NZ618275B2 - Lipid nanoparticle compositions and methods for mrna delivery - Google Patents

Abstract

Disclosed is a composition comprising (a) at least one mRNA molecule at least a portion of which encodes a functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle. Also disclosed is the use of the said composition for the treatment of a subject having a deficiency in a functional polypeptide. y in a functional polypeptide.

Description

LIPID NANOPARTICLE COMPOSITIONS AND METHODS FOR MRNA DELIVERY Novel approaches and iesare still needed for the treatment of protein and enzyme deficiencies. For example, mal storage diseases are a {groupof approximately .50 rare inherited metabolic disorders that result from defects in _ lysosomal function, usually due to a ncy of an enzyme required for metabolism. ‘Fabry disease is a lysosomal storage disease that results from a ncy of the enzyme alphagalactosidase (GLA), Which causes a glycolipid knownas globotriaosylceramide to accumulate in blood vessels and other tissues, . leading to various painful manifestations. For certain diseases, like F.abry disease, there is a need for replacement ofa protein or enzyme that‘is normally secreted by cells into the blood stream. Therapies, such as gene therapy, that increase "the level or production 1 affected n or enzyme could provide a treatment or even a cure for such disorders. However, there have been several limitations to using conventional gene y for this purpose.

Conventional gene therapy involves the use ofDNA for insertion of desired genetic information into host cells. 1116, DNA introduced into the cell is usually integrated to ga'certain extent into the genome of one or more transfected cells, allowing for long—lasting action of the introduced genetic material e host. While there may be substantial benefits to such sustained action, ation of exogenous DNA into a host genome may also have many deleterious effects. For example, it is possible thatthe introduced DNA will be inserted into an intact gene, resulting in a mutation which impedes or even totally eliminates the on ofthe endogenous gene. Thus, gene therapy with DNA may result in the impairment of av'ital genetic function in the treated host, such as e.g_.., ation or deleteriously reduced production of an essential enzyme or interruption of a gene critical for'the regulation of cell growth, resulting in unregulated or cancerous cell eration. - In addition, with conventional DNA based gene therapy it is necessary for effective sion of the desired gene product to include a strong promoter sequence, which again may lead to rable changes in the regulation of normal gene expression in the cell. It is also possible that the DNA based genetic material will result in the induction of undesired anti—DNA antibodies, which in turn, may r a possibly fatal immune in an adverse response. Gene therapy approaches usingtviral vectors can also result immune response. In some circumstances, the viral vector may even integrate into,;the.,_ . host genome. In addition, tion of clinical grade viral vectorsis also expensive and time consuming. Targeting delivery of’the introduced genetic material using viral ._ vectors can also be difficult tocontrol. Thus, while DNA based gene therapy has been evaluated for delivery of secreted proteins using viral vectors (US PatentNo. 6,066,626; USZOO4/O-l 10709), these approaches may be d for these various reasons.

Another obstacle apparent in these prior approaches at delivery'ofnucleic acids encoding secreted proteins, is in the levels of protein that are ultimately produced. It is lt to achieve cant levels of the desired protein in the blood, and the amounts are not sustainedover time. For example, the amount of protein produced by nucleic acid delivery/does not reach normal physiological levels.

See e,g,, USZOO4/0110709.

In st-t0 DNA, the use ofRNA as a gene therapy agent is substantially safer because (1) RNA does volve the risk of being. stably integrated into the genome ofthe transfected cell, thus eliminating the concern e introduced genetic material will t the normal functioning of an essential gene, or cause a mutation that results in deleterious or oncogenic effects; (2:) extraneous promoter sequences are not required for effective translation of the encoded protein, again avoiding possible. deleterious side s; (3) in contrast to: plasmid DNA QpDNA), messenger RNA (mRNA) is devoid of immunogenic CpG’ motifs so that anti—RNA antibodies are not generated; and (4) any- deleterious effects that do result from mRNA based on genetherapy would be of limited duration due to the relatively short half-life of RNA. In on, it is not necessary for mRNA to enter the nucleus to - perform its function, while DNA must me this major barrier.

One reason that mRNAbased gene therapy has not been used more in the past is that mRNA is far less stable than DNA,:es.pecially when it reaches the asm of a cell and is exposed to degrading enzymes. The presence of a hydroxyl group on the second carbon of the sugar moiety in mRNA causes steric hinderance that ts the mRNA from forming the more stable double helix structureof DNAand thus makes the mRNA more prone to hydrolytic degradation. As a result, until recently, it was widely believed that mRNA was too labile to withstand transfection protocols. es in RNA stabilizing modifications have sparked more interest in the use of mRNA in place ofplasmid DNA in gene therapy. Certain delivery vehicles, such as cationic lipid or polymer deli‘Very vehicles may .also help protect the transfected mRNA from nous RNa’ses. "Yet, in spite eased stability ofmodified . ‘ mRNA, delivery ofmRNA to cells in vivo in a manner allowing for therapuetic levels ' of nzp'roductionis still a challenge, particularly for mRNA encoding full length . protein's. While'delivery of inRNA encoding ed proteins has been contemplated “ - (USZOO9/O286852), the levels of a full length secreted protein that would actually he -.. produced via in vivo mRNA delivery are not known and there is not a reason to expect _ the levels would exceed those observed with DNA based gene therapy.

To date, significant progress using mRNA gene therapy'has only been made in . applicationsfor which low‘ levels oftranslation has-not been a limiting factor, such as immunization with mRNA encoding antigens. Clinical trials involving vaccination against tumor antigens by intradermal injection of naked amine-complexed mRNA have demonstrated ility, lack oftoxicity, and ing results. X, Su et al., Mol. Pharmaceutics 8:77-4—787 (2011). Unfortunately, low levels oftranslation has greatly restricted. the exploitation ofmRNA based gene-therapy inother applications which require higher levels of- ned expression of" the mRNA encoded protein to exert a biological or therapeutic: effect.

The invention provides methods for delivery ofmRNA gene therapeutic agents that lead to the production of eutically effective levels of secreted proteins via a “depot effect.” In embodiments ofthe invention, mRNA encoding a secreted n is loaded in lipid nanoparticles and delivered to target cells in vivo.

Target cells then act as a depot source for production of soltible, secreted protein into the atory systemat therapeutic levels. In some embodiments, the levels of secreted protein produced are above normal physiological .

The invention provides compositions and methods for intracellular delivery of mRNA in a liposomal transfer vehicle to one or more target cells for production of therapeutic levels of secreted functional protein.

The compositions and methods ofthe invention are useful in the management and treatment of a large number of diseases, in particular diseases which result from protein and/or enzyme deficiencies, wherein the n or enzyme is normally ed. Individualssuffering from such diseases may have underlying genetic defects that lead to thecompromised expression of a protein or enzyme, including, for example, the non—synthesis of the secreted protein, the reduced synthesis of the secreted protein, or synthesis of a secreted protein g or-having diminished biological activity. .Inparticular, 'thetmethods and compositions ofthe invention are useful for the'treatment of lysosomal storage disorders and/or the urea cyclemetabolic ers that occur as a result of one or more defectsxin the biosynthesis of secreted » enzymes ed in the: urea-cycle.

The‘corhpositions of theinventidn comprise an mRNA, a, transfer vehicle and,._ optionally, an agent to facilitate contact with, and subsequent transfection of-a target cell. The mRN-A can encode a ally useful secreted protein. For-example, the. mRNA may encode a functional secreted urea cycle enzyme or a secreted enzyme implicated in lysosomal storage disorders. The mRNA can encode, for e, erythropoietin (e.g., human EPO) :or d-galacto'sidase (e.~g., human d—galactosidase (human GLA).

In some embodiments the mRNA can comprise one or more modifications that confer stability to the mRNA (e.g., compared to ‘a 'wildetype or native n of the mRNA) and may also comprise one or more modifications relative to the wild- type which corre‘cta defect. implicated in the associated aberrant expresSion of the protein. For example, the nucleic acids of the present invention may comprise modifications to one or both of the 5’ and 3’ untranslated regions. Such modifications may include, but are not limited to, the inclusion ofa partial sequence of a cytomegalovirus (CMV) immediate-early l .(IEI) gene, a poly A tail, a Capl structure or a sequence encoding human growth hormone (hGH)). In some embodiments, the mRNA is. modified to decrease mRNA immunogenecity.

Methods oftreating a subject comprising stering a composition of the invention, are also contemplated. For example, s of‘treating-or preventing conditions in which production of a particular secreted protein 'utilizationzof a particular secreted protein is inadequate or compromised are provided. In one embodiment, the methods provided herein can be used to treat a subject having a deficiency in one or more urea cycle enzymes or in one or more enzymes deficient in a lysosomalstorage disorder.

In a,preferred ment, the mRNA in the compositions of the invention is ated in :a mal transfer vehicle to facilitate delivery to the target cell.

Contemplated transfer vehicles may comprise one or more cationic , non— cationic lipids, and/or PEG-modified lipids. For example, the er vehicle may comprise at least one of the following cationic lipids: (312-200,.DL'in-KC2-DMA, DODAP, HGT4003, ICE, O, or HGTSOOl. .In embodiments, the transfer 1 vehicle comprises Cholesterol .(cho'l) and/era PEG—modified lipid; In some embodimentsgthe errvehicle-s.comprises DMG—PEGZK. In certain . _- ; ‘ ments, the tranfer vehicle comprises one of the ing lipid formulations: 012—200, DOPE, ch01, DMG-PEGZK; DODAP, DOPE, cholesterol, DMG—PEGZK; HGTSOOO, DOPE, chol, DMG'ePEG'ZK, HGTSOO 1-, DOPE, ch01, DMG-PE'GZK.'J ‘ The invention also provides compositions and methods useful for facilitating ‘ the transfection and delivery of'one or more mRNA molecules to target cells capable of exhibiting the “depot effect.” For example, the compositions and methods of the - - present invention contemplate the use of targeting ligands capable of enhancing the - affinity ofthe composition to one or more target cells. In one embodiment, the targeting ligand is apolipoprotei-mB or apolipoprotein—E and corresponding target cells express low-density lipoprotein receptors, thereby facilitating recognition of the targeting . The s and itions of the present invention-may be used to preferentially target a vast number oftarget cells. For e, contemplated target cells include, but are not limited to, hepatoc-ytes, epithelial cells, hematopoietic cells, epithelial cells, endothel-ial-cells, lung cells, bone scells,-stem cells, mesenchymal cells, neural cells, cardiac cells, ytes, vascularzsmooth muscle cells, cardiom-yocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining , ovarian cells, testicular cell-s, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granu'locytes and tumor cells.

In ments, the secreted protein is produced by the target cell for sustained amounts oftime. For example, the secreted protein maybe producted for more than one hour, more than four, more than Six, more than 12, more than {24, more than 48 hours, or more than 72 hours after administration. In some embodiments the polypeptide is expressed at a peak level about six hours after administration. In some embodiments the expression of the polypeptide is sustained at least at a eutic level. In somezembodiments the polypeptidezis expressed at at least a therapeutic level for more than one, more than four, more than six, more than 12, more than 24, more 8 hours, or more than 72 hours after administration. In some " embodiments the polypeptide is detectable at the level in patient serum or tissue (e.g., liver, or lung). In some embodiments, the level of detectable polypeptide is from continuous expession from the mRNA composition over periods of‘time of more than WO 70930 one, more than four, more than six, more than 12, more than 2.4, more than 48 hours, or more than 72-hours’after administration; In certain embodiments, the secreted protein is produced at levels above normal physiological levels. The level of secreted protein may .be-i’ncreased as .- compared to a control... ~ 1 - In some embodiments ,thecontrol is thebasel-ine:physiological level of the polypeptide in a normal'individual or in a population ofnormal individuals. In other »- ments the l is the baseline physiological level of thevpolypeptide in an . indiVidual having a deficiency in the relevant protein or polypeptide or in ‘a population ' of individuals having a deficiency in the relevant protein or ptide. In some embodiments the control can be the normal level of the ntprotein or polypeptide in the individual to whom the» composition is administered In other ments the control is the expression level of the polypeptide upon other therapeutic intervention, e.g., upon direct injection of the corresponding polypeptide, atone or more comparable time .

In certain embodiments the polypeptide is; expressed by the'target cell at a level Which-is at least 15—fold, at least2—fold, at least 5-fold, at least 10—fold, at least -fold, 30-fold, at least l‘OO-fold, at least SOC-fold, :at least 'SOOO-fol'd, at least 50,000- folid .or at least 100,000—fold greater than a control. In some embodiments, the'fold increase of expression greater than control is ned for more than one, more than four, an six, more than 12., an 24,, or more than 48 hours, or more than 7-2 hours after administration. For example, in one embodiment,the. levels of secreted protein are detected in=the serum at least 15-fold, at least 2—fold, at least 5—fold, at least =1 0-fold, at least 20¢:fold, 30-fold, at least ld, at least 500~fold, at least SOOO-fo'ld, at least 50,000~fo'ld or at least 100,000-fold greater than a-control for at least 48 hours or 2 days. In certain embodiments, the levels ofsecreted protein are detectable at ‘3 days, 4days, 5 days, or 1 week or more after administration. Increased levels of secreted protein may beobserved in the serum and/or in a tissue (eg. liver, lung).

In someembodiments, the method yields a ned circulation half-life» of the desired secreted protein. For example, the secreted protein may be detected for hours or days longer than the half—life observed via subcutaneous injection of the secreted protein. In embodiments, the half-life ofthe secreted protein is ned for more than 1 day, 2 days, 3 days, 4 days, 5 days, or 1 week ormore.

WO 70930 In some embodiments administration comprises a single :or repeated doses. In . -c'ertai‘n-embodiments, the dose is administered intravenously, or :by pulmonary.- ‘deliVery; ' The ptide can be, for e, one ormore- of erythropoietin, 0t- ~ galactosidase, LDL receptor, Factor VIII, Factor IX, u-L—iduronidase (for MPS I), . iduronate ase (for MPSIII)“, heparinLN-sulfatase (for MP8 .III'A), a-N- .acetylglucosaminidase (for MP8 IIIB’), galactose 6-sultatase (forMPS , lysos'omal' acid , arYlsulfatase-A. .

Certain embodiments relate "to compositionsand methods that provide to a cell or subject mRNA, at‘least apart ofwhich encodes a functional protein, in art-amount that is substantially less that the amount of ponding onal n generated from that mRNA. Put another way, in certain embodiments themRNA delivered to the-cell duce unt. ofprotein that is substantially greater than "the amount of-mRNA delivered to the cell. For example, ina given amountof time, for example 1, 2, .3, 4, 5, 6, 7, 8, 9, :10, 12, 15,20, or 24 hours from administration ofthe mRNA to a cell or subject, the amount of corresponding protein generated by thatrmRNA can be at least 1.5, ’2, 3., 5, 1-0, .15., 20, 25, '50, 100, 150, 200, 250, 300, 400., 500, or more times greater that the amount ofmRNA actually administered to the cell or subject.

This can be measured on a mass-by=massbasis, ona mole-by-rnole basis, and/or on a moleculeaby—molecule basis The protein can be measured in various ways. FOr example, fora cell, the measured protein can be measured-as intracellularprotein, extracellular protein, or a combination ofthe two. Fora subject, themeasured protein can be protein ediin serum; in a specific tissue or tissues such asthe liver, kidney, heart, or brain; in a specific cell type such. as one of the various cell types of the liver or:brain; or in any combination of serum, tissue, and/or cell type. er, a baseline amount of endogenous protein can be measured in the cell or subject prior to administration of the mRN-A and then subtracted from the protein measured after administration ofthe mRNA to yield the amount of corresponding protein generated from the mRNA. In this way, the mRNA‘can provide a reservoir or depot source of a large amount oftherapeutic material to the cell or subject, for example, as compared to amount ofmRNA delivered to the cell or subject. The-depot source can act asa continuous source for polypeptide expression from the mRNA over sustained periods oftime.

The above discussed and many- other features and ant advantages of the present invention will become better understood by reference tothe following detailed-_ . description of theinvention whentaken in conjunction with the accompanying- examples. The variousembodimentsdeSCribed herein are cemplimentary. and, can be ' combined‘or'used together ina manner-understood by the Skilled person in view of. the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS shows the nucleotide sequence ofa 5’ CNN sequence (SEQ ID N011), wherein X,‘ if t is GGA.

FIG.,2 shoWs the nucleotide ce of a 3’ hGHsequence (SEQ ID N052). shows the nucleotide sequence ofhuman erythropoietic (EPO) mRNA (SEQ ID N023). This sequence-can be flanked on the 51’ end with SEQ ID N0:l and on the 3’ end with SEQ ID N022.

FIG. -4 shows the nucleotide ce of human alpha-galactosidase (GLA) mRNA (SEQ ID N024). This ce can be flanked on the 5’ end with SEQ ID NOIl and on the3’ end with SEQ ID N022.

FIG. '5 shows the nucleotide sequence ofhuman alpha-l antitryps'in.(A1AT) mR-NA (SEQ .ID N025). This sequence can be flanked on theS’ end with SEQ ID N011 and on the 3’ end with SEQ IDN022.

FIG. =6 shows the nucleotide sequence of human factor IX (FIX) mRNA (SEQ ID NO:6). This sequence can be flanked on the 5’ end with SEQ ID N.O:;1 and on the 3’ end with SEQ ID N022.

FIG. ‘7 shows quantification of ed hEPO protein'levels as measured via ELISA. The protein detected is a result- of its production from .hEPO mRNA delivered intravenously via a single dose of various lipid nanoparticle formulations.

The formulations 012-200 ('30 ug), HGT4003 (150 ug), ICE (100 ug), DODAP (.200 ug) are represented as the cationic/ionizable lipid component-of each test e (Formulations 1 -4‘). Values are based on blood sample four hours post- administration. :showsthe hematocrit measurement of mice treated with .a single IV dose of human EPO mRNA-loaded lipid nanoparticles (Formulations 1-4). Whole blood samples were taken at 4 hr (Day I), 24 hr (Day 2), 4 days, 7 days, and 10 days post-administration. shows hematocrit measurements of mice treated with human EPO- ' amRN-‘A-l'oade'd lipid nanoparticl'es with-either a single 1V dose .orthree injections (day: 4 1, day 3-, day '5). Whole bleed samples were taken prior to injection. (day -4),-1day 7,1, - and . “Formulation 1 Was administered: (30 ug, single dose).or (-3 x 10 ,u-g,_-dose V day 1; day 3;, day'S); Formulation 2 was administered: (3 x 50 ug, dose day l,-.day.3-,- day 5). , shows quantification of secreted human ctosidase (hGLA) protein levels :as‘ measured'via ELISA. The protein detected is a result of the production frOm hGLA elivered via lipid nanoparticles (Formulation J; 30-_ ugsingle intravenous dose, based on encapsulated mRNA). hGLA protein is ed through 48 hours.

FIG. ‘11 shows h‘GLA activity in semm. hGLA activity was measured using substrate4-methylumbelliferyl—o~D-galactopyranoside (4-MU-a-ga1) at..37‘-’-C. Data are average ofi6 to 9 individual ements.

FIG. :12 shows quantification ofhGLA n levels in serum as measured via ELISA. Protein is produced from hGLA mRNA delivered via 0- based lipid nanopartic‘les (C-l'2—.20.0:DOP;E:-Chol:DMGPEG2K, 40302255 (Formulation J); 3.0 ugimRNA based on encapsulated mRNA, single IV doseJ. hGLA protein is monitored through 7.2 hours. per single intravenous dose, based on encapsulated mRNA). hGLA protein is monitored through 72 hours. shows quantification of hGLAprotein levels in liver, kidney, and spleen as measured viaELlSA. Protein is produced from hGL-A mRNA delivered Via OO—based lipid nanoparticles (Formulation J; 30 ug mRNA based on encapsulated mRNA, single IV dose). hGLA protein is red through 72 hours. shows a dose response study monitoring protein production of hGLA‘ as secreted MRT~derived human GLA protein in serum (A) and liver (B). Samples were measured 24 hours post—administration (Formulation 1; single dose, IV, N=4 mice/group) and quantified via ELISA. shows the pharmacokinetic s offERT—based Alpha— galactosidase in athymjic nude mice (40 ug/kg dose) and hGLA protein produced from MRT (Formulation J ; 1.0 mg/kg m‘RNA dose). shows the quantification of secreted hGLA protein levels in ”MRT— treated Fabry mice as measured using ELISA. hGLA protein is produced from hGLA mRNA delivered via C12based lipid nanoparticles (Formulation J; 10 ug rnRNA per single enous dose, based on encapsulated mRNA). Serum is monitored through-:72 hours.

, FIG-17 shows.the-quantification.ofthLAirrotsinlevels in liver: kidney: ,,. spleen, and heartof'MR-T—treated Fabry KO mice: as measured. via ELISA. , , Protein is produced from RNA delivered via C'12~200+based.lipid-nanopartic‘les .. ‘ (Formulation 1;'30 ug mRNA baSed on encapsulated m'RNA, Single IV close). hGLAr- protein is monitored through 72 hours. Literature values representing normal» physiological levels are graphed as dashed lines. shows the quantification. of secreted hGLA protein levels in MRTzand Alpha-galactos’idase—treated Fabry mice as measured using ELISA. Both therapies were .dosedzas a single 1.0 mg/kg enous dose. shows the quantification ofhGLA protein levels in liver,,kidnicy., spleen, and heart ofMRT and ERT (Alpha—'galactosidase)-treated Fabry KO mice as ed viaELI-SA. Protein produced from hGLAmRNA delivered via lipid nanoparticles (Formulation 1 ; 1.0 mg/kg . based on encapsulatedmRNA, single IV dose).

FIG. .20 shows the relative quantification of globotrioasylceramide (G703) and lyso~Gb3 in the kidneys oftreated and untreated mice. Male Fabry KO mice were treated with a single dose either GLA mRNA—loaded lipid‘nanopaiticles or Alpha- galactosidase at 1.0 mg/kg, Amounts reflect quantity of'Gb3/lyso-Gb3 one week post-administration.

FIG..21 shows the ve quantification of globotrioasylceramide (GbS) and b3 in the heart oftreated and untreated mice. Male .Fabry KO mice were, treated with a single dose either GLA mRNA—loaded lipid nanopa-rticles or Alpha~ galactosidase at 1.0 ing/kg. Amounts reflect quantity of- Gb3/lyso-Gb3 one week po_st~administration.

FIG..22 shows a dose reSponse-study monitoring n production of- GLA. as ed MRT—derived human GLA protein in serum. Samples were measured 24' hours postradministrat'ion (single dose, IV, N=4 mice/group) of either HGT4003 (Formulation 3) or HGT5000~based lipid nanoparticles (Formulation 5) and quantified via ELISA. shows hGLA protein production as ed in serum (A) or in liver, kidney, and spleen (B). Samples were measured 6 hours and 24 hours post— administration (single dose, IV, N=4 mice/group) of'HGT5001~based lipid nancparti'c'le's (Formulation 6) and quantified via ELISA.

" FIG. .24 shovvs the quantification of secreted human Factor IX protein levels ‘ -' measured‘u’sing ELISA (mean ng/mL :t standard deviation); FIX n is produced:- - from FIX-rmRNA delivered-via C12~ZOO-based lipid nanoparticles (C12— 200:DOP'EtC‘IholiDMGPEGZK, 4023022525 (Formulation 1).; 3:0 ug mRNA per single . intravenous dose, based on encapsulated mRNA). FIX protein is monitored h. ,- - 72 hours. (11:24 mice) FIG; '25 showsthe quantification-of edhuman titrypsin(A1-AT) protein levels measured using ELISA. AlAT protein is produced from AlAT mRNA red via C12—200—base‘d lipid nanopartic‘les (C12-200:DOPE:Chol:DMGPEGZK, , 4030:2525 (Formulation J);'30‘ ug'mRNA per single intravenous dose, based on encapsulated :mRNA).. AlAT protein is monitored through 24 hours.

FIG.Z26 shows an ELISA—based quantification ofhB-PO protein detected in the lungs and serum ated mice after intratracheai administration ofhEPO mRNA— loaded nanoparticles (measured mlU) (012-200, HGTSOOO, or HGT5001-- based lipid, rticles; Formulations 1, 5, E6 respectivebz). Animalswere ced 6 hours post—administration (n=4 mice per group).

PTION OF EXEMPLARY MENTS The invention provides compOSitions and methods for intracellular delivery of mRNA in a liposomal'transfer vehicle to one or more target cells for production of therapeutic levels of secreted functional protein.

The term “functional,” as used herein to y a protein or enzyme, means that the protein or enzyme has biological activity, or alternatively is able to perform the same, or. a similar function as the native or normally—functioning n or . The mRN-A compositions of the invention are useful for the treatment of a various metabolic or genetic disorders, and in particular those genetic or metabolic disorders which involve the non—expression, misexpression or ncy of a protein or enzyme. The term “therapeutic levels”.refer-s to. levels of protein detected in the blood or tissues that are above control levels, wherein the control may be normal physiological levels, or the levels in the subject prior to administration of the .mRNA. composition. The term “secreted” refers to protein that is detected outside the target cell, in extracellular space. The protein may be detected in the blood or in tissues. In it 1 the t ofthe present invention the term “produCed” is used in its broadest Sense ' to refer thetran'slation. of at leaSt. one mRNA into a proteinor enzyme. As. provided} , herein, the compositions include a transfer vehicle; As used herein, the. term f‘transfer “vehicle” includes any of.thexstandard pharmaceutical carriers, diluents, cxcipient-s and; the like which are generally-intended for use in connection with the administration of , biologically active agents,” including nucleic acids. --The compositions-and in particular the transfer es described herein are capable ofdelivering mRNA'to . the target cell. In embodiments, the transfer vehicle is a lipid nanoparticle. mRNA The mMA in the compositions ofthe invention may encode, for e, a secreted e, enzyme, receptor, polypeptide, peptide or other protein of interest that is normally secreted. In one embodiment ofthe invention, the mRNA may optionally have chemical or biological modifications which, for example, improve the stability and/or half~life of such mRN-A or which improve or otherwise facilitate protein production.

The methods of'the invention provide for optional co-delivery ofone or more unique mRNA to target cells, for example, by combining two-unique mRNAs into a single transfer vehicle, In one ment of the t invention, a therapeutic first mRN-A, and a therapeutic second mRNA, may be ated in a single transfer vehicle and administered. The present invention also contemplates co—delivery and/or co~adrninistration of a therapeutic first mRNA and asecond nucleic. acid to tate and/or enhance thefunction or delivery of the therapeutic first mRNA. For example, such a second nucleic acid (e,g., exogenous or synthetic mRNA) may encode a membrane transporter protein that upon expression (e.g., translation of the ous or synthetic mRNA) facilitates the delivery or enhances the biological activity ofthe first‘m‘RNA. atively, the therapeutic first mRNA may be administered with a second nucleic acid that functions as a “chaperone” for example, to direct'the folding of either the eutic first mRNA.

The methods ofthe invention also provide for the delivery of one or more therapeutic nucleic acids to treat .a single disorder ordeficiency, wherein each such . ' therapeutic c acid functions by a ent mechanism of action. For example, the compositions of the present invention may comprise a therapeutic first mRNA which, for e, is administered to correct an endogenous protein or enzyme deficiency, and which is accompanied bya second nucleic. acid, which is administered ‘ to deactivate or “knock-down”{amalfunctioning endogenous nucleic acid and-its -‘ 'p’rOtei‘n' or enzyme product; Such “second” nucleic acids may encode, for example ~' ' - mRNA or si-‘RNA.

‘ Upon-transfeCtion, a- natUral mRNA in‘ the compositions of the invention may ‘ decay With a halfslife be'tWeen'SO'minutes and several days. ThemRNA inthe compositions of the invention preferably retain at least some ability to‘be translated, , thereby producing a functional secreted protein or enzyme. Accordingly, the invention provides compositionscomprising and methods of administering a stabilized mRNA. In some embodiments ofthe invention, the activity ofthe mRNA is prolonged over an extended period of time. For example, the activity of the mRNA may be prolonged such that the compositions .of the present invention are administered to a subject on asemi-weekly or bi—Weekly basis, 'or more preferably on a monthly, bi-amonthly, quarterly or an annual basis. The ed or prolonged activity of the mRNAof the present invention, is directly related to the quantity of secreted functional protein or- enzyme produced from suchmRNA Similarly, the activity of the compositions of the t-invention may be further extended or prolonged by modifications made to improve orenhance translation of the mRNA.

Furthermore, the quantity of functional protein or enzyme produced by the target cell is. a function of the quantity ofmRNA delivered to the target- cells and the stability of such imRNA. To the extent that the stability of the mRNA ofthe t invention may be improved or ed, the half—life, the activity ofthe produced secreted n or enzyme and the dosing frequency of the ition may be further extended. ingly, in some embodiments, the mRNA in the compositions of'the invention comprise at least one modification which confers sed or enhanced stability to the nucleic acid, including, for example, improvedresistance to nuclease digestion in viva. As used herein, the terms ication” and “modified” as such terms relatezto the nucleic acids provided herein, e at, least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to se digestion) than the wild~type or naturally occurring version of the mRNA.

As used herein, the terms “stable” and lity” as such terms'relate to the nucleic acids of the present invention, and ularly with respect .to the mRNA, refer to sed or enhanced ance to degradation by, for example nucleases Ge, endonucleases or exonucleases) which are normally capable of degrading such - niRNA.I Increased stability can include, for example, less sensitivity to ysis or g ‘ other destruotion by endogenous s Cos-a optioned-oases.ornonuclsssod.01‘ conditions within thetarget cell or tissue, thereby increasing or enhancing the - . ‘ residenceoif such mRNA in the target .cell, tissue, subject and/or cytoplasm. The- - stabilized mRNA m‘oleculeszprovidedtherein demonstrate longer half—lives relative to. their naturally occurring, unmodified counterparts (eg. the ype version ofjthe mMA); Also contemplated by the terms “modification?> and “modified” as such terms related'to the mRNA Of the present invention are alterations which improve or enhance ation ofmRNA nucleic acids, including for example, the ion of sequences which function in the initiation of protein ation (e.g., the Kozac- consensus sequence). (Kozak, M., NucleicAcids Res 15 (20); 8125-48 (:1 9.817)).

In some ments, the mRNA of the invention have undergone a chemical or biological modification to ‘render'them more stable. Exemplary modificationsto an mRNA include the depletion of a base (cg, by deletion or by the substitution ofone nucleotide for another) or modification of a base, for example, the chemical modification-0f a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in lly ing mRNA, for example, covalent modifications .such as the introduction of modified tides, (e.g., nucleotide analogs, or the inclusion .ofpendant :grOups which are urally found in such mRNA molecules).

In addition, le modifications includealterations in one. or more nucleotides of at codon such that the codon encodes the same amino acid but is more stable than the codon found in the Wild—type version of the mRNA. 'For e, an inverse relationship between bility ofRNA and a higher number cytidines- (C's) and/or uridines (U's) residues has been demonstrated, and RNA devoid of 1C and U residues have been found to bestable to most RNases (Heidenreich, at al. 'J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and/or U residues in an mRNA sequence is reduced. In a another embodiment, the number of C and/or U residues is reduced by substitution of one codon encoding a particular ' amino acid for another codon encoding the same ora related amino acid.

Contemplated modifications to the mRNA nucleic acids of the present invention also include theincorporatation of pseudouridines. The incorporation ofpseudouridines » into the mRNA nucleic acids of the present ion may enhance stability and translational capacity, as well as diminishing immunogenicity in viva. See, e. g., 'Karik‘o, K,,‘ et ah, Molecular Therapy. 16‘ (113118334 840 (2008).- Substitutions: and modificati’Ons-tothem‘RNA of thepresentinvention may be performed by methods y‘knoWnto one or ordinary/skill in the art. ..

" The'constraints on reducinfgithe number ofC-and U residues in asequence will: -be r Within the coding region ofan mRNA, compared to an untranslated; region, '(i.e.,.'it will "likely not be possible to eliminate all ofthe C and. U residues . present in the message 'While still retaining the ability ofthemessage to encode-the . d amino acid sequence). The degeneracy of the genetic code, r presents an opportunity to allow the number of C and/or U residues that are t in the sequence to be reduced, While'maintaining the same coding capacity (i.e., depending on Which amino acid is encoded by at codon, several differentpossibilities for modification ofRNA sequences may be possible). For example, the codons for Gly can be altered to .GG‘A or GGG d of GGU or GGC.

The term modification also includes, for example, the incorporation ofnon— nucleotide linkages ormodified nucleotides into the mRNA sequences ofthe present invention (-e.»g., modifications to one or both the 3' and 5" ends of an mRNA molecule encoding a functional secreted protein or enzyme). 'Such modifications include the addition of‘bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration ofthe 3' UTR or the 5’ UTR, complexing the mRNA With an agent (e.g., .a protein or a mentary nucleic acid molecule), and inclusion of elements which change the ure of an mRNA molecule (cg, whicheform secondary structures).

The poly .Atail'is thought to stabilize natural messengers. Therefore, in one ment a long poly A tail canibe added to an mRNA molecule thus rendering the mRNA more . Poly .Avtails can be added using :a-variety of art—recognized techniques. For example, long poly A tails can be added to synthetic or in Will-‘0 transcribed mRNA using poly A polymerase (Yokoeb er al. Nature hnology. 199.6; 14: 1252—1256). A transcription vector can also encode long poly A tails. in addition, poly A tails can be added by transcription directly from PCR products. In one embodiment, the length ofthe poly A tail is at least about 90, 200, 300, 400 at . least .500 nucleotides. In one embodiment, the length of the poly A .tail is ed to. control the stability of a modified mRNA molecule of the invention and, thus, the transcription of n. For example, since the length ofthe poly A tail can influence the half—life of an mRNA molecule, the length of the poly A tail can be adjusted to ' modify -ve] of resistance ’ofthe mRN-A-to nucleases and thereby control thetime, course of pretein eXpre'ssionmin a cell. Inon‘e embodiment, theistabilizedtmRNA, ‘ les are suffiCie’n‘tly ant to in vivo degradation (e.g.,by nucleases), such , . '- that they'may be delivered .to the target cell Without a transfer vehicle.

In one embodiment; "an mRNA- can: be modified by the incorporation 3" and/or ' slated (UTR) sequences which are not naturally found in the wild-type mRNA. In one e1nbodiment,‘3' and/or 5' flanking sequence which naturally flanks an mRNA and» encodes a second, unrelated protein can‘be incorporated into the nucleotide'sequence ofan ,mRNA molecule ng a therapeutic or functional protein in: order to modify it. Forzexample, 3’ or 5’ ces from mRN‘A les , which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes”) can be incorporated into the 3' and/or 5' region of a sense mRNA nucleic acid molecule to increase the stability of the senseszNA molecule. See, 'e.g., U-82003/00’83'272.

In. some embodiments,themRNA in the compositions of the. invention include ationofthe 5’ end of the mRNA to include a partial sequence ofa CMV immediate-earlyil (IE1) gene, or a fragment thereof (e.g., SEQ ID NO: I) to improve the nuclease resistance and/or improve the half—life ofthe mRNA. In. addition to increasing-the ity of the mRNA nucleic acid sequence, it has been surprisingly discovered the inclusion of a partial sequence of a CMV immediate-early l (IE1) gene enhances the translation of the mRNA and the sion of'the functional protein or enzyme. Also contemplated is the inclusion of a human growth hormone (hGH); gene sequence, or a fragment thereof (e.g., SEQ ,ID N02) to the 3’ ends of the nucleic acid (cg, 1nRNAj) to further ize the mRNA. Generally, preferred ations improve the stability and/or cokinetic properties (cg, half—life) of the mRNA relative to their unmodified counterparts, and include, forexample modifi cations made to improve such mRNA’s resistance to in viva nuclease-digestion.

- Further contemplated are variants. of the nucelic acid sequence of SEQ ID NO:1 and/or SEQ ID N022, wherein the the variants maintain the functional properties of the nucleic acids including stabilization ofthe-mRNA and/or - pharmacokinetic properties (e..g., half-life). Variants may have greater than 90%, greater than 95%, greater than 98%, or greater than 99% sequence identity to SEQ ID N011 or SEQ ID N02. in some embodiments, the ition can comprise astabilizing reagent.

‘ The compositions can-inelu'de one. or more. formulation reagents that-bind directly » indirectly‘to,':and‘ ize the'mRNA, thereby .enhancin'gresidence time in the -3. cell. Such reagents ably lead to an improvedhalf—life: of the mRNA in the target.

Cellsszor e, the stability of an mRNA .and- efficiency oftransflation may be increaSed by the incorporation "of “stabilizing reagents’.’ that form complexes with the; mRNA that naturallyoccur'within acell (see e.-g;, US. Pat. No. 5,677,124). - Incorporation of a stabilizing reagent can be accomplished for-example,'by combining the poly A-and a protein with the. mRNA to be stabilized in vitro before loading or ulating the mRNA within a transfer vehicle. Exemplary stabilizing reagents include one or more proteins, peptides, aptamers, translational accessory'protein, mRNA binding proteins, and/or’translation initiation factors.

Stabilization ofthe compositions may also be improved by the use of opsonizationeinhibiting moieties, which are typically large hydrophilic-‘po'lymers- that are chemically or ally bound to the transfer e (e.g., by the intercalation of avlipidésoluble anchor into the membrane itself, or by binding directly to'active groups ofmembrane lipids). These opsonization—inhibiting hilic polymers form a protective e layer which significantly decreases the uptake of the liposomes by. the macrophage-monocyte system and reticuloaendothelial SYStem (e,g,, as described in US. Pat. No. 4,920,016, the entire disclosure ofWhich is herein incorporated by reference). Transfer'vehicles modified with zation-inhibition moieties thus remain in. the circulation much longer than their unmodified counterparts.

When RNA is hybridized to a complementary nucleic acid molecule (egg, DNA or RNA) it may be protected :from nucleases. (Krieg, at a]. Melton. Methods in Enzymology. 1987'; 155, 397415). The stability ofhybridized mRNA is likely due to the inherent single strand specificity ofmost RNases. In somezembodiments, the stabilizing reagent selected to complex a mRNA is a eukaryotic protein,._(e.-g., a mammalian protein). In yet another embodiment, the mRNA can be modified by hybridization to asecond c acid molecule. If an entire mRNA molecule were hybridized to a complementary c acid molecule translation initiation may be reduced; In some embodiments the 5' untranslated region and the AUG start region of the mRNA le may optionally be left unhybridized. Following translation initiation, the unwinding activity of the ribosome x can function. even on high affinity duplexes so that ation can proceed. (Liebhaber. J. Mol. Biol. 1.992;.226: -' -2—‘l’3;'l\'lonia,- eta]; J Biol Chem. 1993; 4'Sl4-22._)‘ - » It will be understood that any of the above described methods for enhancing p _ -.__l the stability omeNA may be‘used either alone or in eombination with one e ,- of any of the other'aboveidescribedmethods and/or compositions.- .

The mRNA ofthe present invention may be optionally combined with a . ‘reporter gene (e.g.,. upstream or ream ofthe coding region ofthe mRNA) > which, for example, facilitatesthe determination ofmRNA delivery to the target cells or tissues. Suitable reportergenes may include, for e, Green scent Protein mRN’A '(GFP mMA), Rem‘lla Luciferase mRNA (Luciferase rn'RNA), Firefly Luciferase mRNA, or any Combinations thereof. For example, GFP mRNA may be fused With a mRNA' encoding a secretable protein to facilitate confirmation ofmRNA localization in the target cells that will act as a. depot for protein tion.

As used herein, the terms “transfect” or ‘transfection” mean the intracellular uction of a mRNA into a cell, or-preferably into a'target- cell. The introduced mRNA maybe stably oritransiently maintained in the target. cell. The term “transfection efficiency” refers 'to the relative amount ofmRNA taken up by the target cell which is subject to transfection. In practice, transfection efficiency is estimated by the amount of a reporter c acid product expressed 'by the, target cells following transfection. Preferred ments inelude compositions with high transfecti‘on efficacie's and in particu‘larthose compositions that‘rn'inimize e effects which are mediated by‘transfection ofnon-target cells; The compositions of the present invention that demonstrate high transfection efficacies improve the likelihood that appropriate dosages of thermRNA will be delivered to the target cell, While minimizing potential systemic adverse s. In one embodiment of the present invention, the transfer vehicles of the present invention are capable of ring large mRNA sequences (leg, mRNA of at least lékDa, , 2 kDa, 2.5kDa, SkDa, lOk'Da, l2kDa, lSkDa, 20kDa, 25kDa, SOkDa, or more). The mRNA ‘ can be formulated with one or more acceptable reagents, which provide a vehicle for delivering such mRNA to target cells. Appropriate reagents are generally selected with regard to a number of factors, which include, among other things, the biological or chemical properties of the mRNA, the intended route of administration, the - anticipated biological environment to which such mRNA will be exposed and the Specific properties of the intended target cells, In some embodiments, transfer vehicles, such as liposomes, encapsulate the mRNA'without compromising ical aetiv'ity. 'ln'some embodiments, the transfer vehicle demonstrates preferential and/or ‘ ntial bindingzto'a'target cell» relative to non—target cells. In :a preferred ”embodiment, the transfer vehicle delivers ntents to the target cell such that-the:- ;.- - 'm‘RNA are delivered to thesappropriate subcellular compartment, such as the, ’ cytoplasm. ' ‘ [Transfer Vehicle ' In embodiments, the transfer vehicle in the compositions of the invention is a liposomal er vehicle, e.g. a lipid nanoparticle. In one embodiment, the transfer vehicle may be selected and/or prepared to optimize ry of the mRNA to a target cell. For example, if the target cell is a cyte the properties of the transfer vehicle (e. g., size, charge and/or pH) may be optimized to effectively deliver such transfer vehicle to the target cell, reduce immune clearance and/or promote retention in that target-cell. Alternatively, if the target cell is the centralnervous system (eg, 'm'RNA administered for the treatment ofneurodegenerative diseases may cally target brain or spinal tissue), selection and preparation ofthe transfer vehicle must consider ation of, and retention Within the blood brain barrier and/or the use of alternate means of directlydeliverng such transfer-vehicle to such target cell. In one embodiment, the compositions of the t invention may be combined with agents that facilitate the transfer of exogenous mRNA (e.g” agents which disrupt or improve the permeability of the blood brain barrier and thereby enhancethe transfer of exogenous mRNA to the target cells").

The use of liposomal er vehicles to facilitate the ry of nucleic acids to target cells is contemplated by the present invention. Liposomes (cg, liposomal lipid rticles) are generally useful in a variety of applications in research, industry, and ne, particularly for their use as transfer vehicles of diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol,, 16.: 307-321, 1998,; Drummond at £21., Pharmacol. Rev, 51: 6914743, 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 rs. B'ilayer membranes of liposomes. are typically formed by amphipln'lic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains ('Lasic, Trends Biotechnol., 16: 307—321,, 1998). Bilayer membranes ofthe liposomes can also 2012/041724 be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etct). ' ' In the contextfof- the t invention,-a mal transfer vehicle typically p :-_ ‘ serves to transport'thefimRNA to the target cell.

For'the purposes ofthe present - , invention, the lipo‘soma‘l transfer vehicles are prepared‘to contain the desired nucleic . acids. The process of incOIporation of a desired entity (e.-g., a nucleic acid) into a ‘ liposome is often referred ‘to as “loading” (Lasic, 61'an FEES-Leta, 3 12: 255-25 8, 1992). The liposome-incorporated nucleic acids may be completely or partially located in the interior space ofthe liposome, within the bilayer ne of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into .liposomes is also referred to herein as ‘:‘encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome. The e of incorporating a mRNA into a transfer vehicle, such as a liposome, en to protect the nucleic acid from an environment which may contain enzymesor chemicalsthat degrade nucleic acids and/or systems or receptorsthat-cause the rapid excretion. ofthe nucleic :acids. ingly, in a preferred embodimentofthe present invention, the selected transfer vehicle is e of enhancing the stability 'of’the mRNA contained n. The liposome can allow the encapsulated mRNA to he target cell and/or may preferentially allow the encapsulated mRNA to reaCh the target cell, or alternatively limit the delivery of such mRNA to othersites or cells where the presence of the administered mRNA maybe useless or undesirable. Furthermore, orating the mRNA into a transfer vehicle, such'eas for example, a cationic liposome, also facilitates the delivery ofsuch mRNA into a target cell.

Ideally, liposomal transfer vehicles are prepared to encapsulate one or more desired mRNA such that the compositions demonstrate a high'transfection efficiency and enhanced ity. While liposomes can facilitate introduction of nucleic acids into target cells, the addition of polycations (e.g., poly L—lysine and protamine), as a copolymer can tate, and in some instances markedly enhance the transfection efficiency of several types ofcationic liposomes by. 2—28 fold ina number of cell lines both invitro and'in vivo. (See NJ. , at 611., Gene Ther. 1995:; 2: 603;..8. - Li, at £21., Gene Ther. 1.997; 4, 891.) Lipid Nanogarticle.r In a preferred embodiment ofthe present ion, the transfer vehicle is formulated as a lipid lnanoparticl‘e. As‘ used herein, the phrase ‘ilipid nanoparticle”, refers to altransfer-yeh'icle comprising one or more. lipids (e.g., cationic , non— -‘ cationic lipids, and PEG-modifiedlipids). Preferably, the lipid ,nanoparticles are formulated to deliver- one or more mRNA to one or more target cells. Examples of- , suitable-lipids include, for example,"the phosphatidyl compounds (e.g.., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, osides, and gangliosides). Also , contemplated is the use ofpolymers as transfer es, whether alone or in combination With other er vehicles. Suitable polymers may include, for example, .polyacrylates, :polyalkycyanoacrylates, pol-ylactide, polylactide- polyglycolide copolyrners, polycaprolactones, dextran, albumin, 5gelatin,’alginate, collagen, an, cyciodextrins, dendrimers and polyethylenimine. Incne embodiment, the transfer vehicle is selected based upon its y to facilitate the transfection of a mRNA to a target cell.

The invention contemplates the use of lipid nanoparticles as transfer 'vechicles comprising a cationic lipid to encapsulate and]or enhance the delivery ofmRNA into the target cell that will act as a depot for n production. As used herein, the phrase “Cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected ch,as physiological pH. The contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios ing one or more cationic lipids, non~cationic lipids and PEG- d lipids. l cationic lipids have been described in the literature, many of which are commercially available.

Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publication W0 2010/053‘572, incorporated herein by reference, and most ularly, (312-200 described at paragraph [00225] of ’72. in certain embodiments, the compositions and methods ofthe invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in US. ional patent application 61/617,468, filed March 29, 20.12 (incorporated herein by reference),‘such as, e.g, - (152,1 8Z)~N,N—.dimethy_l~6-(9Z, l 2Z)-octade.ca-9., l 2—dien- l ~yl)tetracosa—l5, l 8-dien—l - amine 00), ( l 5Z, l 8Z)-N,N—dimethyl~6—(V(9Z, l 2Z)~octadeca—9, l 2—dien—l - yl)tetracosa—4,15,18-trienamine 01 ), and (.l SZ,18Z)-N,N~dimeihyl—6~ ((9Z,12Z)-octadeca—9,12-dien~1«yl)tetracosa—35,zl5 ,1 8—trien-l ~amine (HGTS 002).. ~ , in some embodiments, the cationiclipid N—‘ll{12,3-dioleyloxy)p_ropyl]-ljl,N,N—. ; trimet'hylammonium chloride Or “DOTMA” is used. . (Felgner 92‘ al. (Proc. Nat’l Acad,“ - Sci. 84, 7413(1 987); U‘.S.‘ Pat-:-No'.‘4,8‘97,35'5).>.'DOTMA can be formulated alone or can be combined with the neutral lipid, ylphosphatidyl-ethanolamine or, ,“DOPE” or other cationic or non—cationiclipids into a mal transfer vehicle or a lipid nanoparticle, andsuch mes can be used to enhance the delivery o‘fnuc‘leic acids into target cells. Other suitable cationic lipids include, for example, 5,— yspermylglycinedioctadecylamide or “DOGS,” 2,3-dioleyloxy—.N—.[2(spennine- - car‘boxamido)e‘thyl]—N,N-dimethyl—l-propanarninium or “DOSPA” (Behr et al. Proc.

NatL'l Acad. Sci. 86, 69.82 (1989).; US. Pat. No. 5,171,678; US. Pat. No. 5,334,761), 1,2vDioleoyl-3—Dimethylammonium—Propane or ”, l.,'2-Didleoyl—3- Trimethylammonimn—Propane or"‘-DOTA.P”. Contemplated cationic lipids also include 1,2-distearyloxy—NN-dimethfl-B—aminopropane or “-D-SDMA’-?, 1,2— dioleyloxy-N,N—dimethy.l—3-aminop1‘opaner or “DODMA”, 1,2—dilinoleyloxyéN,N— dimethyl:3-..aminopropane or “DLinDMA”, 1,2-dilinolenyloxy—N,N-dimethyl:3- =am'inopropane or “DLenDMA”, N-dioleyl-N,N-dimethy1ammonium de or “DODAC”, fiN—distearyl—N,N—dimethylammonium bromide or “DDAB”, N—(1,2- dimyristyloxyprop—3—-y'l)-N,N~dimethyl-N4hydroxyethyl ammonium bromide or “DMRIE”, 3-dimethylamino-B-(‘cholest—5 senbeta~oxybutanoxy)—1-(ci s,cis—9,1'2.— octadecadienoxy)propane or “CLinDMA”, 2-[5 ’~(cholest¢5-en—3—beta—oxy)~_3 ’— oxapentoxy)dimethy 'l-l-(cis,¢is-9", 1—2 ’«octadecadienoxy)propane or “CpLinDMA”, methyl-3,4-dioleyloxybenzylamine or ‘5DMO'BA’7, l,2—N,N’— charbamyl-B-dimethylaminoprcpane or “DOcarbDAP”, 2,3—Di1inoleoyloxy— N,N-dimethylpropylamine or ‘fDLinDAP”, l,2-N,N1—Dilinoleylca1'bamyl-3— dimethylaminopropane or carbDAP”., 1,2:Dilinoleoylcarbamyl dimethylaminopropane or “DLinCDAP”, 2,2-dilinoleyl—4—dimethylaminomethyl- _ [1.,3]—dioxolane tor “DL'in—K-DMA'”, 2,2-dilinoley-l~4-dimethylaminoethyl—[ 1 ,3} dioxol'ane or ‘fDLin-KaXTCZ—D'MA”, and 2—(2,2-di((9Z,1ZZ)-octadeca—9,12-dien—l- yl)—l ,3 —dioxolan—4—yl)~N,N-dimethylethanam‘ine KCZ-DMA» (See, WO 2010/042877; .Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof.

(Heyes, 1., at 611., J Controlled Release 107: 276-287 (2005); Morrissey, DV., er al., Nat. Biotechnol. 23(8): 11003—1007 (2005); PCT Publication WO2005/121348Al).

The use of cholesterol-based cationic lipids is also contemplated by the - 'preSent- invention. Such cholesterohba'sed"cationic lipids can be used, either aloneor " incombination with. other'cationicor nonecatio‘nic-lipids. Suitable cholesterolrbased- cationic lipids include, for ‘exarnple,’DC-.—thol (N;N-dimethyl—N-- ethylcarbOXamidocholesterol)“, l.,'4-’bis('3 —N—oleylamino—propyl)piperazine (Gao, et al; . B'ioch'em; Biophys'ReS. Comm. "179-, 280 (1991); Wolf etal.rBioCEechniques.23l-,13-9 » (19.97); U.S. Pat. :No. 5,744,335.),cr1CE. - in addition, several ts are commerciallyavailable to enhance _' ’ transfection efficacy; Suitable examples include'LIPOFECTIN (DOTMAzDOPE) - (Invitrogen, Carlsbad, ), LIPOFECT‘AMINE DOPE) (lnvitrogen), CTAMNEZOOO. (Invitrogen‘), 'FUGENE, TRANS'FECTAM (DOGS),- and EFFECTENE.

"Also contemplated are cationic lipids such as the dialkylaminOebased, imidazole-based, and imum—based . For e, certain embodiments are directed to a composition comprising one or more imidazole-based cationic lipids, for example, the imidazole cholesterol ester or “ICE” lipid (3'8, 11,0R, 13R, 17R)-10, methyl-l7-((R)<64methylheptan+2-yl§)~2, 3, 4, 7, ‘8, 9, 10, ll, 12, 13., 14, 15, 16., 17-tetradecahydro-lH-cyclopenta[sa]phenanthren—3eyl 3~(ilHeimidaZOl-4— :yl)propanoate, as represented bystructure (l) below. In a preferred embodiment, :a transfer vehicle for delivery ofmRNA may comprise one or more ole—based cationic lipids, for example, the imidazole terol ester or “ICE” lipid (38, 1-011, 13R, 17R)~10, ‘l3~dimethyl—'l7—((R)-6—methylheptan-2—yl)—2, 3, 4., 7, 8, 9, 10, ll, '12, 1,3,, '14, 15, ‘l 6, l7—tetradecah-ydro—‘l H-cyclopenta['a]p'henanthremB~yl 3~(lH-imidazol~ 4—yl)propanoate, as represented by structure (1).

Without wishing to be bound by a particular'theory, it is believed that the fusogenicity of the imidazole-based cationic lipid ICE is related to the endosomal disruption which is facilitated by the imidazole grOUp, which has a lower pKa relative to traditional 2012/041724 ic lipids. The endosomal disruption in turn promotes osmotic swelling and the disruptionof-the lipo-‘somal membrane, followed by nsfection'or intracellular . e ofthe nucleicxacid(s). contents loaded therein into the targetcell.

Theimidazoleébasedreationic. lipids are also characterized by their reduced " toxicity relative to othercationicliipidsi The imidazoleAbased cationic lipids -(e.g., ICE) may be used 1as *the sole cationic lipid in'the lipidnanoparticle, or alternatively , may be combined with traditional cationic lipids, tionic lipids, and PEG- modified lipids. The cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2%‘toabou’c 70%, about 5% to about 50%, about 10% to about 40% of » the total lipid present in the transfer vehicle, or preferably about 20% to about 70% of the total lipid present in the transfer vehicle.

Similarly, certain embodiments are ed to lipidnanoparticles comprising the HGT4003 cationic lipid .2'~((2,‘3 ~Bis(.(9Z, l tadeca~‘9, l'Z-dien-l- yloxy)propy.l-)disulfanyl):N,N—dimethylethanamine, as represented by structure (II) below, and as further described in US. Provisional Application Noz6‘l /494,745, filed June 8, 2011, the entire teachings of which are incorporated herein by reference in their entirety: In other embodiments the compositions and methods bed herein are directed to lipid nanoparticles comprising one or more cleavable lipids, such as, for example, one or more cationic lipids or compounds that comprise a cl'eavable disulfide (S—S) functional group (egi, .HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as further described in US. Provisional Application No: ,745, the entire ngs of which are incorporated herein by reference in. their entirety.

The use ofpolyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized cerarmides (PEG-GER), including N-Octanoy'l— Sphingosine-l-4[Succinyl(Methoxy Polyethylene Glycol)—2000] (C8 PEG-2000 , ceramide) is also plated by the present invention, either alone or preferably in combination with other lipids together which comprise the transfer vehicle (e.g., a lipid nanoparticle). Contemplated PEG-modified lipids e, but is not limited to, WO 70930 a polyethylene glycol chain of up to '5 kDa in length covalently attached-to a lipid with'alkyl chain(s) of o length; 'Theaddition of such componentsmay prevent» ' ’s'complexaggregation and may also provide :a means .forflincreasing circulation lifetime; and increasing the delivery ‘oftheelipid-nucleic acid ition to the target cell, ., ;- .

(Kli'banov etch-(1990) FEBS :Letters,.268 (1): 7), or they maybe selectedito; :. ' rapidly exchangeout ofthe ation in viva-(see US. Pat. No.»5,8v85,613).

Particularly useful eXChaiigeable lipids are PEG-.ceramides having shorter acyl chains , (erg; C14 "or C18); The PEG-modified olipid and derivitized lipids o'f‘the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.

Thezpresent invention also contemplates the use of non-cationic lipids. As used herein, the phrase “non~cationic lipid” refers to any neutral,.zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any ofa number of lipid species that carry a not ve charge at a selected pH, such as physiological pH. Non—cationic lipids include, but are not d to, distearoylphosphatidylcholine ), ‘dioleoylphosphatidylcholine , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), d'ipalmitoylphosphatidylglycerol (DP'PG'), ylphosph‘atidylethanolamine (DOPE), palmitoyloleoylphosphatidy‘lcholine (POP‘C), palmitoy'lo‘leoyl— phosphatidylethanolamine (POPE), dioleoyl—phosphatidylethanolam'ine 4—(N- maleimidomethyl)-cycloheXane-liecarboxylate (DOPE—mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), di-stearoyl~ phosphatidyl—ethanolamine (DSPE), lé-O—monomethyl PE, l6TO-dimethyl PE, 18—1- trans PE, 1~stearoyl—2~01eoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. Such non~cationic lipids may be used alone, but are ably used in combination with other excipients, forexample, cationic lipids. When used in combination with a cationic lipid, the non—cationic lipid may comprise a molar ratio of . % to about90%, or preferably about 10 % to about 70% ofthe total lipid presentin 4, the transfer vehicle.

Preferably, the transfer vehicle (e.g., a lipid nanoparticle) is prepared by combining multiple lipid and/or polymer components. For example,'a:transfer vehicle may be prepared using .C12--.200, DOPE, ch01, DMG-PEGZK at a molar ratio of 40302255, or DODAP, DOPE, cholesterol, DMG—PEGZK at a molar ratio of' WO 70930 li8:’56:-20:6, 0r HGTSOOG, DOPE, chol, DMG-PEGZK at a molarratio of 40:20:35.:5, or HGTSOO‘l, DOPE, chol, EGZK 'atamolar ratio of 4012023535. The; ‘ selection of cationic ,'nongcationic-g‘lipids and/oz‘iPEG—modified lipidswhic'h; 2 comprise the lipidznanoparticle, as=well as the relativesmolar ratio of‘such lipids to . each other,‘is based upon thecharacteristics oftheselected lipidCs), the nature o-f-the , * intended target: cells, the characteristicsof the .mRNA to be delivered. Additional considerations include, for example, the saturation of- the alkyl chain, as well, as the , ' size, charge, pH, pKa, fusogenicity and toxicity ofthe ed l'ipid(s). Thus the molar- ratios may be adjusted accordingly. For example, in embodiments, the percentage of cationic lipid in the lipid nanoparticle may be» greater than l0%, greater than 20%, greater than 30%, greater than 40%, greater than. 50%, greater-than 60%, or greaterthan 70%. The tage of tionic lipid in the. lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than3 0%, or greater than 40%. The percentage of cholesterol in the lipid nanoparticle.may be r than %, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipid in the lipid nanoparticle may be greaterthan 1%, greater than 2%, greater than 52%, greater than 10%, or greater than 20%; In certain preferred embodiments, the lipid nanoparticiles ofthe invention comprise at least one ofthe following cationic lipids:_:C12:200, DLin-KCZéDMA, DODAP, 3, ICE, HGTSOOO, or 'HGTSOOI. In embodiments, the transfer vehicle comprises cholesterol and/or a PEG—modified lipid. In some: embodiments, the transfer vehicles comprises DMG—PBGZK. In certain embodiments, the tranfer vehicle comprises one-of the folloWing lipid formulations; C'l’2-200, DOPE, chol, DMG—PEGZK; DODjAP, DOPE, terol, DMG—PEG2K; HGTS000, DOPE, c'hol, DMG-PEGIZK, HGTSOO'l, DOPE, c'hol, DMG—PEGZK.

The liposomal transfer es for use in the compositions of the invention can be prepared by various techniques which are presently known in the art. Multi~ lamellar vesiCles (MLV) may be prepared tional techniques, for example, by depositing a selected lipid on the inside wall of a suitable ner or vessel by dissolving the lipid in anappropriate solvent, and then ating the solvent to leave a thin film on the inside ofthe vessel or by spray drying. An aqueous phase ‘may then added to the vessel with a vortexing motion which results in the formation of MLVs. Uni-lamellar es (ULV) can then be formed by homogenization, sonicat-ion or extrusion of the multielamellar vesicles. In addition, ellar vesicles Canbe formed by: detergent removal techniques, ' = in certain embodiments of this ion, the compositions of the present invention comprise a=transfer ewherein the mRNA is associated (on: both the._, _: surface ofthe erivehicle and encapsulated within the same trans'fervehicle.For- "eXam‘ple, during preparation of the-compositions ofthe presentinventidn, cationic omal transfer es may associate with the mRNA through electrostatic . interactions; L In certain embodiments, the compositions ofthe invention may be loaded with diagnostic radionuclide, fluorescent materials or other materials that are detectable in both in vitro and in vivo applications. For example, suitable sticmaterials for use in the present invention may e Rhodamine-dioleoylphospha- tidylethanolamine (Rh-PE), Green Fluorescent Protein mRN‘A (GFP mRNA), Rem’lla Luciferase mRNA and Firefly Luciferase mRNA.

Selection of the appropriate size of a mal transfer vehicle must take into consideration the site of'the target cell or tissue and to some extent the application for . which the liposome is being made. In some embodiments, itmay be desirable to limit transfection of the mRNA to certain cells or tissues. For. example, to target hepatocytes a mal transfer vehicle maybe sized such that its dimensions are smaller than the fenestrations ofthe endothelial layer lining hepatic sinusoids in the liver; accordingly the liposomal transfer vehiCle can readily.penetrate such endothelial fenestrations to reach the target hepatocytes. Alternatively, a-liposomal er vehic1e may be sized such that the dimensions cfthe liposome are of a sufficient diameter to limit or expl'essly avoid distribution into certain cells or tissues. For example, a liposomal er vehicle may be sized such that its dimensions are larger than the fenestrations ofthe endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomal transfer vehicle to hepatocytes. Generally, the size of the transfer vehicle is within the range of about 25 to 250 nm, prefereably less than about: . 250nm, 175nm, 150nm, 125nm, ‘l 00nm, 75mm, 50mm, 2'5nm or lOnm.

A variety rnative methods known in the art are available for -of a population of liposomal transfer vehicles. One such sizing method is described in'U.S.

Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension“ either by bath or probe sonication produces a ssive size reduction , down to small ULV less than about 0.05 microns in diameter. 'Homogenization is another method that relies on. shearing energy to fragment large liposomes into . smaller ones. ’In atypicalhomogenization procedure, MLV are recirculated through a - rd emulsion homo‘geni-zer-until. selected lipdsbmesizes, typically betweenabout 0.1 and 0.5 microns, are observed. The size of the.~lipo‘somal vesicles may be detennined'by'quaSi—electric light ring (QELSr) as. bed in Bloomfield,-Ann.~.

Rev .' Biophys'. Bioeng, 1:0:421—‘450 (1981), incorporated hereinfby reference: Average ‘ome-di-ameter may be reduced :by sonication of formed liposomes.. lntermittentsonication cycles maybe alternated with QELS assessment to guide efficient liposome synthesis. - > Target "Cells As used herein, the term “‘target-cell” refers to a cell or'tissue to which a composition of the'invention is to be directed or targeted. In some em‘bodiments,‘the target cells are nt 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 compositions ofthe invention transfect the target cells on a discriminatory basis (i.e., do. not‘transfect nonatarget cells). The compositions of the invention may also beprepared to preferentially target a variety of‘target cells, which include,but :are not. limited patocytes_, epithelial cells, . hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, hymal cells, neural cells (cg, meninges, astrocytes, motor neurons, cells ofthe dorsal root ganglia and anterior hommotor neurons), photoreceptor cells (e.:g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, ar smooth muscle cells, cardiomyocytes, al muscle cells, beta cells, pituitarycells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, locytes, leukocytes, granulocytes and tumor cells.

The compositions of the invention may be prepared to preferentially distribute to target cells such as in the heart, lungs, kidneys, liver, and spleen. In some embodiments, the compositions of the invention distribute into the cells of the liver to facilitate the deliveryand the uent expression of the mRNA comprised therein by the cells of the-liver (e.g., hepatocytes). The targeted hepatocytes may function as a biological “reservoir” or “depot” capableof ing, and systemically ing a functional protein or-enzyme. Accordingly, in one embodiment ofthe invention the liposomal er vehicle may target hepatocyes and/or preferentially distribute to the cells of the liver upon delivery. Following-transfection ofthe target hepatocytes, the . mRNA loaded in the liposomalvehicle are translated and a functional protein producit~ . 'is produCed, ed and systemicallydistributed. .In other emodiments, cell-s other; than hepatocytes (e.g.’,.-lung,..spleen, heart, ocular, or cells of the central nervous - ._ ) can. serve as adepot location-for protein production.

' 'In' one. embodiment, the: compositions of the ion tate a subj ect-‘s 1 endogenous, production of one or more functional ns and/or enzymes, and in particular-the production of proteins and/or s which demonstrate less . immunogenicity relative to their recombinantly—prepared counterparts, In a preferred embodiment ofthe present invention, the transfer vehicles comprise mRNA which encode adeficient proteinor enzyme. Upon distribution of such compositions to the target tissues and the subsequent transfection of such target cells, the exogenous V mRNA loaded into theliposomal transfer vehicle (eg, a lipid nanoparticle‘) may be translated in viva to produce a functional. protein or enzyme encoded by the exogenousl‘y administered mRNA (e.g., a protein or enzyme in whichthe subject is deficient). Accordingly, the compositions of the present ion exploit a subj ect’s ability to translate exogenously— or inantly—prepared mRNA to produce an nously—translated protein or enzyme, and thereby produce {and where applicable excrete) a. functional protein or enzyme. The expressed or translated proteins or enzymes may also be characterized by the in viva inclusion of native post— translational modifications WhiCh may. often be absent in recombinantly~prepared proteins or enzymes, thereby further reducing the. irnmunogenicity of the translated protein or enzyme.

The stration ofmRNA encoding a deficient protein or enzyme avoids the need to deliver the c acids to specific organelles within a target cell (cg, mitochondria). Rather, upon transfection of a target cell and delivery of the nucleic acids to the cytoplasm of the target cell, the mRNA contents of a ertvehicle may be translated and a functional protein or enzyme expressed.

Thepresent invention also contemplates the discriminatory targeting oftarget cellsand tissues by both passive and active targeting means. The phenomenon of . passive targeting exploits the natural distributions patterns of a transfer e in viva- without relying upon the use of additional excipients or means to enhance recognition- of the er vehicle by target cells. For example, transfer es which are ‘ subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide means to ely direct thei'deliVeriy ofthe Compositions to such target cells. » -_ _ Alternatively, thejpresent invention contemplates active targeting-which involVeS-the use of additional. excipients,’ referre'dftoiherein as “targeting ligands”? that - may be bound'(either covalently or 110n4covalently) to the transfer vehicle to , _- ., . , enCoura‘ge Zation h' transferyehicle at certain target cells or target tissues; . ,5 For example,”targeting may be mediated by ”the inclusionof one or more nous targeting ligands (e.g., apolipoprotein E) in or-on the transfer vehicle to encourage . .- bution to the target cells or'tissues. Recognition of the targeting ligand by the target s actively facilitates tissue distribution and cellular uptake of the transfer vehicle andlor its contents in the target cells and tissues (e.g.,-the inclusion of an apolipoproteian targeting ligand in or on the transfer vehicle- encourages recognition and binding of the transfer vehicle'to endogenous low density lipoprotein receptors expressed by cytes). As ed herein, the composition can comprise a ligand capable of enhancing affinity ofthe composition to the target cell. ing ligands, may: be linked to the outer bilayer- of the lipidjpartieleiduring formulation or post—formulation. These methods are well known in the art. In addition, some lipid particle fonnulat-ionsrmay employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see US, Patent ation Ser. Nos. 08/835,281, and 60/083,294, which are incorporated herein by reference) and other features useful for in vivo and/or intracellular delivery. In. other some embodiments, the compositions of the t invention demonstrate improved transfecti'on ies, and/or demonstrate enhanced selectivity towards target cells or tissues of'interest. Contemplated therefore are itions which comprise one or more ligands (cg, peptides, aptamers, oligonuCleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or s. Suitable ligands may optionally be bound or linked to the e of the transfer vehicle. In some embodiments, the targeting ligand may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle.

Suitable ligands and are selected based upon their physical, al or biological properties (e.g., selective affinity and/or recognition of target cell surface markersor features.) Cell—specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting s are selected such that the unique characteristics of a target cell are-exploited, thus allowing the composition to discriminate between ‘target and rget cells. For example, compositions of the invention may include surfacemarkers '(e.g., "apolipoprotein-B or-apolipopro‘teimE) that selectively enhance,_ recognition of, or- affinity-to hepatocytes. (e. g, by receptor-mediated recognitionof' ; . and binding to suchsurface markers"); . Additionally,,the use ofgalactoseaSatargeting . ligand would be expected to. direct/rhe- compositions ofthe present invention to . parenchymal'hepatocyte’s, :or alternatively. theme of mannose containing sugar, ' residues: as a targetingligand would be expected to direct the compositions of the present invention to liver-endothelial cells , mannose containing sugar residues - that may bind entially to the asialoglycoprotein receptor-present in'hepatocytes).

(See Hillery AM, et a]. “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002) Taylor '& Francis, Inc.) The presentation of such - ; ing ligands thatihave been conjugated to moieties present in the er vehicle (cg, a lipid nanoparticle) ore. facilitate recognition and uptake of the compositions of the present invention in targetcell-‘s and tissues. Examples of suitable ing ligands include one or more peptides, proteins, aptarners, vitamins and oligonuoleotides. tion. inistmtion As used herein,'the term “subject” refers to any animal (egg, a mammal), including, but not limited to, humans, nonhuman primates, rodents, and the like, to which the compositions and methods of”the present invention are administered.

Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

The itions and methods of the invention provide for thedelivery of mRNA to treat .a number of disorders. In particular, the compositionsand methods of the present ion are le for the treatment of diseases or disorders relating to the deficiency of proteins and/or enzymes that are. excreted or secretedby the target cell into the surroundingextracellular fluid (e.g., mRNA encoding hormones and neurotransmitters). ‘ In. embodiments the disease may involve a defect or deficiency in a secreted protein (e.-g. Fabry disease, or ALS). In certain embodiments, the e may not becaused by a defect or deficit in a secreted protein, but may benefit from providing a secreted protein. For example, the symptoms of a disease may be improved by providing the compositions of the invention (e.g. cystic fibrosis).

Disorders for which the t invention are useful include, but are not limited to, WO 70930 disorders such as Huntington’s Disease; Parkinson’s Disease; muscular phies (such as, e.g.'Duc-henne and Becker); hemophelia es (suchvas, e.g., ,hemoph'ilioa B ,__(_FIX),' hemophilia A "(FVIH'X SMNl—relatedspinalmusciilar y: (SMA); .- j‘ -‘ophic lateral sclerosis (AL-S); G-AL‘T—related galactosemia; Cystic Fibrosis.

' (CF);VISLCBAl'Ar'elated- ers-includingcystinuria;‘COL4A5-related disorders including Alport‘synd'mme; galactocerebrosidase deficiencies; X~linlced adrenoleukodyst-rophy and adrenomye'loneuropathy; ‘ Friedreich’s ataxia; Pelizaeus— Merzbaclier disease; T801 and TSCZ—rel‘ated‘tuberous sclerosis; Sanfilippo B - - , .. syndrome (MPS RIB); CTNS-related osis; the FMR] ~related disorders which . include Fragile X syndrome, Fragile X~Associated Tremor/Ataxia Syndrome and .

Fragile X Premature Ovarian Failure Syndrome; ~Willi syndrome; hereditary. hemorrhagic telangiectasia (AT); Niemann-Pick disease Type Cl; the. neuronal ceroid lipofusCinoses-related diseases including Juvenile Neuronal Ceroid Lipofuscinosis , 1uvenil.e.:Batten disease, SantavuorieHaltia disease, ~Bielschowsky disease, and PT‘T—l and TPPl encies;- EIFZBl , EIFZBQ, B, EIF2B4 and 'EIF2B54related childhood ataxia with central nervous system hypomyelination/vanishing white ; CACNAI'A and CACNB4-related Episodic Ataxia Type 2'; the MECPZ-related disorders including Classic Rett Syndrome, MECP2~related Severe Neonatal Encephalopathy and PPM-X Syndrome; CDKLS- related Atypical Rett Syndrome; Kennedy’s disease (SBMA); Notch-3 related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SONIA and SCNlB—related seizure disorders; the Polymerase G-related disorders Which include -HutteriloCher syndrome, POLG- related sensory ataxic neuropathy, dysarthria, and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with mitochondrial DNA deletions; X—Linked adrenal 'hypoplasia; X-linked agammaglobulinemia; Wilson’s disease; and Fabry Disease. In one embodiment, the nucleic acids, and in particular mRNA, ofthe invention may encode functional proteins or enzymes that are secreted into extracellular space. For example, the secreted proteins include clotting factors, components‘of the complement pathway, nes, chemokines, ttractants, protein hormones (e.g. EGF, PDF), protein-components of serum, antibodies, secretable toll—like receptors, and others. In some embodiments, the compositions ofthe present- ion may include mRNA encoding erythropoietin, d]~anti‘t1‘ypsin, carboxypeptidase N or human growth hormone.

In embodiments, the invent-ion encodes a ed protein that is made up of ': *subunits‘that‘are- encoded by more than one gene. For example, the secreted protein inlay belaheterodimer, wherein'each chain-or subunit of the is encoded. by a. separate gene; it is possible that’m'ore than one mRNA le is delivered in the transfer vehicle and the mRNA encodes separate subunit of the, secretedproteinm ‘- -- Alternatively, a- single mRNA'may be engineered to encode-more than one subunit. ,_ ._ ‘ (cg. in-the case of a single-chain Fv antibody”). In certain embodiments, te . mRNA‘ molecules encoding the individual subunits may be administered in separate transfer es. In one embodiment, the :mRNA may encodefull length antibodies (both heavy and light chains of'the variable and constant regions) or fragments of. ‘ antibOdies (e.g. Fab, Fv, or a single chain Fv (scFV) 'to confer ty to a t.

While one embodiment ofthe present invention relates to methods and compositions useful for conferring immunity-to a subject (.e.g., via the translation ofmRNA encoding functional antibodies), the inventions disclosed herein and contemplated hereby are broadly applicable. In an ative embodiment the compositions ofthe present ion encode antibodies that maybe used to transiently or cally effect a functional response in subjects. For example, the mRNA present invention may encode a functional monoclonal or po'lyclonal dy, which upon translationand secretion from target cell may be useful for targeting and/or inactivatinga biOIOgical target (e.g., a stimulatory cytokine such as tumor necrosis factor). Similarly, the mRNA nucleic acids of the present invention mayencode, for example, functional anti—:nephritic factor dies useful for the treatment of membranoproliferative glomerulonephriti'sitype II or acute hemolyticuremic syndrome, orsalternatively may encode anti-vascular.endothelial growth factor (VEGF) antibodies useful for the treatment of VEGFemediated diseases, such. as cancer. In other embodiments, the secreted protein is a .cytokine or other secreted protein comprised of more than one subunit (cg. IL~12, or lL~23).

The compositions of the invention can be administered to a t. In some embodiments, the composition is ated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions Where it is mixed with suitable excipients. For eXample, in one embodiment, the compositions of the invention may be prepared to deliver mRNA encoding two or more distinct proteins or enzymes. Techniques for ation and administration of drugs may be found in “Remington’s ‘ Pharmaceutical'SCi‘enCe‘s,”:MackPublishin‘g (301., Easton, Pa, latest edition.- - A‘jwid'e rangeiof'mo'leculesthat can exert pharmaceutical or therapeuticeffects , ~j “canbedelivered into‘target cells using compositions and methodsof the invention, ‘ “The les can be c or inorganic; Organic les canbe peptides,~- :" .; , ’~ proteins, carbohydrates, .1ipids,~sterols, nucleic acidstincluding peptide nucleic acids), .- x or any combination f, A ation for delivery into target cells can comprise - more than one type'ofmolecule, for e, two different nucleotide sequences, or a -- protein, an enzyme or asteroid.

The compositions of the present invention may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subj ect’s age, sex, body weight and other factors releVant to clinicians of ordinary skill in the art. The “effective amoun ” for the purposes herein may- bedetermined by such relevant considerations as are known‘to those of ordinary skill in experimental clinical research, pharmacological, 'clinicalsand medical arts. In some embodiments, the-amount administered is effective to achieve at least some ization, improvement or elimination of symptoms and other indicators as are selected as appropriate. measures of disease progress, regression or improvement by those ofgskill . in'the art. For example, a le amount and dosing n is one that causes at least ent protein production.

Suitable routes of administration include, for e, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or ed, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal,'intranasal, or intraocular injections.

Alternately, the compositions of the invention may be administered in a local rather than ic manner, for example, via ion of the pharmaceutical composition directlyinto a targeted tissue, preferably in a sustained release formulation.‘ Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing compositions of the present ' ion can be. inhaled (for nasal, tracheal, or bronchial delivery); compositions :of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be ed in liquid, tablet or capsule form for ‘ administration tOrthe‘ stomaCh or intestines, can be supplied in suppository form-for _ rectal or vaginal application; or can even be delivered to the eye by use of creams,- , drops, or even injection. Formulations.cOn‘taining compositions of the present , vinventibn‘ complexed with therapeuticmolecules or ligands can even be surgically: administeredgfor example-in association with a r or otherstructure or j .- substance that can allow the compositionsto diffuse from the site of implantation-to surrounding cells; Alternatively, they can-be applied surgically without theuseof polymers or supports.

In oneembodiment, the compositions ofthe invention are ated such that they-are le for extended—release of the mRNA contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in. one embodiment, the compoSitions ofthe present ion areiadministered to a subject twice day, daily or every other day. In a preferred embodiment, the compositions ofthe present invention are. administered to a subject twice a week, gonce a week, every ten days, every two weeks, every three weeks, or morepreferably every=four week-s, once a month, every six weeks, every weeks, every other month, every three ,:every four , every six months, every eight months, ever-y nine months or armually. Also contemplated are compositions and liposomal vehicles which are formulated for depot administration (.e,g,, intramuscularly, subcutaneously, intravitreally) to either deliver or e a mRNA over extended periods of'time. Preferably, the extended-release means employed are combined with modifications made to the mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of theliposoma‘l nanoparticles disclosed herein and related methods for the use of such lyophilized compositions as sed for example, in United States Provisional Application Noel/494,882, filed June 8, 20:11, the teachings of which are incorporated herein by reference in their entirety. For .- example, lyophilized pharmaceutical compositions according to the ion maybe - reconstituted prior to administration or can be reconstituted in viva. For example, a lyophilized pharmaceutical composition can be formulated in an appropriate . form (e.g.,.an intraderrnal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in viva , individual"s bodily fluids. 2012/041724 While certain compounds, compositions and methods of the present invention ' have been‘de‘Scribed With specificity in. ance with certain embodiments,- the -_ following examples Serve’onlytoillustrate the. compounds-of the invention and are; . not intended to limit the same. Each of the publications, reference materials, accession numbers and the like referenced herein to describe-the background of the invention and to e-additional detailregardingits’ practice are hereby orated by - reference in their entirety.

"The articles nd “an” as used herein in the cation andin the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more'than one, or all of the group members are present in, employed in, or ise relevant to a given product or process unless ted‘to the ry or otherwise evident from the context. The invention includes embodiments in Whichexactly one member ofthe group is present in, ed in, or otherwise relevant to agiven product or process. The invention also includes embodiments in which more than one, or-the entire group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all ions, ations, and permutations in which, one or‘more limitations, elements, clauses, descriptive terms, etc., from one or more ofthe listed claims is introduced into another claim dependent on the same base Claim (or, as relevant, any other claim) unless otherwise indicated or unless it would'be evident to one of ordinary skill in the at a contradiction or istency would arise, Where elements are presented as lists, (e. g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any ,element(s) :can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain ments ofthe invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case beenspecifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exClusion is recited in the specification. The publications and other reference materials referenced herein to describe the background ofthe invention and to provide additional‘de‘tail- ing its practice‘are hereby incorporated by reference.

EXAMPLES. ‘ Exam-p161: Protein Production Depot via enous ry of. it, Polynucleotide Composmons MessengerRNA Human erythropoietin (EPO) (SEQ ID NO: 3; , human alpha- 'galactosidase (GLA) (SEQ ID NO: 4; , human alpha—l antitrypsin (Al-AT) - (SEQ ID NO: ‘5; FIG, 5), and human factor IX (FIX) (SEQ ID NO: 6; FIG. '6‘) were ' synthesized by in vitro transcription from a plasmid DNA template encoding the gene, Which Was fellowed by the addition Of a '5’ cap structure (Capl) (Fechter & Brownlee', J Gen. Virology 8621239-1249 (2005)) and a 3’ poly(A) tail of approXimately 200 nucleotides in length as determined by gel electrophoresis. 5’ and 3’ untranslated regions were present in each mZRNA product in the folloWing examples and are defined by SEQ ID NOS; 1 and 2 (and respectively.

LipidNanopartz‘cle Formulations Formulation J : Aliquots of'50 mg/mL .ethanolic solutions ofC 12-200, DOPE, Chol and 'DMG—PEGZK 55) were mixed and diluted with ethanol‘to 3 mL final volume. Separately, an aqueous bufferedsolution (10 mM citrate/150' mM NaCl? pH 4.5,) ofmRNA was ed from a 1 mg/rn‘L stock. The lipid solution was injected rapidly into the aqueous mRNA solution and shakenrto yielda final suspension in % ethanol. The resulting nanoparticle suspension was filtered, diafiltrated with 1x PBS (pH 7.4), trated and stored at 28°C.

Formulation 2: ts of 50 mg/mL lic ons ofDODAP, DOPE, cholesterol and GZK (18:56.:20t6) were mixed and diluted with ethanol to '3 mL finalvolume. Separately, an aqueous'buffered solution (1 OmM citrate/1.50 mM ' NaCl pH 45) ofEPO mRNA was prepared from a ’1 mg/mL stock The lipid solution was injected y into the aqueous rnRNA solution and shaken to yield a final suspension in 20% ethanol The resulting nanoparticle suspension wasfiltered, diai'iltrated with 1x PBS (pH 7.4), concentrated and stored at 2—89C. Final tration = 1.35 ing/mL EPO mRNA (encapsulated). Zavc =, 75 .9‘ nm (DV(50) = 57.3mm; DV(9('))':~92Ql“~I’lII’I).V ‘ Formulation 3: Aliquots-0:550 mg/mL lic solutions of HGT4003,-DOPE, ., teroltand 'DMG—PEG2K (50:25-20:55) were mixed and diluted with ethanol “(03. mL final volurne.‘ ' Separately, anaqueousbuffered solution (.10 mM citrate/150 mM; NaCl, pH 4.5) ofmRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous niRNA solution and shaken to yield a final _ suspension in 20% ethanol. The ing nanoparticle suspension was filtered, . diafiltrated with lx PBS (pH 7.4), concentrated and stored at 2—8°.C.

Formulation 4: ts of 50 mg/mL ethanolic solutions of ICE, DOPE and DMG— PEGZK (70255) were mixed and diluted with l to 3 mL final volume.

Separately, an aqueous buffered on (10 mM citrate/150 mM NaCl, pH 4.5") of mRNA was prepared from a 1 mg/mL stock. The lipid solution was injected rapidly into the aqueousmRNA on and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticlesuspension was filtered, diafiltrated with 1x,PB‘S (pH 7.4), concentrated and stored at 28°C.

Formulation 5;- Aliquots 01°50 mg/niL ethanolic solutions of HGTSOOO, DOPE, cholesterol and DMG—PEG2K (4022013515) were mixed and diluted with ethanol 'to '3 mL final volume. Separately, an aqueous huffered solution (I OzmM citrate/150 mM NaCl, pH 4.5) of EPO mRNA was prepared from a 1 mg/mL stock. The lipid solution wasinjected rapidly into the aqueous mRNA solution and shaken to yield a final rsuspensiOn in 20% ethanol. The resulting nanoparticle suspension was filtered, rated with 1): PBS (pH 7.4), concentrated and stored at 28°C. Final concentration = 1.82 mg/mL EPO 'mRNA (encapsulated). Zavs = 105.6 nm (Dvwoy: 53.7 nm; Dwgoy = 157 nm). ' Formulation 6: Aliquots of 50 ing/mL ethanolic solutions of HGTSOOI, DOPE, cholesterol and DMG-PEGZK‘ (40:20:35 :5) were mixed and d with ethanol 103‘ . mL final volume. Separately, an aqueous ed solution (10 mM citrate/150 mM NaCl, pH» 4.5) of EPO mRNA was prepared from a 1 mg/mL stock. The lipid . solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in. 20% ethanol. The resulting nanoparticle sion was filtered, di‘afiltrated with 1x PBS (pH 7.4), concentrated- and stored at'2‘—:8°C. delivered mRNA-loadednanopartioles » ,- Analysis ofprotein produced vz'a‘inn'avenously i I I Injection ol fl, , .

Studies were med us1ng.male CD-1 mice of- approximately 6-8 weeks of age atthe beginning of each experiment, unless otherwise ted. Samples were - introduced by a single bolus in injection of an equivalent total dose of30—200 micrograms of encapsulated mRNA. Mice were sacrificed and perfused with saline at the designated time points.

Isolation n tissuesfor analysis The liver and, spleen of each mouse was harvested, apportioned into three parts, and stored in either 10% neutral buffered formalin -frozen and stored at Isolation ofserumfor analysis All s were euthanized by C02 asphyxiation 48 hours post dose administration (i 5%) followed {by thoracotomy and terminal cardiac blood collection.

Whole blood (maximal obtainable ") was collected via cardiac puncture on euthanized animals into serum separator tubes, allowed to clotatroom temperature for at least'30 minutes, fuged at 22°C i 5°C at 9300 g for ‘10 minutes, and the serum extracted. For interim blood collections, approximately 40~SQuL of WhOIe blood was collected via facial vein puncture or tail snip. Samples collected from non treatment animals were used as a baseline for comparison to study animals.

Enzyme-Linked [mmunosorbenlA-ssay (ELISA) Analysis EPO ELISA.' Quantification of:EPO n was performed following procedures reported for human EPO ELISAkit (Quantikine IVD, R&D Systems, Catalog # Dep— OO). Positive controls employed consisted of ultrapure and tissue e grade recombinant human erythropoietin protein _(R&D Systems, Catalog # 286—.EP and . 2'87-TC, respectively). Detection was monitored via absorption (450 nm) on a Molecular Device Flex Station instrument._ GLA ELISA: Standard ELISA procedures were followed ing sheep anti- Alpha—galactosidase (3—1188 he capture-antibody with rabbit anti—Alpha: t galactosidase TK—8’8 IgG as the secondary (detection) antibody (Shire Humaanenetic; ; Therapies). 'Horseradish'peroiidase —COnjugated :goat anti~rabbit lgG waspsed. ’ for activation of the 3;,3',‘5,SY-tetramethylbenzidine (TMB) ate solution. The ~ , reaction was quenched using '2N1H280i'after 20 minutes. Detection was monitored via absorption (1150 run) on a Molecular Device Flex Station instrument. Untreated . mouse serum and human Alpha-galactosidase protein were used as negative and positive controls, respectively.

FIXELISA .‘ Quantification ofFIX n was performed following procedures ‘ reported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog # EF1009—1 ).

AJAT ELISA: Quantification ofAlAT protein was performed following procedures reported for lAT ELISA kit (Innovative Research, Catalog #IRAP-KTOl'S').

Western BlatAnalysz’s (EPO): Western blot analyses were performed using an anti-hEPZO antibody (R&D Systems #MA‘B2871). and ultrapure human EPO protein (R&D Systems #286—E-P) as the control.

Results The work described in this example trates the use of'mRNA— encapsulated lipid nanoparticles as a depot source for the production of protein. Such a depot effect can be achieved in multiple sites within the body (i.-e., liver, , - spleen, and ). Measurement of the desired exogenous—based protein derived from messenger RNA delivered via liposomal rticles was achieved and quantified, and the secretion of protein from a depot using human erythropoietin (hEPO), human alpha—galactosidase (hGLA), human alpha—l ypsin '(hAlAT), and human Factor IX (hFlX) mRNA was demonstrated. 1A. In Vivo Human EPO Protein Production'Results The production ofhEPO protein was demonstrated with various lipid nanopaiti‘cle formulations. Of four different ic lipid systems, C200~based ‘ lipid nanoparticles produced the highest quantity? of hEPO protein after four hours post intravenous..admini'stration asmeasured. by ELISA (. This formulation (Formulation 1) ed in. 18.3 ug/rnL *hEPO n secreted into the bloodstream. .

. Normal hEPO protein in-serum- for human 3.3—l.6-.6 mIU/mL (NCCLS g . Document C28—P;-V'ol.—. 12, No. 2). Based on a specific activity of l20,000 IU/mg of ‘ EPO proteins that yields a quantity of 38 pg/mL {hEPO-gprotein in normal human '_ individuals. Therefore; a single 30 ug dose of a CEIZ-ZOO—based cationic lipid , ation encapsulating hEPO mRNA yielded an increase inrespective protein of - . over 100,000—fold physiological .

Ofthe lipid systems tested, the DODAPabased lipid nanoparticle formulation was the least effective. However, the: observed quantity of human EPO protein derived from delivery via a DODAP-based lipid. nanoparticle encapsulating EPO . mRNA was 4.1 ng/mL, which is still greater than 30-fold over normal physiological levels ofEPO protein (Table '1).

" Cattomc/IomzableLlpld f Secreted.“ immlncrease in Component ’E-nca-psu‘lated Human 3 , Hematocrit mRNA (ug) EP0 Protein (%i) in 1le (112-200 , ‘HGT4003 ,. . i 10-0 . ,, , ICE L :DODAP » 200 Table 1. Raw values of secreted hEPO protein for s cationic lipidrbased nanoparticle systems as measured via ELISA analysis (as depicted in . Doses are based on encapsulated hEPO mRNA. Values ofprotein. are depicted as nanogram ofhuman EPO protein per milliliter of serum. Hematocrit changes are based on comparison of pie—bleed "(Day r-l) and Day 10.

In on, the resulting protein was tested to determine ifit was active» and functioned properly. In the case of mRNA ement therapy (MRT) ing hEPO mR‘NA, hematocrit changes were monitored over a ten day period for five different lipid nanoparticle formulations ( Table 1) to evaluate protein activity. During this time period, two of the'five formulations demonstrated an increase in hematocrit (215%), which is indicative of active hEPO protein being produced from such systems.

WO 70930 In another Experiment, crit changes were monitored over a 15—day ~ I -period~(FIG'. 9, Table.='2');_ The lipid articleformulation(Formulation 1):-was, either as iasingle 30 :ug dose,‘oras- three r '10 ug doses ed. on , administered ‘ day 'l-gda'ys3 and day 5. Similarly;Formulation, 2 wasadministered as?) dosesofSOt pg 011 day 1, day ‘3,‘and day 5. 1C 12-200 produced a significant increase in hematocri-t.

- Overall an increase of-up to ~~25 %‘- change was observed, which is indicative of active, haman 'EPO protein beingproduced from such systems. .

Dose , HctLevels Mean (”/0) :ESEM [TestArticle (gig/animal) —-. Day'l'O 'Day 15" (single'dose) sugars 58;3i3.3 62.8:tl.3 :59;9i3i.3 012-200 biz-200 Q30 (over3 doses) 55;3:l:2.3 63,3i'].6 150 (over 3 doses) 54:8i1.7 Hot ; 'hematocrithEM = standard error of the mean.

”Blood s were collected into non—heparinized hematocrit tubes.

Table 2 crit levels of each group‘over a 15 day observation period (F1Gr 9).

Mice were either dosed as a single injection, or three injections, every other day. N=4, mice per group. 13. In Vivo Human GLA Protein Production Results A second exogenous—based protein system was explored to demonstrate the “depot effect” when employing mRNA—loa'ded lipid nanoparticles. Animals were injected intravenously with a single 30 microgram dose of encapsulated human alpha— galactosidase (hGLA) mRNA using a C12based lipid. nanopart-icle system and sacrificed after six hours (Formulation I). Quantification of secreted hGLA protein was performed via ELiSA. Untreated mouse serum and human Alpha-galactosidase protein were used as. controls. Detection of human alphaagalactosidase protein was monitored over a 4.8 hour period; Measurable levels ofhGLA protein were observed throughout'the time course ofthe experiment witha maximum level of 2.0 ug/mL hGLA protein at six hours (). Table 3 lists the specific quantities of'hGLA found in the serum. Normal ty in y human males has been reported to be approximately 3.05 nanomol/hr/mL. The ty for Alpha~galactcsidase, a recombinant human alpha- galactosidase protein, 3.56 x 1.06 l/hr/mg. Analysis of these values yields a ty of approximately 856. pg/mLof hGL-A‘pro‘tein in normal healthy male ;_ . individuals. ~ The quantity of 2;O ug/mLhGLA protein observed after six hours. when dosing ahGLA inRNA-loaded lipid nanoparticle is Over fold greater than: normalphysiOlcgical levels. >Furthei‘;.1after 48 hours, one can still detect appreciable; ~- levels of liGLA protein (86.2 ng/mL).' This level is entative of almost IOU—fold greater quantities ofhGLA protein over physiological amounts still present at .48 hours. ‘ Time Secreted Human - Protein (ng/mL) , “Post-Administration (hrlmmfl ‘ GLA W ‘6 . 2,038 IIIIIIIIIIIII _ “ ’ 12 1,815 2:4 414 .. l Table 3. Raw values of secreted hGLA protein over time as measured via ELISA analysis (as ed in ). Values are depicted as nanogram of hGLA. protein per milliliter of serum. N=4 mice per group.

In addition, the half-life of Alpha-galactosidase when administered at 0.2 mg/kg is approximately 108 minutes. Production of GLA protein viathe “depot effect” when administering GLA mRNA-loaded lipid nanoparticles shows a substantial increase in blood residence time when compared to direct injection of the naked recombinant protein. As described above, significant quantities ofprotein are t after 48 hours.

The activity profile of the u-galactosidase n produced fromwGLA mRN-A—loaded lipid nanoparticles was measured as a function of 4— umbelliferyl-er~galactopyranoside (4-‘MU-aagal) metabolism. "As shown in , the protein produced from these rt-icle systems is quite active and reflective of the levels of protein ble (, Table 3). AUG comparisons of mRNA therapyubasethLA production versus enzyme replacement therapy (ERT) in mice and humans show a lBZ—fold and 30-fold increase, respectively (Table '4).

, .Dsscrption . lant Patient; Protein Dialysis” _ d—GAL ' Protein .(MMl) 0t ~GAL Protein (MM2) (ll-GAL Mouse mRNA 1 mouse = 5885 (Cat aide ..

. . Table 4. Comparison of Cmax and AUC'infvalues in Fabry ts post-IV dosing 0.2 mg/lcg of Alpha-.galactosidase (pharmacological dose) with those in mice post-IV dosing Alpha-galactosidase andiGLA mRNA. “ Data were from a published paper (Gregory M. Pastores et al. Safety and Pharmacokinetics ofhGLA in patients with Fabry disease and age renal disease. Nephrol Dial Transplant (2007) 2221920- 1925. b d-stage renal disease. ° d—Galactosidase activity at 6 hours after dosing (the earliest time rpointltested in the study).

The ability ofmRNA encapsulated lipid nanopartieles to target organs which can act as a depot for the production ofa desired. protein has been demonstrated. The levels of secreted protein observed have been l orders ofmagnitudeabove normal physiological levels, This “depot effect” is repeatable. Shows again that robust protein production is observed upon dosing wild type (CD—1) mice with a single 30 ug dose ofhGLA mRNA—loaded in C512~200—based lipid rtioles (Formulation 1) . In this experiment, hGLA levels were evaluated over a 72 hour period. A maximum average of 4.0 ug human hGLA protein/min serum is detected six hours post-administration. Based on a value of ~1 ng/mL hGLA protein for normal physiological levels, hGLA MRT es roughly 4000-f01d higher protein levels. As before, hGLA protein could be detected out 'to 48 hr posteadministration ().

An analysis oftissues isolated from this same experiment ed insight into. the bution ofhG’LA protein in'hGLA MRT~treated mice ().

Supraphysiological levels ofhGLA protein were detected in the liver, spleen and. kidneys of all mice treated with a maximum observed between 12 and 24 hour post- administration. Detectable levels of MRT—derived protein could be observed three - days after- a single tiOnothLA-loaded lipid nanoparticles-.

‘In addition, the production ofhGLA upon administration othLA mRNA" ‘ loaded C2OQ-nanoparticles' was ishown to exhibit a dose a response in the serum~ 77 ’ (Aj;_‘,a's well as inthe liver (FIG-.- 1413). .

One inherent characteristic d nanoparticle-mediated mRNA replacement: . therapy would be the phannacokinetic profile ofthe respective protein produced. For example, BERT—based treatment of mice employing Alpha—galactosidase results in a ,- plasma. half~life of approximately 100 minutes. In contrast, MRT—derived — galactosidase has a blood residence time of approximately 72 hrs with a me of 6 hours. This allows for much greater exposure for organs to participate in possible continuous uptake ofthe desired protein. A ison ofPK profilesis shown in FIG; 15 and demonstrates the stark difference in clearance-rates and ultimately a major shift in area under the curve (AUC) can be achieved via MRT-based treatment.

In a separate experiment, hGLA MRT was applied to a mouse disease model, hG'LA KO mice (Fabry mice). A 0.33 mg/kg dose ofhGL-A mRNA—loaded C,l'2~’.200— based lipid rtic-les (Formulation I) was administered} to female KO mice as a single, enous "injection. Substantial quantities of:MRTaderived hGLA protein were ed with a peak at 6hr («660 ng/mL serum) which is approximately 600- fold higher than normal physiological levels. Further, hGLA n was still detectable 72 hr post-administration (FIG. '16).

Quantificationof MRT-derived GLA protein in vital organs demonstrated substantial accumulation as shown in . A ison of observed MRT- derived hGL-A protein to reported normal physiological levels that are found in key organs is plotted (normal levels plotted as dashed lines), While levels. of n at 24 hours are higher than at 72 hours post-administration, the levels of‘hGLA protein detected inthe liver, kidney, spleen and hearts of the d Fabry mice are equivalent to wild type levels. For example, 3.1 ng hGLA protein/mg tissue were found in the kidneys-of treated mice 3 days after a single MRT treatment.

In a subsequent ment, a comparison of ERT—based Alpha-galactosidase treatment versus hGLA 'MRT—based treatment of male Fabry KO mice was conducted.

A single, intravenous dose of 1.0 mg/kg was given for each therapy and the mice were sacrificed one week post-administration. Serum levels ofhGLA protein were monitored at 6 hr and 1 week post—injection. Liver, kidney, spleen, and heart were analyzed for hGLA protein acc’umu‘latiOn one week.post—administration. In addition 7 tothe’ bindiStribution-analyses, a measure ofgefficacy'was determinedlvia ‘. measurement- of globotrioasylceramide'(Gb3) and lyso—Gb3 reductionsin thekidney... and heart FIG 18 shows”the serum levels ofhGLA protein after ent of - ' 'Alpha-galactosidase or GLA mRNA loaded lipid nanoparticles lation 1-)in male Fabry mice; S‘e‘runrsar‘nples were analyzed at-6 hr and 1 week post- administration. A robust signal was detected for. MRT-treated mice after 6 hours, with'hGLAv protein serum levels of«4,0 ug/mL. In contrast, there was no detectable _ galactosidase remaining in the bloodstream at this time.

TheFabry mice in this experiment were sacrificed one week after the l ion and the organs were harvested and analyzed (liver, kidney, Spleen, heart).

FIG. ‘19 shows a comparison ofhuman GLA protein found in each respective organ after either hGILA MRT 'or Alpha-galactosidase ERT treatment. Levels correspond to hGLA present one week post-administration. hGLA=protein was detected in all mice resulted in hGLA protein organs ed. For example, MRT-treated accumulation inthe kidney of2.42 ng hGLA protein/mg protein, While Alpha—~ galactosidaseatreated mice had only residual levels (0.37 rig/mg protein). This correspondsto a ~65-fold higher level of hGL-A protein when treated via h'GZLA MRT. Upon analysis ofthe heart, 11.5 ng hGLA protein/mg protein was found for the MRT—treated cohort as compared to only 1.0 ng/mg protein Alphawgalactosidase.

This corresponds to an ~ll~fold higher accumulation in the heart for ’hGLA MRT- treated mice over ERT—based therapies.

In addition to the biodistribution analyses ted, evaluations of y and b3 were determined via measurement of globotrioasylceramide (Gb3) levels in key organs. A direct comparison of .Gb3 reduction after a single, intravenous /kg GLA MRT treatment as compared to a Alpha—galactosidase ERT-based therapy-of an equivalent dose yielded a sizeable difference in levels of GB?) in the s as well as heart.- For example, (3133 levels for GLA MRT versus Alpha— . galactosidase yielded reductions of 60.2% vs 26.8%, respectively (). Further, Gb3 levels in the heart were reduced by 92.1% vs 66.9% for MRT and Alpha— galactcsidase, respectively (FIG. '21).

A second relevant biomarker for measurement of efficacy is lyso-Gb3. GLA MRT reduced lyso-Gb’3 more efficiently than Alpha-galactosidase as well in the kidneys and heart ( and , respectively). In ular, MRT-treated Fabry mice demonstrated reductions of lyso—Gb3 of 86.1% and 87.9%: in the kidneys . and heart as-compared to Alpha—galactosidase—treated mice yielding a decrease ofg ‘- 47.8% and 613%,- respectively. " The results with for hGLA-in-C-JQQOO based lipid rticles-extend to , other lipid nanopartic’le:formulations. mple, hGLA mRNA loaded into ~ ”HG-T4003 (Formulation 3) or HGTSOOO- based (Formulation '5) lipid nanoparticl'es: ~- ' stedas- a single dose-IV result in production .othiLA at 24 hours post administration ‘ ().. The production ofhGLA exhibited a dose response.» Similarly, hGLA production was ed at 6 hours and 24 hours after administration of-hGLA mRNA loaded into HGTSOOI- based lation 6) lipid nanoparticles administered as a single dose IV. hGLA production was observed in-the serum (A), as well as in organs (B).

Overall, mRNA replacement therapy applied as a depot for protein production produces large quantities of active, fimctionally-therapeutic protein at supraphysiological levels. This method has been demonstrated to yielda sustained circulation fe of the desired protein-and this ri-ved proteinis highly ious for therapy as demonstrated with alphaegalactosidase enzyme in Fabry mice. 1C. In Vivo Human FIXProtein Production Results Studies :were performed administering Factor IX (FIX) mRNA-loade'd lipid nanoparticles in wild type mice (CD-l) and determining FIX protein that is secreted into the tream. Upon intravenous injection of a single dose of 3.0 ug (312—200- based (C1,2-200:DOPE:Chol:PEG at a ratio of4013022525) FIX mRNA—iloa'ded lipid rticles (dose based on encapsulated mRN-A) (Formulation J), a robust protein production was observed ‘(FIG.‘24). > A phannacokineticanalysis over .72 hours showed MRT—derived FIX protein could be detected at all timepoints tested (). The peak serum concentration - was observed at 24 hr post-injection with a value of ~3 ug (29955738 ng/mL) FIX protein/an serum. This represents another successful example of the depot effect.

ID. In Vivo Human .AJAT Protein Produclion Results Studies were performed stering alpha—l ~antitrypsin (AlAT) mRNA— loaded lipid nanoparticles in wild type mice (CD—l) and determining Al AT protein WO 70930 2012/041724 that is secreted into the bloodstream. Upon intravenous injection ofa single doseof ‘ 3.0 ug Cl‘2—‘200-‘based AlAT' niRNA—loa'dedilip’id nanoparticles (dose based on: a; .- encapsulatedtmRNA) (Formulation .1), a robust protein production was observed. , (FIGA‘ZS) ,.

As depicted in ,. detectable levels ofhuman AlATiiprotein derived '3 3ftbr'nAlAT-MR‘1‘ could be observed over a224'hour time period post-administration.

"A’ maximum serum leve‘lbf~48 ug A-l.AT protein/mL serum was detected 12.hours ‘, after inj 'ection.

Example 2: Protein Production Depotzvia Pulmonary Delivery of Polynucleotide .

Compositions z'on Protocol All studies were performed using female CD—l or BALB/C .mice of approXimately 7—1 0 weeks of age at the beginning of each experiment. Test es were introduced Via a single intratracheal aerosolized stration. Mice were sacrificed and perfused with saline at the designated time points The lungs of each mouse were harvested, apportioned into two parts, and stored in either 10% neutral buffered formalin or rozenand stored at ~80°C for analysis. Serum was isolated as described in Example 1. EP0 ELISA .‘ as bed in Example 1.

Results The depot effect can be achieved via ary delivery (eg. intranasala intratracheal, nebulization). Measurement of the deSired exogenous—based protein derived from messenger RNA delivered via nanopaiticle s was achieved and quantified. _ Theproduction ofhuman EPO protein via hEP-O mRNA—loaded lipid nanoparticles was tested in CD-1 mice via a single intratrachea'l administration, Sprayer®). Several formulations were tested using various cationic lipids (Formulations I, 5, 6). All formulations resulted in high encapsulation of human EPO mRNA. Upon stration, animals were sacrificed six hours post- administration and the lungs as well as serum were harvested.

Human EPO protein was detected at the site of administration (lungs) upon treatment via aerosol delivery. Analysis of the serum six hours post—administration showed detectable amounts ofhEPO protein in circulation.‘ These data (shown in ) demonstratethe ability of the lung to act as a ” for the production I (andrsecretion) "of hEPO protein;

Claims (34)

1. A composition sing (a) at least one mRNA le at least a portion of which encodes a functional ed polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle; wherein the lipid nanoparticle comprises one or more cationic , one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids and has a size less than about 100 nm.

2. The composition of claim 1, where in the mRNA encodes an enzyme which is abnormally deficient in an individual with a lysosomal storage disorder.

3. The composition of claim 1, wherein the mRNA encodes a functional erythropoietin or functional α-galactosidase polypeptide

4. The composition of claim 1, wherein the RNA molecule comprises at least one modification which s stability on the RNA molecule.

5. The composition of claim 1, wherein the RNA molecule comprises a modification of the 5’ untranslated region of said RNA molecule.

6. The composition of claim 5, wherein said modification comprises the inclusion of a Cap1 structure.

7. The ition of claim 1, wherein the RNA molecule comprises a modification of the 3’ untranslated region of said RNA molecule.

8. The composition of claim 7, wherein said modification comprises the inclusion of a poly A tail.

9. The composition of claim 1, further comprising an agent for facilitating er of the RNA molecule to an intracellular compartment of a target cell.

10. The composition of claim 1, wherein the lipid rticle comprises C12- 200.

11. The composition of claim 1, wherein the lipid nanoparticle comprises 2DMA, CHOL, DOPE, and DMG-PEG-2000.

12. The composition of claim 1, wherein the lipid nanoparticle comprises C12- 200, DOPE, CHOL, and 2K.

13. The ition of claim 1 , wherein the lipid nanoparticle ses a cleavable lipid.

14. The composition of claim 1 , wherein said composition is lyophilized.

15. The composition of claim 1 , wherein said composition is a reconstituted lyophilized composition.

16. The composition of claim 9, wherein said target cell is selected from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

17. Use of (a) at least one mRNA at least a n of which encodes a functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, in the cture of a composition for the treatment of a subject having a deficiency in a functional polypeptide, wherein the lipid nanoparticle comprises one or more ic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids and has a size less than about 100 nm, wherein treatment comprises administration of the composition to the subject, and wherein following administration of said composition said mRNA is expressed in a target cell to produce said functional ed polypeptide.

18. The use of claim 17, wherein the mRNA encodes a functional erythropoietin, ctosidase, LDL receptor, Factor VIII, Factor IX, α-L- iduronidase, iduronate sulfatase, heparin-N-sulfatase, α-N- acetylglucosaminidase, ose 6-sultatase, β-galactosidase, lysosomal acid lipase or arylsulfatase-A polypeptide.

19. The use of claim 18, wherein the functional secreted polypeptide is an enzyme abnormally deficient in an individual with a lysosomal storage disorder

20. The use of claim 18, wherein the mRNA molecule comprises at least one modification which confers stability to the mRNA molecule.

21. The use of claim 18, wherein the mRNA molecule comprises a modification of the 5’ untranslated region of said mRNA le.

22. The use of claim 21, n said modification comprises the inclusion of a Cap1 ure.

23. The use of claim 18, wherein the mRNA molecule comprises a modification of the 3’ untranslated region of said mRNA molecule.

24. The use of claim 23, wherein said modification comprises the inclusion of a poly A tail.

25. The use of claim 18, further comprising an agent for facilitating transfer of the mRNA molecule to an intracellular compartment of the target cell.

26. The use of claim 18, wherein the lipid nanoparticle comprises C12-200.

27. The use of claim 18, wherein the lipid nanoparticle comprises DLinKC2DMA, CHOL, DOPE, and G-2000.

28. The use of claim 18, wherein the lipid nanoparticle comprises C12-200, DOPE, CHOL, and DMGPEG2K.

29. The use of claim 18, wherein the lipid nanoparticle comprises a cleavable lipid.

30. The use of claim 18, wherein said composition is lyophilized.

31. The use of claim 18, wherein said composition is a reconstituted lyophilized ition.

32. The use of claim 18, wherein said target cell is ed from the group consisting of hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, elial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

33. Use of (a) at least one mRNA at least a portion of which encodes a functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, in the manufacture of a composition for the treatment of a subject having a deficiency in a functional secreted polypeptide, wherein the lipid nanoparticle ses one or more cationic lipids, one or more noncationic lipids, one or more cholesterol-based lipids and one or more PEG- modified lipids and has a size less than about 100 nm, wherein treatment comprises administration of the ition to the subject, and wherein following stration of said composition said mRNA is translated in a target cell to produce the functional polypeptide in said target cell at at least a minimum therapeutic level more than one hour after administration.

34. A composition for use in producing a functional secreted polypeptide in a target cell, wherein the composition comprises (a) at least one mRNA at least a portion of which encodes the functional secreted polypeptide; and (b) a transfer vehicle comprising a lipid nanoparticle, wherein the lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids and has a size less than about 100 nm, and wherein ing administration of said composition said mRNA is translated in a target cell to produce a functional ed polypeptide at at least a minimum therapeutic level more than one hour after administration.