WO2024206465A2 - Agents thérapeutiques d'arnm pour la maturation d'ovocytes - Google Patents

Agents thérapeutiques d'arnm pour la maturation d'ovocytes Download PDF

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WO2024206465A2
WO2024206465A2 PCT/US2024/021708 US2024021708W WO2024206465A2 WO 2024206465 A2 WO2024206465 A2 WO 2024206465A2 US 2024021708 W US2024021708 W US 2024021708W WO 2024206465 A2 WO2024206465 A2 WO 2024206465A2
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mrna
lipid
seq
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WO2024206465A3 (fr
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Pavla BRACHOVA
Nehemiah Seth ALVAREZ
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Eastern Virginia Medical School
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Eastern Virginia Medical School
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0016—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the nucleic acid is delivered as a 'naked' nucleic acid, i.e. not combined with an entity such as a cationic lipid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors

Definitions

  • compositions and methods for fertility treatments relate to compositions and methods for fertility treatments, and more particularly to messenger RNA therapeutics for oocyte maturation.
  • Infertility is a major reproductive health issue that affects -12% of women of reproductive age in the United States.
  • In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) fertility treatments rely on mature oocytes that are retrieved from women and fertilized with sperm in vitro. The ensuing embryos are transplanted into the individual’s uterus to establish pregnancy.
  • IVF in vitro fertilization
  • ICSI intracytoplasmic sperm injection
  • Oocyte maturation occurs when a germinal vesicle (GV) oocyte progresses to metaphase I (MI) and ultimately to metaphase II (Mil).
  • GV germinal vesicle
  • MI metaphase I
  • Mil metaphase II
  • Successful oocyte maturation depends on achieving both nuclear and cytoplasmic maturation to support fertilization and development, which occurs in the absence of transcription.
  • Advances of genome sequencing during the last decade have brought insight into the causes of recurrent oocyte arrest, with 14 described pathologic genetic variants leading to female infertility.
  • several genes involved in meiosis result in recurrent oocyte arrest if they are altered, such as SYCP3, TRIP13, MCM8, STAG3, PATL2, TUBB8, AURKC, and WEE2.
  • RNA drugs make up two general categories of molecules: 1) antisense RNA (RNAi), in which short oligonucleotides bind to complementary sequences in RNA transcripts and typically result in degradation; and 2) mRNA encoding certain peptides or proteins that are transiently expressed in the cytoplasm.
  • RNAi antisense RNA
  • mRNA encoding certain peptides or proteins that are transiently expressed in the cytoplasm.
  • the use of RNA therapeutics requires several hurdles to be overcome: 1) avoiding rapid degradation of the RNA via RNases, 2) delivery of negatively charged RNA molecules that a cannot pass across hydrophobic cell membranes, and 3) strong cellular immunogenicity caused by encountering exogenous RNA.
  • RNA as therapeutic faces several roadblocks linked to the susceptibility of RNA to enzymatic degradation, the immunogenicity of in vitro transcribed RNA, and the efficiency of uptake by the recipient cells.
  • Immune activation of in vitro transcribed mRNA was overcome by incorporating modified nucleosides, and the improvement of the delivery of mRNA therapeutics as well as protection from nucleases has greatly accelerated the route of mRNA therapeutics into the clinic.
  • RNA as a therapy has only recently been applied to a clinical in vitro fertilization setting.
  • the present disclosure provides compositions and methods of making and using said compositions.
  • the present disclosure provides synthetic mRNA and compositions thereof which may find use in inducing oocyte maturation, further finding applications in various fertility treatments.
  • a synthetic mRNA in one aspect, can comprise a coding region encoding a protein involved in oocyte maturation or a variant thereof that is 80% or more homologous. In some aspects, the synthetic mRNA can further comprise at least one chemical modification to a nucleotide. In some aspects, the protein involved in oocyte maturation can be selected from SYCP3, TRIP13, MCM8, STAG3, PATL2, TUBB8, AURKC, and WEE2. In some aspects, the protein involved in oocyte maturation can be PATL2. In some aspects, the coding region can comprise an RNA sequence encoded by a gene selected from Table 1 below, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified.
  • the coding region can comprise an RNA sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide the sequence has been chemically modified.
  • the coding region can comprise a sequence of SEQ ID NO: 7, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide the sequence has been chemically modified.
  • the coding region can encode a polypeptide having a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, or a variant thereof that is 80% or more homologous. In some aspects, the coding region can encode a polypeptide having a sequence of SEQ ID NO: 8, or a variant thereof that is 80% or more homologous.
  • the at least one chemical modification of a nucleoside can comprise: N4-acetylcytidine triphosphate (N4-acetyl-CTP), inosine triphosphate (ITP), 5- methylcytidine-5’ -triphosphate (5-methyl-CTP), pseudouridine-5’ -triphosphate (Pseudo- UTP), Nl-methyladenosine-5’ -triphosphate (N1 -methyl- ATP), Nl-methyl-pseudouridine- 5 ’-triphosphate (Nl-methylpseudo-UTP), 2-thiouridine-5’ -triphosphate (2-Thio-UTP), 5- methoxyuridine-5’ -triphosphate (5 -Methoxy -UTP), 7-m ethylguanosine, 2’-O-7- methylguanosine, or combinations thereof.
  • N4-acetylcytidine triphosphate N4-acetyl-
  • the 3’ UTR can be operably linked to a 3’ end of the coding region.
  • the synthetic mRNA can further comprise a 5’ terminal cap operably linked to a 5’ end of the mRNA.
  • the 5’ terminal cap can have a cap-1 structure.
  • the synthetic mRNA can further comprise a 3’ poly(A) tail operably linked to a 3’ end of the mRNA.
  • therapeutic compositions are provided.
  • the therapeutic composition comprises a synthetic mRNA described herein formulated within a delivery vehicle.
  • the delivery vehicle can be a liposome, a lipoplex, a lipid nanoparticle, a polymer, or a polymeric nanoparticle.
  • a method of inducing maturation in an oocyte comprises contacting the oocyte with a synthetic mRNA or a therapeutic composition described herein.
  • a method of fertilization in another aspect, can comprise contacting an oocyte with a synthetic mRNA or a therapeutic composition described herein, whereupon the oocyte undergoes maturation into an ovum.
  • the method can further comprise fertilizing the ovum with a sperm to form a zygote.
  • fertilizing the ovum with the sperm can comprise contacting the ovum with the sperm in a culture medium.
  • fertilizing the ovum can comprise intracytoplasmic injection of the sperm into the ovum.
  • the oocyte can be collected from an ovary of a first subject.
  • the first subject can be a human.
  • the oocyte is a human oocyte.
  • the method can further comprise the zygote or embryo formed therefrom within a uterus of a second subject.
  • the second subject can be the same as the first subject.
  • the first subject exhibits oocyte maturation arrest.
  • FIG. 1 depicts the experimental approach for analyzing modification of mRNA on oocyte maturation as described in the examples.
  • IVTT in vitro transcription
  • C) Patl2 with unmodified bases or different combinations of modified bases microinjected into Patl2-/- GV oocytes, which are in vitro fertilized, and blastocysts transferred to recipient moms to test fertility (live birth) rates.
  • Ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. Further, the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. There are many values disclosed herein, and each value is also disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value and to “about” another particular value. Similarly, when values are expressed as approximations, using the antecedent “about,” the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a further aspect includes from the one particular value and to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’
  • the phrase “about ‘x’ to ‘y’,” where ‘x’ and ‘y’ are numerical values includes “about ‘x’ to about ‘y’.”
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate, larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, as used herein, “about” and “at or about” mean the nominal value indicated ⁇ 10% variation unless otherwise indicated or inferred.
  • an amount, size, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “at or about,” whether or not expressly stated to be such. Where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself unless expressly stated otherwise.
  • the term “therapeutically effective amount” refers to an amount sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the particular compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts.
  • the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to permanently halt the progression of the disease. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease.
  • the desired response to treatment of the disease or condition can also be delaying the onset or even preventing the onset.
  • the effective daily dose can be divided into multiple doses for administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the individual physician can adjust the dosage in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the disclosure (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. However, a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason.
  • a response to a therapeutically effective dose of a disclosed compound or composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following the administration of the treatment or pharmacological agent.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • the amount of a treatment may be varied, for example, by increasing or decreasing the amount of a disclosed compound or pharmaceutical composition, changing the disclosed compound or pharmaceutical composition administered, changing the route of administration, changing the dosage timing, and so on. Dosage can vary and can be administered in one or more dose administrations daily for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • subject can refer to a vertebrate organism, such as a mammal (e.g., human). “Subject” can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to a human and constituents thereof.
  • treating and “treatment” generally refer to obtaining a desired pharmacological or physiological effect.
  • the effect can be but does not necessarily have to be prophylactic in preventing or partially preventing a disease, symptom, or condition.
  • the effect can be therapeutic regarding a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease, disorder, or condition.
  • treatment as used herein can include any treatment of a disorder in a subject, particularly a human.
  • treatment can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.
  • Those in need of treatment i.e., subjects in need thereof
  • treating can include inhibiting the disease, disorder, or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • dose can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound or a therapeutic composition thereof calculated to produce the desired response or responses in association with its administration.
  • terapéutica can refer to treating, healing, or ameliorating a disease, disorder, condition, or side effect or decreasing the rate of advancement of a disease, disorder, condition, or side effect.
  • the term or phrase “effective,” “effective amount,” or “conditions effective to” refers to such amount or condition that is capable of performing the function or property for which an effective amount or condition is expressed.
  • the exact amount or particular condition required will vary from one aspect to another, depending on recognized variables such as the materials employed and the processing conditions observed. Thus, it is not always possible to specify an exact “effective amount” or “condition effective to.” However, it should be understood that an appropriate effective amount will be readily determined by one of ordinary skill in the art using only routine experimentation.
  • the operations of exemplary aspects of the disclosed method may be described in a particular sequential order for convenient presentation, it should be understood that disclosed aspects can encompass an order of operations other than the particular sequential order disclosed. For example, operations described sequentially may, in some cases, be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular aspect are not limited to that aspect and may be applied to any aspect disclosed.
  • homology refers to overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or polypeptide molecules.
  • polymeric molecules are “homologous” to one another if their sequences are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
  • the term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95, or even 99% identical or similar for at least about 20 amino acids.
  • two polypeptide sequences are considered to be homologous if the polypeptides are at least about 50%, 60%, 70%, 80%, 90%, 95, or even 99% identical or similar for at least about 20 amino acids.
  • Optimal alignment of protein sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. AppL Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
  • encapsulate as used herein means to enclose, surround or encase. As it relates to compositions of the present disclosure, encapsulation may be substantial, complete, or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or greater than 99.999% of the composition may be enclosed, surrounded, or encased within a delivery vehicle. “Partially encapsulated” means that less than 10, 20, 30, 40, 50, or less of the composition may be enclosed, surrounded, or encased within a delivery vehicle.
  • encapsulation may be determined by measuring the escape or the activity of the composition by using fluorescence and/or electron micrography. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or greater than 99.999% of the composition are encapsulated in the delivery vehicle.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • modified refers to a changed state or structure of a molecule of the disclosure. Molecules may be modified in many ways including chemically, structurally, and functionally.
  • the polynucleotides of the present disclosure are “chemically modified” by the introduction of non-natural nucleosides and/or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C.
  • nucleosides and/or nucleotides as used in the present disclosure may be naturally occurring (i.e., comprise a nucleotide and/or nucleoside other than the natural ribonucleotides A, U, G, and C) or may be artificial.
  • Non-canonical nucleotides such as the cap structures are not considered “modified” although they differ from the chemical structure of A, G, C, and U ribonucleotides.
  • a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves.
  • modified nucleotides Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides.
  • modified nucleotides When the polynucleotides of the present disclosure are chemically and/or structurally modified, the polynucleotides may be referred to as “modified nucleotides”.
  • operably linked when referring to a first nucleic acid sequence that is operably linked with a second nucleic acid sequence, means a situation when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • Synthesis of polynucleotides or other molecules of the present disclosure may be chemical or enzymatic.
  • transfection refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments, cationic lipids or mixtures, or the like.
  • “translation” is the process by which mRNA is processed by a ribosome or ribosomal-like machinery, e.g., cellular or artificial, to produce a peptide or polypeptide.
  • “unmodified” refers to any substance, compound, or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequence modification.
  • mRNA Synthetic Messenger RNA
  • Oocyte in vitro maturation is an assisted reproductive technology designed to obtain mature oocytes following culture of immature GV oocytes or cumulus-oocyte complexes collected from antral follicles.
  • IVMM Oocyte in vitro maturation
  • In vitro maturation can be performed clinically for women with or without the ovulatory hCG trigger, by in vitro maturation of GV oocytes prior to hCG treatment, or in vitro maturation of GV oocytes that have failed to mature following the hCG trigger.
  • the present disclosure relates to an mRNA therapeutic that is an in vitro synthesized wild-type mRNA that may contain all the features of an endogenous RNA which include a 5’ and 3’ UTR, coding sequence, and poly(A) tail. Additionally, the mRNA therapeutic may contain modified nucleic acids. The codified nucleic acids could occur as a single/multiple type modified base(s) as a specific location or locations within the synthetic mRNA.
  • the mRNA therapeutic can represent a mixture of modified and unmodified synthetic mRNA molecules, or a pure mixture of modified synthetic mRNA.
  • the modifications to the synthetic mRNA can include: N4-acetyl-CTP, ITP, 5-methyl-CTP, Pseudo-UTP, N1 -methyl -ATP, Nl-methylpseudo-UTP, 2-Thio-UTP, 5 -Methoxy -UTP, 7- methylguanosine, 2' -O-7-methylguanosine.
  • proteins have been experimentally determined to play a role in mammalian oocyte maturation. These include but are not limited to genes listed in Table 1 herein. In humans, the proteins SYCP3, TRIP13, MCM8, STAG3, PATL2, TUBB8, AURKC, and WEE2 have been implicated in oocyte maturation defects, however, oocyte maturation defects are not limited to these proteins.
  • the present disclosure relates to a method to treat oocyte maturation defects with a synthetic mRNA therapeutic containing specific RNA modifications.
  • the mRNA therapeutic can be used to rescue or enhance oocyte maturation by expressing endogenous proteins necessary for oocyte maturation, which include but are not limited to those proteins disclosed here.
  • the present disclosure also relates to the specific RNA modification(s) that regulate the stability and translation of synthetic mRNA therapeutics injected into oocytes regardless of the underlying protein the mRNA therapeutic encodes.
  • RNA modifications impact the translation, stability, and immunogenicity of RNA.
  • Oocytes have a unique RNA environment and particular types of RNA modifications may be required for RNA processing in oocytes.
  • IVM In vitro maturation
  • mRNA therapeutics to promote GV oocyte maturation solves this issue by promoting human GV oocyte maturation by providing oocytes a means to translate proteins necessary for maturation in situations where endogenous mRNA translated products are defective or deficient.
  • the use of mRNA therapeutics is transient; the mRNA is subsequently degraded over the course of hours through natural molecular machinery of the oocyte.
  • compositions and methods can be used in a fertility clinic where assisted reproductive technology (ART) is utilized.
  • ART can be in the form of ICSI or IVF.
  • immature GV or Mil oocytes can be injected with an mRNA therapeutic that enhances maturation rates. This is especially important in clinical situations where patients produce only immature GV oocytes or very few Mil oocytes.
  • the use of mRNA therapy can increase the available Mil oocytes that can be used in ICSI or IVF, increasing the procedure success rate.
  • RNA therapeutics for in vitro maturation: women sensitive to hormones or who are prone to ovarian hyperstimulation syndrome, such as with polycystic ovarian syndrome; women who are recovering or in remission from cancer, since hormonal stimulation could stimulate cancer cells; women seeking alternative options due to financial reasons, since IVM is lower cost and has less associated risks than traditional IVF; women who undergo IVF but all collected eggs are immature, including women with genetic causes of oocyte arrest; women with ovulation disorders, premature ovarian failure, or uterine fibroids; couples or women with unexplained infertility; women with blocked, damaged, or removed fallopian tubes; and male factor infertility.
  • the present disclosure provides messenger RNA (mRNA) which encodes a protein involved in oocyte maturation and natural or artificial variants thereof.
  • mRNA messenger RNA
  • the mRNA may have any of the features described herein.
  • the basic components of an mRNA molecule include at least a coding region, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), a 5' terminal cap and a polyadenylate tail.
  • the mRNA of the present disclosure may include one or more modifications from the naturally occurring mRNA transcript for the polypeptide of interest.
  • the modified mRNA of the present disclosure are distinguished from wild-type mRNA in their functional and/or structural design features, which may serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics, while still maintaining the desired activity in inducing oocyte maturation. It is to be understood that the protein of interest, and natural or artificial variants thereof, may be expressed in a human cell, such as an oocyte, using the modified mRNA as described herein.
  • the mRNA described herein contains a first region of linked nucleotides that is operably linked to a first flanking region on the 5' end and a second flanking region at the 3' end.
  • the first region comprises the encoding sequence for the polypeptide of interest.
  • the first flanking region may comprise a region of linked nucleotides comprising one or more 5' UTR sequences.
  • the first flanking region may include at least one nucleic acid sequence including, for example, translation control sequences, for example a Kozak consensus sequence.
  • the first flanking region may also include a 5' terminal cap at the terminal 5' base location.
  • the 5' terminal capping region may include a naturally occurring cap, a synthetic cap, or an optimized cap.
  • the 5' cap is enzymatically added.
  • optimized caps include the caps taught by Rhoads in U.S. Pat. No. 7,074,596 and International Patent Publication No. WO2008157668, WO2009149253 and WO2013103659, the contents of each of which are herein incorporated by reference in their entirety.
  • the second flanking region may comprise a region of linked nucleotides comprising one or more 3' UTR sequences.
  • the second flanking region may include at least one nucleic acid sequence including, but not limited to, translation control sequences.
  • the second flanking region may also comprise a 3' tailing sequence.
  • the 3' tailing sequence may contain a polyadenylation motif or short poly A tail, for example less than about 100 nucleotides.
  • a first operational region Bridging the 5' terminus of the first region and the first flanking region is a first operational region.
  • this operational region comprises a Start codon.
  • the operational region may alternatively comprise any translation control sequence, for example a Kozak consensus sequence, or signal including a Start codon.
  • this operational region comprises a Stop codon.
  • the operational region may alternatively comprise any translation control sequence or signal including a Stop codon. Multiple serial stop codons may also be used in the polynucleotide.
  • the operational region of the present disclosure may comprise two stop codons. The first stop codon may be “UGA” and the second stop codon may be selected from the group consisting of “UAA,” “UGA” or “UAG.”
  • the present disclosure provides an mRNA comprising a coding region encoding a protein involved in oocyte maturation or a variant thereof that is 80% or more homologous.
  • proteins involved in oocyte maturation include, but are not limited to: AASS, ABCB1, ABCB4, ABCF3, ABHD13, ABHD4, ACBD3, ACCSL,ACOT9, ACOXL, ACP6, ACVR1B, ACVR2B, ADAR, ADCY9, ADK, ADSS2, AIG1, ALDH9A1, ALKBH5, AMFR, AMN1, AP1M1, APIP, ARAP2, ARCN1, ARFIP1, ARHGEF16, ARHGEF18, ARL4A, ARL6, ARNTL, ARPP19, ARRDC1, ARRDC2, ASF1A, ASF1B, ASPM, ASTL, ASZ1, ATG16L1, ATG5, ATL2, ATP2C1, ATP8A2, ATR, AXIN2, B3GALNT2, B3GNT2, B4GALT4, B4GALT6, BANP, BBS4, BBS5, BCAR3, BCL2L11, BI
  • CD7 CDC42SE2, CDCA5, CDK5RAP1, CDK5RAP2, CDO1, CDR2, CDS1, CDYL, CENPH, CENPN, CEP164, CEP55, CHD9, CHST7, CHSY1, CITED2, CMAS, CMKLR2, CMTM8, CMYA5, CNDP1, CNKSR3, CNOT4, CNOT6L, CNOT7, COPB1, COPS8, CORO2B, CPA1, CPSF4L, CRABP2, CRELD2, CRLF2, CRYL1, CSAD, CSF1, CSMD3, CTBP2, CUL1, CUL4B, CWF19L2, CXCR3, CYP19A1, DAZ1, DAZ2, DAZ3, DAZ4, DAZL, DCLK1, DCLK2, DCLRE1A, DCP1A, DDHD1, DDX19A, DDX4, DEFB110, DEGS1, DEPDC7, DGUOK, DHX57, DISP1, DNAJA
  • the coding region comprises an RNA sequence encoded by a gene selected from Table 1 below, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified.
  • the protein involved in oocyte maturation is selected from SYCP3, TRIP13, MCM8, STAG3, PATL2, TUBB8, AURKC, and WEE2.
  • the protein involved in oocyte maturation is SYCP3.
  • the coding region comprises an RNA sequence of SEQ ID NO: 1, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified:
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 2, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous:
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 6, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous:
  • the protein involved in oocyte maturation is PATL2.
  • the coding region comprises an RNA sequence of SEQ ID NO: 7, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified: SEQ ID NO: 7
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 1
  • the protein involved in oocyte maturation is TUBB8.
  • the coding region comprises an RNA sequence of SEQ ID NO: 9, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified:
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 10, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous:
  • the protein involved in oocyte maturation is AURKC.
  • the coding region comprises an RNA sequence of SEQ ID NO: 11, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified:
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 12, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous:
  • the protein involved in oocyte maturation is WEE2.
  • the coding region comprises an RNA sequence of SEQ ID NO: 13, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified:
  • the coding region encodes a polypeptide having a sequence of SEQ ID NO: 14, or a variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous:
  • the mRNA of the present disclosure includes at least one chemical modification.
  • chemical modification or “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides in one or more of their position, pattern, percent, or population. Generally, these terms are not intended to refer to modifications in naturally occurring 5 '-terminal mRNA cap moieties.
  • the chemical modifications may be various distinct modifications.
  • the mRNA may contain one, two, or more of the same or different nucleoside or nucleotide chemical modifications.
  • a modified mRNA may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide.
  • Chemical modifications to the nucleosides as used in the present disclosure may be naturally occurring or may be artificial, i.e. not found in nature and synthesized by man.
  • the one or more chemical modifications include modifications to an adenosine ribonucleoside within the mRNA.
  • adenosine ribonucleoside modifications include, but are not limited to 2-methylthio-N6- (cishydroxyisopentenyl)adenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), N6-glycinylcarbamoyladenosine (g6A), N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), N6- threonylcarbamoyladenosine (t6A), l,2’-O-dimethyladenosine (ml Am), 1-methyladenosine (ml A), 2’-O-methyl
  • from about 5% to about 100% of the adenosine ribonucleosides within the mRNA are modified. In some aspects, from about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, bout 80%, about 90%, or about 100% of the adenine nucleosides within the mRNA are modified.
  • the one or more chemical modifications include modifications to a cytidine ribonucleoside within the mRNA.
  • cytidine ribonucleoside modifications include, but are not limited to, 2-thiocytidine (s2C), 3- methylcytidine (m3C), 5 -formyl cytidine (f5C), 5-hydroxymethylcytidine (hm5C), 5- methylcytidine (m5C), N4-acetylcytidine (ac4C), 2’-O-methylcytidine (Cm), 5,2’ -O- dimethylcytidine (m5Cm), 5-formyl-2’-O-methylcytidine (f5Cm), lysidine (k2C), N4,2’-O- dimethylcytidine (m4Cm), N4-acetyl-2’-O-methylcytidine (ac4Cm), N4-methylcytidine
  • 5-(l-propynyl)ara-cytidine TP 5-(2-chloro-phenyl)-2 -thiocytidine TP, 5-(4-amino-phenyl)-2- thiocytidine TP, 5-aminoallyl-CTP, 5-cyanocytidine TP, 5-ethynylara-cytidine TP, 5- ethynylcytidine TP, 5'-homo-cytidine TP, 5-methoxycytidine TP, 5-trifluoromethyl-cytidine TP, N4-amino-cytidine TP, N4- benzoyl-cytidine TP, and pseudoisocytidine.
  • from about 5% to about 100% of the cytidine ribonucleosides within the mRNA are modified. In some aspects, from about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, bout 80%, about 90%, or about 100% of the cytidine nucleosides within the mRNA are modified.
  • one or more chemical modifications include modifications to a guanosine ribonucleoside within the mRNA.
  • guanosine ribonucleoside modifications include, but are not limited to, 7-methylguanosine (m7G), N2, 2 ’-O-dimethyl guanosine (m2Gm), N2-methylguanosine (m2G), wyosine (imG), 1,2’ -O- dimethylguanosine (mlGm), 1-methylguanosine (mlG), 2 ’-O-m ethylguanosine (Gm), 2’-O- ribosylguanosine (phosphate) (Gr(p)), 7-aminomethyl-7-deazaguanosine (preQi), 7-cyano- 7-deazaguanosine (preQO), archaeosine (G+), methylwyosine (mimG), N2,7- dimethylguanosine (m
  • from about 5% to about 100% of the guanosine ribonucleosides within the mRNA are modified. In some aspects, from about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, bout 80%, about 90%, or about 100% of the guanosine nucleosides within the mRNA are modified.
  • the one or more chemical modifications include modifications to a uridine ribonucleoside within the mRNA.
  • uridine ribonucleoside modifications include, but are not limited to, 2-thiouridine (s2U), 3- methyluridine (m3U), 5-carboxymethyluridine (cm5U), 5-hydroxyuridine (ho5U), 5- methyluridine (m5U), 5-taurinomethyl-2-thiouridine (rm5s2U), 5-taurinomethyluridine (rm5U), Dihydrouridine (D), Pseudouridine ( ), (3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-5-carboxypropyl)pseudouridine (mlacp3'P), 1- methylpseudouridine (ihPP), 2’-O-methyluridine (Um), 2’-O-methylpse
  • aminoalkylaminocarbonylethylenyl pseudouracil
  • aminocarbonylethylenyl-4-(thio)pseudouracil l-(aminocarbonylethylenyl)-pseudouracil, 1 -substituted 2(thio)-pseudouracil, 1 -substituted 2,4-(dithio)pseudouracil, 1 -substituted 4(thio)pseudouracil, 1 -substituted pseudouracil, l-(aminoalkylamino-carbonylethylenyl)-2- (thio)-pseudouracil, l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine TP, l-methyl-3- (3-amino-3- carboxypropyl)pseudo-UTP, 1 -methyl -pseudo-UTP, 2-
  • alkyl)uracil 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(cyanoalkyl)uracil, 5- (dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidinium alkyl)uracil, 5- (halo)uracil, 5-(l,3-diazole-l-alkyl)uracil, 5-(methoxy)uracil, 5-(methoxycarbonylmethyl)-2- (thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(methyl)2(thio)uracil, 5- (methyl)2,4(dithio)uracil, 5-(methyl)4(thio)uracil, 5-(methyl)-2-(thio)pseudouracil, 5- (methyl)-2,4(dithio)pseudouracil, 5-(methyl)-4(thio
  • uridine ribonucleosides within the mRNA are modified. In some aspects, from about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, bout 80%, about 90%, or about 100% of the uridine nucleosides within the mRNA are modified. 5’ Untranslated Regions (5’ UTR) and 3’ Untranslated Regions (3’ UTR)
  • the mRNA of the present disclosure may comprise one or more regions or parts, which act or function as an untranslated region.
  • wild type untranslated regions (UTRs) of a gene are transcribed but not translated.
  • the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon.
  • the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
  • the regulatory features of a UTR can be incorporated into the mRNA of the present disclosure to enhance the stability of the molecule, for example. Specific features can also be incorporated to ensure controlled down-regulation of transcription when present in undesired locations, for example miRNA binding sites and RNA binding protein (RBP) binding sites.
  • RBP RNA binding protein
  • Specific 5' UTR and 3 UTR for use in the present disclosure can be any suitable UTR sequence, for example, a natural UTR sequence, a derivatized naturally occurring UTR, or a synthetic UTR.
  • the 5' UTR and/or the 3' UTR is a naturally occurring human UTR or a human-derived UTR.
  • the use of human-derived UTRs may facilitate the expression of the polypeptide encoded by the coding region in human cells.
  • the 5' UTR and/or the 3' UTR are synthetic, i.e. not completely homologous with a UTR found in any species.
  • the 5' UTR is operably linked to the 5' end of the coding region.
  • the 3' UTR is operably linked to the 3' end of the coding region.
  • Natural 5' UTRs have features which play roles in translation initiation. They can harbor, for example, Kozak consensus sequences which are known to be involved in the process by which the ribosome initiates translation.
  • the Kozak consensus has the sequence GCCNCCAUGG (SEQ ID NO: 15), where N is a purine (adenine or guanine) three nucleobases upstream from the start codon AUG.
  • 5' UTRs have also been known to form secondary structures which are involved in elongation factor binding.
  • Untranslated regions useful in the design and manufacture of mRNA include, but are not limited to, those disclosed in International Application Publication No. WO2014164253, incorporated herein by reference in its entirety.
  • non-UTR sequences may be also used as regions or subregions within the mRNA. Combinations of features may be included in regions flanking the coding region and may be contained within other features.
  • the coding region may be flanked by a 5' UTR which may contain a strong Kozak consensus sequence.
  • WO2014164253 provides a list of exemplary UTRs which may be used as flanking regions and is incorporated herein by reference. Variants of 5' or 3' UTRs may be used wherein one or more nucleotides are added or removed at the termini.
  • Any UTR may be incorporated into the mRNA.
  • multiple wild-type UTRs may be used.
  • UTRs derivatized from a wild-type UTR may be used.
  • artificial UTRs may be used that are not variants of wild-type regions. These UTRs or portions thereof may be placed in the same orientation as the transcript from which they were selected or may be altered in orientation or location.
  • a 5' or 3' UTR may be shortened, lengthened, or made from one or more other 5' or 3' UTRs.
  • a UTR may be “altered”, meaning that the UTR has been changed in some way relative to the reference sequence.
  • a 5' or 3' UTR may be altered relative to the native UTR by a change in orientation or location, by the inclusion of additional nucleotides, deletion of nucleotides, or by swapping or transposing nucleotides.
  • a double, triple, or quadruple UTR such as a 5' or 3' UTR may be used.
  • a double UTR is one in which two copies of the UTR are encoded in series or substantially in series.
  • a patterned 3' or 5' UTR may be used.
  • a patterned UTR are those which reflect a repeating or alternative pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter A, B, or C represents a different UTR at the nucleotide level.
  • flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property.
  • the UTRs of any of these genes may be swapped for any other UTRs of the same or different family of proteins to create a new mRNA.
  • the untranslated region may also include translation enhancer elements (TEEs).
  • TEEs translation enhancer elements
  • the TEE may include those described in U.S. Patent Publication No. 20090226470, which is incorporated herein by reference.
  • AU rich elements can be separated into three classes. Class I AREs contain several dispersed copies of an AUUUA motif within uridine-rich regions. Class II AREs contain two or more UUAUUUA(U/A)(U/A) nonamers. Class III AREs are less well defined; these uridine-rich regions do not contain an AUUUA motif.
  • HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of the mRNA of the present disclosure will lead to HuR binding and subsequent stabilization of the mRNA in vivo.
  • AREs 3' UTR AU rich elements
  • One or more copies of an ARE can be introduced into the 3 UTR of the mRNA to make it less stable, leading to lowered translation and decreased production of the resultant protein.
  • AREs can be identified and removed or mutated to increase the intracellular stability, increasing translation and production of the resultant protein.
  • MicroRNAs are 19-25 nucleotide long noncoding RNAs that bind to the 3' UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
  • the mRNA of the disclosure may comprise one or more microRNA target or binding sequences. microRNA target or binding sequences are well known in the art.
  • a microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence.
  • a microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.
  • a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed- complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed- complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1.
  • A adenine
  • the bases of the microRNA seed have complete complementarity with the target sequence.
  • microRNA target sequences into the polynucleotides (e.g., in a 3'UTR like region or other region) of the disclosuree one can target the molecule for degradation or reduced translation, provided the microRNA in question is available. This process will reduce the hazard of off target effects upon nucleic acid molecule delivery. Identification of microRNA, microRNA target regions, and their expression patterns and role in biology have been reported (Bonauer et ah, Curr Drug Targets 2010 11 :943-949; Anand and Cheresh Curr Opin Hematol 2011 18: 171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi: 10.
  • microRNA target sequence or “microRNA binding sequence” refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.
  • microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they occur, e.g., in order to increase protein expression in specific tissues. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites.
  • binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or to the context of relevant biological processes.
  • microRNA binding sequences can be incorporated into mRNA to decrease expression in certain cells which results in a biological improvement.
  • mRNA can be engineered for more targeted expression in specific cell types or only under specific biological conditions.
  • polynucleotides can be designed that would be optimal for protein expression in a tissue or in the context of a biological condition.
  • Table 2 below provides exemplary 5' UTRs that may be used in the mRNA s of the present disclosure. Variations of these 5' UTRs may be used wherein one or more nucleotides are added to or removed from the termini, including A, U, C, or G.
  • Table 2 Exemplary 5’ Untranslated Regions Table 3 below provides exemplary 3’ UTRs that may be used in the mRNA of the present disclosure. Variations of these 3’ UTRs may be used wherein one or more nucleotides are added to or removed from the termini, including A, U, C, or G.
  • the 5' terminal cap structure of natural mRNA is involved in nuclear transport, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the 5' terminal cap is operably linked to the 5' end of the mRNA as described herein.
  • Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated.
  • 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • mRNA of the present disclosure may be designed to incorporate a cap moiety. Modifications to the polynucleotides of the present disclosure may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with a-thio- guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and/or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring.
  • Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as a polynucleotide which functions as an mRNA molecule.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the disclosure.
  • the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which may equivalently be designated 3'-O-Me-m7G(5')ppp(5')G).
  • the 3'-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide.
  • N7- and 3'-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.
  • Another exemplary cap is mCAP, which is similar to ARC A but has a 2’-0-methyl group on guanosine (i.e., N7, 2’ -O-dimethyl -guanosine-5 '-triphosphate-5 '-guanosine, m7Gm-ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • the cap is a N7-(4-chlorophenoxyethyl)substituted dinucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7- (4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4- chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4-chlorophenoxyethyl)-m3-O G(5')ppp(5')G cap analog (See e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21 :4570-4574, herein incorporated by reference in its entirety).
  • a cap analog of the present disclosure is a 4- chloro/bromophenoxyethyl analog.
  • cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5'-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • mRNA of the disclosure may also be capped post-manufacture (whether by IVT or chemical synthesis), using enzymes, in order to generate more authentic 5'-cap structures, for example to closely mirror or mimic, either structurally or functionally, an endogenous or wild type feature.
  • Non-limiting examples of such 5' cap structures of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping, as compared to synthetic 5' cap structures known in the art (or to a wild-type, natural or physiological 5' cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2 ’-O-m ethyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2’-O-methyl.
  • Capl structure Such a structure is termed the Capl structure.
  • Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).
  • 5'-terminal caps may include endogenous caps or cap analogs.
  • a 5' terminal cap may comprise a guanine analog.
  • Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2’ -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.
  • a long chain of adenine nucleotides may be added to the mRNA molecule in order to increase stability.
  • the 3' end of the transcript may be cleaved to free a 3' hydroxyl.
  • poly(A) polymerase adds a chain of adenine nucleotides to the RNA.
  • the process called polyadenylation, adds a poly(A) tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.
  • the poly(A) tail is operably linked to 3' end of the mRNA as described herein. Poly(A) tails may also be added after the construct is exported from the nucleus.
  • Terminal groups on the poly(A) tail may be incorporated for stabilization into mRNA of the present disclosure.
  • Polynucleotides of the present disclosure may include dess' hydroxyl tails. They may also include structural moieties or 2'-O-methyl modifications as taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, herein incorporated by reference in its entirety).
  • the mRNA of the present disclosure may be designed to encode transcripts with alternative poly(A) tail structures including histone mRNA. According to Norbury, terminal uridylation has also been detected on human replication-dependent histone mRNA. The turnover of these mRNA is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of chromosomal DNA replication.
  • mRNA are distinguished by their lack of a 3' poly(A) tail, the function of which is instead assumed by a stable stem-loop structure and its cognate stem-loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNA” (Norbury, “Cytoplasmic RNA: a case of the tail wagging the dog,” Nature Reviews Molecular Cell Biology; AOP, published online 29 Aug. 2013; doi: 10. 1038/nrm3645, herein incorporated by reference in its entirety).
  • SLBP stem-loop binding protein
  • the length of a poly(A) tail when present, is greater than 30 nucleotides in length.
  • the poly(A) tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).
  • the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides.
  • the poly(A) tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of the coding region, the length of a particular feature or region or based on the length of the ultimate product expressed.
  • the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof.
  • the poly(A) tail may also be designed as a fraction of the polynucleotides to which it belongs.
  • the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly(A) tail.
  • engineered binding sites and conjugation of polynucleotides for Poly(A) binding protein may enhance expression.
  • multiple distinct polynucleotides may be linked together via the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '-terminus of the poly (A) tail.
  • PABP Poly-A binding protein
  • the mRNA of the present disclosure is designed to include a poly(A) G quartet region.
  • the G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA.
  • the G-quartet is incorporated at the end of the poly(A) tail.
  • the resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points.
  • the poly(A) G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly(A) tail of 120 nucleotides alone. Start Codon Region
  • the mRNA of the present disclosure may have regions that are analogous to or function like a start codon region.
  • the translation of the mRNA may initiate on a codon which is not the start codon AUG.
  • Translation of the polynucleotide may initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CUG, GUG, AU A, AUU, UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5: 11, herein incorporated by reference in its entirety).
  • the translation of a polynucleotide begins on the alternative start codon ACG.
  • polynucleotide translation begins on the alternative start codon CUG.
  • the translation of a polynucleotide begins on the alternative start codon GUG.
  • the mRNA of the present disclosure may include at least two stop codons before the 3 ' untranslated region (UTR).
  • the stop codon may be selected from UGA, UAA and UAG.
  • the polynucleotides of the present disclosure include the stop codon UGA and one additional stop codon.
  • the additional stop codon may be UAA.
  • the polynucleotides of the present disclosure include three stop codons.
  • the coding region of the mRNA of the present disclosure and their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, to bias GC content to increase mRNA stability or reduce secondary structures, to minimize tandem repeat codons or base runs that may impair gene construction or expression, to customize translational control regions, to insert or remove protein trafficking sequences, to remove/add post translation modification sites in encoded protein (e.g.
  • Codon optimization tools, algorithms and services are known in the art, non-limiting examples of which include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods.
  • the coding region sequence is optimized using optimization algorithms. mRNA Codon options for each amino acid are given in Table 4.
  • variants of the proteins encoded by the mRNA described herein are also contemplated.
  • Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence.
  • substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
  • Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with Tables 5 and 6 below and are referred to as conservative substitutions.
  • Cremophor polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), di ethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Pol oxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include starch (e.g.
  • cornstarch and starch paste examples include gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g.
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
  • Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, NeoIone, Kathon, and Euxyl.
  • the preservative is an anti-oxidant.
  • the preservative is a chelating agent.
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer
  • Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buck
  • Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and combinations thereof.
  • composition may further comprise a polymer.
  • exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (ELEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, varoius gums, including xanthan gum, guar
  • epsilon. -caprolactone-co-glycolide)- carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid/acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxidepropylene oxide), and a Pluronic polymer, polyoxy ethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG-stearate, l,2-Distearoyl-sn-glycero-3- Phosphoethanolamine-N-[Methoxy(Poly ethylene glycol)-1000], 1,2-Distearoyl-sn-glycero- 3-Phosphoethanolamine-N-[Met
  • composition may further comprise an emulsifying agent.
  • emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g.
  • acacia agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.
  • carboxy polymethylene polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer
  • carrageenan cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • Cremophor polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • the emulsifying agent is cholesterol.
  • Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the liquid compositions can also include adjuvants such as wetting agents, emulsifying and suspending
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80.
  • the injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • lipidoids The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et ak, Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et ak, J Intern Med. 2010 267:9-21; Akinc et ak, Nat Biotechnol. 2008 26:561-569; Love et ak, Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et ak, Proc Natl Acad Sci USA. 2011 108: 12996- 3001, and US 2016/0317647, herein incorporated by reference in their entirety).
  • Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the polynucleotide, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration.
  • Lipidoid complexes of polynucleotides can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
  • nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879, herein incorporated by reference in its entirety).
  • particle size Akinc et al., Mol Ther. 2009 17:872-879, herein incorporated by reference in its entirety.
  • PEG polyethylene glycol
  • Formulations with the different lipidoids including, but not limited to penta[3-(l-laurylaminopropionyl)]- triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401 :61 (2010), herein incorporated by reference in its entirety, and MD1, can be tested for in vivo activity.
  • TETA-5LAP penta[3-(l-laurylaminopropionyl)]- triethylenetetramine hydrochloride
  • the lipidoid referred to herein as “98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879, herein incorporated by reference in its entirety.
  • the lipidoid referred to herein as “02-200” is disclosed by Love et al., Proc Natl Acad Sci USA. 2010 107: 1864- 1869 and Liu and Huang, Molecular Therapy. 2010 669-670, herein incorporated by reference in their entirety.
  • the lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotides.
  • formulations with certain lipidoids include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 alkyl chain length).
  • formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
  • Combinations of different lipidoids may be used to improve the efficacy of polynucleotides directed protein production as the lipidoids may be able to increase cell transfection by the mRNA; and/or increase the translation of encoded protein (see Whitehead et al., Mol. Ther. 2011, 19: 1688- 1694, herein incorporated by reference in its entirety).
  • the mRNA of the present disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
  • the therapeutic compositions described herein include liposomes. Liposomes are artificially prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations.
  • Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unilamellar vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unilamellar vesicle
  • LUV large unilamellar vesicle
  • Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch- to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and
  • therapeutic compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa ).
  • DODMA dioleyloxy-N,N-dimethylaminopropane
  • DLin-DMA 1,2- dilinoleyloxy-3 -
  • compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438- 1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., NatBiotechnol. 2005 2: 1002-1007; Zimmermann et al., Nature.
  • liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy.
  • a liposome can contain, but is not limited to, 55% cholesterol, 20% distcroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2- dioleyloxy-N,N- dimethylaminopropane (DODMA), as described by Jeffs et al.
  • DSPC distcroylphosphatidyl choline
  • PEG-S-DSG 10% PEG-S-DSG
  • DODMA 1,2- dioleyloxy-N,N- dimethylaminopropane
  • certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG- c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or l,2-dilinolenyloxy-3- dimethylaminopropane (DLenDMA), as described by Heyes et al.
  • DSDMA l,2-distearloxy-N,N-dimethylaminopropane
  • DODMA DODMA
  • DLin-DMA DLenDMA
  • DLenDMA l,2-dilinolenyloxy-3- dimethylaminopropane
  • liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and/or from about 48.5% cholesterol to about 60% cholesterol.
  • formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%.
  • formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.
  • therapeutic compositions may include liposomes which may be formed to deliver mRNA of the present disclosure.
  • the polynucleotide may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. W02012031046, WO20 12031043, WO2012030901 and W02012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684; the contents of each of which are herein incorporated by reference in their entirety).
  • liposomes may be formulated for targeted delivery.
  • the liposome used for targeted delivery may include, but is not limited to, the liposomes described in and methods of making liposomes described in US Patent Publication No. US20130195967, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA of the present disclosure may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the polynucleotide anchoring the molecule to the emulsion particle (see International Pub. No. W02012006380; herein incorporated by reference in its entirety).
  • the mRNA of the present disclosure may be formulated in a water-in- oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed.
  • the emulsion may be made by the methods described in International Publication No. WO201087791, the contents of which are herein incorporated by reference in its entirety.
  • the lipid formulation may include at least a cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; the contents of each of which is herein incorporated by reference in their entirety).
  • the mRNA of the present disclosure may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No. 20120177724, the contents of which is herein incorporated by reference in its entirety).
  • the polynucleotides may be formulated in a liposome as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in their entirety.
  • the mRNA may be encapsulated in a liposome using reverse pH gradients and/or optimized internal buffer compositions as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in its entirety.
  • the mRNA therapeutic compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero- 3- phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713, herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
  • liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero- 3- phosphocholine) based liposome
  • the cationic lipid may be a low molecular weight cationic lipid such as those described in US Patent Application No. 20130090372, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
  • the mRNA may be formulated in a liposome comprising a cationic lipid.
  • the liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phosphates in the RNA (N:P ratio) of between 1 : 1 and 20: 1 as described in International Publication No. W02013006825, herein incorporated by reference in its entirety.
  • the liposome may have a N:P ratio of greater than 20: 1 or less than 1 : 1.
  • the mRNA may be formulated in a lipid-polycation complex.
  • the formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326 or US Patent Pub. No. US20130142818; each of which is herein incorporated by reference in its entirety.
  • the mRNA may be formulated in a lipid-polycation complex which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the mRNA may be formulated in an aminoalcohol lipidoid.
  • Aminoalcohol lipidoids which may be used in the present disclosure may be prepared by the methods described in U.S. Pat. No. 8,450,298, herein incorporated by reference in its entirety.
  • the liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Nature Biotech.
  • liposome formulations may comprise from about 35 to about 45% cationic lipid, from about 40% to about 50% cationic lipid, from about 50% to about 60% cationic lipid and/or from about 55% to about 65% cationic lipid.
  • the ratio of lipid to mRNA in liposomes may be from about 5: 1 to about 20: 1, from about 10: 1 to about 25: 1, from about 15: 1 to about 30: 1 and/or at least 30: 1.
  • the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.
  • LNP formulations may contain from about 0.5% to about 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about 3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3- amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol.
  • PEG-c-DOMG R-3-[(co-m ethoxy - poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3- amine
  • the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2- Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).
  • the cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, Cl 2-200 and DLin-KC2- DMA.
  • the mRNA may be formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA formulation is a nanoparticle which may comprise at least one lipid.
  • the lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG- DMG, PEGylated lipids and amino alcohol lipids.
  • the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.
  • the amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in US Patent Publication No. US20130150625, herein incorporated by reference in its entirety.
  • the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- ⁇ [(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl ⁇ propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l-yloxy]-2- ⁇ [(9Z)-octadec-9-en-l- yloxy]methyl ⁇ propan-l-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)
  • Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2- DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)- non-2-en-l-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG- modified lipid.
  • an ionizable cationic lipid for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin
  • the lipid nanoparticle formulation consists essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- di oxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl)-9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid:
  • the formulation includes from about 25% to about 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl)-9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 50% or about 40% on a molar basis.
  • a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dil
  • the formulation includes from about 0.5% to about 15% on a molar basis of the neutral lipid, e.g., from about 3 to about 12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% on a molar basis.
  • exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM.
  • the formulation includes from about 5% to about 50% on a molar basis of the sterol (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis.
  • An exemplary sterol is cholesterol.
  • the formulation includes from about 0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis.
  • the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
  • the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da.
  • Exemplary PEG- modified lipids include, but are not limited to, PEG-di stearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG) and PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), herein incorporated by reference in its entirety).
  • PEG-DMG PEG-di stearoyl glycerol
  • PEG-cDMA further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), herein incorporated by reference in its entirety.
  • the formulations described herein include about 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in its entirety), about 7.5% of the neutral lipid, about 31.5% of the sterol, and about 3.5% of the PEG or PEG-modified lipid on a molar basis.
  • PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in its entirety)
  • about 7.5% of the neutral lipid about 31.5% of the sterol
  • about 3.5% of the PEG or PEG-modified lipid on a molar basis PEG-cDMA
  • lipid nanoparticle formulation consists essentially of a lipid mixture in molar ratios of about 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid; more preferably in a molar ratio of about 20-60% cationic lipid: 5- 25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.
  • the molar lipid ratio is approximately 50/10/38.5/1.5 mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG- DSG or PEG-DPG, 57.2/7.1134.3/1.4 mol % cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA, 40/15/40/5 mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, 50/10/35/4.5/0.5 mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG, 50/10/35/5 cationic lipid
  • Exemplary lipid nanoparticle compositions and methods of making same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51 : 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578, herein incorporated by reference in its entirety.
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid.
  • the lipid nanoparticle may comprise about 40-60% of cationic lipid, about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30- 50% of a structural lipid.
  • the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid.
  • the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles.
  • the lipid nanoparticle may comprise a cationic lipid, a noncationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle may comprise about 40-60% of cationic lipid, about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
  • the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about 38.5% structural lipid.
  • the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural lipid.
  • the cationic lipid may be any cationic lipid described herein such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
  • the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-KC2-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA, about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and about 38.5% of the structural lipid cholesterol.
  • the lipid nanoparticle comprise about 55% of the cationic lipid L319, about 10% of the non-cationic lipid DSPC, about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the structural lipid cholesterol.
  • the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. W02012040184, W02011153120, WO2011149733, W02011090965, W02011043913, W02011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and
  • the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. W02012040184, W02011153120, WO2011149733, W02011090965, W02011043913, W02011022460, WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US Patent Publication Nos. US20130178541 and US20130225836; the contents of each of which is herein incorporated by reference in their entirety.
  • the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No. W02008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLLCLXXXII of U.S. Pat. No. 7,404,969 and formula I- VI of US Patent Publication No. US20100036115, formula I of US Patent Publication No US20130123338; each of which is herein incorporated by reference in their entirety.
  • the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23 -dien- 10-amine, (17Z,20Z)-N,N- dimemylhexacosa- 17,20-dien-9-amine, ( 1 Z , 19Z)-N5N-dimethylpentacosa-16, 19-dien-8- amine, ( 13Z, 16Z)-N,N-dimethyldocosa- 13,16-dien-5-amine, (12Z, 15Z)-N,N- dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-di-l-en-6- amine, (15Z, 18Z)-N,N-dimethyltetracosa- 15,18-dien-7-amine, (18Z,21Z)-N,N- dimethylheptacosa- 18,21
  • the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.
  • the lipid may be a cationic lipid such as, but not limited to, Formula (I) of U.S. Patent Application No. US20130064894, the contents of which are herein incorporated by reference in its entirety.
  • the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. W02012040184, W02011153120, WO2011149733, W02011090965, W02011043913, W02011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, WO2013086373 and WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.
  • the cationic lipid may be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids and methods of making and using the trialkyl cationic lipids are described in International Patent Publication No. WO2013126803, the contents of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticle formulations may contain PEG-c-DOMG at 3% lipid molar ratio. In another aspect, the LNP formulations may contain PEG-c-DOMG at 1.5% lipid molar ratio.
  • the therapeutic compositions may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety.
  • the lipid nanoparticle formulation may contain PEG-DMG 2000 (1,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
  • the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component.
  • the LNP formulation may contain PEG- DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
  • the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PM ID 22908294, herein incorporated by reference in its entirety).
  • the lipid nanoparticle formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276, the contents of each of which is herein incorporated by reference in their entirety.
  • the mRNA described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or W02008103276; each of which is herein incorporated by reference in their entirety.
  • the mRNA described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No. US20120207845; the contents of which are herein incorporated by reference in its entirety.
  • the mRNA may be formulated in a lipid nanoparticle made by the methods described in US Patent Publication No. US20130156845 or International Publication Nos. WO2013093648 or WO2012024526, each of which is herein incorporated by reference in its entirety.
  • lipid nanoparticles described herein may be made in a sterile environment by the system and/or methods described in US Patent Publication No. US20130164400, herein incorporated by reference in its entirety.
  • the mRNA may be formulated in a nanoparticle such as a nucleic acid- lipid particle described in U.S. Pat. No. 8,492,359, the contents of which are herein incorporated by reference in its entirety.
  • the lipid particle may comprise one or more active agents or therapeutic agents; one or more cationic lipids comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.
  • the nucleic acid in the nanoparticle may be the polynucleotides described herein and/or are known in the art.
  • the lipid nanoparticle formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276, the contents of each of which are herein incorporated by reference in their entirety.
  • modified RNA polynucleotide described herein may be encapsulated in lipid nanoparticle formulations as described in WO2011127255 and/or W02008103276; the contents of each of which are herein incorporated by reference in their entirety.
  • lipid nanoparticle formulations described herein may comprise a polycationic composition.
  • the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.
  • the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA described herein in vivo and/or in vitro.
  • the LNP formulations described herein may additionally comprise a permeability enhancer molecule.
  • a permeability enhancer molecule are described in US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.
  • the mRNA therapeutic compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero- 3- phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer as described in Landen et al. Cancer Biology & Therapy 2006 5(12): 1708-1713, herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
  • the mRNA may be formulated in a lyophilized gel-phase liposomal composition as described in US Publication No. US2012060293, herein incorporated by reference in its entirety.
  • the nanoparticle formulations may comprise a phosphate conjugate.
  • the phosphate conjugate may increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle.
  • Phosphate conjugates for use with the present disclosure may be made by the methods described in International Application No. WO2013033438 or US Patent Publication No. US20130196948, the contents of each of which are herein incorporated by reference in its entirety.
  • the phosphate conjugates may include a compound of any one of the formulas described in International Application No. WO2013033438, herein incorporated by reference in its entirety.
  • the nanoparticle formulation may comprise a polymer conjugate.
  • the polymer conjugate may be a water-soluble conjugate.
  • the polymer conjugate may have a structure as described in U.S. Patent Publication No. US20130059360, the contents of which are herein incorporated by reference in its entirety.
  • polymer conjugates with the polynucleotides of the present disclosure may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Publication No. US20130072709, herein incorporated by reference in its entirety.
  • the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. US20130196948, the contents of which is herein incorporated by reference in its entirety.
  • compositions comprising the polynucleotides of the present disclosure and a conjugate which may have a degradable linkage.
  • conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.
  • therapeutic compositions comprising a conjugate with a degradable linkage and methods for delivering such therapeutic compositions are described in US Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in its entirety.
  • the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and an mRNA.
  • the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. W02012109121; the contents of which are herein incorporated by reference in its entirety).
  • Nanoparticle formulations of the present disclosure may be coated with a surfactant or polymer in order to improve the delivery of the particle.
  • the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge.
  • the hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, the polynucleotides within the central nervous system.
  • nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in U.S. Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles.
  • hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the contents of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin- MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain.
  • the internal ester linkage may replace any carbon in the lipid chain.
  • the internal ester linkage may be located on either side of the saturated carbon.
  • Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
  • Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosal tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al.
  • PEG polyethylene glycol
  • the transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT).
  • FRAP fluorescence recovery after photobleaching
  • MPT high resolution multiple particle tracking
  • compositions which can penetrate a mucosal barrier may be made as described in U.S. Patent No. 8,241,670 or International Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.
  • the lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block copolymer.
  • the polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the polymeric material may be biodegradable and/or biocompatible.
  • biocompatible polymers are described in International Publication No. WO2013116804, the contents of which are herein incorporated by reference in its entirety.
  • the polymeric material may additionally be irradiated.
  • the polymeric material may be gamma irradiated (See e.g., International Publication No. WO201282165, herein incorporated by reference in its entirety).
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), 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 cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HP
  • the lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer (such as a branched polyetherpolyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))- (poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., ETS Publication 20120121718 and US Publication No. 20100003337 and U.S. Patent No. 8,263,665; each of which is herein incorporated by reference in their entirety).
  • a block co-polymer such as a branched polyetherpolyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety
  • the copolymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created.
  • the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600, herein incorporated by reference in its entirety).
  • a non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (J Control Release 2013, 170(2):279- 86, herein incorporated by reference in its entirety).
  • the vitamin of the polymer-vitamin conjugate may be vitamin E.
  • the vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
  • the lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carboci steine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin
  • the surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle (see e.g., U.S. Publication Nos. 20100215580, US20080166414, and US20130164343; the contents of each of which is herein incorporated by reference in their entirety).
  • the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein.
  • the polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the particle.
  • the polynucleotide may be covalently coupled to the lipid nanoparticle.
  • Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
  • the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating.
  • the formulation may be hypotonic for the epithelium to which it is being delivered.
  • hypotonic formulations may be found in International Patent Publication No. WO2013110028, the contents of which are herein incorporated by reference in its entirety.
  • the polynucleotide formulation may comprise or be a hypotonic solution, see e.g., Ensign et al. Biomaterials 2013 34(28):6922-9, herein incorporated by reference in its entirety.
  • the mRNA is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, ETnited Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethyleneimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788- 9798; Strumberg et al.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, ETnited Kingdom), STEMFECTTM from STEMGENT® (Cambridge, Mass.), and polyethyleneimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-
  • Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N- acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al., Biomacromolecules.
  • the mRNA is formulated as a solid lipid nanoparticle.
  • a solid lipid nanoparticle may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic aspect and may be stabilized with surfactants and/or emulsifiers.
  • the lipid nanoparticle may be a self- assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702, herein incorporated by reference in its entirety).
  • the SLN may be the SLN described in International Patent Publication No.
  • W02013105101 the contents of which are herein incorporated by reference in its entirety.
  • the SLN may be made by the methods or processes described in International Patent Publication No. W02013105101, the contents of which are herein incorporated by reference in its entirety.
  • Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the mRNA; and/or increase the translation of encoded protein.
  • One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; herein incorporated by reference in its entirety).
  • the liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide.
  • the mRNA of the present disclosure can be formulated for controlled release and/or targeted delivery.
  • controlled release refers to a therapeutic composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the polynucleotide may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the synthetic mRNA of the disclosure, encapsulation may be substantial, complete or partial.
  • substantially encapsulated means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the therapeutic composition or synthetic mRNA of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulated” means that less than 10, 10, 20, 30, 40 50 or less of the therapeutic composition or synthetic mRNA of the disclosure may be enclosed, surrounded or encased within the delivery agent.
  • encapsulation may be determined by measuring the escape or the activity of the therapeutic composition or synthetic mRNA of the disclosure using fluorescence and/or electron micrograph.
  • At least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the therapeutic composition or synthetic mRNA of the disclosure are encapsulated in the delivery agent.
  • the controlled release formulation may include, but is not limited to, tri-block co-polymers.
  • the formulation may include two different types of tri-block co-polymers (International Publication Nos. W02012131104 and W02012131106; the contents of each of which is herein incorporated by reference in its entirety).
  • the mRNA may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL@ (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).
  • the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject.
  • the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
  • the mRNA formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the mRNA controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co- L- lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the mRNA controlled release and/or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and/or PEG related polymer derivatives as described in U.S. Patent No. 8,404,222, herein incorporated by reference in its entirety.
  • the mRNA controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in U.S. Patent Publication No. US20130130348, herein incorporated by reference in its entirety.
  • the mRNA of the present disclosure may be encapsulated in a therapeutic nanoparticle, referred to herein as “therapeutic nanoparticles.”
  • Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Publication Nos. W02010005740, W02010030763, W02010005721, W02010005723, WO2012054923, US Patent Publication Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351 and US20130230567 and U.S. Patent Nos.
  • therapeutic polymer nanoparticles may be identified by the methods described in U.S. Publication No. US20120140790, the contents of which is herein incorporated by reference in its entirety.
  • the therapeutic nanoparticles may be formulated for sustained release.
  • sustained release refers to a therapeutic composition or synthetic mRNA that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides of the present disclosure (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, each of which is herein incorporated by reference in their entirety).
  • the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and/or particulate suspensions (see US Patent Publication No US20130150295, the contents of which is herein incorporated by reference in its entirety).
  • the therapeutic nanoparticles may be formulated to be target specific.
  • the therapeutic nanoparticles may include a corticosteroid (see International Publication No. W02011084518; herein incorporated by reference in its entirety).
  • the therapeutic nanoparticles may be formulated in nanoparticles described in International Publication Nos. WO2008121949, W02010005726, W02010005725, WO2011084521 and US Publication Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in their entirety.
  • the nanoparticles of the present disclosure may comprise a polymeric matrix.
  • the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysinc, polyethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy- L-proline ester) or combinations thereof.
  • polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones
  • the therapeutic nanoparticle comprises a diblock copolymer.
  • the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, polyethylene imine), poly(serine ester), poly(L- lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
  • the diblock copolymer may be a high-X diblock copolymer such as those described in International Patent Publication No.
  • the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is herein incorporated by reference in their entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in its entirety).
  • the therapeutic nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in US Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Patent Nos. 8,263,665 and 8,287,910 and US Patent Publication No. US20130195987; the contents of each of which are herein incorporated by reference in its entirety).
  • the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF- betal gene delivery vehicle in Lee et al.
  • Thermosensitive Hydrogel as a Tgf-b f Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel.
  • Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253, herein incorporated by reference in their entirety).
  • the mRNA of the present disclosure may be formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.
  • the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987; the contents of each of which are herein incorporated by reference in its entirety).
  • a multiblock copolymer See e.g., U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Publication No. US20130195987; the contents of each of which are herein incorporated by reference in its entirety).
  • the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer.
  • a polyion complex comprising a non-polymeric micelle and the block copolymer.
  • the therapeutic nanoparticle may comprise at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the therapeutic nanoparticles may comprise at least one poly(vinyl ester) polymer.
  • the poly(vinyl ester) polymer may be a copolymer such as a random copolymer.
  • the random copolymer may have a structure such as those described in International Publication No. WO2013032829 or US Patent Publication No. US20130121954, the contents of which are herein incorporated by reference in its entirety.
  • the poly (vinyl ester) polymers may be conjugated to the polynucleotides described herein.
  • the therapeutic nanoparticle may comprise at least one diblock copolymer.
  • the diblock copolymer may be, but it not limited to, a poly(lactic) acid- poly(ethylene)glycol copolymer (see e.g., International Patent Publication No. WO2013044219; herein incorporated by reference in its entirety).
  • the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
  • the therapeutic nanoparticles may comprise at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (see e.g., U.S. Patent No. 8,287,849; herein incorporated by reference in its entirety) and combinations thereof.
  • the nanoparticles described herein may comprise an amine cationic lipid such as those described in International Patent Application No. WO2013059496, the contents of which are herein incorporated by reference in its entirety.
  • the cationic lipids may have an amino-amine or an amino-amide moiety.
  • the therapeutic nanoparticles may comprise at least one degradable polyester which may contain poly cationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the therapeutic nanoparticles may be formulated using the methods described by Podobinski et al in U.S. Patent No. 8,404,799, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA may be encapsulated in, linked to and/or associated with synthetic nanocarriers.
  • Synthetic nanocarriers include, but are not limited to, those described in International Publication Nos.
  • the synthetic nanocarriers may be formulated by the methods described in International Publication Nos. W02010005740, W02010030763 and W0201213501 and US Publication Nos. US20110262491, US20100104645, US20100087337 and US2012024422, each of which is herein incorporated by reference in their entirety.
  • the synthetic nanocarrier formulations may be lyophilized by methods described in International Publication No. W02011072218 and U.S. Patent No. 8,211,473; the content of each of which is herein incorporated by reference in their entirety.
  • formulations of the present disclosure may be lyophilized or reconstituted by the methods described in U.S. Patent Publication No. US20130230568, the contents of which are herein incorporated by reference in its entirety.
  • the synthetic nanocarriers may contain reactive groups to release the polynucleotides described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety).
  • the synthetic nanocarriers may be formulated for targeted release.
  • the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle may be formulated to release the mRNA after 24 hours and/or at a pH of 4.5 (see International Publication Nos. W02010138193 and W02010138194 and U.S. Publication Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).
  • the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides described herein.
  • the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Publication No. W02010138192 and U.S. Publication No. 20100303850, each of which is herein incorporated by reference in their entirety.
  • the mRNA may be formulated for controlled and/or sustained release wherein the formulation comprises at least one polymer that is a crystalline side chain (CYSC) polymer.
  • CYSC polymers are described in U.S. Patent No. 8,399,007, herein incorporated by reference in its entirety.
  • the synthetic nanocarrier may include at least one adjuvant.
  • the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g, U.S. Patent No. 8,241,610; herein incorporated by reference in its entirety).
  • the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant.
  • the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Publication No. W02011150240 and U.S. Publication No. US20110293700, each of which is herein incorporated by reference in its entirety.
  • the mRNA may be encapsulated in, linked to and/or associated with zwitterionic lipids.
  • zwitterionic lipids Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in its entirety.
  • the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.
  • the mRNA may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in its entirety.
  • lipid nanoparticles comprise the lipid KL52 (an amino-lipid disclosed in U.S. Patent Publication No. 2012/0295832 expressly incorporated herein by reference in its entirety). Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction) of lipid nanoparticle administration may be improved by incorporation of such lipids.
  • Lipid nanoparticles comprising KL52 may be administered intravenously and/or in one or more doses.
  • administration of lipid nanoparticles comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.
  • the mRNA may be delivered using smaller lipid nanoparticles.
  • Such particles may comprise a diameter from below 0.1 um up to 100 nm such as, but not limited to, less than 0.1 um, less than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than
  • the mRNA may be delivered using smaller lipid nanoparticles which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70
  • microfluidic mixers may include, but are not limited to a slit interdigitial micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir. 2012. 28:3633-40; Belliveau, N. M.
  • methods of lipid nanoparticle generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA).
  • MICA microstructure-induced chaotic advection
  • This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating lipid nanoparticles using SHM include those disclosed in U.S. Patent Publication Nos. 2004/0262223 and 2012/0276209, each of which is expressly incorporated herein by reference in their entirety.
  • the mRNA of the present disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging -jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany).
  • a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging -jet (IJMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany).
  • the mRNA of the present disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, herein incorporated by reference in their entirety).
  • controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (See e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, herein incorporated by reference in its entirety).
  • the mRNA of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the mRNA of the disclosure may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, each of which is herein incorporated by reference in its entirety.
  • the microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in its entirety.
  • the amino acid, peptide, polypeptide, and lipids (APPL) are useful in delivering the mRNA of the disclosure to cells as described in International Patent Publication No. WO2013063468, the contents of which is herein incorporated by reference in its entirety.
  • the mRNA of the disclosure may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50
  • the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in its entirety.
  • the limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and l-palmitoyl-2- oleoyl phosphatidylcholine (POPC).
  • POPC l-palmitoyl-2- oleoyl phosphatidylcholine
  • POPC l-
  • the mRNA may be delivered, localized and/or concentrated in a specific location using the delivery methods described in International Patent Publication No. W02013063530, the contents of which are herein incorporated by reference in its entirety.
  • a subject may be administered an empty polymeric particle prior to, simultaneously with or after delivering the mRNA to the subject.
  • the empty polymeric particle undergoes a change in volume once in contact with the subject and becomes lodged, embedded, immobilized or entrapped at a specific location in the subject.
  • the mRNA may be formulated in an active substance release system (See e.g., ET.S. Patent Publication No. ETS20130102545, the contents of which is herein incorporated by reference in its entirety).
  • the active substance release system may comprise 1) at least one nanoparticle bonded to an oligonucleotide inhibitor strand which is hybridized with a catalytically active nucleic acid and 2) a compound bonded to at least one substrate molecule bonded to a therapeutically active substance (e.g., polynucleotides described herein), where the therapeutically active substance is released by the cleavage of the substrate molecule by the catalytically active nucleic acid.
  • a therapeutically active substance e.g., polynucleotides described herein
  • the mRNA may be formulated in a nanoparticle comprising an inner core comprising a non-cellular material and an outer surface comprising a cellular membrane.
  • the cellular membrane may be derived from a cell or a membrane derived from a virus.
  • the nanoparticle may be made by the methods described in International Patent Publication No. WO2013052167, herein incorporated by reference in its entirety.
  • the nanoparticle described in International Patent Publication No. WO2013052167, herein incorporated by reference in its entirety may be used to deliver the mRNA described herein.
  • the mRNA may be formulated in porous nanoparticle-supported lipid bilayers (protocells).
  • Protocells are described in International Patent Publication No. WO2013056132, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA described herein may be formulated in polymeric nanoparticles as described in or made by the methods described in U.S. Patent Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073848B1, the contents of each of which are herein incorporated by reference in their entirety.
  • the polymeric nanoparticle may have a high glass transition temperature such as the nanoparticles described in or nanoparticles made by the methods described in Ei.S. Patent No. 8,518,963, the contents of which are herein incorporated by reference in its entirety.
  • the polymer nanoparticle for oral and parenteral formulations may be made by the methods described in European Patent No. EP2073848B 1, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA described herein may be formulated in nanoparticles used in imaging.
  • the nanoparticles may be liposome nanoparticles such as those described in EiS Patent Publication No US20130129636, herein incorporated by reference in its entirety.
  • the liposome may comprise gadolinium(III) 2- ⁇ 4,7-bis- carboxymethyl- 10-[(N,N-distearylamidomethyl-N-amido-methyl]- 1 ,4,7,10-tetra- azacyclododec-l-yl ⁇ -acetic acid and a neutral, fully saturated phospholipid component (see e.g., US Patent Publication No E1S20130129636, the contents of which is herein incorporated by reference in its entirety).
  • the nanoparticles which may be used in the present disclosure are formed by the methods described in U.S. Patent Publication No. US20130130348, the contents of which is herein incorporated by reference in its entirety.
  • the nanoparticles of the present disclosure may further include nutrients.
  • the nutrient may be iron in the form of ferrous, ferric salts or elemental iron, iodine, folic acid, vitamins or micronutrients.
  • the mRNA of the present disclosure may be formulated in a swellable nanoparticle.
  • the swellable nanoparticle may be, but is not limited to, those described in U.S. Patent No. 8,440,231, the contents of which is herein incorporated by reference in its entirety.
  • the swellable nanoparticle may be used for delivery of the mRNA of the present disclosure to the pulmonary system (see e.g., U.S. Patent No. 8,440,231, the contents of which is herein incorporated by reference in its entirety).
  • the mRNA of the present disclosure may be formulated in polyanhydride nanoparticles such as, but not limited to, those described in U.S. Patent No. 8,449,916, the contents of which is herein incorporated by reference in its entirety.
  • the nanoparticles and microparticles of the present disclosure may be geometrically engineered to modulate macrophage and/or the immune response.
  • the geometrically engineered particles may have varied shapes, sizes and/or surface charges in order to incorporated the polynucleotides of the present disclosure for targeted delivery (see e.g., International Publication No. W02013082111, the contents of which is herein incorporated by reference in its entirety).
  • Other physical features the geometrically engineering particles may have include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge which can alter the interactions with cells and tissues.
  • nanoparticles of the present disclosure may be made by the methods described in International Publication No W02013082111, the contents of which is herein incorporated by reference in its entirety.
  • the nanoparticles of the present disclosure may be water soluble nanoparticles such as, but not limited to, those described in International Publication No. W02013090601, the contents of which is herein incorporated by reference in its entirety.
  • the nanoparticles may be inorganic nanoparticles which have a compact and zwitterionic ligand in order to exhibit good water solubility.
  • the nanoparticles may also have small hydrodynamic diameters (HD), stability with respect to time, pH, and salinity and a low level of non-specific protein binding.
  • the nanoparticles of the present disclosure may be developed by the methods described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.
  • the nanoparticles of the present disclosure are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Patent Publication No. US20130172406; the contents of which is herein incorporated by reference in its entirety.
  • the nanoparticles of the present disclosure may be made by the methods described in U. S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.
  • the stealth or target-specific stealth nanoparticles may comprise a polymeric matrix.
  • the polymeric matrix may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, poly anhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates or combinations thereof.
  • the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high-density nucleic acid layer.
  • the nanoparticle- nucleic acid hybrid structure may made by the methods described in U.S. Patent Publication No. US20130171646, the contents of which are herein incorporated by reference in its entirety.
  • the nanoparticle may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and/or known in the art.
  • At least one of the nanoparticles of the present disclosure may be embedded in the core a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one payload within or on the surface of the nanostructure.
  • a nanostructure or coated with a low density porous 3-D structure or coating which is capable of carrying or associating with at least one payload within or on the surface of the nanostructure.
  • Non-limiting examples of the nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA of the disclosure can be formulated using natural and/or synthetic polymers.
  • polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (PHASERX®, Seattle, Wash ), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.
  • RONDELTM RNAi/Oligonucleotide Nanoparticle Delivery
  • PHASERX® pH responsive co-block polymers
  • a non-limiting example of a chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (ET.S. Publication No. 20120258176; herein incorporated by reference in its entirety).
  • Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA- chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.
  • the polymers used in the present disclosure have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer.
  • the polymer may be processed by methods known and/or described in the art and/or described in International Publication No. WO20 12150467, herein incorporated by reference in its entirety.
  • PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N- methyl-2- pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • PLGA injectable depots e.g., ELIGARD® which is formed by dissolving PLGA in 66% N- methyl-2- pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
  • NMP N- methyl-2- pyrrolidone
  • the first of these delivery approaches uses dynamic poly conjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et ak, Proc Natl Acad Sci USA. 2007 104: 12982-12887, herein incorporated by reference in its entirety).
  • This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N- acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et ah, Proc Natl Acad Sci USA. 2007 104: 12982-12887, herein incorporated by reference in its entirety).
  • the polymer complex On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer.
  • the polymer Through replacement of the N-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells.
  • Another polymer approach involves using transferrin-targeted cyclodextrin- containing polycation nanoparticles.
  • the polymer formulation can permit the sustained or delayed release of polynucleotides.
  • the altered release profile for the polynucleotide can result in, for example, translation of an encoded protein over an extended period of time.
  • the polymer formulation may also be used to increase the stability of the polynucleotide.
  • Biodegradable polymers have been previously used to protect nucleic acids other than polynucleotide from degradation and been shown to result in sustained release of payloads in vivo (Rozema et ah, Proc Natl Acad Sci USA. 2007 104: 12982- 12887; Sullivan et ah, Expert Opin Drug Deliv.
  • the mRNA therapeutic compositions may be sustained release formulations.
  • Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc.
  • the mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process.
  • tunable release rates e.g., days and weeks
  • EVAc are non- biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters).
  • Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5° C and forms a solid gel at temperatures greater than 15° C.
  • PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days.
  • GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.
  • Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et ak, Biomacromolecules. 201 1 12:2708-2714; Rozema et ah, Proc Natl Acad Sci USA. 2007 104: 12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464: 1067-1070, herein incorporated by reference in their entirety).
  • GalNAc N- acetylgalactosamine
  • the mRNA of the disclosure may be formulated with or in a polymeric compound.
  • the polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4- aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic
  • the mRNA of the disclosure may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Patent No. 6, 177,274; herein incorporated by reference in its entirety.
  • the formulation may be used for transfecting cells in vitro or for in vivo delivery of polynucleotides.
  • the polynucleotide may be suspended in a solution or medium with a cationic polymer, in a dry therapeutic composition or in a solution that is capable of being dried as described in U.S. Publication Nos. 20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.
  • the mRNA of the disclosure may be formulated with a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG- PLGA block copolymers (See U.S. Patent No. 6,004,573, herein incorporated by reference in its entirety).
  • the mRNA of the disclosure may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968, herein incorporated by reference in its entirety).
  • a polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Publication No. 20100260817 (now U.S. Patent No. 8,460,696) the contents of each of which is herein incorporated by reference in its entirety).
  • a therapeutic composition may include the mRNA and the polyamine derivative described in U.S. Publication No. 20100260817 (now U.S. Patent No. 8,460,696; the contents of which are incorporated herein by reference in its entirety.
  • the mRNA of the present disclosure may be delivered using a polyamide polymer such as, but not limited to, a polymer comprising a 1,3 -dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Patent No. 8,236,280; herein incorporated by reference in its entirety).
  • the mRNA of the disclosure may be formulated with at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • the mRNA of the present disclosure may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos. WO2011115862, WO2012082574 and WO2012068187 and U.S. Publication No. 20120283427, each of which are herein incorporated by reference in their entireties.
  • the mRNA of the present disclosure may be formulated with a polymer of formula Z as described in International Patent Publication No. WO2011115862, herein incorporated by reference in its entirety.
  • the mRNA may be formulated with a polymer of formula Z, Z 7 or Z ' as described in International Publication Nos. WO2012082574 or WO2012068187 and U.S. Publication No. 2012028342, each of which are herein incorporated by reference in their entireties.
  • the polymers formulated with the modified RNA of the present disclosure may be synthesized by the methods described in International Publication Nos. WO2012082574 or WO2012068187, each of which are herein incorporated by reference in their entireties.
  • the mRNA of the disclosure may be formulated with at least one acrylic polymer.
  • Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.
  • Formulations of the mRNA of the disclosure may include at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters) or combinations thereof.
  • the poly(amine-co- esters) may be the polymers described in and/or made by the methods described in International Publication No. WO2013082529, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA of the disclosure may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross- linked cationic multi-block copolymer or combinations thereof.
  • the biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Patent No.
  • the poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Publication No. 20100004315, herein incorporated by reference in its entirety.
  • the biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Patent Nos.
  • the linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Patent No. 6,652,886.
  • the PAGA polymer may be made using methods known in the art and/or as described in U.S. Patent No. 6,217,912 herein incorporated by reference in its entirety.
  • the PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L4ysine, polyargine, polyomithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides).
  • the biodegradable crosslinked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Patent Nos. 8,057,821, 8,444,992 or U.S. Publication No. 2012009145 each of which are herein incorporated by reference in their entireties.
  • the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
  • LPEI linear polyethyleneimine
  • the composition or therapeutic composition may be made by the methods known in the art, described herein, or as described in U.S. Publication No. 20100004315 or U.S. Patent Nos. 6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.
  • the mRNA of the disclosure may be formulated with at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the mRNA of the disclosure may be formulated with at least one crosslinkable polyester.
  • Crosslinkable polyesters include those known in the art and described in ET.S. Publication No. 20120269761, the contents of which is herein incorporated by reference in its entirety.
  • the mRNA of the disclosure may be formulated in or with at least one cyclodextrin polymer.
  • Cyclodextrin polymers and methods of making cyclodextrin polymers include those known in the art and described in ET.S. Publication No. 20130184453, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA of the disclosure may be formulated in or with at least one crosslinked cation-binding polymers.
  • Crosslinked cation-binding polymers and methods of making crosslinked cation-binding polymers include those known in the art and described in International Patent Publication Nos. WO2013106072, WO2013106073 and
  • the mRNA of the disclosure may be formulated in or with at least one branched polymer.
  • Branched polymers and methods of making branched polymers include those known in the art and described in International Patent Publication No. WO2013113071, the contents of each of which are herein incorporated by reference in its entirety.
  • the mRNA of the disclosure may be formulated in or with at least PEGylated albumin polymer.
  • PEGylated albumin polymer and methods of making PEGylated albumin polymer include those known in the art and described in U.S. Patent Publication No. US20130231287, the contents of each of which are herein incorporated by reference in its entirety.
  • the polymers described herein may be conjugated to a lipid terminating PEG.
  • PLGA may be conjugated to a lipid- terminating PEG forming PLGA-DSPE-PEG.
  • PEG conjugates for use with the present disclosure are described in International Publication No. W02008103276, herein incorporated by reference in its entirety.
  • the polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its entirety.
  • the mRNA disclosed herein may be mixed with the PEGs or the sodium phosphate/sodium carbonate solution prior to administration.
  • polynucleotides encoding the protein of interest may be mixed with the PEGs and also mixed with the sodium phosphate/sodium carbonate solution.
  • the mRNA described herein may be conjugated with another compound.
  • conjugates are described in ET.S. Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
  • the mRNA of the present disclosure may be conjugated with conjugates of formula 1-122 as described in ET.S. Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties.
  • the mRNA described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Joum. Amer. Chem. Soc. 2009 131(6): 2072-2073).
  • the mRNA described herein may be conjugated and/or encapsulated in gold-nanoparticles.
  • the polymer formulation of the present disclosure may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups.
  • the polymer formulation may be contacted with a cationic lipopolymer using the methods described in ET.S. Publication No. 20090042829 herein incorporated by reference in its entirety.
  • the cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycosidepolyamine, dideoxy-diamino-b- cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3- Trimethylammonium-Propane(DOTAP), N-[l- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3- dioleyl
  • the mRNA of the disclosure may be formulated in a polyplex of one or more polymers (See e.g., U.S. Patent No. 8,501,478, U.S. Publication Nos. 20120237565, 20120270927 and 20130149783 and International Patent Publication No. W02013090861; the contents of each of which is herein incorporated by reference in its entirety).
  • the polyplex may be formed using the noval alpha-aminoamidine polymers described in International Publication No. W02013090861, the contents of which are herein incorporated by reference in its entirety.
  • the polyplex may be formed using the click polymers described in U.S. Patent No.
  • the polyplex comprises two or more cationic polymers.
  • the cationic polymer may comprise a polyethylene imine) (PEI) such as linear PEI.
  • PEI polyethylene imine
  • the polyplex comprises p(TETA/CBA) its PEGylated analog p(TETA/CBA)-g-PEG2k and mixtures thereof (see e.g., US Patent Publication No. US20130149783, the contents of which are herein incorporated by reference in its entirety.
  • the mRNA of the disclosure can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
  • Components may be combined in a core-shell, hybrid, and/or layer- by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the polynucleotide, may be enhanced (Wang et ah, Nat Mater. 2006 5:791-796; Fuller et ah, Biomaterials. 2008 29: 1526- 1532; DeKoker et ah, Adv Drug Deliv Rev.
  • the nanoparticle may comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (International Publication No. WO20120225129; the contents of which is herein incorporated by reference in its entirety).
  • hydrophilic-hydrophobic polymers e.g., PEG-PLGA
  • hydrophobic polymers e.g., PEG
  • hydrophilic polymers International Publication No. WO20120225129
  • the nanoparticle comprising hydrophilic polymers for the mRNA may be those described in or made by the methods described in International Patent Publication No. WO2013119936, the contents of which are herein incorporated by reference in its entirety.
  • the biodegradable polymers which may be used in the present disclosure are poly(ether-anhydride) block copolymers.
  • the biodegradable polymers used herein may be a block copolymer as described in International Patent Publication No. W02006063249, herein incorporated by reference in its entirety, or made by the methods described in International Patent Publication No. W02006063249, herein incorporated by reference in its entirety.
  • the biodegradable polymers which may be used in the present disclosure are alkyl and cycloalkyl terminated biodegradable lipids.
  • the alkyl and cycloalkyl terminated biodegradable lipids may be those described in International Publication No. WO2013086322 and/or made by the methods described in International Publication No. WO2013086322; the contents of which are herein incorporated by reference in its entirety.
  • the biodegradable polymers which may be used in the present disclosure are cationic lipids having one or more biodegradable group located in a lipid moiety.
  • the biodegradable lipids may be those described in U.S. Patent Publication No. US20130195920, the contents of which are herein incorporated by reference in its entirety.
  • Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver polynucleotides in vivo.
  • a lipid coated calcium phosphate nanoparticle which may also contain a targeting ligand such as anisamide, may be used to deliver the mRNA of the present disclosure.
  • a targeting ligand such as anisamide
  • a lipid coated calcium phosphate nanoparticle was used (Li et ah, J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012 158: 108-114; Yang et ah, Mol Then 2012 20:609-615, herein incorporated by reference in their entirety).
  • This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
  • calcium phosphate with a PEG-polyanion block copolymer may be used to deliver the mRNA (Kazikawa et al., J Contr Rel. 2004 97:345- 356; Kazikawa et ah, J Contr Rel. 2006 111 :368-370, herein incorporated by reference in their entirety).
  • a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114, herein incorporated by reference in their entirety) may be used to form a nanoparticle to deliver the mRNA of the present disclosure.
  • the PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
  • a polymer used in the present disclosure may be a pentablock polymer such as, but not limited to, the pentablock polymers described in International Patent Publication No. WO2013055331, herein incorporated by reference in its entirety.
  • the pentablock polymer comprises PGA-PCL-PEG-PCL-PGA, wherein PEG is polyethylene glycol, PCL is poly(E-caprolactone), PGA is poly(glycolic acid), and PLA is poly(lactic acid).
  • the pentablock polymer comprises PEG-PCL-PLA-PCL-PEG, wherein PEG is polyethylene glycol, PCL is poly(E- caprolactone), PGA is poly(glycolic acid), and PLA is poly(lactic acid).
  • a polymer which may be used in the present disclosure comprises at least one diepoxide and at least one aminoglycoside (See e.g., International Patent Publication No. WO2013055971, the contents of which are herein incorporated by reference in its entirety).
  • the di epoxide may be selected from, but is not limited to, 1,4 butanediol diglycidyl ether (l,4B),l,4-cyclohexanedimethanol diglycidyl ether (1,4C),4- vinylcyclohexene diepoxide (4VCD), ethyleneglycol diglycidyl ether (EDGE), glycerol diglycidyl ether (GDE), neopentylglycol diglycidyl ether (NPDGE), poly(ethyleneglycol) diglycidyl ether (PEGDE), poly(propyleneglycol) diglycidyl ether (PPGDE) and resorcinol diglycidyl ether (RDE).
  • 1,4 butanediol diglycidyl ether l,4B
  • EDGE ethyleneglycol diglycidyl ether
  • GDE glycerol diglycidyl ether
  • the aminoglycoside may be selected from, but is not limited to, streptomycin, neomycin, framycetin, paromomycin, ribostamycin, kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin, spectinomycin, hygromycin, gentamicin, netilmicin, sisomicin, isepamicin, verdamicin, astromicin, and apramycin.
  • the polymers may be made by the methods described in International Patent Publication No. WO2013055971, the contents of which are herein incorporated by reference in its entirety.
  • a polymer which may be used in the present disclosure may be a cross- linked polymer.
  • the cross-linked polymers may be used to form a particle as described in U.S. Patent No. 8,414,927, the contents of which are herein incorporated by reference in its entirety.
  • the cross-linked polymer may be obtained by the methods described in U.S. Patent Publication No. US20130172600, the contents of which are herein incorporated by reference in its entirety.
  • a polymer which may be used in the present disclosure may be a cross-linked polymer such as those described in U.S. Patent No. 8,461,132, the contents of which are herein incorporated by reference in its entirety.
  • the cross-linked polymer may be used in a therapeutic composition for the treatment of a body tissue.
  • the therapeutic composition may be administered to damaged tissue using various methods known in the art and/or described herein such as injection or catheterization.
  • a polymer which may be used in the present disclosure may be a dialiphatic substituted pegylated lipid such as, but not limited to, those described in International Patent Publication No. WO2013049328, the contents of which are herein incorporated by reference in its entirety.
  • a block copolymer is PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a gene delivery vehicle in Lee et al. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery System Based on Thermo sensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253), herein incorporated by reference in their entirety, may be used in the present disclosure.
  • the PEG-PLGA-PEG block copolymer is used in the present disclosure to develop a biodegradable sustained release system.
  • the mRNA of the present disclosure is mixed with the block copolymer prior to administration.
  • the mRNA of the present disclosure is co-administered with the block copolymer.
  • the polymer used in the present disclosure may be a multi-functional polymer derivative such as, but not limited to, a multi-functional N-maleimidyl polymer derivatives as described in U.S. Patent No. 8,454,946, the contents of which are herein incorporated by reference in its entirety.
  • core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et ah, Proc Natl Acad Sci USA. 2011 108: 12996-13001, herein incorporated by reference in its entirety).
  • the complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle.
  • a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of the mRNA of the present disclosure.
  • a luciferease-expressing tumor it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated by reference in its entirety).
  • the lipid nanoparticles may comprise a core of the mRNA disclosed herein and a polymer shell.
  • the polymer shell may be any of the polymers described herein and are known in the art.
  • the polymer shell may be used to protect the polynucleotides in the core.
  • Core-shell nanoparticles for use with the mRNA of the present disclosure are described and may be formed by the methods described in U.S. Patent No. 8,313,777 or International Patent Publication No. WO2013124867, the contents of each of which are herein incorporated by reference in their entirety.
  • the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in International Publication No. WO2011 120053, the contents of which are herein incorporated by reference in its entirety.
  • the formulation may be a polymeric carrier cargo complex comprising a polymeric carrier and at least one nucleic acid molecule.
  • Non-limiting examples of polymeric carrier cargo complexes are described in International Patent Publications Nos. WO2013113326, WO2013113501, WO2013113325, WO2013113502 and WO2013113736 and European Patent Publication No. EP2623121, the contents of each of which are herein incorporated by reference in their entireties.
  • the polymeric carrier cargo complexes may comprise a negatively charged nucleic acid molecule such as, but not limited to, those described in International Patent Publication Nos. WO2013113325 and WO2013113502, the contents of each of which are herein incorporated by reference in its entirety.
  • a therapeutic composition may comprise the mRNA of the disclosure and a polymeric carrier cargo complex.
  • the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in International Publication No. WO2011 120053, the contents of which are herein incorporated by reference in its entirety.
  • the synthetic mRNA or therapeutic compositions described may find use in inducing maturation of oocytes.
  • a method is provided for inducing maturation in an oocyte.
  • the method comprises contacting the oocyte with a synthetic mRNA or therapeutic composition described herein.
  • an “oocyte” refers to a female germ cell arrested in prophase of meiosis I of oogenesis.
  • “Maturation,” as used herein in reference to an oocyte, refers to the oocyte attaining the competence to be fertilized and undergo embryogenesis. Maturation allows the formation of an oocyte into an “ovum,” or egg.
  • An “ovum,” as used herein, is a cell that can be fertilized to produce a normal embryo, and, in typical aspects, is arrested in metaphase of meiosis II. An oocyte and ovum are also distinct in several ways.
  • An oocyte is found in the ovary in vivo, has an intact nucleus, an interphase microtubule network, and minimal pervitelline space.
  • an ovum is found in the oviduct in vivo, lacks a nucleus, has a visible first polar body and an enlarged perivitelline space in its vicinity, and have a meiotic spindle.
  • the oocyte as used in the methods herein may be of any mammalian origin.
  • the oocyte is selected from a human oocyte, a bovine oocyte, a porcine oocyte, an equine oocyte, a canine oocyte, a feline oocyte, a murine oocyte (such as a mouse or rate oocyte), an ovine oocyte, and a non-human primate oocyte.
  • an oocyte encompasses an oocyte devoid of companion cells or inclusive of companion cells.
  • the oocyte may be a denuded oocyte where the somatic cell layers (e.g., cumulus cells) that surround the oocyte have been removed.
  • the oocyte may also be part of a follicle, or may be part of a cumulus oocyte complex (COC) in which the cumulus vestments remain intact.
  • COC cumulus oocyte complex
  • the synthetic mRNA and therapeutic compositions thereof may find use in in vitro maturation (IVM) methods.
  • IVM in vitro maturation
  • the oocyte needs to be obtained from a subject.
  • An oocyte can be harvested or collected from an ovary according to standard techniques long known in the art. For example, see Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003, supra).
  • Most oocyte collection techniques involve the insertion of an aspirating needle into an ovarian follicle using transvaginal ultrasound.
  • the aspirating needle is connected by tubing to a material collection trap and the collection trap, in turn, is connected to a suction source such as a manually operated syringe or an electromechanical vacuum source.
  • Oocytes are typically isolated from multiple follicles. As such, harvested oocytes represent a heterogeneous population with regard to their maturity and therefore developmental potential.
  • the oocytes may be primed (such as with follicle-stimulating hormone (FSC) or human chorionic gonadotrophin (hCG)) prior to retrieval. However, in other aspects, priming may not be necessary. In some aspects, the oocytes are classified depending on their condition, with the best oocytes chosen to be maturated.
  • FSC follicle-stimulating hormone
  • hCG human chorionic gonadotrophin
  • priming may not be necessary.
  • the oocytes are classified depending on their condition, with the best oocytes chosen to be maturated.
  • the oocyte is contacts with the synthetic mRNA or therapeutic composition described herein while present in a culture medium.
  • the base medium to which the synthetic mRNA or therapeutic composition thereof is added may be any medium which supports and maintains the viability of an oocyte cultured in the medium in vitro.
  • a suitable base medium for example may include, but is not limited to, Tissue Culture Medium 199 (also known as Media 199, TCM199, and M199) (ThermoFisher Scientific), Minimum Essential Medium Eagle (also known as Eagles' Minimum Essential Medium, EMEM and MEM)(Sigma-Aldrich), Minimum Essential Medium Eagle Alpha Modifications (also known as a-MEM)(Sigma-Aldrich), Dulbecco's Modified Eagle Medium (also known as DMEM or D-MEM)(ThermoFisher Scientific), Ham's F12 (also known as F-12 Ham, Ham's F12 Medium, and F12 Nutrient Mixture)(ThermoFisher Scientific), RPMI Medium 1640 (ThermoFisher Scientific), Isocove's Modified Dulbecco's Medium (also known as IMDM)(ThermoFisher Scientific), Waymouth's MB 752/1 Medium (also known as Waymouth or Waymouth Medium)(Sigma-
  • base media such as ART-1600-B medium.
  • ART-1600-B medium for the maturation of human oocytes, for example, companies such as Origio (Denmark) provide appropriate base media, such as ART-1600-B medium.
  • other base media are contemplated, as would be understood by a person skilled in the art, provided that the base media supports and maintains the viability of an oocyte cultured in the medium in vitro.
  • the base medium to which the synthetic mRNA or therapeutic composition thereof is added may also be supplemented with additional components.
  • additional components include, but are not limited to, inorganic ions (such as cations and anions, e.g., Na + , K + , Mg 2+ , Ca 2+ , Cl", SO4 2 ", PO4 2 ", and HCO3 ), energy substrates (such as glucose, lactate, pyruvate, amino acids), nitrogen sources (such as essential and non-essential amino acids), vitamins, fatty acids or precursors, nucleic acid precursors, chelators (such as EDTA), antioxidants, proteins or macromolecules (such as HSA and hyaluronate), other growth factors or hormones (such as insulin-transferrin-selenium, insulin-like growth factor, epidermal growth factor, follicle stimulating hormone and
  • the method comprises maturing the oocyte into an ovum using the synthetic mRNA or therapeutic composition thereof as described herein.
  • the method comprises fertilizing the ovum with a sperm to form a zygote.
  • fertilizing the ovum with the sperm comprises contacting the ovum with the sperm in a culture medium. In other aspects, fertilizing the ovum comprises intracytoplasmic injection of the sperm into the ovum.
  • the oocyte is collected from an ovary of a first subject.
  • the first subject from which the oocyte is obtained may be an otherwise healthy subject but for their reduced capacity or inability to conceive and/or carry a pregnancy to term.
  • the first subject may be aged and/or obese, thereby also having a reduced capacity or inability to conceive and/or carry a pregnancy to term due to the effect of age and/or obesity on oocyte developmental competence.
  • An aged subject would be considered a subject who is older than the peak fertility age range of the particular species.
  • an otherwise healthy human subject would be expected to have a peak fertility age range between 23 and 35, and thus an aged human subject would be considered a subject older than 35.
  • the peak fertility age range in humans may vary depending on a number of factors, such as genetic background.
  • assisted reproductive technology as used throughout the specification is to be understood to mean any laboratory or clinical technology applied to isolated gametes (oocyte/ovum or sperm) and/or embryos for the purposes on reproduction.
  • Such technologies include in vitro fertilization (IVF; aspiration of an oocyte/ovum, fertilization in the laboratory and transfer of the embryo into a recipient), gamete intrafallopian transfer (GIFT; placement of oocyte/ovum and sperm into the fallopian tube), zygote intrafallopian transfer (ZIFT; placement of fertilized ovum into the fallopian tube), tubal embryo transfer (TET; the placement of cleaving embryos into the fallopian tube), peritoneal oocyte and sperm transfer (POST; the placement of ovum and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA); or any other in vitro technique for producing embryos in humans and/or animals, such as nuclear transfer, parthenogenic activation, embryonic stem cell production, and the use of totipotent cells.
  • IVF in vitro fertilization
  • the assisted reproductive technology comprises in vitro fertilization (IVF).
  • IVF in vitro fertilization
  • IVF relates to the fertilization of an ovum in vitro, wherein an oocyte is isolated from the subject and matured into an ovum as described herein to allow fertilization.
  • methods are well known in the art for collecting oocytes from suitable females and fertilizing in vitro. It is contemplated that fertilization will ideally occur no less than 24 hours, but no later than 60 hours, after collection and maturation as described herein.
  • the sperm may be incubated with the ovum for a period of between 1 to 60 hours.
  • the sperm may be transported into the ovum by a technique called intracytoplasmic sperm injection (ICSI).
  • ICSI intracytoplasmic sperm injection
  • the assisted reproductive technology or the method of assisted reproduction is ICSI.
  • the collected sperm may be maintained in a medium prior to fertilization.
  • a suitable medium would be known in the art and is set out in standard texts, such as the Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003, supra).
  • the medium containing the sperm may be of a constitution so as to minimize any stress placed on the ovum when transferred from the maturation medium to the medium containing the sperm.
  • the medium housing the sperm may have a similar or identical composition of ions and non-essential amino acids as the maturation medium.
  • a suitable medium in which this is conducted i.e. a fertilization medium
  • the fertilization medium may be of a constitution so as to promote sperm function and fertilization.
  • the fertilization medium may comprise an elevated concentration of sodium and/or phosphate compared to the maturation medium.
  • the fertilization medium may also be supplemented with carbohydrates such as glucose, lactate and pyruvate.
  • Specific formulations may involve supplementation of the medium with one or more of bicarbonate, glutathione to promote sperm head decondensation, non-essential amino acids, HSA, hyaluronate, and antibiotics such as penicillin and streptomycin.
  • the collected sperm may be transferred directly into the maturation medium which contains the ovum (for in vitro fertilisation) or may be injected directly into an ovum that is present in the maturation medium (for ICSI).
  • an ICSI medium that can be used to culture the oocyte and can also serve as a carrier for the sperm as it is transported by injection into the ovum.
  • the ICSI medium should preferably be highly compatible with the interior and exterior of the ovum.
  • the ICSI media may be a base medium as described above and may comprise ionic constituents similar to those found in the oocyte maturation medium described herein.
  • phosphate may be omitted to avoid metabolic and homeostatic stress on the ovum. Because ICSI is a clinical procedure performed outside the incubator, a buffering system that is effective in a normal atmosphere is typically used.
  • MOPS and HEPES are accordingly preferred buffers for the ICSI medium. Because the cumulus cells have been removed from the ovum, and the sperm is at the conclusion of its independent life, glucose (the main energy source for cumulus cells and sperm, but not the ovum) may be omitted from the ICSI medium. In order to nourish the ovum, non-essential amino acids most abundant in the ovum (e.g. glycine, proline, serine and taurine) and glutamine can be included in the ICSI medium to avoid osmotic and pH stress.
  • the ICSI medium may also include hyaluronate or polyvinylpyrollidone (PVP) to immobilize or slow the sperm so that they may be captured in the ICSI pipette.
  • PVP polyvinylpyrollidone
  • the zygote may be incubated in a medium which supports development of the embryo (an embryo culture medium).
  • the embryo culture medium may be a base medium as described above and may comprise ionic constituents similar to those found in the oocyte maturation medium.
  • the embryo culture medium may comprise EDTA which is believed to bind and disable toxins that may be deleterious to the early embryo.
  • the embryo culture medium may also comprise HSA and hyaluronate.
  • alanyl-glutamine may be substituted for glutamine to reduce ammonium build up within the medium.
  • the first subject is a human.
  • the oocyte is a human oocyte.
  • the method may further comprise implanting the zygote or an embryo formed therefrom within a uterus of a second subject.
  • the second subject may be the same individual as or a different individual from the first subject.
  • the first subject exhibits oocyte maturation arrest. In some aspects, the oocyte maturation arrest is the result of a genetic anomaly or disorder. In some aspects, the first subject has polycystic ovary syndrome. In some aspects, the first subject exhibits high antral follicle counts. In some aspects, the first subject exhibits ovarian hyperresponsiveness. In some aspects, the first subject may have empty follicle syndrome (EFS).
  • EFS empty follicle syndrome
  • the active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result.
  • the exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the medical disorder, the particular active ingredient, its mode of administration, its mode of activity, and the like.
  • the active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • Useful dosages of the active agents and therapeutic compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary and can be administered in one or more dose administrations daily, for one or several days.
  • a synthetic mRNA comprising a coding region encoding a protein involved in oocyte maturation or a variant thereof that is 80% or more homologous, wherein the synthetic mRNA further comprises at least one chemical modification to a nucleotide.
  • Aspect 2 The synthetic mRNA of aspect 1, wherein the protein involved in oocyte maturation is selected from SYCP3, TRIP13, MCM8, STAG3, PATL2, TUBB8, AURKC, and WEE2.
  • Aspect 3 The synthetic mRNA of aspect 1 or aspect 2, wherein the protein involved in oocyte maturation is PATL2.
  • Aspect 4 The synthetic mRNA of aspect 3, wherein the coding region comprises an RNA sequence encoded by a gene selected from Table 1, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide in the RNA sequence has been chemically modified.
  • the synthetic mRNA of aspect 1, wherein the coding region comprises an RNA sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide the sequence has been chemically modified.
  • Aspect 6 The synthetic mRNA of aspect 1, wherein the coding region comprises a sequence of SEQ ID NO: 7, or a variant thereof that is 80% or more homologous, wherein at least one nucleotide the sequence has been chemically modified.
  • Aspect 7 The synthetic mRNA of aspect 1, wherein the coding region encodes a polypeptide having a sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, or a variant thereof that is 80% or more homologous.
  • Aspect 9 The synthetic mRNA of any one of aspects 1-8, wherein the at least one chemical modification of a nucleoside comprises: N4-acetylcytidine triphosphate (N4- acetyl-CTP), inosine triphosphate (ITP), 5-methylcytidine-5’-triphosphate (5-methyl-CTP), pseudouridine-5’ -triphosphate (Pseudo-UTP), Nl-methyladenosine-5’ -triphosphate (Nl- methyl-ATP), N1 -methyl -pseudouridine-5’ -triphosphate (Nl-methylpseudo-UTP), 2- thiouridine-5’ -triphosphate (2-Thio-UTP), 5-methoxyuridine-5’ -triphosphate (5-Methoxy- UTP), 7-methylguanosine, 2 ’-O-7-m ethylguanosine, or combinations thereof.
  • Aspect 10 The synthetic m
  • Aspect 11 The synthetic mRNA of aspect 10, wherein the 5’ UTR comprises a sequence selected from SEQ ID NO: 16 to SEQ ID NO: 33, or a variant thereof that is 80% or more homologous.
  • Aspect 12 The synthetic mRNA of aspect 10 or aspect 11, wherein the 5’ UTR is operably linked to a 5’ end of the coding region.
  • Aspect 13 The synthetic mRNA of any one of aspects 1-12, further comprising a human-derived or synthetic 3’ untranslated region (3’ UTR).
  • Aspect 14 The synthetic mRNA of aspect 13, wherein the 3’ UTR comprises a sequence selected from SEQ ID NO: 34 to SEQ ID NO: 51, or a variant thereof that is 80% or more homologous.
  • Aspect 15 The synthetic mRNA of aspect 13 or aspect 14, wherein the 3’ UTR is operably linked to a 3’ end of the coding region.
  • Aspect 16 The synthetic mRNA of any one of aspects 1-15, further comprising a 5’ terminal cap operably linked to a 5’ end of the mRNA.
  • Aspect 17 The synthetic mRNA of aspect 16, wherein the 5’ terminal cap has a cap-1 structure.
  • Aspect 18 The synthetic mRNA of any one of aspects 1-17, further comprising a 3’ poly(A) tail operably linked to a 3 ’ end of the mRNA.
  • a therapeutic composition comprising a synthetic mRNA of any one of aspects 1-18 formulated within a delivery vehicle.
  • Aspect 20 The therapeutic composition of aspect 19, wherein the delivery vehicle is a liposome, a lipoplex, a lipid nanoparticle, a polymer, or a polymeric nanoparticle.
  • a method of inducing maturation in an oocyte comprising contacting the oocyte with a synthetic mRNA of any one of aspects 1-18 or a therapeutic composition of any one of aspects 19-20.
  • a method of fertilization comprising: a) contacting an oocyte with a synthetic mRNA of any one of aspects 1-18 or a therapeutic composition of any one of aspects 19-20, whereupon the oocyte undergoes maturation into an ovum; b) fertilizing the ovum with a sperm to form a zygote.
  • Aspect 23 The method of aspect 22, wherein fertilizing the ovum with the sperm comprises contacting the ovum with the sperm in a culture medium.
  • Aspect 24 The method of aspect 22, wherein fertilizing the ovum comprises intracytoplasmic injection of the sperm into the ovum.
  • Aspect 25 The method of any one of aspects 21-24, wherein the oocyte is collected from an ovary of a first subject.
  • Aspect 26 The method of aspect 25, wherein the first subject is a human.
  • Aspect 27 The method of any one of aspects 21-26, wherein the oocyte is a human oocyte.
  • Aspect 28 The method of any one of aspects 25 or 26, further comprising implanting the zygote or an embryo formed therefrom within a uterus of a second subject.
  • Aspect 29 The method of aspect 28, wherein the second subject is the same as the first subject.
  • Aspect 30 The method of any of aspects 25 or 26, wherein the first subject exhibits oocyte maturation arrest.
  • the non-limiting exemplary approach described herein involves generating in vitro transcribed Patl2 without RNA modifications (unmodified), and with combinations of RNA modifications (modified).
  • In vitro transcribed unmodified and modified Patl2 are microinjected into GV oocytes, and rates of oocyte maturation are measured.
  • In vitro fertilization can be performed and embryo development assessed of oocytes injected with unmodified and modified Patl2, and the live birth rates tested following embryo transfer.
  • Combinations of RNA modifications can enhance oocyte maturation due to increased translatability and RNA stability
  • RNA modifications with single types of RNA modifications or multiple types of RNA modifications are microinjected (31 different combinations).
  • Patl2 modified with inosine only vs Patl2 modified with a combination of inosine and pseudouridine.
  • Combinations that promote oocyte maturation are labeled as “active” and further tested.
  • By altering only the RNA modifications it is possible to study RNA modifications in isolation, without confounding factors within the synthetic transcript.
  • Patl2 deficient mouse model was purchased Patl2 FL/FL mice from the EUCOMM Consortium. After crossing to CMVcre+ recombinase mice, the Patl2 gene is truncated at exon 7, so even if a protein product is generated from the modified gene, it would be deficient in the topoisomerase Il-associated protein PAT1 domain and not be functional.
  • the resulting mice (Patl2fl/fl:CMVCre+ ) here are called Patl2-/- mice. This same Patl2 deficient mouse model was previously reported by Christou-Kent et al., who demonstrated female infertility due to meiotic arrest in oocytes.
  • GV oocyte collection' Cumulus-oocyte-complexes are released from large antral follicles into flushing and handling medium (FHM) supplemented with 4 mg/ml BSA (mFHM) and 2.5 pM milrinone to prevent germinal vesicle breakdown using previously established procedures.
  • FHM flushing and handling medium
  • mFHM BSA
  • milrinone 2.5 mg/ml milrinone to prevent germinal vesicle breakdown using previously established procedures.
  • GV oocytes within enclosed cumulus complexes undergo oocyte microinjection, followed by IVM and are collected at 16 hours after Milrinone removal at Mil stage. Confirmation of Mil stage is performed using live cell imaging documented polar body extrusion using Hoechst 33342 imaging. Oocytes at all stages GV, MI, Mil are quantified.
  • Oocyte microinjection' Synthetic mRNA is injected into 40 GV oocytes for each injection paradigm outlined below. Injections are performed on an inverted Nikon Eclipse TE2000-S with a Sutter microinjection system and 0.3 mm ID EggJek microinjection needles (MicroJek, Kansas City, KS). GV oocytes are allotted into three groups: noninjected, injected with either unmodified or modified synthetic mRNA. Oocytes are transferred to Terasaki plates and cultured for 4-5 hr in KSOMaa supplemented with milrinone to maintain GV arrest and to allow for protein expression to occur.
  • GV oocytes are then washed and cultured without milrinone in a live cell EVOS culture system to monitor viability until they reach the Mil stage. After incubation, oocytes are washed in milrinone- free M16 medium, and cultured for 3 hours to observe meiotic resumption (GVBD) and 14 hours to detect the first polar body extrusion.
  • GVBD meiotic resumption
  • Fluorescent signals are imaged and analyzed using QuantStudioTM Absolute Q software. We use the average abundance of Patl2 from 40 GV oocytes to inform the amount of in vitro transcribed Patl2 to microinject into Patl2-/- GV oocytes.
  • Patl2 mRNA therapeutic are generated by first designing a synthetic dsDNA geneBlock (IDT) that contains a T7 promoter at the 5’ end Patl2 mRNA.
  • IVT reaction is carried out using T7 RNA polymerase (NEB) and either unmodified or in combination with modified nucleotides spiked at a molar ratio of 1 :20 (5% modified) to generate RNA that are 10% modified.
  • NEB T7 RNA polymerase
  • a 5% incorporation rate is a standard in RNA therapeutics applications.
  • the modified nucleotides (Jena Biosciences) used in the IVT reactions are; inosine, N4-acetylcytidine, N6- methyladenosine, 5-methylcytidine, and pseudouridine.
  • a 7- methylguanosine (m7G) Cap-1 structure is added to the IVT using the Vaccinia Capping System (NEB) in combination with mRNA Cap 2'-O-Methyltransferase (NEB).
  • the Cap-1 structure has been demonstrated to enhance translation and reduce innate cellular immune response in eukaryotic cells.
  • IVT reactions a poly(A) tail are added using poly(A) polymerase (NEB).
  • RNA is assessed for purity (UV spectrophotometry) and size (electrophoresis) followed by HPLC size selection purification.
  • sequence composition of the final purified and size-selected mRNA is verified by next generation sequencing (iSeq 100, Illumina). Additionally, the proportion of modifications and position of modifications, and poly(A) tail length are determined by direct RNA sequencing (Oxford Nanopore). After verification of mRNA purity and composition final Patl2 RNA therapies are stored at -80°C.
  • Patl2-/- GV oocytes are micro-injected with an unmodified fluorescent reporter Patl2 mRNA (Patl2- eGFP) at physiological abundance, as determined above, to allow the monitoring of adequate PATL2 translation following microinjection.
  • Unmodified Patl2 mRNA fused to eGFP is microinjected into 40 different Patl2-/- GV oocytes and incubated in milrinone in a live cell EVOS culture system to monitor eGFP fluorescence for 24 hours to determine the time at which maximal translation occurs (eGFP fluorescence). This time is the incubation period used in all subsequent experiments. Identify in vitro unmodified and modified Patl2 molecules that promote oocyte maturation.
  • Microinjected Patl2-/- GV oocytes are in vitro matured and assessed for standard oocyte quality measurements. Briefly, groups of 20 oocytes are cultured in mini-drops of M16 medium (M7293; Sigma-Aldrich) covered in mineral oil (M5310; Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere. Eggs are fixed, labeled for tubulin, f- actin, and DNA, and imaged by an EVOS imaging system to assess meiotic maturation (metaphase-II spindle and polar body extrusion) as previously described. All eggs are collected at the end of the oocyte maturation assay to test how the microinjected Patl2 mRNA therapeutic corresponds to translated PATL2 and Patl2 mRNA therapeutic abundance.
  • M16 medium M7293; Sigma-Aldrich
  • mineral oil M5310; Sigma-Aldrich
  • RNA modifications on fertility is tested by measuring live birth rates of microinjected oocytes that undergo in vitro maturation, fertilization, embryo culture, and blastocyst transfer to recipient moms.
  • Patl2-/- GV oocytes microinjected with unmodified or modified Patl2 undergo in vitro maturation, are in vitro fertilized and cultured until the blastocyst stage. Briefly, cauda epididymal sperm from C57B1/6J adult males is released into mTyrode’s solution containing 4 mg/mL BSA. Capacitation is performed for 90 minutes in a swim up column of mTyrodes at 37°C.
  • Groups of hyaluronidase treated Mil eggs are transferred to an IVF culture dish with 500 pL KSOMaa media with 4 mg/mL BSA under mineral oil. Capacitated sperm (2xl0 5 / mL) are added for 4.5 hours, after which inseminated eggs are washed in KSOMaa. Embryos are cultured, and blastocysts are transferred into pseudopregnant females at 2.5 days postcoitus, and in vivo developmental potential measured.
  • RNA-seq short-read Illumina RNA-seq of early embryos that were microinjected with RNA therapeutic along with 4-thiouridine, which labels de novo transcription by the generation of T-to-C mismatches, thus allowing for differentiation of the embryonic genome from the abundant maternal transcriptome.
  • This method called SLAM-seq, is a similar approach to the identification of inosine RNA modifications, which appear as A-to-G mismatches.
  • embryonic genome activation is rapid and transient, beginning at the 1 -cells stage, and continuing during the early and late 2-cell stage, we collect embryos at 1-cell, early 2- cell, late 2-cell, 4-cell, and morula, thus fully encompassing embryonic genome activation.
  • rates of cleavage, and embryos at specific stages are picked off for RNA isolation.
  • Patl2-/- GV oocytes are microinjected with unmodified or modified Patl2 mRNA therapeutic, undergo in vitro fertilization, and are cultured to blastocyst stage. Embryos that fail to progress, exhibit excessive blastomere fragmentation, or lose cellular morphology during in vitro culture are counted as arrested and removed from the culture. Blastocyst stage embryos are transferred to pseudopregnant female mice, and the number of born pups are recorded.
  • RNA modifications in Patl2 mRNA therapeutic on embryonic genome activation are unable to directly distinguish between maternal RNA and embryonic RNA following embryo genome activation.
  • SLAM- seq an approach that uses thiol-(SH)-linked alkylation of RNA for metabolic labeling sequencing, was developed to label newly synthesized RNA within a cell. De novo transcription is labeled through the incorporation of a modified ribonucleotide base, 4- thiouridine (4sU).
  • 4sU 4- thiouridine
  • the reverse transcription step causes misincorporation of cytosine (C) at 4sU bases within the RNA.
  • SLAM-seq has been adopted to mouse single cells, (scSLAM-seq), as well as early embryonic development in zebrafish 33,34.
  • scSLAM-seq mouse single cells
  • Patl2-/- GV oocytes are coinjected with 4-thiouridine and unmodified or modified Patl2 mRNA therapeutic.
  • zona pellucida are removed using acidified Tyrode’s buffer, followed by a brief wash in M2 medium.
  • Embryos are directly lysed in reverse transcription buffer using a Low-input Ovation Solo Total RNA sequencing kit (Tecan) with ERCC spike-in. RNA sequencing is performed using our NextSeq 2000 system.
  • Raw reads are processed to identify T-to-C substitutions in RNA utilizing our previously published approach. Briefly our analysis pipeline is as follows: quality filtered and trimmed reads are aligned to a dbSNP annotated reference genome with ERCC spike in contigs using HISAT2. The elprep software is then used to identify high confidence mismatches between the genome reference and cDNA using known genetic sites of variation identified multiple mouse strains from the Mouse Genomes Project database, Wellcome Trust Sanger Institute mouse strains. Further filtering is performed to remove low converge mismatches.
  • Ensembl Variant Effect Predictor is used to assign mismatches to the reference transcriptome and ERCC spike-in.: Nucleotide substitution positions identified from RNA-seq undergo Wilcoxon test, with Bonferroni-Dunn corrected p value ⁇ 0.5 retained for further analysis.
  • compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims.
  • Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims.
  • other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited.
  • a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

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Abstract

La présente divulgation concerne un ARNm synthétique, une région de codage codant pour une protéine impliquée dans la maturation des ovocytes ou un variant de celle-ci. La divulgation concerne également des procédés d'utilisation de l'ARNm synthétique décrit dans la maturation des ovocytes ou la fécondation in vitro.
PCT/US2024/021708 2023-03-27 2024-03-27 Agents thérapeutiques d'arnm pour la maturation d'ovocytes Ceased WO2024206465A2 (fr)

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