WO2017048770A1 - Systèmes, compositions et procédés de formulation de compositions d'acides nucléiques - Google Patents

Systèmes, compositions et procédés de formulation de compositions d'acides nucléiques Download PDF

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Publication number
WO2017048770A1
WO2017048770A1 PCT/US2016/051614 US2016051614W WO2017048770A1 WO 2017048770 A1 WO2017048770 A1 WO 2017048770A1 US 2016051614 W US2016051614 W US 2016051614W WO 2017048770 A1 WO2017048770 A1 WO 2017048770A1
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Prior art keywords
lipid
nucleic acid
aqueous solution
component
organic solution
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PCT/US2016/051614
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English (en)
Inventor
Priya P. KARMALI
Victor Knopov
Amit Sagi
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Regulus Therapeutics, Inc.
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Publication of WO2017048770A1 publication Critical patent/WO2017048770A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/45—Mixing liquids with liquids; Emulsifying using flow mixing
    • B01F23/453—Mixing liquids with liquids; Emulsifying using flow mixing by moving the liquids in countercurrent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/20—Jet mixers, i.e. mixers using high-speed fluid streams
    • B01F25/23—Mixing by intersecting jets
    • B01F25/231—Mixing by intersecting jets the intersecting jets having the configuration of sheets, cylinders or cones

Definitions

  • compositions, and methods for encapsulating nucleic acids are provided herein.
  • systems, compositions, and methods are provided for automated encapsulating of nucleic acid in lipids.
  • RNA function often employ the use of antisense oligonucleotides that are designed to bind to an RNA target through Watson-Crick base pairing, and, once bound to the target, modulate its function.
  • antisense oligonucleotides are chemically modified to impart desired pharmacokinetic and pharmacodynamic properties to the oligonucleotides.
  • Modified oligonucleotides may modulate a target RNA through a variety of mechanisms, including mechanisms that involve binding of the modified oligonucleotide to the target RNA and interference with its function without promoting degradation of the RNA (e.g., steric hindrance), as well as mechanisms that do promote degradation of the RNA after binding of the modified oligonucleotide, by activities of enzymes such as RNaseH or Argonaute 2.
  • Numerous types of RNAs may be selected as targets of modified oligonucleotides, including messenger RNAs, pre-messenger RNAs, and non-coding RNAs such as microRNAs.
  • microRNAs also known as “mature microRNAs” are small
  • microRNAs (approximately 18-24 nucleotides in length), non-coding RNA molecules encoded in the genomes of plants and animals.
  • highly conserved, endogenously expressed microRNAs regulate the expression of genes by binding to the 3'-untranslated regions (3'-UTR) of specific mRNAs.
  • 3'-UTR 3'-untranslated regions
  • More than 1000 different microRNAs have been identified in plants and animals.
  • Certain mature microRNAs appear to originate from long endogenous primary microRNA transcripts (also known as pri-microRNAs, pri-mirs, pri-miRs or pri-pre- microRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21 (17), 4663-4670). While the biological function of many microRNAs has been identified, to date, there are only a limited number of such molecules that have been sufficiently developed for use as therapuetic molecules.
  • compounds and methods for encapsulating nucleic acids are provided herein.
  • compounds and methods are provided for automated encapsulating of nucleic acid in lipids.
  • the present disclosure provides a system for encapsulating a nucleic acid into a lipid nanoparticle, comprising: a) a first reservoir comprising an aqueous solution comprising a nucleic acid; b) a second reservoir comprising an organic solution comprising lipids; and c) a transport component configured to transport the organic solution and the aqueous solution into a mixing component, wherein the mixing region comprises a connector configured to introduce the aqueous solution and the organic solution at an approximately 180 degree angle relative to each other.
  • the transport component comprises a pump component configured to pump the organic solution and the aqueous solution into the mixing region at equal or different flow rates.
  • the system further comprises a pressurized tank comprising pressurized gas in operable communication with the mixing chamber.
  • the mixing region comprises a connector configured to introduce the aqueous solution and the organic solution at an approximately 180 degree angle relative to each other.
  • the mixing region comprises at least two nozzles configured to atomize the aqueous solution and the organic solution into fine microdroplets using the pressurized gas.
  • the mixing region comprises at least two nozzles configured to atomize the aqueous solution and the organic solution into fine microdroplets without the use of pressurized gas, e.g. where the flow rate of the liquid is sufficiently high to atomize the solution without the use of pressurized gas.
  • the nozzles are atomizing pressure assisted nozzles.
  • the nozzles are between 5-150 mm apart (e.g., approximately 10 mm apart).
  • the nozzles are each connected to a different pressure regulator component, wherein each of the pressure regulator component is individually regulated.
  • each nozzle is at between 3.0 PSI and 70.0 PSI pressure.
  • each nozzle is at between approximately 3 PSI and approximately 70 PSI pressure. In some embodiments, the pressure is 3.0 PSI. In some embodiments, the pressure is approximately 3 PSI. In some embodiments, the system utilizes a flow rate control component for controlling flow rate of the aqueous solution and the lipid solution, wherein the flow rate control component is configured to provide a flow rate between 10 ml/min and 5000 ml/min.
  • the nucleic acid is a miRNA, an antisense nucleic acid, an anti-miRNA, or a siRNA.
  • the lipid comprises one of more of a cationic lipid, a helper lipid, a phospholipid, cholesterol, or a pegylated lipid. In some embodiments, the lipid comprises a cationic lipid comprising one or more of a quaternary ammonium lipid, an ionizable lipid, or a lipidoid. In some embodiments, the cationic lipid has a molar ratio between 20 and 70 mol%. In some embodiments, the cationic lipid and the nucleic acid have a nitrogen/phosphate ratio between 1 and 15. In some embodiments, the organic solution comprises an alcohol (e.g., ethanol, propanol, or t-butanol).
  • an alcohol e.g., ethanol, propanol, or t-butanol.
  • the organic solution is at 20-50 degrees Celsius. In some embodiments, the aqueous solution is a buffer with pH from 3.0-9.0. In some embodiments, the aqueous solution is at 20-50 degrees Celsius. In some embodiments, the organic solution is present in the mixing component at between 20-50% (v/v). In some embodiments, the nucleic acid concentration in the mixing component is between 0.05 and 5 mg/mL. In some embodiments, the lipid concentration in the mixing component is 10 mg/mL.
  • the system further comprises a pump and the organic solution and the aqueous solution are pumped into the mixing component at equal flow rates by the pump. In some embodiments, the system further comprises a pump and the organic solution and the aqueous solution are pumped into the mixing component at different flow rates by the pump.
  • the transport component comprises a connector component that introduces the aqueous solution and the organic solution into the mixing component at an approximately 180 degree angle relative to each other. In some embodiments, the mixing component comprises nucleic acid at a concentration between 0.05-5 mg/mL and lipid at a concentration between 0.5- 50 mg/mL.
  • Additional embodiments provide a lipid nanoparticle comprising a nucleic acid encapsulated therein produced by the methods and systems described herein.
  • Figure 1 shows a schematic of an exemplary system of embodiments of the present disclosure.
  • Figure 2 shows a graph of particle size (y-axis, nm) vs. lipid concentration (x-axis, mM) as resulted from in process analysis.
  • Figure 3 shows a graph of particle size (y-axis, nm) vs. lipid concentration (x-axis, mM) as resulted from in process analysis.
  • Figure 4 shows a graph of particle size (y-axis, nm) vs. Nitrogen to Phosphate ratio (N/P) (x-axis) as resulted from in process analysis.
  • Approximately means a value (e.g., numerical value) plus or minus 10% (e.g., +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +1-2%, +/-1%, or other non-integer values encompassed therein).
  • Helper lipid means a neutral co-lipid in a lipid formulation (e.g. transfection or encapsulation reagent). In some embodiments, helper lipids increase transfection efficiency when included in tranfection complexes. The present disclosure is not limited to a particular helper lipid. In some embodiments, the helper lipid is an electroneutral lipid (e.g., DOPE).
  • DOPE electroneutral lipid
  • PEGated lipid means any lipid comprising one or more PEG molecules. The present disclosure is not limited to a particular molecular weight or length of PEG chain. In some embodiments, PEG chains are branched or straight. PEG is attached any any suitable location of the lipid.
  • Atomizing pressure assisted nozzle means a device configured to disperse liquid into fine microdroplets using pressurized gas.
  • an atomizing pressure assisted nozzle is a two-fluid nozzle.
  • Target nucleic acid means a nucleic acid to which an oligonucleotide is designed to hybridize.
  • Target RNA means an RNA to which an oligonucleotide is complementary.
  • Targeting in the context of designing a sequence means the process of design and selection of nucleobase sequence that will hybridize to a target nucleic acid.
  • “Targeted to” means having a nucleobase sequence that will allow hybridization to a target nucleic acid.
  • Modulation means a perturbation of function, amount, or activity. In certain embodiments, modulation means an increase in function, amount, or activity. In certain embodiments, modulation means a decrease in function, amount, or activity.
  • “Expression” means any functions and steps by which a gene's coded information is converted into structures present and operating in a cell.
  • oligonucleotide means a portion of linked nucleosides within a nucleic acid.
  • an oligonucleotide has a nucleobase sequence that is complementary to a region of a target nucleic acid.
  • an oligonucleotide is
  • an oligonucleotide is fully complementary to a region of a microRNA stem-loop sequence.
  • Segment means a smaller or sub-portion of a region.
  • MicroRNA means an endogenous non-coding RNA between 18 and 25 nucleobases in length, which is the product of cleavage of a pre-microRNA by a Dicer enzyme. Examples of mature microRNAs are found in the microRNA database known as miRBase. In certain embodiments, microRNA is abbreviated as “miRNA” or “miR.”
  • microRNA-regulated transcript means a transcript that is regulated by a microRNA.
  • “Monocistronic transcript” in the context of microRNA means a microRNA precursor containing a single microRNA sequence.
  • Anti-miR means an oligonucleotide having nucleobase sequence complementary to a microRNA. In certain embodiments, an anti-miR is a modified oligonucleotide.
  • Anti-miR-X where "miR-X” designates a particular microRNA, means an
  • oligonucleotide having a nucleobase sequence complementary to miR-X.
  • an anti-miR-X is fully complementary to miR-X. In certain embodiments, an anti- miR-X is at least 80%, at least 85%, at least 90%, or at least 95% complementary to miR-X. In certain embodiments, an anti-miR-X is a modified oligonucleotide.
  • Nucleobase sequence means the order of contiguous nucleobases in an oligomeric compound or nucleic acid, typically listed in a 5 ' to 3' orientation, independent of any sugar, linkage, and/or nucleobase modification.
  • Contiguous nucleobases means nucleobases immediately adjacent to each other in a nucleic acid.
  • Nucleobase complementarity means the ability of two nucleobases to pair non- covalently via hydrogen bonding.
  • “Complementary” means that one nucleic acid is capable of hybridizing to another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide capable of hybridizing to a target nucleic acid.
  • “Fully complementary” means each nucleobase of an oligonucleotide is capable of pairing with a nucleobase at each corresponding position in a target nucleic acid.
  • an oligonucleotide is fully complementary to a microRNA, i.e. each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA.
  • an oligonucleotide wherein each nucleobase has complementarity to a nucleobase within a region of a microRNA stem-loop sequence is fully complementary to the microRNA stem-loop sequence.
  • Percent complementarity means the percentage of nucleobases of an oligonucleotide that are complementary to an equal-length portion of a target nucleic acid. Percent
  • complementarity is calculated by dividing the number of nucleobases of the oligonucleotide that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total number of nucleobases in the oligonucleotide.
  • Percent identity means the number of nucleobases in first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
  • the first nucleic acid is a microRNA and the second nucleic acid is a microRNA.
  • the first nucleic acid is an oligonucleotide and the second nucleic acid is an oligonucleotide.
  • Hybridize means the annealing of complementary nucleic acids that occurs through nucleobase complementarity.
  • Mismatch means a nucleobase of a first nucleic acid that is not capable of pairing with a nucleobase at a corresponding position of a second nucleic acid.
  • nucleobase sequences means having the same nucleobase sequence, independent of sugar, linkage, and/or nucleobase modifications and independent of the methyl state of any pyrimidines present.
  • Oligomeric compound means a compound that comprises a plurality of linked monomeric subunits. Oligomeric compounds included oligonucleotides.
  • Oligonucleotide means a compound comprising a plurality of linked nucleosides, each of which can be modified or unmodified, independent from one another.
  • Subject means a human or non-human animal selected for treatment or therapy.
  • Subject in need thereof means a subject exhibiting one or more clinical indicators consistent with a need of a therapy or treatment.
  • Subject suspected of having means a subject identified as exhibiting one or more clinical indicators of a disease.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self- administering.
  • Parental administration means administration other than through ingestion, such as by injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intracardial administration, or pulmonary administration.
  • Subcutaneous administration means administration just below the skin.
  • Intravenous administration means administration into a vein.
  • Intracardial administration means administration into the heart.
  • intracardial administration occurs by way of a catheter. In certain embodiments, intracardial administration occurs by way of open heart surgery.
  • “Pulmonary administration” means administration to the lungs.
  • administering refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
  • “Therapy” means a disease treatment method.
  • therapy includes, but is not limited to, chemotherapy, radiation therapy, or administration of a pharmaceutical agent.
  • “Treatment” means the application of one or more specific procedures used for the cure or amelioration of a disease. In certain embodiments, the specific procedure is the
  • “Amelioration” means a lessening of severity of at least one indicator of a condition or disease, which may be in terms of duration or intensity of the indicator. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
  • “Therapeutic agent” means a pharmaceutical agent used for the cure, amelioration or prevention of a disease.
  • Dose means a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • Dosage unit means a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial containing lyophilized oligonucleotide.
  • a dosage unit is a vial containing reconstituted oligonucleotide.
  • “Therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to a subject.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent.
  • a pharmaceutical composition may comprise a sterile aqueous solution.
  • “Pharmaceutical agent” means a substance that provides a therapeutic effect when administered to a subject.
  • compositions, and methods for encapsulating nucleic acids are provided for automated encapsulating of nucleic acid in lipds.
  • a system according to this disclosure can be an integrated device or an assembly of operably connected devices.
  • compositions, systems, and methods for performing, for example, a single step process for preparing monodispersive nanoparticles encapsulating oligonucleotides with high efficiency, seamless scale up and reproducibility are described herein.
  • the systems and methods do not require or involve preparing oligonucleotide loaded nanoparticles at high dilution thereby providing a major cost advantage during manufacturing.
  • systems for encapsulating a nucleic acid into a lipid nanoparticle comprising a first reservoir 5 comprising an aqueous solution comprising a nucleic acid; a second reservoir 3 comprising an organic solution comprising lipids; and a transport component 7.
  • the transport component 7 is configured to transport the organic solution and the aqueous solution into a mixing component 2.
  • the transport component 7 comprises a pump configured to pump the organic solution and the aqueous solution into the mixing component 2 at the same or different flow rates.
  • the mixing component 2 comprises a connector 1 configured to introduce the aqueous solution and the organic solution at an approximately 180 degree (e.g., 160 to 200 degrees or values between 160 and 200 degrees (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, etc.)) angle relative to each other.
  • 180 degree e.g., 160 to 200 degrees or values between 160 and 200 degrees (e.g., 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182,
  • the connector 1 comprises or is a plurality of nozzles.
  • the nozzles are atomizing pressure assisted nozzles. Any suitable nozzle may be utilized. Nozzles, including atomizing pressure assisted nozzles are commercially available or are described, for example, in U.S. Pat. App. No. 20110175244 and U.S. Pat. No. 6616068; each of which is herein incorporated by reference in its entirety. In some
  • the nozzles are between 5-150 mm apart (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114
  • each of the nozzles is connected to a differential pressure regulator component that is individually regulated (e.g., each of the nozzles operates at different pressures or flow rates and is independently controlled).
  • a differential pressure regulator component that is individually regulated (e.g., each of the nozzles operates at different pressures or flow rates and is independently controlled).
  • the present disclosure is not limited to particular nozzle flow rates or pressure.
  • each nozzle is at between 3.0 PSI and 70.0 PSI pressure (e.g., 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0. 69.0, 70.0 PSI or fractions thereof), although other pressures are
  • the system comprises a flow rate control component for controlling flow rate of the aqueous solution and the lipid solution.
  • the present disclosure is not limited to particular flow rates.
  • the flow rate control component is configured to maintain a flow rate between 10 ml/min and 5000 ml/min (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 ml/min or fractions thereof), although other flow rates are specifically contemplated.
  • the system comprises a pressurized tank 6 and valve 4 comprising pressurized gas.
  • the nozzles atomize the aqueous solution and the organic solution into fine microdroplets using the pressurized gas.
  • the pressurized gas comprises nitrogen, argon, or compressed air.
  • the pressurized gas comprises nitrogen.
  • the pressurized gas comprises argon.
  • the pressurized gas comprises compressed air.
  • the systems and methods described herein provide the advantage of mixing the nucleic acid at a concentration between 0.05-5 mg/mL (e.g., 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/ml, or fractions thereof) and the lipid at a concentration between 0.5-50 mg/mL (e.g., 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0
  • nucleic acid is, for example, a miRNA, an antisense nucleic acid, an anti-miRNA, or a siRNA.
  • nucleic acids are single stranded, double stranded, comprise modified nucleotides or nucleosides, or a combination thereof.
  • the lipid comprises one of more of a cationic lipid (e.g., one or more of a quaternary ammonium lipid, an ionizable lipid, or a lipidoid), a helper lipid, a phospholipid, cholesterol, or a pegylated lipid.
  • a cationic lipid e.g., one or more of a quaternary ammonium lipid, an ionizable lipid, or a lipidoid
  • the cationic lipid has a molar ratio to total lipid of between 20 and 70 mol%.
  • the cationic lipid and the nucleic acid have a nitrogen/phosphate ratio between 1 and 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or a fraction thereof).
  • the organic solution further comprises an alcohol (e.g., ethanol, propanol, or t-butanol).
  • the lipid component further comprises one or more of a helper lipid, a sterol, a PEG-lipid, an anionic lipid, etc.
  • the aqueous solution is a buffer with pH from 3.0-9.0 (e.g., 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or a fration thereof).
  • the organic and/or aqueous solutions are maintained at 20-50 degrees Celsius (e.g., 20, 21 , 2, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 degrees Celcius or a fraction thereof).
  • the organic solution is present in the mixing component at between 20-50% (v/v) (e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% or a fraction thereof).
  • the nucleic acid concentration in the mixing component is between 0.05 and 5 mg/mL (e.g., 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/ml, or fractions thereof).
  • 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 ,
  • the lipid concentration in the mixing component is between 0.5 mg/mL and 50 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 0.5 mg/mL and 30 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 0.5 mg/mL and 20 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 1 mg/mL and 20 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 2 mg/mL and 20 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 5 mg/mL and 20 mg/mL.
  • the lipid concentration in the mixing component is between 0.5 mg/mL and 15 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 1 mg/niL and 15 mg/niL. In some embodiments, the lipid concentration in the mixing component is between 2 mg/mL and 15 mg/mL. In some embodiments, the lipid concentration in the mixing component is between 5 mg/mL and 15 mg/mL.
  • Exemplary lipid concentrations include 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
  • the lipid concentration in the mixing component is 10 mg/mL.
  • compositions produced by the systems and methods described herein find use in a variety of applications.
  • compositions are used in research, screening, and therapeutic applications.
  • the present disclosure provides therapeutic compositions comprising encapsulated microRNAs or anti-microRNAs and uses thereof.
  • MicroRNAs typically target a set of target genes, resulting in degradation of target gene mRNA and reduction of mRNA levels. Overexpression and/or increased activity of various microRNAs have been associated with diseases and/or conditions, which has led to efforts to reduce the level and/or activity of certain specific microRNAs, e.g., using modified
  • oligonucleotides targeted to the selected microRNA to treat the associated diseases and/or conditions.
  • more than one microRNA is involved in a particular disease or condition, or involved in associated diseases and/or conditions, such that it would be desirable to target more than one microRNA for treatment. It would be beneficial, in such instances, to target more than one microRNA with a single compound, having two regions, each region targeted to a different microRNA, allowing for dosing of a single compound, and targeting of a single compound to the desired cell type.
  • nucleic acids utilized in the compositions and methods described herein are modified.
  • Exemplary modified oligonucleotides and methods for their synthesis are described, for example, in US Patent Nos. 8,765,701 and 8,846,631, each of which is incorporated by reference herein in its entirety for any purpose.
  • a modified oligonucleotide comprises one or more modified nucleosides.
  • a modified nucleoside is a stabilizing nucleoside.
  • An example of a stabilizing nucleoside is a sugar-modified nucleoside.
  • a modified nucleoside is a sugar-modified nucleoside.
  • the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage and may include further modifications independent from the sugar modification.
  • a sugar modified nucleoside is a 2' -modified nucleoside, wherein the sugar ring is modified at the 2' carbon from natural ribose or 2'-deoxy-ribose.
  • a 2'-modified nucleoside has a bicyclic sugar moiety.
  • the bicyclic sugar moiety is a D sugar in the alpha configuration.
  • the bicyclic sugar moiety is a D sugar in the beta configuration.
  • the bicyclic sugar moiety is an L sugar in the alpha configuration.
  • the bicyclic sugar moiety is an L sugar in the beta configuration.
  • the bicyclic sugar moiety comprises a bridge group between the 2' and the 4'-carbon atoms. In certain such embodiments, the bridge group comprises from 1 to 8 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups.
  • the bicyclic sugar moiety is bridged between the 2' and 4' carbon atoms with a biradical group selected from -0-(CH2) P -, -0-CH2-,-0-CH2CH2-, -O-CH(alkyl)-, -NH-(CH 2 )p-, -N(alkyl)-(CH 2 ) P -, -O-CH(alkyl)-, -(CH(alkyl))-(CH 2 ) P -, -NH-0-(CH 2 ) P -, - N(alkyl)-0-(CH2)p-, or -0-N(alkyl)-(CH2) P -, wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be further substituted. In certain embodiments, p is 1, 2 or 3.
  • a bicyclic sugar moiety is -0-(CH2)-, also known as 'locked nucleic acid' or 'LNA.'
  • These 2'-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2'-modified nucleoside comprises a 2 '-substituent group selected from F, O-CH3, and OCH2CH2OCH3.
  • a sugar- modified nucleoside is a 4'-thio modified nucleoside.
  • a sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside.
  • a 4'-thio modified nucleoside has a ⁇ -D-ribonucleoside where the 4'-0 replaced with 4'-S.
  • a 4'-thio-2'- modified nucleoside is a 4'-thio modified nucleoside having the 2'-OH replaced with a 2'- substituent group.
  • Suitable 2'-substituent groups include 2'-0 ⁇ 3 ⁇ 4, 2'-0-(CH2)2-OCH3, and 2'-F.
  • a modified oligonucleotide comprises one or more
  • each intemucleoside linkage of an oligonucleotide is a modified intemucleoside linkage.
  • a modified intemucleoside linkage comprises a phosphorus atom.
  • a modified oligonucleotide comprises at least one
  • each intemucleoside linkage of a modified oligonucleotide is a phosphorothioate intemucleoside linkage.
  • a modified intemucleoside linkage does not comprise a phosphorus atom.
  • an intemucleoside linkage is formed by a short chain alkyl intemucleoside linkage.
  • an intemucleoside linkage is formed by a cycloalkyl intemucleoside linkages.
  • an intemucleoside linkage is formed by a cycloalkyl intemucleoside linkages.
  • intemucleoside linkage is formed by a mixed heteroatom and alkyl intemucleoside linkage. In certain such embodiments, an intemucleoside linkage is formed by a mixed heteroatom and cycloalkyl intemucleoside linkages. In certain such embodiments, an intemucleoside linkage is formed by one or more short chain heteroatomic intemucleoside linkages. In certain such embodiments, an intemucleoside linkage is formed by one or more heterocyclic intemucleoside linkages. In certain such embodiments, an intemucleoside linkage has an amide backbone. In certain such embodiments, an intemucleoside linkage has mixed N, O, S and CH2 component parts.
  • a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, a modified oligonucleotide comprises one or more 5- methylcytosines. In certain embodiments, each cytosine of a modified oligonucleotide comprises a 5-methylcytosine.
  • a modified nucleobase is selected from 5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certain embodiments, a modified nucleobase is selected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certain embodiments, a modified nucleobase is selected from 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5- propynyluracil and 5-propynylcytosine.
  • a modified nucleobase comprises a poly cyclic heterocycle. In certain embodiments, a modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, a modified nucleobase comprises a phenoxazine derivative. In certain embodiments, the phenoxazine can be further modified to form a nucleobase known in the art as a G-clamp.
  • Suitable motifs for modified oligonucleotides of the present disclosure include, but are not limited to, fully modified, uniformly modified, positionally modified, and gapmer.
  • Modified oligonucleotides having a fully modified motif, including a uniformly modified motif may be designed to target mature miRNAs.
  • modified oligonucleotides having a fully modified motif, including a uniformly modified motif may be designed to target certain sites of pri-miRNAs or pre-miRNAs, to block the processing of miRNA precursors into mature miRNAs.
  • Modified oligonucleotides having a fully modified motif or uniformly modified motif are effective inhibitors of miRNA activity.
  • a modified oligonucleotide having a gapmer motif may have an internal region consisting of linked 2'-deoxynucleotides, and external regions consisting of linked 2'-modified nucleosides. Such a gapmer may be designed to elicit RNase H cleavage of a miRNA precursor.
  • the internal 2'-deoxynucleoside region serves as a substrate for RNase H, allowing the cleavage of the miRNA precursor to which a modified oligonucleotide is targeted.
  • each nucleoside of each external region comprises the same 2'-modified nucleoside.
  • one external region is uniformly comprised of a first 2'- modified nucleoside and the other external region is uniformly comprised of a second 2'- modified nucleoside.
  • oligonucleotide The activity of an oligonucleotide is based on the specific hybridization event that occurs between an oligonucleotide and its target RNA and produces a desired pharmacological endpoint. In order for this to occur, certain pharmacokinetic processes must take place, for example, delivery of an intact drug to the target cell or tissue, and entry of the oligonucleotide into the cell containing the target RNA.
  • Oligonucleotides may be conjugated to one or more moieties which improve delivery to the target cell or tissue and/or cellular uptake of the oligonucleotide, ultimately resulting in enhanced potency. For example, increased cellular uptake of compounds may be achieved by utilizing conjugates that are ligands for cell-surface receptors.
  • the binding of a ligand conjugated to an exogenous molecule leads to receptor-mediated endocytosis of the conjugated molecule, thereby facilitating transmembrane transport of the exogenous molecule.
  • an exogenous molecule e.g., a drug
  • the targeted delivery to hepatocyte cells may be achieved by covalently attaching a conjugate comprising a carbohydrate moiety to an oligonucleotide.
  • the conjugated oligonucleotide is transported across the cell membrane into the hepatocyte.
  • the target RNA(s) are associated with a disease. Accordingly, administration of a conjugated oligonucleotide compound to a subject may treat, prevent, or delay the onset of a disease associated with the target RNA(s).
  • the disease is associated with target RNA(s) expressed in a liver cell. In certain embodiments, the disease is associated with target RNA(s) expressed in a hepatocyte. In certain embodiments, the disease is associated with target RNA(s) expressed in a macrophage. In certain embodiments, the disease is associated with target RNA(s) expressed in a dendritic cell. In certain
  • the target RNA(s) are microRNA(s).
  • a target RNA may be any nucleic acid capable of being targeted including, without limitation, microRNAs, pri-microRNAs, pre-microRNAs, pre-messenger RNAs, messenger RNAs, long noncoding RNAs, small transfer RNAs, small nuclear RNAs, small nucleolar RNAs, small ribosomal RNAs, small hairpin RNAs, endogenous antisense RNAs, guide RNAs, tiny noncoding RNAs, small single or double stranded RNAs that are encoded by heterochromatic repeats at centromeres or other chromosomal origin, and any precursors thereof.
  • Target RNAs may be coding or non-coding sequences; single- or double-stranded, or single- stranded with partial double-stranded character; may occur naturally within introns or exons of messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), or transfer RNAs (tRNAs); and can be endogenously transcribed or exogenously produced.
  • mRNAs messenger RNAs
  • rRNAs ribosomal RNAs
  • tRNAs transfer RNAs
  • the function of the target RNA may be inhibited through a nondegradative mechanism, for example RNA antagonism, modulation of RNA splicing, modulation of polyadenylation, disruption of RNA secondary structure, and inhibition of translation, or through a mechanism that promotes degradation of the target RNA, for example RNase H, RNA interference, ribozymes, and double-stranded RNases.
  • a nondegradative mechanism for example RNA antagonism, modulation of RNA splicing, modulation of polyadenylation, disruption of RNA secondary structure, and inhibition of translation
  • a mechanism that promotes degradation of the target RNA for example RNase H, RNA interference, ribozymes, and double-stranded RNases.
  • any of the compounds provided herein may comprise an oligonucleotide comprising a region having a nucleobase sequence complementary to a microRNA.
  • Nucleobase sequences of certain mature microRNA and their corresponding stem-loop sequence are found in miRBase, an online searchable database of microRNA sequences and annotation, found at microrna.sanger.ac.uk.
  • Entries in the miRBase Sequence database represent a predicted hairpin portion of a microRNA transcript (the stem-loop), with information on the location and sequence of the mature microRNA sequence.
  • the microRNA stem-loop sequences in the database are not strictly precursor microRNAs (pre-microRNAs), and may in some instances include the pre-microRNA and some flanking sequence from the presumed primary transcript.
  • sequences of the microRNA targets encompass any version of the microRNA, including the sequences described in Release 15.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database.
  • a sequence database release may result in the re-naming of certain microRNAs.
  • compositions generated by the systems and methods described herein encompass oligonucleotides comprising regions that are complementary to any nucleobase sequence version of the microRNA targets.
  • each nucleobase of a region of an oligonucleotide targeted to a microRNA is capable of undergoing base-pairing with a nucleobase at each corresponding position in the nucleobase sequence of the microRNA, or a precursor thereof.
  • the nucleobase sequence of a region of an oligonucleotide may have one or more mismatched base pairs with respect to its target microRNA or precursor sequence, and remains capable of hybridizing to its target sequence.
  • a region of an oligonucleotide has a nucleobase sequence that is complementary to the nucleobase sequence of a microRNA precursor, such as a microRNA stem-loop sequence.
  • a region of an oligonucleotide having a nucleobase sequence complementary to a microRNA is also complementary to a region of the corresponding microRNA precursor.
  • oligonucleotide is less than the length of a microRNA, or a precursor thereof.
  • the region of the oligonucleotide has a nucleobase sequence that is
  • Such a region of an oligonucleotide has a nucleobase sequence that is 100% complementarity to a 22 nucleobase portion of the microRNA. Further, such a region of an oligonucleotide is considered to be 100% complementary to the microRNA.
  • a region of the nucleobase sequence of an oligonucleotide is fully complementary to a region of the nucleobase sequence of a microRNA.
  • 8 contiguous nucleobases of an oligonucleotide are each complementary to 8 contiguous nucleobases of a microRNA.
  • 9 contiguous nucleobases of an oligonucleotide are each complementary to 9 contiguous nucleobases of a microRNA.
  • 10 contiguous nucleobases of an oligonucleotide are each complementary to 10 contiguous nucleobases of a microRNA.
  • 1 1 contiguous nucleobases of an oligonucleotide are each complementary to 11 contiguous nucleobases of a microRNA.
  • 12 contiguous nucleobases of an oligonucleotide are each complementary to 12 contiguous nucleobases of a microRNA.
  • 13 contiguous nucleobases of an oligonucleotide are each complementary to 13 contiguous nucleobases of a microRNA.
  • 14 contiguous nucleobases of an oligonucleotide are each complementary to 14 contiguous nucleobases of a microRNA.
  • 15 contiguous nucleobases of an oligonucleotide are each complementary to 15 contiguous nucleobases of a microRNA.
  • a nucleobase sequence of a region of an oligonucleotide is 100% complementary to a microRNA nucleobase sequence listed herein, or a precursor thereof.
  • a region of an oligonucleotide has a nucleobase sequence having one mismatch with respect to the nucleobase sequence of a microRNA, or a precursor thereof.
  • a region of an oligonucleotide has a nucleobase sequence having two mismatches with respect to the nucleobase sequence of a microRNA, or a precursor thereof.
  • a region of an oligonucleotide has a nucleobase sequence having no more than two mismatches with respect to the nucleobase sequence of a microRNA, or a precursor thereof.
  • the mismatched nucleobases are contiguous. In certain embodiments, the mismatched nucleobases are not contiguous.
  • Modified or unmodified oligonucleotides may be made with automated, solid phase synthesis methods known in the art. During solid phase synthesis, phosphoramidite monomers are sequentially coupled to a nucleoside that is covalently linked to a solid support. This nucleoside is the 3' terminal nucleoside of the oligonucleotide.
  • the coupling cycle comprises four steps: detritylation (removal of a 5 '-hydroxyl protecting group with acid), coupling (attachment of an activated phosphoroamidite to the support bound nucleoside or oligonucleotide), oxidation or sulfurization (conversion of a newly formed phosphite trimester with an oxidizing or sulfurizing agent), and capping (acetylation of unreacted 5 '-hydroxyl groups).
  • the solid support-bound oligonucleotide is subjected to a detritylation step, followed by a cleavage and deprotection step that simultaneously releases the oligonucleotide from the solid support and removes the protecting groups from the bases.
  • the solid support is removed by filtration, the filtrate is concentrated and the resulting solution is tested for identity and purity.
  • the oligonucleotide is then purified, for example using a column packed with anion-exchange resin.
  • Any of the compounds provided herein may be prepared as a pharmaceutical composition.
  • a pharmaceutical composition is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.).
  • a pharmaceutical composition comprises a compound provided herein at a dose within a range selected from 25 mg to 800 mg, 25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg.
  • such pharmaceutical compositions comprise a compound provided herein present at a dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg,
  • a pharmaceutical composition of the comprises a dose compound provided herein selected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800mg.
  • a pharmaceutical composition comprising a compound provided herein is administered at a dose of 10 mg/kg or less, 9 mg/kg or less, 8 mg/kg or less, 7.5 mg/kg or less, 7 mg/kg or less, 6.5 mg/kg or less, 6 mg/kg or less, 5.5 mg/kg or less, 5 mg/kg or less, 4.5 mg/kg or less, 4 mg/kg or less, 3.5 mg/kg or less, 3 mg/kg or less, 2.5 mg/kg or less, 2 mg/kg or les, 1.5 mg/kg or less, 1 mg/kg or less, 0.75 mg/kg or less, 0.5 mg/kg or less, or 0.25 mg/kg or less.
  • a pharmaceutical composition provided herein comprises a compound in a therapeutically effective amount.
  • the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions described herein.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
  • a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more compounds provided herein to specific tissues or cell types.
  • pharmaceutical compositions include liposomes as described above coated with a tissue-specific antibody.
  • a pharmaceutical composition provided herein is prepared for oral, buccal, transmucosal, injection, or other methods.
  • administering to a subject comprises parenteral administration. In certain embodiments, administering to a subject comprises intravenous administration. In certain embodiments, administering to a subject comprises subcutaneous administration.
  • administering to a subject comprises intraarterial, pulmonary, oral, rectal, transmucosal, intestinal, enteral, topical, transdermal, suppository, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, intramuscular, intramedullary, and intratumoral administration.
  • Treatments for a disease associated with a target RNA may comprise more than one therapy.
  • methods for treating a subject having or suspected of having a disease associated with a target RNA comprising administering at least one therapy in addition to administering an unmodified or modified oligonucleotide or a compound comprising an oligonucleotide.
  • the at least one additional therapy comprises a pharmaceutical agent.
  • pharmaceutical agents include anti-inflammatory agents.
  • an anti-inflammatory agent is a steroidal anti-inflammatory agent.
  • a steroid anti-inflammatory agent is a corticosteroid.
  • a corticosteroid is prednisone.
  • an anti-inflammatory agent is a non-steroidal anti-inflammatory drugs.
  • a non-steroidal antiinflammatory agent is ibuprofen, a COX-I inhibitors, or a COX-2 inhibitors.
  • pharmaceutical agents include, but are not limited to, diuretics (e.g. sprionolactone, eplerenone, furosemide), inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators, angiotensin II converting enzyme (ACE) inhibitors (e.g. are captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril), angiotensin II receptor blockers (ARB) (e.g.
  • diuretics e.g. sprionolactone, eplerenone, furosemide
  • inotropes e.g. dobutamine, milrinone
  • digoxin e.g. dobutamine, milrinone
  • vasodilators e.g. are captopril, enalapril, lisinopril, benaze
  • nitrates e.g. isosorbide mononitrate, isosorbide dinitrate
  • beta-blockers e.g. carvedilol, metoprolol
  • natriuretic peptides e.g. nesiritide
  • pharmaceutical agents include heparinoids.
  • a heparinoid is pentosan polysulfate.
  • an additional therapy may be a pharmaceutical agent that enhances the body's immune system, including low-dose cyclophosphamide, thymostimulin, vitamins and nutritional supplements (e.g., antioxidants, including vitamins A, C, E, beta- carotene, zinc, selenium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g., the immunostimulating complex (ISCOM), which comprises a vaccine formulation that combines a multimeric presentation of antigen and an adjuvant.
  • vitamins and nutritional supplements e.g., antioxidants, including vitamins A, C, E, beta- carotene, zinc, selenium, glutathione, coenzyme Q-10 and echinacea
  • vaccines e.g., the immunostimulating complex (ISCOM), which comprises a vaccine formulation that combines a multimeric presentation of antigen and an adjuvant.
  • ISCOM immunostimulating complex
  • the additional therapy is selected to treat or ameliorate a side effect of one or more pharmaceutical compositions described herein.
  • side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.
  • increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
  • increased bilirubin may indicate liver toxicity or liver function abnormality.
  • additional pharmaceutical agents include, but are not limited to, immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); salicylates; antibiotics; antivirals; antifungal agents; adrenergic modifiers; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents;
  • IVIg intravenous immunoglobulin
  • analgesics e.g., acetaminophen
  • salicylates antibiotics
  • antivirals e.g., antivirals
  • antifungal agents e.g., antifungal agents
  • adrenergic modifiers
  • kits can also contain instructions for using a compound provided herein.
  • a compound provided herein can be present within a vial.
  • a plurality of vials, such as 10, can be present in, for example, dispensing packs.
  • the vial is manufactured so as to be accessible with a syringe.
  • kits may be used for administration a compound provided herein to a subject.
  • the kit can further comprise one or more of the following: syringe, alcohol swab, cotton ball, and/or gauze pad.
  • the compounds can be present in a pre-filled syringe (such as a single-dose syringes with, for example, a 27 gauge, 1 ⁇ 2 inch needle with a needle guard), rather than in a vial.
  • a plurality of pre-filled syringes, such as 10 can be present in, for example, dispensing packs.
  • the kit can also contain instructions for administering the compounds.
  • This example shows encapsulation of anti-miR using the systems and methods described herein.
  • the lipid components were dissolved in absolute ethanol at a final lipid concentration of 1 mM with the following molar ratio and composition: 40% l,2-dioleoyl-3-trimethylammonium- propane (chloride salt) (DOTAP), 25% l ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 33% Cholesterol, 2% l ⁇ -dimyristoyl-OT-glycero-S-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt)(PEG-DMPE).
  • DOTAP l,2-dioleoyl-3-trimethylammonium- propane
  • DOPE 25% l ,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Cholesterol 2% l ⁇ -dimyristoy
  • the anti-miR was dissolved in 25 mM sodium acetate buffer at a concentration to provide an anti miR/cationic lipid molar ratio (mol/mol) of 0.02 (1 :50) and a weight ratio (wt/wt) of 0.074.
  • the final lipid cone. (mM) was 1 mM.
  • Both solutions were pumped at equal flow rates into a mixing component consisting of two atomizing nozzles oriented at 180 degrees relative to each other.
  • the lipid/ethanol and the anti-miR/buffer solutions were atomized into fine microdroplets using pressurized gas.
  • the solvent composition within the mixing chamber was: 50% EtOH/50% Buffer.
  • the anti-miR/lipid particles were immediately diluted and then diafiltered against PBS to remove the ethanol and exchange the buffer.
  • This example describes the effects of ethanol concentration, flow rate, and air pressure withing the mixing component on anti-miR/lipid particle formation.
  • Particles were prepared using the same procedures and composition described in Example 1 except that the flow rates and air pressures of lipid/ethanol and anti-miR/buffer were varied. Distance between the nozzles was kept at 10 mm for all conditions tested.
  • the anti-miR/lipid particles were immediately diluted and then diafiltered against PBS to remove the ethanol and exchange the buffer. The resulting particles were characterized using DLS to determine particle size (Z-ave diameter) and PDI. Mean particle size and PDI were determined both prior to filtration ("In Process") through a 0.2 ⁇ filter.
  • Table 2 shows the effect of flow rate and gas pressure on Z-ave diameter and PDI. Varying the ethanol percentage (EtOH %) from 30-50 %, flow rates, and air pressures did not significantly affect the particle size or polydispersity index (PDI) for this formulation. Table 2
  • This example describes the effect of stabilization temperature and choice of solvent on the formation of anti-miR lipid nanoparticles.
  • Particles were prepared using the same procedures and composition described in Example 1 except that the temperature of the PBS used to stabilized the particles was increased to approximately 40°C.
  • Isopropanol (IP A) was also evaluated as an alternative solvent, replacing ethanol (EtOH) to dissolve the lipid mixture.
  • the flow rates were kept constant at 35 mL/min for the anti-miR/buffer solution and at 18.84 mL/min for the lipid/solvent mixture.
  • the anti-miR/lipid particles were immediately diluted in PBS at approximately 40°C and then diafiltered against PBS to remove the ethanol and exchange the buffer.
  • Table 3 shows the effect of stabilization temperature and lipid solvent.
  • the lowest PDI was observed at elevated temperature, a distance of 10mm distance, and pressure of 3.0 PSI (See Table 3).
  • the use of IP A as the lipid solvent further improved PDI, but also increased particle size compared to EtOH as the lipid solvent. Encapsulation efficiency was >99% for all conditions tested.
  • Particles were prepared using the same procedures and composition described in Example 1. The anti-miR/lipid particles were immediately diluted in PBS at approximately 40°C and then diafiltered against PBS to remove the ethanol and exchange the buffer. Particles were characterized using dynamic light scattering (DLS) to determine mean particle size (Z-ave diameter), polydispersity index (PDI), and Zeta Potential (ZP). The encapsulation efficiency was determined using Ribogreen.
  • DLS dynamic light scattering
  • Z-ave diameter mean particle size
  • PDI polydispersity index
  • ZP Zeta Potential
  • the first experiment identified the maximum lipid concentration compatible with the equipment procedure described in Example 1.
  • the lipid concentration was increased from 20 nM upto 40 mM.
  • the anti-miR concentration in buffer was also increased to maintain an anti- miR/cationic Lipid molar ratio (mol/mol) of 0.0175 and a weight ratio (wt/wt) of 0.073.
  • the N/P ratio was maintained at 3.17.
  • Distance between the nozzles was kept at 10 mm and the flow rates were kept constant at 35 mL/min for the anti-miR/buffer solution and at 18.84 mL/min for the lipid/ethanol mixture.
  • the air pressure for both nozzles was kept constant at 3.0 PSI and the temperature of the PBS for stabilizing the particles was maintained at 35 °C to 40°C.
  • Table 4 and Figure 2 show the effect of lipid concentration on particle size. Increasing concentration increased particle size from 88 nm upto 120 nm. The anti-miR/lipid suspension prepared at 40 mM lipid concentration was too thick to be processed by diafiltration.
  • Lipid concentration was increased from 1 mM up to 20 mM.
  • the anti-miR concentration in buffer was also increased to maintain an anti-miR/cationic Lipid molar ratio (mol/mol) of 0.0175 and a weight ratio (wt/wt) of 0.073.
  • the N/P ratio was maintained at 3.17.
  • Distance between the nozzles was kept at 10 mm and the flow rates were kept constant at 35 mL/min for the anti-miR/buffer solution and at 18.84 mL/min for the lipid/ethanol mixture.
  • the air pressure for both nozzles was kept constant at 3.0 PSI and the temperature of the PBS for stabilizing the particles was maintained at 35 °C to 40°C.
  • Table 5 and Figure 3 show the effect of lipid concentration on particles for lipid concentrations less than or equal to 20 mM. Particle size increased and PDI decreased with increasing lipid concentration. Encapsulation efficiency was >99% for all conditions tested. Table 5
  • N/P nitrogen to phosphorus
  • EE encapsulation efficiency
  • the N/P charge ratio was varied from 1.0 to 2.5 while maintining a constant lipid concentration of 10 mM. Distance between the nozzles was kept at 10 mm and the flow rates were kept constant at 35 mL/min for the anti-miR/buffer solution and at 18.84 mL/min for the lipid/ethanol mixture. The air pressure for both nozzles was kept constant at 3.0 PSI and the temperature of the PBS for stabilizing the particles was maintained at 35 °C to 40°C.
  • Table 6 and Figure 4 show the effect of N/P ratio on particle size and encapsulation efficiency. Particle size increased with decreasing N/P while PDI remained unchanged.
  • Particles were prepared using the same procedures and composition as described in Example 1 with a lipid concentration of 10 mM and an N/P ratio of 2.5.
  • the anti-miR was dissolved in 25 mM sodium acetate buffer at a concentration to provide an anti miR/cationic Lipid molar ratio (mol/mol) of 0.0222 (1 :50) and a weight ratio (wt/wt) of 0.095.
  • Distance between the nozzles was kept at 10 mm and the flow rates were kept constant at 35 mL/min for the anti-miR/buffer solution and at 18.84 mL/min for the lipid/ethanol mixture.
  • the air pressure for both nozzles was kept constant at 3.0 PSI.
  • the anti-miR/lipid particles were immediately diluted in PBS at 35 °C to 40 °C and then diafiltered against PBS to remove the ethanol and exchange the buffer. Final product was filtered through a sterilizing grade, PES, 0.2 ⁇ filter. Particles were characterized (both before and after filtration) using dynamic light scattering (DLS) to determine mean particle size (Z-ave diameter) and polydispersity index (PDI). The encapsulation efficiency (EE) was determined using Ribogreen.
  • DLS dynamic light scattering
  • PDI polydispersity index
  • EE encapsulation efficiency

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Abstract

La présente invention concerne des composés et des procédés d'encapsulation d'acides nucléiques. Dans certains modes de réalisation, des composés et des procédés sont prévus pour l'encapsulation automatisée d'acides nucléiques dans des lipides.
PCT/US2016/051614 2015-09-15 2016-09-14 Systèmes, compositions et procédés de formulation de compositions d'acides nucléiques WO2017048770A1 (fr)

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US11634379B2 (en) 2014-06-25 2023-04-25 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11168051B2 (en) 2015-06-29 2021-11-09 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11712481B2 (en) 2015-10-28 2023-08-01 Acuitas Therapeutics, Inc. Lipid nanoparticle formulations
US11040112B2 (en) 2015-10-28 2021-06-22 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11648324B2 (en) 2015-10-28 2023-05-16 Acuitas Therapeutics, Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11357856B2 (en) 2017-04-13 2022-06-14 Acuitas Therapeutics, Inc. Lipids for delivery of active agents
US11820728B2 (en) 2017-04-28 2023-11-21 Acuitas Therapeutics, Inc. Carbonyl lipids and lipid nanoparticle formulations for delivery of nucleic acids
US11639329B2 (en) 2017-08-16 2023-05-02 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US11542225B2 (en) 2017-08-17 2023-01-03 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US11524932B2 (en) 2017-08-17 2022-12-13 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
US12065396B2 (en) 2017-08-17 2024-08-20 Acuitas Therapeutics, Inc. Lipids for use in lipid nanoparticle formulations
WO2020061426A3 (fr) * 2018-09-21 2020-04-23 Acuitas Therapeutics, Inc. Systèmes et procédés pour la fabrication de nanoparticules lipidiques et de liposomes
CN112888451A (zh) * 2018-10-19 2021-06-01 川斯勒佰尔公司 信使rna的无泵包封
US11453639B2 (en) 2019-01-11 2022-09-27 Acuitas Therapeutics, Inc. Lipids for lipid nanoparticle delivery of active agents
US11976019B2 (en) 2020-07-16 2024-05-07 Acuitas Therapeutics, Inc. Cationic lipids for use in lipid nanoparticles
WO2024040194A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo
WO2024040195A1 (fr) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditionnement pour l'ingénierie de cellules immunitaires in vivo

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