Nucleic acid and hyaluronidase combinations and dosing regimens

Abstract

Provided are combination dosing regimens comprising administering hyaluronidase and a lipid nanoparticle composition. Combinations and compositions containing the hyaluronidase and a lipid nanoparticle composition are provided.

Description

TITLENucleic Acid and Hyaluronidase Combinations and Dosing RegimensCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 702,576, fded October 2, 2024, U.S. Provisional Application No. 63 / 736,538, December 19, 2024, U.S. Provisional Application No. 63 / 767,475, filed March 5, 2025, U.S. Provisional Application No. 63 / 787,232, filed April 11, 2025, U.S. Provisional Application No. 63 / 808,098, filed May 19, 2025, U.S. Provisional Application No. 63 / 810,618, filed May 22, 2025, and U.S. Provisional Application No. 63 / 847,831, filed July 21, 2025, each of which is incorporated by reference herein in its entirety.SEQUENCE LISTING SUBMISSION VIA PATENT CENTER

[0002] The contents of the electronic sequence listing (063995-5129. xml; Size: 388,000 bytes; and Date of Creation: October 2, 2024) is herein incorporated by reference in its entirety.SUMMARY

[0003] In various embodiments, provided herein are compositions and dosing regimens, comprising administering a hyaluronidase and a composition comprising a therapeutic agent selected from an mRNA, nucleic acid conjugate, oligonucleotide, antisense oligonucleotide (ASO), phosphorodiamidate morpholino oligonucleotide (PMO), radiopharmaceutical, aptamer, siRNA, RNAi, miRNA, DNA, self-replicating RNA (replicons), Naked RNA, Circular RNA (circRNA), Trans-activating crRNA (TracrRNA), and gene editing. In some embodiments, the therapeutic agent in the composition is conjugated to a targeting agent or delivered with particles, lipid nanoparticles (LNP), non-lipid nanoparticles, liposomes, cholestosomes, exosomes, viral particles, virus like particles, cochleates, engineered bacterial systems, DNA nanostructures or split-intein systems. In embodiments, the therapeutic agent in the composition is conjugated to a targeting agent; delivered with lipid nanoparticles (LNP) or non-lipid nanoparticles; or incorporated in DOCP neutral liposomes, cholestosome (Cholesteryl esters) particles, cochleate (consisting of phosphatidylserine) particles, virus-like particles (VLPs), adeno-associated virus (AAV), synthetic virus-like particles (sVLPs), lentivirus, adenovirus, orDBl / 162871737.3 1DNA nanostructures (e.g. DNA origami). In other embodiments, the therapeutic agent in the composition is delivered with lipid nanoparticles (LNP) conjugated to a targeting agent. In embodiments, the hyaluronidase and the composition comprising the therapeutic agent are conjugated. In embodiments, the hyaluronidase is conjugated to the therapeutic agent.

[0004] In embodiments of the invention, the composition and combination dosing regimen include hyaluronidase in an amount effective to increase the delivered dose, delivery rate or volume of the therapeutic agent compared to the therapeutic agent administered without hyaluronidase. In other embodiments, the hyaluronidase is administered in an amount effective to increase the delivered dose or volume of the composition compared to the composition administered without hyaluronidase.

[0005] In various embodiments of the invention, the composition and combination dosing regimen include hyaluronidase in an amount effective to increase an immune response to the therapeutic agent or the protein encoded, produced or modified by the therapeutic agent. In some embodiments, the hyaluronidase is in an amount effective to increase activation and proliferation of immune cells. In some embodiments, the hyaluronidase is in an amount effective to increase plasma or serum concentration of the protein produced by the therapeutic agent compared to therapeutic agent administered without hyaluronidase. In other embodiments the hyaluronidase is in an amount effective to decrease plasma or serum concentration of the protein knocked down or modulated by the therapeutic agent compared to the therapeutic agent administered without hyaluronidase. In various embodiments, the hyaluronidase is in an amount effective to decrease the frequency of administration of the therapeutic agent compared to administration of the therapeutic agent without hyaluronidase.

[0006] In various embodiments of the invention described herein the composition and combination dosing regimen is for administration via subcutaneous (SC), intradermal (ID), or intramuscular (IM) administration.

[0007] In some embodiments, the invention described provides an increased in concentration or distribution of the therapeutic agent systemically, in the pancreas, spleen, liver, fat, kidneys, uterus / ovaries, muscle, heart, lungs, endothelial tissue, bone marrow, brain, skin, lymphatic system, lymph nodes, and thymus, when administered with hyaluronidase compared to without hyaluronidase.DBl / 162871737.3 2

[0008] In some embodiments, following administration via ID, SC or IM, with hyaluronidase the invention described results in increased local distribution at the injection site when compared to administration without hyaluronidase.

[0009] 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 co-admini st ering to a mammal (i) a lipid nanoparticle composition comprising a therapeutic selected from a cancer therapy, an antibody nucleic acid conjugate, a monoclonal antibody, a gene therapy, a vaccine, mRNA, antisense oligonucleotide and siRNA wherein the therapeutic is encapsulated in a lipid nanoparticle delivery vehicle; incorporated in DOCP neutral liposomes or Cholestosome (Cholesteryl esters) particles, cochleate (consisting of phosphatidyl serine) particles, virus-like particles (VLPs), adeno-associated virus (AAV), synthetic virus-like particles (sVLPs), lentivirus, adenovirus; or DNA nanostructures (e.g., DNA origami), and (ii) a composition comprising a hyaluronidase, wherein the hyaluronidase is a pure hyaluronidase. Purity of the hyaluronidase can be validated using techniques such as electrophoresis (e.g., further involving a stain, electroblot, immunoblot, Western blot, etc. to confirm a single band located within the range of approximately 50 kDa to approximately 80 kDa), chromatography, centrifugation, mass spectrometry, and other well-known techniques. In some embodiments, the (i) composition comprising the lipid nanoparticle, DOCP neutral liposomes, cholestosome (Cholesteryl esters) particles, cochleate (consisting of phosphatidylserine) particles, virus-like particles (VLPs), adeno-associated virus (AAV), synthetic virus-like particles (sVLPs), lentivirus, adenovirus, or DNA nanostructures (e g. DNA origami) and (ii) hyaluronidase are administered to the mammal as a co-formulation in a single composition. In some embodiments, the (i) composition comprising the lipid nanoparticle, DOCP neutral liposomes, cholestosome (Cholesteryl esters) particles, cochleate (consisting of phosphatidylserine) particles, virus-like particles (VLPs), adeno-associated virus (AAV), synthetic virus-like particles (sVLPs), lentivirus, adenovirus, or DNA nanostructures (e.g. DNA origami) and (ii) the hyaluronidase are administered to the mammal concurrently or sequentially as separate compositions.

[0010] In some embodiments, the present application provides a method of treating a disease in a mammal, comprising co-administering a therapeutically effective amount of a therapeutic and / or a prophylactic agent to the mammal, wherein the therapeutic and / or prophylactic agent is encapsulated in a lipid nanoparticle composition or incorporated in a DOCPDBl / 162871737.3 3neutral liposome, cholestosome (Cholesteryl esters) particle, cochleate (consisting of phosphatidylserine) particle, virus-like particle (VLP), adeno-associated virus (AAV) vector, synthetic virus-like particle (sVLP), lentivirus vector, adenovirus vector, or a DNA nanostructure (e.g. DNA origami). The therapeutic and / or prophylactic agent can be selected from a cancer therapy, an antibody nucleic acid conjugate, a monoclonal antibody, a gene therapy, a vaccine, mRNA, antisense oligonucleotide, and siRNA. The therapeutic and / or prophylactic agent can be encapsulated in a lipid delivery vehicle described herein; administered in a composition comprising a pure hyaluronidase; administered in a composition comprising the therapeutic and / or prophylactic agent encapsulated in a lipid nanoparticle described herein and a pure hyaluronidase; or the pharmaceutical composition described herein and a pure hyaluronidase. In some embodiments, the lipid nanoparticle composition and hyaluronidase are administered to the mammal as a co-formulation in a single composition. In some embodiments, the lipid nanoparticle composition and hyaluronidase are administered to the mammal concurrently or sequentially as separate compositions.

[0011] In embodiments, the hyaluronidase is administered in an amount effective to increase the delivered dose or volume of the therapeutic agent or composition compared to the therapeutic agent or composition administered without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase rate of delivery of the therapeutic agent or composition compared to the therapeutic agent or composition administered without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase plasma or serum concentration of the therapeutic agent or composition compared to the therapeutic agent or composition administered without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase plasma or serum concentration of the protein encoded, produced or modulated by the therapeutic agent compared to therapeutic agent or composition administered without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to decrease plasma or serum concentration of the protein decreased, inhibited, knocked down or modulated by the therapeutic agent compared to the therapeutic agent or composition administered without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to decrease the frequency of administration of the therapeutic agent or composition compared to administration of the therapeutic agent or composition without hyaluronidase.DBl / 162871737.3 4

[0012] In embodiments, following intradermal (ID), subcutaneous (SC), or intramuscular (IM) administration of the therapeutic agent or composition when administered with hyaluronidase, there is an increase in concentration or distribution of the therapeutic agent or composition at the injection site, systemically, and / or in one or more tissues comprising the pancreas, spleen, liver, fat, kidneys, uterus / ovaries, muscle, nerves, heart, lungs, endothelial tissue, bone, bone marrow, brain, skin, gastrointestinal tract, lymphatic system, lymph nodes, or thymus, as compared to an equivalent dose of the therapeutic agent or composition without hyaluronidase. In embodiments, following ID, SC, or IM administration of the therapeutic agent or composition with hyaluronidase, there is an increased concentration or distribution of the protein encoded, produced or modulated by the therapeutic agent at the injection site, systemically, and / or in one or more tissues comprising the pancreas, spleen, liver, fat, kidneys, uterus / ovaries, muscle, nerves, heart, lungs, endothelial tissue, bone, bone marrow, brain, skin, gastrointestinal tract, lymphatic system, lymph nodes, or thymus, as compared to an equivalent dose of the therapeutic agent or composition without hyaluronidase. In embodiments, following ID, SC, or IM administration of the therapeutic agent or composition with hyaluronidase, there is an decreased concentration or distribution of the protein decreased, inhibited, knocked down or modulated by the therapeutic agent at the injection site, systemically, and / or in one or more tissues including the pancreas, spleen, liver, fat, kidneys, uterus / ovaries, muscle, nerves, heart, lungs, endothelial tissue, bone, bone marrow, brain, skin, gastrointestinal tract, lymphatic system, lymph nodes, or thymus, as compared to an equivalent dose of the therapeutic agent or composition without hyaluronidase.

[0013] In embodiments, the total volume administered is less than 1 mL. In embodiments, the total volume administered is between about 1 mL and about 2 mL. In embodiments, the total volume administered is greater than 3 mL. In embodiments, the hyaluronidase and the composition are administered once a day, every other day, every three days, every four days, every five days, or every six days. In embodiments, the hyaluronidase and the composition are administered once a day. In embodiments, the hyaluronidase and the composition are administered once a week, every other week, every three weeks, every four weeks, every five weeks, or every six weeks. In embodiments, the hyaluronidase and the composition are administered once a month, every other month, every two months, every three months, every four months, every five months, every six months, or every twelve months.DBl / 162871737.3 5

[0014] In embodiments, the hyaluronidase is administered in an amount effective to increase the serum, plasma or tissue levels of the protein encoded, produced or modulated by the therapeutic agent as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase duration of the increase of the serum, plasma or tissue levels of the protein encoded, produced or modulated by the therapeutic agent as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to decrease serum, plasma or tissue levels of the protein decreased, inhibited, knocked down or modulated by the therapeutic agent as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase the duration of the decrease in serum, plasma or tissue levels of the protein decreased, inhibited, knocked down or modulated by the therapeutic agent as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to increase the immune response to a vaccine antigen as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the hyaluronidase is administered in an amount effective to reduce the inflammatory response to the therapeutic agent or composition as compared to administration of the therapeutic agent without hyaluronidase.

[0015] In embodiments, the hyaluronidase is administered in an amount effective to increase the levels of the chimeric antigen receptor or immunomodulatory protein encoded, produced or modulated by the therapeutic agent by immune cells in the serum, plasma or lymphatics as compared to administration of the therapeutic agent without hyaluronidase.

[0016] In embodiments, the hyaluronidase is administered in an amount effective to reduce the dose of the therapeutic agent or composition and achieve a similar effect as compared to administration of the therapeutic agent without hyaluronidase. In embodiments, the effect occurs in liver. In embodiments, the effect occurs in the muscle. In embodiments, the effect occurs in the pancreas, spleen, fat, kidneys, uterus, ovaries, heart, lungs, endothelial tissue, bone, bone marrow, brain, nerves, skin, gastrointestinal tract, lymphatic system, lymph nodes, or thymus.BACKGROUND OF THE DISCLOSUREDBl / 162871737.3 6

[0017] The present disclosure provides a formulation comprising a plurality of lipid nanoparticles (LNPs) or non-lipid nanoparticles encapsulating a therapeutic and / or prophylactic agent, or a plurality of conjugation agents conjugated to a therapeutic and / or prophylactic agent wherein the formulation further comprises a hyaluronidase. The therapeutic and / or prophylactic agent may comprise a nucleic acid, an antigen, or a protein. The disclosed formulation is of particular use in the field of gene transfer or gene therapy.

[0018] Messenger RNA therapy (MRT) is becoming an increasingly important approach for the treatment of a variety of diseases. MRT involves administration of a messenger RNA (mRNA) therapeutic and / or prophylactic to a subject in need of the therapy for production of the protein encoded by the mRNA within the patient's body. Similarly, small interfering RNA (siRNA), antisense oligonucleotides (ASOs), PMOs, and other nucleic acids are increasingly used as therapeutic modalities to modulate gene expression or protein levels. DNA-based therapies and self-replicating RNAs also provide opportunities for long-term expression, genome editing, and durable therapeutic benefit. LNPs and / or conjugation agents are commonly used to deliver nucleic acids for efficient in vivo delivery of mRNA, siRNA, or DNA.[00019J Hyaluronan (hyaluronic acid; HA) is a glycosaminoglycan that is found in the extracellular matrix of many cells, especially in soft connective tissues. HA also is found predominantly in skin, cartilage, and in synovial fluid in mammals. Hyaluronan also is the main constituent of the vitreous of the eye.

[0020] Hyaluronidases are enzymes that degrade hyaluronic acid. Six hyaluronidase genes have been identified in the human genome, all of which share homology, both within the paralog and across other vertabrates, including cows. Neutral pH-active hyaluronidases facilitate the dispersion and absorption of molecules through tissue. Furthermore, hyaluronidases can increase the lymphatic absorption profile of co-administered biologies when injected subcutaneously.

[0021] Hyaluronidase has been used for many years for a variety of applications in human clinical medicine, e.g., as an antiedema agent (Lasonil, Thiomucase), as an agent for diffusing medicines which have been injected by the intramuscular or subcutaneous route (Hyaluronidase Choay), as an anti-cancer agent, in formulating local anesthetics (Lewis-Smith, Br. J. Plast. Surg. 1986, 39: 554-558), or else as an agent for reducing myocardial lesions following an infarct.DBl / 162871737.3 7

[0022] There is a need in the art for a formulation comprising a hyaluronidase and a therapeutic and / or prophylactic agent to provide improved absorption and distribution of the therapeutic and / or prophylactic agent, provide reduced inflammation following administration, reduce injection site reactions, enable targeting to the cells in the lymphatic system or other organs, and increase expression and / or knockdown (KD) of a protein modulated by the therapeutic and / or prophylactic agent in a subject in need thereof. There is also an unmet need for formulations that enable larger volumes of LNP or conjugation agent formulations to be administered through various routes of administration, including subcutaneous, intramuscular, and intradermal injections. The present disclosure addresses this unmet need.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Fig. 1 is a graph of TNF-alpha levels over time for the group receiving IV injection of mRNA-LNP.

[0024] Fig. 2 is a graph of TNF-alpha levels over time for the group receiving IM injection of mRNA-LNP.

[0025] Fig. 3 is a graph of TNF-alpha levels over time for the group receiving IM injection of mRNA-LNP + rHuPH20.

[0026] Fig. 4 is a graph of TNF-alpha levels over time for the group receiving ID injection of mRNA-LNP.

[0027] Fig. 5 is a graph of TNF-alpha levels over time for the group receiving ID injection of mRNA-LNP + rHuPH20.

[0028] Fig. 6 is a graph of TNF-alpha levels over time for the group receiving SC injection of mRNA-LNP.

[0029] Fig. 7 is a graph of TNF-alpha levels over time for the group receiving SC injection of mRNA-LNP + rHuPH20.

[0030] Fig. 8 displays images of luminescence over time for the group receiving mRNA and rHuPH20.

[0031] Fig. 9 displays images of luminescence over time for the group receiving mRNA alone.DBl / 162871737.3 8

[0032] Fig. 10 is a graph of total flux values for group dorsally administered mRNA and rHuPH20.

[0033] Fig. 11 is a graph of total flux values for group dorsally administered mRNA alone.

[0034] Fig. 12 is a graph of total flux values for group ventrally administered mRNA and rHuPH20.

[0035] Fig. 13 is a graph of total flux values for group ventrally administered mRNA alone.

[0036] Fig. 14A and 14B are graphs comparing changes in total flux between group dorsally administered mRNA and rHuPH20 and group dorsally administered mRNA alone.

[0037] Fig. 15A and 15B are graphs comparing changes in total flux between group dorsally administered mRNA and rHuPH20 and group dorsally administered mRNA alone.

[0038] Fig. 16 is a perspective view of an auto-injector in accordance with an exemplary embodiment of the present invention.

[0039] Fig. 17 is an exploded view of the auto-injector of FIG. 16.

[0040] Fig. 18 is a perspective view of the auto-injector of FIG. 16.

[0041] Fig. 19 is a cross-sectional view of the auto-injector of FIG. 16.

[0042] Fig. 20 is a partial cross-sectional view of the auto-injector of FIG. 16 in a locked configuration.

[0043] Fig. 21 is a partial cross-sectional view of the auto-injector of FIG. 16 in an unlocked configuration.

[0044] Fig. 22 is a partial cross-sectional view of the auto-injector of FIG. 16 in a discharged configuration.

[0045] Fig. 23 displays dorsal and ventral images of luminescence over time for a group receiving a first injection of mRNA alone (Group 1) and a group receiving a first injection of mRNA and rHuPH20 (Group 2) at day 0.DBl / 162871737.3 9

[0046] Fig. 24 displays dorsal and ventral images of luminescence over time for a group receiving a second injection of mRNA alone (Group 1) and a group receiving a second injection of mRNA and rHuPH20 (Group 2) at day 14.

[0047] Fig. 25 displays dorsal and ventral images of luminescence over time for a group receiving a third injection of mRNA alone (Group 1) and a group receiving a third injection of mRNA and rHuPH20 (Group 2) at day 28, twenty-four (24) hours post injection.

[0048] Fig. 26 displays dorsal and ventral images of luminescence over time for a group receiving a third injection of mRNA alone (Group 1) and a group receiving a third injection of mRNA and rHuPH20 (Group 2) at day 28, forty-eight (48) hours post injection.

[0049] Fig. 27 is a graph of Group Average BLI for a group dorsally receiving mRNA alone (Group 1) and a group dorsally receiving mRNA and rHuPH20 (Group 2).

[0050] Fig. 28 is a graph of Group Average BLI for a group ventrally receiving mRNA alone (Group 1) and a group ventrally receiving mRNA and rHuPH20 (Group 2).

[0051] Fig. 29 is a graph of Group Average BLI of local expression for a group receiving mRNA alone (Group 1) and a group receiving mRNA and rHuPH20 (Group 2).

[0052] Fig. 30 is a graph of Group Average BLI of liver for a group receiving mRNA alone (Group 1 ) and a group receiving mRNA and rHuPH20 (Group 2).

[0053] Fig. 31 is a graph of IL-6 levels in plasma for a group receiving mRNA alone (Group 1) and a group receiving mRNA and rHuPH20 (Group 2).

[0054] Fig. 36 is a graph showing TNF-alpha levels following administration of mRNA- LNP with or without rHuPH20.

[0055] Fig. 37A shows Factor 7 levels for individual mice within the study groups. Fig. 37B is a graph of average Factor 7 levels in the study groups. Fig. 37C is a graph of Factor 7 levels for individual mice within the study groups. Fig. 37D is a graph of pre-bleen serum Factor 7 levels 24 hours and 72 hours post dose. Fig. 37E is a graph of serum factor 7 levels for individual mice within the treatment groups.

[0056] Figs. 38 and 39 are graphs of changes in total flux up to 48 hours post dose for different treatment groups and injection sites.

[0057] Fig. 40 is an image of points of interested on mouse.DBl / 162871737.3 10

[0058] Figs. 41-45 are graphs of total flux measurements for study groups.

[0059] Fig. 46-51 are images of whole body and organs for each group at 8 and 24 hours post injection.

[0060] Figs. 52-56 are graphs depicting BLI measured at study endpoints.

[0061] Fig. 57 is a graph of changes in mIL-6 in plasma for each mouse.

[0062] Fig. 58 is a graph of changes in mlL-lb in plasma for each mouse.

[0063] Fig. 59 is a graph of change in average total flux in the liver after administration.

[0064] Fig. 60A and 60B are graphs of average BLI values for the whole body.

[0065] Fig. 61 A and 6 IB are graphs showing the average BLI values for different organs.

[0066] Figs. 62-67 provide IVIS and organ imaging for each study group: Group 1 (Fig. 62), Group 2 (Fig. 63), Group 3 (Fig. 64), Group 4 (Fig. 65), Group 5 (Fig 66), and Group 6 (Fig. 67).

[0067] Figs. 68-73 are graphs of toal flux measurements for the harvested organs in each group: Group 1 (Fig. 68), Group 2 (Fig. 69), Group 3 (Fig. 70), Group 4 (Fig. 71), Group 5 (Fig 72), and Group 6 (Fig. 73).

[0068] Fig. 74A is a graph of serum EPO concentrations in minipigs detected 6 hours after injection. Fig. 74B is a graph of serum EPO concentration by group. Fig. 74C is a graph of showing mean EPO concnentration for each group before injection through 24 hours post- inhection.

[0069] Figs. 75A-75E are graphs summarizing various PK parameters analyzed including AUC (Fig. 75A), dose-normalized AUC (Fig. 75B), Cmax (Fig. 75C, dose-normalized Cmax (Fig. 75D), and Tmax (Fig. 75E).

[0070] Figs. 76A-76D are graphs of mRNA distribution in the liver, lymph nodes and injection site for individual animals (Fig. 76A) and averages (Fig. 76B). Relative expression mean (Fig. 76C) and absolute EPO copy number mean (Fig. 76D) are also shown.

[0071] Fig. 77A is graphs showing that SC+PH20 resulted in lower cytokine production at 24 hours for IFN-a and TNF-a compared to SC without PH20.DBl / 162871737.3 11

[0072] Fig. 78 presents graphs illustrating minimal crossing reaction of EPO ELISA- DY286, and R&D systems.

[0073] Fig. 79A shows ventral imaging taken 24 hours after each dose for each group. Fig. 79B shows dorsal imaging taken 24 hours after each dose for each group.

[0074] Fig. 80 is a graph showing the average ventral total flux measurements for each group after each dose.

[0075] Fig. 81 is a graph showing the average dorsal total flux measurements for each group after each dose.

[0076] Fig. 82 is a graph showing the average liver sginal total flux measurements for each group after each dose.

[0077] Fig. 83A shows ventral imaging taken after each dose for each group. Fig. 83B shows dorsal imaging taken after each dose for each group.

[0078] Figs. 84A, 84B, 84C, and 84D are graphs showing ventral flux measurements taken 24 hours after each dose for Groups 1, 2, 3, and 4, respectively. Fig. 84E is a graph showing the average ventral total flux measurements for each group after each dose.

[0079] Figs. 85A, 85B, 85C, and 85D are graphs showing dorsal flux measurements taken 24 hours after each dose for Groups 1, 2, 3, and 4, respectively. Fig. 85E is a graph showing the average dorsal total flux measurements for each group after each dose.

[0080] Figs. 86A, 87B, 87C, and 87D are graphs showing liver signal flux measurements taken 24 hours after each dose for Groups 1, 2, 3, and 4, respectively. Fig. 86E is a graph showing the average liver signal total flux measurements for each group after each dose.

[0081] Fig. 87 shows ventral and dorsal imaging taken after the first dose for each group.

[0082] Fig. 88A is a chart showing dorsal BLI measurements taken after the first dose for each group. Fig. 88B is a chart showing ventral BLI measurements taken after the first dose for each group.

[0083] Figs. 90A-90J are graphs of concentrations of IL- 10 (Fig. 90A), KC / GRO (Fig. 90B), IL-4 (Fig. 90C), IL-6 (Fig. 90D), IL-12p70 (Fig. 90E).

[0084] Fig. 91 are flow cytometry plots for control stain lacking the Ly6G Ab.DBl / 162871737.3 12

[0085] Figs. 92A and 92B are graphs showing 1-way ANOVA with Tukey’s test with group averages (Fig. 92A) and individual data points (Fig. 92B).

[0086] Figs. 93A-93C show flow cytometry plots for individual mice in group 1 (Fig. 93 A), group 2 (Fig. 93B), and gropup 3 (Fig. 93C).DETAILED DESCRIPTIONA. Definitions

[0087] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, GenBank® sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. If there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0088] As used herein the term ‘combination dosing regimen’ refers to a regimen in which at least two components administered together to a patient. For example, the at least two components may be administered concurrently as a single formulation, concurrently as separate formulations, sequentially as separate formulations.

[0089] As used herein, “combination therapy” refers to a treatment in which a subject is given two or more therapeutic agents, such as at least two or at least three therapeutic agents, for treating a single disease.

[0090] As used herein, the term ‘treatment’ or ‘treating’ refers to alleviating the specified condition, eliminating, or reducing the symptoms of the condition, slowing or eliminating the progression, invasion, or spread of the condition and reducing or delaying the reoccurrence of the condition in a previously afflicted subject.DBl / 162871737.3 13

[0091] As used herein, the term ‘prevention’ or ‘preventing’ refers to precluding developing a disease, disorder, or condition or reducing the risk of developing the disease, disorder, or condition or reducing the symptoms thereof.

[0092] As used herein the term ‘injection site reaction’ means side effects at or near the spot where the infusion / injection was received. This includes pain or discomfort, redness, swelling, itching, bruising, lumps, infection complications (cellulitis or abscess), and irritation.

[0093] As used herein, “PH20” refers to a type of hyaluronidase that occurs in mammalian 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.

[0094] As used herein, a truncated PH20 hyaluronidase is any C-terminal shortened form thereof, particularly forms that are truncated and neutral active when N-glycosylated and soluble.

[0095] 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-114 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), 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 intoDBl / 162871737.3 14the 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-heterolog ous (i.e. native) signal sequence.

[0096] 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, 36-492, 36-493, 36- 494, 36-495, 36-496 and 36-497 of SEQ ID NO: 1. 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.

[0097] As used herein, “infusion related reaction” means a type of adverse event that occurs during or after the administration of a pharmacological or biological substance via infusion. These reactions can range from mild to severe and may involve various body systems. Common symptoms include itching, flushing, rash, hives, shortness of breath, wheezing, chest discomfort, changes in blood pressure, rapid heartbeat, dizziness nausea, vomiting and abdominal pain. Symptoms may appear within minutes to hours after infusion and usually within 24 hours. Infusion related reactions may also be referred to as hypersensitivity reactions or administration related reactions.

[0098] As used herein the term “rHuPH20” refers to the soluble hyaluronidase composition produced upon expression in a mammalian cell, such as a CHO cell, or other cell that effects glycosylation, of nucleic acid encoding residues 36-482 of SEQ ID NO:1. For expression in cells the encoding nucleic acid is linked to the native (residues 1-35 of SEQ ID NO: 1 ) or a heterologous signal sequence for trafficking and secretion of the encoded polypeptides. The resulting secreted soluble glycoprotein is a heterogeneous mixture of polypeptides, including polypeptides that terminate at residues 479, 480, 481, and 482, and are composed of residues 36-479, 36-480, 36- 481, and 36-482 with reference to SEQ ID NO:1. Shorter C-terminally truncated forms also may be included. Typically, rHuPH20 is produced in cells, such as CHO cells, for example DG44 CHO cells) that facilitate N-glycosylation.DBl / 162871737.3 15

[0099] 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.

[0100] 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.

[0101] 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 moi eties of human PH20 include amino acids N82, N166, N235, N254, N368 and N393 of human PH20 set forth in SEQ ID NO: 1.

[0102] 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 moi eties 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.

[0103] 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 nonhuman 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.DBl / 162871737.3 16Also 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).

[0104] As used herein, “hyaluronidase activity” refers to the ability to enzymatically catalyse 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 to react with the HA for 30 min at 37 °C (USP XXII-NF XVII (1990) 644-645 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 soluble PH20 and esPH20, are known in the art and described herein. Exemplary assays include the micro turbidity assay that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin and the biotinylated-hyaluronic acid assay that measures the cleavage of hyaluronic acid indirectly by detecting the remaining biotinylated-hyaluronic acid non-covalently bound to microtiter plate wells with a streptavidin-horseradish peroxidase conjugate and a chromogenic substrate. Reference Standards can be used, for example, to generate a standard curve to determine the activity in Units of the hyaluronidase being tested. Another exemplary assay includes a hyaluronidase activity assay that uses native (non-biotinylated) HA, non-covalently bound to microtiter plate wells and the remaining substrate after digestion is detected by the use of biotinylated HA binding protein TSG6-Fc, followed by SA-HRP and chromogenic substrate.

[0105] As used herein, specific activity refers to Units (U) of activity per milligram (mg) of protein unless specified otherwise. The concentration of hyaluronidase is defined by the absorption of a solution thereof at 280 nm, assuming a molar extinction coefficient of approximately 1.7, in units of M-1 cm-1.

[0106] As used herein, “neutral active” refers to the ability of a PH20 polypeptide to enzymatically catalyse the cleavage of hyaluronic acid at neutral pH (e.g. at or about pH 7.0 or at a pH between about pH 6.0 to about pH 7.8).DBl / 162871737.3 17

[0107] As used herein, a “GPI-anchor attachment signal sequence” is a C-terminal sequence of amino acids that directs addition of a preformed GPI-anchor to the polypeptide within the lumen of the endoplasmic reticulum (ER). GPI-anchor attachment signal sequences are present in the precursor polypeptides of GPI-anchored polypeptides, such as GPI-anchored PH20 polypeptides. The C-terminal GPI-anchor attachment signal sequence typically contains a predominantly hydrophobic region of 8-20 amino acids, preceded by a hydrophilic spacer region of 8-12 amino acids, immediately downstream of the co-site, or site of GPI-anchor attachment. GPI-anchor attachment signal sequences can be identified using methods well known in the art, such as but not limited to, in silico methods and algorithms (see, e.g. Udenfriend et al. (1995) Methods Enzymol. 250:571-582, Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758, Fankhauser et al., (2005) Bioinformatics 21: 1846-1852, Omaetxebarria et al., (2007) Proteomics 7: 1951-1960, Pierleoni etal., (2008) BMC Bioinformatics 9:392), including those that are readily available on bioinformatic websites, such as the ExPASy Proteomics tools site (e.g. the World Wide Web site expasy.ch / tools / ).

[0108] As used herein, sequence identity refers to the relatedness between or among polypeptides among nucleic acid molecules. Sequence identity can be assessed by aligning two sequences and counting the number of differences between the aligned portion and the sequence to which it is compared. Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” or “homologous” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux (1984) Nucleic Acids Research 12:387), BLASTP, BLASTN, FASTA (Altschul (1990) J. Mol. Biol. 215:403); Guide to Huge Computers, Bishop, ed., Academic Press, 1994, and Carrillo (1988) SIAM J. Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program. Percent homology or identity of proteins and / or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g. Needleman (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. nucleotides or amino acids), which are similar, divided by the totalDBl / 162871737.3 18number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.[00109J Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide.

[0110] As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e. 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10 / 100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.

[0111] As used herein, an aligned sequence refers to the use of homology (similarity and / or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

[0112] As used herein, “denaturing condition” or “denaturation condition” refers to any condition or agent that, when exposed to a protein, affects or influences the degradation or denaturation of the protein, generally as a result of a loss or partial loss of the tertiary or secondaryDBl / 162871737.3 19structure of the protein. Denaturing conditions can result in effects such as loss or reduction in activity, loss or reduction of solubility, aggregation and / or crystallization.

[0113] As used herein, “resistance to a denaturation condition” refers to any amount of decreased reduction or elimination of a property or activity of the protein associated with or caused by denaturation. For example, denaturation is associated with or causes increased crystallization or aggregation, reduced solubility or decreased activity. Hence, resistance to denaturation means that the protein exhibits decreased aggregation or crystallization, increased solubility or increased or greater activity (e.g. hyaluronidase activity) when exposed to a denaturing condition compared to a reference protein (e.g. unmodified enzyme).

[0114] As used herein, stability of a modified PH20 hyaluronidase means that it exhibits resistance to denaturation caused by a denaturation condition or denaturing agent.

[0115] As used herein, a “payload” is an active substance (e.g., a therapeutic agent, prophylactic agent, nucleic acid or a product thereof, or diagnostic compound) that is transported by a delivery system to a target site within the body. The delivery system (which can be thought of as the “vehicle”) may include nanoparticles, viral vectors, liposomes, antibody-drug conjugates, antibody, protein, peptide, or any fragment thereof. The payload, as used herein, is distinct from the structural or functional components of the delivery mechanism and in various embodiments ensures stability, targets specificity, and controls release of the therapeutic agent.

[0116] As used herein, a “linker” is a chemical or molecular segment that covalently or non-covalently joins two or more biomolecules (e.g., proteins, peptides, nucleic acids, and small molecules) into a single functional entity. This connecting component is designed to provide structural flexibility, maintain stability, and often incorporate specific cleavage sites to enable controlled interactions or the release of connected components when required. Linkers can be an essential modular component used to connect two or more biomolecules without interfering with their individual functions. Its design can be customized for flexibility, fixed orientation, or controlled release, depending on the application. Whether in fusion proteins, ADCs, or nucleic acid constructs, the incorporation of an appropriate linker preserves the biological activity, enhancing stability, and achieving targeted interactions in a range of biomedical applications.

[0117] As used herein, a “tissue” is a structurally organized group of similar cells and their associated extracellular matrix that work together to perform specific functions vital for an organism’s survival. The structural and functional complexities of tissues underpin theDBl / 162871737.3 20formation of organs and the integrated systems that sustain life, making them a central subject in biomedical research and tissue engineering.

[0118] As used herein, “blood” or “whole blood” is the complete fluid circulating in the body, composed of formed elements (red blood cells, white blood cells, platelets) suspended in plasma. It is a vital bodily fluid in humans and most animals that delivers essential substances, such as oxygen, nutrients, and hormones, to cells while removing waste products like carbon dioxide and toxins. It is composed of plasma (a liquid that contains water, proteins, and other dissolved substances) and cellular components, including red blood cells, white blood cells, and platelets. Blood circulates through the body via the cardiovascular system, driven by the pumping action of the heart. It plays a critical role in maintaining homeostasis, immune defense, and regulating body temperature. In embodiments of the present disclosure, “blood” may be used to refer to serum or plasma. One of skill in the art would understand that the analytical measurements used herein, including pharmacokinetic measurements such as AUC, Tmax, and Cmax, may use the term “blood” when referring to serum or plasma.

[0119] As used herein, “plasma” is the liquid matrix of blood, primarily composed of water, and containing proteins, salts, hormones, nutrients, waste products, and clotting factors (like fibrinogen) responsible for transporting blood cells and various substances throughout the body. Plasma is the liquid component of blood that serves as the medium for transporting nutrients, hormones, waste products, and other substances throughout the body. It makes up about 55% of total blood volume and consists primarily of water (about 90%), along with dissolved proteins (such as albumin, fibrinogen, and globulins), electrolytes, glucose, lipids, gases (like oxygen and carbon dioxide), and waste products. Plasma also contains clotting factors, which play a crucial role in blood coagulation. It is essential for maintaining blood pressure, regulating body temperature, and supporting the immune system by transporting antibodies and other defense molecules.

[0120]

[0121] As used herein, “serum” refers to the fluid component of blood that remains after coagulation has occurred and the clotting factors have been consumed in the formation of a clot. Serum is distinct from plasma in that it lacks clotting proteins such as fibrinogen, but otherwise contains substantially similar soluble constituents as plasma, including electrolytes, hormones, antibodies, and proteins such as albumin..DBl / 162871737.3 21

[0122] As used herein, “therapeutically effective dose” (TED) is the amount of a drug, therapeutic agent, or prophylactic agent that produces at least one intended beneficial effect in a patient under defined clinical conditions. This dose is determined during clinical development and is critical for achieving the desired therapeutic outcome while minimizing adverse effects. As used herein, the term “therapeutic agent” refers to any compound, composition, molecule, macromolecule, or biological material that produces a beneficial effect in the treatment, management, or amelioration of a disease, disorder, or condition in a subject. Therapeutic agents include, but are not limited to, nucleic acids (e.g., mRNA, siRNA, antisense oligonucleotides, phosphorodiamidate morpholino oligonucleotides, DNA, circular RNA, self-replicating RNA), proteins, peptides, antibodies, antibody fragments, antibody-drug conjugates, aptamers, small molecules, carbohydrates, lipids, and combinations thereof. The term “therapeutic agent” may be used synonymously and interchangeably with the term “therapeutic.”

[0123] As used herein, the term “prophylactic agent” refers to any compound, composition, molecule, macromolecule, or biological material that produces a beneficial effect in preventing or reducing the likelihood, incidence, or severity of a disease, disorder, or condition in a subject prior to the onset of symptoms or pathology. Prophylactic agents include, but are not limited to, vaccines, nucleic acids (e g., mRNA, DNA, self-replicating RNA), proteins, peptides, antigens, antibodies, antibody fragments, aptamers, small molecules, carbohydrates, lipids, and combinations thereof. The term “prophylactic agent” may be used synonymously and interchangeably with the term “prophylactic.” In certain contexts, it is understood that the term “therapeutic agent” may be used broadly as an umbrella term understood to include therapeutic agents and / or prophylactic agents for the sake of clarity and conciseness.

[0124] As used herein, the term “therapeutic agent” or “therapeutic composition” can further refer to an agent or composition that provides a desired biological or pharmacological effect when administered to a human or animal. The therapeutic agent or composition may be a small molecule, biological agent, protein, antibody, mimetibody, mAb, antibody fragment (including a diabody, triabody, or tetrabody), peptide, polypeptide, enzyme, nucleotide, DNA fragment, RNA fragment, plasmid fragment, nucleotide fragment, gene therapy vector, virus, mRNA, nucleic acid conjugate, oligonucleotide, antisense oligonucleotide (ASO), phosphorodiamidate morpholino oligonucleotide (PMO), aptamer, siRNA, RNAi, miRNA,DBl / 162871737.3 22DNA, self-replicating RNA (replicons), Naked RNA, Circular RNA (circRNA), Trans-activating crRNA (TracrRNA), and gene editingor mixtures thereof.

[0125] As used herein, the term “equivalent dose” refers to the amount of a therapeutic agent that, when administered, produces a comparable level of clinical effect — be it efficacy or toxicity — to a reference treatment. This ensures consistent patient outcomes, particularly in the evaluation of generic products and the adjustment of dosing regimens for individualized therapies. An equivalent dose can be the actual equivalent dose to a reference treatment or a similar dose to a reference treatment. In non-limiting examples, a similar dose can be 80% to 125%, 85% to 120%, 90% to 115%, 95% to 110%, 95% to 105%, or 100% of the reference treatment dose.

[0126] As used herein, “area under the curve (AUC)” refers to the area under a curve of the plasma or serum concentration versus time, indicative of the systemic exposure to the drug over a defined period of time. The measured AUC value is dependent on a specific interval of time duration over which plasma or serum concentrations are measured and reflects the extent of drug absorption and bioavailability within that specified duration interval.

[0127] As used herein, “maximum concentration (Cmax)” refers to the maximum systemic exposure of the drug achieved after administration, reflecting the peak plasma or serum concentration of the drug at a single time point. The value of Cmax is dependent on factors that influence the rate and extent of drug absorption, including but not limited to the administered dose and the route of administration (e g., subcutaneous, intravenous, oral).

[0128] As used herein, “subject” refers to an individual, organism, or entity that is being studied, observed, or experimented upon. Subjects can range from mammals (e.g., humans and non-human primates), birds, reptiles, amphibians, and other species.

[0129] As used herein, “antibody” refers to a molecule which comprises or contains: (a) one or more immunoglobulin variable domains; or (b) fragments, variants, modifications or derivatives of such immunoglobulin variable domains irrespective of origin or source, including but not limited to antigen binding portions including Fab, Fab’, F(ab’)2, Fv, dAb and CDR fragments, single chain antibodies (scFv), chimeric antibodies, monospecific antibodies, multispecific antibodies, diabodies and polypeptides (including humanized versions thereof) that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to a polypeptide.DBl / 162871737.3 23

[0130] As used herein, “antibody-drug-conjugate” or “ADC” or “Total ADC” is a specialized molecule composed of an antibody, which is designed to target specific cell types, chemically linked to one or more payloads that can be cytotoxic or have other therapeutic effects. ADCs also include one or more linkers that connect the antibody to the payload(s), enabling precise delivery of the therapeutic agent to the target cells or tissues.

[0131] As used herein, “site of administration” refers to the specific anatomical location where a therapeutic agent, such as a drug or biologic, is introduced into the body for absorption or action. This can encompass various layers of tissue depending on the route of delivery, including the epidermis (outermost layer of the skin), dermis (middle layer containing connective tissue, blood vessels, and nerves), hypodermis (also known as the subcutaneous tissue, which consists of fat and connective tissue beneath the dermis), and the subcutaneous space (the broader region within the hypodermis where subcutaneous injections are typically administered). The site of administration can be chosen based on factors such as the formulation of a drug selected to be administered, the desired absorption rate, and patient-specific considerations.

[0132] As used herein, the term “anionic lipid” means a lipid that is negatively charged at physiological pH. These lipids may include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoylol eyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0133] As used herein, 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 (e.g., positively charged) at a pH below its pKaand 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 amino exemplary amino lipid can include Cis alkyl chains, wherein each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3)DBl / 162871737.3 24double 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 -Bl 1).

[0134] As used herein, term “fully encapsulated” means that the nucleic acid (e.g., mRNA, siRNA, DNA, and so on) in the nucleic acid-lipid particle is adequately sequestered within the particle such that exposure to a serum or nuclease assay (e g.., containing components that would significantly degrade free nucleic acid) does not demonstrate significant degradation of the nucleic acid. The nucleic acid is considered “adequately sequestered” (and accordingly, fully encapsulated) in response to a measured degradation result of preferably less than 25% degradation, more preferably less than 10% degradation, and most preferably, less than 5% degradation of the nucleic acid in the particle following exposure to a treatment that can degrade 100% of a free nucleic acid control. As used herein, “fully encapsulated” can also refer to nucleic acid-lipid particles that do not rapidly decompose into their component parts upon in vivo administration.

[0135] As used herein, the term “delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0136] As used herein, the term “delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide to targeted cells.

[0137] As used herein, 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.

[0138] As used herein, 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.DBl / 162871737.3 25

[0139] As used herein, 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 can be 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.

[0140] As used herein, term “lipid delivery vehicle” means a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA, siRNA, DNA, and so on) 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. The therapeutic nucleic acid (e.g., mRNA, siRNA, DNA, and so on) may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0141] As used herein, 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.

[0142] As used herein, 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 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.

[0143] As used herein, the terms “amphipathic lipid” or “amphiphilic lipid” mean 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 unsaturatedDBl / 162871737.3 26aliphatic 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.

[0144] As used herin, 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.

[0145] As used herein, the term “non-cationic lipid” means an amphipathic lipid or a neutral lipid or anionic lipid and is described herein.

[0146] As used herein, the term “equivalent” means comparable, similar, or equal in value, amount, function, content, substance, material, denomination, number, quality, and any other criterion or characteristic.

[0147] The term “targeting agent” means an agent designed to deliver a drug specifically to a designated site within the body, such as a particular organ, tissue, or type of cell.

[0148] The term “composition” means a mixture of substances suitable for administering to a subject that includes a therapeutic agent.

[0149] The term “particles” means small particles, often in the nanometer range, which transport therapeutic agents through the bloodstream to specific target locations within the body. A “nanoparticle”, as used herein, is a submicron particle having any structure or morphology. Submicron particles may also be referred to as colloids, or colloidal. With respect to the material on which the nanoparticle is based, and to the structure or morphology, a nanoparticle may be classified, for example, as a nanocapsule, a vesicle, a liposome, a lipid nanoparticle, a micelle, a crosslinked micelle, a lipoplex, a polyplex, a mixed or hybrid complex, to mention only a few of the possible designations of specific types of nanoparticles. A “lipid nanoparticle” (LNP) is a nanoparticle formed by lipids, typically including at least one amphiphilic, membrane-forming lipid, and optionally other lipids, further optionally including a cargo material such as a nucleic acid compound. As used herein, the expression “lipid nanoparticles” or “LNP” includes any subtypes and morphologies of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes. A “liposome” is an artificial vesicle composed of one or more concentric phospholipidDBl / 162871737.3 27bilayers. A “cholestosome” is a cholesteryl ester nanoparticle with a hollow compartment. A cholestosome may comprise at least one non-ionic cholesteryl ester and a neutral surface.

[0150] The term “exosome” as used herein, refers to a membranous particle having a size (e.g., as measured by diameter for spheroid particles or a largest dimension for nonspheriod particles) of between about 10 nm to about 5000 nm, more typically between 30 nm and 1000 nm, and most typically between about 50 nm and 750 nm, wherein at least part of the membrane of the exosome is directly obtained from a cell. Most commonly, exosomes will have a size (e.g., as measured by average diameter) that is up to 5% of the size of the donor cell. Therefore, especially contemplated exosomes include those that are shed from a cell.

[0151] As used herein, “viral particles refer to viral structures that are genetically encoded and capable of self-assembling into discrete, generally monodisperse particles that can be classified based on size and / or shape. Viral particles typically contain a viral genome encapsulated within a protein capsid and, in some cases, a lipid envelope. The viral genome may be modified to include heterologic nucleic acid molecules to leverage the virus as a vector for gene modulation. Exemplary viral particles can include, but are not limited to, adenovirus, lentivirus, adeno- associated virus (AAV), retrovirus, and other recognizable naturally occurring or engineered viruses suitable for use as delivery systems.

[0152] As used herein, “virus-like particles” (VLPs) refer to multiprotein assemblies that mimic the structure, organization and conformation of authentic viruses but lack a true viral genome. Because VLPs do not contain replicative genetic material, VLPs are noninfectious and may be used as delivery systems and / or vaccine platforms. Exemplary VLPs can include recombinant hepatitis B surface antigen particles, human papillomavirus (HPV) VLPs, synthetic VLPs, and so on.

[0153] As used herein, “cochleates” refer to solid particulates made up of large continuous lipid bilayer sheets rolled up in a spiral structure with little or no internal aqueous phase. Cochleates are nano-sized or sub-micron sized structures and are generated on fusion of negatively charged liposomes with metal cations.

[0154] As used herein, “DNA nanostructures” refer to nanostructures composed of DNA molecules and are assembled into precise and programmable structures. DNA nanostructures use hydrogen binding or molecular recognition instead of charging interactions to condense functional nucleic acids and form delivery complexes.DBl / 162871737.3 28

[0155] As used herein, the term “delivered dose” means total amount of a therapeutic agent that a subject receives during a treatment period. The delivered dose may refer to the amount of the therapeutic agent transported to an individual organ, tissue, or fluid of interest.

[0156] As used herein, the term “therapeutic dose” or “therapeutic intravenous dose” refers to the amount of a drug, therapeutic agent or active compound that is administered to achieve the desired therapeutic effect in treating a specific condition, disease, or disorder, while minimizing potential side effects or toxicity. A therapeutic dose can include a range of dosages that those practicing in the field would believe to be effective, safe, and / or suitable for administration to a patient. Specific therapeutic doses can be measurable units (e.g., milligrams (mg), micrograms (pg), milligrams per kilogram of body weight (mg / kg), or molarity) for a particular route of administration (e.g., oral, intravenous, topical), at a specific frequency (e.g., once daily, twice weekly), for a specific length of time (e.g., a single dose, over several weeks, or as a continuous regimen), and / or for a specific target patient population (e.g., adults, children, specific disease groups).

[0157] As used herein, the term “residence time” means the time a drug spends in a compartment, tissue or area of the body or in contact with its biological target.

[0158] As used herein, the term “systemic circulation” refers to the overall process of distributing oxygenated blood, nutrients, hormones, and other essential substances throughout the entire body, and simultaneously collecting deoxygenated blood and metabolic waste products for transport back to the heart. Systemic circulation can refer to the overarching circulatory pathway that ensures all body tissues receive the necessary supplies transported by whole blood, which includes both the cellular and fluid components (plasma and serum). This vital process involves blood, plasma and serum.

[0159] As used herein, “site of action” refers to the specific anatomical location, encompassing tissues, organs, or individual cells (including, for instance, tumor tissue or specific target cells), where a drug or therapeutic agent exerts its intended biological or pharmacological effect.

[0160] Unless otherwise stated, the chemical structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds where one or more hydrogen atoms is replaced by deuterium or tritium,DBl / 162871737.3 29or wherein one or more carbon atoms is replaced by13C-enriched or14C-enriched carbons, are within the scope of this invention.

[0161] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.B. Hyaluronidases

[0162] Hyaluronidases are a family of enzymes that degrade hyaluronan. The three main types of hyaluronidases are two classes of eukaryotic endoglycosidase hydrolases and a prokaryotic lyase-type of glycosidase. Hyaluronidases are hyaluronoglucosidases, which means they cleave the (1 — 4)-l inkages between N-acetylglucosamine and glucuronate. The term hyaluronidase may also refer to hyaluronoglucuronidases, which cleave ( l ^3)-linkages. Further, bacterial hyaluronate lyases may also be referred to as hyaluronidases.

[0163] Neutral active 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).

[0164] 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.

[0165] 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.1. Soluble Hyaluronidases

[0166] Soluble hyaluronidases include any that, upon expression, are secreted from a cell and exist in soluble form. Such soluble hyaluronidases include, for example, but are not limited to, bacterial soluble hyaluronidases, non-human soluble hyaluronidases, such as bovine PH20 and ovine PH20, human soluble PH20, and variants thereof. Generally soluble forms of PH20 areDBl / 162871737.3 30produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

[0167] Soluble PH20 hyaluronidase is available and sold, for example, under the trademark ENHANZE®. ENHANZE® technology provides to a drug delivery technology, employing the soluble hyaluronidases to facilitate the delivery of injected drugs and fluids. When co-formulated with other drugs or administered with other drugs, the ENHANZE® technology reduces treatment burden for patients. It can allow for large volume subcutaneous injections with increased dispersion and absorption of co-administered therapies.

[0168] 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: 26, generally linked to the native or a heterologous signal sequence (residues 1-35 of SEQ ID NO: 26). rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO: 26) 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, 36-481, and 36-482 of SEQ ID NO: 26, and some shorter polypeptides, in various abundance. rHuPH20 and forms of soluble hyaluronidase are produced in cells, such as CHO cells, for example DG44 CHO cells, that facilitate N-glycosylation. PH20 is a glycoprotein, and as known in the art, requires glycosylation retain activity. See, e.g. U.S. Patent Nos. 8,927,249 and 9,284,543 (and PCT Publication No. WO 2010 / 077297), which describe the effects of glycosylation and partial glycosylation and elimination of glycosylation on the activity of soluble forms of PH20. These patents and publications also describe and exemplify I soluble C-terminally truncated forms of PH20.2. Forms of Soluble Human PH 20

[0169] Soluble hyaluronidases include bovine and ovine PH20, and recombinant and humanized forms thereof. Human PH20 in nature includes a GPI anchor and exists linked to sperm cells; it is not soluble. C-terminally-truncated forms thereof are soluble. Soluble forms of recombinant human PH20 have been produced and can be used in the compositions, combinations and methods described herein. Descriptions of and production of such solubleDBl / 162871737.3 31forms ofPH20 are described, for example, in U.S. Patent Nos. 7,767,429; 8,202,517; 8,431 ,380; 8,431,124; 8,450,470; 8,765,685; 8,772,246; 7,871,607; 7,846,431; 7,829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061; and 9,677,062, each incorporated by reference herein. The soluble hyaluronidases, thus include forms of human PH20, which are neutral active hyaluronidases and which require glycosylation for activity.

[0170] SEQ ID NO: 1 sets forth the sequence of the precursor polypeptides; the mature PH20 polypeptide (residues 36-509); soluble forms also include those with amino acid truncations at the N-terminal, such as deletions of the first one, two, three, or fours residues, such that the resulting polypeptides have an N-terminus, for example, at residue 36, 37, 38, 39, or 40, and a C-terminus at a residue from 465 to 500, and variants thereof, including, but not limited to, variants discussed below, variants known in the art, and allelic variants.

[0171] Hyaluronidases for use in the compositions, combinations and methods herein are soluble neutral active hyaluronidases. Exemplary thereof are the soluble C-terminally truncated forms of mature human PH20. Soluble forms that have hyaluronidase activity, include but are not limited to, those that are truncated at residues from 465 to 500 of sequence ID No.1, and that are, upon expression, secreted. Exemplary thereof are polypeptides that have sequence 36-465 of SEQ ID NO: 1, 36-466 of SEQ ID NO: 1, 36-467 of SEQ ID NO: 1, 36-468 of SEQ ID NO: 1, 36-469 of SEQ ID NO: 1, 35-470 of SEQ ID NO: 1, 36-471 of SEQ ID NO: 1, 36-472 of SEQ ID NO: 1 , 36-474 of SEQ ID NO: 1, 36-475 of SEQ ID NO: 1 , 36-476 of SEQ ID NO: 1, 35-477 of SEQ ID NO: 1, 36-478 of SEQ ID NO: 1, 36-479 of SEQ ID NO: 1, 36-480 of SEQ ID NO: 1, 36-481 of SEQ ID NO: 1, 36-482 of SEQ ID NO: 1, 36-483 of SEQ ID NO: 1, 35-484 of SEQ ID NO: 1, 36-485 of SEQ ID NO: 1, 36-486 of SEQ ID NO: 1, 36-487 of SEQ ID NO: 1, 36-488 of SEQ ID NO: 1, 36-489 of SEQ ID NO: 1, 36-490 of SEQ ID NO: 1, 35-491 of SEQ ID NO: 1, 36-492 of SEQ ID NO: 1, 36-493 of SEQ ID NO: 1, 36-494 of SEQ ID NO: 1, 36-495 of SEQ ID NO: 1, 36-496 of SEQ ID NO: 1, 36-497 of SEQ ID NO: 1, 35-498 of SEQ ID NO: 1, 36-499 of SEQ ID NO: 1, and 36-500 of SEQ ID NO:1, as well as N-terminally truncated forms of each of the preceding that lack two to five residues at the N-terminus, such as for example 37-368 of SEQ ID NO: 1, 38-468 of SEQ ID NO: 1, and any others that exhibit hyaluronidase activity at neutral pH, such as pH in the range of 7.0-7.4.

[0172] Thus, such soluble forms include truncated forms of the mature form of human PH20 lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase isDBl / 162871737.3 32soluble and retains hyaluronidase activity. Soluble forms are secreted upon expression in mammalian cells, and are encoded with a signal sequence, such are residues 1-35 of SEQ ID NO. 1 or a heterologous signal sequence that is cleaved by the cell to effect secretion. Soluble forms are forms that, when expressed in a cell, lack the signal peptide. Also included among soluble hyaluronidases are variants of the soluble PH20 polypeptides that exhibit hyaluronidase activity. Variants include polypeptides having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the PH20 polypeptides 36-465 of SEQ ID NO: 1, 36-466 of SEQ ID NO: 1, 36-467 of SEQ ID NO: 1, 36-468 of SEQ ID NO: 1, 36-469, 35-470 of SEQ ID NO: 1, 36-471 of SEQ ID NO: 1, 36-472 of SEQ ID NO: 1, 36-474 of SEQ ID NO: 1, 36-475 of SEQ ID NO: 1, 36-476 of SEQ ID NO: 1, 35-477 of SEQ ID NO: 1, 36-478 of SEQ ID NO: 1, 36-479 of SEQ ID NO: 1, 36-480 of SEQ ID NO: 1, 36-481 of SEQ ID NO: 1, 36-482 of SEQ ID NO: 1, 36-483 of SEQ ID NO: 1, 35-484 of SEQ ID NO: 1, 36-485 of SEQ ID NO: 1, 36-486 of SEQ ID NO: 1, 36-487 of SEQ ID NO: 1, 36-488 of SEQ ID NO: 1, 36-489 of SEQ ID NO: 1, 36-490 of SEQ ID NO: 1, 35-491 of SEQ ID NO: 1, 36-492 of SEQ ID NO: 1, 36-493 of SEQ ID NO: 1, 36-494 of SEQ ID NO: 1, 36-495 of SEQ ID NO: 1, 36-496 of SEQ ID NO: 1, 36-497 of SEQ ID NO: 1, 35-498 of SEQ ID NO: 1, 36-499 of SEQ ID NO: 1, and 36-500 of SEQ ID NO: 1. Amino acid variants include conservative and non-conservative insertions, or deletions, or replacements, and include the modifications, singly or combinations of the modifications detailed, for example, in U.S. Patent No. 11,041, 149 and International PCT publication No. WO 2013 / 102144. U.S. Patent No. 11,041,149 and International PCT publication No. WO 2013 / 102144 describe a systematic analysis and results identifying the effects of amino acid modifications at each residue in PH20 to thereby provide a structure / function map of PH20; a skilled person can identify replacement residues and consequent alterations in properties and activities, such as for effecting increases in enzymatic activity, stability in denaturing conditions, and also residues whose replacement or deletion decreases or eliminates enzymatic activity.

[0173] 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 those of skill in the art, are generally invariant and, except for possible conservative amino acid substitutions, cannot be changed. These include, for example, active site residues. For example, amino acid residues 111, 113 and 176 (corresponding to residues in the mature PH20 polypeptide) of a human PH20 polypeptide, or soluble form thereof, are generally invariant and are not altered. Other residuesDBl / 162871737.3 33that confer glycosylation and formation of disulfide bonds required for proper folding also can be invariant.

[0174] The soluble human PH20 hyaluronidase is GPI-anchored and is rendered soluble by truncation at the C-terminus by removal of all or a part of the GPI anchor. Such truncation can remove all of the GPI anchor attachment sequence or can remove only some of the GPI anchor attachment 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).[00175J Extended soluble hyaluronidases, which terminate for example, at residues 495, 496, 497, 498, 499, and 500, with reference to 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, contain amino acid substitutions, and have at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%.

[0176] Typically, for use in the compositions, combinations and methods herein, a soluble human hyaluronidase, such as a soluble human PH20, is used, such as a PH20 and variants having, for example, at least 91% or 95% or 98% sequence identity thereto, including those with 1 to 5 N-terminal residues deleted. Hyaluronidases used in the regimens, combinations, compositions, and methods herein 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.DBl / 162871737.3 34

[0177] 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), 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 of the sequence of amino acids set forth in SEQ ID NO: 1, 468 of the sequence of amino acids set forth in SEQ ID NO: 1, 469 of the sequence of amino acids set forth in SEQ ID NO: 1, 470 of the sequence of amino acids set forth in SEQ ID NO: 1, 471 of the sequence of amino acids set forth in SEQ ID NO: 1, 472 of the sequence of amino acids set forth in SEQ ID NO: 1, 473 of the sequence of amino acids set forth in SEQ ID NO: 1, 474 of the sequence of amino acids set forth in SEQ ID NO: 1, 475 of the sequence of amino acids set forth in SEQ ID NO: 1, 476 of the sequence of amino acids set forth in SEQ ID NO: 1, 477 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 39), 478 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 40), 479 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 41), 480 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 42), 481 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 43), 482 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 3), 483 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 44), 484 of the sequence of amino acids set forth in SEQ ID NO: 1, 485 of the sequence of amino acids set forth in SEQ ID NO: 1, 486 of the sequence of amino acids set forth in SEQ ID NO: 1, 487 of the sequence of amino acids set forth in SEQ ID NO: 1, 488 of the sequence of amino acids set forth in SEQ ID NO: 1, 489 of the sequence of amino acids set forth in SEQ ID NO: 1, 490 of the sequence of amino acids set forth in SEQ ID NO: 1, 491 of the sequence of amino acids set forth in SEQ ID NO: 1, 492 of the sequence of amino acids set forth in SEQ ID NO: 1, 493 of the sequence of amino acids set forth in SEQ ID NO: 1, 494 of the sequence of amino acids set forth in SEQ ID NO: 1, 495 of the sequence of amino acids set forth in SEQ ID NO: 1, 496 of the sequence of amino acids set forth in SEQ ID NO: 1, 497 of the sequence of amino acids set forth in SEQ ID NO: 1, 498 of the sequence of amino acids set forth in SEQ ID NO: 1, 499 of the sequence of amino acids set forth in SEQ ID NO: 1 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, when aligned with the unmodified sequence of the soluble PH20, have activity at neutral pH, and are solubleDBl / 162871737.3 35(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 and terminate at any of residues, 465-500 and optionally include a 1-3 amino acid deletion at the N-terminus (i.e. lack residues 36, 36-37, or 36-38 of SEQ ID NO: 1). For example, when expressed in mammalian cells, the 35 amino acid N-terminal signal sequence (residues 1-35 of SEQ ID NO: 1) is cleaved during processing, and a soluble form of the protein is secreted. Thus, the mature soluble polypeptides include those that contain amino acids 36 to 467 of SEQ ID NO: 1, 468 of SEQ ID NO: 1, 469 of SEQ ID NO: 1, 470 of SEQ ID NO: 1, 471 of SEQ ID NO: 1, 472 of SEQ ID NO: 1, 473 of SEQ ID NO: 1, 474 of SEQ ID NO: 1, 475 of SEQ ID NO: 1, 476 of SEQ ID NO: 1, 477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 9), 478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), 479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), 480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), 481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), 482 of SEQ ID NO: 1 (i.e , SEQ ID NO: 4), 483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46), and up to and including 500 of SEQ ID NO: 1. Exemplary of soluble hyaluronidases are soluble human PH20 polypeptides that are 442 (i.e., SEQ ID NO: 9), 443 (i.e., SEQ ID NO: 8), 444 (i.e., SEQ ID NO: 7), 445 (i.e., SEQ ID NO: 6), 446 (i.e., SEQ ID NO: 5) or 447 (i.e., SEQ ID NO: 4) amino acids in length, such as set forth those set forth above, 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 thereto and retains hyaluronidase activity. The generation of such soluble forms of recombinant human PH20 are described, for example, in U.S. Patent No. 7,767,429; 8,202,517; 8,431,380; 8,431,124; 8,450,470; 8,765,685; 8,772,246; 7,871,607; 7,846,431; 7,829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061; and 9,677,062.

[0178] Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

[0179] The composition that recombinantly produced from mammalian cells, such as CHO cells, has been referred to rHuPH20. It 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 (i.e., SEQ ID NO: 4), generally linked to the native (residues 1-35 of SEQ ID NO: 1; residues 1-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 3) or a heterologous signal sequence. rHuPH20 isDBl / 162871737.3 36produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO: 1; residues 1-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 3) or 36 to 482 (residues 36-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 4) with a heterologous signal sequence. Post translational processing removes the 35 amino acid signal sequence, resulting in polypeptide or a mixture of polypeptides, including those set forth in SEQ ID NOs: 4-8. 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 SEQ ID NOs: 4-8 in various abundance. Generally, the soluble hyaluronidases, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHO cells). Human soluble PH20 hyaluronidase requires glycosylation for activity. When produced recombinantly from a vector encoding residues 36-582, the most abundant species is the 446 amino acid polypeptides corresponding to residues 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5). The particular distribution of resulting polypeptides can depend upon the particular method of production. An exemplary method for production of high levels of PH20 is detailed, for example in U.S. Patent Nos. 8,187,855 and 8,343,487.3. Glycosylation of Hyaluronidases

[0180] Glycosylation, including N- and O-linked glycosylation, of some hyaluronidases, including the soluble PH20 hyaluronidases, can be important for their catalytic activity and stability. For some hyaluronidases, removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N- linked glycans can be important for generating an active enzyme.

[0181] 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-linked oligosaccharides. There are six potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393 of human PH20 exemplified in SEQ ID NO: 1.VariantsDBl / 162871737.3 37

[0182] As discussed above, variants of PH20 are known to those of skill in the art, or readily can be prepared in view of the skill and knowledge in the art. Variants include those with amino acid replacements, insertions, and deletions. Variants of the soluble PH20 polypeptides that have altered properties, such as increased stability and / or activity, have been produced. U.S. Patent No. 9,447,401 and family members U.S. Patent Nos. 10,865,400, 11,041,149 and11,066,656 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 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 those patents, and earlier publications / patents, describing virtually all variants of soluble PH20 polypeptides are known in the art. A skilled person readily can prepare soluble hyaluronidases and variants thereof and know the properties of the resulting hyaluronidase.

[0183] Other variants also are known to those of skill in the art, and can be used in the combinations, regimens, and methods described herein. For example, see, International PCT Publication No. W02020 / 022791 and W02020197230A which are incorporated by reference, and which describe modified PH20 polypeptides. These polypeptides, which include variants of the PH20 polypeptides that generally span residues 38-468, and include replacements, insertions, and deletions. The variants include for example one or more amino acid residues changes S343E, I344N, M345T, M348K, K349E, L353A, L354I, N356E, and I361T (with reference to SEQ ID NO: 1), and others, including about 15 amino acid variations, and truncations at the N-terminus and C-terminus. Variants that contain such modifications and others are set forth in SEQ ID NO: 60-115 of International PCT publication No. W02020 / 022791. Exemplary of these polypeptides is the polypeptide of SEQ ID NO:99, therein. International PCT Publication No.W02021 / 150079 provides variant PH20 polypeptides described as having increased stability relative to unmodified PH20, such as those in rHuPH20. These variant polypeptides have been shown to have PH20 activity and are described as having use for subcutaneous co-administration with other agents.C. Lipid Nanoparticle Formulations

[0184] In an embodiment, the present disclosure provides a lipid nanoparticle (LNP) encapsulating a therapeutic and / or prophylactic. In some embodiments, the therapeutic and / or prophylactic comprises a nucleic acid, an antigen, an antibody-drug conjugate (ADC), a nucleicDBl / 162871737.3 38acid editing system, or a protein. Tn an embodiment, the nucleic acid comprises DNA. In an embodiment, the nucleic acid is antisense oligonucleotide (ASO). In an embodiment, the nucleic acid comprises RNA. In an embodiment, the RNA is messenger RNA (mRNA). In an embodiment, the RNA is small interfering RNA (siRNA). In one embodiment, the RNA is small interfering RNAi. In some embodiments, the nucleic acid, antigen, or protein is commercially available. In some embodiments, the RNA is circular, self-amplifying or micro-amplifying, RNA. In an embodiment, the LNP encapsulates a nucleic acid editing system used in gene editing. In one embodiment, the nucleic acid editing system used in gene editing is a zinc finger nuclease, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing incorporating Cas9, guide RNA (gRNA), base editing, or prime editing.

[0185] In an embodiment, the LNP comprises a liposome. In one embodiment, the liposome comprises a cationic liposome. In an embodiment, the cationic liposome is used for CRISPR / Cas9 delivery. In one embodiment, the liposome comprises a hybrid liposome. Hybrid liposomes include other elements, such as exosomes or mesoporous silica, to improve delivery efficiency. In one embodiment, the liposome comprises a Fusogenic liposome. Fusogenic liposomes are designed to help CRISPR components escape the endosomal and lysosomal compartments. In one embodiment, the liposome comprises a PEGylated liposome. Although not wishing to be limited by theory, PEGylated liposomes have polyethylene glycol (PEG) chains attached to their surfaces, which improves their stability and circulation time in the body. In one embodiment, the liposome comprises a multifunctional liposome. Multifunctional liposomes have multiple compartments and can be engineered for various applications, such as targeting, imaging, and therapy. In one embodiment, the liposome comprises a stimuli- responsive liposome. Stimuli-responsive liposomes can be engineered to respond to stimuli, such as light, ultrasound, magnetic fields, or enzymes, to release drugs, genes, or bioactive gases at the target site. In one embodiment, the liposome comprises an exosome. In an embodiment, the exosome delivers a CRISPR / Cas9 system.I. Lipid Nanoparticles Preparation and Characteristics

[0186] Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in at least WO2017 / 019935, WO2017 / 049074, WO2017 / 201346, WO2017 / 218704, WO2018 / 006052, WO2018 / 013525, WO2018 / 089540, WO2018 / 119115, WO2018 / 126084, WO2018 / 157009, WO2018 / 170336, WO2018 / 222890, WO2019 / 046809, WO2019 / 089828,DBl / 162871737.3 39W02020 / 061284, W02020 / 061317, W02020 / 081938, W02020 / 097511 , W02020 / 097520, W02020 / 097540, W02020 / 097548, W02020 / 214946, W02020 / 219941, WO2020 / 232276, WO2020 / 227615, W02020 / 061295, WO2021 / 007278, W02021 / 016430, WO2021 / 021988, EP Patent No. EP 2 972 360, US20200155691, US20200237671, U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,404,127, 9,504,651, 9,593,077, 9,738,593, 9,868,691, 9,868,692, 9,950,068, 10,138,213, 10,166,298, 10,221,127, 10,238,754, 10,266,485, 10,383,952, 10,730,924, 10,766,852, 11,079,379, 11,141,378 and 11,246,933, which are incorporated herein by reference in their entirety for all purposes.

[0187] In some embodiments, the largest dimension of the LNPs disclosed herein is 1 micrometer or shorter (e.g., 1 micrometer, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method.

[0188] In an embodiment, the LNPs have a diameter of about 300 nm or less, about 275 nm or less, about 250 nm or less, about 225 nm or less, about 200 nm or less, about 175 nm or less, about 150 nm or less, about 125 nm or less, about 100 nm or less, about 75 nm or less, about 50 nm or less, or about 25 nm or less. In various embodiments, a plurality of LNPs have a mean diameter of from about 30 nm to about 250 nm, from about 40 nm to about 225 nm, from about 50 nm to about 225 nm, from about 60 nm to about 225 nm, from about 70 nm to about 225 nm, from about 70 nm to about 200 nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm, from about 100 nm to about 200 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. In one embodiment, the LNPs are substantially non-toxic.2. Lipid Components

[0189] In some embodiments, the LNPs disclosed herein are vesicles including one or more lipid bilayers. In certain embodiments, the vesicles include two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and / or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels. In certain embodiments, the invention described herein is used to increase the amount of monoclonal antibody transported into the lymphatic system and lymphatic nodes. In theseDBl / 162871737.3 40embodiments, upon administration to a subject, the monoclonal antibody delivery system in combination with a hyaluronidase synergistically enhances the immune response by directing the monoclonal antibody to specific cellular targets within the lymphatic system. In this embodiment, administration of the combination modulates the activity of immune cells in a manner that improves the treatment of the disease or condition associated with the immune system. In various embodiments the disease / condition being treated is a cancer. In certain embodiments, the lipid nanoparticles described herein comprise one or more components, including a lipid component, and (optionally) a structural component. The lipid component comprises lipids selected from ionizable and / or cationic lipids (i.e., lipids that may have a positive or partial positive charge at physiological pH), neutral lipids (e.g., phospholipids, or sphingolipids), and polymer-conjugated lipids (e.g., PEGylated lipids). In some embodiments, the lipid component comprises a single ionizable lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 ionizable lipids. In some embodiments, the lipid component comprises a single neutral lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 neutral lipids. In some embodiments, the lipid component comprises a single polymer-conjugated lipid. In other embodiments, the lipid component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 polymer-conjugated lipids. In some embodiments, the structural component comprises a single structural lipid. In other embodiments, the structural component comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 structural lipids. In some embodiments, the lipid component comprises at least one cationic lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. The present disclosure contemplates that the lipid component may comprise any combination of the foregoing constituents. a. lonizable / Cationic Lipids

[0190] In some embodiments, the lipid component comprises an ionizable lipid. In some embodiments, the ionizable lipid is anionic. In other embodiments, the ionizable lipid is a cationic lipid. In some embodiments, the lipid component comprises cationic lipids including, but not limited to, a cationic lipid selected from the group consisting of 3-(didodecylamino)- N1 ,N1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4- tridodecyl- 1 ,4-piperazinediethanamine (KL22), 14,25-ditridecyl- 15,18,21 ,24-tetraaza- octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-DBl / 162871737.3 41tetraen-19-yl 4-(di methyl ami no)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), 1, 2-di oleyloxy -N,N- dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)- -octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)-2- ({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12-dien- l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- , 12Z)-octadeca-9, 12-di en-l-yloxy]propan-l -amine (Octyl-CLinDMA (2S)), a lipid including a cyclic amine group, and mixtures thereof.

[0191] Non-exhaustive and non-limiting examples of cationic lipids include:DBl / 162871737.3 42DB1 / 162871737.3 43DB1 / 162871737.3 44b. Neutral Lipids / Phospholipids

[0192] In some embodiments, the lipid component further comprises neutral lipids including, but not limited to, a phospholipid selected from the group consisting of 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-DBl / 162871737.3 45glycero-3-phosphoethanolamine (ME 16.0 PE), l ,2-distearoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dilinolenoyl-sn- glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1 -glycerol) sodium salt (DOPG), sphingomyelin (SM), and mixtures thereof. c. Polymer-Conjugated Lipids

[0193] In some embodiments, the lipid component further comprises polymer-conjugated lipids, including, but not limited to, a PEGylated lipid selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG2000-C-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DMA or a PEG-DSPE lipid.

[0194] Non-exhaustive and non-limiting examples of PEG lipids include:PEG-C-DMADBl / 162871737.3 46d. Structural Lipids / Sterols

[0195] In some embodiments, the LNP further comprises a structural component. See generally Patel, S., et al. (2020). Nature Communications, 11(1), 1-13. In some embodiments, the structural component comprises a sterol including, but not limited to, a sterol selected from the group consisting of cholesterol, fecosterol, stigmasterol, stigmastanol, sitosterol, P-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, campesterol, fucosterol, brassicasterol, ergosterol, 9, 11 -dehydroergosterol, tomatidine, tomatine, a-tocopherol, and mixtures thereof. In otherDB1 / 162871737.3 47embodiments, the structural lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0196] Non-exhaustive and non-limiting examples of structural lipids include:

[0197] The lipid component of the LNP may include, for example, a cationic lipid, a phospholipid (such as an unsaturated lipid, e g., DOPE or DSPC), a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

[0198] In some embodiments, the lipid component of the LNP includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 0 mol % to about 10 mol % of PEG lipid, and about 17.5 mol % to about 50 mol % structural lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 0 mol % to about 10 mol % PEG lipid, and about 30 mol % to about 40 mol % structural lipid. In a particular embodiment, the lipid component includes about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % PEG lipid. In another embodiment, the lipid component includes about 40 molDBl / 162871737.3 48% ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1 .5 mol % PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and / or the structural lipid may be cholesterol.

[0199] In some embodiments, the ionizable lipids comprise between about 20 and about 60 mol % of the lipid component. In other embodiments, the ionizable lipids comprise between about 35 and about 55 mol % of the lipid component. In various embodiments, the ionizable lipids comprise about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, or 60 mol % of the lipid component.

[0200] In some embodiments, the neutral lipids comprise between about 0 and about 30 mol % of the lipid component. In other embodiments, the neutral lipids comprise between about 5 and about 25 mol % of the lipid component. In various embodiments, the neutral lipids comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mol % of the lipid component.

[0201] In some embodiments, the polymer-conjugated lipids comprise between about 0 and about 15 mol % of the lipid component. In other embodiments, the polymer-conjugated lipids comprise between about 0.5 and about 10 mol % of the lipid component. In various embodiments, the polymer-conjugated lipids comprise about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 9, 9.5, 10, or 15 mol % of the lipid component.

[0202] In some embodiments, the structural component comprises about 17.5 mol % to about 50 mol % of the lipid component. In other embodiments, the structural component comprises about 30 to about 40 mol % of the lipid component. In various embodiments, the structural component comprises about 17.5, 20, 22.5, 25, 27.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mol % of the lipid component.

[0203] The structural component may alternatively be expressed as a ratio relative to the lipid component. In some embodiments, the structural component is in a ratio of about 1 :1 with the lipid component (sterol :lipids). In other embodiments, the structural component is in a ratio of about 1 :5 with the lipid component (sterol :lipids). In various embodiments, the structural component is in a ratio of about 1 : 1, 1 :2, 1:3, 1 :4, 1:5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, 1 : 15, 1 :20, or 1:25 with the lipid component (sterol: lipids).DBl / 162871737.3 49

[0204] In embodiments, the lipid nanoparticles form liposomes. Liposomes are spherical vesicles composed of synthetic or natural phospholipids that self-assemble in aqueous solution. The liposome has an aqueous core surrounded by a hydrophobic membrane and can be loaded with a wide variety of hydrophobic or hydrophilic molecules for therapeutic purposes. Liposomes are typically synthesized with naturally occurring phospholipids, such as phosphatidylcholine. Cholesterol may be included in the formulation. The therapeutic agent can be loaded through liposome formation in aqueous solution, solvent exchange mechanisms, or pH gradients methods. Therapeutic agents can also be chemically conjugated to the surface of the liposome. One exemplary chemical modification can be conjugating polyethyleneglycol (PEG) to the vesicle surface. Liposomes diffuse from the bloodstream into the interstitial space near the target site. As the cell membrane itself is composed of phospholipids, liposomes can directly fuse with the membrane and release the cargo into the cytosol or may enter the cell through phagocytosis or other active transport pathways. Peptides, polymers, and other molecules can be conjugated to the surface of a liposome for targeted delivery. Conjugating various ligands to the surface can facilitate binding to target cells based on the receptor-ligand interaction. Vesicle size and surface chemistry can affect circulation time.3. Physical Properties of LNP Formulations

[0205] The characteristics of the LNPs disclosed herein may depend on the components thereof. For example, LNPs including cholesterol as a structural lipid may have different characteristics than LNPs that include a different structural lipid. Similarly, the characteristics of an LNP may depend on the absolute or relative amounts of its components. For instance, an LNP including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the LNPs or the LNP formulations.

[0206] The LNPs and LNP formulations may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern,DBl / 162871737.3 50Worcestershire, UK) may also be used to measure multiple characteristics of the LNPs or LNP formulations, such as particle size, poly dispersity index, and zeta potential.

[0207] The mean size of a plurality of LNPs may be between 10 nm and 1 micrometer, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 200 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm. In some embodiments, the mean size of a plurality of LNPs have a mean diameter of from about 30 nm to about 250 nm, from about 40 nm to about 225 nm, from about 50 nm to about 225 nm, from about 60 nm to about 225 nm, from about 70 nm to about 225 nm, from about 70 nm to about 200 nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm, or from about 100 nm to about 200 nm. In a particular embodiment, the mean size may be less than about 200 nm or less than about 100 nm.

[0208] A plurality of the LNPs disclosed herein may be relatively homogenous. A poly dispersity index may be used to indicate the homogeneity of the plurality of LNPs, e.g., the particle size distribution of the plurality of LNPs. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. A plurality of the LNPs disclosed herein may have a poly dispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.1 1 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the plurality of nanoparticles may be from about 0.10 to about 0.20.

[0209] The zeta potential of an LNP may be used to indicate its electrokinetic potential. For example, the zeta potential may describe the surface charge of an LNP, a plurality of LNPs, or an LNP formulation. An LNP, a plurality of LNPs, or an LNP formulation with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP, a plurality of LNPs, or the LNP formulation may be from about - 10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0DBl / 162871737.3 51mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0210] The efficiency of encapsulation of a therapeutic and / or prophylactic describes the amount of therapeutic and / or prophylactic that is encapsulated or otherwise associated with an LNP or a plurality of LNPs after preparation, relative to the initial amount provided. In various embodiments of the invention described, encapsulation efficiency is desirably high (e.g., close to or approaching 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic in a solution containing a plurality of LNPs before and after breaking up the plurality of LNPs with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic (e.g., nucleic acid) in a solution. For a plurality of LNPs described herein, the encapsulation efficiency of a therapeutic and / or prophylactic may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0211] An LNP formulation described herein may optionally comprise one or more coatings. For example, the LNP formulation may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including an LNP formulation described herein may have any useful size, tensile strength, hardness, or density.D. Non-Lipid Nanoparticles for Drug Delivery

[0212] In an embodiment, non-lipid nanoparticles are used herein. These non-lipid nanoparticles can be made from natural or synthetic polymers and offer versatility in terms of design and function. Examples of non-lipid nanoparticles include polyethylene glycol (PEG), polylactic acid (PLA), and / or polycaprolactone (PCL).

[0213] In embodiments, the nanoparticles comprise polymer nanoparticles, dendrimers, polymeric micelles, inorganic nanoparticles, organic nanoparticles, inorganic nanocrystals, organic nanocrystals, quantum dots, viral vectors, viral-like nanoparticles, biological nanocarriers, virus-like particles, carbon nanotubes, mesoporous silica nanoparticles, bioceramic nanoparticles, nanogels, or any combination thereof.DBl / 162871737.3 52

[0214] In embodiments, polymer nanoparticles encapsulate the therapeutic. In embodiments, polymeric nanoparticles are synthetic polymers with a size ranging from 10 to 100 nm. Common synthetic polymeric nanoparticles include polyacrylamide, polyacrylate, and chitosan. Therapeutics can be incorporated either during or after polymerization. Depending on the polymerization chemistry, the therapeutic can be covalently bonded, encapsulated in a hydrophobic core, or conjugated electrostatically. Common synthetic strategies for polymeric nanoparticles include microfluidic approaches, electrodropping, high pressure homogenization, and emulsion-based interfacial polymerization. Polymer biodegradability can be considered when choosing a nanoparticle delivery system. Without being bound by any particular theory, nanocarriers composed of biodegradable polymers can undergo hydrolysis in the body, producing biocompatible small molecules such as lactic acid and glycolic acid. Polymeric nanoparticles can be created via self assembly or other methods such as particle replication in nonwetting templates (PRINT) which can allow for customization of composition, size, and shape of the nanoparticle using tiny molds.

[0215] For polymeric nanoparticles, the induction of stimuli-responsiveness has usually relied heavily upon well-known polymers that possess an inherent stimuli-responsiveness. Certain polymers can undergo reversible phase transitions due to changes in temperature or pH. In embodiments, poly(N-isopropylacrylamide) polymers can be used for activation-modulated delivery. Poly(N-isopropylacrylamide) is soluble in water at room temperature but precipitates reversibly from when the temperature is raised above its lower critical solution temperature (LCST), changing from an extended chain conformation to a collapsed chain. Without being bound by any particular theory, this feature can be used to change the hydrophilicity of a polymer via temperature. In embodiments, the polymer nanoparticles can be a dual stimuli- responsive drug delivery system. Dual stimuli-responsive drug delivery systems can be harnessed to control the release of the encapsulated drug. For example, the triblock copolymer of polyethylene glycol)-b-poly(3-aminopropyl-methacrylamide)-b-poly(N-isopropylacrylamide) (PEG-b-PAPMA-b-PNIPAm) can self-assemble to form micelles, possessing a core-shell- corona architecture above the lower critical solution temperature. It is also pH responsive. Drug release using polymer nanoparticles can be tuned by changing either temperature or pH conditions.DBl / 162871737.3 53

[0216] In embodiments, the polymeric nanoparticle is a dendrimer nanoparticle. Dendrimers are unique hyper-branched synthetic polymers with monodispersed size, well- defined structure, and a highly functionalized terminal surface. They can be composed of synthetic or natural amino acid, nucleic acids, and carbohydrates. Therapeutics can be loaded onto the interior of the dendrimers or the terminal surface of the branches via electrostatic interaction, hydrophobic interactions, hydrogen bonds, chemical linkages, or covalent conjugation. Drug-dendrimer conjugation can elongate the half-life of drugs. Currently, dendrimer use in biological systems is limited due to dendrimer toxicity and limitations in their synthesis methods. Dendrimers are also confined within a narrow size range (<15 nm) and current synthesis methods are subject to low yield. The surface groups will reach the de Gennes dense packing limit at high generation level, which seals the interior from the bulk solution which can allow for encapsulation of hydrophobic, poorly soluble drug molecules. The seal can be tuned by intramolecular interactions between adjacent surface groups, which can be varied by the condition of the solution, such as pH, polarity, and temperature, a property which can be tailored for encapsulation and controlled release properties.

[0217] In embodiments, the dendrimer comprises several arms originating from core which are prepared using sugar, nucleotides and amino acids. Dendrimers can be prepared through isothiocyanate-amine coupling and thiol-methacrylate Michael addition reaction to produce one dendrimer generation. In embodiments, PEGylated dendrimers can have lower cytotoxicity compared to unmodified dendrimers.

[0218] In embodiments, the polymeric nanoparticles are polymeric micelles. Polymeric micelles are spherical shell which can self assemble using amphiphilic di- or tri-block copolymers in aqueous media. In embodiments, polymeric micelles can be amphiphilic diblock copolymer micelles having hydrophobic core and hydrophilic shell.

[0219] In embodiments, the therapeutic is encapsulated by inorganic nanoparticles or nanocrystals. Inorganic nanoparticles can have highly tunable properties such as size, shape, and surface functionalization. Inorganic nanoparticles can be composed of inert metals such as gold and titanium that form nanospheres, however, iron oxide nanoparticles have also become an option.

[0220] In embodiments, the inorganic nanocrystals are quantum dots. Quantum dots (QDs), or inorganic semiconductor nanocrystals, have size-dependent optical properties andDBl / 162871737.3 54versatile surface chemistry. Quantum dots can have diameters from 2 - 10 nm which are on the order of the exciton Bohr radius. Without being bound by any particular theory, this diameter can result in quantum confinement effects analogous to the "particle-in-a-box" model. Optical and electronic properties of quantum dots vary with their size: nanocrystals of larger sizes can emit lower energy light upon fluorescence excitation. Surface engineering of QDs can create nanoparticle-biomolecule hybrids capable of participating in biological processes. Manipulation of nanocrystal core composition, size, and structure can change QD photo-physical properties Designing coating materials which encapsulate the QD core in an organic shell make nanocrystals biocompatible, and QDs can be further decorated with biomolecules to enable more specific interaction with biological targets.

[0221] In embodiments, the therapeutic is encapsulated by organic nanocrystals. Organic nanocrystals can consist of pure drugs and stabilizing surface-active agents. They can be carrier- free submicron colloidal drug delivery systems with a mean particle size in the nanometer range. The formulation of drugs into nanocrystals can increase in particle surface area in contact with the dissolution medium, therefore increasing bioavailability.[00222J In embodiments, the therapeutic is encapsulated by viral vectors, viral-like nanoparticles, or biological nanocarriers. In embodiments, the therapeutic is encapsulated in a viral vector. Viral vectors can be used to deliver genes for genetic engineering or gene therapy. The viral vector can be adenoviruses, retroviruses, and various bacteriophages. The surface of the viral particle can also be modified with ligands to increase targeting capabilities. Virusbased nanoparticles comprise strong protein cages prepared by self assembly which can encapsulate material such as a protein, a peptide, a nucleic acid and or a drug. These protein cages are microscopic in size which can lead to difficulty in functionalizing with virus-binding moi eties.

[0223] In embodiments, the therapeutics is encapsulated in protein particles derived from the viral capsid, or virus-like particles (VLPs). VLPs can be manufactured with a structural uniformity allowing VLPs to be produced precisely in large amounts. VLPs also have modifiable surfaces, allowing for targeted delivery. There are various methods of packaging the molecule into the capsid including self-assemble, altering the pH gradient outside the capsid to create pores on the capsid surface to trap the desired molecule, using aggregators such as leucine zippers or polymer-DNA amphiphiles to induce capsid formation and capture drug molecules.DBl / 162871737.3 55Therapeutics can be chemically conjugated directly onto the reactive sites on the capsid surface, often involving the formation of amide bonds. After being introduced to the organism, VLPs often have broad tissue distribution, rapid clearance, and are generally non-toxic. It may, however, like viruses, invoke an immune response, so immune-masking agents can be coadministered.

[0224] In embodiments, the nanoparticles are carbon nanotubes. Carbon nanotubes (CNTs) are prepared by folding grapheme sheet into tube-like structure whose diameter is nanoscale and length is thousand times the diameter. CNTs are flexible and possess electrical conductivity and biocompatibility. A carbon nanotube can be single walled or multiple walled, such as for DOX has stronger bond with multiple walled CNTs and facilitate better drug release from single walled CNTs. Magnetospirillum magneticum bacteria can used to fabricate carbon nanotubes. Carbon nanotubes are capable of holding drug to obtain targeted drug delivery. Carbon nanotubes can be used in design of system capable of reducing systemic side effects.

[0225] In embodiments, the nanoparticles are mesoporous silica nanoparticles. Mesoporous silica nanoparticles (MSNs) are silica material with structure similar to honeycomb and it can be synthesized in varying size consisting of different pore sizes. Sol-gel process is one commonly used approach to synthesize MSNs which involves hydrolysis and condensation of silicon alkoxide precursors. Premature release of a drug before it reaches the intended target location for delivery is a potential challenge, but MSNs offer a solution to this problem by levering stimuli-sensitive pore opening based on exposure to internal or external stimuli, triggering opening and permitting drug release.

[0226] In embodiments, the nanoparticles are bioceramic nanoparticles. Bioceramic nanoparticles can be used as a cargo for targeted drug delivery and prevents enzymatic degradation. Bioceramic nanoparticles can improve sustained release of a therapeutic agent. Alumina, pyrolytic carbons, zirconia, calcium phosphates and silica-based glasses or glass ceramics are non-limiting examples of commonly known bioceramics. Calcium phosphate possess more reaction sites for drug binding due to their high surface-to-volume ratios compared to bulk form and high loading capacity. Calcium phosphate NPs can be synthesized via precipitation, sol-gel method, flame-spray pyrolysis, solid-state reactions etc., with different properties, depending on morphology, size, and surface properties.DBl / 162871737.3 56

[0227] In embodiments, the nanoparticles are nanogels. Nanogels are 3D nanoparticles obtained by crosslinking hydrophilic or amphiphilic polymer chains which can be used for incorporation of drug via hydrogen bonding, salt bond formation or hydrophobic interaction. Nanogels can exhibit reduced toxic effects and / or enhanced therapeutic index of the drug and can be fabricated as stimuli-responsive materials.1. Drug Delivery by Nanoparticles

[0228] In embodiments, nanoparticles are administered via intranasal, intraventricular, and / or intraparenchymal routes. These routes can enable nanoparticles to cross the blood-brain barrier (BBB) due to their small size. When nanoparticles reach the BBB, several mechanisms may be used, including receptor-mediated mechanisms, active transport, and passive transport to deliver nanoparticles into the brain. Nanoparticles are small in size, can diffuse passively across the endothelial cells of the BBB, and can interact favorably with brain receptors and recognize ligands for interaction.

[0229] In embodiments, nanoparticles delivery drugs can be used to treat heart diseases. In embodiments, the nanoparticles comprise liposomes, silica NPs, dendrimers, cerium oxide NPs, micelles, TiO2 NPs, stents with nano-coatings, microbubbles, and polymer-drug conjugates. Magnetic nanoparticles like magnetoliposomes (MLs) are made up of the union of liposomes and magnetic nanoparticles. MLs can be used for magnetic-targeted drug delivery. The PEGylation of MLs increases their rate of flow in the blood, and pairing of the MLs with antibodies can raise active targeting efficacy. Liposomes are used with various modifications, e.g., to load drugs on NPs for delivery inside the cell. Cationic liposomes, perfluorocarbon nanoparticles, poly electrolyte nanoparticles, and polymeric nanoparticles are non-limiting examples of modified nanocarrier forms used in treating heart diseases.

[0230] In embodiments, nanoparticles can deliver drugs to treat skin diseases, e.g., follicular and / or cutaneous skin disease. Nanoparticle delivery for cutaneous disease treatment can be topically delivered through creams, gels, and ointments. Without being bound by any particular theory, polymeric, lipid, and surfactant nanocarriers can enhance drug penetration into the skin tissue to treat skin cancer in one exemplary embodiment. In embodiments, chitosan polymeric NPs, liposomes, and gold nanoparticles can deliver a therapeutic into the dermal and epidermal layers. Gold nanoparticles are extremely small in size and can penetrate skin layers with very low toxicity and little-to-no skin damage.DBl / 162871737.3 57

[0231] In embodiments, nanoparticles can deliver drugs to treat bone diseases. Bone diseases include, for example, bone defects due to fracture, trauma, osteoporosis, arthritis, infections, and many other diseases. In embodiments, nanoparticles can be used to stimulate bone regeneration in order to fuse, strengthen and repair bones. For example, combination of biomaterial and nanomaterial has reduced bone implantation through the development of bone bioscaffolds. A therapeutic agent encapsulated inside the nanoparticle can be delivered through blood to the targeted area in the bones.

[0232] In embodiments, nanoparticles can deliver therapeutics to treat blood diseases such as hemopoietic blood disorder, iron deficiency, leukemia, anemia, hemophilia, platelet diseases, and blood cancer. The conventionally used chemotherapeutic system causes damage to the immune system (e.g., cytokine storm), with high risk of mortality. Bone marrow transplant is also an expensive and intricate process with high risks of immune complications. For example, thalassemia is treated with deferoxamine, a chelating agent to treat excessive iron in the blood. Embodiments of the present disclosure provide therapeutic agents or compositions with reduced immunogenicity, potentially avoiding dangerous immune response or treatment failure due to rejection. The siRNA-coated nanocomposite has the inhibitory activity for tumor cells in vivo.Example Nanoparticles used in Drug DeliveryDBl / 162871737.3 58DB1 / 162871737.3 59DB1 / 162871737.3 60DB1 / 162871737.3 61E. Therapeutic Agents Administered with Nanoparticle or Conjugate Delivery SystemsDBl / 162871737.3 62

[0233] In some embodiments, the invention provides therapeutic agents administered with a nanoparticle or conjugate delivery system. In an embodiment, the present disclosure provides a lipid nanoparticle (LNP) encapsulating a therapeutic and / or prophylactic. Exemplary LNPs can be selected from the group comprising ionizable lipid formulations, liposomes, cholestosomes, and cochleates. In some embodiments, the present disclosure provides nucleic acid-based therapeutic agents and / or prophylactics administered with non-lipid nanoparticles, including examples such as exosomes, viral vectors (e.g., adeno-associated virus (AAV), lentivirus, adenovirus, etc.), virus-like particles (VLPs), synthetic virus-like particles (sVLPs), and DNA nanostructures such as DNA origami.

[0234] In some embodiments, the present disclosure provides a therapeutic agent and / or prophylactic administered as a conjugate independent from the use of a nanoparticle or other particle delivery system. Exemplary conjugates include, but are not limited to, a nucleic acid or other therapeutic agent and / or prophylactic conjugated to a sugar, a fatty acid, a lipid, a protein, a peptide, an antibody fragment (Fab), a monoclonal antibody (mAb), an aptamer, or another targeting ligand. Some embodiments comprise administering a therapeutic agent and / or prophylactic independent of a nanoparticle as a conjugate that results in improved pharmacokinetic parameters, enhanced tissue penetration, increased cellular uptake, or more efficacious receptor-mediated targeting. In some embodiments, the therapeutic and / or prophylactic is conjugated to a sugar moiety. In an embodiment, administration of the sugar conjugate enables receptor-mediated uptake through a hepatocyte asialoglycoprotein receptor or carbohydrate receptor. Non-limting examples of a sugar moiety can include N- acetylgalactosamine (GalNAc), mannose, or sialic acid moieties.

[0235] In some embodiments, the therapeutic and / or prophylactic is conjugated to a fatty acid or lipid moiety. In an embodiment, administration of the fatty acid or lipid conjugate promotes association with a serum lipoporotein and facilitates uptake into tissue such as hepatocytes, muscle, or lymphatic tissue. Non-limiting examples of a fatty acid or lipid moiety can include cholesterol, palmitic acid, stearic acid, or similar hydrophobic moieties. In some embodiments, the therapeutic and / or prophylactic is conjugated to a protein or peptide moiety. In an embodiment, administration of the protein or peptide conjugate improves cell-type targeting or intracellular trafficking. Non-limiting examples of a protein or peptide moiety can include a peptide ligand for a particular cell surface receptor, a signalling peptide, or a fusogenic peptide.DBl / 162871737.3 63Some embodiments comprise conjugation of the therapeutic and / or prophylactic to an antibody or antibody fragment, such as Fabs, single-chain variable fragments (scFv), mAbs, or bispecific antibodies. In an ambodiment, administration of the antibody or antibody fragment conjugate enables targeted delivery to immune cells, tumor cells, or another specific target cell type or tissue.

[0236] In some embodiments, the therapeutic and / or prophylactic is conjugated to an aptamer. In an ambodiment, the aptamer is a nucleic acid aptamer and administration of the aptamer conjugate increases a binding affinity to a particular receptor, protein, or cell surface target. In an embodiment, administration of the conjugate increases nuclease resistance compared to administration of the unconjugated therapeutic and / or prophylactic. In an embodiment, administration of the conjugate prolongs circulation half-life compared to the unconjugated therapeutic and / or prophylactic. In an embodiment, administration of the conjugate reduces immunogenicity compared to administration of the unconjugated therapeutic and / or prophylactic.

[0237] In some embodiments, the present disclosure provides a therapeutic and / or prophylactic administered with a LNP conjugated to a targeting moiety. In an embodiment, the LNP is conjugated with a polyethylene glycol (PEG) lipid, an antibody, an antibody fragment, a peptide, another biomolecule moiety, or another targeting ligand described herein. In an embodiment, administration of the conjugated LNP improves biodistribution compared to an equal dose of the therapeutic and / or prophylactic without use of a conjugated LNP. In an embodiment, administration of the conjugated LNP enhances uptake in a target tissue (e.g., liver, muscle, lymphatic tissues) compared to an equal dose of the therapeutic and / or prophylactic without use of a conjugated LNP.

[0238] In some embodiments, the present disclosure provides vehicle-free delivery of a “naked” nucleic acid therapeutic and / or prophylactic. Exemplary nucleic acids include mRNA, antisense oligonucleotides (ASOs), phosphorodiamidate morpholino oligonucleotides (PMOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), DNA, circular RNAs (circRNAs), self-replicating RNAs (replicons), and other nucleic acid therapeutics. Such nucleic acids may be administered directly, without encapsulation or conjugation, and may be used to achieve expression, knockdown, or modulation of a target gene or protein.DBl / 162871737.3 64

[0239] In some embodiments, the therapeutic and / or prophylactic comprises a nucleic acid, an antigen, an antibody-drug conjugate (ADC), a nucleic acid editing system, or a protein. In an embodiment, the nucleic acid comprises DNA. In an embodiment, the nucleic acid is antisense oligonucleotide (ASO). In an embodiment, the nucleic acid comprises RNA. In an embodiment, the RNA is messenger RNA (mRNA). In an embodiment, the RNA is small interfering RNA (siRNA). In one embodiment, the RNA is small interfering RNA1. In some embodiments, the nucleic acid, antigen, or protein is commercially available. In some embodiments, the RNA is self-amplifying or micro-amplifying, RNA. In an embodiment, the LNP encapsulates a nucleic acid editing system used in gene editing. In one embodiment, the nucleic acid editing system used in gene editing is a zinc finger nuclease, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing incorporating Cas9, guide RNA (gRNA), base editing, or prime editing.1. Nucleic Acids

[0240] In an embodiment, the therapeutic and / or prophylactic is a nucleic acid. In an embodiment, the nucleic acid is administered using a LNP delivery system. In an embodiment, the nucleic acid is administered using a viral delivery system. In an embodiment, the nucleic acid is administered using a non-LNP conjugate, such as an antibody-drug conjugate (ADC). In an embodiment, the nucleic acid is administered using a conjugated LNP delivery system. In an embodiment, the nucleic acid is administered as a naked nucleic acid. In an embodiment, the nucleic acid comprises DNA. In an embodiment, the nucleic acid is antisense oligonucleotide (ASO). In an embodiment, the nucleic acid comprises RNA. In an embodiment, the RNA is messenger RNA (mRNA). In an embodiment, the RNA is small interfering RNA (siRNA). In one embodiment, the RNA is small interfering RNAi.

[0241] Exemplary nucleic acid therapeutics are disclosed in the Table 1 below.Table 1. Exemplary nucleic acids.DBl / 162871737.3 65DB1 / 162871737.3 66DB1 / 162871737.3 67DB1 / 162871737.3 68DB1 / 162871737.3 69DB1 / 162871737.3 70DB1 / 162871737.3 71DB1 / 162871737.3 72DB1 / 162871737.3 73DB1 / 162871737.3 74DB1 / 162871737.3 75DB1 / 162871737.3 76DB1 / 162871737.3 77DB1 / 162871737.3 78DB1 / 162871737.3 79DB1 / 162871737.3 80DB1 / 162871737.3 81DB1 / 162871737.3 82DB1 / 162871737.3 83DB1 / 162871737.3 84DB1 / 162871737.3 85DB1 / 162871737.3 86DB1 / 162871737.3 87DB1 / 162871737.3 88DB1 / 162871737.3 89DB1 / 162871737.3 90DB1 / 162871737.3 91DB1 / 162871737.3 92DB1 / 162871737.3 93DB1 / 162871737.3 94DB1 / 162871737.3 95DB1 / 162871737.3 96DB1 / 162871737.3 97DB1 / 162871737.3 98DB1 / 162871737.3 99DB1 / 162871737.3 100DB1 / 162871737.3 101DB1 / 162871737.3 102DB1 / 162871737.3 103DB1 / 162871737.3 104DB1 / 162871737.3 105DB1 / 162871737.3 106DB1 / 162871737.3 107DB1 / 162871737.3 108DB1 / 162871737.3 109DB1 / 162871737.3 110DB1 / 162871737.3 111DB1 / 162871737.3 112DB1 / 162871737.3 113DB1 / 162871737.3 114DB1 / 162871737.3 115DB1 / 162871737.3 116DB1 / 162871737.3 117DB1 / 162871737.3 118DB1 / 162871737.3 119DB1 / 162871737.3 120DB1 / 162871737.3 121DB1 / 162871737.3 122DB1 / 162871737.3 123DB1 / 162871737.3 124DB1 / 162871737.3 125DB1 / 162871737.3 126DB1 / 162871737.3 127DB1 / 162871737.3 128DB1 / 162871737.3 129DB1 / 162871737.3 130DB1 / 162871737.3 131DB1 / 162871737.3 132DB1 / 162871737.3 133DB1 / 162871737.3 134DB1 / 162871737.3 135DB1 / 162871737.3 136DB1 / 162871737.3 137DB1 / 162871737.3 138DB1 / 162871737.3 139DB1 / 162871737.3 140DB1 / 162871737.3 141DB1 / 162871737.3 142DB1 / 162871737.3 143DB1 / 162871737.3 144DB1 / 162871737.3 145DB1 / 162871737.3 146DB1 / 162871737.3 147DB1 / 162871737.3 148DB1 / 162871737.3 149DB1 / 162871737.3 150DB1 / 162871737.3 151DB1 / 162871737.3 152DB1 / 162871737.3 153DB1 / 162871737.3 154DB1 / 162871737.3 155DB1 / 162871737.3 156DB1 / 162871737.3 157DB1 / 162871737.3 158DB1 / 162871737.3 159DB1 / 162871737.3 160DB1 / 162871737.3 161DB1 / 162871737.3 162DB1 / 162871737.3 163DB1 / 162871737.3 164DB1 / 162871737.3 165DB1 / 162871737.3 166DB1 / 162871737.3 167DB1 / 162871737.3 168DB1 / 162871737.3 169DB1 / 162871737.3 170DB1 / 162871737.3 171DB1 / 162871737.3 172DB1 / 162871737.3 173DB1 / 162871737.3 174DB1 / 162871737.3 175DB1 / 162871737.3 176a. mRNA

[0242] In an embodiment, the LNP encapsulates mRNA that is involved in the synthesis of an inflammatory protein. In an embodiment, the LNP encapsulates mRNA that is involved in the synthesis of a protein that mediates immune response. In an embodiment, the LNP encapsulates mRNA that is involved in the synthesis of a tumor necrosis factor (TNF) protein. In an embodiment, the LNP encapsulates TNF-alpha mRNA. In an embodiment, the LNP encapsulates mRNA that is involved in the synthesis of an enzyme-based reporter. In one embodiment, the LNP encapsulates mRNA that is involved in the synthesis of a bioluminescent enzyme-based reporter. In an embodiment, the LNP encapsulates luciferase mRNA.

[0243] In another embodiment, the LNP encapsulates an mRNA-based vaccine. mRNA- based vaccines provide a promising alternative to traditional subunit vaccines, which contain antigenic proteins derived from a pathogen. Antigenic proteins are usually recombinantly made and require bacterial fermentation and / or cell culture, as well as complex purification. Vaccines based on mRNA allow de novo expression of complex antigens in the vaccinated subject, which in turn allows proper post-translational modification and presentation of the antigen in its natural conformation. Unlike traditional technologies, the manufacture of mRNA vaccines does not require complex and costly bacterial fermentation, tissue culture, and purification processes. Moreover, once established, the manufacturing process for mRNA vaccines can be used for a variety of antigens, enabling rapid development and deployment of mRNA vaccines. Further, mRNA vaccines are inherently safe delivery vectors as they express the antigens only transiently and do not integrate into the host genome. Because antigens encoded by mRNAs are produced in vivo in the vaccinated individual, mRNA vaccines are especially effective in eliciting both humoral and T cell mediated immunity.

[0244] The active ingredient of the present LNP vaccine composition is an mRNA that encodes an antigen of interest. The antigen may be a polypeptide derived from a virus, for example, influenza virus, coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, or MERS -related virus), Ebola virus, Dengue virus, human immunodeficiency virus (HIV), hepatitis A virusDBl / 162871737.3 177(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), rhinovirus, cytomegalovirus (CMV), zika virus, human papillomavirus (HPV), human metapneumovirus (hMPV), human parainfluenza virus type 3 (PIV3), Epstein-Barr virus (EBV), chikungunya virus, or respiratory syncytial virus (RSV).

[0245] The antigen also may be derived from a bacterium, for example, Staphylococcus aureus, Moraxella (e.g., Moraxella catarrhalis,' causing otitis, respiratory infections, and / or sinusitis), Chlamydia trachomatis (causing Chlamydia), Borrelia (e.g., Borrelia burgdorferi causing Lyme Disease), Bacillus anthracis (causing anthrax), Salmonella typhi (causing typhoid fever), Mycobacterium tuberculosis (causing tuberculosis), Propionibacterium acnes (causing acne), or non-typeable Haemophilus influenzae.

[0246] Exemplary mRNA therapeutics are disclosed in Table 1.Table 1. mRNA-based drugs in clinical trialsDBl / 162871737.3 178DB1 / 162871737.3 179

[0247] Where desired, the LNP or the LNP formulation may be multi-valent. In some embodiments, the LNP may carry mRNAs that encode more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten, or more antigens, from the same or different pathogens.For example, the LNP may carry multiple mRNA molecules, each encoding a different antigen; or carry a polycistronic mRNA that can be translated into more than one antigen (e.g., each antigen-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). An LNP carrying different mRNA molecules typically comprises (e g., encapsulates) multiple copies of each mRNA molecule. For example, an LNP carrying or encapsulating two different mRNA molecules typically carries multiple copies of each of the two different mRNA molecules.

[0248] In some embodiments, a single LNP formulation may comprise multiple (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) respective LNPs, and each respective LNP can be carrying a different mRNA.

[0249] Examples of multi-valent LNP vaccines are those containing mRNAs encoding two or more antigens from the above-listed pathogens, such as LNP vaccines comprising mRNAs encoding polypeptides derived from influenza virus. In some embodiments, the multivalent LNP vaccines contain mRNA molecules encoding polypeptides derived from two or more (e.g., three, four, five, six, seven, eight, nine, or ten) influenza viral proteins selected from hemagglutinin (e.g., hemagglutinin 1 (HA1) and hemagglutinin 2 (HA2)), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (Ml), matrix protein 2 (M2), nonstructural protein 1 (NS1), and non- structural protein 2 (NS2). In further embodiments, the multi-valent LNP vaccines containing two or more (e.g., three, four five, six, seven, eight, or more) mRNA molecules encoding antigenic polypeptides derived from an HA protein, from an NA protein, and from bothDBl / 162871737.3 180HA and NA proteins. In some embodiments, the mRNA molecules encoding antigenic polypeptides are derived from different influenza strains.[002501 In certain embodiments, the composition may comprise one or more mRNA molecules encoding antigens of influenza A, B and C viruses. In one embodiment, the composition may comprise one or more mRNA molecules encoding hemagglutinin and / or NA antigens of influenza A and influenza B viruses. In one embodiment, the hemagglutinin antigens of influenza A viruses are selected from subtypes Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl 1, H12, H13, H14, H15, H16, H17, and H18. In one embodiment, the NA antigens of influenza A viruses are selected from subtypes Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Ni l. In one embodiment, the hemagglutinin and NA antigens of Influenza B viruses are from the Influenza B / Yamagata lineage. In one embodiment, the hemagglutinin and NA antigens of Influenza B viruses are from the Influenza B / Victoria lineage. In some embodiments, the one or more hemagglutinin and NA antigens are from influenza virus strains recommended by the World Health Organization (WHO) in their annual recommendation for influenza vaccine formulations.

[0251] In certain embodiments, at least one of the one or more influenza virus proteins comprises an influenza virus hemagglutinin protein and / or an influenza virus NA protein having a molecular sequence identified or designed from a machine learning model, and in certain embodiments, at least one of the one or more ribonucleic acid molecules encode one or more influenza virus proteins having a molecular sequence identified or designed from a machine learning model.

[0252] In certain embodiments, the composition comprises two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding (i) one or more hemagglutinin antigens, (ii) one or more NA antigens, or (iii) a combination of one or more hemagglutinin antigens and NA antigens.

[0253] In one embodiment, the composition comprises two, three, four, five, six, seven, eight, nine, or more mRNA molecules encoding (i) one or more hemagglutinin antigens, (ii) one or more NA antigens, or (iii) a combination of one or more hemagglutinin antigens and NA antigens, selected from H1N1, H3N2, H2N2, H5N1, H7N9, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2, and H10N7 subtypes and / or B / Yamagata and B / Victoria lineages.

[0254] In one embodiment, the composition comprises one mRNA molecule encoding an H3 hemagglutinin antigen, one mRNA molecule encoding an Hl hemagglutinin antigen, oneDBl / 162871737.3 181mRNA molecule encoding a hemagglutinin antigen from the Influenza B / Yamagata lineage, and one mRNA molecule encoding a hemagglutinin antigen from the Influenza B / Victoria lineage.[002551 In one embodiment, the composition comprises one mRNA molecule encoding an H3 hemagglutinin antigen, one mRNA molecule encoding an N2 NA antigen, one mRNA molecule encoding an Hl hemagglutinin antigen, one mRNA molecule encoding an N1 NA antigen, one mRNA molecule encoding a hemagglutinin antigen from the Influenza B / Yamagata lineage, one mRNA molecule encoding an NA antigen from the Influenza B / Yamagata lineage, one mRNA molecule encoding a hemagglutinin antigen from the Influenza B / Victoria lineage, and one mRNA molecule encoding an NA antigen from the Influenza B / Victoria lineage.

[0256] In an embodiment, the composition can further comprise one or more mRNA molecules encoding a machine learning-engineered influenza virus hemagglutinin having a molecular sequence identified, evolved, and / or designed from a machine learning model, wherein the one or more machine learning-engineered influenza virus hemagglutinin may be selected from an Hl hemagglutinin, an H3 hemagglutinin, a hemagglutinin from a B / Victoria lineage, a hemagglutinin from a B / Yamagata lineage, or a combination thereof.

[0257] When selecting one or more machine learning-engineered influenza virus I-lAs, any machine learning algorithm may be used. For example, envisioned herein are any of the machine learning algorithms and methods disclosed in PCT Application Nos. WO 2021 / 080990 Al, entitled Systems and Methods for Designing Vaccines, and WO 2021 / 080999 Al, entitled Systems and Methods for Predicting Biological Responses, both of which are incorporated by reference in their entireties herein.

[0258] The mRNA molecule may be unmodified (e.g., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodi ester bonds), or chemically modified (e.g., including nucleotide analogs such as pseudouridines (e.g., N l-methyl pseudouridine), 2'-fluoro ribonucleotides, and 2'-methoxy ribonucleotides, and / or phosphorothioate bonds). The mRNA molecule may comprise a 5' cap and a polyA tail.

[0259] In certain embodiments, the composition can be used to increase the amount of mRNA transported into the lymphatic system and lymphatic nodes. Upon administration to a subject, the mRNA delivery system in combination with PH20 synergistically enhances the immune response by directing the mRNA to specific cellular targets within the lymphatic system, thereby modulating the activity of immune cells in a manner that is therapeuticallyDBl / 162871737.3 182beneficial for the treatment of diseases or conditions associated with the immune system, that includes the treatments of cancers. b. siRNA

[0260] siRNAs are RNA duplexes that can have an approximate length of 15-30 nucleotides and can associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). RNA interference (RNAi) may be used to disrupt the expression of a gene or polynucleotide of interest. RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts. Therefore, siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA function through a natural mechanism evolved to control gene expression through non-coding RNA. This is generally considered to be the reason why their activity is more potent in vitro and in vivo than either antisense oligonucleotide or ribozymes. RNAi reagents may include DNA sense :RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi. Thus, RNAi molecules comprising any of these different types of double-stranded molecules may be used. In addition, it is understood that RNAi molecules may be used and introduced to cells in a variety of forms. Accordingly, as used herein, RNAi molecules encompasses any and all molecules capable of inducing an RNAi response in cells, including, but not limited to, double-stranded polynucleotides comprising two separate strands(e.g., a sense strand and an antisense strand), small interfering RNA (siRNA), polynucleotides comprising a hairpin loop of complementary sequences, and expression vectors that express one or more polynucleotides capable of forming a double-stranded polynucleotide alone or in combination with another polynucleotide.

[0261] RNA interference (RNAi) may be used to specifically inhibit expression of target polynucleotides. Double-stranded RNA-mediated suppression of gene and nucleic acid expression may be accomplished according to the invention by introducing dsRNA, siRNA or shRNA into cells or organisms. siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand, or siRNA.

[0262] RNAi molecules targeting specific polynucleotides can be readily prepared according to procedures known in the art. Accordingly, one skilled in the art would understand that a wide variety of different siRNA molecules may be used to target a specific gene or transcript. InDBl / 162871737.3 183certain embodiments, siRNA molecules according to the invention are double-stranded and 16- 30 or 18-25 nucleotides in length, including each integer in between.

[0263] Generally, siRNA molecules are complementary to one strand of a target DNA molecule. In other embodiments, siRNAs may have a modified composition, such as, for example, 2'-deoxy or 2'-O-methyl modifications. However, in preferred embodiments, the entire strand of the siRNA is not made with either 2' deoxy or 2'-O-modified bases.

[0264] In certain embodiments, the composition of the invention can be used to increase the amount of siRNA conjugating to a targeting agent, such as GalNAc (N-Acetylgalactosamine) conjugation for targeted delivery to the liver. Upon administration to a subject, the siRNA delivery system in combination with PH20 synergistically enhances the amount transported to a particular organ, tissue, or cell type by directing the siRNA to specific cellular targets (e.g., cell surface markers or receptors), thereby modulating the activity of the cellular target in a manner that is therapeutically beneficial for the treatment of diseases or conditions.

[0265] In some embodiments, the composition of the invention can be used to increase the amount of siRNA transported to target organs. Upon administration to a subject, the siRNA delivery system in combination with PH20 synergistically enhances the amount reaching the target organ by directing the siRNA to specific cellular targets, thereby modulating the activity of the target organ cells in a manner that is therapeutically beneficial.

[0266] Exemplary, non-limiting, siRNA therapeutics are disclosed in the table below:

[0267] In particular embodiments, the lipid nanoparticles encapsulate siRNA. siRNAs are RNA duplexes normally 15-30 nucleotides long that can associate with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC). RNA interference (RNAi) methods using RNAi molecules may be used to disrupt the expression of a gene or polynucleotide of interest. RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts. Therefore, siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA can function through a natural mechanism evolved to control gene expression through non-coding RNA. This is generallyDBl / 162871737.3 184considered to be the reason why their activity is more potent in vitro and in vivo than either antisense oligonucleotide or ribozymes. RNAi reagents may include DNA sense :RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi. Thus, RNAi molecules comprising any of these different types of double-stranded molecules may be used. In addition, it is understood that RNAi molecules may be used and introduced to cells in a variety of forms. Accordingly, as used herein, RNAi molecules encompasses any and all molecules capable of inducing an RNAi response in cells, including, but not limited to, double-stranded polynucleotides, polynucleotides comprising a hairpin loop of complementary sequences, thereby forming at least a partial double-stranded region, and expression vectors that express one or more polynucleotides capable of forming a doublestranded polynucleotide alone or in combination with another polynucleotide.

[0268] RNA interference (RNAi) may be used to specifically inhibit expression of target polynucleotides. Double-stranded RNA-mediated suppression of gene and nucleic acid expression may be accomplished according to the present disclosure by introducing dsRNA, siRNA or shRNA into cells or organisms. siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand, or siRNA.

[0269] RNAi molecules targeting specific polynucleotides can be readily prepared according to procedures known in the art. Accordingly, one skilled in the art would understand that a wide variety of different siRNA molecules may be used to target a specific gene or transcript. In certain embodiments, siRNA molecules according to the invention are double-stranded and approximately 16-30 or 18-25 nucleotides in length, including each integer in between.

[0270] Generally, siRNA molecules are completely complementary to one strand of a target DNA molecule. In other embodiments, siRNAs may have a modified composition, such as, for example, 2'-deoxy or 2'-O-methyl modifications. However, in preferred embodiments, the entire strand of the siRNA is not made with either 2' deoxy or 2'-O-modified bases.

[0271] In certain embodiments, the present invention relates to methods and compositions for producing lipid-encapsulated nucleic acid particles in which nucleic acids are encapsulated within a lipid nanoparticle and are administered in combination with a hyaluronidase enzyme. Such lipid nanoparticles, encapsulating siRNA oligonucleotides, are characterized using a variety of biophysical parameters including: (1) nucleic acid to lipid ratio; (2) encapsulation efficiency; and (3) particle size. High encapsulation efficiency, good nuclease resistance and serum stability and controllable particle size (e.g., generally less than 200 nm in diameter) are desirable. InDBl / 162871737.3 185addition, the nature of the nucleic acid polymer is of significance, since the modification of nucleic acids in an effort to impart nuclease resistance adds to the cost of therapeutics while in many cases providing only limited resistance.

[0272] In certain embodiments, the composition can be used to increase the amount of siRNA transported into the lymphatic system and lymphatic nodes. Upon administration to a subject, the siRNA delivery system in combination with PH20 synergistically enhances the immune response by directing the siRNA to specific cellular targets within the lymphatic system, thereby modulating the activity of immune cells in a manner that is therapeutically beneficial for the treatment of diseases or conditions associated with the immune system, that includes the treatments of cancers.

[0273] Exemplary siRNA therapeutics are disclosed in Table 2.Table 2. siRNA-based drugsDBl / 162871737.3 186DB1 / 162871737.3 187c. Chemically Modified Nucleic Acids

[0274] In an embodiment, the disclosed formulations comprise a chemically modified nucleic acid. In embodiments, the chemically modified nucleic acid comprises one or more of aDBl / 162871737.3 188backbone modification, a sugar modification, a termini modification, a conjugation of a targeting ligand, and a conjugation of a fatty acid. In another embodiment, the disclosed conjugated formulations comprise a chemically modified nucleic acid conjugated to a conjugation agent and / or targeting moiety, such as nucleic acid-antibody conjugates, antibody-drug conjugates (ADCs), peptide-nucleic acid conjugates, aptamer conjugates, sugar-antibody conjugates, and / or synthetic small molecule conjugates. In some embodiments, the present disclosure provides a formulation comprising the LNP encapsulating the chemically modified nucleic acid and the LNP further conjugated to a targeting moiety such as a PEG lipid, an antibody, an antibody fragment, a peptide, a sugar moiety, and / or a synthetic small molecule conjugation agent.

[0275] In embodiments, the chemically modified nucleic acid comprises a backbone modification. In embodiments, the backbone modification comprises the inclusion of a phosphorothioate linkage. In a phosphorothioate (PS) linkage, a non-bridging phosphodiester oxygen is replaced with sulfur. In embodiments, the backbone modification comprises a boranophosphate intemucleotide linkage. In embodiments, the backbone modification comprises a phosphonoacetate linkage or a thiophosphonoacetate linkage incorporated into the phosphate backbone.

[0276] In embodiments, the chemically modified nucleic acid comprises a modified sugar moiety. In embodiments, the modified sugar moiety is a modified furanose sugar moiety. In embodiments, the furanose sugar moiety is modified at the 2’-hydroxyl (2’-OH) position. In embodiments, the 2’-OH modified furanose sugar moiety is a 2’-O-methyl (2’0Me) furanose sugar moiety, 2’-O-methoxyethyl (2’MOE) furanose sugar moiety, 2’ -fluoro (2’F) furanose sugar moiety, or 2’-fluoro-P-d-arabinonucleic acid (2’F-ANA) furanose sugar moiety. In embodiments, the modified sugar moiety is a conformationally constrained sugar moiety. In embodiments, the conformationally constrained sugar moiety is a locked nucleic acid (LNA). In embodiments, the licked nucleic acid is a bicyclic RNA analog that covalently links the 2’- and 4’ -positions on the furanose ring with a methylene bridge. In embodiments, the conformationally constrained sugar moiety is a tricyclo-DNA (tcDNA) nucleoside. In embodiments, the modified sugar moiety is a less conformationally constrained sugar moiety. In embodiments, the less conformationally constrained sugar moiety is an unlocked nucleic acid. In embodiments, the unlocked nucleic acid is a non-nucleotide residue whereby the covalent C2’- C3’ bond of the ribose ring is cleaved. In embodiments, the less conformationally constrainedDBl / 162871737.3 189sugar moiety is a glycol nucleic acid. In embodiments, the glycol nucleic acid is a flexible three- carbon, acyclic nucleic acid analog where the (S)-isomer will specifically hybridize with A / T- rich RNA sequences.

[0277] In embodiments, the chemically modified nucleic acid comprises a termini modification. In embodiments, the termini modification comprises a 5 ’-phosphate group, a 5’- (E)-vinyl-phosphonate, or a 5’-C-malonyl group.

[0278] In embodiments, the chemically modified nucleic acid comprises a conjugated targeting ligand or conjugated hydrophobic moiety. In embodiments, the conjugated targeting ligand has a high affinity to a target receptor. In embodiments, the conjugated targeting ligand is triantennaiy N-acetylgalactosamine (GalNAc) or an eGPLl. In embodiments, the conjugated hydrophobic moiety is lipid-based moiety or fatty acid moiety. In embodiments, the lipid-based moiety is cholesterol. In embodiments, the fatty acid moiety is palmitate, a-tocopherol, docosanoic acid, oleic acid, a-linolenic acid, linoleic acid, y-linolenic acid, linolelaidic acid, dihomo-y-linolenic acid, palmitoleic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, paullinic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatri acontylic acid, nonatriacontylic acid, tetracontylic acid, 2-hydroxy fatty acids including 2-hydroxy fatty acids with chain-lengths from about C16 to C26, including saturated and unsaturated versions, perdeuterated fatty acids where some or all hydrogen atoms are replaced with deuterium, nitro fatty acids which contain a nitro group (NO2), branched-chain fatty acids, and fatty acids with substituted groups where various functional groups are attached to the fatty acid chain such as selenium or epimino groups).2. Nucleic acid editing systems

[0279] In an embodiment, the LNP encapsulates a nucleic acid editing system. In one embodiment, the nucleic acid editing system is a Zinc-Finger nuclease, Clustered RegularlyDBl / 162871737.3 190Interspaced Short Palindromic Repeats (CRISPR) editing incorporating Cas9, guide RNA (gRNA), base editing, or prime editing.

[0280] Cas9 (CRISPR associated protein 9) is an RNA-guided DNA nuclease enzyme associated with Streptococcus pyogenes CRISPR immunity system. Cas9 can be used to induce site-directed double strand breaks in DNA, which can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively. mRNA systems for expressing Cas9 are commercially available from TriLink Biotechnologies (San Diego, Calif). The mRNA may be codon optimized for human or other mammalian systems. The expressed Cas9 protein may contain a nuclear localization signal at the C-terminus. The RNA encoding Cas9 may be capped and polyadenylated to support expression in mammalian cells and may contain modifications to reduce immune stimulation. The amino acid sequence and encoding nucleic acid sequence for Cas9 and functional derivatives and homologs (which can be used in accordance with the disclosure) include those described in U.S. Pat. No. 8,697,359, which is hereby incorporated by reference in its entirety.[00281J The Cas9 may be delivered in conjunction with a gRNA, which directs the Cas9 editing system to the nucleotide sequence recognized by the gRNA. The term “gRNA” is used interchangeably herein with “gRNA” “single gRNA,” and “sgRNA.” In general, a gRNA can be designed to target any nucleotide sequence. Generally, for Cas9, gRNAs guide the Cas9 endonuclease to the complementary 20 nucleotide (nt) genomic sequences with a downstream NGG protospacer-adjacent motif (PAM). Cas9 generates double-stranded breaks, which can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR).

[0282] In some embodiments, a Cas9 nickase version is employed. Cas9 nickase can generate single stranded breaks, and a double nickase can generate double stranded breaks. The nickase can provide for reduced off-target effects. Further, by delivering two gRNAs and a Cas9 nickase, off target effects can be further reduced.

[0283] In place of a CRISPR-Cas9 system, alternate nucleic acid editing systems may be used. For example, suitable systems include any CRISPR / cas system (e g., any Cascade-like CRISPR / cas, Type I CRISPR / cas, Type II CRISPR / cas, and type III CRISPR / cas), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and engineered meganuclease re-engineered homing endonucleases.DBl / 162871737.3 191

[0284] RNA encoding the nucleic acid editing system can be modified, and the modification selected from one or more of modifications of the phosphate backbone (e.g., phosphorothioate linkages or boranophosphate linkages), ribose ring modifications such as 2'-O- methyl and / or 2'-fluoro and / or 4'-thio modifications, and locked or unlocked nucleic acids. Other modifications may include pseudouridine, 2-thiouridine, 4-thiouridine, 5 -azauridine, 5- hydroxyuridine, 5 -aminouridine, 5-methyluridine, 2-thiopseudouridine, 4-thiopseudouridine, 5- hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, 5- methylcytidine, N4-methylcytidine, 2-thiocytidine, 5 -azacytidine, 5 -hydroxy cytidine, 5- aminocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5- hydroxypseudoisocytidine, 5-aminopseudoisocytidine, 5-methylpseudoisocytidine, N6- methyladenosine, 7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio- 7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8- azaguanosine. Generally, modifications are selected to reduce immune stimulation and stabilize the RNA and improve expression of the encoded protein. For example, the RNA may have a combination of 2-thiouridine and 5-methyl-cytidine, which has been shown to reduce immune stimulation through pattern recognition receptors, such as TLR3, TLR7, TLR8 and RIG-I (retinoic-acid-inducible protein I). In some embodiments, the mRNA has one or more pseudouridine (preventing activation of pattern recognition receptors and 2 '-5 '-oligoadenylate synthetase). These modifications can also stabilize the mRNA against cleavage, and ultimately improve expression rates.

[0001] Artificial nucleases, such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), the CRISPR / Cas system with an engineered crRNA / tracr RNA ('single guide RNA'), comprise DNA binding domains (nucleotide or polypeptide) associated with or operably linked to cleavage domains, and have been used for targeted alteration of genomic sequences.

[0002] In other embodiments, the nuclease is a zinc finger nuclease (ZFN) or TALE DNA binding domain-nuclease fusion (TALEN). ZFNs and TALENs comprise a DNA binding domain (zinc finger protein or TALE DNA binding domain) that has been engineered to bind to a target site in a gene of choice and cleavage domain or a cleavage half-domain (e.g., from a restriction and / or meganuclease as described herein).DBl / 162871737.3 192

[0003] As described in detail above, zinc finger binding domains and TALE DNA binding domains can be engineered to bind to a sequence of choice. An engineered zinc finger binding domain or TALE protein can have a novel binding specificity, compared to a naturally-occurring protein.

[0004] Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual zinc finger or TALE amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers or TALE repeat units which bind the particular triplet or quadruplet sequence.

[0005] Selection of target sites; and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Patent Nos. 7,888,121 and 8,409,861, incorporated by reference in their entireties herein.

[0006] In addition, as disclosed in these and other references, zinc finger domains, TALEs and / or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0007] Thus, nucleases such as ZFNs, TALENs and / or meganucleases can comprise any DNA-binding domain and any nuclease (cleavage) domain (cleavage domain, cleavage halfdomain). As noted above, the cleavage domain may be heterologous to the DNA-binding domain, for example a zinc finger or TAL-effector DNA-binding domain and a cleavage domain from a nuclease or a meganuclease DNA-binding domain and cleavage domain from a different nuclease. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. Additional enzymes which cleave DNA are known (e.g., SI Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.DBl / 162871737.3 193

[0008] The term "zinc finger nuclease," as used herein, refers to a nuclease comprising a nucleic acid cleavage domain conjugated to a binding domain that comprises a zinc finger array. In some embodiments, the cleavage domain is the cleavage domain of the type II restriction endonuclease Fokl. Zinc finger nucleases can be designed to target virtually any desired sequence in a given nucleic acid molecule for cleavage, and the possibility to design zinc finger binding domains to bind unique sites in the context of complex genomes allows for targeted cleavage of a single genomic site in living cells, for example, to achieve a targeted genomic alteration of therapeutic value. Targeting a double- strand break to a desired genomic locus can be used to introduce frame- shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway. Zinc finger nucleases can be generated to target a site of interest by methods well known to those of skill in the art. For example, zinc finger binding domains with a desired specificity can be designed by combining individual zinc finger motifs of known specificity. In some embodiments, separate zinc fingers that each recognizes a 3 base pair DNA sequence are combined to generate 3-, 4-, 5-, or 6-fmger arrays that recognize target sites ranging from 9 base pairs to 18 base pairs in length. In some embodiments, longer arrays are contemplated. In other embodiments, 2-finger modules recognizing 6-8 nucleotides are combined to generate 4-, 6-, or 8- zinc finger arrays. In some embodiments, bacterial or phage display is employed to develop a zinc finger domain that recognizes a desired nucleic acid sequence, for example, a desired nuclease target site of 3-30 bp in length. Zinc finger nucleases, in some embodiments, comprise a zinc finger binding domain and a cleavage domain fused or otherwise conjugated to each other via a linker, for example, a polypeptide linker. The length of the linker determines the distance of the cut from the nucleic acid sequence bound by the zinc finger domain. If a shorter linker is used, the cleavage domain will cut the nucleic acid closer to the bound nucleic acid sequence, while a longer linker will result in a greater distance between the cut and the bound nucleic acid sequence. In some embodiments, the cleavage domain of a zinc finger nuclease has to dimerize in order to cut a bound nucleic acid. In some such embodiments, the dimer is a heterodimer of two monomers, each of which comprises a different zinc finger binding domain. For example, in some embodiments, the dimer may comprise one monomer comprising zinc finger domain A conjugated to a Fokl cleavage domain, and one monomer comprising zinc finger domain B conjugated to a Fokl cleavage domain. In this non-limiting example, zinc finger domain A binds a nucleic acid sequence on one side of the target site, zinc finger domain B binds a nucleic acidDBl / 162871737.3 194sequence on the other side of the target site, and the dimerize Fokl domain cuts the nucleic acid in between the zinc finger domain binding sites.

[0009] The term "zinc finger," as used herein, refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold. Zinc fingers encompass a wide variety of differing protein structures. Zinc fingers can be designed to bind a specific sequence of nucleotides, and zinc finger arrays comprising fusions of a series of zinc fingers, can be designed to bind virtually any desired target sequence. Such zinc finger arrays can form a binding domain of a protein, for example, of a nuclease, e.g., if conjugated to a nucleic acid cleavage domain. Different types of zinc finger motifs are known to those of skill in the art, including, but not limited to, CyS2His2, Gag knuckle, Treble clef, Zinc ribbon, Zn2 / Cyse, and TAZ2 domain-like motifs. Typically, a single zinc finger motif binds 3 or 4 nucleotides of a nucleic acid molecule. Accordingly, a zinc finger domain comprising 2 zinc finger motifs may bind 6-8 nucleotides, a zinc finger domain comprising 3 zinc finger motifs may bind 9- 12 nucleotides, a zinc finger domain comprising 4 zinc finger motifs may bind 12-16 nucleotides, and so forth. Any suitable protein engineering technique can be employed to alter the DNA -binding specificity of zinc fingers and / or design novel zinc finger fusions to bind virtually any desired target sequence from 3 - 30 nucleotides in length. Fusions between engineered zinc finger arrays and protein domains that cleave a nucleic acid can be used to generate a “zinc finger nuclease.” A zinc finger nuclease typically comprises a zinc finger domain that binds a specific target site within a nucleic acid molecule, and a nucleic acid cleavage domain that cuts the nucleic acid molecule within or in proximity to the target site bound by the binding domain. Typical engineered zinc finger nucleases comprise a binding domain having between 3 and 6 individual zinc finger motifs and binding target sites ranging from 9 base pairs to 18 base pairs in length. Longer target sites are particularly attractive in situations where it is desired to bind and cleave a target site that is unique in a given genome.

[0010] Base editing is a genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double-stranded DNA breaks (DSB), or single stranded breaks (i.e., nicking). To date, other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repairDBl / 162871737.3 195enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g. typically 0.1% to 5%), with the major genome editing products being indels.

[0011] A base editor (BE) is a CRISPR-mediated fusion protein utilized in base editing methods. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase which binds a nucleic acid in a guide RNA-programmed manner via the formation of an R-loop but does not cleave the nucleic acid. For example, the dCas9 domain of the fusion protein may include a D10A and a H840A mutation (which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex). The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA (the “targeted strand”, or the strand in which editing, or deamination occurs), whereas the RuvCl subdomain cleaves the non-complementary strand containing the PAM sequence (the “non-edited strand”). The RuvC 1 mutant D10A generates a nick in the targeted strand, while the HNH mutant H840A generates a nick on the non-edited strand.

[0012] In some embodiments, a base editor is a macromolecule or macromolecular complex that results primarily (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, more than 99.9%, or 100%) in the conversion of a nucleobase in a polynucleic acid sequence into another nucleobase (e.g., a transition or transversion) using a combination of 1) a nucleotide-, nucleoside-, or nucleobase-modifying enzyme and 2) a nucleic acid binding protein that can be programmed to bind to a specific nucleic acid sequence.

[0013] In some embodiments, the base editor comprises a DNA binding domain (e.g., a programmable DNA binding domain such as a dCas9 or nCas9) that directs it to a target sequence. In some embodiments, the base editor comprises a nucleobase modifying enzyme fused to a programmable DNA binding domain (e.g., a dCas9 or nCas9). A “nucleobase modifying enzyme” is an enzyme that can modify a nucleobase and convert one nucleobase to another (e.g., a deaminase such as a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor may target cytosine (C) bases in a nucleic acid sequence and convert the C to thymine (T) base. In some embodiments, the C to T editing is carried out by aDBl / 162871737.3 196deaminase, e g., a cytidine deaminase. Base editors that can carry out other types of base conversions (e.g., adenosine (A) to guanine (G), C to G) are also contemplated.

[0014] In principle, there are twelve possible base-to-base changes that may occur via individual or sequential use of transition (i.e., a purine-to-purine change or pyrimidine-to- pyrimidine change) or transversion (i.e., a purine-to-pyrimidine or pyrimidine-to-purine) editors. These include transition base editors such as a C-to-T base editor (or “CTBE”). This type of editor converts a C:G Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a G-to-A base editor (or “GABE”). In other embodiments, the transition base editor is an A-to-G base editor (or “AGBE”). This type of editor converts a A:T Watson-Crick nucleobase pair to a G:C Watson- Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to-C base editor (or “TCBE”).

[0015] Transversion base editors include G-to-T base editors (or “GTBE”), C-to- G base editors (or “CGBE”), A-to-T base editors (or “ATBE”), and A-to-C base editors (or “ACBE”). A G-to-T base editor (or “GTBE”) converts a G:C Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a C-to-A base editor (or “CABE”). A C-to-G base editor (or (“CGBE”) converts a C:G Watson-Crick nucleobase pair to a G:C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a G-to-C base editor (or “GCBE”). A, A-to-T base editor (or “ATBE”) converts a A:T Watson-Crick nucleobase pair to a T:A Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to- A base editor (or “TABE”). An A-to-C base editor (or “ACBE”) converts a A:T Watson-Crick nucleobase pair to a C:G Watson-Crick nucleobase pair. Because the corresponding Watson- Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a T-to-G base editor (or “TGBE”).DBl / 162871737.3 197

[0016] Prime editing (PE) is a nucleic acid editing platform that enables the targeted and programmable installation of defined changes in a nucleotide sequence at a desired locus. It involves targeting of a prime editor to a target site in the genome, wherein the prime editor comprises a nucleic acid programmable DNA binding protein (napDNAbp) fused to a polymerase (e.g., a reverse transcriptase (RT)) associated with a prime editing guide RNA (pegRNA). The pegRNA comprises a scaffold (which binds to the napDNAbp), a spacer sequence (which is complementary to the genomic site), and an extension arm at the 3' or 5' end of the pegRNA. The extension arm includes a DNA synthesis template which includes the sequence of the desired edit. During prime editing, once the prime editor complexed with the pegRNA localizes to the genomic site, the polymerase (e.g., reverse transcriptase) synthesizes a new strand of DNA containing a desired edit using the DNA synthesis template. The new strand of DNA then replaces the corresponding endogenous DNA strand at the genomic site, thereby installing the desired, edited nucleotide sequence into the genome at the edit site.

[0017] The process of prime editing may introduce at least one or more of the following genetic changes into a nucleic acid (e.g., genome): transversions, transitions, deletions, and insertions. In addition, prime editing may be implemented for specific applications. For example, prime editing can be used to (a) install mutation-correcting changes to a nucleotide sequence, (b) install protein and RNA tags, (c) install immunoepitopes on proteins of interest, (d) install dimerization domains in proteins, (e) install or remove sequences that alter the activity of a biomolecule, (f) install recombinase target sites to direct specific genetic changes, and / or (g) mutagenize a target sequence by using an error-prone RT, as well as other purposes. And, with the modified pegRNAs described herein, these applications of prime editing may be conducted with high efficiency and / or reduced occurrence of indels.

[0018] In another aspect, the disclosure provides pegRNAs for prime editing comprising (i) a guide RNA comprising a spacer and (ii) at least one nucleic acid extension arm comprising a DNA synthesis template, a primer binding site, a toehold motif, and an additional nucleic acid moiety, wherein the toehold motif occludes interaction of the primer binding site and the spacer when the pegRNA is not bound by a prime editor, but does not occlude interaction of the primer binding site and a protospacer sequence on a target DNA molecule when the pegRNA is bound by a prime editor. In some embodiments, the toehold motif and the additional nucleic acid moiety are attached to the 3' end of the extension arm. In some embodiments, the toehold motifDBl / 162871737.3 198is attached to the 3' end of the extension arm, and the additional nucleic acid moiety is attached to the 3' end of the toehold motif. In some embodiments, the toehold motif is attached to the pegRNA by a linker.

[0019] In various embodiments, prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA).

[0020] In certain embodiments, the delivery system comprises” lipid” nanoparticles or non-lipid nanoparticles. In some embodiments, the lipid-based vector is a “lipid” nanoparticle, which is a lipid particle between about 1 and about 100 nanometers in size. In certain embodiments, the composition of the invention can be used to increase the amount of nucleic acid editing system transported into the lymphatic system and lymphatic nodes. Upon administration to a subject, the nucleic acid editing system delivery system in combination with PH20 synergistically enhances the immune response by directing the nucleic acid editing system to specific cellular targets within the lymphatic system, thereby modulating the activity of immune cells in a manner that is therapeutically beneficial for the treatment of diseases or conditions associated with the immune system, that includes the treatments of cancers.

[0021] In some embodiments, the lipid-based vector is a lipid or liposome. Liposomes are artificial spherical vesicles comprising a lipid bilayer.

[0022] In some embodiments, the lipid-based vector is a small nucleic acid-lipid particle (SNALP). SNALPs comprise small (e.g., less than 200 nm in diameter) lipid-based nanoparticles that encapsulate a nucleic acid. In some embodiments, the SNALP is useful for delivery of an RNA molecule such as siRNA. In some embodiments, SNALP formulations deliver nucleic acids to a particular tissue in a subject, such as the liver.

[0023] In some embodiments, the gRNA and / or nucleic acid editing system (or the RNA encoding the same) is delivered via polymeric vectors. In some embodiments, the polymeric vector is a polymer or polymerosome. Polymers encompass any long repeating chain of monomers and include, for example, linear polymers, branched polymers, dendrimers, and polysaccharides. Linear polymers comprise a single line of monomers, whereas branched polymers include side chains of monomers. Dendrimers are also branched molecules, which are arranged symmetrically around the core of the molecule. Polysaccharides are polymericDBl / 162871737.3 199carbohydrate molecules and are made up of long monosaccharide units linked together. Polymersomes are artificial vesicles made up of synthetic amphiphilic copolymers that form a vesicle membrane and may have a hollow or aqueous core within the vesicle membrane.

[0024] Various polymer-based systems can be adapted as a vehicle for administering RNA encoding the nucleic acid editing machinery. Exemplary polymeric materials include poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(L -lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), PLGA-b-poly(ethylene glycol)-PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-m al eimide (PLGA-PEG-mal), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide- co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co- PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (polyacrylic acids), and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes, Poly([beta]-amino esters (PBAE), and polyphosphoesters, and blends and / or block copolymers of two or more such polymers. Polymer-based systems may also include Cyclodextrin polymer (CDP)-based nanoparticles such as, for example, CDP- admantane (AD)-PEG conjugates and CDP-AD-PEG-transferrin conjugates.DBl / 162871737.3 200

[0025] In one embodiment, nanoparticles are formulated with Cas9 mRNA chemically modified to reduce TLR responses. In a further embodiment, the nanoparticles are formulated using controlled microfluidic mixing systems.

[0026] In one embodiment, the delivery system is a layer-by-layer particle system comprising two or more layers. In a further embodiment, the guide RNA and the nucleic acid editing system are present in different layers within the layer-by-layer particle. In a yet further embodiment, the guide RNA and nucleic acid editing system may be administered to a subject in a layer-by-layer particle system such that the release of the guide RNA and nucleic acid editing system from the particles can be controlled in a cell-specific and / or temporal fashion. In one embodiment, the layer-by-layer particle system is designed to allow temporally controlled expression of the guide RNA and the nucleic acid editing system as disclosed herein.

[0027] In some embodiments, the lipid-based delivery system comprises a lipid encapsulation system. The lipid encapsulation system can be designed to drive the desired tissue distribution and cellular entry properties, as well as to provide the requisite circulation time and biodegrading character. The lipid encapsulation may involve reverse micelles and / or further comprise polymeric matrices. In some embodiments, the particle includes a lipophilic delivery compound to enhance delivery of the particle to tissues, including in a preferential manner. Such compounds may generally include lipophilic groups and conjugated amino acids or peptides, including linear or cyclic peptides, and isomers thereof. Compounds can be engineered for targeting various tissues, including pancreas, spleen, liver, fat, kidneys, uterus / ovaries, muscle, heart, lungs, endothelial tissue, bone marrow, brain, skin, lymphatic system, lymph nodes, and thymus.3. Nucleic Acid Conjugates

[0028] In an embodiment, the disclosed LNPs and LNP formulations comprise a nucleic acid conjugate. In embodiments, the nucleic acid conjugate is a covalent biochemical conjugate. In embodiments, the nucleic acid conjugate is heterobifunctional. In embodiments, the nucleic conjugate is a short-interfering RNA (siRNA), oligonucleotide, antisense oligonucleotides (ASO), aptamers, and CpG deoxynucleotides.

[0029] In embodiments, the nucleic acid conjugate is a short interfering RNA (siRNA). Natural and synthetic siRNA can be taken up into a multiprotein complex, RISC (RNA-inducedDBl / 162871737.3 201silencing complex) consisting minimally of Dicer, Tar RNA Binding Protein (TRBP) and Ago2. Therapeutic siRNA can hence be optimized to cleave target specific mRNA, leading to the “knockdown” of the encoded protein. In an embodiment, the siRNA is conjugated to a small molecule ligand which targets and / or binds to a specific protein or proteins. This conjugation directs the siRNA to specific cells and enhances its cellular uptake, leading to more efficient knockdown.[00030J In an embodiment, the nucleic acid conjugate is a microRNA (miRNA) which are small, non-coding RNA molecules that regulate gene expression after transcription.

[0031] In embodiments, the nucleic acid conjugate is an antisense oligonucleotide (ASO). Antisense oligonucleotides can work by altering the metabolism of RNAs by degrading or triggering the synthesis of precursor RNAs or mature mRNAs via base complementarity through a variety of mechanisms broadly classed as enzyme-active and splice-switching. Spliceswitching or enzyme-inactive ASOs possess backbones invisible to this enzymatic machinery and thus, can mask specific oligonucleotide sequences altering splicing decisions, reading frames, and other key steps in translation. Splice-switching ASOs can be used to restore the function of a defective gene, inhibit nonsense-mediated decay, and increase transcript stability.

[0032] In embodiments, the nucleic acid conjugate is an aptamer. Aptamers are three- dimensional DNA or RNA structures selected from a larger pool of sequences to bind a specific target, a small molecule or protein. Aptamers can possess significant advantages over their protein counterparts such as ease of screening and synthesis, smaller size, and increased permeability and stability.

[0033] In embodiments, the nucleic acid conjugate is a CpG Deoxynucleotide. CpG deoxynucleotides are short single-stranded DNAs that produce an immunostimulatory response. CG deoxynucleotides can activate dendritic cells and B cells by interacting with Toll-like receptor 9 resulting in a proinflammatory cascade.Table 3. List of Approved ASO, siRNA, Aptamer, and CpG Nucleic Acid TherapeuticsDBl / 162871737.3 202DB1 / 162871737.3 203When not stated, the sugar chemistry is full 2'-H, apart from PMOs that have altered sugars themselves. PO, phosphate; PS, phosphorothioate; PMO, phosphorodiamidate morpholino oligomer; OMe, methoxyl; MOE, methoxyethyl; PEG, polyethylene glycol; GalNAc, N- acetylgalactosamine.4. AntigensDBl / 162871737.3 204

[0034] In an embodiment, the disclosed formulations comprise an antigen for a traditional subunit vaccine. In certain embodiments, the antigen is a protein (including recombinant proteins), polypeptide, or peptide (including synthetic peptides). In certain embodiments, the antigen is a lipid or a carbohydrate (polysaccharide). In certain embodiments, the antigen is a protein extract, cell (including tumor cell), or tissue. The compositions provided herein can contain one or more antigens (e.g., at least two, three, four, five, or six antigens).[00035J In specific embodiments, antigens can be selected from the group consisting of the following: (a) polypeptides suitable to induce an immune response against cancer cells; (b) polypeptides suitable to induce an immune response against infectious diseases; (c) polypeptides suitable to induce an immune response against allergens; and (d) polypeptides suitable to induce an immune response in farm animals or pets.

[0036] In certain embodiments, the compositions of the invention can be used in combination with an immunoregulatory therapy to target either activating receptors or inhibitory receptors. See, e.g., Mellman et al., 2013, Nature 480:480-489. The immunoregulatory therapy can be, for example, a T cell engaging agent selected from agonistic antibodies which bind to human 0X40, to G1TR, to CD27, or to 4-IBB, and T-cell bispecific antibodies (e.g. T cellengaging BiTE™ antibodies CD3-CD19, CD3-EpCam, CD3-EGFR), IL-2 (Proleukin), Interferon (IFN) alpha, antagonizing antibodies which bind to human CTLA-4 (e.g. ipilimumab), to PD-1 , to PD-L1 , to TIM-3, to BTLA, to VISTA, to LAG-3, or to CD25.

[0037] In certain embodiments, the composition can be used to increase the amount of nucleic acid antigen transported into the lymphatic system and lymphatic nodes. Whereby, upon administration to a subject, the nucleic acid antigen delivery system in combination with PH20 synergistically enhances the immune response by directing the nucleic acid antigens to specific cellular targets within the lymphatic system, thereby modulating the activity of immune cells in a manner that is therapeutically beneficial for the treatment of diseases or conditions associated with the immune system, that includes the treatments of cancers.

[0038] Exemplary antigens for the traditional subunit vaccine include those from a pathogen (e.g. virus, bacterium, parasite, fungus) and tumors (especially tumor-associated antigens or “tumor markers”). Other exemplary antigens include autoantigens.

[0039] In some embodiments, the antigen or antigenic determinant is one that is useful for the prevention of infectious disease. Such treatment will be useful to treat a wide variety ofDBl / 162871737.3 205infectious diseases affecting a wide range of hosts, preferably human, but including cow, sheep, pig, dog, cat, and other mammalian species and non-mammalian species. Thus, antigens or antigenic determinants selected for the compositions will be well known to those in the medical art.

[0040] Examples of antigens or antigenic determinants include the following: the RSV F or G antigens, Chlamydia antigens such as the Major outer membrane protein (mOMP), the Dengue type 1 to 4 envelope proteins, the HIV antigens gp!40 and gp!60; the influenza antigens hemagglutinin, M2 protein, and neuraminidase; hepatitis B surface antigen or core; and circumsporozoite protein of malaria, or fragments thereof.

[0041] Appropriate antigens for use with this LNP technology may be derived from, but not limited to, pathogenic bacterial, fungal, or viral organisms, Streptococcus species, Candida species, Brucella species, Salmonella species, Shigella species, Pseudomonas species, Bordetella species, Clostridium species, Norwalk virus, Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zoster virus, Epstein- Barr virus, Hepatitis viruses, Plasmodium species, Trichomonas species, Ebola, sexually transmitted disease agents, viral encephalitis agents, protozoan disease agents, fungal disease agents, cancer cells, or mixtures thereof. Other appropriate molecules incorporated in the LNP vaccines may include self-antigens, adhesins, or surface exposed cell signalling receptors or ligands. A variety of diseases and disorders may be treated by such LNPvaccine constructs or assemblies, including: inflammatory diseases, infectious diseases, cancer, genetic disorders, organ transplant rejection, autoimmune diseases and immunological disorders.

[0042] Examples of infectious disease include, but are not limited to, viral infectious diseases, such as AIDS, Respiratory Syncytial Virus (RSV), Chickenpox (Varicella), Common cold, Cytomegalovirus Infection, Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hand, foot and mouth disease, Hepatitis, Herpes simplex, Herpes zoster, HPV, Influenza (Flu), Lassa fever, Measles, Marburg hemorrhagic fever, Infectious mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viral gastroenteritis, Viral meningitis, Viral pneumonia, West Nile disease and Yellow fever; bacterial infectious diseases, such as Anthrax, Bacterial Meningitis, Botulism, Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,DBl / 162871737.3 206Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy (Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease, Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis (Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever, Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia, Typhoid Fever, Typhus and Urinary Tract Infections; parasitic infectious diseases, such as African trypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis, Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Free-living amebic infection, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Kalaazar, Leishmaniasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Pinworm Infection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungal infectious disease, such as Aspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis (Athlete's Foot) and Tinea cruris; prion infectious diseases, such as Alpers' disease, Fatal Familial Insomnia, Gerstmann- Straussler-Scheinker syndrome, Kuru and Variant Creutzfeldt-Jakob disease.

[0043] Examples of cancers include, but are not limited to breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and meduUoblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia / lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor.DBl / 162871737.3 207

[0044] Any antigen associated with any of the diseases or conditions provided herein can be used in the compositions and methods described herein. These include antigens associated with cancer, infections or infectious disease or degenerative or non-autoimmune disease. Antigens associated with HIV, malaria, leischmaniasis, a human filovirus infection, a togavirus infection, a alphavirus infection, an arenavirus infection, a bunyavirus infection, a flavivirus infection, a human papillomavirus infection, a human influenza A virus infection, a hepatitis B infection or a hepatitis C infection are also included.

[0045] Examples of cancer antigens include HER 2 (pi 85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG-72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and M0vl8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Further examples include MAGE, MART-l / Melan-A, gplOO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, Colorectal associated antigen (CRC) — C017-1A / GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, prostatic acid phosphatase (PAP), Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, pro state-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-I or MAGE-II families) (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE- AI I, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE- 64), MAGE-CI, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, a-fetoprotein, E-cadherin, a-catenin, P-catenin and y-catenin, pl20ctn, gplOOPmell 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pl5, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PIA, EBV- encoded nuclear antigen (EBNA)-l, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL- 40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 and c-erbB-2.

[0046] In another embodiment, antigens associated with infection or infectious disease are associated with any of the infectious agents provided herein. In one embodiment, theDBl / 162871737.3 208infectious agent is a virus of the Adenoviridae, Picomaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae or Paroviridae family. In still another embodiment, the infectious agent is adenovirus, coxsackievirus, hepatitis A virus, poliovirus, Rhinovirus, Herpes simplex virus, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus, Hepatitis B virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenza virus, Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Human papillomavirus, Rabies virus, Rubella virus, Human bocarivus or Parvovirus Bl 9. In yet another embodiment, the infectious agent is a bacterium of the Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, TreponemaVibrio or Yersinia genus. In a further embodiment, the infectious agent is Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersinia pestis. In another embodiment, the infectious agent is a fungus of the Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrys genus. In still another embodiment, the infectious agent is C. albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neof ormans, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii or Stachybotrys chartarum.

[0047] In yet another embodiment, the antigen associated with infection or infectious disease is one that comprises VI, VII, El A, E3-19K, 52K, VP1, surface antigen, 3A protein,DBl / 162871737.3 209capsid protein, nucleocapsid, surface projection, transmembrane proteins, UL6, ULI 8, UL35, UL38, UL19, early antigen, capsid antigen, Pp65, gB, p52, latent nuclear antigen-1, NS3, envelope protein, envelope protein E2 domain, gpl20, p24, lipopeptides Gag (17-35), Gag (253- 284), Nef (66-97), Nef (116-145), Pol (325-355), neuraminidase, nucleocapsid protein, matrix protein, phosphoprotein, fusion protein, hemagglutinin, hemagglutinin-neuraminidase, glycoprotein, E6, E7, envelope lipoprotein or non-structural protein (NS). In another embodiment, the antigen comprises pertussis toxin (PT), fdamentous hemagglutinin (FHA), pertactin (PRN), fimbriae (FIM 2 / 3), VlsE; DbpA, OspA, Hia, PrpA, MltA, L7 / L12, D15, 0187, VirJ, Mdh, AfuA, L7 / L12, out membrane protein, LPS, antigen type A, antigen type B, antigen type C, antigen type D, antigen type E, FliC, FliD, Cwp84, alpha-toxin, theta-toxin, fructose 1,6- biphosphate-aldolase (FBA), glyceraldehydes-3 -phosphate dehydrogenase (GPD), pyruvate:ferredoxin oxidoreductase (PFOR), elongation factor-G (EF-G), hypothetical protein (HP), T toxin, Toxoid antigen, capsular polysaccharide, Protein D, Mip, nucleoprotein (NP), RD1, PE35, PPE68, EsxA, EsxB, RD9, EsxV, Hsp70, lipopolysaccharide, surface antigen, Spl, Sp2, Sp3, Glycerophosphodiester Phosphodiesterase, outer membrane protein, chaperone-usher protein, capsular protein (Fl) or V protein. In yet another embodiment, the antigen is one that comprises capsular glycoprotein, Yps3P, Hsp60, Major surface protein, MsgCl, MsgC3, MsgC8, MsgC9 or SchS34.

[0048] In certain embodiments, one or more antigens are physically encapsulated in the LNP during or after LNP preparation. Antigens physically encapsulated in the LNP can be prepared via confined-volume ethanol desolvation method as described, or via alternative techniques known in the art, including, but not limited to thin-film hydration, emulsion diffusion, or homogenization.

[0049] In certain embodiments of the invention, one or more of the antigens is adsorbed, covalently coupled, ionically-interacted, or formulated onto surfaces of the LNP adjuvant.

[0050] In an alternative embodiment, the LNP adjuvant may be co-administered with one or more antigens and / or one or more agonists.5. Antibody-Drug Conjugates

[0051] In another embodiment, the LNP encapsulates an ADC. In some embodiments, the ADC is administered independent of a LNP delivery system.DBl / 162871737.3 210

[0052] In embodiments herein, antibody-drug conjugates (ADC) are comprised of a monoclonal antibody (mAb), a cytotoxic payload, and a chemical linker. Once the ADC reaches the target cells, the mAb component recognizes and binds to the cell surface antigens, and the ADC-antigen complex is then internalized within the cancer cell by endocytosis to form an early endosome, which, following a maturation, forms late endosomes and finally fuses with lysosomes. The cytotoxic drug payload is then released from the mAb via either a chemical reaction or enzyme digestion in the lysosomes, and exerts its cytotoxic effect, causing cell apoptosis or death.

[0053] In addition to the cytotoxic properties from the payload, the Fc portion of the monoclonal antibody aids in immune-related cytotoxicities, such as antibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), and complementdependent cytotoxicity (CDC). Genetic engineering technologies have advanced to enhance the effector function of the antibody in the Fc region. Additionally, the binding of the antibody component of ADC with the specific antigen epitope of cancer cells can inhibit the downstream signal transduction of the antigen receptor.

[0054] An appropriate selection of target antigen is central to the design of an ADC. First, the antigen should be expressed, either exclusively or predominantly, in the tumor cells to reduce the off-target toxicity. Secondly, the binding to the target antigen should ideally lead to the internalization of the antigen-antibody complex. Additionally, it should ideally be on the surface rather than intracellular for it to be recognized, and lastly, it should not be secretory since a secreted antigen in the circulation would cause the undesirable ADC to bind outside of the tumor sites. Exemplary target antigens for ADC include but are not limited to, CD 19, CD22, CD30, CD33, and CD79b in hematological malignancies and HER2, trop2, nectin4, tissue factor, and folate receptor alpha (FRa) in solid cancers.

[0055] An ideal antibody moiety should facilitate an effective internalization, have high antigen affinity, preserve long plasma half-life, and demonstrate low immunogenicity. The mAb are large-sized and account for over 90% of the mass of any given ADC. This is favorable because it encounters reduced distribution or permeation into healthy tissue, including those normally functioning as metabolizing and eliminating organs. No such problem is encountered at the tumor site as the vasculature in the tumor is characteristically leaky and allows the distribution and permeation of the ADC to the tumor cells.DBl / 162871737.3 211

[0056] In an embodiment, there are two types of linkers in the ADC, including cleavable and non-cleavable. The cleavable linkers are either chemically labile (hydrazone bond and disulfide bond) or enzymatically labile. Hydrazone linkers are generally stable in alkaline environments and are hydrolyzed in low pH environments, such as that in the lysosome and endosome. Hence, the cleavage of ADC with hydrazone linkers occurs predominantly in the lysosome and endosome upon internalization, with occasional hydrolysis in the plasma, resulting in off-target, systemic toxicity. Similarly, a disulfide bond linker can be stable in the plasma or serum while specifically releasing the active payloads in the cancer cells with an elevated reductive glutathione level. The enzyme sensitive linkers are sensitive to the lysosomal protease that is generally overexpressed in cancer cells, enabling an accurate drug release in the cells after internalization. ADCs with non-cleavable linkers are resistant to chemical or enzymatic digestion in the plasma or serum and will require complete degradation of the antibody within the late endosomes and lysosome to release the payload. Therefore, ADC with non-cleavable linkers may have the lowest off-target systemic toxicity due to increased plasma or serum stability and thus they are most suitable in the treatment of tumors with homogenous antigen expression. In an embodiment, some of the ADC have been engineered to have desirable “off-target effect” for “by-stander killing” extending the cytotoxic effect to the low or negative antigen-expressing cells in the tumor proximity. For this mechanism to work, several characteristics of the ADC molecules are crucial: namely, a cleavable linker and a non-polar, freely membrane-permeable payload. Conversely, to reduce the undesirable systemic toxicity from payload molecules permeating out of the tumor cells, ionizable payloads (e.g. containing carboxylic acids) can be used.

[0057] In an embodiment, the cytotoxic payloads can preferably have the following properties. High potency, in vitro high cytotoxic activity (e.g., sub-nanomolar half maximal inhibitory concentration (IC50) value), high stability in the systemic circulation, sufficient solubility in the aqueous environment of antibody and biochemical properties to allow easier conjugation to the antibody, low immunogenicity, small molecular weight, and a long half-life. In an embodiment, there are mainly two classes of cytotoxic drugs used as payloads, comprising microtubule inhibitors or DNA damaging agents.

[0058] In an embodiment, auristatins and maytansines payloads are both cytotoxic agents that work as tubulin inhibitors. Auristatin is a dolastatin synthetic analog. There are twoDBl / 162871737.3 212auristatin derivatives: one is monomethyl auristatin E (MMAE) and the other is monomethyl auristatin F (MMAF). These two cytotoxic agents differ structurally wherein the phenylalanine present at the C-terminus renders MMAF membrane-impermeable, whereas the MMAE can exit the cell and thus diffuse to nearby cells and kill them through the bystander effects. In an embodiment, the cytoxic agent is selected from maytansinoids which are natural cytotoxic agents isolated from the cortex of Maytenus serrata, which possesses a macrolide structure.[00059J In an embodiment, the cytotoxic agents are selected from calicheamicins, pyrrolobenzodiazepines and topoisomerase inhibitors which are DNA-damaging agents that act through DNA double strand breaks, crosslinking, and intercalation, respectively. Both gemtuzumab ozogamicin and inotuzumab Ozogamicin have N-acetyl gamma calicheamicin as a payload. Calicheamicins belong to a class of potent anti-tumor antibiotics that cleave the DNA in a site-specific, double-stranded manner. Pyrrolobenzodiazepines are another class of antibiotics derived from Streptomyces species and is used as a cytotoxic payload in Loncastuximab Tesirine. SN-38 and Deruxtecan are topoisomerase inhibitors that are the cytotoxic components of Sacituzumab Govitecan and Trastuzumab Deruxtecan, respectively. Any of the above cytotoxic agents are embodied in the ADC.

[0060] In addition to the choice of the antibody, the linker, and the payload, the method of conjugation is also important for the successful structure of ADC. In an embodiment, the lysine and cysteine residues on the antibody provide the accessible reaction sites for conjugation. In an embodiment, a varying number (0-8) of small-molecule toxins may be attached to an antibody, as the conventional conjugation methods are random, resulting in a wide drugantibody ratio (DAR) distribution. In an embodiment, the ideal DAR is 2-4. A low DAR can lower the efficacy, while a high DAR may increase the drug potency.

[0061] Exemplary ADC, antigen targets, linkers and cytotoxins for use in the present disclosure are included in Table 4 below.Table 4. Exemplary ADC, antigen targets, linkers, and cytotoxins.DBl / 162871737.3 213DB1 / 162871737.3 214

[0062] In embodiments herein, an antibody-drug conjugate is provided in a suspension. The suspension includes any suitable suspension of an antibody-drug conjugate, such as those exemplified. Combinations and treatment regimens are provided herein in which an antibodydrug conjugate is administered in combination with a soluble hyaluronidase.

[0063] In certain embodiments, the composition of the invention can be used to increase the amount of ADC transported into the lymphatic system and lymphatic nodes. Upon administration to a subject, the ADC delivery system in combination with PH20 synergistically enhances the immune response by directing the ADC to specific cellular targets within the lymphatic system, thereby modulating the activity of immune cells in a manner that is therapeutically beneficial for the treatment of diseases or conditions associated with the immune system, that includes the treatments of cancers.6. Monoclonal Antibodies

[0064] In an embodiment, the LNP encapsulates a therapeutic monoclonal antibody.

[0065] The term “antibody” is used in the broadest sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), multi-specific antibodies (e g. bispecific antibodies), and antibody fragments that exhibit a desired biological activity or function.

[0066] An antibody generally comprises two heavy chains and two light chains, each comprising a variable domain and a constant domain. Each variable domain contains a hypervariable region containing three complementarity determining regions (CDR) flanked by four segments of the framework region. As used herein, the “framework region” contains all four segments FR1, FR2, FR3, and FR4 that flank a set of three hypervariable regions.DBl / 162871737.3 215

[0067] Antibodies can be chimeric, humanized, or human, for example, and can be antigen-binding fragments of these. Antibodies are generally produced by immunizing an animal with an antigen, and can be produced by recombinant technology, or by synthesis of the amino acid sequence, for example. “Antibody fragments” comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies such as bispecific antibodies, for example formed from antibody fragments. “Functional fragments” substantially retain binding to an antigen of the full- length antibody and retain a biological activity.

[0068] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler (1975), Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries.

[0069] Chimeric” antibodies (immunoglobulins) contain a portion of a heavy and / or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567). A “humanized antibody” as the term is used herein, is a subset of chimeric antibodies.DBl / 162871737.3 216

[0070] ‘Humanized” forms of non-human (e.g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which variable domain hypervariable region residues of the recipient antibody are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. The hypervariable regions can be complementarity-determining regions (CDR) defined by sequence, or hypervariable loops (HVL) defined by structure, or both. In some embodiments, the variable domain framework regions are derived from a consensus sequence variable domain, for example, containing at each residue an amino acid compiled as most abundant at that position in a class or subclass of human immunoglobulin variable domains, for example, in a Kabat compilation. In some instances, one or more amino acids of the variable domain framework region (FR) of the human immunoglobulin or consensus sequence is replaced with one or more corresponding residues of the non-human donor antibody and / or one or more amino acids of the donor antibody hypervariable regions is replaced with one or more corresponding human residues of the human recipient variable domain. In some instances, one or more residues of the variable domain framework regions and / or hypervariable regions is a residue not found at the corresponding position in the recipient antibody or in the donor antibody. Modifications to the amino acid sequence of the variable domain framework regions and hypervariable regions are generally made to further refine antibody performance, for example, improve binding affinity. In general, the humanized antibodies used to produce the pan-specific antibodies described herein will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable region residues (CDR or HVL) correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework region (FR) residues correspond to those of a human variable domain consensus sequence, and may include one or more amino acid substitutions. In some embodiments, the number of amino acid substitutions in the human consensus framework region is typically no more than 10, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the heavy chain variable domain framework regions, and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the light chain variable domain framework region. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin.DBl / 162871737.3 217

[0071] An “Fv” fragment is an antibody fragment that contains a complete antigen recognition and binding site and generally comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in a single chain variable domain fragment (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain comprising only three hypervariable regions specific for an antigen has the ability to recognize and bind antigen.

[0072] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0073] A “Fab” fragment includes a variable domain and a constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. A Fab1fragment includes one or more cysteine carboxy terminal linkages to the heavy or light chains. F(ab')2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.

[0074] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two variable domains on the same chain, the variable domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites.

[0075] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity-determining region” or “CDR” (defined by sequence as residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health) and / or those residues from a hypervariable loop. In one example, HVL residues can include, 26-32 (LI),DBl / 162871737.3 21850-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain.

[0076] “Framework region” or “FR” residues are those variable domain residues flanking the hypervariable region residues as herein defined. In general, a variable domain contains three hypervariable regions flanked by four sequences of the framework region (FR1, FR2, FR3, and FR4).

[0077] The term “consensus sequence” as used herein, refers to an artificial variable domain sequence comprising at each position the residue that is most abundant at that position in the variable domains of a group of antibodies of a particular class. The consensus variable domain sequences do not have any known antibody binding specificity or affinity.

[0078] Exemplary monoclonal antibodies, target antigens, and antibody type for use in the present disclosure are included in Table 5 below.Table 5. Exemplary monoclonal antibodies, target antigens, and antibody type.DBl / 162871737.3 219DB1 / 162871737.3 220DB1 / 162871737.3 221DB1 / 162871737.3 222DB1 / 162871737.3 223DB1 / 162871737.3 224

[0079] In embodiments herein, a monoclonal antibody is provided in a suspension. The suspension includes any suitable suspension of a monoclonal antibody, such as thoseDB1 / 162871737.3 225exemplified. Combinations and treatment regimens are provided herein in which a monoclonal antibody is administered in combination with a soluble hyaluronidase.

[0080] In certain embodiments, disclosure describes a monoclonal antibody delivery system used to increase the amount of a monoclonal antibody transported into the lymphatic system and lymphatic nodes. In these embodiments, upon administration to a subject, the monoclonal antibody delivery system in combination with a hyaluronidase synergistically enhances the immune response by directing the monoclonal antibody to specific cellular targets within the lymphatic system. In this embodiment, upon administration the combination modulates the activity of immune cells in a manner that improves the treatment of the disease or condition associated with the immune system. In various embodiments the disease / condition being treated is a cancer.7. Cancer Therapies

[0081] In an embodiment, the LNP encapsulates a cancer therapy. In some embodiments, the cancer therapy is an antitumor agent. In some embodiments, the LNP is selected from liposomes, cubosomes, niosomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), microemulsions (ME), nanoemulsions (NE), and Pickering emulsions (PE). In some embodiments, the cancer therapy is encapsulated by liposomes, cubosomes, niosomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), microemulsions (ME), nanoemulsions (NE), or Pickering emulsions (PE).

[0082] In some embodiments, the LNP encapsulated cancer therapy is administered to treat a cancer selected from pancreatic cancer, intestinal cancer, myelodysplastic syndrome, kidney cancer, liver cancer, thyroid cancer, renal cell carcinoma, adrenal cell carcinoma, head and neck cancer, pharyngeal cancer, esophageal cancer, retinoblastoma, glioblastoma, medulloblastoma, neuroblastoma, low grade glioma, colorectal cancer, lung cancer, mesothelioma, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, testicular cancer, melanoma, osteosarcoma, triple negative breast cancer (TNBC), urothelial carcinoma, bladder cancer, gastric cancer, cholangiocarcinoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL), chronic lymphocytic leukemia (CLL), mast cell leukemia (MCL), lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cellDBl / 162871737.3 226lymphomas, extranodal marginal zone B-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), high risk myelodysplastic syndrome, acute myelodysplastic syndrome, myeloproliferative neoplasm myelodysplastic syndrome, myelofibrosis, uveal melanoma, synovial sarcoma, non-Hodgkin lymphoma, pancreatic ducal adenocarcinoma, advanced adenoid cystic carcinoma, oral cancer, gastroesophageal malignancy, glioblastoma multiforme, giant cell glioblastoma, brain cancer, glioma, oligodendrogliomas, colon cancer, colorectal adenocarcinoma, KRAS-driven lung cancer, lung adenocarcinoma, non-small cell lung cancer, extensive stage lung small cell carcinoma, astrocytoma, metastatic castration-prostate cancer, advanced sarcoma, metastatic sarcoma, Ewing sarcoma, breast cancer, acute leukemia, myelofibrosis leukemia, eosinophilic leukemia, and T-cell acute lymphoblastic lymphoma.

[0083] In some embodiments, the LNP encapsulated cancer therapy is administered to treat a KRAS-driven cancer selected from KRAS-driven pancreatic cancer, KRAS-driven intestinal cancer, KRAS-driven myelodysplastic syndrome, KRAS-driven kidney cancer, KRAS-driven liver cancer, KRAS-driven thyroid cancer, KRAS-driven renal cell carcinoma, KRAS-driven adrenal cell carcinoma, KRAS-driven head and neck cancer, KRAS-driven colorectal cancer, KRAS-driven lung cancer, KRAS-driven ovarian cancer, KRAS-driven endometrial cancer, KRAS-driven cervical cancer, KRAS-driven prostate cancer, KRAS-driven testicular cancer, KRAS-driven melanoma, KRAS-driven osteosarcoma, KRAS-driven triple negative breast cancer (TNBC), KRAS-driven urothelial carcinoma, KRAS-driven bladder cancer, KRAS-driven gastric cancer, KRAS-driven leukemia, KRAS-driven lymphoma, KRAS-driven multiple myeloma, KRAS-driven uveal melanoma, KRAS-driven synovial sarcoma, KRAS-driven oral cancer, KRAS-driven brain cancer, KRAS-driven colon cancer, KRAS-driven colorectal adenocarcinoma, KRAS-driven lung cancer, KRAS-driven prostate cancer, KRAS-driven sarcoma, and KRAS-driven breast cancer.

[0084] As described herein, cubosomes are lipid vesicles that are comparable to vesicular systems like liposomes. They are dispersions of one of the bicontinuous cubic phases that have a lamellar wall. Cubosomes may be created with certain amphiphilic lipids in the presence of a suitable stabilizer. In some embodiments, cubosomes are nanostructured liquid-crystalline particles formed of specific amphiphilic lipids in different ratios.DBl / 162871737.3 227

[0085] As described herein, niosomes are delivery devices that can be used to deliver the compositions disclosed herein. Niosomes are multilamellar or unilamellar vesicles involving non-ionic surfactants. An aqueous solution of solute is enclosed by a bilayer resulting from the organization of surfactant macromolecules. Similar to liposomes, niosomes are used in targeted delivery of, for example, anticancer drugs, including methotrexate, doxorubicin, and immunoadjuvants. They are generally understood to be different from transferosomes, vesicles prepared from amphiphilic carbohydrate and amino group containing polymers, e.g., chitosan.

[0086] As used herein, solid lipid nanoparticles (SLN) are colloidal drug carriers and dynamic structures that are typically synthesized from phospholipids, lipids, and excipients. They are composed of an outer phase membrane of lipids and / or phospholipids and an inner phase solid lipid inner core. SLNs have a mean particle size in the nanometer range. SLNs combine the advantages of emulsions, liposomes and polymeric nanoparticles. The solid matrix can protect incorporated active ingredients against chemical degradation and provide the highest flexibilities in the modulation of the drug release profiles. SLNs provide controlled release, efficient targeting, and stability. SLNs are particulates structurally related to polymeric nanoparticles. However, in contrast to polymeric systems, SLNs can be composed of biocompatible lipids that are physiologically well tolerated when administered in vivo and may also be prepared without organic solvents.

[0087] As used herein, nanostructured lipid carriers (NLC) are colloidal carriers and a second generation evolvement of SLNs. NLCs are characterized by an outer phase phospholipid and / or lipid membrane and an inner phase lipid core consisting of a mixture of solid and liquid lipids. NLCs have a mean particle size in the nanometer range. NLC controlled nanostructuring of the lipid matrix is performed due to the mixture of solid and liquid lipids, in order to increase drug loading and prevent its expulsion. In addition, the NLC nanostructured lipid matrix gives more flexibility in modulation of drug release. NLCs are composed of a lipid matrix of cannabinoids with a nanostructure that improves cannabinoid loading and firmly retains the cannabinoids during storage.

[0088] As used herein, a microemulsion is an aqueous suspension of pseudomicelles which contain relatively hydrophobic lipid centers surrounded by a monolayer of amphipatic (sometimes spelled amphipathic) molecules bearing hydrophilic moieties. The concentration of pseudomicelles in the aqueous suspension is typically about 0.1 mg / ml-10 mg / ml. In theDBl / 162871737.3 228microemulsions of the invention, the diameter of the pseudomicelles is approximately 500-1.500 A. preferably 700-1.300 A. The nature of the pseudomicellular particle is different from that of liposomes, which are unilamellar or multilamellar particles of bilayers, wherein the bilayers have hydrophilic surfaces on either side. Thus, liposomes have hydrophilic interiors as well as exteriors and are often of smaller dimension, i.e. of the order of 50-300 A. Liposomes are appropriate for delivery of hydrophilic, rather than hydrophobic, active ingredients.[00089J As used herein, a nanoemulsion (NE) is a nanosized colloidal system that consists of poorly water soluble compounds, suspended in an appropriate dispersion medium (oil-in- water emulsion) stabilized by surfactants. NEs are carrier systems in the nanometer size comprising a continuous aqueous phase and at least one dispersed oily phase, in which the oily phase comprises at least one amphiphilic lipid such as phospholipids and at least one solubilizing lipid with a monolayer around an amorphous core.

[0090] In certain embodiments, the composition of the invention can be used to increase the amount of cancer therapy transported into the lymphatic system and lymphatic nodes. In these embodiments, upon administration to a subject, the cancer therapy delivery system in combination with PH20 synergistically enhances the immune response by directing the cancer therapy to specific cellular targets within the lymphatic system. In some embodiments, the cancer therapy delivery system modulates, thereby modulating the activity of immune cells in a manner that is therapeutically beneficial for the treatment of diseases or conditions associated with the treatments of cancers.

[0091] In some embodiments, the composition of the invention is used to improve the pharmacokinetic parameters of the cancer therapy. In particular embodiments, the administration of a cancer therapy encapsulated in an LNP in combination with a hyaluronidase enzyme increase the Cmax of the cancer therapy. In embodiments, the Cmax is increased by at least 5%, at least 10%, at least 15%, at least 20%>, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In particular embodiments, the administration of a cancer therapy encapsulated in an LNP in combination with a hyaluronidase enzyme increase the Tmax of the cancer therapy. In embodiments, the Tmax is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atDBl / 162871737.3 229least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0092] In some embodiments, the composition of the invention is used to improve the solubility of the cancer therapy. In particular embodiments, the administration of a cancer therapy encapsulated in an LNP in combination with a hyaluronidase enzyme increase the solubility of the cancer therapy. In embodiments, the solubility is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0093] Exemplary cancer therapies for use in the present invention are included in the Table 5 below.Table 5DBl / 162871737.3 230DB1 / 162871737.3 2318. Radiopharmaceuticals

[0094] In an embodiment, the LNP encapsulates a radiopharmacetuical. In some embodiments, the radiopharmaceutical comprises one or more of a radioisotiope and a radioligand. In some embodiments, the LNP is selected from liposomes, cubosomes, niosomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), microemulsions (ME), nanoemulsions (NE), and Pickering emulsions (PE). In some embodiments, the radiopharmceutical is encapsulated by liposomes, cubosomes, niosomes, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), microemulsions (ME), nanoemulsions (NE), or Pickering emulsions (PE).

[0095] In certain embodiments, radioisotope (also referred to in the art as a radionuclide) can be selected such that its decay chain releases one or more of alpha radiation, beta radiation, gamma radiation, Auger electrons, and / or x-rays. The radioactive isotope of the present invention can be selected to enable imaging and / or radiotherapy. The radioactive isotopes of the present invention may include radioactive metal or semi-metal isotopes. Preferably, the radioactive isotopes are water soluble metal cations. Exemplary radioisotopes include1SF,32P,

[0096] The radioisotope may be bound to a pharmaceutical compound to provide a radiopharmaceutical compound that transports it to a required location in the bodyDB1 / 162871737.3 232after administration. Radiopharmaceuticals have been used for a variety of therapeutic and diagnostic indications. Among others, radiolabeled molecules have been useful to treat various malignant tumors. The majority of radiopharmaceuticals are used for in vivo imaging, and comprise a radionuclide having emissions suitable for detection, typically by single-photon emission computed tomography (SPECT) or positron emission tomography (PET).

[0097] In embodiments, the radiopharmaceutical comprises a chelating moiety. The chelating moiety can comprise any chelator known in the art that is suitable for chelating a particular radioisotope, see, e.g., Parus et al., “Chemistry and bifunctional chelating agents for binding (177) Lu,” Curr Radi opharm. 2015; 8(2):86-94; Wangler et al., “Chelating agents and their use in radiopharmaceutical sciences,” Mini Rev Med Chem. 201 1 October; 1 1(11 ):968-83; Liu, “Bifunctional Coupling Agents for Radiolabeling of Biomolecules and Target-Specific Delivery of Metallic Radionuclides,” Adv Drug Deliv Rev. 2008 September; 60(12): 1347-1370. Examples of chelating moieties are included in Table 6below.

[0098] Table 6. Chelating Moieties.DBl / 162871737.3 233DB1 / 162871737.3 234DB1 / 162871737.3 235DB1 / 162871737.3 236

[0099] Examples of metal isotopes and chelators that are suitably form a complex are shown in Table 7 below.Table 7. Exemplary metal isotopes and chelators.DBl / 162871737.3 237

[0100] Examples of radiolabelled pharmaceutical compounds are: In-111 Oxy quinoline, Tc- 99m Disofenin, Tc-99m Lidofenin, Tc-99m Mebrofenin, Tc-99m Disida, Sodium Chromate Cr- 51, Sodium Pertechnetate Tc-99m, lofetamine 1-123, Sodium 1-131, Sodium Fluoride F-18, Tc- 99m Pyrophosphate, Tc-99m (Pyro- and trimeta-) Phosphates, Tc-99m Albumin Colloid, Tc- 99m Sulfur Colloid, Fluodeoxyglucose F-18, In-111 Pentetreotide, Tc-99m Exametazime, Tc- 99m Gluceptate, Tc-99m Arcitumomab, Tc-99m Nofetumomab Merpentan, Ferrous Citrate Fe- 59, Tc-99m Teboroxime, Tc-99m Tetrofosmin, Thallous Chloride Tl-201, lodohippurate Sodium 1-123, lodohippurate Sodium 1-131, Iothalamate Sodium 1-125, Tc-99m Succimer, Cyanocobalamin Co-57, lobenguane, Sodium 1-123, lobenguane, Sodium 1-131, F-18 florbetapir, F-18 florbetaben, F-18 NAV4694, F-18 Flutemetamol, 1-123 loflupane, 1-131 tositumomab, Sm-153 EDTMP, Ho-166 DOTMP, Re-186-HEDP, Sr-89 chloride Y-90 chloride, Y-90 ibritumomab tiuxetan, Re-188-HEDP, Tc-99m-HEDP, Zr-89 DFO-J549, Cu-64 ATSM, P- 32 sodium phosphate, Ga-68 DOTATATE, Ga-68 RGD, Ga-68 UBI, Ga-68 citrate, Lu-177 DOTATATE, Lu-177 ibritumomab tiuxetan, Lu-177-EDTMP, F-18 maltose, F-18- maltohexaose, F-18-2-fluorodeoxy sorbitol, Pt-195m cisplatinum, Pt-195m carboplatinum, Pt- 195m satraplatin, Pt-195m eloxatin, 1-123 Deoxyuridine, 1-125 Deoxyuridine, Technescan™ HDP (Tc-99m oxidronate), CARDIOLITE® (Tc-99m sestamibi), AN-DTPA® (Tc-99m Pentetate), Technescan™ MAG3 (Tc-99m mertiatide), Gluscan® (F-18 FOG), Xofigo® (Ra-223 Chloride), Gallium Citrate Ga-67 Injection.DBl / 162871737.3 238F. Methods of Administration, Regimens, and Combinations1. Methods of Administration

[0101] In an embodiment, each of the hyaluronidase and the LNP formulation can be administered to a patient via injection. In an embodiment the hyaluronidase and the LNP formulation is administered subcutaneously. For example, the hyaluronidase and the LNP formulation can be administered to a patient subcutaneously in the abdominal tissue, leg or arm. The hyaluronidase and the LNP formulation can be administered separately or in the same composition.

[0102] The compositions for administration to a patient via an injection (e.g. subcutaneously) also may comprise suitable inert additives, stabilizers, carriers, or excipients. In an embodiment, the injectable composition comprises histidine. In an embodiment, the injectable composition comprises sodium chloride. In an embodiment, the injectable composition comprises polysorbate. In an embodiment, the polysorbate comprises polysorbate 80. In an embodiment, the injectable composition comprises an antioxidant. In an embodiment, the antioxidant comprises methionine.

[0103] It is shown and described herein that when an LNP formulation is administered in combination with the hyaluronidase, dispersion of the co-injected drugs or co-delivered is enhanced. By depolymerizing hyaluronan, hyaluronidase temporarily facilitates dispersion by reducing the viscosity of interstices. The permeability barrier in these tissues is restored to preinjection levels within 24 to 48 hours after injection of hyaluronidase. This allows for higher volumes in a single injection of the LNP formulation to be administered to the patient.

[0104] When administered in separate compositions, the hyaluronidase and the LNP formulation are injected as close to the same site as possible. For example, in an embodiment, hyaluronidase is first injected into a patient at a first injection site and subsequently the LNP formulation is injected at the same injection site or at an injection site as close to the first injection site as possible.

[0105] 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 thanDBl / 162871737.3 2398000U, 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 8OOU, less than 700U, less than 600U, or less than 5OOU. 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, at least 5OU, at least 60U, at least 70U, at least 8OU, at least 1OOU, or at least 15OU. 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.

[0106] 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 (ODgoo) and one unit yields a 0.33 ODeoo change 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).

[0107] 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 fromDBl / 162871737.3 24080% 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 at least 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%.

[0108] 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.

[0109] 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.

[0110] In embodiments, the hyaluronidase may be a recombinant hyaluronidase.

[0111] In some embodiments of the combination dosing regimen, the hyaluronidase is administered in an amount effective to increase duration of the production of the functional protein encoded by the mRNA or DNA.

[0112] In some embodiments of the combination dosing regimen, the hyaluronidase is administered in an amount effective to increase the duration of the decrease in the production of the protein inhibited by the siRNA or miRNA.2. Administration of Co-Formulation of Hyaluronidase and LNP FormulationsDBl / 162871737.3 241

[0113] In an embodiment, the hyaluronidase and the LNP formulation are co-formulated for administration. In some embodiments, the co-formulation of the hyaluronidase and LNP formulation is administered to a patient via injection. In particular embodiments, the coformulation is administered subcutaneously, intramuscularly, intradermally, by intratissue injection, or by intratumoral injection.

[0114] In one embodiment, the co-formulation is self-administered. In another embodiment, the co-formulation is administered to the subject by a healthcare professional. In yet another embodiment, the co-formulation is administered to the subject by a layperson, such as a caregiver or patient. In one embodiment, the co-formulation is administered using an autoinjector. In one embodiment, the co-formulation is administered using a HVAI. In one embodiment, the co-formulation is administered from a prefilled syringe using a HVAI. In another embodiment, the co-formulation is administered manually using a manually triggered injection device. In another embodiment, the co-formulation is administered from an on-body device. In another embodiment, the co-formulation is administered from an off-body device. In various embodiments, the off-body device comprises one or more components.

[0115] In one embodiment, the co-formulation is subcutaneously administered to the abdomen, leg, or arm of the patient. In particular embodiments, the formulation is administered to the abdomen or thigh of the patient.3. Administration of Separate Hyaluronidase and LNP Formulations

[0116] In an embodiment, the hyaluronidase and the LNP formulations are each administered concurrently. In some embodiments, each of the hyaluronidase and LNP formulations are administered to a patient via injection. In particular embodiments, the hyaluronidase formulation is administered subcutaneously, intramuscularly, intradermally, by intratissue injection, or by intratumor injection. In particular embodiments, the LNP formulation is administered subcutaneously, intramuscularly, intradermally, by intratissue injection, or by intratumor injection.

[0117] In one embodiment, each of the hyaluronidase and LNP formulations are selfadministered. In another embodiment, each of the hyaluronidase and LNP formulations are administered to the subject by a healthcare professional. In yet another embodiment, each of the hyaluronidase and LNP formulations are administered to the subject by a layperson, such as a caregiver. In one embodiment, the hyaluronidase formulation is administered using anDBl / 162871737.3 242autoinjector. In one embodiment, the hyaluronidase formulation is administered using a HVAI. In one embodiment, the hyaluronidase formulation is administered from a prefilled syringe using a HVAI. In another embodiment, the hyaluronidase formulation is administered manually using a manually triggered injection device. In another embodiment, the hyaluronidase formulation is administered from an on-body device. In another embodiment, the co-formulation is administered from an off-body device. In various embodiments, the off-body device comprises one or more components.

[0118] In one embodiment, the hyaluronidase formulation is subcutaneously administered to the abdomen, leg, or arm of the patient. In particular embodiments, the hyaluronidase formulation is administered to the abdomen or thigh of the patient. In one embodiment, the LNP formulation is subcutaneously administered to the abdomen, leg, or arm of the patient. In particular embodiments, the LNP formulation is administered to the abdomen or thigh of the patient.4. Dosage Regimens

[0119] The amount of the disclosed LNP formulation administered to the subject is dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds, and / or the discretion of the prescribing physician. In various embodiments, an effective dosage of the active ingredient in the disclosed LNP formulation is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg / kg / day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g / day, such as about 0.05 to about 2.5 g / day. In some embodiments, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other embodiments larger doses may be employed without causing any harmful side effects, for example by dividing such larger doses into several small doses for administration throughout the day.

[0120] In one embodiment, the disclosed LNP formulation is administered to the subject in multiple doses. In some embodiments, the disclosed LNP formulation is administered once a day, every other day, every three days, every four days, every five days, or every six days. In other embodiments, the disclosed LNP formulation is administered once a week, every other week, every three weeks, every four weeks, every five weeks, or every six weeks. In otherDBl / 162871737.3 243embodiments, the disclosed LNP formulation is administered once a month, every other month, every two months, every three months, every four months, every five months, or every six months.

[0121] In various embodiments, the disclosed hyaluronidase formulation is administered to the subject in multiple doses. In some embodiments, the disclosed hyaluronidase formulation is administered once a day, every other day, every three days, every four days, every five days, or every six days. In other embodiments, the disclosed hyaluronidase formulation is administered once a week, every other week, every three weeks, every four weeks, every five weeks, or every six weeks. In other embodiments, the disclosed hyaluronidase formulation is administered once a month, every other month, every two months, every three months, every four months, every five months, or every six months.

[0122] In various embodiments, the composition described herein includes a lipid nanoparticle (LNP) therapeutic containing a nucleotide payload in a dosage exceeding 1 milligram, and a hydrogenase enzyme. In some embodiments, the hydrogenase enzyme is formulated to facilitate the dispersion and absorption of the LNP therapeutic. In various embodiments this composition is administered to a subject in need and the method of administering the composition described enables the subcutaneous or intramuscular delivery of the LNP therapeutic in a single administration volume greater than 1 milliliter. In some embodiments, the administration does not cause welts, pain, or irritation to the recipient. In various embodiments, the LNP therapeutic and the hydrogenase enzyme are co-administered in a manner that allows for the efficient uptake of the high-dose nucleotides by target cells. In these embodiments the composition and method described overcomes conventional volume limitations associated with traditional vaccine LNP formulations (typically limited to microgram doses and volumes smaller than 1 milliliter).

[0123] In one embodiment, the disclosed co-formulation is administered to the subject in multiple doses. In some embodiments, the disclosed co-formulation is administered once a day, every other day, every three days, every four days, every five days, or every six days. In other embodiments, the disclosed co-formulation is administered once a week, every other week, every three weeks, every four weeks, every five weeks, or every six weeks. In other embodiments, the disclosed co-formulation is administered once a month, every other month, every two months, every three months, every four months, every five months, or every six months, or annually.DBl / 162871737.3 2445. Auto-Injector or Delivery System

[0124] Referring to the drawings in detail, wherein like reference numerals indicate like elements throughout, there is shown in FIG. 16-22 an auto-injector or injector, generally designated 10, in accordance with a first exemplary embodiment of the present invention. Suitable autoinjectors are described, for example, in U.S. Patent Application Publication No. US 2024-0207375 Al, the entire disclosure of which is hereby incorporated by reference. While an autoinjector is depicted in the accompanying figures for illustrative purposes, it should be understood that the injector 10 described herein is not limited to autoinjector configurations. Injector 10 can encompass any of the following variations: manually operated injectors, pen injectors, prefilled syringes, needle-free injectors, pump-based infusion devices, or any other medical delivery system capable of administering a therapeutic agent.

[0125] Referring to FIG. 16, an injector 10 is shown having a housing 12 configured for allowing a user to grip or handle the injector 10. The housing 12 may be shaped to fit into a user’s hand for single-handed function. The housing 12 may have a generally ovular crosssection to help position the injector 10 in the user’s hand. The housing 12 may further include a ridge extending along a longitudinal axis L of the housing (as shown in FIG. 16-18) to help align or position the injector 10 in the user’s hand. The housing 12 may substantially house the components shown in FIG. 17 and 19-22.

[0126] Referring to FIG. 17, an injector 10 as described below in more detail. The injector 10 may have a button 24, a latch 34, a spring 42, a ram 38, a housing 12, a container support 46, a stopper 18, a primary container 14, a flange 20 and a plug 54.

[0127] A primary container 14 containing an injectable fluid may be at least partially retained within the housing 12. As used herein, the fluid may comprise medicaments, drugs, biologies, solutions, gels, suspensions or other substances that may be delivered via a syringe or needle, and such terms may be used interchangeably as appearing in the specification and claims. The primary container 14 may be a prefilled syringe. In one embodiment, the primary container 14 is one of a prefilled cartridge, prefilled staked needle syringe, ampules, vials, collapsible reservoirs, multi-chamber containers, or other injectable fluid containing vessel. The primary container 14 has a distal portion and a proximal portion opposite the distal portion. The primary container 14 may comprise a container portion 16 defining a fluid chamber containing a medicament. In one embodiment, the container portion 16 of the primary container 14 has aDBl / 162871737.3 245maximum volume of approximately 5 mL. In one embodiment, the container portion 16 of the primary container 14 has a maximum volume selected from approximately: 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL, 10 mL, 10.5 mL, l l mL, 11.5 mL, 12 mL, 12.5 mL, 13 mL, 13.5 mL, 14 mL, 14.5 mL, 15 mL, 15.5 mL, 16 mL, 16.5 mL, 17 mL, 17.5 mL, 18 mL, 18.5 mL, 19 mL, 19.5 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, and 50 mL.[00128J As shown in FIG. 19, the primary container 14 may further comprise a stopper 18 movable within the fluid chamber relative to the primary container 14. Prior to use or triggering of the injector 10, the stopper 18 may be disposed at a distal end of the container portion 16. The stopper 18 may be a plunger that seals the medicament in the container portion 16. The stopper 18 may be made of a rubber material. In one embodiment, the stopper 18 is made of a plastic. In one embodiment, the stopper 18 is made of butyl rubber, polyisoprene, polytetrafluorethylene, high density polyethylene or other thermoset elastomers.

[0129] As shown in FIG. 18 and 19, a flange 20 may extent outwardly from the distal portion of the primary container 14. In one embodiment, the flange 20 is a Luer. In one embodiment, the flange 20 is a Luer-Lock. In another embodiment, the flange 20 may be couplable to a needle 19 in fluid communication with the medicament in the container portion 16. While needle 19 is shown in the figures coupled to the flange 20, in some embodiments, an infusion line, a jet injector, a microneedle array, a luer-lock port, a diaphragm or valve mechanism, an integrated cannula, or a needle hub assembly may be coupled to the flange. In some embodiments, the needle 19 may be a regular walled needle. In some embodiments, the needle 19 may be a thin walled needle.

[0130] The needle 19 may be a 21-30 gauge needle. In one embodiment, the flange 20 may be couplable to a tubing set 21 in fluid communication with the medicament in the container portion 16. In one embodiment, a staked needle 27 is pre-attached and extending from the distal portion of the primary container 14. In one embodiment, a double-hub pen needle is attached to a drug cartridge with the needle piercing the septum of the cartridge to deliver the fluid. In one embodiment, the primary container bearing a septum is inserted into a stationary needle hub.

[0131] The needle 19 may be a 20 gauge needle, the needle 19 may be a 21 gauge needle, the needle 19 may be a 22 gauge needle, the needle 19 may be a 23 gauge needle, the needle 19 may be a 24 gauge needle, the needle 19 may be a 25 gauge needle, the needle 19 may be a 26DBl / 162871737.3 246gauge needle, the needle 19 may be a 27 gauge needle, the needle 19 may be a 28 gauge needle, the needle 19 may be a 29 gauge needle, the needle 19 may be a 30 gauge needle, the needle 19 may be a 31 gauge needle.

[0132] The needle 19 may have a length selected from: 1 / 8”, 5 / 32”, 3 / 16”, 7 / 32”, 1 / 4", 9 / 32”, 5 / 16”, 11 / 32”, 3 / 8”, 13 / 32”, 7 / 16”, 15 / 32”, 1 / 2", 17 / 32”, 9 / 16”, 19 / 32”, 5 / 8”, 21 / 32”, 11 / 16”, 23 / 32”, 3 / 4", 25 / 32”, 13 / 16”, 27 / 32”, 7 / 8”, 29 / 32”, 15 / 16”, 31 / 32”, 1”, 1 1 / 16”, 1 1 / 8”, 1 5 / 32”, 1 3 / 16”, 1 7 / 32”, 1 1 / 4", 1 9 / 32”, 1 5 / 16”, 1 11 / 32”, 1 3 / 8”, 1 13 / 32”, 1 7 / 16”, 1 15 / 32”, 1 1 / 2"

[0133] The housing 12 may house at least a portion of the primary container 14. In one embodiment, the housing 12 only houses a proximal portion of the primary container 14. In one embodiment, the housing 12 may house the entire primary container 14. The portion of the housing 12 that receives the primary container 14 may have a shape generally the same as a proximal portion of the primary container 14 to prevent rotation of the primary container 14 relative to the housing 12. The primary container 14 may be prevented from moving relative to the housing 12 as described below in more detail.

[0134] The primary container 14 may be selected from: a luer-fit cyclic olefin copolymer (COC) syringe, a glass staked needle syringe, a polymer staked needle syringe, a glass cartridge syringe, a cyclic olefin polymer (COP) syringe, a collapsible polymer reservoir, a dual-chamber cartridge for reconstitution, or an ampule.

[0135] The primary container 14 may be sized and shaped to hold a volume corresponding to a volume selected from: a) 1 mL to 5 mL, 2 mL to 5 mL, 3 mL to 5 mL, 3 mL to 10 mL, 3 mL to 15 mL, 3 mL to 20 mL, 3 mL to 25 mL, 3 ml to 30 mL, 3 mL to 35 mL, 3 mL to 40 mL, 3 mL to 45 mL, 3 mL to 50 mL, 5 mL to 10 mL, 5 mL to 15 mL, 5 mL to 20 mL, 5 mL to 25 mL, 5 mL to 30 mL, 5 mL to 35 mL, 5 mL to 40 mL; 5 mL to 45 mL, 5 mL to 50 mL, 10 mL to 15 mL; 10 mL to 20 mL;10 mL to 25 mL; 10 mL to 30 mL; 10 mL to 35 mL; 10 mL to 40 mL, 10 mL to 50 mL; b) about 3 mL to about 5 mL, about 3 mL to about 10 mL, about 3 mL to about 15 mL, about 3 mL to about 20 mL, about 3 mL to about 25 mL, about 3 ml to about 30 mL, about 3 mL to about 35 mL, about 3 mL to about 40 mL, about 3 mL to about 45 mL, about 3 mL to about 50 mL, about 5 mL to about 10 mL, about 5 mL to about 15 mL, about 5 mL to about 20 mL, about 5 mL to about 25 mL, about 5 mL to about 30 mL, about 5 mL to about 35 mL, about 5 mL toDBl / 162871737.3 247about 40 mL; about 5 mL to about 45 mL, about 5 mb to about 50 mL, about 10 mL to about 15 mL; about 10 mL to about 20 mL; about 10 mL to about 25 mL; about 10 mL to about 30 mL; about 10 mL to about 35 mL; about 10 mL to about 40 mL, about 10 mL to about 50 mL; c) at least about 3 mL, at least about 3.5 mL, at least about 4 mL, at least about 4.5 mL, at least about 5.5 mL, at least about 6 mL, at least about 6.5 mL, at least about 7 mL, at least about 7.5 mL, at least about 8 mL, at least about 8.5 mL, at least about 9 mL, at least about 9.5 mL, at least about 10 mL, at least about 10.5 mL, at least about 11 mL, at least about 11.5 mL, at least about 12 mL, at least about 12.5 mL, at least about 13 mL, at least about 13.5 mL, at least about 14 mL, at least about 14.5 mL, at least about 15 mL, at least about 15.5 mL, at least about 16 mL, at least about 16.5 mL, at least about 17 mL, at least about 17.5 mL, at least about 18 mL, at least about 18.5 mL, at least about 19 mL, at least about 19.5 mL, at least about 20 mL, at least about 25 mL, at least about 30 mL, at least about 35 mL, at least about 40 mL, at least about 45 mL, at least about 50 mL; and d) at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, at least 5.5 mL, at least 6 mL, at least 6.5 mL, at least 7 mL, at least 7.5 mL, at least 8 mL, at least 8.5 mL, at least 9 mL, at least 9.5 mL, at least 10 mL, at least 10.5 mL, at least 11 mL, at least 11.5 mL, at least 12 mL, at least 12.5 mL, at least 13 mL, at least 13.5 mL, at least 14 mL, at least 14.5 mL, at least 15 mL, at least 15.5 mL, at least 16 mL, at least 16.5 mL, at least 17 mL, at least 17.5 mL, at least 18 mL, at least 18.5 mL, at least 19 mL, at least 19.5 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 45 mL, at least 50 mL.

[0136] The injector 10 may be configured to deliver the entire amount or a portion of the predetermined amount of the medicament within primary container 14. The predetermined amount of the medicament may correspond with the volume contained in the primary container 14. In one embodiment, the injector 10 may expel an initial portion of the volume (priming volume) followed by a second step to expel the remaining portion of the volume (deliverable volume). In one embodiment, the medicament contained in the primary container 14 corresponds to a volume selected from: a) 1 mL to 5 mL, 2 mL to 5 mL, 3 mL to 5 mL, 3 mL to 10 mL, 3 mL to 15 mL, 3 mL to 20 mL, 3 mL to 25 mL, 3 ml to 30 mL, 3 mL to 35 mL, 3 mL to 40 mL, 3 mL to 45 mL, 3 mL to 50 mL, 5 mL to 10 mL, 5 mL to 15 mL, 5 mL to 20 mL, 5 mL to 25 mL, 5 mL to 30 mL, 5 mL to 35 mL, 5 mL to 40 mL; 5 mL to 45 mL, 5 mL to 50 mL, 10 mL to 15 mL; 10 mL to 20 mL; 10 mL to 25 mL; 10 mL to 30 mL; 10 mL to 35 mL; 10 mL to 40 mL, 10 mL to 50 mL;DBl / 162871737.3 248b) about 3 mb to about 5 mL, about 3 mL to about 10 mL, about 3 mL to about 15 mL, about 3 mL to about 20 mL, about 3 mL to about 25 mL, about 3 ml to about 30 mL, about 3 mL to about 35 mL, about 3 mL to about 40 mL, about 3 mL to about 45 mL, about 3 mL to about 50 mL, about 5 mL to about 10 mL, about 5 mL to about 15 mL, about 5 mL to about 20 mL, about 5 mL to about 25 mL, about 5 mL to about 30 mL, about 5 mL to about 35 mL, about 5 mL to about 40 mL; about 5 mL to about 45 mL, about 5 mL to about 50 mL, about 10 mL to about 15 mL; about 10 mL to about 20 mL; about 10 mL to about 25 mL; about 10 mL to about 30 mL; about 10 mL to about 35 mL; about 10 mL to about 40 mL, about 10 mL to about 50 mL; c) at least about 3 mL, at least about 3.5 mL, at least about 4 mL, at least about 4.5 mL, at least about 5.5 mL, at least about 6 mL, at least about 6.5 mL, at least about 7 mL, at least about 7.5 mL, at least about 8 mL, at least about 8.5 mL, at least about 9 mL, at least about 9.5 mL, at least about 10 mL, at least about 10.5 mL, at least about 11 mL, at least about 11.5 mL, at least about 12 mL, at least about 12.5 mL, at least about 13 mL, at least about 13.5 mL, at least about 14 mL, at least about 14.5 mL, at least about 15 mL, at least about 15.5 mL, at least about 16 mL, at least about 16.5 mL, at least about 17 mL, at least about 17.5 mL, at least about 18 mL, at least about 18.5 mL, at least about 19 mL, at least about 19.5 mL, at least about 20 mL, at least about 25 mL, at least about 30 mL, at least about 35 mL, at least about 40 mL, at least about 45 mL, at least about 50 mL; and d) at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, at least 5.5 mL, at least 6 mL, at least 6.5 mL, at least 7 mL, at least 7.5 mL, at least 8 mL, at least 8.5 mL, at least 9 mL, at least 9.5 mL, at least 10 mL, at least 10.5 mL, at least 11 mL, at least 11.5 mL, at least 12 mL, at least 12.5 mL, at least 13 mL, at least 13.5 mL, at least 14 mL, at least 14.5 mL, at least 15 mL, at least 15.5 mL, at least 16 mL, at least 16.5 mL, at least 17 mL, at least 17.5 mL, at least 18 mL, at least 18.5 mL, at least 19 mL, at least 19.5 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 45 mL, at least 50 mL.[00137J Flow rate of the injector 10 is heavily dependent on the viscosity and volume of the medicament. However, the injector 10 may deliver the full volume of the medicament at a rate of approximately 0.08-0.75 mL / sec. For example, this would provide target delivery rate ranges of 13-120 seconds for a 10 mL dose volume. The injector 10 may deliver 10 mL of the medicament at a rate of 0.33 mL / sec. In one embodiment, the injector 10 delivers the full deliverable volume of the medicament at a rate of:DBl / 162871737.3 249a) 0.5 mL / 10 sec., 0.75 mL / 10 sec., 1 mL / 10 sec., 1 .25 mL / 10 sec., 1 .5 mL / 10 sec., 1 .75 mL / 10 sec, 2 mL / 10 sec., 2.25 mL / 10 sec, 2.5 mL / 10 sec., 2.75 mL / 10 sec, 3 mL / 10 sec., 3.25 mL / 10 sec, 3.5 mL / 10 sec., 3.75 mL / 10 sec, 4 mL / 10 sec., 4.25 mL / 10 sec, 4.5 mL / 10 sec., 4.75 mL / 10 sec, 5 mL / 10 sec; b) 2 mL / 30 sec., 2.5 mL / 30 sec., 3 mL / 30 sec., 3.5 mL / 30 sec., 4 mL / 30 sec., 4.5 mL / 30 sec., 5 mL / 30 sec., 5.5 mL / 30 sec., 6 mL / 30 sec., 6.5 mL / 30 sec., 7 mL / 30 sec., 7.5 mL / 30 sec., 8 mL / 30 sec., 8.5 mL / 30 sec., 9 mL / 30 sec., 9.5 mL / 30 sec., 10 mL / 30 sec., 10.5 mL / 30 sec.; and c) 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min, 20 mL / min, 21 mL / min.

[0138] In one embodiment, the viscosity of the medicament may be selected from: a) 5 centipoise (cP), 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30; b) about 5 cP to about 7 cP, about 5 cP to about 9 cP, about 5 cP to about 11 cP, about 5 cP to about 13 cP, about 5 cP to about 15 cP, about 5 cP to about 17 cP, about 5 cP to about 19 cP, about 5 cP to about 21 cP, about 5 cP to about 23 cP, about 5 cP to about 25 cP, about 5 cP to about 27 cP, about 5 cP to about 29 cP, about 10 cP to about 15 cP, about 10 cP to about 20 cP, about 10 cP to about 25 cP, about 10 cP to about 30 cP, c) at least about 5 cP, at least about 6 cP, at least about 7 cP, at least about 8 cP, at least about 9 cP, at least about 10 cP, at least about 11 cP, at least about 12 cP, at least about 13 cP, at least about 14 cP, at least about 15 cP, at least about 16 cP, at least about 17 cP, at least about 18 cP, at least about 19 cP, at least about 20 cP, at least about 21 cP, at least about 22 cP, at least about 23 cP, at least about 24 cP, at least about 25 cP, at least about 26 cP, at least about 27 cP, at least about 28 cP, at least about 29 cP, at least about 30; and d) at least about 5 cP, at least 6 cP, at least 7 cP, at least 8 cP, at least 9 cP, at least 10 cP, at least 11 cP, at least 12 cP, at least 13 cP, at least 14 cP, at least 15 cP, at least 16 cP, at least 17 cP, at least 18 cP, at least 19 cP, at least 20 cP, at least 21 cP, at least 22 cP, at least 23 cP, at least 24 cP, at least 25 cP, at least 26 cP, at least 27 cP, at least 28 cP, at least 29 cP, at least 30.

[0139] The user’s experience may be improved if the injector 10 can deliver the full volume of the medicament as fast as possible. A faster delivery may result in less pain and discomfort for the patient. The injector 10 may deliver the full deliverable volume of theDBl / 162871737.3 250medicament in 5 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 10 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 15 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 20 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 25 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 30 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 35 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 40 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 45 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 50 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 55 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 60 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 70 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 80 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 90 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 100 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 110 seconds. The injector 10 may deliver the full deliverable volume of the medicament in 120 seconds.

[0140] Any number of indicia may be displayed on the injector 10. For example, a symbol 23 (as shown in FIG. 18) may be displayed on the housing 12 or an indicator regarding the status of the injector 10 may be displayed on a button 24 (as shown in FIG. 18). Referring to FIG. 16-18, the housing 12 may include a cutout 13 extending therethrough to allow an indicator on the button 24 disposed within the housing 12 to be viewed. The cutout 13 may be located at a proximal portion of the housing 12. The cutout 13 may be a generally oval shape. The cutout 13 may expose a portion of the button 24 containing an indicia. The button 24 may be at least partially received within the proximal portion of the housing 12. The button 24 may include a lock indicator 15 thereon for indicating a lock status of the button and / or for indicating the injector 10 is in a locked configuration. The lock indicator 15 may be engraved, etched, printed or molded in the button 24. In one embodiment, the lock indicator 15 is a decal fixed to the button 24 with an adhesive. In one embodiment, the lock indicator 15 is applied onto the button 24 via spray painting, powder coating, silk screen, laser marking, pad printing, or heat staking.DBl / 162871737.3 2 1The lock indicator 15 may be a graphic of a lock signifying that the injector 10 is in the locked configuration. The lock indicator 15 may be any combination of shapes and / or words.

[0141] The button 24 may further include a rotation indicator 17 for indicating a direction the button 24 is movable about the longitudinal axis L. The rotation indicator 17 may be engraved in the button 24. In one embodiment, the rotation indicator 17 is a decal fixed to the button 24 with an adhesive. In one embodiment, the rotation indicator 17 is applied onto the button 24 via spray painting, powder coating, silk screen, laser marking, pad printing, or heat staking. The rotation indicator 17 may be an arrow signifying the direction the button 24 must be rotated relative to the housing 12 to transition from a locked configuration to an unlocked configuration. The rotation indicator 17 may be any combination of shapes and / or words. In one embodiment, the button contains an indication for partial dosing of the medicament, such as priming volume in the location of the lock indicator 15. The partial dosing indicator may be engraved, etched, printed or molded in the button 24. In one embodiment, the partial dosing indicator is a decal fixed to the button 24 with an adhesive. In one embodiment, the partial dosing indicator is applied onto the button 24 via spray painting, powder coating, silk screen, laser marking, pad printing, or heat staking.

[0142] To expel the medicament from the primary container 14, the injector 10 undergoes a series of sequential movements that results in a triggering event. The triggering event is initiated by a user moving the button 24 relative to the housing 12. Referring to FIG. 19-22, the injector 10 may further comprise a trigger mechanism 22. While a button-actuated injector is shown in the figures, it should be understood that trigger mechanism 22 may be any or all of slide switches, rotary mechanisms, pull tabs, levers, squeeze grips, push-and-twist locks, touch sensors, voice activation systems, pressure-sensitive triggers, proximity sensors, spring- loaded mechanisms, motorized or servo-driven systems, and systems triggered by timers, wireless signals, or preset conditions.

[0143] The trigger mechanism 22 may comprise the button 24, latch 34, ram 38 and spring 42 located at the proximal portion of the housing 12. The button 24 may be rotatably coupled to the housing 12 about a longitudinal axis L thereof. The button 24 may be rotatable between the unlocked configuration and the locked configuration, which may be indicated to the user by an indicia visible through the cutout 13. Rotation of the button 24 between the locked configuration and the unlocked configuration may not breach a sterile barrier of the primaryDBl / 162871737.3 252container 14. This may allow the user to rotate the button 24 back to the locked configuration for use at a later time.

[0144] In the locked configuration, the button 24 may be prevented from moving distally along the longitudinal axis L of the housing 12 by a rim along an inner surface of the housing 12. In certain applications (i.e., lab testing), it may be necessary to remove the button 24. In one embodiment, the button 24 may be moved proximally along the longitudinal axis L of the housing 12. The button 24 may include a passthrough hole 59 extending through a proximal end thereof that allows for disassembly using appropriate equipment. In one embodiment, the button 24 is removable by inserting a disassembly tool (not shown) into the passthrough hole 59, thereby releasing the button 24 from the rim. In the unlocked configuration, the button 24 may be movable distally along the longitudinal axis L of the housing 12 to initiate a triggering event. The button 24 may be a generally cylindrical shape. A proximal end of the button 24 may be closed and a distal end of the button 24 may be open. The button 24 may have an internal cavity 28 defined therein. The button 24 may be the only feature of the injector 10 that is moveable relative to the housing 12 prior to the triggering event. An outer surface of the button 24 may have one or more ridges extending along a length thereof. In one embodiment, rotation of the button 24 to the unlocked position causes the device to expel the priming volume.

[0145] Referring to FIG. 19-22, the button 24 may comprise a barrel 26 extending distally from the proximal end within the internal cavity 28. The barrel 26 may have a proximal side and a distal side thereof. The barrel 26 may include a depression 30 extending radially inward on the barrel 26. The barrel 26 may be a generally cylindrical shape at the distal side. The barrel 26 may be a non-uniform generally cylindrical shape at the proximal side. The barrel 26 may have a radius less than a radius of the button 24. The radius of the barrel 26 may be one third the radius of the button 24. The barrel 26 may have a radius 0.1 to 0.5 inches. The depression 30 may be located on the proximal side of the barrel 26. In one embodiment, the barrel 26 includes two depressions 30 on opposite sides of the barrel 26 from each other. The distal side of the barrel 26 may have a smaller diameter than the proximal side of the barrel 26. The barrel 26 may extend distally only a portion of a length of the button 24. In one embodiment, the barrel 26 extends distally substantially along a length of the button 24.

[0146] Referring to FIG. 17 and 19-22, the trigger mechanism 22 may further comprise a latch 34 for facilitating the triggering event. The latch 34 may have a proximal end and a distalDBl / 162871737.3 253end thereof. The latch 34 may have a generally cylindrical shape. The latch 34 may be disposed within the housing 12. The latch 34 may be fixed to the housing 12. The proximal end of the latch 34 may be disposed within the button 24.

[0147] The latch 34 may further comprise a latch arm 32. The latch arm 32 may extend distally along the longitudinal axis L from a proximal end of the latch 34. The latch arm 32 may be coupled to the latch 34. The latch arm 32 may be biased in an inward radial direction. The latch arm 32 may be prevented from deflecting in the inward radial direction by the barrel 26 prior to the triggering event. The latch arm 32 may include a protrusion 36. The protrusion 36 may extend radially outwardly from the latch arm 32. The depression 30 may be configured to align with the latch arm 32 in the unlocked configuration. When the button 24 is moved distally a predetermined distance along the longitudinal axis L in the unlocked configuration, the latch arm 32 may be received in the depression 30. In one embodiment, the latch arm 32 comprises two diametrically opposed latches.

[0148] Referring to FIG. 17 and 19-22, the trigger mechanism 22 may further comprise a ram 38. The ram 38 may comprise a proximal side and a distal side opposite the proximal side. The ram 38 may be disposed within the latch 34. The ram 38 may be configured to engage the stopper 18 at the distal side thereof. The ram 38 may be a generally cylindrical shape defining an internal cavity 40 therein. The spring 42 may be disposed in the internal cavity 40. In one embodiment, the spring 42 may be disposed on an outside of the ram 38. The spring 42 may have a proximal end and a distal end opposite the proximal end. The proximal end of the spring 42 may engage a collar 98 of the latch 34. The collar 98 may be a generally cylindrical shape. The collar 98 may be sized such that the barrel 26 is able to pass therethrough. A radius of the collar 98 may be the same as the spring 42 to ensure the collar 98 engages the spring 42. The distal end of spring 42 may engage a ram collar 96 located within the internal cavity 40 at the distal side of the ram 38. During a triggering event, the spring 42 may bias the ram 38 distally along the longitudinal axis L relative to the housing 12. In an exemplary embodiment, spring 42 includes a compression spring, however, other suitable energy source can be used, such as an electric pump, elastomer or compressed-gas spring, a compressed-gas cylinder, a gas generator, or other suitable energy storage members. The ram 38 may cause the stopper 18 to move distally along the longitudinal axis L relative to the primary container 14.DBl / 162871737.3 254

[0149] In some embodiments, the compressed-gas cylinder (not shown) is used in combination with or in place of spring 42 as an energy source of the injector 10. The compressed-gas cylinder may be disposed in the injector 10 at a proximate end. The compressed-gas cylinder may store energy therein which may be selectively released upon a user’s movement of the button 24 distally along the longitudinal axis L relative to the housing 12. A distal end of the compressed-gas cylinder may engage the ram 38 to move the ram 38 relative to the primary container 14 thereby ejecting the medicament. In some embodiments, the compressed-gas cylinder includes a pin 45 movable relative to the compressed-gas cylinder and extendable from a distal end thereof when the injector 10 is actuated. The pin 45 may engage the ram 38 and move the ram 38 distally relative to the housing 12 when the injector 10 is actuated. The compressed-gas cylinder may increase precision of medicament delivery by precisely controlling the force acting on the ram 38. The compressed-gas cylinder may reduce vibration and noise during use compared to alternate embodiments (e.g., spring 42). In some embodiments, an injector 10 using the compressed-gas cylinder has a length smaller along the longitudinal axis L than an injector 10 using an alternate embodiment of an energy source (e.g., spring 42).

[0150] Referring to FIG. 17 and 19-22, the ram 38 may comprise an aperture 44 extending therethrough. The aperture 44 may be located on the proximal side of the ram 38. The ram 38 may be prevented from moving distally relative to the housing 12 by the latch arm 32. The protrusion 36 may engage the aperture 44 to prevent the spring 42 from biasing the ram 38 distally.

[0151] Referring to FIG. 21, in the unlocked configuration, movement of the button 24 distally along the longitudinal axis L relative to the housing 12 may allow the latch arm 32 to deflect radially inward into the depression 30 of the barrel 26 thereby initiating a triggering event. Radial deflection of the latch arm 32 may disengage the protrusion 36 from the aperture 44. The latch arm 32 disengaging the protrusion 36 may allow the spring 42 to bias the ram 38 distally along the longitudinal axis L relative to the housing 12. The ram 38 may move the stopper 18 distally along the longitudinal axis L relative to the primary container 14 to eject the medicament in a discharged configuration.

[0152] Referring to FIG. 19-22, the spring 42 may be a compression spring. The spring 42 may have a 5 mm diameter. The spring 42 may have a 6 mm diameter. The spring 42 mayDBl / 162871737.3 255have a 7 mm diameter. The spring 42 may have an 8 mm diameter. The spring 42 may have a 9 mm diameter. The spring 42 may have a 10 mm diameter. The spring 42 may have a l l mm diameter. The spring 42 may have a 12 mm diameter. The spring 42 may have a 13 mm diameter. The spring 42 may have a 14 mm diameter. The spring 42 may have a 15 mm diameter. The spring 42 may have a 0.75 mm wire diameter. The spring 42 may have a 1 mm wire diameter. The spring 42 may have a 1.25 mm wire diameter. The spring 42 may have a 1.5 mm wire diameter. The spring 42 may have a 1.75 mm wire diameter. The spring 42 may have a 2 mm wire diameter.

[0153] The spring may produce 8 Ibf of force. The spring may produce 9 Ibf of force. The spring may produce 10 Ibf of force. The spring may produce 11 Ibf of force. The spring may produce 12 Ibf of force. The spring may produce 13 Ibf of force. The spring may produce 14 Ibf of force. The spring may produce 15 Ibf of force. The spring may produce 16 Ibf of force. The spring may produce 17 Ibf of force. The spr...

Claims

WHAT IS CLAIMED IS:

1. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein a detectable amount of a protein encoded, produced or modulated by the therapeutic agent reaches systemic circulation, and wherein serum or plasma levels of the protein are greater than 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

2. The combination dosing regimen of Claim 1, wherein the serum or plasma levels of the protein are greater than the protein levels prior to administration of the therapeutically effective amount of the therapeutic agent or the composition; and administering the soluble hyaluronidase.

3. The combination dosing regimen of any of Claims 1-2, wherein plasma or serum concentration is at least 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

4. The combination dosing regimen of any of Claims 1-3, wherein AUC of the protein is at least 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

5. The combination dosing regimen of any of Claims 1-4, wherein Cmin of the protein is at least 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

6. The combination dosing regimen of any of Claims 1-5, wherein Cmax of the protein is less than 90% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

7. The combination dosing regimen of any of Claims 1-6, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with an equal or higher AUC of the protein.

8. The combination dosing regimen of any of Claims 1-7, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with an equal or higher Cmin of the protein.DBl / 162871737.3 4529. The combination dosing regimen of any of Claims 1 -8, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with an equal or lower Cmax of the protein.

10. The combination dosing regimen of any of Claims 1-9, wherein the amount of dose delivered subcutaneously is at least 10% higher than a therapeutically effective intravenous dose.

11. The combination dosing regimen of any of Claims 1-10, wherein serum or plasma levels of anti-inflammatory cytokines are increased compared to an equivalent intravenous dose of the therapeutic agent or the composition.

12. The combination dosing regimen of any of Claims 1-11, wherein serum or plasma levels of IL- 10 are increased compared to an equivalent intravenous dose of the therapeutic agent or the composition.

13. The combination dosing regimen of any of Claims 1-12, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with equal or higher serum or plasma levels of antiinflammatory cytokines.

14. The combination dosing regimen of any of Claims 1-13, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with equal or higher serum or plasma levels of IL-10.

15. The combination dosing regimen of any of claims 1-14, wherein neutrophil levels are equal or reduced compared to an equivalent intravenous dose of the therapeutic agent or the composition.

16. The combination dosing regimen of any of Claims 1-15, wherein plasma or serum concentration is at least 20% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

17. The combination dosing regimen of any of Claims 1-16, wherein plasma or serum concentration is at least 30% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45318. The combination dosing regimen of any of Claims 1-17, wherein plasma or serum concentration is at least 40% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

19. The combination dosing regimen of any of Claims 1-18, wherein plasma or serum concentration is at least 50% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

20. The combination dosing regimen of any of Claims 1-19, wherein plasma or serum concentration is at least 60% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

21. The combination dosing regimen of any of Claims 1-20, wherein plasma or serum concentration is at least 70% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

22. The combination dosing regimen of any of Claims 1-21, wherein plasma or serum concentration is at least 80% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

23. The combination dosing regimen of any of Claims 1-22, wherein plasma or serum concentration is at least 90% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

24. The combination dosing regimen of any of Claims 1-23, wherein plasma or serum concentration is at least 100% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

25. The combination dosing regimen of any of Claims 1-24, wherein plasma or serum concentration is at least 110% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

26. The combination dosing regimen of any of Claims 1-25, wherein plasma or serum concentration is at least 120% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45427. The combination dosing regimen of any of Claims 1-26, wherein plasma or serum concentration is at least 130% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

28. The combination dosing regimen of any of Claims 1-27, wherein plasma or serum concentration is at least 140% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

29. The combination dosing regimen of any of Claims 1-28, wherein plasma or serum concentration is at least 150% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

30. The combination dosing regimen of any of Claims 1-29, wherein plasma or serum concentration is at least 160% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

31. The combination dosing regimen of any of Claims 1-30, wherein plasma or serum concentration is at least 170% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

32. The combination dosing regimen of any of Claims 1-31, wherein plasma or serum concentration is at least 180% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

33. The combination dosing regimen of any of Claims 1-32, wherein plasma or serum concentration is at least 190% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

34. The combination dosing regimen of any of Claims 1-33, wherein plasma or serum concentration is at least 200% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

35. The combination dosing regimen of any of Claims 1-34, wherein AUC of the protein is at least 20% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45536. The combination dosing regimen of any of Claims 1-35, wherein AUC of the protein is at least 30% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

37. The combination dosing regimen of any of Claims 1-36, wherein AUC of the protein is at least 40% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

38. The combination dosing regimen of any of Claims 1-37, wherein AUC of the protein is at least 50% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

39. The combination dosing regimen of any of Claims 1-38, wherein AUC of the protein is at least 60% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

40. The combination dosing regimen of any of Claims 1-39, wherein AUC of the protein is at least 70% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

41. The combination dosing regimen of any of Claims 1-40, wherein AUC of the protein is at least 80% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

42. The combination dosing regimen of any of Claims 1-41, wherein AUC of the protein is at least 90% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

43. The combination dosing regimen of any of Claims 1-42, wherein AUC of the protein is at least 100% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

44. The combination dosing regimen of any of Claims 1-43, wherein AUC of the protein is at least 110% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45645. The combination dosing regimen of any of Claims 1-44, wherein AUC of the protein is at least 120% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

46. The combination dosing regimen of any of Claims 1-45, wherein AUC of the protein is at least 130% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

47. The combination dosing regimen of any of Claims 1-46, wherein AUC of the protein is at least 140% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

48. The combination dosing regimen of any of Claims 1-47, wherein AUC of the protein is at least 150% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

49. The combination dosing regimen of any of Claims 1-48, wherein AUC of the protein is at least 160% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

50. The combination dosing regimen of any of Claims 1-49, wherein AUC of the protein is at least 170% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

51. The combination dosing regimen of any of Claims 1-50, wherein AUC of the protein is at least 180% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

52. The combination dosing regimen of any of Claims 1-51, wherein AUC of the protein is at least 190% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

53. The combination dosing regimen of any of Claims 1-52, wherein AUC of the protein is at least 200% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45754. The combination dosing regimen of any of Claims 1-53, wherein Cmin of the protein is at least 20% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

55. The combination dosing regimen of any of Claims 1-54, wherein Cmin of the protein is at least 30% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

56. The combination dosing regimen of any of Claims 1-55, wherein Cmin of the protein is at least 40% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

57. The combination dosing regimen of any of Claims 1-56, wherein Cmin of the protein is at least 50% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

58. The combination dosing regimen of any of Claims 1-57, wherein Cmin of the protein is at least 60% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

59. The combination dosing regimen of any of Claims 1-58, wherein Cmin of the protein is at least 70% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

60. The combination dosing regimen of any of Claims 1-59, wherein Cmin of the protein is at least 80% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

61. The combination dosing regimen of any of Claims 1-60, wherein Cmin of the protein is at least 90% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

62. The combination dosing regimen of any of Claims 1-61, wherein Cmin of the protein is at least 100% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45863. The combination dosing regimen of any of Claims 1-62, wherein Cmin of the protein is at least 110% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

64. The combination dosing regimen of any of Claims 1-63, wherein Cmin of the protein is at least 120% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

65. The combination dosing regimen of any of Claims 1-64, wherein Cmin of the protein is at least 130% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

66. The combination dosing regimen of any of 1-65, wherein Cmin of the protein is at least 140% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

67. The combination dosing regimen of any of Claims 1-66, wherein Cmin of the protein is at least 150% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

68. The combination dosing regimen of any of Claims 1-67, wherein Cmin of the protein is at least 160% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

69. The combination dosing regimen of any of Claims 1-68, wherein Cmin of the protein is at least 170% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

70. The combination dosing regimen of any of Claims 1-69, wherein Cmin of the protein is at least 180% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

71. The combination dosing regimen of any of Claims 1-70, wherein Cmin of the protein is at least 190% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 45972. The combination dosing regimen of any of Claims 1-71, wherein Cmin of the protein is at least 200% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

73. The combination dosing regimen of any of Claims 1-72, wherein Cmax of the protein is less than 80% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

74. The combination dosing regimen of any of Claims 1-73, wherein Cmax of the protein is less than 70% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

75. The combination dosing regimen of any of Claims 1-74, wherein Cmax of the protein is less than 60% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

76. The combination dosing regimen of any of Claims 1-75, wherein Cmax of the protein is less than 50% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

77. The combination dosing regimen of any of Claims 1-76, wherein Cmax of the protein is less than 40% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

78. The combination dosing regimen of any of Claims 1-77, wherein Cmax of the protein is less than 30% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

79. The combination dosing regimen of any of Claims 1-78, wherein Cmax of the protein is less than 20% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

80. The combination dosing regimen of any of Claims 1-79, wherein Cmax of the protein is less than 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

81. The combination dosing regimen of any of Claims 1-80, wherein the amount of dose delivered subcutaneously is at least 50% higher than a therapeutically effective intravenous dose.DBl / 162871737.3 46082. The combination dosing regimen of any of Claims 1-81, wherein the amount of dose delivered subcutaneously is at least 100% higher than a therapeutically effective intravenous dose.

83. The combination dosing regimen of any of Claims 1-82, wherein the amount of dose delivered subcutaneously is at least 200% higher than a therapeutically effective intravenous dose.

84. The combination dosing regimen of any of Claims 1-83, wherein the amount of dose delivered subcutaneously is at least 300% higher than a therapeutically effective intravenous dose.

85. The combination dosing regimen of any of Claims 1-84, wherein the amount of dose delivered subcutaneously is at least 400% higher than a therapeutically effective intravenous dose.

86. The combination dosing regimen of any of Claims 1-85, wherein the amount of dose delivered subcutaneously is at least 500% higher than a therapeutically effective intravenous dose.

87. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein a detectable amount of the protein encoded, produced or modulated by the therapeutic agent reaches systemic circulation, and wherein serum or plasma levels of the protein are greater than 100% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

88. The combination dosing regimen of Claim 87, wherein plasma or serum concentration is at least 100% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

89. The combination dosing regimen of any of Claims 87-88, wherein plasma or serum concentration is at least 150% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

90. The combination dosing regimen of any of Claims 87-89, wherein plasma or serum concentration is at least 500% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.DBl / 162871737.3 46191 . The combination dosing regimen of any of Claims 87-90, wherein plasma or serum concentration is at least 1000% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

92. The combination dosing regimen of any of Claims 87-91, wherein AUC of the protein is at least 100% of an equivalent dose of the therapeutic agent or the composition or administered without the hyaluronidase.

93. The combination dosing regimen of any of Claims 87-92, wherein AUC of the protein is at least 150% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

94. The combination dosing regimen of any of Claims 87-93, wherein AUC of the protein is at least 500% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

95. The combination dosing regimen of any of Claims 87-94, wherein AUC of the protein is at least 1000% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

96. The combination dosing regimen of any of Claims 87-95, wherein Cmin of the protein is at least 100% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

97. The combination dosing regimen of any of Claims 87-96, wherein the serum or plasma levels of anti-inflammatory cytokines are increased compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

98. The combination dosing regimen of any of Claims 87-97, wherein the serum or plasma levels of IL-10 are increased compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

99. The combination dosing regimen of any of Claims 87-98, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or higher serum or plasma levels of anti-inflammatory cytokines.DBl / 162871737.3 462100. The combination dosing regimen of any of Claims 87-99, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or higher serum or plasma levels of IL- 10.

101. The combination dosing regimen of any of Claims 87-100, wherein neutrophil levels are equal or reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

102. The combination dosing regimen of any of Claims 87-101, wherein the serum or plasma levels of pro-inflammatory cytokines are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

103. The combination dosing regimen of any of Claims 87-102, wherein serum or plasma levels of IL-6 are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

104. The combination dosing regimen of any of Claims 87-103, wherein serum or plasma levels of KC / GRO are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

105. The combination dosing regimen of any of Claims 87-104, wherein serum or plasma levels of IL-12p70 are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

106. The combination dosing regimen of any of Claims 87-105, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered without the hyaluronidase with equal or reduced serum or plasma levels of pro-inflammatory cytokines.

107. The combination dosing regimen of any of Claims 87-106, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of IL-6.

108. The combination dosing regimen of any of Claims 87-107, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of KC / GRO.DBl / 162871737.3 463109. The combination dosing regimen of any of Claims 87-108, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of IL-12p70.

110. The combination dosing regimen of any of Claims 1-109, wherein the therapeutic agent is selected from an mRNA, nucleic acid conjugate, oligonucleotide, radiopharmaceutical, aptamer, DNA, self-replicating RNA (replicons), Naked RNA, and Circular RNA (circRNA).

111. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein a detectable amount of protein decreased, inhibited, knocked down or modulated by the therapeutic agent reaches systemic circulation, and wherein serum or plasma levels of the protein are at least 60% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

112. The combination dosing regimen of Claim 111, wherein the serum or plasma levels of the protein are at least 120% of the equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

113. The combination dosing regimen of Claim 111 or 112, wherein the serum or plasma levels of the protein are between 120-400% of the equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

114. The combination dosing regimen of Claim 111-113, wherein the serum or plasma levels of the protein are less than 90% of the protein levels prior to the administration of the therapeutically effective amount of the therapeutic agent or the composition; and administering a soluble hyaluronidase.

115. The combination dosing regimen of any of Claims 111-114, wherein AUC of the protein is at least 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

116. The combination dosing regimen of any of Claims 111 -1 15, wherein AUC of the protein is at least 100% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 464117. The combination dosing regimen of any of Claims 111 -1 16, wherein Cmin of the protein is at least 10% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

118. The combination dosing regimen of any of Claims 111-117, wherein Cmin of the protein is at least 100% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

119. The combination dosing regimen of any of claims 111-118, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose administered intravenously with an equal or lower AUC of the protein.

120. The combination dosing regimen of any of claims 111-119, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose administered intravenously with an equal or lower Cmin of the protein.

121. The combination dosing regimen of any of Claims 111-120, wherein the amount of dose delivered is at least 10% higher than a therapeutically effective intravenous dose.

122. The combination dosing regimen of any of Claims 111-121, wherein the amount of dose delivered is at least 100% higher than a therapeutically effective intravenous dose.

123. The combination dosing regimen of any of Claims 111-122, wherein the serum or plasma levels of anti-inflammatory cytokines are increased compared to an equivalent intravenous dose of the therapeutic agent or the composition.

124. The combination dosing regimen of any of Claims 111-123, wherein the serum or plasma levels of IL-10 are increased compared to an equivalent intravenous dose of the therapeutic agent or the composition.

125. The combination dosing regimen of any of Claims 111-124, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose administered intravenously with equal or higher serum or plasma levels of anti-inflammatory cytokines.

126. The combination dosing regimen of any of Claims 111-125, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than aDBl / 162871737.3 465therapeutically effective dose administered intravenously with equal or higher serum or plasma levels of IL-10.

127. The combination dosing regimen of any of Claims 111-126, wherein neutrophil levels are equal or reduced compared to an equivalent intravenous dose of the therapeutic agent or the composition administered.

128. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein a detectable amount of protein decreased, inhibited, knocked down or modulated by the therapeutic agent reaches systemic circulation, and wherein plasma or serum concentration of the protein is less than 90% of an equivalent dose of the therapeutic agent or the composition administered without hyaluronidase.

129. The combination dosing regimen of Claim 128, wherein AUC of the protein is less than 100% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

130. The combination dosing regimen of any of Claims 128-129, wherein AUC of the protein is less than 80% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

131. The combination dosing regimen of any of Claims 128-130, wherein Cmax of the protein is less than 100% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

132. The combination dosing regimen of any of Claims 128-131, wherein Cmax of the protein is less than 80% of an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

133. The combination dosing regimen of any of Claims 128-132, wherein the serum or plasma levels of anti-inflammatory cytokines are increased compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

134. The combination dosing regimen of any of Claims 128-133, wherein the serum or plasma levels of IL-10 are increased compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.DBl / 162871737.3 466135. The combination dosing regimen of any of Claims 128-134, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or higher serum or plasma levels of anti-inflammatory cytokines.

136. The combination dosing regimen of any of Claims 128-135, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously than a therapeutically effective dose without the hyaluronidase with equal or higher serum or plasma levels of LL-10.

137. The combination dosing regimen of any of Claims 128-136, wherein neutrophil levels are equal or reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

138. The combination dosing regimen of any of Claims 128-137, wherein the serum or plasma levels of pro-inflammatory cytokines are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

139. The combination dosing regimen of any of Claims 128-138, wherein serum or plasma levels of IL-6 are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

140. The combination dosing regimen of any of Claims 128-139, wherein serum or plasma levels of KC / GRO are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

141. The combination dosing regimen of any of Claims 128-140, wherein serum or plasma levels of IL-12p70 are reduced compared to an equivalent dose of the therapeutic agent or the composition administered without the hyaluronidase.

142. The combination dosing regimen of any of Claims 128-141, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose administered without the hyaluronidase with equal or reduced serum or plasma levels of pro-inflammatory cytokines.

143. The combination dosing regimen of any of Claims 128-142, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of IL-6.DBl / 162871737.3 467144. The combination dosing regimen of any of Claims 128-143, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of KC / GRO.

145. The combination dosing regimen of any of Claims 128-144, wherein a higher dose of the therapeutic agent or the composition is delivered subcutaneously or intramuscularly than a therapeutically effective dose without the hyaluronidase with equal or reduced serum or plasma levels of IL-12p70.

146. The combination dosing regimen of any of Claims 111-145, wherein the therapeutic agent is selected from a nucleic acid conjugate, oligonucleotide, antisense oligonucleotide (ASO), phosphorodiamidate morpholino oligonucleotide (PMO), radiopharmaceutical, aptamer, siRNA, RNAi, miRNA, and Trans-activating crRNA (TracrRNA).

147. The combination dosing regimen of any of Claims 1-146, wherein the dosing regimen is a co-formulation comprising the hyaluronidase and a composition comprising the therapeutic agent.

148. The combination dosing regimen of any of Claims 1-147, wherein the therapeutic agent in the composition is conjugated to one or more molecules or delivered with particles, lipid nanoparticles (LNP), non-lipid nanoparticles, liposomes, cholestosomes, exosomes, viral particles, virus-like particles, cochleates, engineered bacterial systems, DNA nanostructures or split-intein systems.

149. The combination dosing regimen of any of Claims 1-148, wherein the therapeutic agent in the composition is delivered with particles, lipid nanoparticles (LNP), non-lipid nanoparticles, liposomes, cholestosomes, exosomes, viral particles, virus like particles or cochleates conjugated to one more molecules.

150. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein concentration of the therapeutic agent or the composition in the systemic, regional or local lymphatics is greater than 200% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.DBl / 162871737.3 468151. The combination dosing regimen of Claim 150, wherein levels of a chimeric antigen receptor or an immunomodulatory protein encoded, produced or modulated by the therapeutic agent by immune cells in the serum, plasma or lymphatics is greater than 200% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

152. The combination dosing regimen of any of Claims 1, 87, 111, 128, or 150-151, wherein the hyaluronidase is administered in an amount effective to increase activation and proliferation of immune cells.

153. The combination dosing regimen of any of Claims 1, 87, 111, 128, or 150-152, wherein the hyaluronidase is administered in an amount effective to increase an immune response to a vaccine antigen as compared to administration of the therapeutic agent without hyaluronidase.

154. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein concentration of the therapeutic agent or the composition in the muscle, adipose or subcutaneous tissue proximal to the site of administration is greater than 150% of an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

155. A combination dosing regimen comprising administering to a human subject a therapeutic agent or a composition; and administering a soluble hyaluronidase, wherein concentration of the therapeutic agent or the composition in the pancreas, spleen, liver, fat, kidneys, uterus, ovaries, muscle, nerves, heart, lungs, endothelial tissue, bone, bone marrow, brain, skin, gastrointestinal tract, lymphatic system, lymph nodes, or thymus is greater than 120% of an equivalent dose of the therapeutic agent without the hyaluronidase.

156. The combination dosing regimen of any of Claims 1, 87, 111, 128, or 154-155, wherein the hyaluronidase is administered in an amount effective to reduce the immune response to the therapeutic agent or composition antigen as compared to administration of the therapeutic agent without the hyaluronidase.

157. The combination dosing regimen of any of Claims 1, 87, 111, 128, or 154-156, wherein following the administration with the hyaluronidase there is a reduced incidence or severity of injection site reactions when compared to administration of the therapeutic agent or the composition without the hyaluronidase.DBl / 162871737.3 469158. The combination dosing regimen of any of Claims 1, 87, 1 11, 128, or 154-157, wherein following the administration with the hyaluronidase there is a reduced incidence or severity of infusion related reactions when compared to an equivalent intravenous dose or a therapeutic intravenous dose of the therapeutic agent or the composition.

159. The combination dosing regimen of any of Claims 1-158, wherein the combination dosing regimen is administered via subcutaneous (SC) administration.

160. The combination dosing regimen of any of Claims 1-158, wherein the combination dosing regimen is administered via intramuscular (IM) administration.DBl / 162871737.3 470

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