modulating
`a cell's
`function
`and/or activity.
`
`1 t
`
`» T
`
`Figure 2
`
`s
`
`00
`o
`
`© oo
`
`ARBUTUS - EXHIBIT 2015
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

`w o 2013/090648 Ai
`
`llll I I I I 11III
`
`III
`
`II 11 II II
`
`III I III II I II
`
`IS, JP, KE, KG, KM, KN, KP,
`IL, IN,
`ID,
`HN, HR, HU,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS,
`RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA, ZM, ZW.
`
`(AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`TM), European
`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,
`SM, TR), OAPI
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`— with international search report (Art. 21(3))
`(84) Designated States (unless otherwise indicated, for even-
`kind of regional protection available): ARIPO (BW, GH, — with sequence listing part of description (Rule 5.2(a))
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian
`(AM, AZ, BY, KG, KZ, RU, TJ,
`
`

`

`MODIFIED NUCLEOSIDE, NUCLEOTIDE, AND NUCLEIC ACID COMPOSITIONS
`
`REFERENCE TO SEQUENCE LISTING
`
`[0001]
`
`The present application is being filed along with a Sequence Listing in electronic format.
`
`The Sequence Listing file, entitled M l lPCTSQLST.txt, was created on December 14, 2012 and is
`
`25,579 bytes in size. The information in electronic format of the Sequence Listing is incorporated
`
`herein by reference in its entirety.
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`[0002]
`
`This application claims the benefit of U.S. Provisional Patent Application No. 61/576,705,
`
`filed December 16, 201 1, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid
`
`Compositions, U.S. Provisional Patent Application No. 61/618,957, filed April 2, 2012, entitled
`
`Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent
`
`Application No. 61/648,244, filed May 17, 2012, entitled Modified Nucleoside, Nucleotide, and
`
`Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/681,712, filed August
`
`10,2012, entitled Modified Nucleoside, Nucleotide, Nucleic Acid Compositions and U.S. Provisional
`
`Patent Application No. 61/696,381 filed September 4, 2012, entitled Modified Nucleoside,
`
`Nucleotide and Nucleic Acid Compositions, and Nucleic Acid Compositions, U.S. Provisional
`
`Patent Application No. 61/709,303 filed October 3, 2012, entitled Modified Nucleoside, Nucleotide
`
`and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61/712,490 filed October
`
`11, 2012, entitled Modified Nucleoside, Nucleotide and Nucleic Acid Compositions and
`
`International Pub. No. PCT/US2012/058519 filed October 3, 2012 Modified Nucleosides,
`
`Nucleotides, and Nucleic Acids, And Uses Thereof, the contents of which are incorporated herein by
`
`reference in their entirety.
`
`BACKGROUND OF THE INVENTION
`
`[0003]
`
`In general, exogenous unmodified nucleic acid molecules, particularly viral nucleic acids,
`
`introduced into the cell induce an innate immune response which results in cytokine and interferon
`
`(IFN) production and ultimately cell death. It is of great interest for therapeutics, diagnostics,
`
`reagents and for biological assays to be able to deliver a nucleic acid, e.g., a ribonucleic acid (RNA),
`
`into a cell, such as to cause intracellular translation of the nucleic acid and production of the encoded
`
`

`

`protein instead of generating an innate immune response. Thus, there is a need to develop
`
`formulation compositions comprising a delivery agent that can effectively facilitate the in vivo
`
`delivery of nucleic acids to targeted cells without generating an innate immune response.
`
`SUMMARY OF THE INVENTION
`
`[0004]
`
`The present disclosure provides, inter alia, formulation compositions comprising modified
`
`nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully
`
`processed form of the protein or a protein precursor. The formulation compositions may further
`
`include a modified nucleic acid molecule and a delivery agent. The present invention further
`
`provides nucleic acids useful for encoding polypeptides capable of modulating a cell's function
`
`and/or activity.
`
`[0005]
`
`In one aspect a method of producing a polypeptide of interest in a mammalian cell or tissue
`
`is described. The method comprises contacting the mammalian cell or tissue with a formulation
`
`comprising a modified mRNA encoding a polypeptide of interest. The formulation may be, but is
`
`not limited to, nanoparticles, poly(lactic-co-glycolic acid)(PLGA) microspheres, lipidoids, lipoplex,
`
`liposome, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin
`
`hydrogel, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles
`
`(reLNPs) and combinations thereof. The modified mRNA may comprise a purified IVT transcript.
`
`[0006]
`
`In one embodiment, the formulation comprising the modified mRNA is a nanoparticle
`
`which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-
`
`DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3 -DMA, DLin-KC2-DMA, DODMA, PLGA,
`
`PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as,
`
`but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3 -DMA, DLin-KC2-DMA and DODMA.
`
`[0007]
`
`The lipid to modified mRNA ration in the formulation may be between 10:1 and 30:10.
`
`The mean size of the nanoparticle formulation may comprise the modified mRNA between 60 and
`
`225 nm. The PDI of the nanoparticle formulation comprising the modified mRNA is between 0.03
`
`and 0.15. The zeta potential of the lipid may be from -10 to +10 at a pH of 7.4
`
`[0008]
`
`The formulations of modified mRNA may comprise a fusogenic lipid, cholesterol and a
`
`PEG lipid. The formulation may have a molar ratio 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic
`
`lipid: cholesterol: PEG lipid). The PEG lipid may be selected from, but is not limited to PEG-c-
`
`DOMG, PEG-DMG. The fusogenic lipid may be DSPC.
`
`

`

`[0009]
`
`The mammalian cell or tissue may be contacted using a device such as, but not limited to, a
`
`syringe pump, internal osmotic pump and external osmotic pump.
`
`The formulation of modified mR A may be a PLGA microsphere which may be between 4
`
`[0010]
`and 20 mih
`48 hour time period. The PLGA microsphere formulation may be stable in serum. Stability may be
`
`in size. The modified mRNA may be released from the formulation at less than 50% in a
`
`determined relative to unformulated modified mRNA in 90%.
`
`[0011]
`
`The loading weight percent of the modified mRNA PLGA microsphere may be at least
`
`0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4% or at least 0.5%. The encapsulation
`
`efficiency of the modified mRNA in the PLGA microsphere may be at least 50%, at least 70%, at
`
`least 90% or at least 97%.
`
`[0012] A lipid nanoparticle of the present invention may be formulated in a sealant such as, but not
`
`limited to, a fibrin sealant.
`
`[0013]
`
`The mammalian cells or tissues may be contacted by a route of administration such as, but
`
`not limited to, intravenous, intramuscular, intravitreal, intrathecal, intratumoral, pulmonary and
`
`subcutaneous. The mammalian cells or tissues may be contacted using a split dosing schedule.
`
`The mammalian cell or tissue may be contacted by injection. The injection may be made to tissue
`
`selected from the group consisting of intradermal space, epidermis, subcutaneous tissue and muscle.
`
`The polypeptide of interest may be produced in the cell or tissue in a location systemic from the
`
`location of contacting.
`
`[0014]
`
`The polypeptide of interest may be detectable in serum for up to 72 hours after contacting.
`
`The level of the polypeptide of interest can be higher than the levels prior to dosing. The level of the
`
`polypeptide of interest may be greater in the serum of female subjects than in the serum of male
`
`subjects.
`
`[0015]
`
`The formulation of modified mRNA may comprise more than one modified mRNA. The
`
`formulation may have two or three modified mRNA.
`
`[0016]
`
`The formulation comprising the modified mRNA may comprise a rapidly eliminated lipid
`
`nanoparticle (reLNP) which may comprise a reLNP lipid, fusogenic lipid, cholesterol and a PEG
`
`lipid at a molar ratio of 50: 10: 38.5: 1.5 (reLNP lipid:fusogenic lipid: cholesterol: PEG lipid). The
`
`fusogenic lipid may be DSPC and the PEG lipid may be PEG-c-DOMG. The reLNP lipid may be
`
`DLin-DMA with an internal or terminal ester or DLin-MC3-DMA with an internal or terminal ester.
`
`The total lipid to modified mRNA weight ration may be between 10:1 and 30:1.
`
`

`

`[0017]
`
`The formulation comprising modified mRNA may comprise a fibrin sealant.
`
`[0018]
`
`The formulation comprising modified mRNA may comprise a lipidoid where the lipid is
`
`selected from the group consisting of CI2-200 and 98N12-5.
`
`[0019]
`
`The formulation comprising modified mRNA may include a polymer. The polymer may
`
`be coated, covered, surrounded, enclosed or comprise a layer of a hydrogel or surgical sealant. The
`
`polymer may be selected from the group consisting of PLGA, ethylene vinyl acetate, poloxamer and
`
`GELSITE®.
`
`[0020] A polypeptide of interest may be produced in a mammalian cell or tissue by contacting the
`
`mammalian cell or tissue with a buffer formulation comprising a modified mRNA encoding the
`
`polypeptide of interest. The buffer formulation may be selected from, but is not limited to, slaine,
`
`phosphate buffered saline and Ringer's lactate. The buffer formulation may comprise a calcium
`
`concentration of between 1 to 10 mM. The modified mRNA in the buffer formulation may comprise
`
`a purified IVT transcript.
`
`[0021] A pharmacologic effect in a primate may be produced by contacting the primate with a
`
`composition comprising a formulated modified mRNA encoding a polypeptide of interest. The
`
`modified mRNA may comprise a purified IVT transcript and/or may be formulated in nanoparticles,
`
`poly(lactic-co-glycolic acid)(PLGA) microspheres, lipidoids, lipoplex, liposome, polymers,
`
`carbohydrates (including simple sugars), cationic lipids, fibrin gel, fibrin hydrogel, fibrin glue, fibrin
`
`sealant, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNPs) and combinations
`
`thereof. The pharmacological effect may be greater than the pharmacologic effect associated with a
`
`therapeutic agent and/or composition known to produce said pharmacologic effect. The composition
`
`may comprise a formulated or unformulated modified mRNA. The pharmacologic effect may result
`
`in a therapeutically effective outcome of a disease, disorder, condition or infection. Such
`
`therapeutically effective outcome may include, but is not limited to, treatment, improvement of one
`
`or more symptoms, diagnosis, prevention, and delay of onset. The pharmacologic effect may
`
`include, but is not limited to, change in cell count, alteration in serum chemistry, alteration of
`
`enzyme activity, increase in hemoglobin, and increase in hematocrit.
`
`[0022]
`
`In one embodiment, the present disclosure provides a formulation composition which
`
`comprises a modified nucleic acid molecule and a delivery agent. The modified nucleic acid
`
`molecule may be selected from the group consisting of DNA, complimentary DNA (cDNA), RNA,
`
`messenger RNA (mRNA), RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense
`
`

`

`R A, ribozymes, catalytic DNA, R A that induce triple helix formation, aptamers, vectors and
`
`combinations thereof. If the modified nucleic acid molecule is mR A the mR A may be derived
`
`from cDNA.
`
`[0023]
`
`In one embodiment, the modified nucleic acid molecule may comprise at least one
`
`modification and a translatable region. In some instances, the modified nucleic acid comprises at
`
`least two modifications and a translatable region. The modification may be located on the backbone
`
`and/or a nucleoside of the nucleic acid molecule. The modification may be located on both a
`
`nucleoside and a backbone linkage.
`
`[0024]
`
`In one embodiment, a modification may be located on the backbone linkage of the
`
`modified nucleic acid molecule. The backbone linkage may be modified by replacing of one or more
`
`oxygen atoms. The modification of the backbone linkage may comprise replacing at least one
`
`phosphodiester linkage with a phosphorothioate linkage.
`
`[0025]
`
`In one embodiment, a modification may be located on a nucleoside of the modified nucleic
`
`acid molecule. The modification on the nucleoside may be located on the sugar of said nucleoside.
`
`The modification of the nucleoside may occur at the 2' position on the nucleoside.
`
`[0026]
`
`The nucleoside modification may include a compound selected from the group consisting
`
`of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-
`
`pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,
`
`1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-
`
`taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-
`
`taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-
`
`pseudouridine, 2-thio- 1-methyl-pseudouridine, 1-methyl- 1-deaza-pseudouridine, 2-thio- 1-methyl- 1-
`
`deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-
`
`dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-
`
`methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-
`
`acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-
`
`pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-
`
`cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza-
`
`pseudoisocytidine, 1-methyl- 1-deaza-pseudoisocytidine, 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-
`
`methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-
`
`N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-
`
`threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-
`
`dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 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-methylguanosine, N2-methylguanosine,
`
`N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-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-methylcytosine,
`
`pseudouridine and 1-methylpseudouridine
`
`[0027]
`
`In one embodiment, a modification may be located on a nucleobase of the modified nucleic
`
`acid molecule. The modification on the nucleobase may be selected from the group consisting of
`
`cytosine, guanine, adenine, thymine and uracil. The modification on the nucleobase may be selected
`
`from the group consisting of deaza-adenosine and deaza-guanosine, and the linker may be attached
`
`at a C-7 or C-8 position of said deaza-adenosine or deaza-guanosine. The modified nucleobase may
`
`be selected from the group consisting of cytosine and uracil, and the linker may be attached to the
`
`modified nucleobase at an N-3 or C-5 position. The linker attached to the nucleobase may be
`
`selected from the group consisting of diethylene glycol, dipropylene glycol, triethylene glycol,
`
`tripropylene glycol, tetraethylene glycol, tetraethylene glycol, divalent alkyl, alkenyl, alkynyl
`
`moiety, ester, amide, and ether moiety.
`
`[0028]
`
`In one embodiment, two modifications of the nucleic acid molecule may be located on
`
`nucleosides of the modified nucleic acid molecule. The modified nucleosides may be selected from
`
`5-methylcytosine and pseudouridine.
`
`[0029]
`
`In one embodiment, two modifications of the modified nucleic acid molecule may be
`
`located on a nucleotide or a nucleoside. In one embodiment, the present disclosure provides a
`
`formulation comprising a nucleic acid molecule such as, but not limited to, SEQ ID NO: 6, SEQ ID
`
`NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10 and a delivery agent. The nucleic acid molecule may
`
`comprise a polyA tail about 160 nucleotides in length. Further, the nucleic acid molecule may
`
`

`

`comprise at least one 5' terminal cap such as, but not limited to, CapO, Capl, ARCA, inosine, Nl-
`
`methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,
`
`LNA-guanosine, and 2-azido-guanosine.
`
`[0030]
`
`In one embodiment, the present disclosure provides a nucleic acid of SEQ ID NO: 6, a 5'
`
`terminal cap which is Capl, a poly A tail of approximately 160 nucleotides in length and a delivery
`
`agent.
`
`[0031]
`
`In one embodiment, the present disclosure provides a nucleic acid of SEQ ID NO: 7, a 5'
`
`terminal cap which is Capl, a poly A tail of approximately 160 nucleotides in length and a delivery
`
`agent.
`
`[0032]
`
`In one embodiment, the present disclosure provides a nucleic acid of SEQ ID NO: 9, a 5'
`
`terminal cap which is Capl, a poly A tail of approximately 160 nucleotides in length and a delivery
`
`agent.
`
`[0033]
`
`In one embodiment, the present disclosure provides a nucleic acid of SEQ ID NO: 10, a 5'
`
`terminal cap which is Capl, a poly A tail of approximately 160 nucleotides in length and a delivery
`
`agent.
`
`[0034]
`
`In one embodiment, the delivery agent comprises at least one method to improve delivery
`
`selected from the group consisting of lipidoids, liposomes, lipid nanoparticles, rapidly eliminated
`
`lipid nanoparticles (reLNPs), polymers, lipoplexes, peptides, proteins, hydrogels, sealants, chemical
`
`modifications, conjugation, cells and enhancers. The lipidoid, lipid nanoparticle and rapidly
`
`eliminated lipid nanoparticles which may be used as a delivery agent may include a lipid which may
`
`be selected from the group consisting of C12-200, MD1, 98N12-5, DLin-DMA, DLin-K-DMA,
`
`DLin-KC2-DMA, DLin-MC3 -DMA, PLGA, PEG, PEG-DMG, PEGylated lipids and analogs
`
`thereof. The rapidly eliminated lipid nanoparticle may have an ester linkage at the terminal end of
`
`the lipid chain, or an ester linkage may be an internal linkage located to the right or left of a
`
`saturated carbon in the lipid chain. The rapidly eliminated lipid nanoparticle which may be used as a
`
`delivery agent may be, but is not limited to, DLin-MC3-DMA and DLin-DMA.
`
`[0035]
`
`In one embodiment, the lipid nanoparticle may comprise PEG and at least one component
`
`such as, but not limited to, cholesterol, cationic lipid and fusogenic lipid.
`
`[0036]
`
`In one embodiment, the lipid nanoparticle may comprise at least one of a PEG, cholesterol,
`
`cationic lipid and fusogenic lipid.
`
`

`

`[0037]
`
`In one embodiment, the fusogenic lipid is disteroylphophatidyl choline (DSPC). In
`
`another embodiment, the PEG lipid is PEG-DMG. In yet another embodiment, the cationic lipid
`
`may be, but not limited to, DLin-DMA, DLin-MC3 -DMA, C12-200, 98N12-5 and DLin-KC2-
`
`DMA.
`
`[0038]
`
`In one embodiment, the lipid nanoparticle composition may comprise 50 mol% cationic
`
`lipid, 10 mol% DSPC, 1.5-3.0 mol% PEG and 37-38.5 mol% cholesterol.
`
`[0039]
`
`In one embodiment, a modified nucleic acid may be formulated with PLGA to form a
`
`sustained release formulation. In another embodiment, a modified nucleic acid may be formulated
`
`with PLGA and other active and/or inactive components to form a sustained release formulation. In
`
`one embodiment, the modified nucleic acid molecule may include, but is not limited to, SEQ ID NO:
`
`9 and SEQ ID NO: 10.
`
`[0040]
`
`In one embodiment, a sustained release formulation may comprise a sustained release
`
`microsphere. The sustained release microsphere may be about 10 to about 50 um in diameter. In
`
`another embodiment, the sustained release microsphere may contain about 0.001 to about 1.0 weight
`
`percent of at least one modified nucleic acid molecule.
`
`[0041]
`
`In one embodiment, the modified nucleic acids of the present invention may include at least
`
`one stop codon before the 3' untranslated region (UTR). The stop codon may be selected from
`
`TGA, TAA and TAG. In one embodiment, the modified nucleic acids of the present invention
`
`include the stop codon TGA and one additional stop codon. In a further embodiment the addition
`
`stop codon may be TAA. In another embodiment, the modified nucleic acid of the present invention
`
`includes three stop codons.
`
`[0042]
`
`In one embodiment, the present disclosure provides a controlled release formulation
`
`comprising a modified nucleic acid which may encode a polypeptide of interest. The modified
`
`nucleic acid may be encapsulated or substantially encapsulated in a delivery agent. The delivery
`
`agent may be coated, covered, surrounded, enclosed or comprise a layer of polymer, hydrogel and/or
`
`surgical sealant. In a further embodiment, the controlled release formulation may comprise a
`
`second layer of polymer, hydrogel and/or surgical sealant.
`
`[0043]
`
`In one embodiment, the delivery agent of the controlled release formulation may include,
`
`but is not limited to, lipidoids, liposomes, lipid nanoparticles, rapidly eliminated lipid nanoparticles,
`
`lipoplexes and self-assembled lipid nanoparticles.
`
`[0044]
`
`

`

`[0045]
`
`The polymer which may be used in the controlled release formulation may include, but is
`
`not limited to, PLGA, ethylene vinyl acetate, poloxamer and GELSITE®. The surgical sealant
`
`which may be used in the controlled release formulation may include, but is not limited to,
`
`fibrinogen polymers, TISSEELL®, PEG-based sealants and COSEAL®.
`
`[0046]
`
`In one embodiment, the delivery agent of the controlled release formulation comprises a
`
`lipid nanoparticle or a rapidly eliminated lipid nanoparticle delivery agent. In one aspect, the lipid
`
`nanoparticle or rapidly eliminated lipid nanoparticle may be coated, substantially coated, covered,
`
`substantially covered, surrounded, substanitally surrounded, enclosed, substantially enclosed or
`
`comprises a layer of polymer, hydrogel and/or surgical sealant. In another aspect, the delivery agent
`
`may be a lipid nanoparticle which may be coated, substantially coated, covered, substantially
`
`covered, surrounded, substantially surrounded, enclosed, substantially enclosed or comprises a layer
`
`of PLGA.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0047]
`
`The foregoing and other objects, features and advantages will be apparent from the
`
`following description of particular embodiments of the invention, as illustrated in the accompanying
`
`drawings in which like reference characters refer to the same parts throughout the different views.
`
`The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the
`
`principles of various embodiments of the invention.
`
`[0048]
`
`FIG. 1 illustrates lipid structures in the prior art useful in the present invention. Shown are
`
`the structures for 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-DMA (2,2-Dilinoleyl-4-
`
`dimethylaminomethyl-[l,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA and C12-200.
`
`[0049]
`
`FIG. 2 is a representative plasmid useful in the IVT reactions taught herein. The plasmid
`
`contains Insert 64818, designed by the instant inventors.
`
`[0050]
`
`FIG. 3 is a gel profile of modified mR A encapsulated in PLGA microspheres.
`
`DETAILED DESCRIPTION
`
`[0051]
`
`The delivery of nucleic acids into cells has many undesired complications including the
`
`integration of the nucleic acid into the target cell genome which may result in imprecise expression
`
`levels, the deleterious transfer of the nucleic acid to progeny and neighbor cells and a substantial risk
`
`of causing mutations. The modified nucleic acid molecules of the present disclosure are capable of
`
`

`

`reducing the innate immune activity of a population of cells into which they are introduced, thus
`
`increasing the efficiency of protein production in that cell population. Further, one or more
`
`additional advantageous activities and/or properties of the nucleic acids and proteins of the present
`
`disclosure are described herein.
`
`[0052]
`
`In addition, provided herein are methods of treating a subject having or being suspected of
`
`having a disease, disorder and/or condition the methods comprising administering to a subject in
`
`need of such treatment a composition described herein in an amount sufficient to treat the disease,
`
`disorder and/or condition.
`
`[0053] Unless otherwise defined, all technical and scientific terms used herein have the same
`
`meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
`
`Although methods and materials similar or equivalent to those described herein can be used in the
`
`practice or testing of methods featured in the invention, suitable methods and materials are described
`
`below.
`
`Modified Nucleic Acid Molecules
`
`[0054]
`
`The present disclosure provides nucleic acids, including RNA such as mRNA, which
`
`contain one or more modified nucleosides or nucleotides (termed "modified nucleic acid molecules,"
`
`"modified mRNA" or "modified mRNA molecules") as described herein. The modification of the
`
`nucleic acid molecules of the present invention may have useful properties including, but not limited
`
`to, a significant decrease in or a lack of a substantial induction of the innate immune response of a
`
`cell into which the modified mRNA is introduced. The modified nucleic acid molecules may also
`
`exhibit enhanced efficiency of protein production, intracellular retention of nucleic acids, and
`
`viability of contacted cells, as well as having reduced immunogenicity as compared to unmodified
`
`nucleic acid molecules.
`
`[0055]
`
`Provided are modified nucleic acid molecules containing a translatable region and one,
`
`two, or more than two different nucleoside modifications Exemplary nucleic acids for use in this
`
`disclosure include ribonucleic acids (RNA), deoxyribonucleic acids (DNAs), threose nucleic acids
`
`(TNAs), glycol nucleic acids (GNAs), locked nucleic acids (LNAs) or a hybrid thereof. In preferred
`
`embodiments, the modified nucleic acid molecules include messenger RNA (mRNA). As described
`
`herein, the modified nucleic acid molecules of the present disclosure may not substantially induce an
`
`innate immune response of a cell into which the modified mRNA is introduced. In another
`
`embodiment, the modified nucleic acid molecule may exhibit reduced degradation, as compared to a
`
`

`

`nucleic acid that has not been modified, in a cell where the modified nucleic acid molecule is
`
`introduced.
`
`[0056]
`
`The term "nucleic acid" includes any compound and/or substance that is or can be
`
`incorporated into an oligonucleotide chain. Exemplary nucleic acids for use in accordance with the
`
`present disclosure include, but are not limited to, one or more of DNA, cDNA, RNA including
`
`messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNA, shRNA,
`
`miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induce triple helix formation,
`
`aptamers, vectors and the like.
`
`[0057]
`
`In certain embodiments, it is desirable to intracellularly degrade a modified nucleic acid
`
`molecule introduced into the cell. For example it would be desirable to degrade a modified nucleic
`
`acid molecule if precise timing of protein production was desired. Thus, the present disclosure
`
`provides a modified nucleic acid molecule containing a degradation domain, which is capable of
`
`being acted on in a directed manner within a cell.
`
`[0058]
`
`In some embodiments, the modified nucleic acid molecules may be chemically modified on
`
`the sugar, nucleobase (e.g.,
`
`in the 5' position of the nucleobase), or phosphate backbone (e.g.,
`
`replacing the phosphate with another moiety such as a thiophospate). In some embodiments, the
`
`modification may result in a disruption of a major groove binding partner interaction, which may
`
`contribute to an innate immune response. In some embodiments, the formulation composition, when
`
`administered to a subject, can result in improved bioavailability, therapeutic window, or volume of
`
`distribution of the modified nucleic acid molecule relative to administration of the modified nucleic
`
`acid molecule without the incorporation of the delivery agent. In some embodiments, the modified
`
`nucleosides and nucleotides of the modified nucleic acid molecules of the present invention may be
`
`synthesized using the O-protected compounds described in International Pub. No. WO2012138530,
`
`the contents of which is herein incorporated by reference in its entirety.
`
`[0059]
`
`In certain embodiments, the modified nucleic acid molecule may comprise mRNA. In
`
`particular embodiments, the modified mRNA (mmRNA) may be derived from cDNA. In certain
`
`embodiments, mmRNA may comprise at least two nucleoside modifications. In one embodiment,
`
`the nucleoside modifications may be selected from 5-methylcytosine and pseudouridine. In another
`
`embodiment, at least one of the nucleoside modifications is not 5-methylcytosine and/or
`
`pseudouridine. In certain embodiments the delivery agent may comprise formulations allowing for
`
`localized and systemic delivery of mmRNA. The formulations of the modified nucleic acids
`
`

`

`molecules and/or mmR A may be selected from, but are not limited to, lipidoids, liposomes and
`
`lipid nanoparticles, rapidly eliminated lipid nanoparticles, polymers, lipoplexes, peptides and
`
`proteins, at least one chemical modification and conjugation, enhancers, and/or cells.
`
`[0060]
`
`In one embodiment, the modified nucleic acid molecules of the present invention may
`
`include at least two stop codons before the 3' untranslated region (UTR). The stop codon may be
`
`s