MRNA THERAPEUTIC COMPOSITIONS AND USE TO TREAT DISEASES AND DISORDERS

Abstract
Disclosed are compositions and methods for producing therapeutic fusion proteins in vivo. The compositions and methods disclosed herein are capable of ameliorating diseases by providing therapeutic protein delivery.
Description
SEQUENCE LISTING

The instant application contains a ST26 Sequence Listing in XML format which is hereby incorporated by reference in its entirety. Said XML copy, entitled “MRT-1105US3_ST26.xml”, created on Mar. 20, 2023 and 19 kilobytes in size is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION

Conventional gene therapy involves the use of DNA for insertion of desired genetic information into host cells. The DNA introduced into the cell is usually integrated into the genome of one or more transfected cells, allowing for long-lasting action of the introduced genetic material in the host. While there may be perceived benefits to such sustained action, integration of exogenous DNA into a host genome may also have many deleterious effects. For example, it is possible that the introduced DNA will be inserted into an intact gene, resulting in a mutation which impedes or even totally eliminates the function of the endogenous gene. Thus, gene therapy with DNA may result in the impairment of a vital genetic function in the treated host, such as e.g., elimination 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 proliferation. In addition, with conventional DNA based gene therapy it is necessary for effective expression of the desired gene product to include a strong promoter sequence, which again may lead to undesirable 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 trigger a possibly fatal immune response.


In contrast to DNA, the use of RNA as a gene therapy agent is substantially safer because RNA is not integrated into the genome of the transfected cell, thus eliminating the concern that the introduced genetic material will disrupt the normal functioning of an essential gene, or cause a mutation that results in deleterious or oncogenic effects. RNA therapy also does not require extraneous promoter for effective translation of the encoded protein, again avoiding possible deleterious side effects. In addition, any deleterious effects that do result from mRNA based on gene therapy would be of limited duration due to the relatively short half-life Consequently, for many applications the transient nature of mRNA and short life span of the resulting protein can be desirable, compared to the longer lasting stable integration achieved using DNA based gene therapy.


However, in some cases, mRNA instability and short half-life limits its therapeutic effects. Therefore, there is a need for enhancing mRNA stability and prolong half-life for more effective and successful therapeutic use.


SUMMARY OF THE INVENTION

The invention provides improved mRNA therapy that has increased mRNA stability and prolonged half-life, among other things. In particular, the invention is based on mRNA encoding a therapeutic protein fused to a polypeptide that is capable of binding to an Fc receptor (“Therapeutic Fusion Protein”), for delivery to one or more target cells for production of therapeutic levels of functional protein. Without wishing to be bound by any theory, it is contemplated that such therapeutic fusion protein is readily transported from the target cell into systemic circulation via an Fc receptor and/or secreted from the cell, recaptured by an Fc receptor and then transcytosed into the systemic circulation. In certain embodiments, the therapeutic protein encoded is naturally secreted and thus naturally associated with an appropriate signal sequence. In other embodiments mRNA encoding a protein that is not normally secreted may be operatively linked to an appropriate signal sequence that results in the secretion of the translated protein.


In certain embodiments, the compositions of the invention are able to translocate, i.e., move intact by either active or passive means from initial target cells (e.g., lung cells) to the systemic blood supply where they are then deposited in different tissues (e.g., liver cells). In these embodiments, the cells where the mRNA is deposited act as a depot for the production of therapeutic protein, which is then readily transported out of the depot cells into systemic circulation via an Fc receptor.


In some embodiments, the compositions of the invention are administered to the lung by inhalation, aerosolization, nebulization, or instillation. Pulmonary delivery provides significant advantages over intravenous infusions or local injections. It eliminates injection site or infusion reactions and should reduce pain upon administration.


Thus, the invention provides compositions and methods for delivery of therapeutic proteins through non-invasive pulmonary applications that result in the production of therapeutically effective levels of protein in both lung and non-lung cells, which is then readily transported into systemic circulation via an Fc receptor. This results in the accumulation of therapeutically effective systemic concentrations of the encoded protein by simple inhalation of the synthetic mRNA compositions of the invention. In addition to facilitating delivery of the fusion protein to the circulatory system, the polypeptide that is capable of binding to an Fc receptor also improves systemic exposure by extending protein half-life.


The compositions and methods of the invention are useful in the management and treatment of a large number of diseases, including but not limited to diseases which result from protein and/or enzyme deficiencies or malfunction. In some embodiments, individuals suffering from such diseases may have underlying genetic defects that lead to the compromised 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 lacking or having diminished biological activity. In some embodiments, the methods and compositions of the invention are useful for the treatment of lysosomal storage disorders and/or urea cycle metabolic disorders that occur as a result of one or more defects in the biosynthesis of secreted enzymes involved in the urea cycle.


The compositions of the invention comprise an mRNA encoding a therapeutic protein fused to a polypeptide that is capable of binding to an Fc receptor (i.e., mRNA that encodes a Therapeutic Fusion Protein). Optionally, the mRNA may include one or more untranslated regions. The compositions of the invention may further comprise a transfer vehicle, such as, e.g., a lipid nanoparticle or a polymeric carrier. The mRNA can encode any clinically useful secreted protein or any clinically useful protein that has been engineered to include a signal sequence that allows the protein to be secreted. In one aspect of the invention, the therapeutic protein is chosen from proteins listed in Tables 1-3, mammalian homologs thereof, and homologs from animals of veterinary or industrial interest thereof. The polypeptide that binds to an Fc receptor can be, e.g., an immunoglobulin Fc domain or an FcRn binding peptide.


Another aspect of the invention provides a method of treating a subject that will benefit from in vivo expression of a therapeutic protein, comprising administering a composition comprising at least one mRNA that encodes a Therapeutic Fusion Protein, wherein following administration of said composition the mRNA is translated in a target cell to produce the Therapeutic Fusion Protein, which is then transported into circulation via an Fc receptor. In some embodiments administration comprises single or repeated doses. In certain embodiments, the dose is administered intravenously, or by pulmonary delivery.


Therapeutic Fusion Proteins produced from mRNA in vivo provide significant advantages over administration of recombinant proteins. Proteins produced from mRNA in endogenous cells, such as, e.g., endogenous epithelial cells, reflect post-translational modifications present normally in the body as opposed to proteins manufactured in common commercially used non-human host systems such as Chinese Hamster Ovary, cells, bacterial cells or yeast cells. Endogenous human glycosylation patterns, protein folding or other native posttranslational modifications may improve tolerability, potency, and reduce immunogenicity.


In addition, the mRNA production process is simplified and improved compared to typical recombinant protein production. The process development, manufacturing, and cost profile compared to typical protein manufacturing is improved. The mRNA process is interchangeable among constructs; only the mRNA sequence changes. mRNA manufacturing also eliminates the need for costly fermentation in bioreactors and large footprint manufacturing facilities and staffing.


The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the accompanying examples. The various embodiments described herein are complimentary and can be combined or used together in a manner understood by the skilled person in view of the teachings contained herein.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1A is a nucleotide sequence of a 5′ CMV untranslated sequence (SEQ ID NO:1), wherein X, if present is GGA. FIG. 1B is a nucleotide sequence of a 3′ hGH sequence (SEQ ID NO:2).



FIG. 2A shows an nucleotide sequence encoding human erythropoietin (EPO) mRNA fused to a human IgG Fc domain and flanked on the 5′ end with CMV UTR and on the 3′ end with hGH UTR (SEQ ID NO:3). FIG. 2B is a graphic representation of the mRNA construct shown in FIG. 2A.



FIG. 3 shows an mRNA encoding human alpha-galactosidase (GLA) fused to an FcRn binding peptide (SEQ ID NO:4). This mRNA construct can optionally be flanked on the 5′ end with SEQ ID NO:1 and on the 3′ end with SEQ ID NO:2.



FIG. 4 shows an mRNA encoding human alpha-1 antitrypsin (A1AT) fused to an FcRn binding peptide (SEQ ID NO:5). This mRNA construct can optionally be flanked on the 5′ end with SEQ ID NO:1 and on the 3′ end with SEQ ID NO:2.



FIG. 5 shows an mRNA encoding human Factor IX (FIX) fused to an FcRn binding peptide (SEQ ID NO:6). This sequence can be flanked on the 5′ end with SEQ ID NO:1 and on the 3′ end with SEQ ID NO:2.



FIG. 6 is a graphic representation of aerosolized and inhaled mRNA encoding a Therapeutic Fusion Protein delivered to the lung epithelial cells and the encoded fusion protein is transported across the lung epithelium to the bloodstream.





DESCRIPTION OF EXEMPLARY EMBODIMENTS

The invention provides compositions and methods for intracellular delivery of mRNA encoding a Therapeutic Fusion Protein for production of therapeutic levels of functional protein in vivo. The invention further provides methods of treatment of various diseases and conditions by administering the compositions of the invention.


Administration of the compositions of the invention results in the production of functional protein in vivo. The term “functional,” as used herein to qualify 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 protein or enzyme. The mRNA compositions of the invention are useful for the treatment of various metabolic or genetic disorders, and in particular those genetic or metabolic disorders that involve the non-expression, mis-expression or deficiency of a protein or enzyme.


The term “therapeutic levels” refers 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.


As provided herein, the compositions may include a transfer vehicle. As used herein, the terms “transfer vehicle,” “delivery vehicle,” “carrier” and the like refer to standard pharmaceutical carriers, diluents, excipients 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 vehicles described herein are capable of delivering mRNA to the target cell. In certain embodiments, the transfer vehicle is a lipid nanoparticle. In other embodiments, the transfer vehicle is a polymeric carrier, such as, e.g., polyethyleneimine.


mRNA


The mRNA in the compositions of the invention encodes a therapeutic protein, (including a functional polypeptide or peptide), such as, e.g., a hormone, enzyme, or receptor. The therapeutic protein of interest may be one that is normally secreted or excreted. In alternate embodiments, the mRNA is engineered to encode a protein that is not normally secreted or excreted, operably linked to a signal sequence that will allow the protein to be secreted when it is expressed in the cells.


In some embodiments of the invention, the mRNA may optionally have chemical or biological modifications which, for example, improve the stability and/or half-life of such mRNA or which improve or otherwise facilitate protein production. Upon transfection, a natural mRNA in the compositions of the invention may decay with a half-life of between 30 minutes and several days. The mRNAs in the compositions of the invention preferably retain at least some ability to be translated, thereby producing a functional protein or enzyme. Accordingly, the invention provides compositions comprising and methods of administering a stabilized mRNA. In some embodiments of the invention, the activity of the 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 a semi-weekly or bi-weekly basis, or more preferably on a monthly, bi-monthly, quarterly or an annual basis. The extended or prolonged activity of the mRNA of the present invention is directly related to the quantity of protein or enzyme produced from such mRNA. Similarly, the activity of the compositions of the present invention may be further extended or prolonged by modifications made to improve or enhance translation of the mRNA. Furthermore, the quantity of functional protein or enzyme produced by the target cell is a function of the quantity of mRNA delivered to the target cells and the stability of such mRNA. To the extent that the stability of the mRNA of the present invention may be improved or enhanced, the half-life, the activity of the produced protein or enzyme and the dosing frequency of the composition may be further extended.


Accordingly, in some embodiments, the mRNA in the compositions of the invention comprise at least one modification which confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of the mRNA. As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly with respect to the mRNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such mRNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such mRNA in the target cell, tissue, subject and/or cytoplasm. The stabilized mRNA molecules provided herein demonstrate longer half-lives relative to their naturally occurring, unmodified counterparts (e.g. the wild-type version of the mRNA). 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 translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).


In some embodiments, the mRNAs used in the compositions of the invention have undergone a chemical or biological modification to render them more stable. Exemplary modifications to an mRNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring mRNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).


In addition, suitable modifications include alterations in one or more nucleotides of a 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 example, an inverse relationship between the stability of RNA and a higher number cytidines (C's) and/or uridines (U's) residues has been demonstrated, and RNA devoid of C and U residues have been found to be stable to most RNases (Heidenreich, et 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 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 or a related amino acid. Contemplated modifications to the mRNA nucleic acids of the present invention also include the incorporation of pseudouridines. The incorporation of pseudouridines into the mRNA nucleic acids of the present invention may enhance stability and translational capacity, as well as diminishing immunogenicity in vivo. See, e.g., Kariko, K., et al., Molecular Therapy 16 (11): 1833-1840 (2008). Substitutions and modifications to the mRNA of the present invention may be performed by methods readily known to one of ordinary skill in the art.


The constraints on reducing the number of C and U residues in a sequence may be greater within the coding region of an mRNA, compared to an untranslated region, (i.e., it will likely not be possible to eliminate all of the C and U residues present in the message while still retaining the ability of the message to encode the desired amino acid sequence). The degeneracy of the genetic code, however presents an opportunity to allow the number of C and/or U residues that are present in the sequence to be reduced, while maintaining the same coding capacity (i.e., depending on which amino acid is encoded by a codon, several different possibilities for modification of RNA sequences may be possible). For example, the codons for Gly can be altered to GGA or GGG instead of GGU or GGC.


Other suitable polynucleotide modifications that may be incorporated into the mRNA used in the compositions of the invention include, but are not limited to, 4′-thio-modified bases: 4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine, 4′-thio-5-methyl-cytidine, 4′-thio-pseudouridine, and 4′-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof. The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both of the 3′ and 5′ ends of an mRNA molecule encoding a functional 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 of the 3′ UTR or the 5′ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures).


Cap Structure


In some embodiments, mRNAs include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.


Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.


A common cap for mRNA produced by in vitro transcription is m7G(5′)ppp(5′)G, which has been used as the dinucleotide cap in transcription with T7 or SP6 RNA polymerase in vitro to obtain RNAs having a cap structure in their 5′-termini. The prevailing method for the in vitro synthesis of capped mRNA employs a pre-formed dinucleotide of the form m7G(5′)ppp(5′)G (“m7GpppG”) as an initiator of transcription.


To date, a usual form of a synthetic dinucleotide cap used in in vitro translation experiments is the Anti-Reverse Cap Analog (“ARCA”) or modified ARCA, which is generally a modified cap analog in which the 2′ or 3′ OH group is replaced with —OCH3.


Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).


In some embodiments, a suitable cap is a 7-methyl guanylate (“m7G”) linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in m7G(5′)ppp(5′)N, where N is any nucleoside. A preferred embodiment of a m7G cap utilized in embodiments of the invention is m7G(5′)ppp(5′)G.


In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a 2′-O-methyl residue of the ribose attached to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. Cap1 structures have a 2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. Cap2 structures have a 2′-O-methyl residue attached to both bases 2 and 3.


A variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as the ARCA 3′-OCH3 and 2′-OCH3 cap analogs (Jemielity, J. et al., RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodiments of the invention include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529, incorporated by reference herein.


Tail Structure


Typically, the presence of a “tail” serves to protect the mRNA from exonuclease degradation. A poly A or poly U tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A or poly U tail can be added to an mRNA molecule thus rendering the RNA more stable. Poly A or poly U tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 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. Poly A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).


Typically, the length of a poly A or poly U tail can be at least about 10, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail.


In some embodiments, the length of the poly A or poly C tail is adjusted to control the stability of a modified sense mRNA molecule of the invention and, thus, the transcription of protein. For example, since the length of the poly A tail can influence the half-life of a sense mRNA molecule, the length of the poly A tail can be adjusted to modify the level of resistance of the mRNA to nucleases and thereby control the time course of polynucleotide expression and/or polypeptide production in a target cell.


Signal Peptide Sequence


In some embodiments, an mRNA according to the present invention incorporates a nucleotide sequence encoding a signal peptide. As used herein, the term “signal peptide” refers to a peptide present at a newly synthesized protein that can target the protein towards the secretory pathway. In some embodiments, the signal peptide is cleaved after translocation into the endoplasmic reticulum following translation of the mRNA. Signal peptide is also referred to as signal sequence, leader sequence or leader peptide. Typically, a signal peptide is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptide. A signal peptide may be present at the N-terminus of a newly synthesized protein. Without wishing to be bound by any particular theory, the incorporation of a signal peptide encoding sequence on an mRNA may facilitate the secretion and/or production of the encoded protein in vivo.


A suitable signal peptide for the present invention can be a heterogeneous sequence derived from various eukaryotic and prokaryotic proteins, in particular secreted proteins. In some embodiments, a suitable signal peptide is a leucine-rich sequence. See Yamamoto Y et al. (1989), Biochemistry, 28:2728-2732, which is incorporated herein by reference. A suitable signal peptide may be derived from a human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, Azurocidin preproprotein, cystatin-S precursor, trypsinogen 2 precursor, potassium channel blocker, alpha conotoxin 1p1.3, alpha conotoxin, alfa-galactosidase, cellulose, aspartic proteinase nepenthesin-1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and Cholera toxin. Exemplary signal peptide sequences are described in Kober, et al., Biotechnol. Bioeng., 110: 1164-73, 2012, which is incorporated herein by reference.


In some embodiments, an mRNA according to the present invention may incorporate a sequence encoding a signal peptide derived from human growth hormone (hGH), or a fragment thereof. A non-limiting nucleotide sequence encoding a hGH signal peptide is show below.









5′ human growth hormone (hGH) sequence (SEQ ID


NO: 7):


AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUU


GCCUGCCCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUC


C





Alternative 5′ human growth hormone (hGH)


sequence (SEQ ID NO: 8):


AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCU


GUCUCCCAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUC


G






In some embodiments, an mRNA according to the present invention may incorporate a signal peptide encoding sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7 or SEQ ID NO:8.


5′ and 3′ Untranslated Region


In one embodiment, an mRNA can be modified by the incorporation of 3′ and/or 5′ untranslated (UTR) sequences that are not naturally found in the wild-type mRNA. In one embodiment, a 3′ and/or 5′ flanking sequence that naturally flanks an mRNA and encodes a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein in order to modify it. For example, 3′ or 5′ sequences from mRNA molecules that 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 sense mRNA molecule. See, e.g., US2003/0083272.


In some embodiments, the mRNA in the compositions of the invention include modification of the 5′ end of the mRNA to include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof (e.g., SEQ ID NO:1) to improve the nuclease resistance and/or improve the half-life of the mRNA. In addition to increasing the stability of the mRNA nucleic acid sequence, it has been surprisingly discovered that the inclusion of a partial sequence of a CMV immediate-early 1 (IE1) gene enhances the translation of the mRNA and the expression 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 NO:2) to the 3′ ends of the nucleic acid (e.g., mRNA) to further stabilize the mRNA. Generally, preferred modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example modifications made to improve such mRNA's resistance to in vivo nuclease digestion.


Further contemplated are variants of the nucleic acid sequence of SEQ ID NO:1 and/or SEQ ID NO:2, wherein the variants maintain the functional properties of the nucleic acids including stabilization of the mRNA and/or pharmacokinetic properties (e.g., half-life). Variants may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.


In some embodiments, the composition can comprise a stabilizing reagent. The compositions can include one or more formulation reagents that bind directly or indirectly to, and stabilize the mRNA, thereby enhancing residence time in the target cell. Such reagents preferably lead to an improved half-life of the mRNA in the target cells. For example, the stability of an mRNA and efficiency of its translation may be increased by the incorporation of “stabilizing reagents” that form complexes with the mRNA that naturally occur within a cell (see e.g., U.S. 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 encapsulating 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 of the compositions may also be improved by the use of opsonization-inhibiting moieties, which are typically large hydrophilic polymers that are chemically or physically bound to the transfer vehicle (e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids). These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system and reticulo-endothelial system (e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference). Transfer vehicles modified with opsonization-inhibition moieties thus remain in the circulation much longer than their unmodified counterparts.


When RNA is hybridized to a complementary nucleic acid molecule (e.g., DNA or RNA) it may be protected from nucleases. (Krieg, et al. Melton. Methods in Enzymology. 1987; 155, 397-415). The stability of hybridized mRNA is likely due to the inherent single strand specificity of most RNases. In some embodiments, the stabilizing reagent selected to complex an mRNA is a eukaryotic protein, (e.g., a mammalian protein). In yet another embodiment, the mRNA can be modified by hybridization to a second nucleic acid molecule. If an entire mRNA molecule were hybridized to a complementary nucleic acid molecule translation initiation may be reduced. In some embodiments the 5′ untranslated region and the AUG start region of the mRNA molecule may optionally be left unhybridized. Following translation initiation, the unwinding activity of the ribosome complex can function even on high affinity duplexes so that translation can proceed. (Liebhaber. J. Mol. Biol. 1992; 226: 2-13; Monia, et al. J Biol Chem. 1993; 268: 14514-22.)


It will be understood that any of the above described methods for enhancing the stability of mRNA may be used either alone or in combination with one or more of any of the other above-described methods and/or compositions.


The mRNA of the present invention may be optionally combined with a reporter gene (e.g., upstream or downstream of the coding region of the mRNA) which, for example, facilitates the determination of mRNA delivery to the target cells or tissues. Suitable reporter genes may include, for example, Green Fluorescent Protein mRNA (GFP mRNA), Renilla Luciferase mRNA (Luciferase mRNA), Firefly Luciferase mRNA, or any combinations thereof. For example, GFP mRNA may be fused with an mRNA encoding a secretable protein to facilitate confirmation of mRNA localization in the target cells that will act as a depot for protein production.


mRNA Synthesis


mRNAs according to the present invention may be synthesized according to any of a variety of known methods. For example, mRNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application.


In some embodiments, for the preparation of mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired mRNA and a termination signal.


Desired mRNA sequence(s) according to the invention may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. Typically, the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.


Therapeutic Fusion Proteins


Compositions of the invention comprise mRNA encoding a Therapeutic Fusion Protein. A Therapeutic Fusion Protein comprises a therapeutic protein fused to a polypeptide capable of binding to an Fc receptor. The Therapeutic Fusion Protein may optionally comprise a linker sequence between the therapeutic protein and the polypeptide capable of binding to an Fc receptor. Upon delivery of the compositions of the invention to target cells, the mRNA is translated to produce the encoded Therapeutic Fusion Protein, which is secreted and taken up again by the cells and transported to the circulatory system via the Fc receptor.


Therapeutic Protein


The therapeutic protein portion of the Therapeutic Fusion Protein can be chosen from any protein or polypeptide that can be expressed to provide a therapeutic effect. In some embodiments, the therapeutic protein may be a functional protein or enzyme that is normally secreted into extracellular space. For example, such secreted proteins include clotting factors, components of the complement pathway, cytokines, chemokines, chemoattractants, protein hormones (e.g. EGF, PDF), protein components of serum, secretable toll-like receptors, and others. In some embodiments, the therapeutic protein is erythropoietin, al-antitrypsin, carboxypeptidase N or human growth hormone. In some embodiments, the therapeutic protein is one that is not normally secreted. In such cases, the mRNA in the compositions of the invention may be engineered to comprise a secretory leader sequence operatively linked to the sequence encoding the Therapeutic Fusion Protein to direct secretion of the encoded protein. Suitable secretory leader sequences are described, for example, in US 2008/0286834.


In some embodiments, the therapeutic protein in the Therapeutic Fusion Protein is chosen from the secreted proteins listed in Table 1; thus, compositions of the invention may comprise an mRNA encoding a protein listed in Table 1 (or a homolog thereof, as discussed below) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a protein listed in Table 1 (or a homolog thereof, as discussed below) along with other components set out herein.









TABLE 1







Secreted Proteins









Uniprot ID
Protein Name
Gene Name





A1E959
Odontogenic ameloblast-associated protein
ODAM


A1KZ92
Peroxidasin-like protein
PXDNL


A1L453
Serine protease 38
PRSS38


A1L4H1
Soluble scavenger receptor cysteine-rich domain-
SSC5D



containing protein SSC5D


A2RUU4
Colipase-like protein 1
CLPSL1


A2VDF0
Fucose mutarotase
FUOM


A2VEC9
SCO-spondin
SSPO


A3KMH1
von Willebrand factor A domain-containing
VWA8



protein 8


A4D0S4
Laminin subunit beta-4
LAMB4


A4D1T9
Probable inactive serine protease 37
PRSS37


A5D8T8
C-type lectin domain family 18 member A
CLEC18A


A6NC86
phospholipase A2 inhibitor and Ly6/PLAUR
PINLYP



domain-containing protein


A6NCI4
von Willebrand factor A domain-containing
VWA3A



protein 3A


A6ND01
Probable folate receptor delta
FOLR4


A6NDD2
Beta-defensin 108B-like


A6NE02
BTB/POZ domain-containing protein 17
BTBD17


A6NEF6
Growth hormone 1
GH1


A6NF02
NPIP-like protein LOC730153


A6NFB4
HCG1749481, isoform CRA_k
CSH1


A6NFZ4
Protein FAM24A
FAM24A


A6NG13
Glycosyltransferase 54 domain-containing protein


A6NGN9
IgLON family member 5
IGLON5


A6NHN0
Otolin-1
OTOL1


A6NHN6
Nuclear pore complex-interacting protein-like 2
NPIPL2


A6NI73
Leukocyte immunoglobulin-like receptor
LILRA5



subfamily A member 5


A6NIT4
Chorionic somatomammotropin hormone 2
CSH2



isoform 2


A6NJ69
IgA-inducing protein homolog
IGIP


A6NKQ9
Choriogonadotropin subunit beta variant 1
CGB1


A6NMZ7
Collagen alpha-6(VI) chain
COL6A6


A6NNS2
Dehydrogenase/reductase SDR family member 7C
DHRS7C


A6XGL2
Insulin A chain
INS


A8K0G1
Protein Wnt
WNT7B


A8K2U0
Alpha-2-macroglobulin-like protein 1
A2ML1


A8K7I4
Calcium-activated chloride channel regulator 1
CLCA1


A8MTL9
Serpin-like protein HMSD
HMSD


A8MV23
Serpin E3
SERPINE3


A8MZH6
Oocyte-secreted protein 1 homolog
OOSP1


A8TX70
Collagen alpha-5(VI) chain
COL6A5


B0ZBE8
Natriuretic peptide
NPPA


B1A4G9
Somatotropin
GH1


B1A4H2
HCG1749481, isoform CRA_d
CSH1


B1A4H9
Chorionic somatomammotropin hormone
CSH2


B1AJZ6
Protein Wnt
WNT4


B1AKI9
Isthmin-1
ISM1


B2RNN3
Complement C1q and tumor necrosis factor-
C1QTNF9B



related protein 9B


B2RUY7
von Willebrand factor C domain-containing
VWC2L



protein 2-like


B3GLJ2
Prostate and testis expressed protein 3
PATE3


B4DI03
SEC11-like 3 (S. cerevisiae), isoform CRA_a
SEC11L3


B4DJF9
Protein Wnt
WNT4


B4DUL4
SEC11-like 1 (S. cerevisiae), isoform CRA_d
SEC11L1


B5MCC8
Protein Wnt
WNT10B


B8A595
Protein Wnt
WNT7B


B8A597
Protein Wnt
WNT7B


B8A598
Protein Wnt
WNT7B


B9A064
Immunoglobulin lambda-like polypeptide 5
IGLL5


C9J3H3
Protein Wnt
WNT10B


C9J8I8
Protein Wnt
WNT5A


C9JAF2
Insulin-like growth factor II Ala-25 Del
IGF2


C9JCI2
Protein Wnt
WNT10B


C9JL84
HERV-H LTR-associating protein 1
HHLA1


C9JNR5
Insulin A chain
INS


C9JUI2
Protein Wnt
WNT2


D6RF47
Protein Wnt
WNT8A


D6RF94
Protein Wnt
WNT8A


E2RYF7
Protein PBMUCL2
HCG22


E5RFR1
PENK(114-133)
PENK


E7EML9
Serine protease 44
PRSS44


E7EPC3
Protein Wnt
WNT9B


E7EVP0
Nociceptin
PNOC


E9PD02
Insulin-like growth factor I
IGF1


E9PH60
Protein Wnt
WNT16


E9PJL6
Protein Wnt
WNT11


F5GYM2
Protein Wnt
WNT5B


F5H034
Protein Wnt
WNT5B


F5H364
Protein Wnt
WNT5B


F5H7Q6
Protein Wnt
WNT5B


F8WCM5
Protein INS-IGF2
INS-IGF2


F8WDR1
Protein Wnt
WNT2


H0Y663
Protein Wnt
WNT4


H0YK72
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


H0YK83
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


H0YM39
Chorionic somatomammotropin hormone
CSH2


H0YMT7
Chorionic somatomammotropin hormone
CSH1


H0YN17
Chorionic somatomammotropin hormone
CSH2


H0YNA5
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


H0YNG3
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


H0YNX5
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


H7BZB8
Protein Wnt
WNT10A


H9KV56
Choriogonadotropin subunit beta variant 2
CGB2


I3L0L8
Protein Wnt
WNT9B


J3KNZ1
Choriogonadotropin subunit beta variant 1
CGB1


J3KP00
Choriogonadotropin subunit beta
CGB7


J3QT02
Choriogonadotropin subunit beta variant 1
CGB1


O00175
C-C motif chemokine 24
CCL24


O00182
Galectin-9
LGALS9


O00187
Mannan-binding lectin serine protease 2
MASP2


O00230
Cortistatin
CORT


O00253
Agouti-related protein
AGRP


O00270
12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid
GPR31



receptor


O00292
Left-right determination factor 2
LEFTY2


O00294
Tubby-related protein 1
TULP1


O00295
Tubby-related protein 2
TULP2


O00300
Tumor necrosis factor receptor superfamily
TNFRSF11B



member 11B


O00339
Matrilin-2
MATN2


O00391
Sulfhydryl oxidase 1
QSOX1


O00468
Agrin
AGRN


O00515
Ladinin-1
LAD1


O00533
Processed neural cell adhesion molecule L1-like
CHL1



protein


O00584
Ribonuclease T2
RNASET2


O00585
C-C motif chemokine 21
CCL21


O00602
Ficolin-1
FCN1


O00622
Protein CYR61
CYR61


O00626
MDC(5-69)
CCL22


O00634
Netrin-3
NTN3


O00744
Protein Wnt-10b
WNT10B


O00755
Protein Wnt-7a
WNT7A


O14498
Immunoglobulin superfamily containing leucine-
ISLR



rich repeat protein


O14511
Pro-neuregulin-2, membrane-bound isoform
NRG2


O14594
Neurocan core protein
NCAN


O14625
C-X-C motif chemokine 11
CXCL11


O14638
Ectonucleotide pyrophosphatase/phosphodiesterase
ENPP3



family member 3


O14656
Torsin-1A
TOR1A


O14657
Torsin-1B
TOR1B


O14786
Neuropilin-1
NRP1


O14788
Tumor necrosis factor ligand superfamily member
TNFSF11



11, membrane form


O14791
Apolipoprotein L1
APOL1


O14793
Growth/differentiation factor 8
MSTN


O14904
Protein Wnt-9a
WNT9A


O14905
Protein Wnt-9b
WNT9B


O14944
Proepiregulin
EREG


O14960
Leukocyte cell-derived chemotaxin-2
LECT2


O15018
Processed PDZ domain-containing protein 2
PDZD2


O15041
Semaphorin-3E
SEMA3E


O15072
A disintegrin and metalloproteinase with
ADAMTS3



thrombospondin motifs 3


O15123
Angiopoietin-2
ANGPT2


O15130
Neuropeptide FF
NPFF


O15197
Ephrin type-B receptor 6
EPHB6


O15204
ADAM DEC1
ADAMDEC1


O15230
Laminin subunit alpha-5
LAMA5


O15232
Matrilin-3
MATN3


O15240
Neuroendocrine regulatory peptide-1
VGF


O15263
Beta-defensin 4A
DEFB4A


O15335
Chondroadherin
CHAD


O15393
Transmembrane protease serine 2 catalytic chain
TMPRSS2


O15444
C-C motif chemokine 25
CCL25


O15467
C-C motif chemokine 16
CCL16


O15496
Group 10 secretory phospholipase A2
PLA2G10


O15520
Fibroblast growth factor 10
FGF10


O15537
Retinoschisin
RS1


O43157
Plexin-B1
PLXNB1


O43184
Disintegrin and metalloproteinase domain-
ADAM12



containing protein 12


O43240
Kallikrein-10
KLK10


O43278
Kunitz-type protease inhibitor 1
SPINT1


O43320
Fibroblast growth factor 16
FGF16


O43323
Desert hedgehog protein C-product
DHH


O43405
Cochlin
COCH


O43508
Tumor necrosis factor ligand superfamily member
TNFSF12



12, membrane form


O43555
Progonadoliberin-2
GNRH2


O43557
Tumor necrosis factor ligand superfamily member
TNFSF14



14, soluble form


O43692
Peptidase inhibitor 15
PI15


O43699
Sialic acid-binding Ig-like lectin 6
SIGLEC6


O43820
Hyaluronidase-3
HYAL3


O43827
Angiopoietin-related protein 7
ANGPTL7


O43852
Calumenin
CALU


O43854
EGF-like repeat and discoidin I-like domain-
EDIL3



containing protein 3


O43866
CD5 antigen-like
CD5L


O43897
Tolloid-like protein 1
TLL1


O43915
Vascular endothelial growth factor D
FIGF


O43927
C-X-C motif chemokine 13
CXCL13


O60218
Aldo-keto reductase family 1 member B10
AKR1B10


O60235
Transmembrane protease serine 11D
TMPRSS11D


O60258
Fibroblast growth factor 17
FGF17


O60259
Kallikrein-8
KLK8


O60383
Growth/differentiation factor 9
GDF9


O60469
Down syndrome cell adhesion molecule
DSCAM


O60542
Persephin
PSPN


O60565
Gremlin-1
GREM1


O60575
Serine protease inhibitor Kazal-type 4
SPINK4


O60676
Cystatin-8
CST8


O60687
Sushi repeat-containing protein SRPX2
SRPX2


O60844
Zymogen granule membrane protein 16
ZG16


O60882
Matrix metalloproteinase-20
MMP20


O60938
Keratocan
KERA


O75015
Low affinity immunoglobulin gamma Fc region
FCGR3B



receptor III-B


O75077
Disintegrin and metalloproteinase domain-
ADAM23



containing protein 23


O75093
Slit homolog 1 protein
SLIT1


O75094
Slit homolog 3 protein
SLIT3


O75095
Multiple epidermal growth factor-like domains
MEGF6



protein 6


O75173
A disintegrin and metalloproteinase with
ADAMTS4



thrombospondin motifs 4


O75200
Nuclear pore complex-interacting protein-like 1
NPIPL1


O75339
Cartilage intermediate layer protein 1 C1
CILP


O75354
Ectonucleoside triphosphate diphosphohydrolase 6
ENTPD6


O75386
Tubby-related protein 3
TULP3


O75398
Deformed epidermal autoregulatory factor 1
DEAF1



homolog


O75443
Alpha-tectorin
TECTA


O75445
Usherin
USH2A


O75462
Cytokine receptor-like factor 1
CRLF1


O75487
Glypican-4
GPC4


O75493
Carbonic anhydrase-related protein 11
CA11


O75594
Peptidoglycan recognition protein 1
PGLYRP1


O75596
C-type lectin domain family 3 member A
CLEC3A


O75610
Left-right determination factor 1
LEFTY1


O75629
Protein CREG1
CREG1


O75636
Ficolin-3
FCN3


O75711
Scrapie-responsive protein 1
SCRG1


O75715
Epididymal secretory glutathione peroxidase
GPX5


O75718
Cartilage-associated protein
CRTAP


O75829
Chondrosurfactant protein
LECT1


O75830
Serpin I2
SERPINI2


O75882
Attractin
ATRN


O75888
Tumor necrosis factor ligand superfamily
TNFSF13



member 13


O75900
Matrix metalloproteinase-23
MMP23A


O75951
Lysozyme-like protein 6
LYZL6


O75973
C1q-related factor
C1QL1


O76038
Secretagogin
SCGN


O76061
Stanniocalcin-2
STC2


O76076
WNT1-inducible-signaling pathway protein 2
WISP2


O76093
Fibroblast growth factor 18
FGF18


O76096
Cystatin-F
CST7


O94769
Extracellular matrix protein 2
ECM2


O94813
Slit homolog 2 protein C-product
SLIT2


O94907
Dickkopf-related protein 1
DKK1


O94919
Endonuclease domain-containing 1 protein
ENDOD1


O94964
N-terminal form
SOGA1


O95025
Semaphorin-3D
SEMA3D


O95084
Serine protease 23
PRSS23


O95150
Tumor necrosis factor ligand superfamily
TNFSF15



member 15


O95156
Neurexophilin-2
NXPH2


O95157
Neurexophilin-3
NXPH3


O95158
Neurexophilin-4
NXPH4


O95388
WNT1-inducible-signaling pathway protein 1
WISP1


O95389
WNT1-inducible-signaling pathway protein 3
WISP3


O95390
Growth/differentiation factor 11
GDF11


O95393
Bone morphogenetic protein 10
BMP10


O95399
Urotensin-2
UTS2


O95407
Tumor necrosis factor receptor superfamily
TNFRSF6B



member 6B


O95428
Papilin
PAPLN


O95445
Apolipoprotein M
APOM


O95450
A disintegrin and metalloproteinase with
ADAMTS2



thrombospondin motifs 2


O95460
Matrilin-4
MATN4


O95467
LHAL tetrapeptide
GNAS


O95631
Netrin-1
NTN1


O95633
Follistatin-related protein 3
FSTL3


O95711
Lymphocyte antigen 86
LY86


O95715
C-X-C motif chemokine 14
CXCL14


O95750
Fibroblast growth factor 19
FGF19


O95760
Interleukin-33
IL33


O95813
Cerberus
CER1


O95841
Angiopoietin-related protein 1
ANGPTL1


O95897
Noelin-2
OLFM2


O95925
Eppin
EPPIN


O95965
Integrin beta-like protein 1
ITGBL1


O95967
EGF-containing fibulin-like extracellular matrix
EFEMP2



protein 2


O95968
Secretoglobin family 1D member 1
SCGB1D1


O95969
Secretoglobin family 1D member 2
SCGB1D2


O95970
Leucine-rich glioma-inactivated protein 1
LGI1


O95972
Bone morphogenetic protein 15
BMP15


O95994
Anterior gradient protein 2 homolog
AGR2


O95998
Interleukin-18-binding protein
IL18BP


O96009
Napsin-A
NAPSA


O96014
Protein Wnt-11
WNT11


P00450
Ceruloplasmin
CP


P00451
Factor VIIIa light chain
F8


P00488
Coagulation factor XIII A chain
F13A1


P00533
Epidermal growth factor receptor
EGFR


P00709
Alpha-lactalbumin
LALBA


P00734
Prothrombin
F2


P00738
Haptoglobin beta chain
HP


P00739
Haptoglobin-related protein
HPR


P00740
Coagulation factor IXa heavy chain
F9


P00742
Factor X heavy chain
F10


P00746
Complement factor D
CFD


P00747
Plasmin light chain B
PLG


P00748
Coagulation factor XIIa light chain
F12


P00749
Urokinase-type plasminogen activator long
PLAU



chain A


P00750
Tissue-type plasminogen activator
PLAT


P00751
Complement factor B Ba fragment
CFB


P00797
Renin
REN


P00973
2′-5′-oligoadenylate synthase 1
OAS1


P00995
Pancreatic secretory trypsin inhibitor
SPINK1


P01008
Antithrombin-III
SERPINC1


P01009
Alpha-1-antitrypsin
SERPINA1


P01011
Alpha-1-antichymotrypsin His-Pro-less
SERPINA3


P01019
Angiotensin-1
AGT


P01023
Alpha-2-macroglobulin
A2M


P01024
Acylation stimulating protein
C3


P01031
Complement C5 beta chain
C5


P01033
Metalloproteinase inhibitor 1
TIMP1


P01034
Cystatin-C
CST3


P01036
Cystatin-S
CST4


P01037
Cystatin-SN
CST1


P01042
Kininogen-1 light chain
KNG1


P01127
Platelet-derived growth factor subunit B
PDGFB


P01135
Transforming growth factor alpha
TGFA


P01137
Transforming growth factor beta-1
TGFB1


P01138
Beta-nerve growth factor
NGF


P01148
Gonadoliberin-1
GNRH1


P01160
Atrial natriuretic factor
NPPA


P01178
Oxytocin
OXT


P01185
Vasopressin-neurophysin 2-copeptin
AVP


P01189
Corticotropin
POMC


P01210
PENK(237-258)
PENK


P01213
Alpha-neoendorphin
PDYN


P01215
Glycoprotein hormones alpha chain
CGA


P01222
Thyrotropin subunit beta
TSHB


P01225
Follitropin subunit beta
FSHB


P01229
Lutropin subunit beta
LHB


P01233
Choriogonadotropin subunit beta
CGB8


P01236
Prolactin
PRL


P01241
Somatotropin
GH1


P01242
Growth hormone variant
GH2


P01243
Chorionic somatomammotropin hormone
CSH2


P01258
Katacalcin
CALCA


P01266
Thyroglobulin
TG


P01270
Parathyroid hormone
PTH


P01275
Glucagon
GCG


P01282
Intestinal peptide PHM-27
VIP


P01286
Somatoliberin
GHRH


P01298
Pancreatic prohormone
PPY


P01303
C-flanking peptide of NPY
NPY


P01308
Insulin
INS


P01344
Insulin-like growth factor II
IGF2


P01350
Big gastrin
GAST


P01374
Lymphotoxin-alpha
LTA


P01375
C-domain 1
TNF


P01562
Interferon alpha-1/13
IFNA1


P01563
Interferon alpha-2
IFNA2


P01566
Interferon alpha-10
IFNA10


P01567
Interferon alpha-7
IFNA7


P01568
Interferon alpha-21
IFNA21


P01569
Interferon alpha-5
IFNA5


P01570
Interferon alpha-14
IFNA14


P01571
Interferon alpha-17
IFNA17


P01574
Interferon beta
IFNB1


P01579
Interferon gamma
IFNG


P01583
Interleukin-1 alpha
IL1A


P01584
Interleukin-1 beta
IL1B


P01588
Erythropoietin
EPO


P01591
Immunoglobulin J chain
IGJ


P01732
T-cell surface glycoprotein CD8 alpha chain
CD8A


P01833
Polymeric immunoglobulin receptor
PIGR


P01857
Ig gamma-1 chain C region
IGHG1


P01859
Ig gamma-2 chain C region
IGHG2


P01860
Ig gamma-3 chain C region
IGHG3


P01861
Ig gamma-4 chain C region
IGHG4


P01871
Ig mu chain C region
IGHM


P01880
Ig delta chain C region
IGHD


P02452
Collagen alpha-1(I) chain
COL1A1


P02458
Chondrocalcin
COL2A1


P02461
Collagen alpha-1(III) chain
COL3A1


P02462
Collagen alpha-1(IV) chain
COL4A1


P02647
Apolipoprotein A-I
APOA1


P02649
Apolipoprotein E
APOE


P02652
Apolipoprotein A-II
APOA2


P02654
Apolipoprotein C-I
APOC1


P02655
Apolipoprotein C-II
APOC2


P02656
Apolipoprotein C-III
APOC3


P02671
Fibrinogen alpha chain
FGA


P02675
Fibrinopeptide B
FGB


P02679
Fibrinogen gamma chain
FGG


P02741
C-reactive protein
CRP


P02743
Serum amyloid P-component(1-203)
APCS


P02745
Complement C1q subcomponent subunit A
C1QA


P02746
Complement C1q subcomponent subunit B
C1QB


P02747
Complement C1q subcomponent subunit C
C1QC


P02748
Complement component C9b
C9


P02749
Beta-2-glycoprotein 1
APOH


P02750
Leucine-rich alpha-2-glycoprotein
LRG1


P02751
Ugl-Y2
FN1


P02753
Retinol-binding protein 4
RBP4


P02760
Trypstatin
AMBP


P02763
Alpha-1-acid glycoprotein 1
ORM1


P02765
Alpha-2-HS-glycoprotein chain A
AHSG


P02766
Transthyretin
TTR


P02768
Serum albumin
ALB


P02771
Alpha-fetoprotein
AFP


P02774
Vitamin D-binding protein
GC


P02775
Connective tissue-activating peptide III
PPBP


P02776
Platelet factor 4
PF4


P02778
CXCL10(1-73)
CXCL10


P02786
Transferrin receptor protein 1
TFRC


P02787
Serotransferrin
TF


P02788
Lactoferroxin-C
LTF


P02790
Hemopexin
HPX


P02808
Statherin
STATH


P02810
Salivary acidic proline-rich phosphoprotein 1/2
PRH2


P02812
Basic salivary proline-rich protein 2
PRB2


P02814
Peptide D1A
SMR3B


P02818
Osteocalcin
BGLAP


P03950
Angiogenin
ANG


P03951
Coagulation factor XIa heavy chain
F11


P03952
Plasma kallikrein
KLKB1


P03956
27 kDa interstitial collagenase
MMP1


P03971
Muellerian-inhibiting factor
AMH


P03973
Antileukoproteinase
SLPI


P04003
C4b-binding protein alpha chain
C4BPA


P04004
Somatomedin-B
VTN


P04054
Phospholipase A2
PLA2G1B


P04085
Platelet-derived growth factor subunit A
PDGFA


P04090
Relaxin A chain
RLN2


P04114
Apolipoprotein B-100
APOB


P04118
Colipase
CLPS


P04141
Granulocyte-macrophage colony-stimulating
CSF2



factor


P04155
Trefoil factor 1
TFF1


P04180
Phosphatidylcholine-sterol acyltransferase
LCAT


P04196
Histidine-rich glycoprotein
HRG


P04217
Alpha-1B-glycoprotein
A1BG


P04275
von Willebrand antigen 2
VWF


P04278
Sex hormone-binding globulin
SHBG


P04279
Alpha-inhibin-31
SEMG1


P04280
Basic salivary proline-rich protein 1
PRB1


P04628
Proto-oncogene Wnt-1
WNT1


P04745
Alpha-amylase 1
AMY1A


P04746
Pancreatic alpha-amylase
AMY2A


P04808
Prorelaxin H1
RLN1


P05000
Interferon omega-1
IFNW1


P05013
Interferon alpha-6
IFNA6


P05014
Interferon alpha-4
IFNA4


P05015
Interferon alpha-16
IFNA16


P05019
Insulin-like growth factor I
IGF1


P05060
GAWK peptide
CHGB


P05090
Apolipoprotein D
APOD


P05109
Protein S100-A8
S100A8


P05111
Inhibin alpha chain
INHA


P05112
Interleukin-4
IL4


P05113
Interleukin-5
IL5


P05120
Plasminogen activator inhibitor 2
SERPINB2


P05121
Plasminogen activator inhibitor 1
SERPINE1


P05154
Plasma serine protease inhibitor
SERPINA5


P05155
Plasma protease C1 inhibitor
SERPING1


P05156
Complement factor I heavy chain
CFI


P05160
Coagulation factor XIII B chain
F13B


P05161
Ubiquitin-like protein ISG15
ISG15


P05230
Fibroblast growth factor 1
FGF1


P05231
Interleukin-6
IL6


P05305
Big endothelin-1
EDN1


P05408
C-terminal peptide
SCG5


P05451
Lithostathine-1-alpha
REG1A


P05452
Tetranectin
CLEC3B


P05543
Thyroxine-binding globulin
SERPINA7


P05814
Beta-casein
CSN2


P05997
Collagen alpha-2(V) chain
COL5A2


P06276
Cholinesterase
BCHE


P06307
Cholecystokinin-12
CCK


P06396
Gelsolin
GSN


P06681
Complement C2
C2


P06702
Protein S100-A9
S100A9


P06727
Apolipoprotein A-IV
APOA4


P06734
Low affinity immunoglobulin epsilon Fc receptor
FCER2



soluble form


P06744
Glucose-6-phosphate isomerase
GPI


P06850
Corticoliberin
CRH


P06858
Lipoprotein lipase
LPL


P06881
Calcitonin gene-related peptide 1
CALCA


P07093
Glia-derived nexin
SERPINE2


P07098
Gastric triacylglycerol lipase
LIPF


P07225
Vitamin K-dependent protein S
PROS1


P07237
Protein disulfide-isomerase
P4HB


P07288
Prostate-specific antigen
KLK3


P07306
Asialoglycoprotein receptor 1
ASGR1


P07355
Annexin A2
ANXA2


P07357
Complement component C8 alpha chain
C8A


P07358
Complement component C8 beta chain
C8B


P07360
Complement component C8 gamma chain
C8G


P07477
Alpha-trypsin chain 2
PRSS1


P07478
Trypsin-2
PRSS2


P07492
Neuromedin-C
GRP


P07498
Kappa-casein
CSN3


P07585
Decorin
DCN


P07911
Uromodulin
UMOD


P07942
Laminin subunit beta-1
LAMB1


P07988
Pulmonary surfactant-associated protein B
SFTPB


P07998
Ribonuclease pancreatic
RNASE1


P08118
Beta-microseminoprotein
MSMB


P08123
Collagen alpha-2(I) chain
COL1A2


P08185
Corticosteroid-binding globulin
SERPINA6


P08217
Chymotrypsin-like elastase family member 2A
CELA2A


P08218
Chymotrypsin-like elastase family member 2B
CELA2B


P08253
72 kDa type IV collagenase
MMP2


P08254
Stromelysin-1
MMP3


P08294
Extracellular superoxide dismutase [Cu—Zn]
SOD3


P08476
Inhibin beta A chain
INHBA


P08493
Matrix Gla protein
MGP


P08572
Collagen alpha-2(IV) chain
COL4A2


P08581
Hepatocyte growth factor receptor
MET


P08603
Complement factor H
CFH


P08620
Fibroblast growth factor 4
FGF4


P08637
Low affinity immunoglobulin gamma Fc region
FCGR3A



receptor III-A


P08697
Alpha-2-antiplasmin
SERPINF2


P08700
Interleukin-3
IL3


P08709
Coagulation factor VII
F7


P08833
Insulin-like growth factor-binding protein 1
IGFBP1


P08887
Interleukin-6 receptor subunit alpha
IL6R


P08949
Neuromedin-B-32
NMB


P08F94
Fibrocystin
PKHD1


P09038
Fibroblast growth factor 2
FGF2


P09228
Cystatin-SA
CST2


P09237
Matrilysin
MMP7


P09238
Stromelysin-2
MMP10


P09341
Growth-regulated alpha protein
CXCL1


P09382
Galectin-1
LGALS1


P09466
Glycodelin
PAEP


P09486
SPARC
SPARC


P09529
Inhibin beta B chain
INHBB


P09544
Protein Wnt-2
WNT2


P09603
Processed macrophage colony-stimulating factor 1
CSF1


P09681
Gastric inhibitory polypeptide
GIP


P09683
Secretin
SCT


P09919
Granulocyte colony-stimulating factor
CSF3


P0C091
FRAS1-related extracellular matrix protein 3
FREM3


P0C0L4
C4d-A
C4A


P0C0L5
Complement C4-B alpha chain
C4B


P0C0P6
Neuropeptide S
NPS


P0C7L1
Serine protease inhibitor Kazal-type 8
SPINK8


P0C862
Complement C1q and tumor necrosis factor-
C1QTNF9



related protein 9A


P0C8F1
Prostate and testis expressed protein 4
PATE4


P0CG01
Gastrokine-3
GKN3P


P0CG36
Cryptic family protein 1B
CFC1B


P0CG37
Cryptic protein
CFC1


P0CJ68
Humanin-like protein 1
MTRNR2L1


P0CJ69
Humanin-like protein 2
MTRNR2L2


P0CJ70
Humanin-like protein 3
MTRNR2L3


P0CJ71
Humanin-like protein 4
MTRNR2L4


P0CJ72
Humanin-like protein 5
MTRNR2L5


P0CJ73
Humanin-like protein 6
MTRNR2L6


P0CJ74
Humanin-like protein 7
MTRNR2L7


P0CJ75
Humanin-like protein 8
MTRNR2L8


P0CJ76
Humanin-like protein 9
MTRNR2L9


P0CJ77
Humanin-like protein 10
MTRNR2L10


P0DJD7
Pepsin A-4
PGA4


P0DJD8
Pepsin A-3
PGA3


P0DJD9
Pepsin A-5
PGA5


P0DJI8
Amyloid protein A
SAA1


P0DJI9
Serum amyloid A-2 protein
SAA2


P10082
Peptide YY(3-36)
PYY


P10092
Calcitonin gene-related peptide 2
CALCB


P10124
Serglycin
SRGN


P10145
MDNCF-a
IL8


P10147
MIP-1-alpha(4-69)
CCL3


P10163
Peptide P-D
PRB4


P10451
Osteopontin
SPP1


P10599
Thioredoxin
TXN


P10600
Transforming growth factor beta-3
TGFB3


P10643
Complement component C7
C7


P10645
Vasostatin-2
CHGA


P10646
Tissue factor pathway inhibitor
TFPI


P10720
Platelet factor 4 variant(4-74)
PF4V1


P10745
Retinol-binding protein 3
RBP3


P10767
Fibroblast growth factor 6
FGF6


P10909
Clusterin alpha chain
CLU


P10912
Growth hormone receptor
GHR


P10915
Hyaluronan and proteoglycan link protein 1
HAPLN1


P10966
T-cell surface glycoprotein CD8 beta chain
CD8B


P10997
Islet amyloid polypeptide
IAPP


P11047
Laminin subunit gamma-1
LAMC1


P11150
Hepatic triacylglycerol lipase
LIPC


P11226
Mannose-binding protein C
MBL2


P11464
Pregnancy-specific beta-1-glycoprotein 1
PSG1


P11465
Pregnancy-specific beta-1-glycoprotein 2
PSG2


P11487
Fibroblast growth factor 3
FGF3


P11597
Cholesteryl ester transfer protein
CETP


P11684
Uteroglobin
SCGB1A1


P11686
Pulmonary surfactant-associated protein C
SFTPC


P12034
Fibroblast growth factor 5
FGF5


P12107
Collagen alpha-1(XI) chain
COL11A1


P12109
Collagen alpha-1(VI) chain
COL6A1


P12110
Collagen alpha-2(VI) chain
COL6A2


P12111
Collagen alpha-3(VI) chain
COL6A3


P12259
Coagulation factor V
F5


P12272
PTHrP[1-36]
PTHLH


P12273
Prolactin-inducible protein
PIP


P12544
Granzyme A
GZMA


P12643
Bone morphogenetic protein 2
BMP2


P12644
Bone morphogenetic protein 4
BMP4


P12645
Bone morphogenetic protein 3
BMP3


P12724
Eosinophil cationic protein
RNASE3


P12821
Angiotensin-converting enzyme, soluble form
ACE


P12838
Neutrophil defensin 4
DEFA4


P12872
Motilin
MLN


P13232
Interleukin-7
IL7


P13236
C-C motif chemokine 4
CCL4


P13284
Gamma-interferon-inducible lysosomal thiol
IFI30



reductase


P13500
C-C motif chemokine 2
CCL2


P13501
C-C motif chemokine 5
CCL5


P13521
Secretogranin-2
SCG2


P13591
Neural cell adhesion molecule 1
NCAM1


P13611
Versican core protein
VCAN


P13671
Complement component C6
C6


P13688
Carcinoembryonic antigen-related cell adhesion
CEACAM1



molecule 1


P13725
Oncostatin-M
OSM


P13726
Tissue factor
F3


P13727
Eosinophil granule major basic protein
PRG2


P13942
Collagen alpha-2(XI) chain
COL11A2


P13987
CD59 glycoprotein
CD59


P14138
Endothelin-3
EDN3


P14174
Macrophage migration inhibitory factor
MIF


P14207
Folate receptor beta
FOLR2


P14222
Perforin-1
PRF1


P14543
Nidogen-1
NID1


P14555
Phospholipase A2, membrane associated
PLA2G2A


P14625
Endoplasmin
HSP90B1


P14735
Insulin-degrading enzyme
IDE


P14778
Interleukin-1 receptor type 1, soluble form
IL1R1


P14780
82 kDa matrix metalloproteinase-9
MMP9


P15018
Leukemia inhibitory factor
LIF


P15085
Carboxypeptidase A1
CPA1


P15086
Carboxypeptidase B
CPB1


P15151
Poliovirus receptor
PVR


P15169
Carboxypeptidase N catalytic chain
CPN1


P15248
Interleukin-9
IL9


P15291
N-acetyllactosamine synthase
B4GALT1


P15309
PAPf39
ACPP


P15328
Folate receptor alpha
FOLR1


P15374
Ubiquitin carboxyl-terminal hydrolase isozyme L3
UCHL3


P15502
Elastin
ELN


P15509
Granulocyte-macrophage colony-stimulating
CSF2RA



factor receptor subunit alpha


P15515
Histatin-1
HTN1


P15516
His3-(31-51)-peptide
HTN3


P15692
Vascular endothelial growth factor A
VEGFA


P15814
Immunoglobulin lambda-like polypeptide 1
IGLL1


P15907
Beta-galactoside alpha-2,6-sialyltransferase 1
ST6GAL1


P15941
Mucin-1 subunit beta
MUC1


P16035
Metalloproteinase inhibitor 2
TIMP2


P16112
Aggrecan core protein 2
ACAN


P16233
Pancreatic triacylglycerol lipase
PNLIP


P16442
Histo-blood group ABO system transferase
ABO


P16471
Prolactin receptor
PRLR


P16562
Cysteine-rich secretory protein 2
CRISP2


P16619
C-C motif chemokine 3-like 1
CCL3L1


P16860
BNP(3-29)
NPPB


P16870
Carboxypeptidase E
CPE


P16871
Interleukin-7 receptor subunit alpha
IL7R


P17213
Bactericidal permeability-increasing protein
BPI


P17538
Chymotrypsinogen B
CTRB1


P17931
Galectin-3
LGALS3


P17936
Insulin-like growth factor-binding protein 3
IGFBP3


P17948
Vascular endothelial growth factor receptor 1
FLT1


P18065
Insulin-like growth factor-binding protein 2
IGFBP2


P18075
Bone morphogenetic protein 7
BMP7


P18428
Lipopolysaccharide-binding protein
LBP


P18509
PACAP-related peptide
ADCYAP1


P18510
Interleukin-1 receptor antagonist protein
IL1RN


P18827
Syndecan-1
SDC1


P19021
Peptidylglycine alpha-hydroxylating
PAM



monooxygenase


P19235
Erythropoietin receptor
EPOR


P19438
Tumor necrosis factor-binding protein 1
TNFRSF1A


P19652
Alpha-1-acid glycoprotein 2
ORM2


P19801
Amiloride-sensitive amine oxidase [copper-
ABP1



containing]


P19823
Inter-alpha-trypsin inhibitor heavy chain H2
ITIH2


P19827
Inter-alpha-trypsin inhibitor heavy chain H1
ITIH1


P19835
Bile salt-activated lipase
CEL


P19875
C-X-C motif chemokine 2
CXCL2


P19876
C-X-C motif chemokine 3
CXCL3


P19883
Follistatin
FST


P19957
Elafin
PI3


P19961
Alpha-amylase 2B
AMY2B


P20061
Transcobalamin-1
TCN1


P20062
Transcobalamin-2
TCN2


P20142
Gastricsin
PGC


P20155
Serine protease inhibitor Kazal-type 2
SPINK2


P20231
Tryptase beta-2
TPSB2


P20333
Tumor necrosis factor receptor superfamily
TNFRSF1B



member 1B


P20366
Substance P
TAC1


P20382
Melanin-concentrating hormone
PMCH


P20396
Thyroliberin
TRH


P20742
Pregnancy zone protein
PZP


P20774
Mimecan
OGN


P20783
Neurotrophin-3
NTF3


P20800
Endothelin-2
EDN2


P20809
Interleukin-11
IL11


P20827
Ephrin-A1
EFNA1


P20849
Collagen alpha-1(IX) chain
COL9A1


P20851
C4b-binding protein beta chain
C4BPB


P20908
Collagen alpha-1(V) chain
COL5A1


P21128
Poly(U)-specific endoribonuclease
ENDOU


P21246
Pleiotrophin
PTN


P21583
Kit ligand
KITLG


P21741
Midkine
MDK


P21754
Zona pellucida sperm-binding protein 3
ZP3


P21781
Fibroblast growth factor 7
FGF7


P21802
Fibroblast growth factor receptor 2
FGFR2


P21810
Biglycan
BGN


P21815
Bone sialoprotein 2
IBSP


P21860
Receptor tyrosine-protein kinase erbB-3
ERBB3


P21941
Cartilage matrix protein
MATN1


P22003
Bone morphogenetic protein 5
BMP5


P22004
Bone morphogenetic protein 6
BMP6


P22079
Lactoperoxidase
LPO


P22105
Tenascin-X
TNXB


P22301
Interleukin-10
IL10


P22303
Acetylcholinesterase
ACHE


P22352
Glutathione peroxidase 3
GPX3


P22362
C-C motif chemokine 1
CCL1


P22455
Fibroblast growth factor receptor 4
FGFR4


P22466
Galanin message-associated peptide
GAL


P22692
Insulin-like growth factor-binding protein 4
IGFBP4


P22749
Granulysin
GNLY


P22792
Carboxypeptidase N subunit 2
CPN2


P22891
Vitamin K-dependent protein Z
PROZ


P22894
Neutrophil collagenase
MMP8


P23142
Fibulin-1
FBLN1


P23280
Carbonic anhydrase 6
CA6


P23352
Anosmin-1
KAL1


P23435
Cerebellin-1
CBLN1


P23560
Brain-derived neurotrophic factor
BDNF


P23582
C-type natriuretic peptide
NPPC


P23946
Chymase
CMA1


P24043
Laminin subunit alpha-2
LAMA2


P24071
Immunoglobulin alpha Fc receptor
FCAR


P24347
Stromelysin-3
MMP11


P24387
Corticotropin-releasing factor-binding protein
CRHBP


P24592
Insulin-like growth factor-binding protein 6
IGFBP6


P24593
Insulin-like growth factor-binding protein 5
IGFBP5


P24821
Tenascin
TNC


P24855
Deoxyribonuclease-1
DNASE1


P25067
Collagen alpha-2(VIII) chain
COL8A2


P25311
Zinc-alpha-2-glycoprotein
AZGP1


P25391
Laminin subunit alpha-1
LAMA1


P25445
Tumor necrosis factor receptor superfamily
FAS



member 6


P25940
Collagen alpha-3(V) chain
COL5A3


P25942
Tumor necrosis factor receptor superfamily
CD40



member 5


P26022
Pentraxin-related protein PTX3
PTX3


P26927
Hepatocyte growth factor-like protein beta chain
MST1


P27169
Serum paraoxonase/arylesterase 1
PON1


P27352
Gastric intrinsic factor
GIF


P27487
Dipeptidyl peptidase 4 membrane form
DPP4


P27539
Embryonic growth/differentiation factor 1
GDF1


P27658
Vastatin
COL8A1


P27797
Calreticulin
CALR


P27918
Properdin
CFP


P28039
Acyloxyacyl hydrolase
AOAH


P28300
Protein-lysine 6-oxidase
LOX


P28325
Cystatin-D
CST5


P28799
Granulin-1
GRN


P29122
Proprotein convertase subtilisin/kexin type 6
PCSK6


P29279
Connective tissue growth factor
CTGF


P29320
Ephrin type-A receptor 3
EPHA3


P29400
Collagen alpha-5(IV) chain
COL4A5


P29459
Interleukin-12 subunit alpha
IL12A


P29460
Interleukin-12 subunit beta
IL12B


P29508
Serpin B3
SERPINB3


P29622
Kallistatin
SERPINA4


P29965
CD40 ligand, soluble form
CD40LG


P30990
Neurotensin/neuromedin N
NTS


P31025
Lipocalin-1
LCN1


P31151
Protein S100-A7
S100A7


P31371
Fibroblast growth factor 9
FGF9


P31431
Syndecan-4
SDC4


P31947
14-3-3 protein sigma
SFN


P32455
Interferon-induced guanylate-binding protein 1
GBP1


P32881
Interferon alpha-8
IFNA8


P34096
Ribonuclease 4
RNASE4


P34130
Neurotrophin-4
NTF4


P34820
Bone morphogenetic protein 8B
BMP8B


P35030
Trypsin-3
PRSS3


P35052
Secreted glypican-1
GPC1


P35070
Betacellulin
BTC


P35225
Interleukin-13
IL13


P35247
Pulmonary surfactant-associated protein D
SFTPD


P35318
ADM
ADM


P35542
Serum amyloid A-4 protein
SAA4


P35555
Fibrillin-1
FBN1


P35556
Fibrillin-2
FBN2


P35625
Metalloproteinase inhibitor 3
TIMP3


P35858
Insulin-like growth factor-binding protein complex
IGFALS



acid labile subunit


P35916
Vascular endothelial growth factor receptor 3
FLT4


P35968
Vascular endothelial growth factor receptor 2
KDR


P36222
Chitinase-3-like protein 1
CHI3L1


P36952
Serpin B5
SERPINB5


P36955
Pigment epithelium-derived factor
SERPINF1


P36980
Complement factor H-related protein 2
CFHR2


P39059
Collagen alpha-1(XV) chain
COL15A1


P39060
Collagen alpha-1(XVIII) chain
COL18A1


P39877
Calcium-dependent phospholipase A2
PLA2G5


P39900
Macrophage metalloelastase
MMP12


P39905
Glial cell line-derived neurotrophic factor
GDNF


P40225
Thrombopoietin
THPO


P40967
M-alpha
PMEL


P41159
Leptin
LEP


P41221
Protein Wnt-5a
WNT5A


P41222
Prostaglandin-H2 D-isomerase
PTGDS


P41271
Neuroblastoma suppressor of tumorigenicity 1
NBL1


P41439
Folate receptor gamma
FOLR3


P42127
Agouti-signaling protein
ASIP


P42702
Leukemia inhibitory factor receptor
LIFR


P42830
ENA-78(9-78)
CXCL5


P43026
Growth/differentiation factor 5
GDF5


P43251
Biotinidase
BTD


P43652
Afamin
AFM


P45452
Collagenase 3
MMP13


P47710
Casoxin-D
CSN1S1


P47929
Galectin-7
LGALS7B


P47972
Neuronal pentraxin-2
NPTX2


P47989
Xanthine oxidase
XDH


P47992
Lymphotactin
XCL1


P48023
Tumor necrosis factor ligand superfamily
FASLG



member 6, membrane form


P48052
Carboxypeptidase A2
CPA2


P48061
Stromal cell-derived factor 1
CXCL12


P48304
Lithostathine-1-beta
REG1B


P48307
Tissue factor pathway inhibitor 2
TFPI2


P48357
Leptin receptor
LEPR


P48594
Serpin B4
SERPINB4


P48645
Neuromedin-U-25
NMU


P48740
Mannan-binding lectin serine protease 1
MASP1


P48745
Protein NOV homolog
NOV


P48960
CD97 antigen subunit beta
CD97


P49223
Kunitz-type protease inhibitor 3
SPINT3


P49747
Cartilage oligomeric matrix protein
COMP


P49763
Placenta growth factor
PGF


P49765
Vascular endothelial growth factor B
VEGFB


P49767
Vascular endothelial growth factor C
VEGFC


P49771
Fms-related tyrosine kinase 3 ligand
FLT3LG


P49862
Kallikrein-7
KLK7


P49863
Granzyme K
GZMK


P49908
Selenoprotein P
SEPP1


P49913
Antibacterial protein FALL-39
CAMP


P50607
Tubby protein homolog
TUB


P51124
Granzyme M
GZMM


P51512
Matrix metalloproteinase-16
MMP16


P51654
Glypican-3
GPC3


P51671
Eotaxin
CCL11


P51884
Lumican
LUM


P51888
Prolargin
PRELP


P52798
Ephrin-A4
EFNA4


P52823
Stanniocalcin-1
STC1


P53420
Collagen alpha-4(IV) chain
COL4A4


P53621
Coatomer subunit alpha
COPA


P54108
Cysteine-rich secretory protein 3
CRISP3


P54315
Pancreatic lipase-related protein 1
PNLIPRP1


P54317
Pancreatic lipase-related protein 2
PNLIPRP2


P54793
Arylsulfatase F
ARSF


P55000
Secreted Ly-6/uPAR-related protein 1
SLURP1


P55001
Microfibrillar-associated protein 2
MFAP2


P55056
Apolipoprotein C-IV
APOC4


P55058
Phospholipid transfer protein
PLTP


P55075
Fibroblast growth factor 8
FGF8


P55081
Microfibrillar-associated protein 1
MFAP1


P55083
Microfibril-associated glycoprotein 4
MFAP4


P55107
Bone morphogenetic protein 3B
GDF10


P55145
Mesencephalic astrocyte-derived neurotrophic
MANF



factor


P55259
Pancreatic secretory granule membrane major
GP2



glycoprotein GP2


P55268
Laminin subunit beta-2
LAMB2


P55773
CCL23(30-99)
CCL23


P55774
C-C motif chemokine 18
CCL18


P55789
FAD-linked sulfhydryl oxidase ALR
GFER


P56703
Proto-oncogene Wnt-3
WNT3


P56704
Protein Wnt-3a
WNT3A


P56705
Protein Wnt-4
WNT4


P56706
Protein Wnt-7b
WNT7B


P56730
Neurotrypsin
PRSS12


P56851
Epididymal secretory protein E3-beta
EDDM3B


P56975
Neuregulin-3
NRG3


P58062
Serine protease inhibitor Kazal-type 7
SPINK7


P58215
Lysyl oxidase homolog 3
LOXL3


P58294
Prokineticin-1
PROK1


P58335
Anthrax toxin receptor 2
ANTXR2


P58397
A disintegrin and metalloproteinase with
ADAMTS12



thrombospondin motifs 12


P58417
Neurexophilin-1
NXPH1


P58499
Protein FAM3B
FAM3B


P59510
A disintegrin and metalloproteinase with
ADAMTS20



thrombospondin motifs 20


P59665
Neutrophil defensin 1
DEFA1B


P59666
Neutrophil defensin 3
DEFA3


P59796
Glutathione peroxidase 6
GPX6


P59826
BPI fold-containing family B member 3
BPIFB3


P59827
BPI fold-containing family B member 4
BPIFB4


P59861
Beta-defensin 131
DEFB131


P60022
Beta-defensin 1
DEFB1


P60153
Inactive ribonuclease-like protein 9
RNASE9


P60827
Complement C1q tumor necrosis factor-related
C1QTNF8



protein 8


P60852
Zona pellucida sperm-binding protein 1
ZP1


P60985
Keratinocyte differentiation-associated protein
KRTDAP


P61109
Kidney androgen-regulated protein
KAP


P61278
Somatostatin-14
SST


P61366
Osteocrin
OSTN


P61626
Lysozyme C
LYZ


P61769
Beta-2-microglobulin
B2M


P61812
Transforming growth factor beta-2
TGFB2


P61916
Epididymal secretory protein E1
NPC2


P62502
Epididymal-specific lipocalin-6
LCN6


P62937
Peptidyl-prolyl cis-trans isomerase A
PPIA


P67809
Nuclease-sensitive element-binding protein 1
YBX1


P67812
Signal peptidase complex catalytic subunit
SEC11A



SEC11A


P78310
Coxsackievirus and adenovirus receptor
CXADR


P78333
Secreted glypican-5
GPC5


P78380
Oxidized low-density lipoprotein receptor 1
OLR1


P78423
Processed fractalkine
CX3CL1


P78509
Reelin
RELN


P78556
CCL20(2-70)
CCL20


P80075
MCP-2(6-76)
CCL8


P80098
C-C motif chemokine 7
CCL7


P80108
Phosphatidylinositol-glycan-specific
GPLD1



phospholipase D


P80162
C-X-C motif chemokine 6
CXCL6


P80188
Neutrophil gelatinase-associated lipocalin
LCN2


P80303
Nucleobindin-2
NUCB2


P80511
Calcitermin
S100A12


P81172
Hepcidin-25
HAMP


P81277
Prolactin-releasing peptide
PRLH


P81534
Beta-defensin 103
DEFB103A


P81605
Dermcidin
DCD


P82279
Protein crumbs homolog 1
CRB1


P82987
ADAMTS-like protein 3
ADAMTSL3


P83105
Serine protease HTRA4
HTRA4


P83110
Serine protease HTRA3
HTRA3


P83859
Orexigenic neuropeptide QRFP
QRFP


P98088
Mucin-5AC
MUC5AC


P98095
Fibulin-2
FBLN2


P98160
Basement membrane-specific heparan sulfate
HSPG2



proteoglycan core protein


P98173
Protein FAM3A
FAM3A


Q00604
Norrin
NDP


Q00796
Sorbitol dehydrogenase
SORD


Q00887
Pregnancy-specific beta-1-glycoprotein 9
PSG9


Q00888
Pregnancy-specific beta-1-glycoprotein 4
PSG4


Q00889
Pregnancy-specific beta-1-glycoprotein 6
PSG6


Q01523
HD5(56-94)
DEFA5


Q01524
Defensin-6
DEFA6


Q01955
Collagen alpha-3(IV) chain
COL4A3


Q02297
Pro-neuregulin-1, membrane-bound isoform
NRG1


Q02325
Plasminogen-like protein B
PLGLB1


Q02383
Semenogelin-2
SEMG2


Q02388
Collagen alpha-1(VII) chain
COL7A1


Q02505
Mucin-3A
MUC3A


Q02509
Otoconin-90
OC90


Q02747
Guanylin
GUCA2A


Q02763
Angiopoietin-1 receptor
TEK


Q02817
Mucin-2
MUC2


Q02985
Complement factor H-related protein 3
CFHR3


Q03167
Transforming growth factor beta receptor type 3
TGFBR3


Q03403
Trefoil factor 2
TFF2


Q03405
Urokinase plasminogen activator surface receptor
PLAUR


Q03591
Complement factor H-related protein 1
CFHR1


Q03692
Collagen alpha-1(X) chain
COL10A1


Q04118
Basic salivary proline-rich protein 3
PRB3


Q04756
Hepatocyte growth factor activator short chain
HGFAC


Q04900
Sialomucin core protein 24
CD164


Q05315
Eosinophil lysophospholipase
CLC


Q05707
Collagen alpha-1(XIV) chain
COL14A1


Q05996
Processed zona pellucida sperm-binding protein 2
ZP2


Q06033
Inter-alpha-trypsin inhibitor heavy chain H3
ITIH3


Q06141
Regenerating islet-derived protein 3-alpha
REG3A


Q06828
Fibromodulin
FMOD


Q07092
Collagen alpha-1(XVI) chain
COL16A1


Q07325
C-X-C motif chemokine 9
CXCL9


Q07507
Dermatopontin
DPT


Q075Z2
Binder of sperm protein homolog 1
BSPH1


Q07654
Trefoil factor 3
TFF3


Q07699
Sodium channel subunit beta-1
SCN1B


Q08345
Epithelial discoidin domain-containing receptor 1
DDR1


Q08380
Galectin-3-binding protein
LGALS3BP


Q08397
Lysyl oxidase homolog 1
LOXL1


Q08431
Lactadherin
MFGE8


Q08629
Testican-1
SPOCK1


Q08648
Sperm-associated antigen 11B
SPAG11B


Q08830
Fibrinogen-like protein 1
FGL1


Q10471
Polypeptide N-acetylgalactosaminyltransferase 2
GALNT2


Q10472
Polypeptide N-acetylgalactosaminyltransferase 1
GALNT1


Q11201
CMP-N-acetylneuraminate-beta-galactosamide-
ST3GAL1



alpha-2,3-sialyltransferase 1


Q11203
CMP-N-acetylneuraminate-beta-1,4-galactoside
ST3GAL3



alpha-2,3-sialyltransferase


Q11206
CMP-N-acetylneuraminate-beta-galactosamide-
ST3GAL4



alpha-2,3-sialyltransferase 4


Q12794
Hyaluronidase-1
HYAL1


Q12805
EGF-containing fibulin-like extracellular matrix
EFEMP1



protein 1


Q12836
Zona pellucida sperm-binding protein 4
ZP4


Q12841
Follistatin-related protein 1
FSTL1


Q12904
Aminoacyl tRNA synthase complex-interacting
AIMP1



multifunctional protein 1


Q13018
Soluble secretory phospholipase A2 receptor
PLA2R1


Q13072
B melanoma antigen 1
BAGE


Q13093
Platelet-activating factor acetylhydrolase
PLA2G7


Q13103
Secreted phosphoprotein 24
SPP2


Q13162
Peroxiredoxin-4
PRDX4


Q13201
Platelet glycoprotein Ia*
MMRN1


Q13214
Semaphorin-3B
SEMA3B


Q13219
Pappalysin-1
PAPPA


Q13231
Chitotriosidase-1
CHIT1


Q13253
Noggin
NOG


Q13261
Interleukin-15 receptor subunit alpha
IL15RA


Q13275
Semaphorin-3F
SEMA3F


Q13291
Signaling lymphocytic activation molecule
SLAMF1


Q13316
Dentin matrix acidic phosphoprotein 1
DMP1


Q13361
Microfibrillar-associated protein 5
MFAP5


Q13410
Butyrophilin subfamily 1 member A1
BTN1A1


Q13421
Mesothelin, cleaved form
MSLN


Q13429
Insulin-like growth factor I
IGF-I


Q13443
Disintegrin and metalloproteinase domain-
ADAM9



containing protein 9


Q13519
Neuropeptide 1
PNOC


Q13751
Laminin subunit beta-3
LAMB3


Q13753
Laminin subunit gamma-2
LAMC2


Q13790
Apolipoprotein F
APOF


Q13822
Ectonucleotide pyrophosphatase/phosphodiesterase
ENPP2



family member 2


Q14031
Collagen alpha-6(IV) chain
COL4A6


Q14050
Collagen alpha-3(IX) chain
COL9A3


Q14055
Collagen alpha-2(IX) chain
COL9A2


Q14112
Nidogen-2
NID2


Q14114
Low-density lipoprotein receptor-related protein 8
LRP8


Q14118
Dystroglycan
DAG1


Q14314
Fibroleukin
FGL2


Q14393
Growth arrest-specific protein 6
GAS6


Q14406
Chorionic somatomammotropin hormone-like 1
CSHL1


Q14507
Epididymal secretory protein E3-alpha
EDDM3A


Q14508
WAP four-disulfide core domain protein 2
WFDC2


Q14512
Fibroblast growth factor-binding protein 1
FGFBP1


Q14515
SPARC-like protein 1
SPARCL1


Q14520
Hyaluronan-binding protein 2 27 kDa light chain
HABP2


Q14563
Semaphorin-3A
SEMA3A


Q14623
Indian hedgehog protein
IHH


Q14624
Inter-alpha-trypsin inhibitor heavy chain H4
ITIH4


Q14667
UPF0378 protein KIAA0100
KIAA0100


Q14703
Membrane-bound transcription factor site-1
MBTPS1



protease


Q14766
Latent-transforming growth factor beta-binding
LTBP1



protein 1


Q14767
Latent-transforming growth factor beta-binding
LTBP2



protein 2


Q14773
Intercellular adhesion molecule 4
ICAM4


Q14993
Collagen alpha-1(XIX) chain
COL19A1


Q14CN2
Calcium-activated chloride channel regulator 4,
CLCA4



110 kDa form


Q15046
Lysine--tRNA ligase
KARS


Q15063
Periostin
POSTN


Q15109
Advanced glycosylation end product-specific
AGER



receptor


Q15113
Procollagen C-endopeptidase enhancer 1
PCOLCE


Q15166
Serum paraoxonase/lactonase 3
PON3


Q15195
Plasminogen-like protein A
PLGLA


Q15198
Platelet-derived growth factor receptor-like protein
PDGFRL


Q15223
Poliovirus receptor-related protein 1
PVRL1


Q15238
Pregnancy-specific beta-1-glycoprotein 5
PSG5


Q15363
Transmembrane emp24 domain-containing protein 2
TMED2


Q15375
Ephrin type-A receptor 7
EPHA7


Q15389
Angiopoietin-1
ANGPT1


Q15465
Sonic hedgehog protein
SHH


Q15485
Ficolin-2
FCN2


Q15517
Corneodesmosin
CDSN


Q15582
Transforming growth factor-beta-induced protein ig-h3
TGFBI


Q15661
Tryptase alpha/beta-1
TPSAB1


Q15726
Metastin
KISS1


Q15782
Chitinase-3-like protein 2
CHI3L2


Q15828
Cystatin-M
CST6


Q15846
Clusterin-like protein 1
CLUL1


Q15848
Adiponectin
ADIPOQ


Q16206
Protein disulfide-thiol oxidoreductase
ENOX2


Q16270
Insulin-like growth factor-binding protein 7
IGFBP7


Q16363
Laminin subunit alpha-4
LAMA4


Q16378
Proline-rich protein 4
PRR4


Q16557
Pregnancy-specific beta-1-glycoprotein 3
PSG3


Q16568
CART(42-89)
CARTPT


Q16610
Extracellular matrix protein 1
ECM1


Q16619
Cardiotrophin-1
CTF1


Q16623
Syntaxin-1A
STX1A


Q16627
HCC-1(9-74)
CCL14


Q16651
Prostasin light chain
PRSS8


Q16661
Guanylate cyclase C-activating peptide 2
GUCA2B


Q16663
CCL15(29-92)
CCL15


Q16674
Melanoma-derived growth regulatory protein
MIA


Q16769
Glutaminyl-peptide cyclotransferase
QPCT


Q16787
Laminin subunit alpha-3
LAMA3


Q16842
CMP-N-acetylneuraminate-beta-galactosamide-
ST3GAL2



alpha-2,3-sialyltransferase 2


Q17RR3
Pancreatic lipase-related protein 3
PNLIPRP3


Q17RW2
Collagen alpha-1(XXIV) chain
COL24A1


Q17RY6
Lymphocyte antigen 6K
LY6K


Q1L6U9
Prostate-associated microseminoprotein
MSMP


Q1W4C9
Serine protease inhibitor Kazal-type 13
SPINK13


Q1ZYL8
Izumo sperm-egg fusion protein 4
IZUMO4


Q29960
HLA class I histocompatibility antigen, Cw-16
HLA-C



alpha chain


Q2I0M5
R-spondin-4
RSPO4


Q2L4Q9
Serine protease 53
PRSS53


Q2MKA7
R-spondin-1
RSPO1


Q2MV58
Tectonic-1
TCTN1


Q2TAL6
Brorin
VWC2


Q2UY09
Collagen alpha-1(XXVIII) chain
COL28A1


Q2VPA4
Complement component receptor 1-like protein
CR1L


Q2WEN9
Carcinoembryonic antigen-related cell adhesion
CEACAM16



molecule 16


Q30KP8
Beta-defensin 136
DEFB136


Q30KP9
Beta-defensin 135
DEFB135


Q30KQ1
Beta-defensin 133
DEFB133


Q30KQ2
Beta-defensin 130
DEFB130


Q30KQ4
Beta-defensin 116
DEFB116


Q30KQ5
Beta-defensin 115
DEFB115


Q30KQ6
Beta-defensin 114
DEFB114


Q30KQ7
Beta-defensin 113
DEFB113


Q30KQ8
Beta-defensin 112
DEFB112


Q30KQ9
Beta-defensin 110
DEFB110


Q30KR1
Beta-defensin 109
DEFB109P1


Q32P28
Prolyl 3-hydroxylase 1
LEPRE1


Q3B7J2
Glucose-fructose oxidoreductase domain-
GFOD2



containing protein 2


Q3SY79
Protein Wnt
WNT3A


Q3T906
N-acetylglucosamine-1-phosphotransferase
GNPTAB



subunits alpha/beta


Q495T6
Membrane metallo-endopeptidase-like 1
MMEL1


Q49AH0
Cerebral dopamine neurotrophic factor
CDNF


Q4G0G5
Secretoglobin family 2B member 2
SCGB2B2


Q4G0M1
Protein FAM132B
FAM132B


Q4LDE5
Sushi, von Willebrand factor type A, EGF and
SVEP1



pentraxin domain-containing protein 1


Q4QY38
Beta-defensin 134
DEFB134


Q4VAJ4
Protein Wnt
WNT10B


Q4W5P6
Protein TMEM155
TMEM155


Q4ZHG4
Fibronectin type III domain-containing protein 1
FNDC1


Q53H76
Phospholipase A1 member A
PLA1A


Q53RD9
Fibulin-7
FBLN7


Q53S33
BolA-like protein 3
BOLA3


Q5BLP8
Neuropeptide-like protein C4orf48
C4orf48


Q5DT21
Serine protease inhibitor Kazal-type 9
SPINK9


Q5EBL8
PDZ domain-containing protein 11
PDZD11


Q5FYB0
Arylsulfatase J
ARSJ


Q5FYB1
Arylsulfatase I
ARSI


Q5GAN3
Ribonuclease-like protein 13
RNASE13


Q5GAN4
Ribonuclease-like protein 12
RNASE12


Q5GAN6
Ribonuclease-like protein 10
RNASE10


Q5GFL6
von Willebrand factor A domain-containing
VWA2



protein 2


Q5H8A3
Neuromedin-S
NMS


Q5H8C1
FRAS1-related extracellular matrix protein 1
FREM1


Q5IJ48
Protein crumbs homolog 2
CRB2


Q5J5C9
Beta-defensin 121
DEFB121


Q5JS37
NHL repeat-containing protein 3
NHLRC3


Q5JTB6
Placenta-specific protein 9
PLAC9


Q5JU69
Torsin-2A
TOR2A


Q5JXM2
Methyltransferase-like protein 24
METTL24


Q5JZY3
Ephrin type-A receptor 10
EPHA10


Q5K4E3
Polyserase-2
PRSS36


Q5SRR4
Lymphocyte antigen 6 complex locus protein G5c
LY6G5C


Q5T1H1
Protein eyes shut homolog
EYS


Q5T4F7
Secreted frizzled-related protein 5
SFRP5


Q5T4W7
Artemin
ARTN


Q5T7M4
Protein FAM132A
FAM132A


Q5TEH8
Protein Wnt
WNT2B


Q5TIE3
von Willebrand factor A domain-containing
VWA5B1



protein 5B1


Q5UCC4
ER membrane protein complex subunit 10
EMC10


Q5VST6
Abhydrolase domain-containing protein
FAM108B1



FAM108B1


Q5VTL7
Fibronectin type III domain-containing protein 7
FNDC7


Q5VUM1
UPF0369 protein C6orf57
C6orf57


Q5VV43
Dyslexia-associated protein KIAA0319
KIAA0319


Q5VWW1
Complement C1q-like protein 3
C1QL3


Q5VXI9
Lipase member N
LIPN


Q5VXJ0
Lipase member K
LIPK


Q5VXM1
CUB domain-containing protein 2
CDCP2


Q5VYX0
Renalase
RNLS


Q5VYY2
Lipase member M
LIPM


Q5W186
Cystatin-9
CST9


Q5W5W9
Regulated endocrine-specific protein 18
RESP18


Q5XG92
Carboxylesterase 4A
CES4A


Q63HQ2
Pikachurin
EGFLAM


Q641Q3
Meteorin-like protein
METRNL


Q66K79
Carboxypeptidase Z
CPZ


Q685J3
Mucin-17
MUC17


Q68BL7
Olfactomedin-like protein 2A
OLFML2A


Q68BL8
Olfactomedin-like protein 2B
OLFML2B


Q68DV7
E3 ubiquitin-protein ligase RNF43
RNF43


Q6B9Z1
Insulin growth factor-like family member 4
IGFL4


Q6BAA4
Fc receptor-like B
FCRLB


Q6E0U4
Dermokine
DMKN


Q6EMK4
Vasorin
VASN


Q6FHJ7
Secreted frizzled-related protein 4
SFRP4


Q6GPI1
Chymotrypsin B2 chain B
CTRB2


Q6GTS8
Probable carboxypeptidase PM20D1
PM20D1


Q6H9L7
Isthmin-2
ISM2


Q6IE36
Ovostatin homolog 2
OVOS2


Q6IE37
Ovostatin homolog 1
OVOS1


Q6IE38
Serine protease inhibitor Kazal-type 14
SPINK14


Q6ISS4
Leukocyte-associated immunoglobulin-like
LAIR2



receptor 2


Q6JVE5
Epididymal-specific lipocalin-12
LCN12


Q6JVE6
Epididymal-specific lipocalin-10
LCN10


Q6JVE9
Epididymal-specific lipocalin-8
LCN8


Q6KF10
Growth/differentiation factor 6
GDF6


Q6MZW2
Follistatin-related protein 4
FSTL4


Q6NSX1
Coiled-coil domain-containing protein 70
CCDC70


Q6NT32
Carboxylesterase 5A
CES5A


Q6NT52
Choriogonadotropin subunit beta variant 2
CGB2


Q6NUI6
Chondroadherin-like protein
CHADL


Q6NUJ1
Saposin A-like
PSAPL1


Q6P093
Arylacetamide deacetylase-like 2
AADACL2


Q6P4A8
Phospholipase B-like 1
PLBD1


Q6P5S2
UPF0762 protein C6orf58
C6orf58


Q6P988
Protein notum homolog
NOTUM


Q6PCB0
von Willebrand factor A domain-containing
VWA1



protein 1


Q6PDA7
Sperm-associated antigen 11A
SPAG11A


Q6PEW0
Inactive serine protease 54
PRSS54


Q6PEZ8
Podocan-like protein 1
PODNL1


Q6PKH6
Dehydrogenase/reductase SDR family member 4-
DHRS4L2



like 2


Q6Q788
Apolipoprotein A-V
APOA5


Q6SPF0
Atherin
SAMD1


Q6UDR6
Kunitz-type protease inhibitor 4
SPINT4


Q6URK8
Testis, prostate and placenta-expressed protein
TEPP


Q6UW01
Cerebellin-3
CBLN3


Q6UW10
Surfactant-associated protein 2
SFTA2


Q6UW15
Regenerating islet-derived protein 3-gamma
REG3G


Q6UW32
Insulin growth factor-like family member 1
IGFL1


Q6UW78
UPF0723 protein C11orf83
C11orf83


Q6UW88
Epigen
EPGN


Q6UWE3
Colipase-like protein 2
CLPSL2


Q6UWF7
NXPE family member 4
NXPE4


Q6UWF9
Protein FAM180A
FAM180A


Q6UWM5
GLIPR1-like protein 1
GLIPR1L1


Q6UWN8
Serine protease inhibitor Kazal-type 6
SPINK6


Q6UWP2
Dehydrogenase/reductase SDR family member 11
DHRS11


Q6UWP8
Suprabasin
SBSN


Q6UWQ5
Lysozyme-like protein 1
LYZL1


Q6UWQ7
Insulin growth factor-like family member 2
IGFL2


Q6UWR7
Ectonucleotide pyrophosphatase/phosphodiesterase
ENPP6



family member 6 soluble form


Q6UWT2
Adropin
ENHO


Q6UWU2
Beta-galactosidase-1-like protein
GLB1L


Q6UWW0
Lipocalin-15
LCN15


Q6UWX4
HHIP-like protein 2
HHIPL2


Q6UWY0
Arylsulfatase K
ARSK


Q6UWY2
Serine protease 57
PRSS57


Q6UWY5
Olfactomedin-like protein 1
OLFML1


Q6UX06
Olfactomedin-4
OLFM4


Q6UX07
Dehydrogenase/reductase SDR family member 13
DHRS13


Q6UX39
Amelotin
AMTN


Q6UX46
Protein FAM150B
FAM150B


Q6UX73
UPF0764 protein C16orf89
C16orf89


Q6UXB0
Protein FAM131A
FAM131A


Q6UXB1
Insulin growth factor-like family member 3
IGFL3


Q6UXB2
VEGF co-regulated chemokine 1
CXCL17


Q6UXF7
C-type lectin domain family 18 member B
CLEC18B


Q6UXH0
Hepatocellular carcinoma-associated protein TD26
C19orf80


Q6UXH1
Cysteine-rich with EGF-like domain protein 2
CRELD2


Q6UXH8
Collagen and calcium-binding EGF domain-
CCBE1



containing protein 1


Q6UXH9
Inactive serine protease PAMR1
PAMR1


Q6UXI7
Vitrin
VIT


Q6UXI9
Nephronectin
NPNT


Q6UXN2
Trem-like transcript 4 protein
TREML4


Q6UXS0
C-type lectin domain family 19 member A
CLEC19A


Q6UXT8
Protein FAM150A
FAM150A


Q6UXT9
Abhydrolase domain-containing protein 15
ABHD15


Q6UXV4
Apolipoprotein O-like
APOOL


Q6UXX5
Inter-alpha-trypsin inhibitor heavy chain H6
ITIH6


Q6UXX9
R-spondin-2
RSPO2


Q6UY14
ADAMTS-like protein 4
ADAMTSL4


Q6UY27
Prostate and testis expressed protein 2
PATE2


Q6W4X9
Mucin-6
MUC6


Q6WN34
Chordin-like protein 2
CHRDL2


Q6WRI0
Immunoglobulin superfamily member 10
IGSF10


Q6X4U4
Sclerostin domain-containing protein 1
SOSTDC1


Q6X784
Zona pellucida-binding protein 2
ZPBP2


Q6XE38
Secretoglobin family 1D member 4
SCGB1D4


Q6XPR3
Repetin
RPTN


Q6XZB0
Lipase member I
LIPI


Q6ZMM2
ADAMTS-like protein 5
ADAMTSL5


Q6ZMP0
Thrombospondin type-1 domain-containing
THSD4



protein 4


Q6ZNF0
Iron/zinc purple acid phosphatase-like protein
PAPL


Q6ZRI0
Otogelin
OTOG


Q6ZRP7
Sulfhydryl oxidase 2
QSOX2


Q6ZWJ8
Kielin/chordin-like protein
KCP


Q75N90
Fibrillin-3
FBN3


Q765I0
Urotensin-2B
UTS2D


Q76B58
Protein FAM5C
FAM5C


Q76LX8
A disintegrin and metalloproteinase with
ADAMTS13



thrombospondin motifs 13


Q76M96
Coiled-coil domain-containing protein 80
CCDC80


Q7L1S5
Carbohydrate sulfotransferase 9
CHST9


Q7L513
Fc receptor-like A
FCRLA


Q7L8A9
Vasohibin-1
VASH1


Q7RTM1
Otopetrin-1
OTOP1


Q7RTW8
Otoancorin
OTOA


Q7RTY5
Serine protease 48
PRSS48


Q7RTY7
Ovochymase-1
OVCH1


Q7RTZ1
Ovochymase-2
OVCH2


Q7Z304
MAM domain-containing protein 2
MAMDC2


Q7Z3S9
Notch homolog 2 N-terminal-like protein
NOTCH2NL


Q7Z4H4
Intermedin-short
ADM2


Q7Z4P5
Growth/differentiation factor 7
GDF7


Q7Z4R8
UPF0669 protein C6orf120
C6orf120


Q7Z4W2
Lysozyme-like protein 2
LYZL2


Q7Z5A4
Serine protease 42
PRSS42


Q7Z5A7
Protein FAM19A5
FAM19A5


Q7Z5A8
Protein FAM19A3
FAM19A3


Q7Z5A9
Protein FAM19A1
FAM19A1


Q7Z5J1
Hydroxysteroid 11-beta-dehydrogenase 1-like
HSD11B1L



protein


Q7Z5L0
Vitelline membrane outer layer protein 1 homolog
VMO1


Q7Z5L3
Complement C1q-like protein 2
C1QL2


Q7Z5L7
Podocan
PODN


Q7Z5P4
17-beta-hydroxysteroid dehydrogenase 13
HSD17B13


Q7Z5P9
Mucin-19
MUC19


Q7Z5Y6
Bone morphogenetic protein 8A
BMP8A


Q7Z7B7
Beta-defensin 132
DEFB132


Q7Z7B8
Beta-defensin 128
DEFB128


Q7Z7C8
Transcription initiation factor TFIID subunit 8
TAF8


Q7Z7H5
Transmembrane emp24 domain-containing protein 4
TMED4


Q86SG7
Lysozyme g-like protein 2
LYG2


Q86SI9
Protein CEI
C5orf38


Q86TE4
Leucine zipper protein 2
LUZP2


Q86TH1
ADAMTS-like protein 2
ADAMTSL2


Q86U17
Serpin A11
SERPINA11


Q86UU9
Endokinin-A
TAC4


Q86UW8
Hyaluronan and proteoglycan link protein 4
HAPLN4


Q86UX2
Inter-alpha-trypsin inhibitor heavy chain H5
ITIH5


Q86V24
Adiponectin receptor protein 2
ADIPOR2


Q86VB7
Soluble CD163
CD163


Q86VR8
Four-jointed box protein 1
FJX1


Q86WD7
Serpin A9
SERPINA9


Q86WN2
Interferon epsilon
IFNE


Q86WS3
Placenta-specific 1-like protein
PLAC1L


Q86X52
Chondroitin sulfate synthase 1
CHSY1


Q86XP6
Gastrokine-2
GKN2


Q86XS5
Angiopoietin-related protein 5
ANGPTL5


Q86Y27
B melanoma antigen 5
BAGE5


Q86Y28
B melanoma antigen 4
BAGE4


Q86Y29
B melanoma antigen 3
BAGE3


Q86Y30
B melanoma antigen 2
BAGE2


Q86Y38
Xylosyltransferase 1
XYLT1


Q86Y78
Ly6/PLAUR domain-containing protein 6
LYPD6


Q86YD3
Transmembrane protein 25
TMEM25


Q86YJ6
Threonine synthase-like 2
THNSL2


Q86YW7
Glycoprotein hormone beta-5
GPHB5


Q86Z23
Complement C1q-like protein 4
C1QL4


Q8IU57
Interleukin-28 receptor subunit alpha
IL28RA


Q8IUA0
WAP four-disulfide core domain protein 8
WFDC8


Q8IUB2
WAP four-disulfide core domain protein 3
WFDC3


Q8IUB3
Protein WFDC10B
WFDC10B


Q8IUB5
WAP four-disulfide core domain protein 13
WFDC13


Q8IUH2
Protein CREG2
CREG2


Q8IUK5
Plexin domain-containing protein 1
PLXDC1


Q8IUL8
Cartilage intermediate layer protein 2 C2
CILP2


Q8IUX7
Adipocyte enhancer-binding protein 1
AEBP1


Q8IUX8
Epidermal growth factor-like protein 6
EGFL6


Q8IVL8
Carboxypeptidase O
CPO


Q8IVN8
Somatomedin-B and thrombospondin type-1
SBSPON



domain-containing protein


Q8IVW8
Protein spinster homolog 2
SPNS2


Q8IW75
Serpin A12
SERPINA12


Q8IW92
Beta-galactosidase-1-like protein 2
GLB1L2


Q8IWL1
Pulmonary surfactant-associated protein A2
SFTPA2


Q8IWL2
Pulmonary surfactant-associated protein A1
SFTPA1


Q8IWV2
Contactin-4
CNTN4


Q8IWY4
Signal peptide, CUB and EGF-like domain-
SCUBE1



containing protein 1


Q8IX30
Signal peptide, CUB and EGF-like domain-
SCUBE3



containing protein 3


Q8IXA5
Sperm acrosome membrane-associated protein 3,
SPACA3



membrane form


Q8IXB1
DnaJ homolog subfamily C member 10
DNAJC10


Q8IXL6
Extracellular serine/threonine protein kinase
FAM20C



Fam20C


Q8IYD9
Lung adenoma susceptibility protein 2
LAS2


Q8IYP2
Serine protease 58
PRSS58


Q8IYS5
Osteoclast-associated immunoglobulin-like
OSCAR



receptor


Q8IZC6
Collagen alpha-1(XXVII) chain
COL27A1


Q8IZJ3
C3 and PZP-like alpha-2-macroglobulin domain-
CPAMD8



containing protein 8


Q8IZN7
Beta-defensin 107
DEFB107B


Q8N0V4
Leucine-rich repeat LGI family member 2
LGI2


Q8N104
Beta-defensin 106
DEFB106B


Q8N119
Matrix metalloproteinase-21
MMP21


Q8N129
Protein canopy homolog 4
CNPY4


Q8N135
Leucine-rich repeat LGI family member 4
LGI4


Q8N145
Leucine-rich repeat LGI family member 3
LGI3


Q8N158
Glypican-2
GPC2


Q8N1E2
Lysozyme g-like protein 1
LYG1


Q8N2E2
von Willebrand factor D and EGF domain-
VWDE



containing protein


Q8N2E6
Prosalusin
TOR2A


Q8N2S1
Latent-transforming growth factor beta-binding
LTBP4



protein 4


Q8N302
Angiogenic factor with G patch and FHA domains 1
AGGF1


Q8N307
Mucin-20
MUC20


Q8N323
NXPE family member 1
NXPE1


Q8N387
Mucin-15
MUC15


Q8N3Z0
Inactive serine protease 35
PRSS35


Q8N436
Inactive carboxypeptidase-like protein X2
CPXM2


Q8N474
Secreted frizzled-related protein 1
SFRP1


Q8N475
Follistatin-related protein 5
FSTL5


Q8N4F0
BPI fold-containing family B member 2
BPIFB2


Q8N4T0
Carboxypeptidase A6
CPA6


Q8N5W8
Protein FAM24B
FAM24B


Q8N687
Beta-defensin 125
DEFB125


Q8N688
Beta-defensin 123
DEFB123


Q8N690
Beta-defensin 119
DEFB119


Q8N6C5
Immunoglobulin superfamily member 1
IGSF1


Q8N6C8
Leukocyte immunoglobulin-like receptor
LILRA3



subfamily A member 3


Q8N6G6
ADAMTS-like protein 1
ADAMTSL1


Q8N6Y2
Leucine-rich repeat-containing protein 17
LRRC17


Q8N729
Neuropeptide W-23
NPW


Q8N8U9
BMP-binding endothelial regulator protein
BMPER


Q8N907
DAN domain family member 5
DAND5


Q8NAT1
Glycosyltransferase-like domain-containing
GTDC2



protein 2


Q8NAU1
Fibronectin type III domain-containing protein 5
FNDC5


Q8NB37
Parkinson disease 7 domain-containing protein 1
PDDC1


Q8NBI3
Draxin
DRAXIN


Q8NBM8
Prenylcysteine oxidase-like
PCYOX1L


Q8NBP7
Proprotein convertase subtilisin/kexin type 9
PCSK9


Q8NBQ5
Estradiol 17-beta-dehydrogenase 11
HSD17B11


Q8NBV8
Synaptotagmin-8
SYT8


Q8NCC3
Group XV phospholipase A2
PLA2G15


Q8NCF0
C-type lectin domain family 18 member C
CLEC18C


Q8NCW5
NAD(P)H-hydrate epimerase
APOA1BP


Q8NDA2
Hemicentin-2
HMCN2


Q8NDX9
Lymphocyte antigen 6 complex locus protein G5b
LY6G5B


Q8NDZ4
Deleted in autism protein 1
C3orf58


Q8NEB7
Acrosin-binding protein
ACRBP


Q8NES8
Beta-defensin 124
DEFB124


Q8NET1
Beta-defensin 108B
DEFB108B


Q8NEX5
Protein WFDC9
WFDC9


Q8NEX6
Protein WFDC11
WFDC11


Q8NF86
Serine protease 33
PRSS33


Q8NFM7
Interleukin-17 receptor D
IL17RD


Q8NFQ5
BPI fold-containing family B member 6
BPIFB6


Q8NFQ6
BPI fold-containing family C protein
BPIFC


Q8NFU4
Follicular dendritic cell secreted peptide
FDCSP


Q8NFW1
Collagen alpha-1(XXII) chain
COL22A1


Q8NG35
Beta-defensin 105
DEFB105B


Q8NG41
Neuropeptide B-23
NPB


Q8NHW6
Otospiralin
OTOS


Q8NI99
Angiopoietin-related protein 6
ANGPTL6


Q8TAA1
Probable ribonuclease 11
RNASE11


Q8TAG5
V-set and transmembrane domain-containing
VSTM2A



protein 2A


Q8TAL6
Fin bud initiation factor homolog
FIBIN


Q8TAT2
Fibroblast growth factor-binding protein 3
FGFBP3


Q8TAX7
Mucin-7
MUC7


Q8TB22
Spermatogenesis-associated protein 20
SPATA20


Q8TB73
Protein NDNF
NDNF


Q8TB96
T-cell immunomodulatory protein
ITFG1


Q8TC92
Protein disulfide-thiol oxidoreductase
ENOX1


Q8TCV5
WAP four-disulfide core domain protein 5
WFDC5


Q8TD06
Anterior gradient protein 3 homolog
AGR3


Q8TD33
Secretoglobin family 1C member 1
SCGB1C1


Q8TD46
Cell surface glycoprotein CD200 receptor 1
CD200R1


Q8TDE3
Ribonuclease 8
RNASE8


Q8TDF5
Neuropilin and tolloid-like protein 1
NETO1


Q8TDL5
BPI fold-containing family B member 1
BPIFB1


Q8TE56
A disintegrin and metalloproteinase with
ADAMTS17



thrombospondin motifs 17


Q8TE57
A disintegrin and metalloproteinase with
ADAMTS16



thrombospondin motifs 16


Q8TE58
A disintegrin and metalloproteinase with
ADAMTS15



thrombospondin motifs 15


Q8TE59
A disintegrin and metalloproteinase with
ADAMTS19



thrombospondin motifs 19


Q8TE60
A disintegrin and metalloproteinase with
ADAMTS18



thrombospondin motifs 18


Q8TE99
Acid phosphatase-like protein 2
ACPL2


Q8TER0
Sushi, nidogen and EGF-like domain-containing
SNED1



protein 1


Q8TEU8
WAP, kazal, immunoglobulin, kunitz and NTR
WFIKKN2



domain-containing protein 2


Q8WTQ1
Beta-defensin 104
DEFB104B


Q8WTR8
Netrin-5
NTN5


Q8WTU2
Scavenger receptor cysteine-rich domain-
SRCRB4D



containing group B protein


Q8WU66
Protein TSPEAR
TSPEAR


Q8WUA8
Tsukushin
TSKU


Q8WUF8
Protein FAM172A
FAM172A


Q8WUJ1
Neuferricin
CYB5D2


Q8WUY1
UPF0670 protein THEM6
THEM6


Q8WVN6
Secreted and transmembrane protein 1
SECTM1


Q8WVQ1
Soluble calcium-activated nucleotidase 1
CANT1


Q8WWA0
Intelectin-1
ITLN1


Q8WWG1
Neuregulin-4
NRG4


Q8WWQ2
Inactive heparanase-2
HPSE2


Q8WWU7
Intelectin-2
ITLN2


Q8WWY7
WAP four-disulfide core domain protein 12
WFDC12


Q8WWY8
Lipase member H
LIPH


Q8WWZ8
Oncoprotein-induced transcript 3 protein
OIT3


Q8WX39
Epididymal-specific lipocalin-9
LCN9


Q8WXA2
Prostate and testis expressed protein 1
PATE1


Q8WXD2
Secretogranin-3
SCG3


Q8WXF3
Relaxin-3 A chain
RLN3


Q8WXI7
Mucin-16
MUC16


Q8WXQ8
Carboxypeptidase A5
CPA5


Q8WXS8
A disintegrin and metalloproteinase with
ADAMTS14



thrombospondin motifs 14


Q92484
Acid sphingomyelinase-like phosphodiesterase 3a
SMPDL3A


Q92485
Acid sphingomyelinase-like phosphodiesterase 3b
SMPDL3B


Q92496
Complement factor H-related protein 4
CFHR4


Q92520
Protein FAM3C
FAM3C


Q92563
Testican-2
SPOCK2


Q92583
C-C motif chemokine 17
CCL17


Q92626
Peroxidasin homolog
PXDN


Q92743
Serine protease HTRA1
HTRA1


Q92752
Tenascin-R
TNR


Q92765
Secreted frizzled-related protein 3
FRZB


Q92819
Hyaluronan synthase 2
HAS2


Q92820
Gamma-glutamyl hydrolase
GGH


Q92824
Proprotein convertase subtilisin/kexin type 5
PCSK5


Q92832
Protein kinase C-binding protein NELL1
NELL1


Q92838
Ectodysplasin-A, membrane form
EDA


Q92874
Deoxyribonuclease-1-like 2
DNASE1L2


Q92876
Kallikrein-6
KLK6


Q92913
Fibroblast growth factor 13
FGF13


Q92954
Proteoglycan 4 C-terminal part
PRG4


Q93038
Tumor necrosis factor receptor superfamily
TNFRSF25



member 25


Q93091
Ribonuclease K6
RNASE6


Q93097
Protein Wnt-2b
WNT2B


Q93098
Protein Wnt-8b
WNT8B


Q95460
Major histocompatibility complex class I-related
MR1



gene protein


Q969D9
Thymic stromal lymphopoietin
TSLP


Q969E1
Liver-expressed antimicrobial peptide 2
LEAP2


Q969H8
UPF0556 protein C19orf10
C19orf10


Q969Y0
NXPE family member 3
NXPE3


Q96A54
Adiponectin receptor protein 1
ADIPOR1


Q96A83
Collagen alpha-1(XXVI) chain
EMID2


Q96A84
EMI domain-containing protein 1
EMID1


Q96A98
Tuberoinfundibular peptide of 39 residues
PTH2


Q96A99
Pentraxin-4
PTX4


Q96BH3
Epididymal sperm-binding protein 1
ELSPBP1


Q96BQ1
Protein FAM3D
FAM3D


Q96CG8
Collagen triple helix repeat-containing protein 1
CTHRC1


Q96DA0
Zymogen granule protein 16 homolog B
ZG16B


Q96DN2
von Willebrand factor C and EGF domain-
VWCE



containing protein


Q96DR5
BPI fold-containing family A member 2
BPIFA2


Q96DR8
Mucin-like protein 1
MUCL1


Q96DX4
RING finger and SPRY domain-containing protein 1
RSPRY1


Q96EE4
Coiled-coil domain-containing protein 126
CCDC126


Q96GS6
Abhydrolase domain-containing protein
FAM108A1



FAM108A1


Q96GW7
Brevican core protein
BCAN


Q96HF1
Secreted frizzled-related protein 2
SFRP2


Q96I82
Kazal-type serine protease inhibitor domain-
KAZALD1



containing protein 1


Q96ID5
Immunoglobulin superfamily member 21
IGSF21


Q96II8
Leucine-rich repeat and calponin homology
LRCH3



domain-containing protein 3


Q96IY4
Carboxypeptidase B2
CPB2


Q96JB6
Lysyl oxidase homolog 4
LOXL4


Q96JK4
HHIP-like protein 1
HHIPL1


Q96KN2
Beta-Ala-His dipeptidase
CNDP1


Q96KW9
Protein SPACA7
SPACA7


Q96KX0
Lysozyme-like protein 4
LYZL4


Q96L15
Ecto-ADP-ribosyltransferase 5
ART5


Q96LB8
Peptidoglycan recognition protein 4
PGLYRP4


Q96LB9
Peptidoglycan recognition protein 3
PGLYRP3


Q96LC7
Sialic acid-binding Ig-like lectin 10
SIGLEC10


Q96LR4
Protein FAM19A4
FAM19A4


Q96MK3
Protein FAM20A
FAM20A


Q96MS3
Glycosyltransferase 1 domain-containing protein 1
GLT1D1


Q96NY8
Processed poliovirus receptor-related protein 4
PVRL4


Q96NZ8
WAP, kazal, immunoglobulin, kunitz and NTR
WFIKKN1



domain-containing protein 1


Q96NZ9
Proline-rich acidic protein 1
PRAP1


Q96P44
Collagen alpha-1(XXI) chain
COL21A1


Q96PB7
Noelin-3
OLFM3


Q96PC5
Melanoma inhibitory activity protein 2
MIA2


Q96PD5
N-acetylmuramoyl-L-alanine amidase
PGLYRP2


Q96PH6
Beta-defensin 118
DEFB118


Q96PL1
Secretoglobin family 3A member 2
SCGB3A2


Q96PL2
Beta-tectorin
TECTB


Q96QH8
Sperm acrosome-associated protein 5
SPACA5


Q96QR1
Secretoglobin family 3A member 1
SCGB3A1


Q96QU1
Protocadherin-15
PCDH15


Q96QV1
Hedgehog-interacting protein
HHIP


Q96RW7
Hemicentin-1
HMCN1


Q96S42
Nodal homolog
NODAL


Q96S86
Hyaluronan and proteoglycan link protein 3
HAPLN3


Q96SL4
Glutathione peroxidase 7
GPX7


Q96SM3
Probable carboxypeptidase X1
CPXM1


Q96T91
Glycoprotein hormone alpha-2
GPHA2


Q99062
Granulocyte colony-stimulating factor receptor
CSF3R


Q99102
Mucin-4 alpha chain
MUC4


Q99217
Amelogenin, X isoform
AMELX


Q99218
Amelogenin, Y isoform
AMELY


Q99435
Protein kinase C-binding protein NELL2
NELL2


Q99470
Stromal cell-derived factor 2
SDF2


Q99542
Matrix metalloproteinase-19
MMP19


Q99574
Neuroserpin
SERPINI1


Q99584
Protein S100-A13
S100A13


Q99616
C-C motif chemokine 13
CCL13


Q99645
Epiphycan
EPYC


Q99674
Cell growth regulator with EF hand domain
CGREF1



protein 1


Q99715
Collagen alpha-1(XII) chain
COL12A1


Q99727
Metalloproteinase inhibitor 4
TIMP4


Q99731
C-C motif chemokine 19
CCL19


Q99748
Neurturin
NRTN


Q99935
Proline-rich protein 1
PROL1


Q99942
E3 ubiquitin-protein ligase RNF5
RNF5


Q99944
Epidermal growth factor-like protein 8
EGFL8


Q99954
Submaxillary gland androgen-regulated protein 3A
SMR3A


Q99969
Retinoic acid receptor responder protein 2
RARRES2


Q99972
Myocilin
MYOC


Q99983
Osteomodulin
OMD


Q99985
Semaphorin-3C
SEMA3C


Q99988
Growth/differentiation factor 15
GDF15


Q9BPW4
Apolipoprotein L4
APOL4


Q9BQ08
Resistin-like beta
RETNLB


Q9BQ16
Testican-3
SPOCK3


Q9BQ51
Programmed cell death 1 ligand 2
PDCD1LG2


Q9BQB4
Sclerostin
SOST


Q9BQI4
Coiled-coil domain-containing protein 3
CCDC3


Q9BQP9
BPI fold-containing family A member 3
BPIFA3


Q9BQR3
Serine protease 27
PRSS27


Q9BQY6
WAP four-disulfide core domain protein 6
WFDC6


Q9BRR6
ADP-dependent glucokinase
ADPGK


Q9BS86
Zona pellucida-binding protein 1
ZPBP


Q9BSG0
Protease-associated domain-containing protein 1
PRADC1


Q9BSG5
Retbindin
RTBDN


Q9BT30
Probable alpha-ketoglutarate-dependent
ALKBH7



dioxygenase ABH7


Q9BT56
Spexin
C12orf39


Q9BT67
NEDD4 family-interacting protein 1
NDFIP1


Q9BTY2
Plasma alpha-L-fucosidase
FUCA2


Q9BU40
Chordin-like protein 1
CHRDL1


Q9BUD6
Spondin-2
SPON2


Q9BUN1
Protein MENT
MENT


Q9BUR5
Apolipoprotein O
APOO


Q9BV94
ER degradation-enhancing alpha-mannosidase-like 2
EDEM2


Q9BWP8
Collectin-11
COLEC11


Q9BWS9
Chitinase domain-containing protein 1
CHID1


Q9BX67
Junctional adhesion molecule C
JAM3


Q9BX93
Group XIIB secretory phospholipase A2-like
PLA2G12B



protein


Q9BXI9
Complement C1q tumor necrosis factor-related
C1QTNF6



protein 6


Q9BXJ0
Complement C1q tumor necrosis factor-related
C1QTNF5



protein 5


Q9BXJ1
Complement C1q tumor necrosis factor-related
C1QTNF1



protein 1


Q9BXJ2
Complement C1q tumor necrosis factor-related
C1QTNF7



protein 7


Q9BXJ3
Complement C1q tumor necrosis factor-related
C1QTNF4



protein 4


Q9BXJ4
Complement C1q tumor necrosis factor-related
C1QTNF3



protein 3


Q9BXJ5
Complement C1q tumor necrosis factor-related
C1QTNF2



protein 2


Q9BXN1
Asporin
ASPN


Q9BXP8
Pappalysin-2
PAPPA2


Q9BXR6
Complement factor H-related protein 5
CFHR5


Q9BXS0
Collagen alpha-1(XXV) chain
COL25A1


Q9BXX0
EMILIN-2
EMILIN2


Q9BXY4
R-spondin-3
RSPO3


Q9BY15
EGF-like module-containing mucin-like hormone
EMR3



receptor-like 3 subunit beta


Q9BY50
Signal peptidase complex catalytic subunit
SEC11C



SEC11C


Q9BY76
Angiopoietin-related protein 4
ANGPTL4


Q9BYF1
Processed angiotensin-converting enzyme 2
ACE2


Q9BYJ0
Fibroblast growth factor-binding protein 2
FGFBP2


Q9BYW3
Beta-defensin 126
DEFB126


Q9BYX4
Interferon-induced helicase C domain-containing
IFIH1



protein 1


Q9BYZ8
Regenerating islet-derived protein 4
REG4


Q9BZ76
Contactin-associated protein-like 3
CNTNAP3


Q9BZG9
Ly-6/neurotoxin-like protein 1
LYNX1


Q9BZJ3
Tryptase delta
TPSD1


Q9BZM1
Group XIIA secretory phospholipase A2
PLA2G12A


Q9BZM2
Group IIF secretory phospholipase A2
PLA2G2F


Q9BZM5
NKG2D ligand 2
ULBP2


Q9BZP6
Acidic mammalian chitinase
CHIA


Q9BZZ2
Sialoadhesin
SIGLEC1


Q9C0B6
Protein FAM5B
FAM5B


Q9GZM7
Tubulointerstitial nephritis antigen-like
TINAGL1


Q9GZN4
Brain-specific serine protease 4
PRSS22


Q9GZP0
Platelet-derived growth factor D, receptor-
PDGFD



binding form


Q9GZT5
Protein Wnt-10a
WNT10A


Q9GZU5
Nyctalopin
NYX


Q9GZV7
Hyaluronan and proteoglycan link protein 2
HAPLN2


Q9GZV9
Fibroblast growth factor 23
FGF23


Q9GZX9
Twisted gastrulation protein homolog 1
TWSG1


Q9GZZ7
GDNF family receptor alpha-4
GFRA4


Q9GZZ8
Extracellular glycoprotein lacritin
LACRT


Q9H0B8
Cysteine-rich secretory protein LCCL domain-
CRISPLD2



containing 2


Q9H106
Signal-regulatory protein delta
SIRPD


Q9H114
Cystatin-like 1
CSTL1


Q9H173
Nucleotide exchange factor SIL1
SIL1


Q9H1E1
Ribonuclease 7
RNASE7


Q9H1F0
WAP four-disulfide core domain protein 10A
WFDC10A


Q9H1J5
Protein Wnt-8a
WNT8A


Q9H1J7
Protein Wnt-5b
WNT5B


Q9H1M3
Beta-defensin 129
DEFB129


Q9H1M4
Beta-defensin 127
DEFB127


Q9H1Z8
Augurin
C2orf40


Q9H239
Matrix metalloproteinase-28
MMP28


Q9H2A7
C-X-C motif chemokine 16
CXCL16


Q9H2A9
Carbohydrate sulfotransferase 8
CHST8


Q9H2R5
Kallikrein-15
KLK15


Q9H2X0
Chordin
CHRD


Q9H2X3
C-type lectin domain family 4 member M
CLEC4M


Q9H306
Matrix metalloproteinase-27
MMP27


Q9H324
A disintegrin and metalloproteinase with
ADAMTS10



thrombospondin motifs 10


Q9H336
Cysteine-rich secretory protein LCCL domain-
CRISPLD1



containing 1


Q9H3E2
Sorting nexin-25
SNX25


Q9H3R2
Mucin-13
MUC13


Q9H3U7
SPARC-related modular calcium-binding protein 2
SMOC2


Q9H3Y0
Peptidase inhibitor R3HDML
R3HDML


Q9H4A4
Aminopeptidase B
RNPEP


Q9H4F8
SPARC-related modular calcium-binding protein 1
SMOC1


Q9H4G1
Cystatin-9-like
CST9L


Q9H5V8
CUB domain-containing protein 1
CDCP1


Q9H6B9
Epoxide hydrolase 3
EPHX3


Q9H6E4
Coiled-coil domain-containing protein 134
CCDC134


Q9H741
UPF0454 protein C12orf49
C12orf49


Q9H772
Gremlin-2
GREM2


Q9H7Y0
Deleted in autism-related protein 1
CXorf36


Q9H8L6
Multimerin-2
MMRN2


Q9H9S5
Fukutin-related protein
FKRP


Q9HAT2
Sialate O-acetylesterase
SIAE


Q9HB40
Retinoid-inducible serine carboxypeptidase
SCPEP1


Q9HB63
Netrin-4
NTN4


Q9HBJ0
Placenta-specific protein 1
PLAC1


Q9HC23
Prokineticin-2
PROK2


Q9HC57
WAP four-disulfide core domain protein 1
WFDC1


Q9HC73
Cytokine receptor-like factor 2
CRLF2


Q9HC84
Mucin-5B
MUC5B


Q9HCB6
Spondin-1
SPON1


Q9HCQ7
Neuropeptide NPSF
NPVF


Q9HCT0
Fibroblast growth factor 22
FGF22


Q9HD89
Resistin
RETN


Q9NNX1
Tuftelin
TUFT1


Q9NNX6
CD209 antigen
CD209


Q9NP55
BPI fold-containing family A member 1
BPIFA1


Q9NP70
Ameloblastin
AMBN


Q9NP95
Fibroblast growth factor 20
FGF20


Q9NP99
Triggering receptor expressed on myeloid cells 1
TREM1


Q9NPA2
Matrix metalloproteinase-25
MMP25


Q9NPE2
Neugrin
NGRN


Q9NPH0
Lysophosphatidic acid phosphatase type 6
ACP6


Q9NPH6
Odorant-binding protein 2b
OBP2B


Q9NQ30
Endothelial cell-specific molecule 1
ESM1


Q9NQ36
Signal peptide, CUB and EGF-like domain-
SCUBE2



containing protein 2


Q9NQ38
Serine protease inhibitor Kazal-type 5
SPINK5


Q9NQ76
Matrix extracellular phosphoglycoprotein
MEPE


Q9NQ79
Cartilage acidic protein 1
CRTAC1


Q9NR16
Scavenger receptor cysteine-rich type 1
CD163L1



protein M160


Q9NR23
Growth/differentiation factor 3
GDF3


Q9NR71
Neutral ceramidase
ASAH2


Q9NR99
Matrix-remodeling-associated protein 5
MXRA5


Q9NRA1
Platelet-derived growth factor C
PDGFC


Q9NRC9
Otoraplin
OTOR


Q9NRE1
Matrix metalloproteinase-26
MMP26


Q9NRJ3
C-C motif chemokine 28
CCL28


Q9NRM1
Enamelin
ENAM


Q9NRN5
Olfactomedin-like protein 3
OLFML3


Q9NRR1
Cytokine-like protein 1
CYTL1


Q9NS15
Latent-transforming growth factor beta-binding
LTBP3



protein 3


Q9NS62
Thrombospondin type-1 domain-containing
THSD1



protein 1


Q9NS71
Gastrokine-1
GKN1


Q9NS98
Semaphorin-3G
SEMA3G


Q9NSA1
Fibroblast growth factor 21
FGF21


Q9NT22
EMILIN-3
EMILIN3


Q9NTU7
Cerebellin-4
CBLN4


Q9NVR0
Kelch-like protein 11
KLHL11


Q9NWH7
Spermatogenesis-associated protein 6
SPATA6


Q9NXC2
Glucose-fructose oxidoreductase domain-
GFOD1



containing protein 1


Q9NY56
Odorant-binding protein 2a
OBP2A


Q9NY84
Vascular non-inflammatory molecule 3
VNN3


Q9NZ20
Group 3 secretory phospholipase A2
PLA2G3


Q9NZC2
Triggering receptor expressed on myeloid cells 2
TREM2


Q9NZK5
Adenosine deaminase CECR1
CECR1


Q9NZK7
Group IIE secretory phospholipase A2
PLA2G2E


Q9NZP8
Complement C1r subcomponent-like protein
C1RL


Q9NZV1
Cysteine-rich motor neuron 1 protein
CRIM1


Q9NZW4
Dentin sialoprotein
DSPP


Q9P0G3
Kallikrein-14
KLK14


Q9P0W0
Interferon kappa
IFNK


Q9P218
Collagen alpha-1(XX) chain
COL20A1


Q9P2C4
Transmembrane protein 181
TMEM181


Q9P2K2
Thioredoxin domain-containing protein 16
TXNDC16


Q9P2N4
A disintegrin and metalloproteinase with
ADAMTS9



thrombospondin motifs 9


Q9UBC7
Galanin-like peptide
GALP


Q9UBD3
Cytokine SCM-1 beta
XCL2


Q9UBD9
Cardiotrophin-like cytokine factor 1
CLCF1


Q9UBM4
Opticin
OPTC


Q9UBP4
Dickkopf-related protein 3
DKK3


Q9UBQ6
Exostosin-like 2
EXTL2


Q9UBR5
Chemokine-like factor
CKLF


Q9UBS5
Gamma-aminobutyric acid type B receptor subunit 1
GABBR1


Q9UBT3
Dickkopf-related protein 4 short form
DKK4


Q9UBU2
Dickkopf-related protein 2
DKK2


Q9UBU3
Ghrelin-28
GHRL


Q9UBV4
Protein Wnt-16
WNT16


Q9UBX5
Fibulin-5
FBLN5


Q9UBX7
Kallikrein-11
KLK11


Q9UEF7
Klotho
KL


Q9UFP1
Protein FAM198A
FAM198A


Q9UGM3
Deleted in malignant brain tumors 1 protein
DMBT1


Q9UGM5
Fetuin-B
FETUB


Q9UGP8
Translocation protein SEC63 homolog
SEC63


Q9UHF0
Neurokinin-B
TAC3


Q9UHF1
Epidermal growth factor-like protein 7
EGFL7


Q9UHG2
ProSAAS
PCSK1N


Q9UHI8
A disintegrin and metalloproteinase with
ADAMTS1



thrombospondin motifs 1


Q9UHL4
Dipeptidyl peptidase 2
DPP7


Q9UI42
Carboxypeptidase A4
CPA4


Q9UIG4
Psoriasis susceptibility 1 candidate gene 2 protein
PSORS1C2


Q9UIK5
Tomoregulin-2
TMEFF2


Q9UIQ6
Leucyl-cystinyl aminopeptidase, pregnancy serum
LNPEP



form


Q9UJA9
Ectonucleotide pyrophosphatase/phosphodiesterase
ENPP5



family member 5


Q9UJH8
Meteorin
METRN


Q9UJJ9
N-acetylglucosamine-1-phosphotransferase
GNPTG



subunit gamma


Q9UJW2
Tubulointerstitial nephritis antigen
TINAG


Q9UK05
Growth/differentiation factor 2
GDF2


Q9UK55
Protein Z-dependent protease inhibitor
SERPINA10


Q9UK85
Dickkopf-like protein 1
DKKL1


Q9UKJ1
Paired immunoglobulin-like type 2 receptor alpha
PILRA


Q9UKP4
A disintegrin and metalloproteinase with
ADAMTS7



thrombospondin motifs 7


Q9UKP5
A disintegrin and metalloproteinase with
ADAMTS6



thrombospondin motifs 6


Q9UKQ2
Disintegrin and metalloproteinase domain-
ADAM28



containing protein 28


Q9UKQ9
Kallikrein-9
KLK9


Q9UKR0
Kallikrein-12
KLK12


Q9UKR3
Kallikrein-13
KLK13


Q9UKU9
Angiopoietin-related protein 2
ANGPTL2


Q9UKZ9
Procollagen C-endopeptidase enhancer 2
PCOLCE2


Q9UL52
Transmembrane protease serine 11E non-
TMPRSS11E



catalytic chain


Q9ULC0
Endomucin
EMCN


Q9ULI3
Protein HEG homolog 1
HEG1


Q9ULZ1
Apelin-13
APLN


Q9ULZ9
Matrix metalloproteinase-17
MMP17


Q9UM21
Alpha-1,3-mannosyl-glycoprotein 4-beta-N-
MGAT4A



acetylglucosaminyltransferase A soluble form


Q9UM22
Mammalian ependymin-related protein 1
EPDR1


Q9UM73
ALK tyrosine kinase receptor
ALK


Q9UMD9
97 kDa linear IgA disease antigen
COL17A1


Q9UMX5
Neudesin
NENF


Q9UN73
Protocadherin alpha-6
PCDHA6


Q9UNA0
A disintegrin and metalloproteinase with
ADAMTS5



thrombospondin motifs 5


Q9UNI1
Chymotrypsin-like elastase family member 1
CELA1


Q9UNK4
Group IID secretory phospholipase A2
PLA2G2D


Q9UP79
A disintegrin and metalloproteinase with
ADAMTS8



thrombospondin motifs 8


Q9UPZ6
Thrombospondin type-1 domain-containing
THSD7A



protein 7A


Q9UQ72
Pregnancy-specific beta-1-glycoprotein 11
PSG11


Q9UQ74
Pregnancy-specific beta-1-glycoprotein 8
PSG8


Q9UQC9
Calcium-activated chloride channel regulator 2
CLCA2


Q9UQE7
Structural maintenance of chromosomes protein 3
SMC3


Q9UQP3
Tenascin-N
TNN


Q9Y223
UDP-N-acetylglucosamine 2-epimerase
GNE


Q9Y240
C-type lectin domain family 11 member A
CLEC11A


Q9Y251
Heparanase 8 kDa subunit
HPSE


Q9Y258
C-C motif chemokine 26
CCL26


Q9Y264
Angiopoietin-4
ANGPT4


Q9Y275
Tumor necrosis factor ligand superfamily member
TNFSF13B



13b, membrane form


Q9Y287
BRI2 intracellular domain
ITM2B


Q9Y2E5
Epididymis-specific alpha-mannosidase
MAN2B2


Q9Y334
von Willebrand factor A domain-containing
VWA7



protein 7


Q9Y337
Kallikrein-5
KLK5


Q9Y3B3
Transmembrane emp24 domain-containing protein 7
TMED7


Q9Y3E2
BolA-like protein 1
BOLA1


Q9Y426
C2 domain-containing protein 2
C2CD2


Q9Y4K0
Lysyl oxidase homolog 2
LOXL2


Q9Y4X3
C-C motif chemokine 27
CCL27


Q9Y5C1
Angiopoietin-related protein 3
ANGPTL3


Q9Y5I2
Protocadherin alpha-10
PCDHA10


Q9Y5I3
Protocadherin alpha-1
PCDHA1


Q9Y5K2
Kallikrein-4
KLK4


Q9Y5L2
Hypoxia-inducible lipid droplet-associated protein
HILPDA


Q9Y5Q5
Atrial natriuretic peptide-converting enzyme
CORIN


Q9Y5R2
Matrix metalloproteinase-24
MMP24


Q9Y5U5
Tumor necrosis factor receptor superfamily
TNFRSF18



member 18


Q9Y5W5
Wnt inhibitory factor 1
WIF1


Q9Y5X9
Endothelial lipase
LIPG


Q9Y625
Secreted glypican-6
GPC6


Q9Y646
Carboxypeptidase Q
CPQ


Q9Y6C2
EMILIN-1
EMILIN1


Q9Y6F9
Protein Wnt-6
WNT6


Q9Y6I9
Testis-expressed sequence 264 protein
TEX264


Q9Y6L7
Tolloid-like protein 2
TLL2


Q9Y6N3
Calcium-activated chloride channel regulator
CLCA3P



family member 3


Q9Y6N6
Laminin subunit gamma-3
LAMC3


Q9Y6R7
IgGFc-binding protein
FCGBP


Q9Y6Y9
Lymphocyte antigen 96
LY96


Q9Y6Z7
Collectin-10
COLEC10









The Uniprot IDs set forth in Table 1 refer to the human versions of the listed proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also generally available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding a Therapeutic Fusion Protein, wherein the encoded therapeutic protein is chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of the secreted proteins listed in Table 1. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.


In some embodiments, the therapeutic protein is chosen from the putative secreted proteins listed in Table 2; thus, compositions of the invention may comprise an mRNA encoding a Therapeutic Fusion Protein, wherein the encoded therapeutic protein is one listed in Table 2 (or a homolog thereof, as discussed below) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a Therapeutic Fusion Protein, wherein the therapeutic protein is chosen from the proteins listed in Table 2 (or a homolog thereof, as discussed below) along with other components set out herein.









TABLE 2







Putative Secreted Proteins.









Uniprot
Protein
Gene


ID
Name
Name





A6NGW2
Putative stereocilin-like protein
STRCP1


A6NIE9
Putative serine protease 29
PRSS29P


A6NJ16
Putative V-set and immunoglobulin domain-
IGHV4OR15-8



containing-like protein IGHV4OR15-8


A6NJS3
Putative V-set and immunoglobulin domain-
IGHV1OR21-1



containing-like protein IGHV1OR21-1


A6NMY6
Putative annexin A2-like protein
ANXA2P2


A8MT79
Putative zinc-alpha-2-glycoprotein-like 1


A8MWS1
Putative killer cell immunoglobulin-like
KIR3DP1



receptor like protein KIR3DP1


A8MXU0
Putative beta-defensin 108A
DEFB108P1


C9JUS6
Putative adrenomedullin-5-like protein
ADM5


P0C7V7
Putative signal peptidase complex catalytic
SEC11B



subunit SEC11B


P0C854
Putative cat eye syndrome critical region
CECR9



protein 9


Q13046
Putative pregnancy-specific beta-1-
PSG7



glycoprotein 7


Q16609
Putative apolipoprotein(a)-like protein 2
LPAL2


Q2TV78
Putative macrophage-stimulating protein
MST1P9



MSTP9


Q5JQD4
Putative peptide YY-3
PYY3


Q5R387
Putative inactive group IIC secretory
PLA2G2C



phospholipase A2


Q5VSP4
Putative lipocalin 1-like protein 1
LCN1P1


Q5W188
Putative cystatin-9-like protein CST9LP1
CST9LP1


Q6UXR4
Putative serpin A13
SERPINA13P


Q86SH4
Putative testis-specific prion protein
PRNT


Q86YQ2
Putative latherin
LATH


Q8IVG9
Putative humanin peptide
MT-RNR2


Q8NHM4
Putative trypsin-6
TRY6


Q8NHW4
C-C motif chemokine 4-like
CCL4L2


Q9H7L2
Putative killer cell immunoglobulin-like
KIR3DX1



receptor-like protein KIR3DX1


Q9NRI6
Putative peptide YY-2
PYY2


Q9UF72
Putative TP73 antisense gene protein 1
TP73-AS1


Q9UKY3
Putative inactive carboxylesterase 4
CES1P1









The Uniprot IDs set forth in Table 2 refer to the human versions the listed putative proteins and the sequences of each are available from the Uniprot database. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding a Therapeutic Fusion Protein, wherein the therapeutic protein is chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 2. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.


In some embodiments, the therapeutic protein is chosen from the lysosomal and related proteins listed in Table 3; thus, compositions of the invention may comprise an mRNA encoding a Therapeutic Fusion Protein, wherein the therapeutic protein is one listed in Table 3 (or a homolog thereof, as discussed below) along with other components set out herein, and methods of the invention may comprise preparing and/or administering a composition comprising an mRNA encoding a Therapeutic Fusion Protein, wherein the therapeutic protein is chosen from the proteins listed in Table 3 (or a homolog thereof, as discussed below) along with other components set out herein.









TABLE 3





Lysosomal and Related Proteins.

















α-fucosidase



α-galactosidase



α-glucosidase



α-Iduronidase



α-mannosidase



α-N-acetylgalactosaminidase (α-galactosidase B)



β-galactosidase



β-glucuronidase



β-hexosaminidase



β-mannosidase



3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase



3-methylcrotonyl-CoA carboxylase



3-O-sulfogalactosyl cerebroside sulfatase (arylsulfatase A)



acetyl-CoA transferase



acid alpha-glucosidase



acid ceramidase



acid lipase



acid phosphatase



acid sphingomyelinase



alpha-galactosidase A



arylsulfatase A



beta-galactosidase



beta-glucocerebrosidase



beta-hexosaminidase



biotinidase



cathepsin A



cathepsin K



CLN3



CLN5



CLN6



CLN8



CLN9



cystine transporter (cystinosin)



cytosolic protein beta3A subunit of the adaptor protein-3



complex, AP3



formyl-Glycine generating enzyme (FGE)



galactocerebrosidase



galactose-1-phosphate uridyltransferase (GALT)



galactose 6-sulfate sulfatase (also known as



N-acetylgalactosamine-6-sulfatase)



glucocerebrosidase



glucuronate sulfatase



glucuronidase



glycoprotein cleaving enzymes



glycosaminoglycan cleaving enzymes



glycosylasparaginase (aspartylglucosaminidase)



GM2-AP



Heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT,



TMEM76)



Heparan sulfatase



hexosaminidase A lysosomal proteases methylmalonyl-CoA mutase



hyaluronidase



Iduronate sulfatase



LAMP-2



lysosomal α-mannosidase



Lysosomal p40 (C2orf18)



Major facilitator superfamily domain containing 8 protein



(MFSD8 or CLN7)



N-acetylgalactosamine 4-sulfatase



N-acetyl glucosamine 6-sulfatase



N-acetyl glucosaminidase



N-acetylglucosamine-1-phosphate transferase



NPC1



NPC2



palmitoyl-protein thioesterase



palmitoyl-protein thioesterase (CLN1)



Saposin A (Sphingolipid activator protein A)



Saposin B (Sphingolipid activator protein B)



Saposin C (Sphingolipid activator protein C)



Saposin D (Sphingolipid activator protein D)



sialic acid transporter (sialin)



sialidase



Sialin



sulfatase



Transmembrane protein 74 (TMEM74)



tripeptidyl-peptidase



tripeptidyl-peptidase I (CLN2)



UDP-N-acetylglucosamine- phosphotransferase










Information regarding lysosomal proteins is available from Lubke et al., “Proteomics of the Lysosome,” Biochim Biophys Acta. (2009) 1793: 625-635. In some embodiments, the protein listed in Table 3 and encoded by mRNA in the compositions and methods of the invention is a human protein. Sequences of the listed proteins are also available for various animals, including various mammals and animals of veterinary or industrial interest. Accordingly, in some embodiments, compositions and methods of the invention provide for the delivery of one or more mRNAs encoding a Therapeutic Fusion Protein, wherein the therapeutic protein chosen from mammalian homologs or homologs from an animal of veterinary or industrial interest of a protein listed in Table 3. In some embodiments, mammalian homologs are chosen from mouse, rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat, ferret, sheep, goat, or camel homologs. In some embodiments, the animal of veterinary or industrial interest is chosen from the mammals listed above and/or chicken, duck, turkey, salmon, catfish, or tilapia.


In some embodiments, the therapeutic protein is erythropoietin, α-galactosidase, low density lipoprotein receptor (LDLR), Factor VIII, Factor IX, α-L-iduronidase, iduronate sulfatase, heparin-N-sulfatase, α-N-acetylglucosaminidase, galactose 6-sulfatase, lysosomal acid lipase, arylsulfatase-A, IL-12, IL-23, α-galactosidase, erythropoietin (EPO), α-1-antitrypsin (A1AT), follistatin, glucocerebrosidase, interferon-β, hemoglobin, collagen type 4 (COL4A5), arginosuccinate synthase (AS), surfactant protein B (SPB), methylmalonyl-coA mutase (MCM), proprionyl-coA carboxylase (PCC), phenylalanine hydroxylase (PAH), apolipoprotein E (APOE), glucose-6-phosphatase (G6P), human growth hormone (hGH), urate oxidase, or granulocyte colony stimulating factor (GCSF).


Polypeptides Capable of Binding to an Fc Receptor


The Therapeutic Fusion Proteins of the invention comprise a therapeutic protein fused to a polypeptide capable of binding to an Fc receptor. In some embodiments, the polypeptide capable of binding to an Fc receptor comprises a portion of an immunoglobulin constant region that includes an Fc fragment. An Fc fragment can be comprised of the CH2 and CH3 domains of an immunoglobulin and the hinge region of the immunoglobulin. The immunoglobulin may be IgG, IgM, IgA, IgD, or IgE. In certain embodiments, the polypeptide capable of binding to an Fc receptor comprises an Fc fragment of an IgG1, an IgG2, an IgG3 or an IgG4. In one embodiment, the immunoglobulin is an Fc fragment of an IgG1. In one embodiment, the immunoglobulin is an Fc fragment of an IgG2.


The portion of an immunoglobulin constant region may include an Fc variant. “Fc variant” refers to a polypeptide or amino acid sequence that is modified from a native Fc but still comprises a binding site for an Fc receptor, such as, e.g., the FcRn. (See, e.g., WO 97/34631). “Native Fc” refers to an Fc that has not been modified. WO 96/32478 describes exemplary Fc variants, as well as interaction with an Fc receptor. Thus, the term “Fc variant” includes a polypeptide or amino acid sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that can and/or should be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, Fc variant may comprise a polypeptide or amino acid sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a target cell (3)N-terminal heterogeneity upon expression in a target cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than FcRn, or (7) antibody-dependent cellular cytotoxicity (ADCC).


In some embodiments, the polypeptide capable of binding to an Fc receptor binds to the neonatal Fc receptor, FcRn. FcRn is active in adult epithelial tissue and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). Chimeric proteins comprised of FcRn binding partners (e.g., IgG-Fc fragments) can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to administer the desired therapeutic protein. Additionally, Therapeutic Fusion Proteins comprising an FcRn binding partner will be endocytosed by cells expressing the FcRn. But instead of being marked for degradation, proteins bound to the FcRn are recycled out into circulation again, thus increasing the in vivo half-life of these proteins.


Thus, in some embodiments, the polypeptide capable of binding to an Fc receptor is an FcRn binding partner. An FcRn binding partner is any polypeptide, peptide, or amino acid sequence that specifically binds to the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner and any associated therapeutic protein. The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos. 6,030,613 and 6,086,875) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282: F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.


FcRn binding partners useful in the Therapeutic Fusion Proteins in the compositions of the invention may encompass any polypeptide, peptide, or amino acid sequence that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, MD


The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example the following single amino acid residues in human IgG1 Fc (Fcγ1) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where, for example, P238A represents wild type proline substituted by alanine at position number 238. In addition to alanine, other amino acids may be substituted for the wild type amino acids at the positions specified above. Mutations may be introduced singly into Fc, giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more FcRn binding partners.


Certain of the above mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG1 “ELLG” to the corresponding sequence from IgG2 “PVA” (with one amino acid deletion). It has been shown that FcγRI, FcγRII, and FcγRIII, which mediate various effector functions will not bind to IgG1 when such mutations have been introduced (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased “on” rate, a decreased “off” rate or both an increased “on” rate and a decreased “off” rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).


In one embodiment, the FcRn binding partner is a polypeptide including the sequence PKNSSMISNTP and optionally further including a sequence selected from HQSLGTQ, HQNLSDGK, HQNISDGK, or VISSHLGQ (See, U.S. Pat. No. 5,739,277).


Optional Linkers


The Therapeutic Fusion Protein encoded by the mRNA in the compositions of the invention may optionally comprise one or more linker sequences. In certain embodiments, the linker can comprise 1-5 amino acids, 1-10 amino acids, 1-15 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, or 100-200 amino acids. In one embodiment, the linker may comprise only glycine residues. In other embodiments, the linker can comprise the sequence (GGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Examples of suitable linkers include, but are not limited to, GGG, SGGSGGS, GGSGGSGGSGGSGGG, GGSGGSGGSGGSGGSGGS. In some embodiments, the linker is encoded by the sequence ccc aag agc ugu gac aag acc cac acc ugc ccu ccg ugu ccc.


Transfer Vehicle


In certain embodiments, the mRNA molecules of the invention may be administered as naked or unpackaged mRNA. In some embodiments, the administration of the mRNA in the compositions of the invention may be facilitated by inclusion of a suitable carrier. In certain embodiments, the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more mRNAs. As used herein, the terms “transfect” or “transfection” mean the intracellular introduction of an mRNA encoding a Therapeutic Fusion Protein into a cell, and preferably into a target cell. The introduced mRNA may be stably or transiently maintained in the target cell. The term “transfection efficiency” refers to the relative amount of mRNA taken up by the target cell which is subject to transfection. In practice, transfection efficiency can be estimated by the amount of a reporter nucleic acid product expressed by the target cells following transfection. The mRNA in the compositions of the invention may be introduced into target cells with or without a carrier or transfer vehicle.


As used herein, the terms “transfer vehicle,” “carrier,” and the like include any of the standard pharmaceutical carriers, vehicles, diluents, excipients and the like which are generally intended for use in connection with the administration of biologically active agents, including mRNA.


In certain embodiments, the carriers employed in the compositions of the invention may comprise a liposomal vesicle, or other means to facilitate the transfer of a mRNA to target cells and/or tissues. Preferred embodiments include compositions with high transfection efficacies and in particular those compositions that minimize adverse effects which are mediated by transfection of non-target cells. The compositions of the present invention that demonstrate high transfection efficacies improve the likelihood that appropriate dosages of the mRNA will be delivered to the target cell, while minimizing potential systemic adverse effects. In one embodiment of the present invention, the transfer vehicles of the present invention are capable of delivering large mRNA sequences (e.g., mRNA of a size ranging from 0.2 kilobases (kb) to 10 kb or more, e.g., mRNA of a size greater than or equal to 0.2 kb, 0.5 kb, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, or 4.5 kb, and/or having a size of up to 5 kb, 5.5 kb, 6 kb, 7 kb, 8 kb, 9 kb, or 10 kb).


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 biological activity. In some embodiments, the transfer vehicle demonstrates preferential and/or substantial binding to a target cell relative to non-target cells. In a preferred embodiment, the transfer vehicle delivers its contents to the target cell such that the mRNA is delivered to the appropriate subcellular compartment, such as the cytoplasm.


In some embodiments, the compositions of the invention employ a polymeric carrier alone or in combination with other carriers. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL, polyethylenimine (PEI), including, but not limited to branched PEI (25 kDa) and multi-domain-block polymers. Alternatively, suitable carriers include, but are not limited to, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, dry powders, nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, small-molecule targeted conjugates, and other vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable carrier. (Hum. Gene Ther. 2008 Sep; 19(9):887-95).


Lipid Nanoparticles


In certain embodiments, the transfer vehicle in the compositions of the invention is a liposomal transfer vehicle, e.g. a lipid nanoparticle or a lipidoid nanoparticle. In one embodiment, the transfer vehicle may be selected and/or prepared to optimize delivery of the mRNA to a target cell. For example, if the target cell is a hepatocyte 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 in the central nervous system (e.g., mRNA administered for the treatment of neurodegenerative diseases may specifically target brain or spinal tissue), selection and preparation of the transfer vehicle must consider penetration of, and retention within, the blood brain barrier and/or the use of alternate means of directly delivering such transfer vehicle to such target cell. In one embodiment, the compositions of the present 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 enhance the transfer of exogenous mRNA to the target cells).


Liposomes (e.g., liposomal lipid nanoparticles) are known to be particularly for their use as transfer vehicles of diagnostic or therapeutic compounds in vivo (Lasic, Trends Biotechnol., 16: 307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).


In the context of the present invention, a liposomal transfer vehicle typically serves to transport the mRNA to the target cell. For the purposes of the present invention, the liposomal transfer vehicles are prepared to contain mRNA encoding a Therapeutic Fusion Protein. The process of incorporation of the desired mRNA into a liposome is referred to as “loading” and is described in Lasic, et al., FEES Lett., 312: 255-258, 1992. The liposome-incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane. The incorporation of a nucleic acid into liposomes is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the liposome.


The purpose of incorporating an mRNA into a transfer vehicle, such as a liposome, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in a preferred embodiment of the present invention, the selected transfer vehicle is capable of enhancing the stability of the mRNA contained therein. The liposome can allow the encapsulated mRNA to reach the target cell and/or may preferentially allow the encapsulated mRNA to reach the target cell, or alternatively limit the delivery of such mRNA to other sites or cells where the presence of the administered mRNA may be useless or undesirable. Furthermore, incorporating the mRNA into a transfer vehicle, such as for example, a cationic liposome, also facilitates the delivery of such mRNA into a target cell.


Ideally, liposomal transfer vehicles are prepared to encapsulate mRNA encoding a Therapeutic Fusion Protein such that the compositions demonstrate high transfection efficiency and enhanced stability. 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 facilitate, and in some instances markedly enhance, the transfection efficiency of several types of cationic liposomes by 2-28 fold in a number of cell lines both in vitro and in vivo. (See N.J. Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997; 4, 891.) Thus, in certain embodiments of the present invention, the transfer vehicle is formulated as a lipid nanoparticle.


In certain embodiments, the mRNA encoding a Therapeutic Fusion Protein is combined with a multi-component lipid mixture of varying ratios employing one or more cationic lipids, non-cationic lipids, helper lipids, and PEG-modified or PEGylated lipids designed to encapsulate various nucleic acid-based materials. 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 pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.


Cationic lipids may include, but are not limited to ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1,3]dioxol-5-amine)), DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety), HGT5000 (U.S. Provisional Patent Application No. 61/617,468, the teachings of which are incorporated herein by reference in their entirety) or HGT5001(cis or trans) (Provisional Patent Application No. 61/617,468), aminoalcohol lipidoids such as those disclosed in WO2010/053572, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane)(Heyes, et al., J. Contr. Rel. 107:276-287(2005)), DLin-KC2-DMA (Semple, et al., Nature Biotech. 28:172-176 (2010)), C12-200 (Love, et al., Proc. Nat'l. Acad. Sci. 107:1864-1869(2010)).


In some embodiments, DOTMA can be formulated alone or can be combined with the neutral lipid, DOPE (dioleoylphosphatidyl-ethanolamine), or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, DOGS (5-carboxyspermyl glycinedioctadecylamide), DOSPA (2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium) (Behr et al. Proc. Nat'l Acad. Sci. 86, 6982 (1989); U.S. Pat. Nos. 5,171,678; 5,334,761), DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane). Contemplated cationic lipids also include DSDMA (1,2-distearyloxy-N,N-dimethyl-3-aminopropane, DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane), DLenDMA (1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane), DODAC (N-dioleyl-N,N-dimethylammonium chloride), DDAB (N,N-distearyl-N,N-dimethylammonium bromide), DMRIE (N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide), CLinDMA (3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane), CpLinDMA (2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy 1-1-(cis,cis-9′, 1-2′-octadecadienoxy)propane), DMOBA (N,N-dimethyl-3,4-dioleyloxybenzylamine), DOcarbDAP (1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane), DLinDAP (2,3-Dilinoleoyloxy-N,N-dimethylpropylamine), DLincarbDAP (1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane), DLinCDAP (1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-K-XTC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), or mixtures thereof.


Specific biodegradable lipids suitable for use in the compositions and methods of the invention include:




embedded image


and their salts.


Additional specific cationic lipids for use in the compositions and methods of the invention are XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-11,31-dioxolane) and, MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate):




embedded image


both of which are described in detail in US 20100267806.


Another cationic lipid that may be used in the compositions and methods of the invention is NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide):




embedded image


which is described in WO06138380A2.


Suitable helper lipids include, but are not limited to DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and cholesterol. Cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate)(WO/2011/068810).


Non-cationic lipids may also be used in the compositions of the invention. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. “Anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, DSPC (distearoylphosphatidyl-choline), DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidyl-choline), DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidyl-glycerol), DOPE (dioleoylphosphatidylethanolamine), POPC (palmitoyloleoyl-phosphatidylcholine), POPE (palmitoyloleoyl-phosphatidylethanolamine), DOPE-mal (dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), DDPE (dipalmitoyl phosphatidyl ethanolamine), DMPE (dimyristoyl-phosphoethanolamine), DSPE (distearoylphosphatidylethanolamine), SOPE (16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipid present in the transfer vehicle.


Polyethylene glycol (PEG)-modified phospholipids and derivatized lipids for use in nanoparticle formulations include, but are not limited to a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length, DMG-PEG2K, PEG-DSG, PEG-DMG, and PEG-derivatized ceramides (PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000], (C8 PEG-2000 ceramide). The use of PEG-modified lipids is contemplated for use the compositions of the invention, either alone or preferably in combination with other lipids which together comprise the transfer vehicle (e.g., a lipid nanoparticle). The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 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.


In addition, several reagents are commercially available to enhance transfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), and EFFECTENE.


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 comprise C12-200, DSPC, CHOL, and DMG-PEG or MC3, DSPC, chol, and DMG-PEG or C12-200, DOPE, chol, DMG-PEG2K. The selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. For example, a transfer vehicle may be prepared using C12-200, DOPE, cholesterol, DMG-PEG2K at a molar ratio of or DODAP, DOPE, cholesterol, DMG-PEG2K at a molar ratio of 18:56:20:6; or HGT5000, DOPE, cholesterol, DMG-PEG2K at a molar ratio of or HGT5001, DOPE, cholesterol, DMG-PEG2K at a molar ratio of or XTC, DSPC, cholesterol, PEG-DMG at a molar ratio of 57.5:7.5:31.5:3.5 or a molar ratio of 60:7.5:31:1.5; or MC3, DSPC, cholesterol, PEG-DMG in a molar ratio of 50:10:38.5:1.5 or a molar ratio of 40:15:40:5; or MC3, DSPC, cholesterol, PEG-DSG/GalNAc-PEGDSG in a molar ratio of 50:10:35:4.5:0.5; or ALNY-100, DSPC, cholesterol, PEG-DSG.


Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(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 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%. The percentage of non-cationic lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%. The percentage of PEG-modified lipid in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.


In certain preferred embodiments, the lipid nanoparticles of the invention comprise at least one of the following cationic lipids: XTC, MC3, NC98-5, ALNY-100, C12-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001. In some embodiments, the transfer vehicle comprises cholesterol and/or a PEG-modified lipid. In some embodiments, the transfer vehicles comprise DMG-PEG2K.


The liposomal transfer vehicles 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 via conventional techniques, for example, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the 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 vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.


In certain embodiments of this invention, the compositions of the present invention comprise a transfer vehicle wherein the mRNA is associated on both the surface of the transfer vehicle and encapsulated within the same transfer vehicle. For example, during preparation of the compositions of the present invention, cationic liposomal transfer vehicles may associate with the mRNA through electrostatic interactions.


Selection of the appropriate size of a liposomal 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, it may be desirable to limit transfection of the mRNA to certain cells or tissues. For example, to target hepatocytes a liposomal transfer vehicle may be sized such that its dimensions are smaller than the fenestrations of the 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 transfer vehicle may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. For example, a liposomal transfer vehicle may be sized such that its dimensions are larger than the fenestrations of the 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, preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or 10 nm.


A variety of alternative methods known in the art are available for sizing 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 progressive 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 a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the liposomal vesicles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein by reference. Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be 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 embodiments, the target cells are epithelial cells found e.g., in the lung, intestine, renal proximal tubes, nasal passages, vaginal surfaces, and bilary tree surfaces, which contain the Fc neonatal receptor.


In some embodiments, the target cells are deficient in a protein or enzyme of interest. For example, where it is desired to deliver a nucleic acid to a hepatocyte, the hepatocyte represents the target cell. In some embodiments, the compositions of the invention transfect the target cells on a discriminatory basis (i.e., do not transfect non-target cells). The compositions of the invention may be prepared to preferentially target a variety of target cells, which include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, 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 delivery and the subsequent 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” capable of producing, and systemically excreting a functional protein or enzyme. Accordingly, in one embodiment of the invention the liposomal transfer vehicle may target hepatocyes and/or preferentially distribute to the cells of the liver upon delivery. Following transfection of the target hepatocytes, the mRNA loaded in the liposomal vehicle is translated and a functional protein product is produced, excreted and systemically distributed. In other embodiments, cells other than hepatocytes (e.g., lung, spleen, heart, ocular, or cells of the central nervous system) can serve as a depot location for protein production.


In one embodiment, the compositions of the invention facilitate a subject's endogenous production of one or more functional proteins and/or enzymes, and in particular the production of proteins and/or enzymes which demonstrate less immunogenicity relative to their recombinantly-prepared counterparts. In a preferred embodiment of the present invention, the transfer vehicles comprise mRNA which encode a protein or enzyme for which the subject is deficient. Upon distribution of such compositions to the target tissues and the subsequent transfection of such target cells, the exogenous mRNA loaded into the liposomal transfer vehicle (e.g., a lipid nanoparticle) may be translated in vivo to produce a functional protein or enzyme encoded by the exogenously administered mRNA (e.g., a protein or enzyme for which the subject is deficient). Accordingly, the compositions of the present invention exploit a subject's ability to translate exogenously- or synthetically-prepared mRNA to produce an endogenously-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 vivo inclusion of native post-translational modifications which may often be absent in recombinantly-prepared proteins or enzymes, thereby further reducing the immunogenicity of the translated protein or enzyme.


The present invention also contemplates the discriminatory targeting of target cells and tissues by both passive and active targeting means. The phenomenon of passive targeting exploits the natural distributions patterns of a transfer vehicle in vivo without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells. For example, transfer vehicles 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 passively direct the delivery of the compositions to such target cells.


Alternatively, the present invention contemplates active targeting, which involves the use of additional excipients, referred to herein as “targeting ligands” that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues. For example, targeting may be mediated by the inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the transfer vehicle to encourage distribution to the target cells or tissues. Recognition of the targeting ligand by the target tissues actively facilitates tissue distribution and cellular uptake of the transfer vehicle and/or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle encourages recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes).


As provided herein, the composition may comprise a ligand capable of enhancing affinity of the composition to the target cell. Targeting ligands may be linked to the outer bilayer of the lipid particle during formulation or post-formulation. These methods are well known in the art. In addition, some lipid particle formulations may employ fusogenic polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S. Patent Application 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 present invention demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest. Contemplated therefore are compositions which comprise one or more ligands (e.g., 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 tissues. Suitable ligands may optionally be bound or linked to the surface 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, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features.) Cell-specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting ligands are selected such that the unique characteristics of a target cell are exploited, thus allowing the composition to discriminate between target and non-target cells. For example, compositions of the invention may include surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers). Additionally, the use of galactose as a targeting ligand would be expected to direct the compositions of the present invention to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor present in hepatocytes). (See Hillery A M, et al. “Drug Delivery and Targeting: For Pharmacists and Pharmaceutical Scientists” (2002) Taylor & Francis, Inc.) The presentation of such targeting ligands that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present invention in target cells and tissues. Examples of suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.


Methods of Administration and Treatment


As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human 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 compositions and methods of the invention provide for the delivery of mRNA encoding a Therapeutic Fusion Protein to treat a number of disorders. In some embodiments, the compositions and methods of the present invention are suitable for the treatment of diseases or disorders relating to the deficiency of proteins and/or enzymes that are excreted or secreted by the target cell into the surrounding extracellular fluid (e.g., mRNA encoding hormones and neurotransmitters). In some embodiments the disease may involve a defect or deficiency in a secreted protein (e.g. Fabry disease, or ALS). In certain embodiments, the disease may not be caused 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. Disorders for which the present invention are useful include, but are not limited to, disorders such as Pompe Disease, Gaucher Disease, beta-thalassemia, Huntington's Disease; Parkinson's Disease; muscular dystrophies (such as, e.g. Duchenne and Becker); hemophilia diseases (such as, e.g., hemophilia B (FIX), hemophilia A (FVIII); SMN1-related spinal muscular atrophy (SMA); amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; SLC3A1-related disorders including cystinuria; COL4A5-related disorders including Alport syndrome; galactocerebrosidase deficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy; Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-related tuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-related cystinosis; the FMR1-related disorders which include Fragile X syndrome, Fragile X-Associated Tremor/Ataxia Syndrome and Fragile X Premature Ovarian Failure Syndrome; Prader-Willi syndrome; hereditary hemorrhagic telangiectasia (AT); Niemann-Pick disease Type Cl; the neuronal ceroid lipofuscinoses-related diseases including Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies; EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia with central nervous system hypomyelination/vanishing white matter; CACNA1A and CACNB4-related Episodic Ataxia Type 2; the MECP2-related disorders including Classic Rett Syndrome, MECP2-related Severe Neonatal Encephalopathy and PPM-X Syndrome; CDKL5-related Atypical Rett Syndrome; Kennedy's disease (SBMA); Notch-3 related cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); SCN1A and SCN1B-related seizure disorders; the Polymerase G-related disorders which include Alpers-Huttenlocher 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 some embodiments, the compositions of the invention provide for the in vivo delivery of one or more of Alpha 1-antitrypsin (A1AT), follistatin (e.g., for treatment of Duchenne's Muscular Dystrophy or A1At deficiency), acid alpha-glucosidase (GAA) (e.g., for treatment of Pompe Disease), glucocerebrosidase (e.g., for treatment of Gaucher Disease), Interferon Beta (IFN-β), hemoglobin (e.g., for treatment of beta-thalassemia), Collagen Type 4 (COL4A5) (e.g., for treatment of Alport Syndrome) and Granulocyte colony-stimulating factor (GCSF).


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 subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein production.


Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Pulmonary administration by aerosolization or nebulization is particularly preferred for its non-invasive features and because of the ability of the Therapeutic Fusion Protein to be easily transported across the lung epithelium into the circulatory system.


Alternately, the compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into 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 invention 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 supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can be delivered to the eye by use of creams, drops, or even injection. Formulations containing compositions of the present invention complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.


In one embodiment, the compositions of the invention are formulated such that they are suitable 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 of the present invention are administered to a subject twice day, daily or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually. Also contemplated are compositions and liposomal vehicles which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release an 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 the liposomal nanoparticles disclosed herein and related methods for the use of such lyophilized compositions as disclosed for example, in PCT Application Publication No. WO 2012/170889, the teachings of which are incorporated herein by reference in their entirety. For example, lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo. For example, a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.


Apparatuses Loaded with a Pharmaceutical Composition


In some embodiments, the compositions of the invention, such as a cationic lipid-based or PEI-based composition comprising an mRNA encoding a Therapeutic Fusion Protein, is provided within an apparatus for administration to the respiratory system of a subject. The apparatus can be, e.g., an instillation, aerosolization, or nebulization apparatus. Suitable apparatuses include, for example, a PARI Boy jet nebulizer, Aeroneb® Lab nebulizer, MicroSprayer®, or EFlow mesh nebulizer. Alternatively, dry powder inhalers or aerosolization apparatuses such as portable inhalers may be used.


While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate 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 provide additional detail regarding its practice are hereby incorporated by reference in their entirety.


The articles “a” and “an” as used herein in the specification and in 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 otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given 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 variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the 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 art that a contradiction or inconsistency 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 embodiments of the 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 been specifically 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 of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.


EXAMPLES
Example 1: Messenger RNA and Lipid Nanoparticle Formulations

mRNAs encoding human erythropoietin•IgG Fc (SEQ ID NO: 3; FIG. 2A), human alpha-galactosidase•IgG Fc (SEQ ID NO: 4; FIG. 3), human alpha-1 antitrypsin•IgG Fc(SEQ ID NO: 5; FIG. 4), and human factor IX•IgG Fc (SEQ ID NO: 6; FIG. 5) are synthesized by in vitro transcription from plasmid DNA template encoding the fusion protein, with subsequent addition of a 5′ cap structure (Cap1) (Fechter & Brownlee, J. Gen. Virology 86:1239-1249 (2005)) and a 3′ poly(A) tail of approximately 200 nucleotides in length. The poly(A) tail length is determined by gel electrophoresis. 5′ and 3′ untranslated regions as defined by SEQ ID NOs 1 and 2 (FIG. 1A and FIG. 1B) are present in each mRNA construct.


Formulation 1: Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, Chol and DMG-PEG2K (40:30:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C.


Formulation 2: Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol and DMG-PEG2K (18:56:20:6) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.35 mg/mL EPO mRNA (encapsulated). Zave=75.9 nm (Dv(50)=57.3 nm; Dv(90)=92.1 nm).


Formulation 3: Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE, cholesterol and DMG-PEG2K (50:25:20:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C.


Formulation 4: Aliquots of 50 mg/mL ethanolic solutions of ICE, DOPE and DMG-PEG2K (70:25:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C.


Formulation 5: Aliquots of 50 mg/mL ethanolic solutions of HGT5000, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C. Final concentration=1.82 mg/mL EPO mRNA (encapsulated). Zave=105.6 nm (Dv(50)=53.7 nm; Dv(90)=157 nm).


Formulation 6: Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE, cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mL final volume. Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of mRNA is prepared from a 1 mg/mL stock. The lipid solution is injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension is filtered, diafiltrated with 1× PBS (pH 7.4), concentrated and stored at 2-8° C.


Example 2: Administration of mRNA and Harvesting Samples for Analysis

Studies are performed using either female BALB/C mice or (therapeutic protein deficient) KO mice. Samples are introduced via either direct instillation (MicroSprayer®) or nebulization (PARI Boy or Aeroneb) respective dose of encapsulated FFL mRNA. Mice are sacrificed and perfused with saline at the designated time points.


Intratracheal Administration. Test materials are administered by a single intratracheal aerosol administration via a Microsprayer™ (50 μL/animal) while animals are anesthetized with intraperitoneal injection of a mixture of ketamine mg/kg and xylazine 5-15 mg/kg.


Nebulization (Aerosol) Administration. FFL test materials are administered to all animals by a single aerosol inhalation via Aeroneb® Lab nebulizer (nominal dose volume of up to 8 mL/group). The test material is delivered to a box containing the whole group of animals (n=4) and connected to oxygen flow and scavenger system.


Euthanasia. Animals are euthanized by CO 2 asphyxiation at representative times post-dose administration (±5%) followed by thoracotomy and exsanguinations. Whole blood (maximal obtainable volume) is collected via cardiac puncture.


Perfusion. Following exsanguination, animals undergo cardiac perfusion with saline. In brief, whole body intracardiac perfusion is performed by inserting 23/21 gauge needle attached to 10 mL syringe containing saline set into the lumen of the left ventricle for perfusion. The right atrium is incised to provide a drainage outlet for perfusate. Gentle and steady pressure is applied to the plunger to perfuse the animal after the needle has been positioned in the heart. Adequate flow of the flushing solution is ensured when the exiting perfusate flows clear (free of visible blood) indicating that the body is saturated with flushing solution and the procedure is complete.


Tissue Collection. Following perfusion, the liver, lungs (right and left) and spleen are harvested from each animal, snap frozen, and stored at −80° C. or stored in 10% neutral buffered formalin for analysis.


Isolation of serum for analysis. Whole blood (maximal obtainable volume) is collected from animals euthanized by CO 2 asphyxiation 48 hours post dose administration (±5%) followed by thoracotomy and terminal cardiac blood collection via cardiac puncture on euthanized animals into serum separator tubes, allowed to clot at room temperature for at least 30 minutes, centrifuged at 22° C.±5° C. at 9300 g for 10 minutes, and the serum is extracted. For interim blood collections, approximately 40-50 μL of whole blood is collected via facial vein puncture or tail snip. Samples collected from non treatment animals are used as a baseline for comparison to study animals.


Example 3: Enzyme-Linked Immunosorbent Assay (ELISA) Analysis

EPO ELISA: Quantification of EPO protein is performed following procedures reported for human EPO ELISA kit (Quantikine IVD, R&D Systems, Catalog #Dep-00). Positive controls are ultrapure and tissue culture grade recombinant human erythropoietin protein (R&D Systems, Catalog #286-EP and 287-TC, respectively). Detection is monitored via absorption (450 nm) on a Molecular Device Flex Station instrument.


GLA ELISA: Standard ELISA procedures are followed employing sheep anti-Alpha-galactosidase G-188 IgG as the capture antibody with rabbit anti-Alpha-galactosidase TK-88 IgG as the secondary (detection) antibody (Shire Human Genetic Therapies). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG is used for activation of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reaction is quenched using 2N H2SO4 after 20 minutes. Detection is monitored via absorption (450 nm) on a Molecular Device Flex Station instrument. Untreated mouse serum and human Alpha-galactosidase protein are used as negative and positive controls, respectively.


FIX ELISA: Quantification of FIX protein is performed following procedures reported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog #EF1009-1).


A1AT ELISA: Quantification of A1AT protein is performed following procedures reported for human A1AT ELISA kit (Innovative Research, Catalog #IRAPKT015).


Western Blot Analysis (EPO): Samples can also be analyzed via Western blot. For example, Western blot analyses of the EPO fusion protein are performed using an anti-hEPO antibody (R&D Systems #MAB2871) and ultrapure human EPO protein (R&D Systems #286-EP) as the control.


Results: The results will demonstrate that administration of mRNA encoding Therapeutic Fusion Proteins result in the production of protein in vivo and delivery of significant levels of therapeutic protein into the circulatory system. Such a depot effect can be achieved in multiple sites within the body (i.e., lung, liver, kidney, spleen, and muscle).

Claims
  • 1. A composition comprising (a) at least one mRNA molecule, at least a portion of which encodes a therapeutic polypeptide fused to a polypeptide capable of binding to an Fc receptor; and (b) a transfer vehicle.
  • 2. The composition of claim 1, wherein the polypeptide capable of binding to an Fc receptor is an FcRn binding peptide, or an immunoglobulin Fc domain.
  • 3. (canceled)
  • 4. The composition of claim 2, wherein the immunoglobulin Fc domain is from IgG.
  • 5. The composition of claim 1, wherein the Fc receptor is the neonatal Fc receptor, FcRn.
  • 6. (canceled)
  • 7. The composition of claim 1, wherein the mRNA molecule comprises a 5′ untranslated region, optionally wherein the 5′ untranslated region is from CMV IE1.
  • 8. (canceled)
  • 9. The composition of claim 1, wherein the mRNA molecule comprises a Cap1 structure, or a m7GpppG cap.
  • 10. (canceled)
  • 11. The composition of claim 1, wherein the mRNA molecule comprises a 3′ untranslated region, optionally wherein the 3′ untranslated region is from hGH.
  • 12. (canceled)
  • 13. (canceled)
  • 14. The composition of claim 1, wherein the mRNA molecule comprises one or more modified nucleosides, optionally wherein the modified nucleosides are selected from pseudouridine, 5-methylcytidine, 2′-O-methyluridine, 5-methyluridine, 2-thio-uridine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine cytosine, 4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine, 4′thio-5-methylcytidine, 4′-thio-pseudouridine, and 2,4′-dithiouridine.
  • 15. (canceled)
  • 16. The composition of claim 1, wherein the mRNA molecule has been modified to reduce or eliminate CpG motifs, repeat sequences, inverted sequences, cryptic promoter sequences, and to ensure a GC content less than the GC content of the wild type sequence.
  • 17. The composition of claim 1, wherein the mRNA encodes a protein which is abnormal or deficient in an individual, the mRNA encodes a protein that is not normally secreted by a cell, operably linked to a secretory leader sequence that is capable of directing secretion of the encoded fusion protein,the mRNA encodes a protein that is normally secreted by a cell,the mRNA encodes an enzyme which is abnormally deficient in an individual with a lysosomal storage disorder,the mRNA encodes a polypeptide selected from the group of polypeptides/proteins listed in Tables 1-3, and/orthe mRNA encodes a therapeutic protein selected from α-galactosidase, erythropoietin (EPO), α-1-antitrypsin (A1 AT), follistatin, glucocerebrosidase, interferon-β, hemoglobin, collagen type 4 (COL4A5), low density liprotein (LDL) receptor, Factor VIII, Factor IX, α-L-iduronidase, iduronate sulfatase, heparin-N-sulfatase, α-N-acetylglucosaminidase, galactose 6-sulfatase, lysosomal acid lipase, arylsulfatase-A, granulocyte colony-stimulating factor (GCSF), anti-vascular endothelial growth factor (VEGF), interleukin 12 (IL-12), interleukin 23 (IL-23), arginosuccinate synthase (AS), surfactant protein B (SPB), methylmalonyl-coA mutase (MCM), proprionyl-coA carboxylase (PCC), phenylalanine hydroxylase (PAH), apolipoprotein E (APOE), glucose-6-phosphatase (G6P), human growth hormone (hGH), and urate osidase.
  • 18-23. (canceled)
  • 24. The composition of claim 1, wherein the composition is formulated for pulmonary administration.
  • 25. The composition of claim 1, wherein the delivery vehicle is polyethyleneimine (PEI), and/or wherein the PEI has a molecular weight between 20 and 30 kD.
  • 26. (canceled)
  • 27. The composition of claim 1, wherein the delivery vehicle is a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG modified lipid, or the lipid nanoparticle comprises one or more cationic lipids, one ore more neutral lipids, one or more cholesterols, and/or one or more PEG modified lipids.
  • 28. (canceled)
  • 29. The composition of claim 27, wherein the lipid nanoparticle comprises a cationic lipid nanoparticle selected from: C12-200, XTC, MC3, NC98-5, DLinDMA, HGT5001cis, HGT5001trans, HGT5000, HGT4003, DLinKC2DMA, ALNY100, ICE, DOTAP, DODAP, DOTMA.
  • 30-35. (canceled)
  • 36. The composition of 27, wherein the lipid nanoparticle comprises DLinKC2DMA, CHOL, DOPE, and DMG-PEG-2000, or wherein the lipid nanoparticle comprises C12-200, DOPE, CHOL, and DMG-PEG-2000.
  • 37. (canceled)
  • 38. The composition of claim 1, wherein said composition is lyophilized, or wherein said composition is a reconstituted lyophilized composition.
  • 39. (canceled)
  • 40. A method of inducing expression of a functional polypeptide in a subject, comprising administering to the subject a composition comprising (a) at least one mRNA molecule, at least a portion of which encodes a therapeutic polypeptide fused to a polypeptide capable of binding to an Fc receptor, and (b) a transfer vehicle.
  • 41. The method of claim 40, wherein the subject has a deficiency in a therapeutic protein encoded by the mRNA in the composition and/or wherein the therapeutic protein is an enzyme abnormally deficient in an individual with a lysosomal storage disorder.
  • 42. (canceled)
  • 43. The method of claim 40, wherein the composition is administered by nebulization, aerosolization, or instillation.
  • 44. A composition comprising (a) at least one mRNA molecule at least a portion of which encodes a therapeutic protein fused to a polypeptide capable of binding to an Fc receptor; and (b) a transfer vehicle comprising a lipid nanoparticle or a lipidoid nanoparticle, wherein the polypeptide is chosen from proteins listed in table S1, table S2, and table S3, mammalian homologs thereof, and homologs from animals of veterinary or industrial interest.
CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation application of U.S. application Ser. No. 16/422,403, filed on May 24, 2019, which is a continuation of application of U.S. application Ser. No. 14/774,263, filed on Feb. 19, 2016, which is a U.S. National Entry claiming priority to International Application No. PCT/US14/28330, filed on Mar. 14, 2014, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/784,766, filed on Mar. 14, 2013, the entirety of which is incorporated herein by reference.

Provisional Applications (1)
Number Date Country
61784766 Mar 2013 US
Continuations (2)
Number Date Country
Parent 16422403 May 2019 US
Child 18186856 US
Parent 14774263 Feb 2016 US
Child 16422403 US