Subcutaneous delivery of lipid nanoparticle formulations

Abstract

Provided herein, inter alia, are methods and compositions related to subcutaneous delivery of nucleic acids encapsulated by a lipid delivery vehicle.

Description

Attorney Docket No. 049386-549001WOSUBCUTANEOUS DELIVERY OF LIPID NANOPARTICLE FORMULATIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 753,233, filed Feb 3, 2025, the contents of which are hereby incorporated by reference in their entirety for all purposes.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence Listing, which is incorporated by reference in its entirety. The accompanying Sequence Listing text file, name, “049386-549001 W0_2026-02-03_Scqucncc_Listing_ST26.xml” was created on February 3, 2026, and is 59,187 bytes.BACKGROUND

[0003] Lipid nanoparticle (LNP) formulations are effective agents for hepatocyte delivery of nucleic acid therapeutics. Innovation in this field led to the development of lipid formulations of nucleic acid therapeutics for parenteral administration via intravenous (IV), and intramuscular (IM) administration routes. Intravenous (IV) administration requires access to a medical facility with trained medical professionals, which is inconvenient or even prohibitive to many patients due to logistical constraints including travel to medical facilities. IV administration is associated with the risk of systemic infection and potential exposure to hospital-acquired infections.Subcutaneous administration has been proven to be effective in several therapeutic modalities and allows therapeutics to be self-administered by patients. Subcutaneous drug administration is also known to produce a depot effect whereby drug is slowly released into circulation and hence can produce a sustained pharmacodynamic effect over an extended time. Thus, many chronic diseases can be treated in a convenient and self-administered fashion in a home rather than in a clinical setting. Subcutaneous or intradermal administration of a lipid nanoparticle (LNP) and concomitant release of encapsulated nucleic acid therapeutics with a meaningful therapeutic outcome has not yet been shown. One premise for the lack of lipid nanoparticle release into systemic circulation upon subcutaneous administration is the inability for lipid nanoparticles to penetrate the hyaluronic acid polymer mesh work that barriers the administration site. The compositions and methods provided herein, inter alia, address these and other problems in the art.SUMMARY

[0004] In an aspect, provide herein is a method of delivering a therapeutically effective amount of a nucleic acid encapsulated in a lipid delivery vehicle comprising a mammal (i) a composition comprising the nucleic acid encapsulated in the lipid delivery vehicle and (ii) hyaluronidase, wherein the hyaluronidase is a pure hyaluronidase as determined by the presence of a single band located within the range of about 50 kDa to about 80 kDa.

[0005] In some embodiments, the present application provides a pharmaceutical composition comprising the compound described herein or the lipid nanoparticle described herein, and a pharmaceutically acceptable excipient.

[0006] In some embodiments, the present application provides a method of treating a disease in a mammal in need thereof, comprising co-administering a therapeutically effective amount to the mammal the nucleic acid encapsulated in a lipid delivery vehicle described herein and a pure hyaluronidase, the nucleic acid encapsulated in a lipid nanoparticle described herein and a pure hyaluronidase, or the pharmaceutical composition described herein and a pure hyaluronidase.

[0007] In another aspect, provided herein is a method of delivering a nucleic acid comprising administering to a patient in need thereof (i) a composition comprising a therapeutically effective amount of nucleic acid encapsulated in a lipid delivery vehicle and (ii) a soluble hyaluronidase.

[0008] In some embodiments, the purity of hyaluronidase is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.8%.

[0009] In some embodiments, the nucleic acid is selected from an siRNA (small interfering RNA), an mRNA (messenger RNA), a self-replicating RNA, a DNA plasmid, an antisense oligonucleotide, a single strand guide RNA, and combinations thereof.

[0010] In some embodiments, the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein. In some embodiments, the protein is an enzyme, and antibody, an antigen, a receptor, or a transporter. In some embodiments, the protein is a gene-editing enzyme. In some embodiments, the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

[0011] In some embodiments, the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a viral protein that when expressed elicits an immunological response. In some embodiments, the immunological response comprises production of antibodies against the viral protein.

[0012] In some embodiments, the lipid delivery vehicle is selected from a lipid nanoparticle (LNP), a liposome, and a lipoplex. In some embodiments, the lipid delivery vehicle is a lipid nanoparticle.

[0013] In some embodiments, the lipid nanoparticle has an average particle size of less than about 100 nm. In some embodiments, the lipid nanoparticle has an average particles size of about 55 nm to about 85 nm.

[0014] In some embodiments, the lipid nanoparticle further comprises a helper lipid. In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC).

[0015] In some embodiments, the lipid nanoparticle further comprises cholesterol.

[0016] In some embodiments, the lipid nanoparticle further comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0017] In some embodiments, the lipid nanoparticle comprises about 45 mol% to 65 mol% of a cationic lipid or an ionizable lipid, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate. In some embodiments, the lipid nanoparticle comprises about 50 mol% to about 61 mol% of the cationic lipid or ionizable lipid, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate. In some embodiments, the lipid nanoparticle comprises about 56 mol% to about 58 mol% of the cationic lipid or ionizable lipid, about 6 mol% to about 8 mol% of DSPC, about31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

[0018] In some embodiments, the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1.

[0019] In some embodiments, the composition further comprises one or more cryoprotectants. In some embodiments, the one or more cryoprotectants is selected from sucrose, glycerol, and a combination of sucrose and glycerol. In some embodiments, the composition is reconstituted from a lyophilized composition.

[0020] In some embodiments, the purity of hyaluronidase is determined by gel electrophoresis, optionally wherein the gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0021] In some embodiments, the pure hyaluronidase is free from impurities selected from denatured hyaluronidase, degraded hyaluronidase, hyaluronidase fragment, extraneous protein, nucleic acid, and a combination thereof.

[0022] In some embodiments, the co-administration results in an increased systemic delivery of the nucleic acid.

[0023] In some embodiments, the hyaluronidase has the sequence set forth as residues 36-482 or 36-483 or has at least 98% sequence identity to the sequence set forth as residues 36-482 or 36-483 ofSEQ ID NO:1.

[0024] In some embodiments, the hyaluronidase is a soluble PH20 hyaluronidase. In some embodiments, the soluble hyaluronidase is the composition designated rHuPH20.

[0025] In some embodiments, the soluble hyaluronidase comprises amino acids 36-464 ofSEQ ID NO:1 or comprises a sequence of amino acids that has at least 85% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 ofSEQ ID NO:1, and retains hyaluronidase activity.

[0026] In some embodiments, the soluble hyaluronidase comprises a sequence of amino acids that has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO:1 and retains hyaluronidase activity.

[0027] In some embodiments, (a) the soluble hyaluronidase has a sequence of amino acids set forth as residues 36-465, 36-466, 36-467, 36-468, 36-469, 35-470, 36-471, 36-472, 36-474, 36-475, 36-476, 35-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483 35-484, 36-485, 36-486, 36-487, 36-488, 36-489, 36-490, 35-491, 36-492, 36-493, 36-494, 36-495, 36-496, 36-497, 35-498, 36-499, and 36-500 of SEQ ID NO:1, or an N-terminally truncated variant thereof lacking residues 36, 36-37, 36-38, 36-39, or 36-40; or (b) the soluble hyaluronidase comprises a variant soluble hyaluronidase that has at least 91% sequence identity to the soluble hyaluronidase of (a).

[0028] In some embodiments, the soluble hyaluronidase is a soluble human hyaluronidase.

[0029] In some embodiments, the hyaluronidase is administered in an amount effective to increase bioavailability of the nucleic acid.

[0030] In some embodiments, the soluble hyaluronidase and composition are administered via subcutaneous (SC), intradermal (ID), or intramuscular (IM) administration. In some embodiments, the soluble hyaluronidase and composition are administered via SC administration. In some embodiments, the soluble hyaluronidase and composition are administered via ID administration. In some embodiments, the soluble hyaluronidase and composition are administered via IM administration.

[0031] In some embodiments, the soluble hyaluronidase and the nucleic acid are both encapsulated in the lipid delivery vehicle.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the effect on Factor VII (FVII) knockdown when hyaluronidase was included in lipid nanoparticle administrations having the indicated dose of FVII siRNA. Lipid nanoparticles containing the indicated amount of FVII siRNA were administered intravenously (IV) and intradermally (ID) in 6–8 weeks old female Balb / C mice. For the ID group, lipid nanoparticles were co-administered with or without 100 U of hyaluronidase enzyme.Measurement of FVII protein in plasma was determined using the colorimetric Biophen VIIassay kit. Data is shown as the relative ratio to control (PBS). The hyaluronidase was Millipore Sigma’s hyaluronidase from bovine testes, CAS Number: 37326-33-3 (“Vendor 1”).

[0033] FIG.2 shows hEPO expression after co-administration of hyaluronidase and EPO mRNA formulations. Lipid nanoparticles containing the indicated amount of hEPO siRNA were administered intravenously (IV) and intradermally (ID) in 6–8 weeks old female Balb / C mice.For the ID group, lipid nanoparticle was co-administered with or without 100 U of hyaluronidase enzyme. Measurement of hEPO protein in the serum was determined using the hEPO Elisa kit. The hyaluronidases used were Creative Biomart’s recombinant human hyaluronidase, catalog # THP-0123 (“Vendor 2”); Creative Enzymes’s Native Bovine Hyaluronidase, catalog # NATE-0347 (“Vendor 3”); Worthington Biochemical’s Hyaluronidase from Bovine Testes, CAS Number: 37326-33-3 (“Vendor 4”); and Halozyme’s recombinant human hyaluronidase PH20 enzyme, rHuPH20 (“Vendor 5”).

[0034] FIG.3 shows comparison of frozen lipid nanoparticle vs lipid nanoparticle colyophilized with hyaluronidase. Experiment was performed as described for FIG. 2, except lipid nanoparticle was either frozen or co-lyophilized with hyaluronidase prior to thawing / reconstitution for treatment. The hyaluronidase used in this example was from Vendor 1.

[0035] FIG. 4 is a SDS-PAGE gel of different sources of hyaluronidases to confirm the purity of the enzymes. Gel was stained with Coomassie R-250. The results showed that the hyaluronidase from Vendors 2, 3, and 5 contained less impurities than the hyaluronidase from Vendors 1 and 4.DETAILED DESCRIPTION

[0036] In an aspect, provided herein is a method of delivering a therapeutically effective amount of a nucleic acid encapsulated in a lipid delivery vehicle comprising subcutaneously coadministering to a mammal (i) a composition comprising the nucleic acid encapsulated in the lipid delivery vehicle and (ii) hyaluronidase, wherein the hyaluronidase is a pure hyaluronidase as determined by the presence of a single band located within the range of about 50 kDa to about 80 kDa.

[0037] In an embodiment, the purity of hyaluronidase is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.8%.

[0038] In an embodiment, the nucleic acid is selected from an siRNA (small interfering RNA), an mRNA (messenger RNA), a self-replicating RNA, a DNA plasmid, an antisense oligonucleotide, a single strand guide RNA, and combinations thereof.

[0039] In an embodiment, the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein. In an embodiment, wherein the protein is an enzyme, and antibody, an antigen, a receptor, or a transporter.

[0040] In an embodiment, the protein is a gene-editing enzyme. In embodiments, the geneediting enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

[0041] In an embodiment, the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a viral protein that when expressed elicits an immunological response. In an embodiment, the immunological response comprises production of antibodies against the viral protein.

[0042] In an embodiment, the lipid delivery vehicle is selected from a lipid nanoparticle (LNP), a liposome, and a lipoplex. In an embodiment, the lipid delivery vehicle is a lipid nanoparticle.

[0043] In an embodiment, the lipid nanoparticle has an average particle size of less than about 100 nm. In an embodiment, the lipid nanoparticle has an average particles size of about 55 nm to about 85 nm.

[0044] In an embodiment, the lipid nanoparticle further comprises a helper lipid. In embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In an embodiment, the helper lipid is distearoylphosphatidylcholine (DSPC).

[0045] In an embodiment, the lipid nanoparticle further comprises cholesterol.

[0046] In an embodiment, the lipid nanoparticle further comprises a polyethylene glycol (PEG)-lipid conjugate. In an embodiment, PEG-lipid conjugate is PEG-DMG. In a further embodiment, the PEG-DMG is PEG2000-DMG.

[0047] In an embodiment, the lipid nanoparticle comprises about 45 mol% to 65 mol% of a cationic lipid or an ionizable lipid, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

[0048] In an embodiment, the lipid nanoparticle comprises about 50 mol% to about 61 mol% of the cationic lipid or ionizable lipid, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate.

[0049] In an embodiment, the lipid nanoparticle comprises about 56 mol% to about 58 mol% of the cationic lipid or ionizable lipid, about 6 mol% to about 8 mol% of DSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

[0050] In an embodiment, the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1.

[0051] In an embodiment, the composition further comprises one or more cryoprotectants. In an embodiment, the one or more cryoprotectants is selected from sucrose, glycerol, and a combination of sucrose and glycerol.

[0052] In an embodiment, the composition is reconstituted from a lyophilized composition.

[0053] In an embodiment, the gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0054] In an embodiment, the pure hyaluronidase is free from impurities selected from denatured hyaluronidase, degraded hyaluronidase, hyaluronidase fragment, extraneous protein, nucleic acid, and a combination thereof.

[0055] In an embodiment, the co-administration results in a systemic delivery of the nucleic acid.DEFINITIONS

[0056] Before the present disclosure is further described, it is to be understood that this disclosure is not strictly limited to particular embodiments described, as such may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the claims.

[0057] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It should further be understood that as used herein, the term “a” entity or “an” entity refers to one or more of that entity. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly the terms “comprising”, “including” and “having” can be used interchangeably.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0059] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by thepresent disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0060] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0061] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0062] The term “anionic lipid” means a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0063] The term “cationic lipid” means amphiphilic lipids and salts thereof having a positive, hydrophilic head group; one, two, three, or more hydrophobic fatty acid or fatty alkyl chains; and a connector between these two domains. An ionizable or protonatable cationic lipid is typically protonated (z.e., positively charged) at a pH below its pKa and is substantially neutral at a pH above the pKa. Preferred ionizable cationic lipids are those having a pKa that is less than physiological pH, which is typically about 7.4. The cationic lipids of the disclosure may also be termed titratable cationic lipids. The cationic lipids can be an “amino lipid” having a protonatable tertiary amine (e.g., pH-titratable) head group. Some exemplary amino lipid can include Cis alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and alkyl chains. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, y-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3 -DM A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3 -DMA (also known as MC3) and (DLin-MP-DMA)(also known as 1-B1 1).

[0064] The term “complementary nucleotide bases” means a pair of nucleotide bases that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or with uracil (U) in RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acid that hybridize (i.e. join by hydrogen bonding) with each other. By “complementary” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence either by traditional Watson-Crick or by other non-traditional modes of binding.

[0065] The term “cryoprotectant” refers to a substance that prevents degradation of physical characteristics upon freezing. Cryoprotectants are added before lyophilization (e.g., the freezing cycle). Generally, a cryoprotectant may increase the viscosity and the volume of the unfrozen phase, reducing the likelihood of particle interaction during freezing. Cryoprotectants reduce or eliminate agglomeration of solute or suspended materials.

[0066] The term “CRISPR” refers to clustered regularly interspaced short palindromic repeats”). The technology leverages these repeats to selectively modify the DNA of living organisms. CRISPR-Cas is a gene-editing technique that combines CRISPR with a Cas protein, such as Cas-9.

[0067] The term “fully encapsulated” means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free RNA. When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particle is degraded. “Fully encapsulated” also means that the nucleic acid-lipid particles do not rapidly decompose into their component parts upon in vivo administration.

[0068] The term “nucleic acid” means deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0069] The term “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0070] The term “delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide to targeted cells.

[0071] The term “engineered” refers to a molecule designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

[0072] The term “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0073] The term “guide RNA,” “signal guide RNA” or “sgRNA” comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, nonlimiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at worldwideweb.novocraft.com), ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA). In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0074] The term “hydrophobic lipids” means compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkyl glycerol, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane, and 1,2-dialkyl -3 -aminopropane.

[0075] The term “lipid” means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0076] The term “lipid delivery vehicle” means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). The lipid delivery vehicle can be a nucleic acid-lipid particle, which can be formed from a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0077] The term “lipid encapsulated” means a lipid particle that provides a therapeutic nucleic acid such as an mRNA with full encapsulation, partial encapsulation, or both. In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated in the lipid particle.

[0078] The term “lipid conjugate” means a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as, e.g., PEG coupled to dialkyl oxypropyls (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, and mixtures thereof. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In certain embodiments, non-ester-containing linker moieties, such as amides or carbamates, are used.

[0079] The term “amphipathic lipid” or “amphiphilic lipid” means the material in which the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0080] The term “mammal” means a human or other mammal or means a human being.

[0081] The term “meganuclease” refers to endodeoxyribonucleases that function as molecular DNA “scissors.”

[0082] The term “messenger RNA” (mRNA) refers to any polynucleotide which encodes a protein or polypeptide and which is capable of being translated to produce the encoded protein or polypeptide in vitro, in vivo, in situ or ex vivo.

[0083] The term “modified” refers to a changed state or structure of a molecule of the disclosure. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the mRNA molecules of the present disclosure are modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Noncanonical nucleotides such as the cap structures are not considered “modified” although they may differ from the chemical structure of the A, C, G, U ribonucleotides.

[0084] The term “naturally occurring” means existing in nature without artificial aid.

[0085] The term “nucleotide” means natural bases (standard) and modified bases well known in the art. Such bases are generally located at the T position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar, and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, nonstandard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No. WO 92 / 07065; Usman, et al., International PCT Publication No. WO 93 / 15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include: inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5 -alkyl cytidines (e.g., 5 -methylcytidine), 5 -alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5 -bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). By “modified bases” in this aspect is meantnucleotide bases other than adenine, guanine, cytosine, thymine and uracil at 1 ' position or their equivalents.

[0086] The term “open reading frame” or “ORF” to a nucleic acid sequence (DNA or RNA) which is capable of encoding a polypeptide. ORFs often begin with the start codon ATG, and end with a nonsense or termination codon or signal.

[0087] The term “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

[0088] The term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0089] The term “RNA” means a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an interfering RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. As used herein, the terms “ribonucleic acid” and “RNA” refer to a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single stranded RNA, microRNA, mRNA, noncoding RNA, and multivalent RNA.

[0090] The term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (c.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

[0091] The terms “significant” or “significantly” are used synonymously with the term “substantially.”

[0092] The phrase “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0093] The term “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

[0094] The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0095] The term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0096] The term “monomer” refers to a single unit, e.g., a single nucleic acid, which may be joined with another molecule of the same or different type to form an oligomer. In some embodiments, a monomer may be an unlocked nucleic acid, i.e., a UNA monomer.

[0097] The term “neutral lipid” means a lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example,diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0098] The term “non-cationic lipid” means an amphipathic lipid or a neutral lipid or anionic lipid and is described herein.

[0099] The term “oligomer” may be used interchangeably with “polynucleotide” and refers to a molecule comprising at least two monomers and includes oligonucleotides such as DNAs and RNAs. In the case of oligomers containing RNA monomers and / or unlocked nucleic acid (UNA) monomers, the oligomers of the present disclosure may contain sequences in addition to the coding sequence (CDS). These additional sequences may be untranslated sequences, i.e., sequences which are not converted to protein by a host cell. These untranslated sequences can include a 5* cap, a 5' untranslated region (5* UTR), a 3' untranslated region (3' UTR), and a tail region, e.g., a poly-A tail region. As described in further detail herein, any of these untranslated sequences may contain one or more UNA monomers - these UNA monomers are not capable of being translated by a host cell's machinery. In the context of the present disclosure, a “mRNA sequence”, “translatable polynucleotide”, or “translatable compound” refers to a sequence that comprises a region, e.g., the coding region of an RNA, that is capable of being converted to a protein or a fragment thereof.

[0100] The term “plasmid” is a type of vector that is capable of transporting a nucleic acid. Generally, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0101] The term “subcutaneous administration” refers to administration of a medicament beneath the skin or infusion. Subcutaneous administration may be used interchangeably with “subdermal administration.” The medicament is administered into the subcutis, the layer of skin directly below the dermis and epidermis. A subcutaneous injection refers to an injection administered in the fatty tissue under the dermis and epidermis. This type of administration is advantageous for medications that cannot be administered by mouth or where the medication cannot be absorbed by the gastrointestinal tract. A subcutaneous injection is absorbed slower than a substance injected intravenously or into a muscle, but faster than a medication administered by mouth.

[0102] The term “TALEN” refers to transcription activator-like effector nucleases that are restriction enzymes engineered to cut specific sequences of DNA.

[0103] The term “translatable” may be used interchangeably with the term “expressible” and refers to the ability of polynucleotide, or a portion thereof, to be converted to a polypeptide by a host cell. As is understood in the art, translation is the process in which ribosomes in a cell's cytoplasm create polypeptides. In translation, messenger RNA (mRNA) is decoded by tRNAs in a ribosome complex to produce a specific amino acid chain, or polypeptide. Furthermore, the term “translatable” when used in this specification in reference to an oligomer, means that at least a portion of the oligomer, e.g. the coding region of an oligomer sequence (also known as the coding sequence or CDS), is capable of being converted to a protein or a fragment thereof.

[0104] The term “translation efficiency” refers to a measure of the production of a protein or polypeptide by translation of an mRNA sequence in vitro or in vivo. This disclosure provides a range of mRNA sequence molecules, which can contain one or more UNA monomers, and anumber of nucleic acid monomers, wherein the mRNA sequence can be expressible to provide a polypeptide or protein.

[0105] The term “therapeutically effective outcome,” as used herein, refers to an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0106] As used herein, a “hyaluronan degrading enzyme” refers to an enzyme that catalyzes the cleavage of a hyaluronan polymer (also referred to as hyaluronic acid or HA) into smaller molecular weight fragments. Exemplary of hyaluronan degrading enzymes are hyaluronidases, and some chondroitinases and lyases that have the ability to depolymerize hyaluronan. Reference to hyaluronan degrading enzymes includes precursor hyaluronan degrading enzyme polypeptides and mature hyaluronan degrading enzyme polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have activity, and includes allelic variants and species variants, variants encoded by splice variants, and other variants that retain activity.

[0107] As used herein, hyaluronidase refers to a class of hyaluronan degrading enzymes.Hyaluronidases include bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases include human hyaluronidases as described herein, and any of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Also included amongst hyaluronidases are soluble hyaluronidases, including, ovine and bovine PH20, soluble human PH20 and soluble rHuPH20. Examples of commercially available bovine and ovine soluble hyaluronidases include those sold under the following trademarks VITRASE® (ovine hyaluronidase), AMPHADASE® (bovine hyaluronidase) and HYDASE® (bovine hyaluronidase).

[0108] As used herein, hyaluronidase activity refers to the ability to enzymatically catalyze the cleavage of hyaluronic acid. The United States pharmacopeia (USP) xxii assay for hyaluronidase determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronic acid, or hyaluronan, (ha) substrate remaining after the enzyme is allowed toreact with the hyaluronan for 30 min at 37 °C (United States Pharmacopeia Convention, Inc, Rockville, Md). A reference standard solution can be used in an assay to ascertain the relative activity, in units, of any hyaluronidase. In vitro assays to determine the hyaluronidase activity of hyaluronidases, such as PH20, including modified PH20 polypeptides, are known in the art and described herein. Exemplary assays include the microturbidity assay, which measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin. Reference standards can be used, for example, to generate a standard curve to determine the activity in units of the hyaluronidase being tested.

[0109] As used herein, specific activity refers to units of activity per mg protein. The milligrams of hyaluronidase is defined by the absorption of a solution of at 280 nm assuming a molar extinction coefficient of approximately 1.7, in units of m-1 cm-1.

[0110] As used herein, neutral active refers to the ability of a PH20 polypeptide to enzymatically catalyze the cleavage of hyaluronic acid at neutral pH, such as at a pH between or about between pH 6.0 to pH 7.8.

[0111] As used herein, “PH20” refers to a type of hyaluronidase that occurs in sperm and is neutral-active, as well as active at lower pH. PH20 occurs on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH20 includes those of any origin including, but not limited to, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine, ovine, guinea pig, rabbit and rat origin. In some embodiments, the variants or homologs of PH20 have at least 90%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PH20 protein. In some embodiments, the PH20 protein is substantially identical to the protein identified by UniProt No. P38567 (SEQ ID No:1) or a variant or homolog having substantial identity thereto.

[0112] As used herein, a truncated PH20 hyaluronidase is any C-terminal shortened form of PH20 (SEQ ID No:1) thereof, particularly forms that are truncated and neutral active when N-glycosylated and soluble.

[0113] As used herein, a “soluble PH20” refers to any form of PH20 that is soluble under physiologic conditions. A soluble PH20 can be identified, for example, by its partitioning into the aqueous phase of a Triton® X-l 14 solution at 37 °C (Bordier et al., (1981) J. Biol. Chem., 256:1604-7). Membrane-anchored PH20, such as lipid-anchored PH20, including GPI-anchored PH20, will partition into the detergent-rich phase, but will partition into the detergent-poor or aqueous phase following treatment with Phospholipase-C. Included among soluble PH20 are membrane-anchored PH20 in which one or more regions associated with anchoring of the PH20 to the membrane has been removed or modified, where the soluble form retains hyaluronidase activity. Soluble PH20 also includes recombinant soluble PH20 and those contained in or purified from natural sources, such as, for example, testes extracts from sheep or cows.Exemplary of such soluble PH20 is soluble human PH20, which include C-terminally truncated human PH20 (truncated at residue 465, 466, 467, 468,... 500, with reference to SEQ ID NO:1, which sets forth full-length PH20). The soluble forms do not include the signal sequence (residues 1-35, SEQ ID No:2), and can start at residue 36, 37, 38, 39, 40, 41, and 42 and terminate at the C-terminus as noted above (with reference to SEQ ID NO: 1). Soluble human PH20 polypeptides, however, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. Reference to secretion by CHO cells is definitional. Hence, if a polypeptide could be expressed and secreted by CHO cells and is soluble, i.e. partitions into the aqueous phase when extracted with Triton® X-l 14, it is a soluble PH20 polypeptide whether or not it is so-produced. The precursor polypeptides for sHuPH20 polypeptides can include a signal sequence, such as a heterologous or non-heterologous (i.e. native) signal sequence. In some embodiments, the soluble PH20 can have a sequence starting at any residue 36 to 42 as set forth in SEQ ID NO:1 and ending in any residue 465 to 500 as set forth in SED ID No:l. For example, the soluble PH20 can have a sequence starting at residue 36 as set forth in SEQ ID NO: 1 and ending in any residue 468 to 484 as set forth in SEQ ID NO:1. For example, the soluble PH20 can have a sequence starting at residue 36 as set forth in SEQ ID NO:1 and ending in any residue 478 to 484 as set forth in SEQ ID NO:1. For example, soluble PH20 polypeptides can have amino acids sequence corresponding to residues 36-469 (SEQ ID NO: 5), 36-470 (SEQ ID NO: 6), 36-471 (SEQ ID NO: 7), 36-478 (SEQ ID NO: 14), 36-479 (SEQ ID NO: 15), 36-480 (SEQ ID NO: 16), 36-481 (SEQ ID NO: 17), 36-482 (SEQ ID NO: 18), 36-483 (SEQ ID NO: 19), or 36-484 (SEQID NO:20). In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 5. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO:6. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 7. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 14. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 15. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 16. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO:17. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 18. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO: 19. In some embodiments, soluble PH20 polypeptides can have amino acids sequence corresponding to SEQ ID NO:20.

[0114] As used herein, an “extended soluble PH20” or “esPH20” includes soluble PH20 polypeptides that contain residues up to the GPI anchor-attachment signal sequence and one or more contiguous residues from the GPI-anchor attachment signal sequence such that the esPH20 is soluble under physiological conditions. Exemplary human esPH20 soluble polypeptides are those that have amino acids residues corresponding to amino acids 36-491 (SEQ ID NO:23), 36-492 (SEQ ID NO:24), 36-493 (SEQ ID NO:25), 36-494 (SEQ ID NO:26), 36-495 (SEQ ID NO:27), 36-496 (SEQ ID NO:28), 36-497 (SEQ ID NO:29), 36 to 498 (SEQ ID NO:30), 36 to 499 (SEQ ID NO: 31), and 36 to 500 (SEQ ID NO:32) of SEQ ID NO: 1.

[0115] Also included are allelic variants and other variants, such as any with 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the corresponding reference polypeptide and that retain neutral activity and are soluble. Reference to sequence identity refers to variants with amino acid substitutions, insertions, and / or deletions. In some embodiments, the esPH20 has an amino acid corresponding to SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In some embodiments, the esPH20 has an amino acid corresponding to SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:23. In some embodiments, the esPH20 includesan amino acid sequence of SEQ ID NO:24. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:25 In some embodiments, the csPH20 includes an amino acid sequence of SEQ ID NO:26. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:27. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:28. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:29. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:30. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:31. In some embodiments, the esPH20 includes an amino acid sequence of SEQ ID NO:32.

[0116] As used herein, “soluble recombinant human PH20” refers to a composition containing a soluble form of human PH20 as recombinantly expressed and secreted in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. Soluble recombinant human PH20 can be encoded by nucleic acid molecule that includes the signal sequence (SEQ ID NO:2) and that includes the sequence set forth in SEQ ID NO: 18 (corresponding to residues 36-482 of SEQ ID NO:1). The signal sequence (residues 1 to 35, SEQ ID NO:2) is cleaved upon post-translational processing. The nucleic acid encoding soluble recombinant human PH20 is expressed in CHO cells which secrete the mature polypeptide. As produced in the culture medium, there is heterogeneity at the C-terminus so that the product includes a mixture of species that can include any one or more of polypeptide that terminate in residue 478, 479, 480, 481 and 482 of SEQ ID NO:1 in various abundance. Accordingly, in some embodiments, the term “soluble recombinant human PH20” also includes variants that have at least 90%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity across the whole sequence of SEQ ID NO: 18. For example, the soluble recombinant human PH20 can include one or more polypeptides of SEQ ID NO: 14, 15, 16, 17, and 18, and variants thereof.

[0117] Similarly, for other forms of PH20, such as the extended soluble PH20s (esPH20s), recombinantly expressed polypeptides and compositions thereof can include a plurality of species whose C-terminus exhibits heterogeneity. For example, compositions of recombinantly expressed esPH20 produced by expression of a nucleic acid that encodes an esPH20 that has amino acids 36-497 (SEQ ID No:29), with reference to SEQ ID NO:1, can include forms with fewer amino acids, such as 36-496 (SEQ ID NO:28) or 36-495 (SEQ ID NO:27).

[0118] The term “recombinant Human PH20” or “rHuPH20” refers to the composition produced upon expression in a cell, such as CHO cell, of nucleic acid encoding at least residues 36-482 of SEQ ID NO:1 (SEQ ID NO:18), generally linked to the native or a heterologous signal sequence (residues 1-35 of SEQ ID NO: 1). For example, rHuPH20 can be produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (SEQ ID NO:33) or 36-483 (SEQ ID NO: 19) linked to a heterologous signal sequence in a mammalian cell.Translational processing removes the 35 amino acid signal sequence. As produced in the culture medium, there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of the polypeptides 36-480 (SEQ ID NO: 16), 36-481 (SEQ ID NO: 17), and 36-482 (SEQ ID NO: 18) of SEQ ID NO:1, and some shorter polypeptides, in various abundance. Typically, rHuPH20 is produced in cells, such as CHO cells, for example DG44 CHO cells that facilitate correct N-glycosylation to retain activity. In some embodiments, rHuPH20 includes the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19. In some embodiments, rHuPH20 includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, rHuPH20 includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, rHuPH20 includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, rHuPH20 includes the amino acid sequence of SEQ ID NO: 18.

[0119] In embodiments, the truncated PH20 hyaluronidase is a soluble PH20, an extended soluble PH20, a soluble recombinant human PH20, or a rHuPH20. In embodiments, the truncated PH20 hyaluronidase is a soluble PH20. In embodiments, the truncated PH20 hyaluronidase is an extended soluble PH20. In embodiments, the truncated PH20 hyaluronidase is a soluble recombinant human PH20. In embodiments, the truncated PH20 hyaluronidase is a rHuPH20. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:3. In embodiments, the truncated PH20 hyaluronidase includes the amino acidsequence of SEQ ID NO:4. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:5. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:6. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:7. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:8. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:9. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 10. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 12. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 13. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 14. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 15. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 16. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 17. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 18. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 19. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:20. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:21. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:22. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:23. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:24. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:25. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:26. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:27. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:28. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO: 29. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:30. In embodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:31. Inembodiments, the truncated PH20 hyaluronidase includes the amino acid sequence of SEQ ID NO:32.

[0120] As used herein, “purified bovine testicular hyaluronidase” refers to a bovine hyaluronidase purified from bovine testicular extracts (see U. S. Patent Nos. 2,488,564, 2,488,565, 2,806,815, 2,808,362, 2,676,139, 2,795,529, 5,747,027 and 5,827,721). Examples of commercially available purified bovine testicular hyaluronidases include those sold as Amphadase®and Hydase™, and bovine hyaluronidases, including, but not limited to, those available from Sigma Aldrich, Abnova, EMD Chemicals, GenWay Biotech, Inc., Raybiotech, Inc., and Calzyme. Also included are recombinantly produced bovine hyaluronidases.

[0121] As used herein, “purified ovine testicular hyaluronidase” refers to an ovine hyaluronidase purified from ovine testicular extracts (see U. S. Patent Nos. 2,488,564, 2,488,565 and 2,806,815 and International PCT Publication No. W02005 / 118799). Examples of commercially available purified ovine testicular extract include VITRASE®, and ovine hyaluronidases, including, but not limited to, those available from Sigma Aldrich, Cell Sciences, EMD Chemicals, GenWay Biotech, Inc., Mybiosource.com and Raybiotech, Inc. Also included are recombinantly produced ovine hyaluronidases.

[0122] As used herein, an “N-linked moiety” refers to an asparagine (N) amino acid residue of a polypeptide that is capable of being glycosylated by post-translational modification of a polypeptide. Exemplary N-linked moieties of human PH20 include amino acids N82, N166, N235, N254, N368 and N393 of human PH20 set forth in SEQ ID NO:1.

[0123] As used herein, an “N-glycosylated polypeptide” refers to a PH20 polypeptide or truncated form thereto containing oligosaccharide linkage of at least three N-linked amino acid residues, for example, N-linked moieties corresponding to amino acid residues N235, N368 and N393 of SEQ ID NO: 1. An N-glycosylated polypeptide can include a polypeptide where three, four, five and up to all of the N-linked moieties are linked to an oligosaccharide. The N-linked oligosaccharides can include oligo-mannose, complex, hybrid or sulfated oligosaccharides, or other oligosaccharides and monosaccharides.Detailed DescriptionLipid-Based Formulations

[0124] Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. Indeed, naked nucleic acid materials cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocyte uptake and their ability in activating the immune response, all features that preclude their clinical development. When exogenous nucleic acid material (e.g., mRNA) enters the human biological system, it is recognized by the reticuloendothelial system (RES) as foreign pathogens and cleared from blood circulation before having the chance to encounter target cells within or outside the vascular system. It has been reported that the half-life of naked nucleic acid in the blood stream is around several minutes (KawabataK, TakakuraY, Hashida MPharm Res. 1995 Jun; 12(6):825-30). Chemical modification and a proper delivery method can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increase stability and efficacy of nucleic acid-based therapies. In addition, RNAs or DNAs are anionic hydrophilic polymers that are not favorable for uptake by cells, which are also anionic at the surface. The success of nucleic acidbased therapies thus depends largely on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0125] Moreover, upon internalization into a target cell, nucleic acid delivery vectors are challenged by intracellular barriers, including endosome entrapment, lysosomal degradation, nucleic acid unpacking from vectors, translocation across the nuclear membrane (for DNA), and release at the cytoplasm (for RNA). Successful nucleic acid-based therapy thus depends upon the ability of the vector to deliver the nucleic acids to the target sites inside of the cells to obtain sufficient levels of a desired activity such as expression of a gene.

[0126] While several gene therapies have been able to successfully utilize a viral delivery vector (e.g., AAV), lipid-based formulations have been increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and their ease of large-scale production. One of the most significant advances in lipid-based nucleic acid therapies happened in August 2018 when Patisiran (ALN-TTR02) was the first siRNA therapeutic approved by both the Food and Drug Administration (FDA) and theEuropean Commission (EC). ALN-TTR02 is an siRNA formulation based upon the so-called Stable Nucleic Acid Lipid Particle (SNALP) transfecting technology. Despite the success of Patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still undergoing development.

[0127] Some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics include, according to various embodiments, polymer based carriers, such as polyethyleneimine (PEI), lipidoid-containing formulations, lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, micelles, and emulsions.

[0128] These lipid formulations vary in their structure and composition, and as can be expected in a rapidly evolving field, several different terms have been used in the art to describe a single type of delivery vehicle. At the same time, the terms for lipid formulations have frequently been conflated throughout the scientific literature, and this inconsistent use has caused confusion as to the exact meaning of several terms for lipid formulations. Among the several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail and defined herein for the purposes of the present disclosure.Liposomes

[0129] Conventional liposomes are vesicles that consist of at least one bilayer and an internal aqueous compartment. 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.). They generally present as spherical vesicles and can range in size from 20 nm to several microns. Liposomal formulations can be prepared as a colloidal dispersion or they can be lyophilized to reduce stability risks and to improve the shelf-life for liposome-based drugs. Methods of preparing liposomal compositions are known in the art and are within the skill of an ordinary artisan.

[0130] Liposomes that have only one bilayer are referred to as being unilamellar, and those having more than one bilayer are referred to as multilamellar. The most common types ofliposomes are small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), and multilamellar vesicles (MLV). In contrast to liposomes, lysosomes, micelles, and reversed micelles are composed of monolayers of lipids. Generally, a liposome is thought of as having a single interior compartment, however some formulations can be multivesicular liposomes (MVL), which consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers.

[0131] Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes and can be prepared from both natural and synthetic phospholipids (Int. J. Nanomedicine. 2014; 9:1833-1843). In their use as drug delivery vehicles, hydrophilic solutes dissolved in the liposomal core cannot readily pass through the bilayer’s hydrophobic membrane, and hydrophobic compounds will associate with the bilayer. Thus, a liposome can be loaded with hydrophobic and / or hydrophilic molecules. When a liposome is used to carry a nucleic acid such as RNA, the nucleic acid is contained within the liposomal compartment in an aqueous phase.Cationic Liposomes

[0132] Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. In addition to the general characteristics profiled above for liposomes, the positively charged moieties of cationic lipids used in cationic liposomes provide several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and thus will direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety will associate with aqueous media and more importantly with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being researched for use in gene therapy due to their favorability towards negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed hereinbelow.Lipid Nanoparticles

[0133] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While the scientific literature varies on what size qualifies a lipid particle as being nanoparticulate, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0134] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid’s negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0135] In embodiments, lipids of the present application are compounds of Formula Iwherein:Riand R2 are the same or different, each a linear or branched alkyl, alkenyl, or alkynyl,Li and L2 are the same or different, each a linear alkyl having at least five carbon atoms, or form a heterocycle with the N,Xi is a bond, or is -C(0)-0 where L2-C(O)-O-R2 is formedX2 is S or O,L3 is a bond or a lower alkyl,R3 is a lower alkyl,R4 and R5 are the same or different, each a lower alkyl.

[0136] In embodiments, lipids of the present application are compounds of Formula (II) ouRB O-L6 R4H L3RR5O — L7 L^\-L5-0 0RL,° —L9 R3T (inwherein:R1and R2are each independently H or Ci -6 alkyl; orR1and R2are joined to form a saturated heterocyclic ring, wherein:R1is a linear C1-4 alkylene; andR2is -(CH2)m(X)n-, whereinX is O, S, or NR9, wherein R9is H or Ci-6 alkyl;m is 1, 2, 3 or 4, andn is 0 or 1;LI is a linear C1-6 alkylene optionally substituted with one to three methyl groups; Y is selected from the group consisting of:° o °, ^'N*XX>-§, •sS'N*'^XN— §,™ ’ wWRWO Ohjjhn•^'c* — O^ J5“d ^'C* — ’*wwherein:each asterisk (*) indicates the atom attached to L2 and L3; and R10is H or Ci-6 alkyl;L2 and L3 are each independently a linear Ci-8 alkylene;L4, L5, L6, L7, L8 and L9 are each independently absent or -CH2-, provided that:at least two of L4, L6 and L8 are -CH2-; andat least two of L5, L7 and L9 are -CH2-;R3and R4are each independently H, methyl or ethyl; andR5, R6, R7and R8are each independently selected from the group consisting of:linear C1-20 alkyl, wherein each said linear C1-20 alkyl is optionally substituted with one or more substituents selected from the group consisting of:C1-6 alkyl, C1-6 alkoxy and -F, wherein each said C1-6 alkyl substituent is optionally substituted with one or more groups selected from the group consisting of C1-3 alkoxy and -F;C3-8 monocycloalkyl, wherein each said C3-8 monocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; C7- 12 bicycloalkyl, wherein each said C7-12 bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; andCe-ioaryl, wherein each said Ce-io aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of Ci-6 alkyl, Ci -6 alkoxy and -F;C3-8 monocycloalkyl, wherein each said C3-8 monocycloalkyl is optionally substituted with one or more substituents selected from the group consisting of Ci -6 alkyl, Ci -6 alkoxy and -F;C7-12 bicycloalkyl, wherein each said C7-12 bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F; andCe-io aryl, wherein each said Ce-io aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of C1-6 alkyl, C1-6 alkoxy and -F.

[0137] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation.Lipid-mRNA Formulations

[0138] An mRNA as disclosed herein or a pharmaceutically acceptable salt thereof can be incorporated into a lipid formulation (i.e., a lipid-based delivery vehicle).

[0139] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing an mRNAof the present disclosure. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and an mRNA of the present disclosure. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or a polymer. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or a polymer. In some embodiments, the lipid formulation preferably encapsulates the nucleic acid.

[0140] The description provides lipid formulations comprising one or more therapeutic mRNA molecules encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises liposomes. In some embodiments, the lipid formulation comprises cationic liposomes. In some embodiments, the lipid formulation comprises lipid nanoparticles.

[0141] In some embodiments, the mRNA is fully encapsulated within the lipid portion of the lipid formulation such that the mRNA in the lipid formulation is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals such as humans.

[0142] The lipid formulations of the disclosure also typically have a total lipid: RNA ratio (mass / mass ratio) of from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 to about 45:1, from about 3:1 to about 40:1, from about 5:1 to about 38:1, or from about 6:1 to about 40:1, or from about 7:1 to about 35:1, or from about 8:1 to about 30:1; or from about 10:1 to about 25:1; or from about 8:1 to about 12:1; or from about 13:1 to about 17:1; or from about 18:1 to about 24:1; or from about 20:1 to about 30:1. In some embodiments, the total lipid: RNA ratio (mass / mass ratio) is from about 10:1 to about 25:1. The ratio may be any value or subvalue within the recited ranges, including endpoints.

[0143] The lipid formulations of the present disclosure typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited ranges, including endpoints. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.

[0144] In embodiments, the lipid formulations comprise an mRNA, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid formulations can also include cholesterol.

[0145] In the nucleic acid-lipid formulations, the mRNA may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In embodiments, a lipid formulation comprising an mRNA is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the mRNA in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes. In certain other instances, the mRNA in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the mRNA is complexed with the lipid portion of the formulation.

[0146] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with a nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent -mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (Io - I) / Io, where I and Io refer to the fluorescence intensities before and after the addition of detergent.

[0147] In other embodiments, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleicacid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-lipid nanoparticles.

[0148] In some embodiments, the lipid formulations comprise mRNA that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or range therein) of the particles have the mRNA encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0149] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0150] According to some embodiments, the expressible polynucleotides and mRNA constructs described herein are lipid formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In one embodiment, a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising: (a) an mRNA of the present disclosure,(b) a cationic lipid,(c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG- modified lipid),(d) optionally a non-cationic lipid (such as a neutral lipid), and(e) optionally, a sterol.

[0151] In some embodiments, the cationic lipid is an ionizable cationic lipid. In one embodiment, the lipid nanoparticle formulation consists of (i) at least one cationic lipid; (ii) a helper lipid; (iii) a sterol (e.g., cholesterol); and (iv) a PEG-lipid, in a molar ratio of about 20% to about 40% ionizable cationic lipid: about 25% to about 45% helper lipid: about 25% to about 45% sterol; about 0.5-5% PEG-lipid. Example cationic lipids (including ionizable cationic lipids), helper lipids (e.g., neutral lipids), sterols, and ligand-containing lipids (e.g., PEG-lipids) are described herein below.

[0152] The selection of specific lipids and their relative % compositions depends on several factors including the desired therapeutic effect, the intended in vivo delivery target, and the planned dosing regimen and frequency. Generally, lipids that correspond to both high potency (i.e, therapeutic effect such as knockdown activity or translation efficiency) and biodegradability resulting in rapid tissue clearance are most preferred. However, biodegradability may be less important for formulations that are intended for only one or two administrations within the subject. In addition, the lipid composition may require careful engineering so that the lipid formulation preserves its morphology during in vivo administration and its journey to the intended target, but will then be able to release the active agent upon uptake into target cells. Thus, several formulations typically need to be evaluated in order to find the best possible combination of lipids in the best possible molar ratio of lipids as well as the ratio of total lipid to active ingredient.

[0153] Suitable lipid components and methods of manufacturing lipid nanoparticles are well known in the art and are described for example in PCT / US2020 / 023442, U. S. 8,058,069, U. S.8,822,668, U. S. 9,738,593, U. S. 9,139,554, PCT / US2014 / 066242, PCT / US2015 / 030218, PCT / 2017 / 015886, and PCT / US2017 / 067756, the contents of which are incorporated by reference.Cationic Lipids

[0154] The lipid formulation preferably includes a cationic lipid suitable for forming a cationic liposome or lipid nanoparticle. Cationic lipids are widely studied for nucleic acid deliverybecause they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids arc amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a steroid portion and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at about physiological pH. Cationic liposomes have been traditionally the most commonly used non-viral delivery systems for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids, such as DOTAP, (l,2-dioleoyl-3- trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N, N, N-trimethyl- ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids by electrostatic interaction, providing high in vitro transfection efficiency.

[0155] In the presently disclosed lipid formulations, the cationic lipid may be, for example, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride and 1.2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1.2-DiLinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N, N-dimethylaminopropane (y-DLenDMA), l,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N, N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N, N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N, N- dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N, N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 l-tetraen-19-yl4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28 31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,3 l-tetraen-19-yloxy)-N, N-dimethylpropan-l-amine (MC3 Ether), 4-((6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N, N-dimethylbutan-l-amine (MC4 Ether), or any combination thereof. Other cationic lipids include, but are not limited to, N, N-distearyl-N, N-dimethylammonium bromide (DDAB), 3P-(N-(N', N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N, N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dileoyl-sn-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3 -dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), and 2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (XTC). Additionally, commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL), and Lipofectamine (comprising DOSPA and DOPE, available from GIBCO / BRL).

[0156] Other suitable cationic lipids are disclosed in International Publication Nos. WO 2021 / 030701, WO 2015 / 074085, WO 2016 / 081029, WO 2017 / 117530, WO 2018 / 118102, WO 2018 / 119163, WO 2023 / 086514, WO 2022 / 235935, WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 2023 / 133946, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493; U. S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U. S. Patent Nos. 8,158,601 and 10,227,302; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are herein incorporated by reference.

[0157] Other suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those, in which the alkyl substituents are different (e.g., N-ethyl- N-methylamino-, and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids referred to as amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids having less saturated acyl chains are more easily sized, particularly when the complexes must be sized below about 0.3 microns, for purposes of filter sterilization. Amino lipids containing unsaturated fattyacids with carbon chain lengths in the range of C14to C22may be used. Other scaffolds can also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0158] In some embodiments, amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Of course, it will be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Lipids that have more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded from use in the disclosure. In certain embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of an ionizable cationic lipid is about 6 to about 7.Helper Lipids and Sterols

[0159] The mRNA-lipid formulations of the present disclosure can comprise a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, non-cationic lipid, non-cationic helper lipid, anionic lipid, anionic helper lipid, or a zwitterionic lipid. It has been found that lipid formulations, particularly cationic liposomes and lipid nanoparticles have increased cellular uptake if helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9):882-92). For example, some studies have indicated that neutral and zwitterionic lipids such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), Di-Oleoyl-Phosphatidyl-Ethanoalamine (DOPE) and 1,2-DiStearoyl-sn-glycero-3-PhosphoCholine (DSPC), being more fusogenic (i.e., facilitating fusion) than cationic lipids, can affect the polymorphic features of lipid-nucleic acid complexes, promoting the transition from a lamellar to a hexagonal phase, and thus inducing fusion and a disruption of the cellular membrane. (Nanomedicine (Lond). 2014 Jan; 9(1): 105-20). In addition, the use of helper lipids can help to reduce any potential detrimental effects from using many prevalent cationic lipids such as toxicity and immunogenicity.

[0160] Non-limiting examples of non-cationic lipids suitable for lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine,lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0161] Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof. One study concluded that as a helper lipid, cholesterol increases the spacing of the charges of the lipid layer interfacing with the nucleic acid making the charge distribution match that of the nucleic acid more closely. (J. R. Soc. Interface. 2012 Mar 7; 9(68): 548–561). Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[0162] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.

[0163] Other examples of helper lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0164] In some embodiments, the helper lipid comprises from about 20 mol% to about 50 mol%, from about 22 mol% to about 48 mol%, from about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, from about 26 mol% to about 42 mol%, from about 27 mol% to about 41 mol%, from about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0165] In some embodiments, the total of helper lipid in the formulation comprises two or more helper lipids and the total amount of helper lipid comprises from about 20 mol% to about 50 mol%, from about 22 mol% to about 48 mol%, from about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, from about 26 mol% to about 42 mol%, from about 27 mol% to about 41 mol%, from about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipids are a combination of DSPC and DOTAP. In some embodiments, the helper lipids are a combination of DSPC and DOTMA.

[0166] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.

[0167] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.

[0168] A lipid formulation containing a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 20-40% cationic lipid compound, about 25-40 % cholesterol, about 25-50% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation. In some embodiments, the composition is about 22-30% cationic lipid compound, about 30- 40% cholesterol, about 30-40% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.Lipid Conjugates

[0169] The lipid formulations described herein may further comprise a lipid conjugate. The conjugated lipid is useful for preventing the aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic-polymer-lipid conjugates, and mixtures thereof. Furthermore, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (Front. Pharmacol. 2015 Dec 1; 6:286).

[0170] In an embodiment, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) in a lipid formulation as a coating or surface ligand, a technique referred to as PEGylation, helps protect nanoparticles from the immune system and their escape from RES uptake (Nanomedicine (Lond). 2011 Jun; 6(4):715-28). PEGylation has been widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Detergent-like PEG lipids (e.g., PEG-DSPE) can enter the lipid formulation to form a hydrated layer and steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types, brush-like and mushroom-like layers. For PEG-DSPE-stabilized formulations, PEG will take on the mushroom conformation at a low degree of PEGylation (usually less than 5 mol%) and will shift to brush conformation as the content of PEG-DSPE is increased past a certain level (J. Nanomaterials. 2011; 2011:12). It has been shown that increased PEGylation leads to a significant increase in the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15; 13():507-30; J. Control Release. 2010 Aug 3; 145(3): 178-81 ).

[0171] Suitable examples of PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG coupled tophospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.

[0172] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycolsuccinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a terminal hydroxyl group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0173] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons). In embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited ranges, including endpoints.

[0174] In certain instances, the PEG monomers can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In an embodiment, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea(-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In an embodiment, a carbamate linker is used to couple the PEG to the lipid.

[0175] In other embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, e.g., carbonate(-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0176] Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those of skill in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of C10to C20are preferred. Phosphatidylethanolamines with mono- or di -unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine (DSPE).

[0177] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) conjugate. In these embodiments, the PEG preferably has an average molecular weight of about 750 to about 2,000 daltons. In particular embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.

[0178] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0179] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 0.9 mol% to about 1.6 mol%, from about 0.9 mol% to about 1.8 mol%, from about 1 mol% to about 1.8 mol%, from about 1 mol% to about1.7 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, or from about 1.4 mol% to about 1.6 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5%, (or any fraction thereof or range therein) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0180] In some embodiments, the PEG-lipid is PEG550-PE. In some embodiments, the PEG-lipid is PEG750-PE. In some embodiments, the PEG-lipid is PEG2000-DMG. PEG2000-DMG is used or known interchangeably with DMG-PEG2000. DMG-PEG 2000 is a synthetic lipid formed by the PEGylation of myristoyl diglyceride. When DMG-PEG 2000 is commercially sourced from Avanti Lipids, DMG-PEG 2000 is a mixture of 1,2-DMG PEG2000 and 1,3-DMG PEG2000 in -97:3 ratio.

[0181] In some embodiments, the lipid conjugate comprises a peptide-lipid conjugate, or a pharmaceutically acceptable salt thereof. In some embodiments, the peptide-lipid conjugate comprises a lipid conjugated via a linking moiety to a peptide of Formula (III):Y A1-A2-A3-A4-(A5) - zyn (III),wherein,A1can be serine, threonine, O-C1-6alkyl serine, and O-C1-6alkyl threonine;A2can be from serine, threonine, O-C1-6alkyl serine, and O-C1-6alkyl threonine; A3can be glutamic acid, glutamine, asparagine, and aspartic acid;A4is proline;each A5is independently selected from a natural or modified amino acid;Y is absent or selected from A2-A3-A4-(A5)m-, A3-A4-(A5)m-, A4-(A5)m-, and (A5)m-;Z is absent or selected from -A1-A2-A3-A4, -A1-A2-A3, -A1-A2, and -A1;m is 0-5;n is 1 to 12;wherein the lipid is conjugated to the N-terminus, C-terminus, or an amino acid side chain of the peptide of Formula (III); andwherein the peptide of Formula (III) is optionally protected with a neutral group selected from an amide and a Ci-6 alkyl ester at its C-terminus when conjugated at its N-terminus or an amino acid side chain.

[0182] In some embodiments, the peptide-lipid conjugate is selected fromoooo

[0183] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ± 0.5 mol%. One of ordinary skill in the art will appreciate that the concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid formulation is to become fusogenic.Mechanism of Action for Cellular Uptake of Lipid Formulations

[0184] Lipid formulations for the intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating target cells through exploitation of the target cells’ endocytic mechanisms where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3): 146-157, 2018). Specifically, in the case of a mRNA-lipid formulation targeting hepatocytes described herein, the mRNA-lipid formulation enters hepatocytes through receptor mediated endocytosis. Prior to endocytosis, functionalized ligands such as PEG-lipid at the surface of the lipid delivery vehicle are shed from the surface, which triggers internalization into the target cell. During endocytosis, some part of the plasma membrane of the cell surrounds the vector and engulfs it into a vesicle that then pinches off from the cell membrane, enters the cytosol and ultimately undergoes the endolysosomal pathway. For ionizable cationic lipid-containing delivery vehicles, the increased acidity as the endosome ages results in a vehicle witha strong positive charge on the surface. Interactions between the delivery vehicle and the endosomal membrane then result in a membrane fusion event that leads to cytosolic delivery of the payload. For mRNA payloads, the cell’s own internal translation processes will then translate the mRNA into the encoded protein. The encoded protein can further undergo post-translational processing, including transportation to a targeted organelle or location within the cell.

[0185] By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid formulation and, in turn, the rate at which the lipid formulation becomes fusogenic. In addition, other variables including, e.g., pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid formulation becomes fusogenic. Other methods which can be used to control the rate at which the lipid formulation becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, one can control the liposomal or lipid particle size.Lipid Formulation Manufacture

[0186] There are many different methods for the preparation of lipid formulations comprising a nucleic acid. (Curr. Drug MetaboL 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, dual asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are known to one of skill in the art.Microfluidic Preparation

[0187] The micro fluidic method, unlike other bulk techniques, gives the possibility of controlling the lipid hydration process. The method can be classified in continuous-flow microfluidic and droplet-based micro fluidic, according to the way in which the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in a continuous flow mode, lipids are dissolved in isopropyl alcohol which is hydrodynamically focused in a microchannel cross junction between two aqueous buffer streams. Vesicles size can be controlled by modulating the flow rates, thus controlling the lipids solution / buffer dilution process. The method can be used for producing oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three-inlet and one-outlet ports.Dual Asymmetric Centrifugation

[0188] Dual Asymmetric Centrifugation (DAC) differs from more common centrifugation as it uses an additional rotation around its own vertical axis. An efficient homogenization is achieved due to the two overlaying movements generated: the sample is pushed outwards, as in a normal centrifuge, and then it is pushed towards the center of the vial due to the additional rotation. By mixing lipids and an NaCl-solution a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be regulated by optimizing DAC speed, lipid concentration and homogenization time.Lipid-Encapsulated RNA Nanoparticles

[0189] The lipid-encapsulated RNA nanoparticles disclosed herein comprise a nanoparticle or a bilayer of lipid molecules. In addition to the cationic lipid (e.g., an ionizable cationic lipid), the lipid-encapsulated RNA nanoparticle comprises a neutral lipid or a polymer.

[0190] In some embodiments, the RNA is fully encapsulated within the lipid portion of the lipid nanoparticle such that the RNA in the lipid-encapsulated RNA nanoparticles is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid-encapsulated RNA nanoparticles described herein are substantially non-toxic to mammals such as humans. The lipid-encapsulated RNA nanoparticles typically have a mean diameter of from 30 nni to 150 nm, from 40 nm to 150 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 70 nm to 110 nm, or from 70 to 90 nm. The lipid-encapsulated RNA nanoparticles described herein also typically have a lipid: RNA ratio (mass / mass ratio) of from 1:1 to 100:1, from 1:1 to 50:1, from 5:1 to 45:1, from 10:1 to 40:1, from 12:1 to 38:1, or from 15:1 to 45:1, or from 25:1 to 40:1, or from 30:1 to 40:1. In some embodiments, the composition has a total lipid: RNA weight ratio of between about 50:1 and 10:1. In some embodiments, the composition has a total lipid: RNA weight ratio of between about 40:1 and 20:1. In some embodiments, the composition has a total lipid: RNA weight ratio of between about 45:1 and 30:1. In some embodiments, the composition has a total lipid: RNA weight ratio of between about 38:1 and 30:1.

[0191] In embodiments, the lipid particles comprise an RNA, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid-encapsulated RNA nanoparticles can also include cholesterol. The lipid-encapsulated RNAnanoparticles may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNA that express one or more polypeptides.

[0192] In the lipid-encapsulated RNA nanoparticles the RNA may be fully encapsulated within the lipid portion of the particle, thereby protecting the RNA from nuclease degradation. In embodiments, the lipid-encapsulated RNA nanoparticles comprise an RNA that is fully encapsulated within the lipid portion of the particle, thereby protecting the RNA from nuclease degradation. In certain instances, the RNA in the lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37 °C for at least 20, 30, 45, or 60 minutes. In certain other instances, the RNA in the lipid particle is not substantially degraded after incubation of the particle in serum at 37 °C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the RNA is complexed with the cationic lipid of the lipid-encapsulated RNA nanoparticles. One of the benefits of the formulations of the present disclosure is that the lipid-encapsulated RNA nanoparticles are substantially non-toxic to mammals such as humans.

[0193] The lipid particle comprises RNA that is fully encapsulated within the lipid portion of the particles, such that from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, from 90% to 100%, from 30% to 95%, from 40% to 95%, from 50% to 95%, from 60% to 95%, from 70% to 95%, from 80% to 95%, from 85% to 95%, from 90% to 95%, from 30% to 90%, from 40% to 90%, from 50% to 90%, from 60% to 90%, from 70% to 90%, from 80% to 90%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range therein) of the particles have the RNA encapsulated therein.

[0194] Depending on the intended use of the lipid-encapsulated RNA nanoparticles, the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using assays know in the art.Dilution of the Lipid-Encapsulated RNA Nanoparticles

[0195] After mixing the organic lipid solution into the aqueous RNA solution, the extent of RNA encapsulation can be enhanced if the suspension of lipid-encapsulated RNA nanoparticles is further diluted prior to removal of free RNA. This can be done via one or more bufferdilutions, for example, via one or more Y-connectors that flow into the output line. The buffers flowing into the one or more Y-connectors do not have to be the same.

[0196] The diluted lipid-encapsulated RNA nanoparticles can then be optionally collected in a vessel maintained at 15-20 °C and allowed to incubate from a few minutes to two hours prior to a further dilution step or a concentration step.Sample Concentration

[0197] Diluted lipid-encapsulated RNA nanoparticles can be concentrated, e.g., by tangential flow filtration (TFF) using hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories, Inc., Rancho Dominguez, California), optionally via a peristaltic pump or a 4-piston-diaphragm pump or a centrifugal pump (based on principle of magnetic levitation).Methods for such concentration techniques are known in the art and would be readily apparent to a person of ordinary skill.Removal of Free RNA and Buffer Replacement

[0198] Concentration can be followed by diafiltration against 7-10 volumes of 10 mM Tris, 50 mM NaCl, 9% sucrose, pH 7.5 to remove organic solvent and unbound RNA. Preferably, the diafiltration buffer is added via a heat exchanger such that product temperature is maintained at 15-20 °C. The formulation can be further concentrated to target a total formulated RNA concentration of > 3 mg / mL.Sterile Filtration and Fill

[0199] The RNA concentration in the formulation of lipid-encapsulated RNA nanoparticles can then be measured by IPRP-HPLC (Ion Pair Reverse Phase-High Performance Liquid Chromatography) and adjusted to ~2 mg / mL (1.85 to 2.3 mg / mL) by diluting with a buffer as described hereinbelow optionally containing glycerol such that the final concentration of glycerol in the formulation is 5%. The diafiltered lipid-encapsulated RNA nanoparticles are sterile filtered through a 0.2 pm sterilizing grade filter (PES). The filtered formulation can then be aseptically filled into glass vials, stoppered, capped and placed at -20 or -70 ± 5 °C.Hyaluronidases

[0200] Hyaluronidases are a family of enzymes that degrade hyaluronan. The three main types of hyaluronidases are two classes of eukaryotic endoglycosidase hydrolases and a prokaryoticlyase-type of glycosidase. Hyaluronidases are hyaluronoglucosidases, which means they cleave the ( 1 — >4)-l inkages between N-acctylglucosaminc and glucuronatc. The term hyaluronidase may also refer to hyaluronoglucuronidases, which cleave (1— >3)-linkages. Further, bacterial hyaluronate lyases may also be referred to as hyaluronidases.

[0201] Hyaluronidases are endoglycosidases used to increase the dispersion and absorption of other co-administered drugs when administered subcutaneously (e.g., subcutaneous injections, subcutaneous infusion such as hypodermoclysis). Hyaluronidases include, but are not limited to, bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), hyaluronidases from leeches, other parasites and crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35).

[0202] Hyaluronidases include those of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Exemplary human hyaluronidases include HYAL1, HYAL2, HYAL3, HYAL4, and PH20.

[0203] Soluble hyaluronidase polypeptides, such as soluble forms of PH20, are of interest herein. Soluble PH20 hyaluronidases include, ovine and bovine PH20, and soluble forms of human PH20. Exemplary hyaluronidases are the soluble human PH20 polypeptides and variants that are soluble and active.Soluble Hyaluronidases

[0204] Provided in the co-formulations, combinations, and methods herein that contain or employ soluble hyaluronidases. Soluble hyaluronidases include any, that, upon expression, are secreted from a cell, and exist in soluble form. Such soluble hyaluronidases include, but are not limited to, bacterial soluble hyaluronidases, non-human soluble hyaluronidases, such as bovine PH20, ovine PH20, human soluble PH20, and variants thereof. Included among the soluble hyaluronidases are human PH20 polypeptides that have been been modified so that they are soluble. Human PH20 mature set forth in SEQ ID NO:34 (corresponding to residues 36-509 of SEQ ID NO:1) is not soluble. Hyaluronidases, such as human PH20, that contain a glycophophatidylinositol (GPI) anchor can be made soluble by truncation of and removal of all or a portion of the GPI anchor. Human hyaluronidase PH20, which is normally membrane anchored via a GPI anchor, is made soluble by truncation of and removal of all or a portion of the GPI anchor at the C-terminus.

[0205] Soluble PH20 hyaluronidases, such as the soluble human PH20 polypeptides, are active at neutral pH, as well as at lower pH, including pH 5.5 to 7.4, and require glycoslyation for activity. Soluble hyaluronidases include a soluble form of a PH20 from any species, such as a soluble form of a human PH20, including those that have sequences, such as residues 36-469 (SEQ ID NO:5), 36-470 (SEQ ID NO:6), 36-471 (SEQ ID NO:7), 36-472 (SEQ ID NO:8), 36-473 (SEQ ID NO:9), 36-474 (SEQ ID NO: 10), 36-475 (SEQ ID NO: 11), 36-476 (SEQ ID NO:12), 36-477 (SEQ ID NO:13), 36-478 (SEQ ID NO:14), 36-479 (SEQ ID NO:15), 36-480 (SEQ ID NO: 16), 36-481 (SEQ ID NO: 17), 36-482 (SEQ ID NO: 18), 36-483 (SEQ ID NO:19), 36-484 (SEQ ID NO:20), 36-485 (SEQ ID NO:21), 36-486 (SEQ ID NO:22) of SEQ ID NO:1, and longer soluble forms, including the extended soluble forms discussed below. Also included are a N-terminally truncated forms with an N-terminus at 37, 38, 39, 40, 41, and 42. These include the C-terminal truncated forms thereof lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase is secreted upon expression, is soluble, and retains hyaluronidase activity. Such forms also typically are mature forms that, when expressed in a cell, lack the signal peptide.

[0206] Included among soluble hyaluronidases are soluble of any of the PH20s from any species, including the human truncated forms, and precursor and mature forms there of that exhibit hyaluronidase activity, and variants thereof. Variants include polypeptides having at least 60%, 70%, 80%, 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% (when aligned for maximal identity and not including terminal gaps) or more sequence identity to any truncated forms that are soluble and neutral active. Amino acid variants include conservative and nonconservative mutations. It is understood that residues that are important or otherwise required for the activity of a hyaluronidase, such as any described above or known to skill in the art, are generally invariant and cannot be changed. These include, for example, active site residues. Thus, for example, amino acid residues 111, 113 and 176 (with reference to residues in the PH20 polypeptide set forth in SEQ ID NO:1) of a human PH20 polypeptide, and soluble form thereof, generally are invariant and are not altered. Other residues that confer glycosylation and formation of disulfide bonds required for proper folding also can be invariant.

[0207] Human PH20 normally is GPI-anchored. Human PH20, which occurs as GPI-anchored is rendered soluble by truncation at the C-terminus. Such truncation can remove all of the GPIanchor attachment signal sequence, or can remove only some of the GPI anchor attachment signal sequence. The resulting polypeptide, however, is soluble. In instances where the soluble hyaluronidase retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI-anchor attachment signal sequence can be retained, provided the polypeptide is soluble. Polypeptides containing one or more amino acids of the GPI anchor are termed extended soluble hyaluronidases. One of skill in the art can determine whether a polypeptide is GPI-anchored using methods well known in the art. Such methods include, but are not limited to, using known algorithms to predict the presence and location of the GPI-anchor attachment signal sequence and co-site, and performing solubility analyses before and after digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

[0208] Extended soluble hyaluronidases are soluble PH20 polypeptides having residues 36-495 (SEQ ID NO: 27), 36-496 (SEQ ID NO: 28), 36-497 (SEQ ID NO: 29), 36-498 (SEQ ID NO: 30), 36-499 (SEQ ID NO: 31), and 36-500 (SEQ ID NO: 32) of SEQ ID NO:1, can be produced by making C-terminal truncations to any naturally GPI-anchored hyaluronidase such that the resulting polypeptide is soluble and contains one or more amino acid residues from the GPI-anchor attachment signal sequence (see, e.g., U. S. Patent No. 8,927,249). These include hyaluronidases that are neutral active, soluble, and can contain amino acid substitutions.

[0209] Typically, for use in the compositions, and methods and uses herein, a soluble human hyaluronidase, such as a soluble human PH20, is used, such as a PH20 polypeptide that has residues 36-469 (SEQ ID NO:5), 36-470 (SEQ ID NO:6), 36-471 (SEQ ID NO:7), 36-472 (SEQ ID NO:8), 36-473 (SEQ ID NO:9), 36-474 (SEQ ID NO: 10), 36-475 (SEQ ID NO:11), 36-476 (SEQ ID NO: 12), 36-477 (SEQ ID NO: 13), 36-478 (SEQ ID NO: 14), 36-479 (SEQ ID NO: 15), 36-480 (SEQ ID NO:16), 36-481 (SEQ ID NO:17), 36-482 (SEQ ID NO:18), 36-483 (SEQ ID NO:19), 36-484 (SEQ ID NO:20), 36-485 (SEQ ID NO:21), 36-486 (SEQ ID NO:22), with reference to SEQ ID NO:1 and variants having, for examples, at least 90% or more sequence identity thereto, where the variants are soluble and neutral active. Hyaluronidases can be recombinantly produced or can be purified or partially-purified from natural sources, such as, for example, from testes extracts. Methods for production of recombinant proteins, including recombinant hyaluronidases, are well known in the art.

[0210] PH20 (also known as sperm surface protein, sperm adhesion molecule 1 or SPAM1) is a hyaluronidase that hydrolyzes hyaluronan (also called hyaluronic acid, hyaluronate or HA) found in connective tissues such as the extracellular matrix. Hyaluronan polymers are composed of repeating disaccharide units, D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), linked together via alternating 0-1— >4 and 0-1— >3 glycosidic bonds. Hyaluronan chains can reach about 25,000 disaccharide repeats or more in length, and polymers of hyaluronan can range in size from about 5,000 to 20,000,000 Da in vivo. Hyaluronan, also called hyaluronic acid or hyaluronate, is a non-sulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissues. Hyaluronan is an essential component of the extracellular matrix and a major constituent of the interstitial barrier. PH20 is an endo-0-N-acetyl-hexosaminidase that hydrolyzes the 01— >4 glycosidic bond of hyaluronic acid into various oligosaccharide lengths such as tetrasaccharides and hexasaccharides. PH20 has both hydrolytic and transglycosidase activities. In addition to degrading hyaluronic acid, PH20 also can degrade chondroitin sulfates, such as C4-S and C6-S. PH20 can exhibit hyaluronidase activity at acidic pH and neutral pH.Structure

[0211] PH20 cDNA has been cloned from numerous mammalian species, including, for example, human, bovine, rabbit, and non-human primates. The mRNA transcript is typically translated to generate a precursor protein containing a 35 amino acid signal sequence in human at the N-terminus. Following transport to the ER, the signal peptide is removed to yield a mature PH20 polypeptide to produce in human 474 amino acid mature polypeptide with an amino acid sequence set forth as residues 36-509 (SEQ ID NO:34) in SEQ ID NO:1. Allelic variants and other variants of PH20 are known. Other sequences of PH20 have been reported. For example, an allelic PH20 variant contains an Ala at position 48 and a Trp at position 499 with reference to SEQ ID NO:1. A natural variant of PH20 has been identified containing a Glutamine (Gin; Q) at position 5 compared to the precursor sequence of amino acids set forth in SEQ ID NO: 1.Another natural variant contains an Alanine (Ala; A) at position 47 of SEQ ID No: 1.

[0212] The sequence and structure of PH20 polypeptides is highly conserved among species. Sequence identity between and among PH20 proteins from various species is about 50% to about 98%. The hydrophobic N-terminal signal sequence of 35 amino acids in length is generallyconserved among PH20 hyaluronidase polypeptides. PH20 hyaluronidases contain a common core hyaluronidase domain region of about 340 amino acids in length that corresponds to amino acid residues 38-374 of the precursor human PH20 sequence set forth in SEQ ID NO:1. A mature PH20 polypeptide lacking the signal sequence and containing a contiguous sequence of amino acids having a C-terminal amino acid residue corresponding to amino acid residue 464 of SEQ ID NO:1 (e.g., amino acid residues corresponding to positions 36-464 of the amino acid sequence set forth in SEQ ID NO:1) is the minimal sequence required for hyaluronidase activity (see e.g., U. S. Patent Application No. 10 / 795,095, which is issued as U. S. Patent No. 7,767,429; see also U. S. Publication No. US20100143457).

[0213] Within the common hyaluronidase domain region, at least 57 amino acids are conserved between and among species (see e.g., Arming et al. (1997) Eur. J. Biochem., 247:810-814; ten Have et al. (1998) Reprod. Fertil. Dev., 10:165-72; Chowpongpang et al. (2004) Biotechnology Letters, 26:1247-1252). Cysteine residues corresponding to 25 and 316 and cysteine residues corresponding to 189 and 203 form disulfide bridges. The other cysteine residues also form disulfide bridges, are involved in posttranslational protein maturation and / or in activity modulation. For example, four disulfide bonds are formed between the cysteine residues. Amino acid residues corresponding to amino acid residue D111, E113 and E249 of the sequence of amino acids, with reference to the mature polypeptide not including the signal sequence are acidic residues part of the enzyme active site and are conserved between and among PH20 species amino acid residues R176, R246, R252 of the also conserved between and among species and contribute to substrate binding and / or hyaluronidase activity. Amino acid mutations Dll IN, El 13Q, R176G, E249N and R252T (with reference to mature numbering without the signal sequence) result in enzymes that have no detectable enzymatic activity or residual enzymatic activity (sec e.g., Arming et al. (1997) Eur. J. Biochem., 247:810-814).(a) Exemplary Soluble Human PH20

[0214] Exemplary of a soluble hyaluronidase is soluble human PH20. Soluble forms of recombinant human PH20 have been produced and can be used in the compositions, combinations and methods described herein. The production of such soluble forms of PH20 is described in U. S. Patent Nos. 7,767,429, 8,202,517, 8,431,380, 8,431,124, 8,450,4708,765,685, 8,772,246, 7,871,607, 7,846,431, 7,829,081, 7,829,081, 8,105,586, 8,257,699, and 8,580,252,which are incorporated by reference herein and are entitled “Soluble Hyaluronidase Glycoprotein (sHASEGP), Process for Preparing the Same, Uses and Pharmaceutical Compositions Comprising Thereof.”

[0215] Recombinant soluble forms of human PH20 have been generated and can be used in the compositions, combinations and methods provided herein. For example, with reference to SEQ ID NO:1, which sets forth the sequence of full length precursor PH20, which includes a signal sequence (residues 1-35 SEQ ID NO:2), soluble forms include, but are not limited to, C-terminal truncated polypeptides of human PH20 set forth in SEQ ID NO:1 having a C-terminal amino acid residue 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acids set forth in SEQ ID NO:1, or polypeptides that exhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, have activity at neutral pH, and are soluble (secreted into the medium when expressed in a mammalian cell). Soluble forms of human PH20 generally include those that contain amino acids 36-464 set forth in SEQ ID NO:1. For example, when expressed in mammalian cells, the 35 amino acid N-terminal signal sequence is cleaved during processing, and the mature form of the protein is secreted. Thus, the mature soluble polypeptides include those that contain amino acids 36 to 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 and 483 (SEQ ID Nos: 3 to 19 respectively) of SEQ ID NO:1. Exemplary of soluble hyaluronidases are soluble human PH20 polypeptides that are 442, 443, 444, 445, 446 or 447 amino acids in length, and variants thereof that have, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding residues (i.e., residues 36-482 of SEQ ID NO:1 is a soluble PH20 polypeptide that is 474 amino acids in length) and retains hyaluronidase activity. The generation of such soluble forms of recombinant human PH20 are described, for example, in U. S. Patent Nos. 7,767,429, 8,202,517, 8,431,380, 8,431,124, 8,450,4708,765,685, 8,772,246, 7,871,607, 7,846,431, 7,829,081, 7,829,081, 8,105,586, 8,257,699, and 8,580,252.

[0216] Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation isimportant for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).(b) rHuPH20

[0217] rHuPH20 refers to the composition produced upon expression in a cell, such as CHO cell, of nucleic acid encoding residues 36-482 of SEQ ID NO:1, generally linked to the native or a heterologous signal sequence (residues 1-35 of SEQ ID NO: 1). rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO:1) in a mammalian cell. Translational processing removes the 35 amino acid signal sequence. As produced in the culture medium there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of the polypeptides 36-480 (SEQ ID NO:16), 36-481 (SEQ ID NO:17), and 36-482 (SEQ ID NO: 18) of SEQ ID NO: 1, and some shorter polypeptides, in various abundance. Typically, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHO cells). In some instances, the most abundant species is the 446 amino acid polypeptide corresponding to residues 36-481 (SEQ ID NO: 17) of SEQ ID NO:1, in the most abundant species are shorter. The approximate molecular weight of the resulting product is about 61 kD.(c) Glycosylation of hyaluronidases

[0218] Glycosylation, including N- and O-linked glycosylation, of some hyaluronidases, including the soluble PH20 hyaluronidases, can be important for their catalytic activity and stability. Sialylation also can increase activity and / or stability. For some hyaluronidases, removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. Thus, for such hyaluronidases, the presence of N-linked glycans can be important for generating an active enzyme.

[0219] N-linked oligosaccharides fall into several primary types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc- cores attached via the amide nitrogen of Asn residues that fall within -Asn-Xaa-Thr / Ser-sequences (where Xaa is not Pro). Glycosylation at an -Asn-Xaa-Cys-site has been reported for coagulation protein C. In some instances, a hyaluronidase, such as a PH20 hyaluronidase, can contain N-glycosidic and O-glycosidic linkages. For example, PH20 has O-linked oligosaccharides as well as N-linkedoligosaccharides. There are six potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393 of human PH20 exemplified in SEQ ID NO:1.Variants

[0220] Variants of the soluble PH20 polypeptides that have altered properties, such as increased stability and / or activity have been produced. U. S. Patent Nos. 9,447,401, 10,865,400, 11,066,656, and 11,041,149, which are incorporated by reference, describe and provide a structure / function map of human PH20 detailing the effects of amino acid replacements at every residue in the catalytic domain of human PH20. These patents provide about 7000 examples in which the effects of replacing each amino acid with 15 other amino acids on activity and stability were identified and described. By virtue of that publication and earlier publications / patents virtually all variants of soluble PH20 polypeptides, including those with amino acid replacements, deletions, and insertions, are known in the art. A skilled person readily can prepare soluble hyaluronidases and variants thereof and know the properties of the resulting hyaluronidase.

[0221] Other variants are known to those of skill in the art. See, International PCT application Nos. W02020 / 022791, W02020 / 197230, and WO2021 / 150079, which are incorporated by reference, and which describe modified PH20 polypeptides. These polypeptides, which are variants of the PH20 polypeptides and of the mature PH20 polypeptide of (residues 36-482 of SEQ ID NO:1), include replacements, insertions, and deletions of about 15-20 residues, including, one or more of amino acid residues S343E, M345T, K349E, L353A, L354I, N356E, and 136 IT with reference to SEQ ID NO:1, and sequence numbers therein.Administration of hyaluronidase

[0222] In some embodiments, a hyaluronidase is used, at a dose amount ranging between 1 -50,000 Units for subcutaneous injection. The administered subcutaneous dose of hyaluronidase is about 1 Unit to 50,000 Units. The hyaluronidase is administered at a dose amount of less than 40,000U, less than 30,000U, less than 20,000U, less than 10,000U, less than 9000U, less than 8000U, less than 7000U, less than 6000U, less than 5000U less than 4000U, less than 3000U, less than 2000U, less than 1000U, less than 900U, less than 800U, less than 700U, less than 600U, or less than 500U. In some embodiments, the hyaluronidase enzyme is administered at a dose amount of at least 1U, at least 5U, at least 10U, at least 20U, at least 30U, at least 40U, atleast 50U, at least 60U, at least 70U, at least 80U, at least 100U, or at least 150U. In some other embodiments, the hyaluronidase enzyme is administered at a dose amount of at least 160U, at least 180U, at least 200U, at least 220U, at least 240U, at least 260U, at least 280U, at least 300U, at least 320U, at least 340U, at least 360U, at least 380U, or at least 400U. In one or more embodiments, a porcine (pig) hyaluronidase is used at a dose ranging between 1-50,000 Units. The hyaluronidase enzyme is administered at a dose amount of less than 40,000U, less than 30,000U, less than 20,000U, less than 10,000U, less than 9000U, less than 8000U, less than 7000U, less than 6000U, less than 5000U less than 4000U, less than 3000U, less than 2000U, less than 1000U, less than 900U, less than 800U, less than 700U, less than 600U, or less than 500U. In embodiments the hyaluronidase enzyme is administered at a dose amount of at least 1U, at least 5U, at least 10U, at least 20U, at least 30U, at least 40U, at least 50U, at least 60U, at least 70U, at least 80U, at least 100U, or at least 150U. In embodiments, the hyaluronidase enzyme is administered at a dose amount of at least 160U, at least 180U, at least 200U, at least 220U, at least 240U, at least 260U, at least 280U, at least 300U, at least 320U, at least 340U, at least 360U, at least 380U, or at least 400U.

[0223] In embodiments, an International Unit for hyaluronidase may be defined as the activity of 0.1 mg of the International Standard Preparation and is equal to one turbidity reducing unit (TRU). As used herein, enzyme units are defined by a turbimetric assay in which hyaluronic acid, a complex carbohydrate polymer, is hydrolyzed to di- and mono-saccharides by hyaluronidase. Turbidity is assayed at an optical density of 600 nm (OD600) and one unit yields a 0.33 OD600change per minute at pH 5.35, T=37 °C in a 2 mL volume, where the reaction time is over a 45 minute period. Accordingly, high purity hyaluronidase has a higher specific activity (Units / mg of protein).

[0224] In embodiments, the hyaluronidase purity may be quantified as a function of turbidity, which is measured through optical density. In embodiments, the hyaluronidase purity may be quantified by HPLC (high-performance liquid chromatography). In embodiments, the hyaluronidase purity is from 75% to 100%. In embodiments, the hyaluronidase purity is from 80% to 100%. In embodiments, the hyaluronidase purity is from 85% to 100%. In embodiments, the hyaluronidase purity is from 90% to 100%. In embodiments, the hyaluronidase purity is from 95% to 100%. In embodiments, the hyaluronidase purity is from 98% to 100%. In embodiments, the hyaluronidase purity is from 99% to 100%. In embodiments, the hyaluronidase purity is atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84 %, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%.

[0225] In embodiments, hyaluronidase is administered simultaneously with the nucleic acid encapsulated by the lipid nanoparticle. In some embodiments, hyaluronidase may be administered prior to the administration of the nucleic acid encapsulated by the lipid nanoparticle. In some embodiments, the nucleic acid encapsulated by the lipid nanoparticle and the hyaluronidase enzyme are part of the same formulation. In some embodiments, the nucleic acid encapsulated by the lipid nanoparticle and the hyaluronidase enzyme are injected as separate formulations.

[0226] In embodiments, the hyaluronidase enzyme may be administered in an aqueous solution. In embodiments, the enzyme is administered in saline solution. In some embodiments the hyaluronidase enzyme is part of the lipid nanoparticle formulation and is present in the same solution, the solution comprising nucleic acid encapsulated lipid nanoparticles. In some embodiments a lyophilized preparation comprising the nucleic acid encapsulated lipid nanoparticles and the hyaluronidase enzyme is formulated for therapeutic use.

[0227] In embodiments, the hyaluronidase may be a recombinant hyaluronidase.EXAMPLESExample 1Preparation of siRNA / mRNA Formulations:

[0228] The lipid nanoparticles (LNPs) were prepared by mixing appropriate volumes of lipids in ethanol with an aqueous phase containing small interfering RNA (siRNA) using a microfluidic device, followed by downstream processing. siRNA or mRNA was dissolved in 5 mM citrate buffer (pH 3.5). Lipids at the desired molar ratio were dissolved in ethanol. The lipids used were ionizable amino lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids), cholesterol (Avanti Polar Lipids), and DMG-PEG2000 (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol 2000, NOF America Corporation). At a flow rate ratio of 1:3ethanol / aqueous phases, the solutions were combined in the microfluidic device (Nanoassemblr, Precision NanoSystems). The mixed material was then diluted three times with Tris buffer (pH 7.4) containing 50 mM sodium chloride and 9% sucrose after leaving the micromixer outlet, reducing the ethanol content to 6.25%. The diluted LNP formulation was concentrated and diafdtered by tangential flow filtration using hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories) and Tris buffer (pH 7.4) containing 50 mM sodium chloride and 9% sucrose. A total of 10 diavolumes were exchanged, effectively removing the ethanol. The particle size and polydispersity index (PDI) were determined using a Zen3600 (Malvern Instruments, with Zetasizer 7.1 software, Malvern, U. K.). A volume of 50 pL of formulations were diluted into 950 pL of Tris buffer (tris(hydroxymethyl)aminomethane) at pH = 7.4 and equilibrated to 25 °C prior to analysis in a 1 mL cuvette using the following settings for measurement: material refractive index of 1.5, dispersant viscosity of 1.1 cP, and refractive index of 1.3. Each sample was analyzed for up to 30 runs. Encapsulation efficiency was calculated by determining the unencapsulated RNA content by measuring the fluorescence intensity (Fi) upon the addition of RiboGreen (Molecular Probes) to the lipid nanoparticle and comparing this value to the total fluorescence intensity (Ft) of the RNA content that is obtained upon lysis of the lipid nanoparticles by 1% Triton X-100, where % encapsulation = (Ft - Fi) / Ft xl00). Lipid 1 was used below and refers to di((Z)-non-2-en-l-yl) 8,8'-((((2-(dimethylamino)ethyl)thio)- carbonyl)azanediyl)dioctanoate and was associated with encapsulation efficiencies of >90%. The characterization results are shown in Table 1.Table 1: FVII formulations characterization dataTarget Lipid Mol Dia % Lipid Composition PDI Ratio% (nm) Encap Lipid 1: DSPC: CHOL: DMG- 58:7:33.5:1.5 80.1 0.052 96 PEG2000Sourcing and Characterization of Commercial Hyaluronidase Samples:

[0229] Commercially available hyaluronidases were sourced from several vendors for comparison as follows: from vendors Sigma-Aldrich (St. Louis, MO USA), Creative Enzymes (Upton, NY, USA), Halozyme, Inc. (San Diego, CA USA), Worthington Biochemical(Lakewood, NJ USA) and Creative Biomart (Shirley NY, USA) were sourced. These enzymes were supplied at different International Units representing their potency. Each vendor supplied their Certificate of Analysis indicating the purity and total units per gram or volume supplied. Upon receipt, the purity of these enzymes was further analyzed using gel electrophoresis before being used in the mouse studies described herein. Based on the total number of units per volume or gram reported in the vendor supplied hyaluronidases, each sample of hyaluronidase was diluted to have 100 units per each dose administered in the studies described herein.

[0230] In vivo Factor VII knockdown study. Lipid nanoparticles containing FVII siRNA were administered intravenously (IV) and intradermally (ID) in 6-8 weeks old female Balb / C mice. For IV, 0.03 and 0.01 mg / kg doses were used with a dosing volume of 10 mL / kg and for the ID administration group, 0.3, 1, and 3 mg / kg doses were used with a dosing volume of 50 L. For the ID group, the lipid nanoparticle formulation was co-administered with 100 U of hyaluronidase enzyme. For each dose of 0.03, 0.1, 0.3, 1 and 3 mg / kg corresponding dosing volume was made by mixing the LNP formulation with a specified volume of 100 U of hyaluronidase in water for injection (WFI) prior to ID administration. After 48 h postadministration, mice were anesthetized with 2.5% isoflurane and whole blood was collected retro-orbitally into microtiter tubes coated with 0.109 M sodium citrate buffer (BD Biosciences, San Diego, CA, USA) and processed to plasma. Plasma was stored at -80 °C for later analysis for FVII levels. Measurement of FVII protein in plasma was determined using the colorimetric Biophen VII assay kit (Aniara Diagnostica, West Chester Township, OH, USA). Absorbance was measured at 405 nm and a calibration curve was generated using the serially diluted control plasma to determine levels of FVII in plasma from treated animals, relative to the PBS-treated control animals.

[0231] Referring to Figure 1, Figure 1 shows the effect of co-administration of hyaluronidase on Factor VII (FVII) knockdown. Lipid nanoparticles containing the indicated amount of FVII siRNA were administered intravenously (IV) and intradermally (ID) in 6-8 weeks old female Balb / C mice. For the ID group, the lipid nanoparticle was co-administered with or without 100 U of hyaluronidase enzyme. Measurement of FVII protein in plasma was determined using the colorimetric Biophen VII assay kit. Data is shown as the relative ratio to control (PBS). As canbe seen for the ID administration, FVII expression was significantly reduced with coadministration of hyaluronidase as compared to LNP administered without hyaluronidase. Preparation of mRNA Formulations:

[0232] The lipid nanoparticles were prepared by mixing appropriate molar amounts of lipids dissolved in ethanol with an aqueous solution containing mRNA using a mixing device (Holland Applied Technology), followed by downstream processing. For the encapsulation, mRNA was dissolved in 5 mM citrate buffer (pH 3.5). Lipids at the desired molar ratio were dissolved in ethanol. The molar percentage ratios 50:7:41.5:1.3:0.2 ionizable amino lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids, Alabaster, AL, USA), cholesterol (Avanti Polar Lipids), DMG-PEG2000 (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol 2000 (NOF America Corporation, Irvine, CA, USA) and DSPE-PEG2000-GalNAc lipids. The buffer solution of RNA and ethanolic lipid mixture were combined in the micro fluidic device at a flow ratio of 1:3 ethanol / aqueous phases and the total lipid-to RNA weight ratio was ~23: 1. The mixed material was then diluted 3 times with HEPES buffer ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) (pH 8) containing 50 mM sodium chloride and 9% sucrose after leaving the micromixer outlet, reducing the ethanol content to 6.25%. The diluted LNP formulation was concentrated and diafiltered by tangential flow filtration using hollow fiber membranes (mPES Kros membranes, Spectrum Laboratories, Rancho Dominguez, CA, USA) and HEPES buffer (pH 8) containing 50 mM sodium chloride and 9% sucrose. A total of 10 diavolumes were exchanged, effectively removing the ethanol. The particle size and PDI were characterized using a Zen3600 (Malvern Instruments, with Zetasizer 7.1 software, Malvern, U. K). A volume of 50 pL of formulations were diluted into 950 pL of Tris buffer at pH 7.4 and equilibrated to 25 °C prior to analysis in a 1 mL cuvette using the following settings for measurement: material refractive index of 1.5, dispersant viscosity of 1.1 cP, and refractive index of 1.3. Each sample was analyzed for up to 30 runs. Encapsulation efficiency was calculated by determining the unencapsulated siRNA content by measuring the fluorescence intensity (Fi) upon the addition of RiboGreen (Molecular Probes, Eugene, OR, USA) to the LNP and comparing this value to the total fluorescence intensity (Ft) of the RNA content that is obtained upon lysis of the lipid nanoparticles by 1% Triton X-100, where % encapsulation = (Ft - Fi) / Ft x 100). Lipid 2 was used below and refers to ((4,4'-(((((3-dimethylamino)propyl)thio)carbonyl)azanediyl)-bis(butanoyl))bis(oxy))bis(propane-2,l,3-triyl)tetranonanoate and was associated with encapsulation efficiencies of >90%. The characterization results are shown in Table 2.Table 2. EPO formulations characterization dataTarget Lipid Mol RatioLipid Composition Dia (nm) PDI Encap %%Lipid 2:DSPC: Chol: DMG- 50:7:41.5:1.3:0.2 71.5 0.06 94PEG2000: GalNAc

[0233] In vivo hEPO mRNA expression study. Lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA were administered intravenously (IV) and intradermally (ID) in 6-8 weeks old female Balb / C mice. For IV administration, 0.1 mg / kg or 0.3 mg / kg doses were used with a dosing volume of 10 mL / kg. For ID administered groups, 0.3, 1, and 3 mg / kg doses were used with and without hyaluronidase enzyme (100 U). A dosing volume of 50 μL was used in both ID groups. For each dose of 0.1 and 0.3 mg / kg, corresponding dosing volume (50 uL) was made by mixing the LNP formulation with a specified volume of 100 U of hyaluronidase in water for injection (WFI) prior to ID administration. After 6 h post-administration, mice were anesthetized via 2.5% isoflurane and blood was collected retro-orbitally into BD MICROTAINER® Tube with BD MICROGARD™ Closure (BD Biosciences, San Diego, CA, USA) and processed to serum. The serum was stored at -80 °C for later analysis for hEPO protein expression levels. Measurement of hEPO protein in the serum was determined using the hEPO Elisa kit (ThermoFisher Scientific, USA). Absorbance was measured at 450 nm and a calibration curve was generated using the serially diluted control serum to determine levels of hEPO in the serum from nanoparticles or phosphate-buffered saline (PBS-)treated animals.

[0234] Turning to Figure 2, the figure shows hEPO expression after co-administration of hyaluronidase and EPO mRNA formulations. Lipid nanoparticles containing the indicated amount of hEPO mRNA were administered intravenously (IV) and intradermally (ID) in 6–8 weeks old female Balb / C mice.For the ID group, lipid nanoparticle was co-administered with or without 100 U of hyaluronidase enzyme. Measurement of hEPO protein in the serum was determined using the hEPO Elisa kit. In this study, the data from Vendors 1 and 4 are not presented due to low signals at the 0.3 and 1 mg / kg levels, however the results from Vendors 2, 3, and 5, which were those displaying a higher degree of purity as shown by gel analysis, allshowed significantly increased expression relative to ID-administered samples lacking hyaluronidase.

[0235] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): For gel electrophoresis, 5 pg of hyaluronidase in 15 pL solution was mixed with 5 pL 4X Laemmli sample buffer (Licor, Lincoln, Nebraska, USA). CHAMELEON® Duo Pre-stained Protein Ladder (10 pL, Licor, Lincoln, Nebraska, USA) and 20 pL of samples were loaded in each well of the 4-12% Bis-Tris Precast protein gels (NuPAGE Gel, Invitrogen, Waltham, MA, USA). Electrophoresis was performed in the XCell SureLock Mini -Cell (Invitrogen, Waltham, MA, USA) for 90 minutes at a constant voltage of 100 V and IX MES (2-ethanesulfonic acid) buffer was used as the running buffer. The gels were subsequently stained with Coomassie R-250 for at least 3 h and de-stained with a solution of 40% ethanol and 10% acetic acid for more than 4 h. Two gels were scanned and analyzed with Azure Imaging Systems (Azure Biosystems, Dublin, CA, USA).

[0236] Lyophilization of mRNA lipid nanoparticle: Lipid nanoparticles were mixed with 0.05% PVA10, 0.1M Proline, and 9% iodixanol to yield a lipid nanoparticle suspension with a final concentration of 1.2 mg / mL mRNA. Glass vials (8R, borosilicate, SCHOTT® FIOLAX® Vial, Schott, USA) were filled with 1 mL of mRNA lipid nanoparticle suspension containing 1.2 mg / mL of mRNA and 2000 U of hyaluronidase in 1.2 mg / mL of mRNA containing lipid nanoparticles. Samples were lyophilized using Millrock LyoStar-OlU Lyophilizer. First, samples were frozen at -50 °C for 4.5 h, this being followed by a primary dry cycle at -25 °C / 20 h, gradually increasing the temperature to -15 °C for 1 h, and kept at this temperature for 20 h, then gradually increasing the temperature to 0 °C for 1 h and kept at this temperature for 15 h. This was followed by a secondary drying cycle by increasing the temperature from 0 to 10 °C for 1 h and maintaining at 10 °C for 17 h and further increasing the temperature from 0 °C to 15 °C to 20 °C in 2 h and maintaining at 20 °C for 11 h. Finally, the temperature was brought to 25 °C and kept for 6 h to end the cycle. The pressure was maintained at 38 Torr for the entire drying cycle. Vials were backfilled with nitrogen, capped, and transferred to -80 °C storage temperature for stability assessment.Table 3. Formulation characterization data for frozen and lyophilized formulationsDia (nm) PDI % EncapFrozen lipid nanoparticle 66.47 0.094 92.2 Lyophilized lipid nanoparticle 77.62 0.181 91.1Co-lyophilized lipid nanoparticle76.02 0.201 92with hyaluronidase

[0237] Figure 3 shows the comparison of frozen lipid nanoparticles versus lipid nanoparticles co-lyophilized with hyaluronidase. The experimental conditions from figure 2 were repeated except the lipid nanoparticle was either frozen or co-lyophilized with hyaluronidase prior to thawing / reconstitution for treatment. The results show that lyophilized formulations show comparable expression as frozen / thawed formulations when coadministered with hyaluronidase.

[0238] Table 4 provides the formulation characterization data that was used in the SDS Page gel evaluating the purity of different hyaluronidases from different commercial sources. The results are illustrated in Figure 4. Lipid nanoparticles containing the indicated amount of hEPO mRNA were administered intravenously (IV) and intradermally (ID) in 6–8 weeks old female Balb / C mice.For the ID group, lipid nanoparticle was co-administered with or without 100 U of hyaluronidase enzyme from vendor 2 or vendor 3. Measurement of hEPO protein in the serum was determined using the hEPO Elisa kit.Table 4. Formulation characterization data for LNP used in screening different vendor hyaluronidasesTarget Lipid Mol RatioLipid Composition Dia (nm) PDI % Encap %Lipid 2:DSPC: Chol: DMG- 50:7:41.5:1.3:0.2 63.54 0.16 94PEG2000: GalNAc

[0239] The results show that purities of the equipotent (e.g., 100 U) hyaluronidases administered have a direct impact on the protein expression for a given dose of mRNA. The lower the purity of the commercially supplied or proprietary hyaluronidase, the lower the protein expression at similar enzyme units.Table 5. Tables of Sequences (Informal Sequence Listing)SEQ ID NO. Name of Sequence Sequence MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTL NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG VTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQ QQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLG KLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNPrecursor Full1 VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAA length human PH20TLYVRNRVREAIRVSKIPDAKSPLPVFAYTRTVFT DQVLKFLSQDELVYTFGETVALGASGIVIWGTLSI MRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQ VLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKG GKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKA DVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY NASPSTLSATMFIVSILFLIISSVASLSignal sequence ofprecursor fulllength PH202 MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLT corresponding toresidues 1 to 35 ofSEQ ID NO:1LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH203 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-467 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDASEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH204 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-468 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAF LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH205 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-469 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFL LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH206 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-470 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH207 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-471 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKP LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH208 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-472 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPP LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH209 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-473 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2010 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-474 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPME LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2011 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-475 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMET LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2012 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-476 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETESEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2013 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-477 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEE LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2014 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-478 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEP LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2015 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-479 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2016 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-480 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQI LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSChuman PH2017 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV (residue 36-481 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1)FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 18(residue 36-482 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 19(residue 36-483 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YN LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 20(residue 36-484 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNA LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 21(residue 36-485 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 22(residue 36-486 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASP LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 23(residue 36-491 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSA LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 24(residue 36-492 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 25(residue 36-493 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATM LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 26(residue 36-494 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATMF LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 27(residue 36-495 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFISEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 28(residue 36-496 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATMFIV LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 29(residue 36-497 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATMFIVS LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 30(residue 36-498 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSISEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 31(residue 36-499 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATMFIVSIL LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKExemplary mature LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 32(residue 36-500 of AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV SEQ ID No:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIF YNASPSTLSATMFIVSILF MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTL NFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDM SLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITG VTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLG MAVIDWEEWRPTWARNWKPKDVYKNRSIELVQExemplaryQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGtruncated humanKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFN33 PH20 (residue 1 to VEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAA482 of SEQ IDTLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFT NO:1)DQVLKFLSQDELVYTFGETVALGASGIVIWGTLSI MRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQ VLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKG GKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYSEQ ID NO. Name of Sequence Sequence LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSI TGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDN LGMAVIDWEEWRPTWARNWKPKDVYKNRSIEL VQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKMature full length LGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSC Human PH20 FNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPV 34(residues 36 to 509 AATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIV of SEQ ID NO:1) FTDQVLKFLSQDELVYTFGETVALGASGIVIWGTL SIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEK GGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEK ADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL

Claims

Attorney Docket No. 049386-549001 WOWHAT IS CLAIMED IS:

1. A method of delivering a therapeutically effective amount of a nucleic acid encapsulated in a lipid delivery vehicle comprising subcutaneously co-administering to a mammal (i) a composition comprising the nucleic acid encapsulated in the lipid delivery vehicle and (ii) hyaluronidase, wherein the hyaluronidase is a pure hyaluronidase as determined by the presence of a single band located within the range of about 50 kDa to about 80 kDa.

2. The method of claim 1, wherein the purity of hyaluronidase is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.8%.

3. The method of claim 1 or 2, wherein the nucleic acid is selected from an siRNA (small interfering RNA), an mRNA (messenger RNA), a self-replicating RNA, a DNA plasmid, an antisense oligonucleotide, a single strand guide RNA, and combinations thereof.

4. The method of claim 3, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a protein.

5. The method of claim 4, wherein the protein is an enzyme, and antibody, an antigen, a receptor, or a transporter.

6. The method of claim 4 or 5, wherein the protein is a gene-editing enzyme.

7. The method of claim 6, wherein the gene-editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, or a zinc finger nuclease.

8. The method of claim 3 or 4, wherein the nucleic acid is a mRNA or a self-replicating RNA comprising a coding region that encodes a viral protein that when expressed elicits an immunological response.

9. The method of claim 8, wherein the immunological response comprises production of antibodies against the viral protein.

10. The method of any of claims 1 to 9, wherein the lipid delivery vehicle is selected from a lipid nanoparticle (LNP), a liposome, and a lipoplex.

11. The method of claim 10, wherein the lipid delivery vehicle is a lipid nanoparticle.

12. The method of claim 11, wherein the lipid nanoparticle has an average particle size of less than about 100 nm.

13. The method of claim 11 or 12, wherein the lipid nanoparticle has an average particles size of about 55 nm to about 85 nm.

14. The method of any one of claims 11 to 13, wherein the lipid nanoparticle further comprises a helper lipid.

15. The method of claim 14, wherein the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC).

16. The method of claim 15, wherein the helper lipid is distearoylphosphatidylcholine (DSPC).

17. The method of any one of claims 11 to 16, wherein the lipid nanoparticle further comprises cholesterol.

18. The method of any one of claims 11 to 17, wherein the lipid nanoparticle further comprises a polyethylene glycol (PEG)-lipid conjugate.

19. The method of claim 18, wherein PEG-lipid conjugate is PEG-DMG.

20. The method of claim 19, wherein the PEG-DMG is PEG2000-DMG.

21. The method of any one of claims 11 to 20, wherein the lipid nanoparticle comprises about 45 mol% to 65 mol% of a cationic lipid or an ionizable lipid, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

22. The method of claim 21, wherein the lipid nanoparticle comprises about 50 mol% to about 61 mol% of the cationic lipid or ionizable lipid, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate.

23. The method of claim 21 or 22, wherein the lipid nanoparticle comprises about 56 mol% to about 58 mol% of the cationic lipid or ionizable lipid, about 6 mol% to about 8 mol% ofDSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

24. The method of any one of claims 11 to 23, wherein the lipid nanoparticle has a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1.

25. The method of any one of claims 1 to 24, wherein the composition further comprises one or more cryoprotectants.

26. The method of claim 25, wherein the one or more cryoprotectants is selected from sucrose, glycerol, and a combination of sucrose and glycerol.

27. The method of any one of claims 1 to 26, wherein the composition is reconstituted from a lyophilized composition.

28. The method of any one of claims 1 to 27, wherein the purity of hyaluronidase is determined by gel electrophoresis, optionally wherein the gel electrophoresis is sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

29. The method of any one of claims 1 to 28, wherein the pure hyaluronidase is free from impurities selected from denatured hyaluronidase, degraded hyaluronidase, hyaluronidase fragment, extraneous protein, nucleic acid, and a combination thereof.

30. The method of any one of claims 1 to 29, wherein the co-administration results in an increased systemic delivery of the nucleic acid.

31. The method of any one of claims 1 to 30, wherein the hyaluronidase has the sequence set forth as residues 36-482 or 36-483 or has at least 98% sequence identity to the sequence set forth as residues 36-482 or 36-483 of SEQ ID NO:1.

32. The method of any one of claims 1 to 31, wherein the hyaluronidase is a soluble PH20 hyaluronidase.

33. The method of claim 32, wherein the soluble hyaluronidase is the composition designated rHuPH20.

34. The method of claim 32 or 33, wherein the soluble hyaluronidase comprises amino acids 36- 464 of SEQ ID NO:1 or comprises a sequence of amino acids that has at least 85% sequenceidentity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO:1, and retains hyaluronidase activity.

35. The method of claim 32 or 33, wherein the soluble hyaluronidase comprises a sequence of amino acids that has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO:1 and retains hyaluronidase activity.

36. The method of claim 32 or 33, wherein (a) the soluble hyaluronidase has a sequence of amino acids set forth as residues 36-465, 36-466, 36-467, 36-468, 36-469, 35-470, 36-471, 36-472, 36-474, 36-475, 36-476, 35-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483 35-484, 36-485, 36-486, 36-487, 36-488, 36-489, 36-490, 35-491, 36-492, 36-493, 36-494, 36-495, 36-496, 36-497, 35-498, 36-499, and 36-500 of SEQ ID NO:1, or an N-terminally truncated variant thereof lacking residues 36, 36-37, 36-38, 36-39, or 36-40; or (b) the soluble hyaluronidase comprises a variant soluble hyaluronidase that has at least 91% sequence identity to the soluble hyaluronidase of (a).

37. A method of delivering a nucleic acid comprising administering to a patient in need thereof (i) a composition comprising a therapeutically effective amount of nucleic acid encapsulated in a lipid delivery vehicle and (ii) a soluble hyaluronidase.

38. The method of claim 37, wherein the soluble hyaluronidase is a soluble human hyaluronidase.

39. The method of claim 37 or 38, wherein the hyaluronidase is administered in an amount effective to increase bioavailability of the nucleic acid.

40. The method of any one of claims 37-39, wherein the soluble hyaluronidase and composition are administered via subcutaneous (SC), intradermal (ID), or intramuscular (IM) administration.

41. The method of any one of claims 37-40, wherein the soluble hyaluronidase and the nucleic acid are both encapsulated in the lipid delivery vehicle.

42. The method of any one of claims 37-41, wherein the soluble hyaluronidase and composition are administered via SC administration.

43. The method of any one of claims 37-41, wherein the soluble hyaluronidase and composition are administered via ID administration.

44. The method of any one of claims 37-41, wherein the soluble hyaluronidase and composition are administered via IM administration.

45. The method of any one of claims 37-44, wherein the hyaluronidase is a soluble PH20 hyaluronidase.

46. The method of any one of claims 37-44, wherein the soluble hyaluronidase is the composition designated rHuPH20.

47. The method of any one of claims 37-44, wherein the hyaluronidase has the sequence set forth as residues 36-482 or 36-483 or has at least 98% sequence identity to the sequence set forth as residues 36-482 or 36-483 of SEQ ID NO: 1.

48. The method of any one of claims 37-44, wherein the soluble hyaluronidase comprises amino acids 36-464 of SEQ ID NO: 1 or comprises a sequence of amino acids that has at least 85% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO:1, and retains hyaluronidase activity.

49. The method of any one of claims 37-44, wherein the soluble hyaluronidase comprises a sequence of amino acids that has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO:1 and retains hyaluronidase activity.

50. The method of any one of claims 37-44, wherein (a) the soluble hyaluronidase has a sequence of amino acids set forth as residues 36-465, 36-466, 36-467, 36-468, 36-469, 35- 470, 36-471, 36-472, 36-474, 36-475, 36-476, 35-477, 36-478, 36-479, 36-480, 36-481, 36- 482, 36-483 35-484, 36-485, 36-486, 36-487, 36-488, 36-489, 36-490, 35-491, 36-492, 36- 493, 36-494, 36-495, 36-496, 36-497, 35-498, 36-499, and 36-500 of SEQ ID NO:1, or an N-terminally truncated variant thereof lacking residues 36, 36-37, 36-38, 36-39, or 36-40; or (b) the soluble hyaluronidase comprises a variant soluble hyaluronidase that has at least 91% sequence identity to the soluble hyaluronidase of (a).

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