WO2013078199A2 - Méthodes pour une administration in vivo améliorée d'arn synthétiques modifiés - Google Patents

Méthodes pour une administration in vivo améliorée d'arn synthétiques modifiés Download PDF

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Publication number
WO2013078199A2
WO2013078199A2 PCT/US2012/066055 US2012066055W WO2013078199A2 WO 2013078199 A2 WO2013078199 A2 WO 2013078199A2 US 2012066055 W US2012066055 W US 2012066055W WO 2013078199 A2 WO2013078199 A2 WO 2013078199A2
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Prior art keywords
unit dose
synthetic
modified
body weight
per
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PCT/US2012/066055
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WO2013078199A3 (fr
Inventor
Derrick Rossi
Morag Stewart
Pankaj MANDAL
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Children's Medical Center Corporation
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Publication of WO2013078199A3 publication Critical patent/WO2013078199A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • 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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the field of the invention relates to unit dose compositions comprising synthetic, modified RNAs and methods of delivery thereof.
  • Methodologies for deliverying pharmaceutical compositions comprising nucleic acids in order to achieve effective protein expression for therapeutics and bioprocessing applications can be problematic.
  • introduced DNA can integrate into host cell genomic DNA and/or can be inherited by daughter cells.
  • multiple steps occur after delivery but before the encoded protein is made, which can impact protein expression, including, for example, transportation into the nucleus for transcription into RNA, followed by transcribed RNA entering the cytoplasm where it is translated into protein.
  • expression levels and rates of expression can vary.
  • effective delivery and achievement of therapeutically relevant levels of proteins for a time sufficient to produce clinical results can be problematic.
  • the present invention provides novel unit doses of synthetic, modified
  • RNAs for optimizing protein expression from delivered pharmaceutical modalities provide, in part, novel unit doses of synthetic, modified RNAs for intramuscular administration.
  • the inventors have discovered particularly effective unit dosages for intramuscular administration of synthetic modified RNAs using a combination of a synthetic modified RNAs and a cationic lipid.
  • the RNAs provided using the dosages as provided have been shown to provide a dose- dependent response both in terms of protein expression and function.
  • unit dose compositions comprising at least one synthetic, modified RNA encoding a therapeutic agent, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight.
  • the synthetic, modified RNA comprises at least two modified nucleosides.
  • the therapeutic agent is a polypeptide or a non-translated RNA molecule.
  • the polypeptide is a somatotrophic agent.
  • the somatotrophic agent is a growth hormone.
  • the polypeptide is a cytokine.
  • the polypeptide is a cellular growth factor.
  • the unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight.
  • the unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight.
  • the unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight.
  • the unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight.
  • the unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight.
  • the unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, N2,
  • the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.
  • the unit dose compositions further comprise a cationic lipid.
  • compositions for intramuscular delivery comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent and a pharmaceutically acceptable carrier, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight.
  • the synthetic, modified RNA comprises at least two modified nucleosides.
  • the therapeutic agent is a polypeptide or a non-translated RNA molecule.
  • the polypeptide is a somatotrophic agent.
  • the somatotrophic agent is a growth hormone.
  • the polypeptide is a cytokine.
  • the polypeptide is a cellular growth factor.
  • the unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight.
  • the unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight.
  • the unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight.
  • the unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight.
  • the unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight.
  • the unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, N2,
  • the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.
  • the pharmaceutical compositions further comprise a cationic lipid.
  • kits comprising (a) a container or vial containing a unit dose of a synthetic, modified RNA encoding a therapeutic agent, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight, or a pharmaceutical composition for intramuscular delivery comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent and a pharmaceutically acceptable carrier, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight; and (b) packaging and instructions therefor.
  • the synthetic, modified RNA comprises at least two modified nucleosides.
  • the unit dose is divided into at least two containers or vials.
  • intramuscular delivery devices comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight, or a pharmaceutical composition for intramuscular delivery comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent and a pharmaceutically acceptable carrier, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight.
  • the intramuscular delivery device is a non-implantable delivery device or an implantable delivery device.
  • the intramuscular delivery device is a syringe.
  • enhanced methods for delivering synthetic, modified RNA into a subject comprising administering intramuscularly to a subject at least one unit dose of a synthetic, modified RNA encoding a therapeutic agent, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight, or a pharmaceutical composition for intramuscular delivery comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent and a pharmaceutically acceptable carrier, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight.
  • the unit dose is divided into at least two separate unit dosages and administered simultaneously into at least two muscular locations.
  • the unit dose is divided into at least two separate unit dosages and administered sequentially into the same or a different muscular locations.
  • the therapeutic agent is a polypeptide or a non-translated RNA molecule.
  • the polypeptide is a somatotrophic agent.
  • the somatotrophic agent is a growth hormone.
  • the polypeptide is a cytokine.
  • the polypeptide is a cellular growth factor.
  • the unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight.
  • the unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight.
  • the unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight.
  • the unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight. [0040] In some embodiments of these methods and all such methods described herein, the unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight.
  • the unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, N2,
  • the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.
  • the unit dose or pharmaceutical compositions further comprise a cationic lipid.
  • a pharmaceutical composition for intramuscular delivery comprising a unit dose of a synthetic, modified RNA encoding a therapeutic agent and a pharmaceutically acceptable carrier, wherein the unit dose ranges from between 0.03 mg/kg of body weight and 10 mg/kg of body weight.
  • the therapuetic agent is erythropoietin.
  • the unit dose is divided into at least two separate unit dosages and administered simultaneously into at least two muscular locations.
  • the unit dose is divided into at least two separate unit dosages and administered sequentially into the same or a different muscular locations.
  • the unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight.
  • the unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight.
  • the unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight.
  • the unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight. [0053] In some embodiments of these methods and all such methods described herein, the unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight.
  • the unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, N2,
  • the at least two modified nucleosides are 5-methylcytidine (5mC) and pseudouridine.
  • the unit dose or pharmaceutical compositions further comprise a cationic lipid.
  • FIG. 1 demonstrates dose-dependency of protein expression upon intramuscular injection of synthetic, modified RNAs.
  • Various doses of a synthetic, modified RNA encoding Erythropoietin (EPO) were injected intramuscularly, and thirteen hours later plasma Erythropoietin levels were examined using ELISA.
  • EPO Erythropoietin
  • FIG. 1 demonstrates dose-dependency of protein expression upon intramuscular injection of synthetic, modified RNAs.
  • EPO Erythropoietin
  • FIG. 2 demonstrates that 1 week following injection of a synthetic, modified RNA encoding erythropoietin, functional parameters mediated by erythropoietin increase in a dose- dependent fashion.
  • Synthetic, modified RNA encoding erythropoietin was injected with (+) or without (-) lipofectamine, with unmodified RNA with lipofectamine used as a control.
  • the upper left, upper right, and lower left panels demonstrates that red blood cell (RBC) counts, hematocrit, and hemoglobin levels were increased in a dose-dependent fashion 1 week following injection of the synthetic, modified RNA encoding erythropoietin in the presence of lipfectamine. In the absence of liptofectamine or when unmodified RNAs were used, a dose-dependent effect was not observed for any of these functional parameters.
  • RBC red blood cell
  • FIG. 3 demonstrates that parameters and cells not responsive to erythropoietin are not impacted by the injection of the synthetic, modified RNA encoding erythropoietin, therefore demonstrating the specificity of the effects of the synthetic, modified RNA.
  • Synthetic, modified RNA encoding erythropoietin was injected with (+) or without (-) lipofectamine, with unmodified RNA with lipofectamine used as a control.
  • the upper left panel shows white blood cell (WBC) counts
  • the upper right panel shows lymphocyte counts
  • the lower left panel shows neutrophil counts
  • the lower right paner shows platelet numbers 1 week following injection.
  • WBC white blood cell
  • unit dose compositions, kits, delivery devices, and methods for enhancing delivery of a synthetic, modified RNAs encoding a therapeutic agent to a subject can be used to express a desired therapeutic agent without the introduction of any exogenous DNA or viral vectors, and thus, do not cause permanent modification of the genome or have the potential for unintended mutagenic effects.
  • erythropoietin as an exemplary therapeutic agent, that intramuscular administration of a unit dose of a synthetic, modified RNA encoding a therapeutic agent, such as a growth factor, particularly when combined with a lipid carrier, permits dose-dependent protein expression that can be measured in the systemic circulation.
  • a synthetic, modified RNA encoding Erythropoietin (EPO) were injected intramuscularly, and thirteen hours later plasma erythropoietin levels were examined using ELISA.
  • EPO Erythropoietin
  • Increasing doses of the synthetic, modified RNA is demonstrated herein to result in a proportional increase in plasma levels of a therapeutic agent, such as erythropoietin.
  • the inventors further demonstrate the functional impact of intramuscular administration of unit dose compositions comprising synthetic, modified RNAs encoding a therapeutic agent.
  • unit dose compositions comprising synthetic, modified RNAs encoding a therapeutic agent.
  • the inventors show that there is a dose -dependent functional response to the increased levels of erythropoietin. More specifically, one week following the injection of a synthetic, modified RNA encoding erythropoietin, functional parameters mediated by erythropoietin were found to increase in a dose-dependent fashion.
  • Synthetic, modified RNA encoding erythropoietin was injected with or without a cationic lipid agent, lipofectamine, with an unmodified RNA injected with lipofectamine used as a control. It was found that red blood cell (RBC) counts, hematocrit, and hemoglobin levels were increased in a dose- dependent fashion that was measurable one week following injection of the synthetic, modified RNA encoding erythropoietin in the presence of lipofectamine. In the absence of liptofectamine or when unmodified RNAs were used, a dose -dependent effect was not observed for any of these functional parameters. Thus, expression of a therapeutic agent encoded by a unit dose composition comprising synthetic, modified RNA is demonstrated herein to have functional implications that occur in a dose- dependent fashion.
  • the inventors also demonstrated that cells not responsive to a therapeutic agent, such as erythropoietin, are not impacted by the intramuscular injection of the synthetic, modified RNA encoding the therapeutic agent, therefore demonstrating the specificity of the effects of the unit dose composition comprising synthetic, modified RNAs.
  • a therapeutic agent such as erythropoietin
  • the unit dose compositions comprising synthetic, modified RNA encoding erythropoietin were injected with or without a cationic lipid agent, lipofectamine, and with unmodified RNA plus lipofectamine used as a control.
  • the inventors demonstrated that the dose dependent response in protein expression as well as the functional effects of the protein expression was only seen when the intramuscular administration was performed with the modified RNA combined with the cationic lipid agent.
  • RNA therapies comprising a unit dose of synthetic modified RNA encoding a protein of interest for the delivery of such synthetic modified RNAs to a subject.
  • the unit dose compositions comprising synthetic, modified RNAs encoding therapeutic agents can be used to provide dose-dependent effects on specific cell types in vivo.
  • RNA encoding EPO protein The specific dosage ranges have been shown to work for an exemplary RNA encoding EPO protein. However, these dosage ranges may be extrapolated to any other protein with the knowledge that the method allows a dose-dependent titration of the RNAs. Similarly, while the preliminary results were obtained in a mouse model, the results may be extrapolated into other animals, including humans based on the per kg amount calculated from the exemplary mouse models. Moreover, the discovery of a unit dose-dependent expression of the RNA allows one to more easily titrate effective dosages for any protein.
  • compositions comprising synthetic, modified RNAs encoding therapeutic agents for enhancing delivery of such agents to a subject.
  • unit dose when used in reference to a therapeutic composition, refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material, such as a synthetic, modified RNA calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e. , carrier, or vehicle.
  • active material such as a synthetic, modified RNA calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e. , carrier, or vehicle.
  • the unit dose compositions are administered intramuscularly in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired.
  • compositions comprising synthetic, modified RNAs encoding therapeutic agents described herein can be evaluated by the ordinarily skilled clinician by monitoring one or more symptoms or markers of the disease or disorder being treated by administration of the cells.
  • Effective treatment includes any statistically significant improvement in one or more indicia of the disease or disorder.
  • a clinically accepted grade or scaling system for the given disease or disorder can be applied, with an
  • Dosages of the unit dose compositions comprising synthetic, modified RNAs encoding therapeutic agents can vary depending upon the approach taken and the disease to be treated. For example, intramuscular administration without a targeting approach will generally require greater amounts of the unit dose compositions comprising synthetic, modified RNA than intramuscular administration that employs a targeting or homing approach.
  • unit dose compositions comprising synthetic, modified RNAs encoding therapeutic agents provide dose-dependent expression of the therapeutic agent.
  • effective dosages of unit dose compositions comprising synthetic, modified RNA can include, for example, 1 ng/kg of body weight up to a gram or more per kg of body weight and any amount in between.
  • unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight; unit dose ranges from about 0.05 mg per kg of body weight to about 0.2 mg per kg of body weight; unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight; unit dose ranges from about 0.3 mg per kg of body weight to about 1.0 mg per kg of body weight; unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight; unit dose ranges from about 1.5 mg per kg of body weight to about 2.0 mg per kg of body weight; unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight; unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight; unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight; unit dose ranges from about 8.0 mg per kg of body weight to about 20 mg per kg of body weight
  • Dosages in such ranges can be administered once, twice, three times, four times or more per day, or every two days, every three days, every four days, once a week, twice a month, once a month or less frequently over a duration of days, weeks or months, depending on the condition being treated - where the therapeutic approach treats or ameliorates but does not permanently cure the disease or disorder, e.g., where the unit dose compositions comprising synthetic, modified RNAs effects treatment of a growth or metabolic disorder by expression of a protein that is deficient in the subject, administration of the unit dose compositions comprising synthetic, modified RNAs can be repeated over time as needed.
  • unit dose compositions comprising synthetic, modified RNAs where, instead, the expression of the therapeutic agent encoded by the unit dose compositions comprising synthetic, modified RNAs leads to the establishment of a cell compartment that maintains itself and treats the disease or disorder, readministration can become unnecessary.
  • Sustained release formulations of unit dose compositions comprising synthetic, modified RNAs are specifically contemplated herein. Continuous, relatively low doses are contemplated after an initial higher therapeutic dose.
  • a unit dose composition comprising at least one synthetic, modified RNA as described herein can be delivered to or administered to a subject by an intramuscular delivery route or intramuscular injection.
  • a unit dose composition comprising at least one synthetic, modified RNAs can be incorporated into pharmaceutical compositions suitable for intramuscular administration.
  • unit dose compositions can include one or more synthetic, modified RNAs and a
  • Unit dose compositions for intramuscular administration of synthetic, modified RNAs can include sterile aqueous solutions that can also contain buffers, diluents and other suitable additives.
  • the effective dose of a synthetic, modified RNA can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances.
  • the effective dose of a synthetic, modified RNA can be administered as two or more separate unit dosages and administered simultaneously into at least two different muscular locations. In some embodiments, the effective dose of a synthetic, modified RNA can be administered as two or more separate unit dosages and administered sequentially into the same or different muscular locations.
  • a non-implantable intramuscular delivery device e.g., needle, syringe, pen device, or implantatable intramuscular delivery device, e.g., a pump, semi-permanent stent, or reservoir
  • the delivery device can include a mechanism to dispense a unit dose of the pharmaceutical composition
  • the device releases the pharmaceutical composition comprising a synthetic, modified RNAcontinuously, e.g., by diffusion.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • a unit dose composition comprising at least one synthetic, modified RNA can be modified such that it is capable of traversing the blood brain barrier.
  • the synthetic, modified RNA can be conjugated to a molecule that enables the agent to traverse the barrier.
  • conjugated synthetic, modified RNA can be administered by intramuscular injection.
  • a unit dose composition comprising a synthetic, modified RNA described herein can also be delivered through the use of implanted, indwelling catheters that provide a means for injecting small volumes of fluid containing the synthetic, modified RNAs described herein directly into the muscle.
  • the proximal end of these catheters can be connected to an implanted, access port surgically affixed to the patient's body.
  • implantable delivery devices such as an implantable pump can be employed.
  • the delivery of the unit dose compositions comprising at least one synthetic, modified RNA as described herein can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference.
  • U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579 all of which are incorporated herein by reference.
  • these and other devices and systems can be suitable for intramuscular delivery of unit dose compositions comprising the synthetic, modified RNAs described herein.
  • the delivery system further comprises implanting a pump outside the body, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of a unit dose composition comprising a synthetic, modified RNA described herein through the discharge portion of the catheter.
  • a further embodiment comprises periodically refreshing a supply of the unit dose composition comprising a synthetic, modified RNA to the pump outside the body.
  • Intramuscular administration of a unit dose composition comprising at least one synthetic, modified RNA can be provided by the subject or by another person, e.g., a another caregiver.
  • a caregiver can be any entity involved with providing care to the human: for example, a hospital, hospice, doctor's office, outpatient clinic; a healthcare worker such as a doctor, nurse, or other practitioner; or a spouse or guardian, such as a parent.
  • the dose of cells administered will also vary with the therapeutic approach.
  • a unit dose composition expresses a therapeutic agent for targeting a tumor cell
  • the dosage of cells administered will vary with, for example, the size of the tumor being treated - generally more cells or more frequent administration is warranted for larger tumors versus smaller ones.
  • the unit dose composition amount administered will also vary with the level of expression of the polypeptide or polypeptides encoded by the synthetic, modified RNA - this is equally true of the administration of cells expressing proteins encoded by modified RNA for any purpose described herein.
  • unit dose compositions and methods described herein are that where, for example, more than one factor or polypeptide is expressed from a unit dose composition comprising at least one synthetic, modified RNA administered to a subject, the relative dosage of the expressed proteins can be tuned in a straightforward manner by adjusting the relative amounts of the unit dose composition administered to the subject. This is in contrast to the difficulty of tuning the expression of even a single gene product in a cell transduced with a viral or even a plasmid vector.
  • compositions & Formulations for intramuscular delivery comprising the unit dose compositions comprising synthetic, modified RNAs dissolved or dispersed as an active ingredient and a physiologically tolerable carrier.
  • the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes, unless so desired.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable or unacceptable physiological effects such as toxicity, nausea, dizziness, gastric upset, immune reaction and the like.
  • a pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired.
  • compositions that contains active ingredients dissolved or dispersed therein are well understood in the art and need not be limited based on formulation.
  • Such compositions are prepared as injectable either as liquid solutions or suspensions, however, particularly where synthetic, modified RNA itself is administered, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the preparation can also be emulsified or presented as a liposome composition.
  • the active ingredient i.e., unit dose compositions comprising synthetic, modified RNAs, can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the excipients are, for example, water, saline
  • composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate -buffered saline. Saline -based carriers are most useful for the administration of cells or cell preparations. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.
  • compositions comprising synthetic, modified RNAs described herein can be formulated in conjunction with one or more penetration enhancers, surfactants and/or chelators for intramuscular delivery.
  • Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenodeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic acid deoxycholic acid
  • glucholic acid glycholic acid
  • glycodeoxycholic acid taurocholic acid
  • taurodeoxycholic acid sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate.
  • Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium).
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
  • combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts.
  • One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene-9-lauryl ether,
  • the unit dose compositions comprising synthetic, modified RNAs described herein can be formulated into any of many possible administration forms, including a sustained release form.
  • formulations comprising a plurality of different synthetic, modified RNAs are prepared by first mixing all members of a plurality of different synthetic, modified RNAs, and then complexing the mixture comprising the plurality of different synthetic, modified RNAs with a desired ligand or targeting moiety, such as a lipid.
  • the unit dose compositions comprising synthetic, modified RNAs can be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension can also contain stabilizers.
  • compositions comprising synthetic, modified RNAs described herein can be prepared and formulated as emulsions for the delivery of synthetic, modified RNAs.
  • Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ in diameter (see e.g. , Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • aqueous phase When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion.
  • oil-in- water (o/w) emulsion When an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in- water (o/w) emulsion.
  • Emulsions can contain further components in addition to the dispersed phases, and the active drug (i.e., synthetic, modified RNA) which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
  • Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed.
  • Emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxy vinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and carboxy
  • liposomes can optionally be prepared to contain surface groups to facilitate delivery of liposomes and their contents to specific cell populations.
  • a liposome can comprise a surface groups such as antibodies or antibody fragments, small effector molecules for interacting with cell-surface receptors, antigens, and other like compounds.
  • Surface groups can be incorporated into the liposome by including in the liposomal lipids a lipid derivatized with the targeting molecule, or a lipid having a polar-head chemical group that can be derivatized with the targeting molecule in preformed liposomes.
  • a targeting moiety can be inserted into preformed liposomes by incubating the preformed liposomes with a ligand-polymer-lipid conjugate.
  • a number of liposomes comprising nucleic acids are known in the art. WO 96/40062
  • RNAi molecules targeted to the raf gene discloses methods for encapsulating high molecular weight nucleic acids in liposomes.
  • U.S. Pat. No. 5,264,221 discloses protein-bonded liposomes and asserts that the contents of such liposomes can include an RNA molecule.
  • U.S. Pat. No. 5,665,710 describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
  • WO 97/04787 discloses liposomes comprising RNAi molecules targeted to the raf gene.
  • a liposome composition comprising a nucleic acid
  • Each of these approaches can provide delivery of a synthetic, modified RNA as described herein to a cell.
  • the unit dose compositions comprising synthetic, modified RNAs described herein can be encapsulated in a nanoparticle.
  • the unit dose compositions comprising synthetic, modified
  • RNAs and methods thereof can comprise the use of other agents or measures to prevent or reduce any cytotoxicity caused by the administration procedure, the unit dose compositions, or a combination thereof.
  • the cytotoxicity of synthetic, unmodified RNAs involves a cellular innate immune response designed to recognize a foreign pathogen (e.g. , virus) and to produce interferons, which in turn stimulates the activity of the protein kinase PKR, Toll-like receptors (TLRs) and RIG-1, among others, to mediate anti-viral actions.
  • PKR protein kinase
  • TLRs Toll-like receptors
  • RIG-1 receptor anti-viral actions
  • the synthetic, modified RNA described herein comprises a 5'-monophosphate, a 5'-diphosphate, or a 5' 7-methyl guanosine cap to escape the immune response initiated by PKR.
  • the synthetic, modified RNA as described herein is treated to remove the 5'-triphosphate using an alkaline phosphatase, e.g., calf intestinal phosphatase.
  • RNA interference agents e.g. , siRNA, shRNA, etc.
  • RNA interference agents can be administered to a subject and used to inhibit expression of RIG-1, MYD88, VISA, PKR, TRIF, TRL7, or TLR8, which will result in a lower innate immune mediated response in the cells.
  • a unit dose composition comprising a synthetic, modified RNA encoding a therapeutic agent can further comprise or be administered to a subject with a modified RNA encoding an interferon scavenging agent (e.g. , a soluble interferon receptor) to further reduce the innate immune response of the cells.
  • an interferon scavenging agent e.g. , a soluble interferon receptor
  • TLR signaling inhibitor a TLR signaling inhibitor
  • 2-aminopurine a PKR inhibitor
  • TLR-signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available for purchase from INVIVOGENTM).
  • PRR pattern recognition receptors
  • 2-aminopurine, BX795, chloroquine, and H-89 can also be used in the compositions and methods described herein.
  • unit dose compositions comprising synthetic, modified RNAs encoding inhibitors of the innate immune system can be used to avoid the innate immune response generated in the cell.
  • Unit dose compositions comprising synthetic, modified RNAs can be intramuscularly administered or delivered into a subject using, for example, a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system.
  • a drug delivery system such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system.
  • Positively charged cationic delivery systems facilitate binding of a synthetic, modified RNA (negatively charged polynucleotides) and also enhances interactions at the negatively charged cell membrane to permit efficient cellular uptake by the subject.
  • Cationic lipids, dendrimers, or polymers can either be bound to synthetic, modified RNAs, or induced to form a vesicle or micelle (see e.g.
  • RNA complexes are well within the abilities of those skilled in the art (see e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9: 1291-1300; Arnold, AS et al (2007) J. Hypertens. 25: 197-205, which are incorporated herein by reference in their entirety).
  • unit dose compositions comprising a first and second synthetic, modified RNA respectively are administered in a separate and temporally distinct manner.
  • each of a plurality of unit dose compositions comprising synthetic, modified RNAs can be administered at a separate time or at a different frequency interval to achieve the desired expression of a given set of therapeutic agents.
  • the unit dose compositions comprising synthetic, modified RNAs further comprise a delivery reagent that facilitates uptake of a synthetic, modified RNA into a cell of a subject to which it is adminstered, such as an emulsion, a liposome, a cationic lipid, a non-cationic lipid, an anionic lipid, a charged lipid, a penetration enhancer, a modification to the synthetic, modified RNA to attach e.g. , a ligand, peptide, lipophillic group, or targeting moiety, or any combination thereof.
  • a delivery reagent that facilitates uptake of a synthetic, modified RNA into a cell of a subject to which it is adminstered, such as an emulsion, a liposome, a cationic lipid, a non-cationic lipid, an anionic lipid, a charged lipid, a penetration enhancer, a modification to the synthetic, modified RNA to attach e.g. , a ligand,
  • Suitable delivery agents that can be added to the unit dose compositions comprising synthetic, modified RNAs described herein include, for example, llPOFECTIN, LIPOFECT AMINe , DIMRIE CTM, SUPERFECTTM, and EFFECTINTM (QIAGENTM), UNIFECTINTM, MAXIFECTINTM, DOTMA, DOGSTM (Transfectam; dioctadecylamidoglycylspermine), DOPE (l,2-dioleoyl-sn-glycero-3- phosphoethanolamine), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecylammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB (N-n-hexadecyl-N,N-dihydroxye
  • PEI poly(ethylenimine)
  • the unit dose compositions comprising synthetic, modified
  • RNAs can further comprise cationic lipid carriers (e.g. , OLIGOFECTAMF ETM) or non-cationic lipid- based carriers (e.g., TRANSIT-TKOTMTM, Minis Bio LLC, Madison, WI) as delivery agents.
  • cationic lipid carriers e.g. , OLIGOFECTAMF ETM
  • non-cationic lipid- based carriers e.g., TRANSIT-TKOTMTM, Minis Bio LLC, Madison, WI
  • RNA expression levels of the therapeutic agent encoded by the synthetic, modified RNA can be monitored using various known methods.
  • transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • Successful transfection of a modified RNA can also be determined by measuring the protein expression level of the therapeutic agent by e.g., Western Blotting or immunocytochemistry, or ELISA, as described herein for the unit dose compositions encoding erythropoietin.
  • the unit dose compositions comprising synthetic, modified
  • RNAs can further comprise a transfection reagent as a delivery agent.
  • transfection reagents contemplated for the administration of the unit dose compositions comprising synthetic, modified RNAs include, for example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731).
  • transfection reagents for use in delivery include, but are not limited to, for example RNAlMAX (Invitrogen; Carlsbad, CA), LlPOFECTAMINETM (Invitrogen; Carlsbad, CA), LlPOFECT AMINE 2000TM (Invitrogen; Carlsbad, CA), 293FECTINTM (Invitrogen; Carlsbad, CA), CELLFECTINTM (Invitrogen; Carlsbad, CA), DMRIE-CTM (Invitrogen; Carlsbad, CA), FREESTYLETM MAX (Invitrogen; Carlsbad, CA), LlPOFECTAMINETM 2000 CD (Invitrogen; Carlsbad, CA), LlPOFECTAMINETM (Invitrogen; Carlsbad, CA), OLIGOFECTAMTNETM (Invitrogen; Carlsbad, CA), OPTIFECTTM (Invitrogen; Carlsbad, CA), X-TREMEGENE Q2 Transfection Reagent (Roche; Grenz
  • TROGANPORTERTM transfection Reagent Genlantis; San Diego, CA, USA ), RlBOFECT (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UNIFECTOR (B-Bridge International; Mountain View, CA, USA), SUREFECTOR (B-Bridge International; Mountain View, CA, USA), or HIFECTTM (B-Bridge International, Mountain View, CA, USA), among others.
  • unit dose compositions comprising synthetic, modified RNAs can further comprise highly branched organic compounds, termed "dendrimers,” that can bind the synthetic, modified RNAs.
  • nucleic acids may be utilized to enhance the penetration of the administered nucleic acids, including glycols, such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes, such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions comprising synthetic, modified RNAs for expressing a therapeutic agent, such as a polypeptide or non-translated RNA.
  • synthetic, modified RNA refers to a nucleic acid molecule encoding a factor, such as a polypeptide, to be expressed in a host cell, which comprises at least one modified nucleoside and has at least the following characteristics as the term is used herein: (i) it can be generated by in vitro transcription and is not isolated from a cell; (ii) it is translatable in a mammalian (and preferably human) cell; and (iii) it does not provoke or provokes a significantly reduced innate immune response or interferon response in a cell to which it is introduced or contacted relative to a synthetic, non- modified RNA of the same sequence .
  • a synthetic, modified RNA can be generated by in vitro transcription, using any method known to one of skill in the art, such as "splint-mediated ligation" technique described in International Publication WO 2011/130624 or using the IVT templates described in US 61/558,563.
  • the transcribed, synthetic, modified RNA polymer can be modified further post-transcriptionally, e.g., by adding a cap or other functional group.
  • the modified nucleoside(s) must be recognized as substrates by at least one RNA polymerase enzyme.
  • RNA polymerase enzymes can tolerate a range of nucleoside base modifications, at least in part because the naturally occurring G, A, U, and C nucleoside bases differ from each other quite significantly.
  • the structure of a modified nucleoside base for use in generating the synthetic, modified RNAs described herein can generally vary more than the sugar-phosphate moieties of the modified nucleoside. That said, ribose and phosphate-modified nucleosides or nucleoside analogs are known in the art that permit transcription by RNA polymerases.
  • the RNA polymerase is a phage RNA polymerase.
  • the modified nucleotides pseudouridine, m5U, s2U, m6A, and m5C are known to be compatible with transcription using phage RNA polymerases, while Nl-methylguanosine, Nl-methyladenosine, N7-methylguanosine, 2'-)-methyluridine, and 2'-0- methylcytidine are not.
  • Polymerases that accept modified nucleosides are known to those of skill in the art.
  • modified polymerases can be used to generate synthetic, modified RNAs, as described herein.
  • a polymerase that tolerates or accepts a particular modified nucleoside as a substrate can be used to generate a synthetic, modified RNA including that modified nucleoside.
  • the synthetic, modified RNA must be translatable by the translation machinery of a eukaryotic, preferably mammalian, and more preferably, human cell. Translation generally requires at least a ribosome binding site, a methionine start codon, and an open reading frame encoding a polypeptide.
  • the synthetic, modified RNA also comprises a 5' cap, a stop codon, a Kozak sequence, and a polyA tail.
  • mRNAs in a eukaryotic cell are regulated by degradation, thus a synthetic, modified RNA as described herein can be further modified to extend its half-life in the cell by incorporating modifications to reduce the rate of RNA degradation ⁇ e.g.
  • Nucleoside modifications can interfere with translation. To the extent that a given modification interferes with translation, those modifications are not encompassed by the synthetic, modified RNA as described herein.
  • an in vitro translation assay e.g. , a rabbit reticulocyte lysate assay, a reporter activity assay, or measurement of a radioactive label in the translated protein
  • the translation of a synthetic, modified RNA comprising a candidate modification is compared to the translation of an RNA lacking the candidate modification, such that if the translation of the synthetic, modified RNA having the candidate modification remains the same or is increased then the candidate modification is contemplated for use with the compositions and methods described herein. It is noted that fluoro-modified nucleosides are generally not translatable and can be used herein as a negative control for an in vitro translation assay.
  • the synthetic, modified RNA provokes a reduced (or absent) innate immune response or interferon response by a transfected cell or population of cells thereof, or in the subject to whom the unit dose compositions are administered.
  • mRNA produced in eukaryotic cells e.g. , mammalian or human cells
  • the cell responds by shutting down translation or otherwise initiating an innate immune or interferon response.
  • an exogenously added RNA can be modified to mimic the modifications occurring in the endogenous RNAs produced by a target cell
  • the exogenous RNA can avoid at least part of the target cell's defense against foreign nucleic acids.
  • synthetic, modified RNAs as described herein include in vitro transcribed RNAs including modifications as found in eukaryotic/mammalian/human RNA in vivo. Other modifications that mimic such naturally occurring modifications can also be helpful in producing a synthetic, modified RNA molecule that will be tolerated by a cell.
  • compositions comprising synthetic, modified RNA molecules encoding therapeutic agents, comprise one or more nucleoside modifications, such that administering the synthetic, modified RNA molecules to a subject results in a reduced innate immune response relative to a subject contacted with synthetic RNA molecules encoding the therapeutic agents not comprising the one or more nucleoside modifications.
  • the unit dose compositions comprising synthetic, modified RNAs described herein include modifications to prevent rapid degradation by endo- and exo-nucleases and to avoid or reduce a subject's cellular innate immune or interferon response to the RNA.
  • Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.)
  • base modifications e.g., replacement with modified bases, stabilizing bases, destabilizing
  • compositions comprising synthetic, modified RNA compositions described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages.
  • Synthetic, modified RNAs having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage.
  • the synthetic, modified RNA has a phosphorus atom in its internucleoside linkage(s).
  • Non-limiting examples of modified internucleoside linkages include
  • aminoalkylphosphotriesters methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,
  • thionoalkylphosphotriesters having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified internucleoside linkages that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • RNAs described herein include nucleic acids with phosphorothioate internucleoside linkages and oligonucleosides with heteroatom internucleoside linkage, and in particular— CH2-NH-CH2-, -CH2- N(CH3)-0-CH2- [known as a methylene (methylimino) or MMI ], -CH2-0-N(CH3)-CH2-, -CH2- N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the native phosphodiester internucleoside linkage is represented as -0-P-0-CH2-] of the above -referenced U.S. Pat. No.
  • nucleic acid sequences featured herein have morpholino backbone structures of the above -referenced U.S. Pat. No. 5,034,506, herein incorporated by reference in its entirety.
  • Unit dose compositions comprising synthetic, modified RNAs described herein can also contain one or more substituted sugar moieties.
  • the nucleic acids featured herein can include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted CI to CIO alkyl or C2 to CIO alkenyl and alkynyl.
  • Exemplary modifications include 0[(CH2)nO] mCH3, 0(CH2).nOCH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)nONH2, and 0(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10.
  • synthetic, modified RNAs include one of the following at the 2' position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of a synthetic, modified RNA, and other substituents having similar properties.
  • the modification includes a 2' methoxyethoxy (2'-0-CH2CH20CH3, also known as 2'- 0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e. , an alkoxy- alkoxy group.
  • a 2' methoxyethoxy (2'-0-CH2CH20CH3
  • 2'-MOE 2'-MOE
  • Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e. , a
  • 0(CH2)20N(CH3)2 group also known as 2'-DMAOE
  • 2'-dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE
  • OCH2CH2CH2NH2 OCH2CH2CH2NH2
  • 2'-fluoro (2'-F) Similar modifications can also be made at other positions on the nucleic acid sequence, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked nucleotides and the 5' position of 5' terminal nucleotide.
  • a synthetic, modified RNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • unit dose compositions comprising synthetic, modified
  • RNAs described herein can include at least one modified nucleoside including a 2'-0-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a 2'-amino-modified nucleoside, 2'- alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof.
  • the at least one modified nucleoside is selected from the group consisting of 5-methylcytidine (5mC), N6- methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2' deoxyuridine (2' dU), 4-thiouridine (s4U), 5- methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'- O-trimethyladenosine (m6 2 Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0- methylguanosine (Gm), N2,7-dimethylguanosine (m2,7G), N2, N2, N2,
  • unit dose compositions comprising synthetic, modified RNA can comprise at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the oligonucleotide.
  • a synthetic, modified RNA molecule comprising at least one modified nucleoside comprises a single nucleoside with a modification as described herein. It is not necessary for all positions in a given synthetic, modified RNA to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single synthetic, modified RNA or even at a single nucleoside within a synthetic, modified RNA.
  • each occurrence of a given nucleoside in a molecule is modified (e.g. , each cytosine is a modified cytosine e.g. , 5mC).
  • each cytosine is a modified cytosine e.g. , 5mC.
  • different occurrences of the same nucleoside can be modified in a different way in a given synthetic, modified RNA molecule (e.g. , some cytosines modified as 5mC, others modified as 2'-0-methylcytidine or other cytosine analog). The modifications need not be the same for each of a plurality of modified nucleosides in a synthetic, modified RNA.
  • a unit dose composition comprising synthetic, modified RNA comprises at least two different modified nucleosides.
  • the at least two different modified nucleosides are 5-methylcytidine and pseudouridine.
  • a synthetic, modified RNA can also contain a mixture of both modified and unmodified nucleosides.
  • nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • a synthetic, modified RNA comprises at least one nucleoside ("base") modification or substitution.
  • Modified nucleosides include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine, 2-(aminoalkyll)adenine, 2 (aminopropyl)adenine, 2 (methylthio) N6 (isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine, 8- (halo)adenine, 8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine, N6-
  • Modified nucleosides also include natural bases that comprise conjugated moieties, e.g. a ligand.
  • the RNA containing the modified nucleosides must be translatable in a subject's cell (i.e., does not prevent translation of the polypeptide encoded by the modified RNA).
  • transcripts containing s2U and m6A are translated poorly in rabbit reticulocyte lysates, while pseudouridine, m5U, and m5C are compatible with efficient translation.
  • 2'-fluoro-modified bases useful for increasing nuclease resistance of a transcript leads to very inefficient translation. Translation can be assayed by one of ordinary skill in the art using e.g., a rabbit reticulocyte lysate translation assay.
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley- VCH, 2008; those disclosed in Int. Appl. No. PCT/US09/038425, filed March 26, 2009; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613.
  • RNA molecules can be chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the RNA.
  • Ligands can be particularly useful where, for example, a synthetic, modified RNA is directly administered in vivo, as described herein.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556, herein incorporated by reference in its entirety), cholic acid (Manoharan et al., Biorg. Med. Chem.
  • a thioether e.g. , beryl-S- tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3:2765-2770, each of which is herein incorporated by reference in its entirety), a thiocholesterol (Oberhauser et al, Nucl.
  • the synthetic, modified RNAs described herein can further comprise a 5' cap.
  • the synthetic, modified RNAs comprise a 5' cap comprising a modified guanine nucleotide that is linked to the 5' end of an RNA molecule using a 5'- 5 'triphosphate linkage.
  • 5' cap is also intended to encompass other 5' cap analogs including, e.g., 5' diguanosine cap, tetraphosphate cap analogs having a methylene - bis(phosphonate) moiety (see e.g., Rydzik, AM et al., (2009) Org Biomol Chem 7(22):4763-76), dinucleotide cap analogs having a phosphorothioate modification (see e.g., Kowalska, J. et al., (2008) RNA 14(6): 1119-1131), cap analogs having a sulfur substitution for a non-bridging oxygen (see e.g., Grudzien-Nogalska, E.
  • 5' diguanosine cap tetraphosphate cap analogs having a methylene - bis(phosphonate) moiety
  • dinucleotide cap analogs having a phosphorothioate modification see e.g., Kowalska, J. et al
  • the 5' cap analog is a 5' diguanosine cap.
  • the synthetic, modified RNA does not comprise a 5' triphosphate.
  • the 5' cap is important for recognition and attachment of an mRNA to a ribosome to initiate translation.
  • the 5' cap also protects the synthetic, modified RNA from 5' exonuclease mediated degradation. It is not an absolute requirement that a synthetic, modified RNA comprise a 5' cap, and thus in other embodiments the synthetic, modified RNAs lack a 5' cap. However, due to the longer half -life of synthetic, modified RNAs comprising a 5' cap and the increased efficiency of translation, synthetic, modified RNAs comprising a 5' cap are preferred herein.
  • the synthetic, modified RNAs described herein can further comprise a 5' and/or 3' untranslated region (UTR).
  • Untranslated regions are regions of the RNA before the start codon (5') and after the stop codon (3'), and are therefore not translated by the translation machinery.
  • Modification of an RNA molecule with one or more untranslated regions can improve the stability of an mRNA, since the untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation.
  • modification of an RNA with a 5' and/or 3' untranslated region can enhance translational efficiency by binding proteins that alter ribosome binding to an mRNA.
  • Modification of an RNA with a 3' UTR can be used to maintain a cytoplasmic localization of the RNA, permitting translation to occur in the cytoplasm of the cell.
  • the synthetic, modified RNAs described herein do not comprise a 5' or 3' UTR.
  • the synthetic, modified RNAs comprise either a 5' or 3' UTR.
  • the synthetic, modified RNAs described herein comprise both a 5' and a 3' UTR.
  • the 5' and/or 3' UTR is selected from an mRNA known to have high stability in the cell (e.g., a murine alpha-globin 3' UTR).
  • the 5' UTR, the 3' UTR, or both comprise one or more modified nucleosides.
  • the synthetic, modified RNAs described herein further comprise a Kozak sequence.
  • the "Kozak sequence” refers to a sequence on eukaryotic mRNA having the consensus (gcc)gccRccAUGG (SEQ ID NO: 1), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.
  • the Kozak consensus sequence is recognized by the ribosome to initiate translation of a polypeptide. Typically, initiation occurs at the first AUG codon encountered by the translation machinery that is proximal to the 5' end of the transcript.
  • the synthetic, modified RNAs described herein further comprise a Kozak consensus sequence at the desired site for initiation of translation to produce the correct length polypeptide.
  • the Kozak sequence comprises one or more modified nucleosides.
  • the synthetic, modified RNAs described herein further comprise a "poly (A) tail", which refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides).
  • a poly (A) tail refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenine nucleotides).
  • the inclusion of a 3' poly(A) tail can protect the synthetic, modified RNA from degradation in the cell, and also facilitates extra-nuclear localization to enhance translation efficiency.
  • the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 adenine nucleotides or more.
  • the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides.
  • one or more modifications to the synthetic, modified RNAs described herein permit greater stability of the synthetic, modified RNA in a subject to which the unit dose compositions comprising one or more synthetic, modified RNAs is being administered.
  • modifications permit translation and either reduce or do not exacerbate a cell's innate immune or interferon response to the synthetic, modified RNA with the modification, such modifications are specifically contemplated for use herein.
  • the greater the stability of a synthetic, modified RNA the more protein can be produced from that synthetic, modified RNA.
  • a synthetic, modified RNA as described herein does not comprise an AU-rich region.
  • the 3' UTR substantially lacks AUUUA sequence elements.
  • the unit dose compositions comprising synthetic, modified RNAs and methods of enhancing delivery described herein permit the long-term, safe, and efficient expression of therapeutic agents, such as polypepeptides and non-translated RNAs, without the risk of permanent genomic alterations.
  • therapeutic agents such as polypepeptides and non-translated RNAs
  • Such unit dose compositions and methods are useful for a variety of applications, indications, and modalities, including, but not limited to, gene therapy, regenerative medicine, and cancer therapies.
  • Unit dose compositions comprising synthetic, modified RNAs as described herein can be made that direct the expression of essentially any gene product or open reading frame whose coding sequences can be cloned, such as those regulating cellular differentiation and growth, transcription factor, hormones, cytokines, as well as non-translated RNA products.
  • the synthetic, modified RNA encodes a mRNA that undergoes translation into a peptide or polypeptide.
  • the synthetic, modified RNA encodes inhibitory RNAs, such as small interfering RNAs (siRNA) or micro RNAs (miRNA).
  • siRNA small interfering RNAs
  • miRNA micro RNAs
  • an interfering RNA that prevents expression of an mRNA an RNA that is a pre-RNA, for example pre-miRNA, or a mature RNA, for example mature miRNA.
  • the mRNA can encode or be translated into essentially any polypeptide or peptide that is desired to be expressed.
  • polypeptides include, but are not limited to, transcription factors, targeting moieties and other cell-surface polypeptides, cell-type specific polypeptides, differentiation factors, death receptors, death receptor ligands, structural proteins, enzymes, hormones, reprogramming factors, de- differentiation factors, cytokines, and any combination thereof.
  • polypeptides or peptides to be expressed can include fusion proteins, truncated variants, protein domains, allelic variants and the like of any polypeptide.
  • the synthetic, modified RNA can encode essentially any non-translated RNA molecule that it is desired to synthesize, including, for example, shRNA molecules, siRNA molecules, dsRNA molecules, ribozymes, and any combinations thereof.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a cytokine, a hormone or a cellular growth factor such as, for example, somatrophic hormone, insulin, glucagon, erythropoietin, a glucocorticoid, epidermal growth factor (EGF), insulin, transforming growth factors (TGF-OC and TGF- ⁇ ), heparin, hepatocyte growth factors (HGF), interleukins (IL-1 and IL-6), insulin-like growth factors (IGF-I and IGF-II), heparin-binding growth factors (HBGF-1), angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein- 13, bone morphogenic protein- 14, bone morph
  • epidermal growth factor hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma colony stimulating factors (CSFs) such as macrophage -CSF (M-CSF); granulocyte -macrophage-CSF (GM-CSF); and granulocyte-CSF (G- CSF);
  • RNA that encodes for a mitogen or growth factor receptor include those that bind ligands including, but not limited to: insulin, insulin-like growth factor (e.g., IGF1, IGF2), platelet derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), fibroblast growth factor (FGF), bone morphogenic proteins (BMPs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha and -beta, among others.
  • IGF1, IGF2 platelet derived growth factor
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • NGF nerve growth factor
  • FGF fibroblast growth factor
  • BMPs bone morphogenic proteins
  • G-CSF granulocyte colony
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a transcription factor.
  • transcription factor refers to a protein that binds to specific DNA sequences and thereby controls the transfer (or transcription) of genetic information from DNA to mRNA.
  • the transcription factor encoded by the synthetic, modified RNA is a human transcription factor, such as those described in e.g., Messina DM, et al. (2004) Genome Res. 14(10B):2041-2047, which is herein incorporated by reference in its entirety.
  • the unit dose composition comprises a synthetic, modified
  • CD molecules and their mRNA IDs and sequence identifiers) for use in the aspects and embodiments described herein include those listed in Tables 2 and 3 of International Patent Publication PCT/US2011/032679, the contents of which are herein incorporated by reference in their entireties.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a ligand or ligand receptor on the surface of a cell e.g. , a homing moiety.
  • a ligand or ligand receptor moiety attached to a cell surface permits the cell to have a desired biological interaction with a tissue or an agent in vivo.
  • a ligand can be an antibody, an antibody fragment, an aptamer, a peptide, a vitamin, a carbohydrate, a protein or polypeptide, a receptor, e.g., cell-surafce receptor, an adhesion molecule, a glycoprotein, a sugar residue, a therapeutic agent, a drug, a glycosaminoglycan, or any combination thereof.
  • a ligand can be an antibody that recognizes a cancer-cell specific antigen, rendering the cell capable of preferentially interacting with tumor cells to permit tumor-specific localization of a modified cell.
  • a ligand can confer the ability of a cell composition to accumulate in a tissue to be treated, since a preferred ligand is capable of interacting with a target molecule on the external face of a tissue to be treated. Ligands having limited cross-reactivity to other tissues are generally preferred.
  • a ligand can act as a homing moiety which permits the cell to target to a specific tissue or interact with a specific ligand.
  • Such homing moieties can include, for example, any member of a specific binding pair, antibodies, monoclonal antibodies, or derivatives or analogs thereof, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab') 2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as disulfide stabilized Fv fragments, scFv tandems ((scFv) 2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e. , leucine zipper or helix stabilized) scFv fragments; and other homing moieties include for example, aptamers, receptors, and fusion proteins.
  • the homing moiety is a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site.
  • multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of homing interactions.
  • an estrogen receptor ligand such as tamoxifen
  • ligand/receptor interactions include CCR1 (e.g. , for treatment of inflamed joint tissues or brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8 (e.g. , targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g. , to target to intestinal tissue), CCR4, CCR10 (e.g.
  • CXCR4 e.g. , for general enhanced transmigration
  • HCELL e.g. , for treatment of inflammation and inflammatory disorders, bone marrow
  • Alpha4beta7 e.g. , for intestinal mucosa targeting
  • VLA-4 / VCAM-1 e.g. , targeting to endothelium.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a reprogramming factor.
  • a "reprogramming factor” refers to a developmental potential altering factor, such as a protein, RNA, or small molecule, the expression of which contributes to the reprogramming of a cell, e.g. a somatic cell, to a less differentiated or undifferentiated state, e.g. to a cell of a pluripotent state or partially pluripotent state.
  • a reprogramming factor can be, for example, transcription factors that can reprogram cells to a pluripotent state, such as SOX2, OCT3/4, KLF4, NANOG, LIN-28, c-MYC, and the like, including as any gene, protein, RNA or small molecule, that can substitute for one or more of these in a method of reprogramming cells in vitro.
  • a reprogramming factor can also be termed a "de -differentiation factor,” which refers to a developmental potential altering factor, such as a protein or RNA, that induces a cell to de-differentiate to a less differentiated phenotype, or, in other words, increases the developmental potential of a cell.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a differentiation factor refers to a developmental potential altering factor, such as a protein, RNA, or small molecule, that induces a cell to differentiate to a desired cell-type, i.e. , a differentiation factor reduces the developmental potential of a cell.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a cell-type specific polypeptide refers to a polypeptide that is expressed in a cell having a particular phenotype (e.g., a muscle cell) but is not generally expressed in other cell types with different phenotypes.
  • MyoD is expressed specifically in muscle cells but not in non-muscle cells, thus MyoD is a cell-type specific polypeptide.
  • albumin is expressed in hepatocytes and is thus an hepatocyte-specific polypeptide.
  • Such cell-specific polypeptides are well known in the art or can be identified using a gene array analysis and comparison of at least two different cell types. Methods for gene expressional array analysis is well known in the art.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a death receptor or death receptor ligand RNA that encodes for a death receptor or death receptor ligand.
  • death receptor a receptor that induces cellular apoptosis once bound by a ligand.
  • Death receptors include, for example, tumor necrosis factor (TNF) receptor superfamily members having death domains (e.g., TNFRI, Fas, DR3, 4, 5, 6) and TNF receptor superfamily members without death domains LTbetaR, CD40, CD27, HVEM. Death receptors and death receptor ligands are well known in the art.
  • TNF tumor necrosis factor
  • death receptors include FAS (CD95, Apol), TNFRl (p55, CD120a), DR3 (Apo3, WSL-1, TRAMP, LARD), DR4, DR5 (Apo2, TRAIL-R2, TRICK2, KILLER), CAR1, and the adaptor molecules FADD, TRADD, and DAXX.
  • death receptor ligands include FASL (CD95L), TNF, lymphotoxin alpha, Apo3L (TWEAK), and TRAIL (Apo2L).
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for an RNA molecule found in a non-human species, including other mammalian RNAs, avian RNA, reptilian RNAs, bacterial RNA, and viral RNAs.
  • Such sequences can encode for protein or peptides that have a desirable function, such as a reporter molecule, a secreted antimicrobial peptide, and the like.
  • the unit dose composition comprises a synthetic, modified
  • RNA that encodes for a tRNA transfer RNA
  • an snRNA small nuclear RNA
  • an rRNA ribosomal RNA
  • an anti-sense RNA anti-sense RNA
  • siRNA small interfering RNA
  • miRNA micro RNA
  • an “antisense RNA” comprises one or more nucleotide sequences sufficient in identity, number and size to effect specific hybridization with a preselected nucleic acid sequence.
  • ribozymes refer to RNA molecules having enzymatic activities usually associated with cleavage, splicing or ligation of nucleic acid sequences to which the ribozyme binds. Typical substrates for ribozymes include RNA molecules, although ribozymes can also catalyze reactions in which DNA molecules serve as substrates.
  • ribozyme Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence, and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing.
  • RNA refers to a 1-50 nucleotide double stranded RNA (dsRNA) molecule that has sequence-specific homology to its "target" nucleic acid sequences (Caplen, N. J., et al., Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001)) and is derived from the processing of a larger dsRNA by an RNase known as Dicer (Bernstein, E., et al., Nature 409:363-366 (2001)).
  • Dicer RNase
  • shRNA molecules are single stranded nucleic acid molecules that comprise two sequences complementary to each other, oriented such that one of the sequences is inverted relative to the other, which allows the two complementary sequences to base pair with each other, thereby forming a hairpin structure.
  • the two copies of the inverted repeat need not be contiguous.
  • microRNA refers to molecules which are structurally similar to shRNA molecules, as described herein, but, typically, contain one or more mismatches or insertion/deletions in their regions of sequence complementary. The binding of miRNA of perfect complementarity to a target sequence results in mRNA degradation; single base mismatches can block translation.
  • the unit dose compositions comprising synthetic, modified
  • RNAs further comprise a ligand.
  • a ligan can, for example, alter the cellular uptake, intracellular targeting or half-life of a synthetic, modified RNA into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, intracellular compartment, e.g., mitochondria, cytoplasm, peroxisome, lysosome, as, e.g., compared to a composition absent such a ligand.
  • a selected target e.g., molecule, cell or cell type
  • intracellular compartment e.g., mitochondria, cytoplasm, peroxisome, lysosome
  • Preferred ligands do not interfere with expression of a polypeptide from the synthetic, modified RNA.
  • Ligands can include a naturally occurring substance, such as a protein ⁇ e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate ⁇ e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a. synthetic polyamino acid.
  • polyamino acids examples include polylysine (PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether- maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer examples include poly(L-lactide-co-glycolied) copolymer, divinyl ether- maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copoly
  • polyethylenimine polylysine (PLL)
  • PLL polylysine
  • spermine spermidine
  • polyamine pseudopeptide-polyamine
  • peptidomimetic polyamine dendrimer polyamine
  • arginine amidine
  • protamine cationic lipid
  • cationic porphyrin quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g. , a cell targeting agent, (e.g. , a lectin, glycoprotein, lipid or protein), or an antibody, that binds to a specified cell type such as a fibroblast cell.
  • a cell targeting agent e.g. , a lectin, glycoprotein, lipid or protein
  • an antibody that binds to a specified cell type such as a fibroblast cell.
  • a targeting group can be, for example, a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic, among others.
  • ligands include dyes, intercalating agents (e.g. acridines), cross- linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g. , phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g.
  • intercalating agents e.g. acridines
  • cross- linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g. , phenazine, dihydrophenazine
  • artificial endonucleases e.g. EDTA
  • lipophilic molecules e.g.
  • alkylating agents amino, mercapto, PEG (e.g. , PEG-40K), MPEG, [MPEGJ2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), and transport/absorption facilitators (e.g. , aspirin, vitamin E, folic acid).
  • Ligands can be proteins, e.g. , glycoproteins, or peptides, e.g. , molecules having a specific affinity for a co-ligand, or antibodies e.g. , an antibody, that binds to a specified cell type such as a fibroblast cell, or other cell useful in the production of polypeptides.
  • Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be a substance, e.g. , a drug, which can increase the uptake of the unit dose compositions comprising synthetic, modified RNAs into a cell of a subject to which it is adminstered, for example, by disrupting the cell's cytoskeleton, e.g. , by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • One exemplary ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation, and/or (b) increase targeting or transport into a target cell or cell membrane.
  • a lipid based ligand can be used to modulate, e.g., binding of the modified RNA composition to a target cell.
  • the ligand is a moiety, e.g. , a vitamin, which is taken up by a host cell.
  • exemplary vitamins include vitamin A, E, and K.
  • Other exemplary vitamins include B vitamin, e.g. , folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up, for example, by cancer cells.
  • the ligand is a cell-permeation agent, preferably a helical cell- permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • a "cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell.
  • a microbial cell- permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, ⁇ -defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin).
  • a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
  • targeting moiety refers to an agent that directs a composition to a particular tissue, cell type, receptor, or other area of interest.
  • a targeting moiety can be attached directly to a synthetic, modified RNA or indirectly to a composition used for delivering a synthetic, modified RNA (e.g., a liposome, polymer etc) to direct expression in a particular cell etc.
  • a targeting moiety can also be encoded or expressed by a synthetic, modified-NA as described herein, such that a cell expresses a targeting moiety on it surface, permitting a cell to be targeted to a desired tissue, organ etc.
  • targeting moieties expressed on a cell surface are referred to herein as "homing moieties.”
  • Non-limiting examples of a targeting moiety or homing moiety include, but are not limited to, an oligonucleotide, an antigen, an antibody or functional fragment thereof, a ligand, a cell- surface receptor, a membrane -bound molecule, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative, a hormone, e.g., estradiol or histamine, a hormone-mimic, e.g. , morphine, or hormone -receptor, or other compound having binding specificity for a target.
  • a targeting moiety or homing moiety include, but are not limited to, an oligonucleotide, an antigen, an antibody or functional fragment thereof, a ligand, a cell- surface receptor, a membrane -bound molecule, one member of a specific binding pair,
  • a targeting moiety promotes transport or preferential localization of a synthetic, modified RNA to a target cell, while a homing moiety permits the targeting of a cell modified using the synthetic, modified RNAs described herein to a particular tissue in vivo. It is contemplated herein that the homing moiety can be also encoded in a cell by a synthetic, modified RNA as described herein.
  • a synthetic, modified RNA or unit dose composition thereof can be targeted by means of a targeting moiety, including, e.g., an antibody or targeted liposome technology.
  • a synthetic, modified RNA or unit dose composition thereof is targeted to a specific tissue by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • bispecific antibodies for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • molecular conjugates of antibodies can be used for production of recombinant, bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules.
  • the addition of an antibody to a synthetic, modified RNA composition permits the agent attached to accumulate additively at the desired target site.
  • Antibody-based or non- antibody-based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site.
  • a natural binding agent for an unregulated or disease associated antigen is
  • a unit dose composition can comprise a plurality of different synthetic, modified RNA.
  • a unit dose composition can comprise two or more synthetic, modified RNAs, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more synthetic, modified RNAs.
  • the two or more synthetic, modified RNAs are capable of increasing expression of a desired therapeutic agent (e.g. , a transcription factor, a cell surface marker, a death receptor, etc.).
  • the plurality of synthetic, modified RNAs when a plurality of different synthetic, modified RNAs are used to modulate expression of a desired set of therapeutic agents, can be administered to a subject simultaneously in a single unit dose composition, for example.
  • the plurality of synthetic, modified RNAs can be administered to a subject individually in separate unit dose compositions.
  • each synthetic, modified RNA can be administered according to its own dosage regime.
  • a unit dose composition can be prepared comprising a plurality of synthetic, modified RNAs, in differing relative amounts or in equal amounts, that is administered to a subject such that the plurality of synthetic, modified RNAs are administered simultaneously.
  • one synthetic, modified RNA at a time can be administered to a subject.
  • the expression desired for each target therapeutic agent can be easily tailored by altering the frequency of administration and/or the amount of a particular synthetic, modified RNA administered.
  • adminstereing unit dose composition comprising a unique synthetic, modified RNA separately, interactions between the synthetic, modified RNAs that can reduce efficiency of expression can be prevented.
  • the unit dose compositions and methods described herein permit the expression of one or more therapeutic agents to be tuned to a desired level by varying the amount of each synthetic, modified RNA adminstered.
  • One of skill in the art can easily monitor the expression level of the therapeutic agent encoded by a synthetic, modified RNA using e.g., Western blotting techniques, immunocytochemistry techniques, or ELISA, as described herein for erythropoietin.
  • a unit dose composition comprising one or more synthetic, modified RNAs can be administered at a frequency and dose that permit a desired level of expression of the therapeutic agent encoded by the synthetic, modified RNA.
  • the synthetic, modified RNAs administered toa subject are transient in nature (i.e., ate degraded over time) one of skill in the art can easily remove or stop expression of a synthetic, modified RNA by halting further administrations and permitting the subject to degrade the synthetic, modified RNA over time.
  • the synthetic, modified RNAs will degrade in a manner similar to cellular mRNAs.
  • RNAs described herein can be synthesized and/or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference in its entirety. Transcription methods are described further herein in the Examples.
  • a template for a synthetic, modified RNA is synthesized using "splint-mediated ligation," which allows for the rapid synthesis of DNA constructs by controlled concatenation of long oligos and/or dsDNA PCR products and without the need to introduce restriction sites at the joining regions. It can be used to add generic untranslated regions (UTRs) to the coding sequences of genes during T7 template generation. Splint mediated ligation can also be used to add nuclear localization sequences to an open reading frame, and to make dominant-negative constructs with point mutations starting from a wild-type open reading frame.
  • single-stranded and/or denatured dsDNA components are annealed to splint oligos which bring the desired ends into conjunction, the ends are ligated by a thermostable DNA ligase and the desired constructs amplified by PCR.
  • a synthetic, modified RNA is then synthesized from the template using an RNA polymerase in vitro. After synthesis of a synthetic, modified RNA is complete, the DNA template is removed from the transcription reaction prior to use with the methods described herein.
  • the synthetic, modified RNAs are further treated with an alkaline phosphatase.
  • the synthetic modified RNAs can be synethesized using the
  • kits and intramuscular delivery devices comprising unit dose compositions comprising synthetic, modified RNAs encoding therapeutic agents as described herein.
  • kits comprising (a) a container or vial containing a unit dose comprising between 0.03 mg/kg of body weight and 10 mg/kg of body weight of a synthetic, modified RNA encoding a therapeutic agent, wherein the synthetic, modified RNA comprises at least two modified nucleosides, or a pharmaceutical composition thereof and (b) packaging and instructions therefor.
  • the therapeutic agent is a polypeptide or a non- translated RNA molecule.
  • the polypeptide is a somatotrophic agent.
  • the somatotrophic agent is a growth hormone.
  • the polypeptide is a cytokine.
  • the unit dose ranges from about 0.03 mg per kg of body weight to about 0.0625 mg per kg of body weight. In some embodiments of these kits, the unit dose ranges from about 0.1 mg per kg of body weight to about 0.4 mg per kg of body weight. In some embodiments of these kits, the unit dose ranges from about 0.9 mg per kg of body weight to about 1.6 mg per kg of body weight. In some embodiments of these kits, the unit dose ranges from about 1.8 mg per kg of body weight to about 3.2 mg per kg of body weight.In some embodiments of these kits, the unit dose ranges from about 3.0 mg per kg of body weight to about 6.5 mg per kg of body weight. In some embodiments of these kits, unit dose ranges from about 5.5 mg per kg of body weight to about 10 mg per kg of body weight.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-0- dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0-methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-0-methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'-0-trimethyladenosine (m62Am), 2'-0- methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0-methylguanosine (Gm), N2,7- dimethylguanosine (m2,7G), N2, N2, 7-trimethylguanosine (m2,2,2, 7-trimethylguanosine (m
  • the at least two modified nucleosides are 5- methylcytidine (5mC) and pseudouridine.
  • the unit dose composition further comprises a cationic lipid or lipid carrier.
  • the unit dose is divided into at least two containers or vials.
  • intramuscular delivery devices comprising a unit dose comprising between 0.03 mg/kg of body weight and 10 mg/kg of body weight of a synthetic, modified RNA encoding a therapeutic agent, wherein the synthetic, modified RNA comprises at least two modified nucleosides, or a pharmaceutical composition thereof
  • the intramuscular delivery device is a non-implantable delivery device or an implantable delivery device.
  • the intramuscular delivery device is a syringe.
  • kits and/or intramuscular delivery devices described herein can further comprise one or more buffers, cell culture mediums, transfection medium and/or a media supplement.
  • the buffers, cell culture mediums, transfection mediums, and/or media supplements are RNase-free.
  • the unit dose compositions comprising synthetic, modified RNAs provided in the kits can be in a non-solution form of specific quantity or mass, e.g., 20 ⁇ g, such as a lyophilized powder form, such that the end-user adds a suitable amount of buffer or medium to bring the synthetic, modified RNAs to a desired unit dose concentration, e.g., 100 ng/ ⁇ .
  • kits and/or intramuscular delivery devices described herein can further comprise devices to facilitate single-adminstration or repeated or frequent infusions of a unit dose of a synthetic, modified RNA, such as a non-implantable delivery device, e.g., needle, syringe, pen device, or an implantatable delivery device, or reservoir.
  • the delivery device can include a mechanism to dispense a unit dose composition comprising a synthetic, modified RNA.
  • the device releases the unit dose composition comprising a synthetic, modified RNA continuously, e.g., by diffusion.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g., and, optionally, associated electronics.
  • a unit dose comprising between 0.03 mg/kg of body weight and 10 mg/kg of body weight of a synthetic, modified RNA encoding a therapeutic agent, wherein the synthetic, modified RNA comprises at least two modified nucleosides.
  • the at least two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-0-dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-0- methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-0- methyladenosine (m6A), N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'-0-trimethyladenosine (m62Am), 2'-0-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-0-methylguanosine (Gm), N2,7- dimethylguanosine (m2,7G), N2, 7-trimethylgua
  • a pharmaceutical composition for intramuscular delivery comprising the unit dose of a synthetic, modified RNA encoding a therapeutic agent of any one of paragraphs 1-15 and a pharmaceutically acceptable carrier.
  • a kit comprising (a) a container or vial containing the unit dose of a synthetic, modified RNA encoding a therapeutic agent of any one of paragraphs 1-15 or the pharmaceutical composition of paragraph 16, and (b) packaging and instructions therefor.
  • An intramuscular delivery device comprising the unit dose of a synthetic, modified RNA encoding a therapeutic agent of any one of paragraphs 1-15 or the pharmaceutical composition of paragraph 16.
  • intramuscular delivery device of paragraph 19 wherein the intramuscular delivery device is a non-implantable delivery device or an implantable delivery device.
  • An enhanced method for delivering synthetic, modified RNA into a subject comprising administering intramuscularly to a subject at least one unit dose of any one of paragraphs 1-15 or the pharmaceutical composition of paragraph 16.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the singular forms "a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
  • references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • the term 'cell' can be construed as a cell population, which can be either heterogeneous or homogeneous in nature, and can also refer to an aggregate of cells.
  • FIG. 1 Dose-dependency of protein expression upon intramuscular injection of synthetic, modified RNAs is shown in FIG. 1.
  • Various doses of a synthetic, modified RNA encoding Erythropoietin (EPO) were injected intramuscularly, and thirteen hours later plasma erythropoietin levels were examined using ELISA.
  • EPO Erythropoietin
  • increasing the doses of the synthetic, modified RNA leads to a proportional increase in plasma levels of erythropoietin.

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Abstract

L'invention concerne des compositions monodoses, des kits, des dispositifs d'administration, et des méthodes pour améliorer l'administration d'ARN synthétiques modifiés codant pour un agent thérapeutique à un patient. Ces compositions monodoses, kits, dispositifs d'administration, et méthodes pour améliorer l'administration peuvent être utilisés pour exprimer un agent thérapeutique désiré sans introduire d'ADN exogène ou des vecteurs viraux, et ainsi, ne provoquent pas de modifications permanentes du génome et n'ont pas un potentiel pour produire des effets mutagènes inattendus. Les ARN fournis au moyen des dosages de l'invention se sont avérés pouvoir produire une réaction dépendante de la dose en termes d'expression et de fonction de protéine.
PCT/US2012/066055 2011-11-23 2012-11-20 Méthodes pour une administration in vivo améliorée d'arn synthétiques modifiés WO2013078199A2 (fr)

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