Formulation and delivery of PLGA microspheres

• US 9,295,689 B2
• Filed: 05/18/2013
• Issued: 03/29/2016
• Est. Priority Date: 12/16/2011
• Status: Active Grant

• Current Assignee: Moderna TX, Inc. (Moderna, Inc.)
• Original Assignee: Moderna Therapeutics, Inc. (Moderna, Inc.)
• Inventors: de Fougerolles, Antonin, Wood, Kristy M., Valencia, Pedro
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US13/897,375
• Publication Number:
  US 20130252281A1
• Time in Patent Office: 1,046 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/4833 Thrombin (3.4.21.5)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0033 the non-active part being non-polymeric
• CPC Class Codes: A61K 48/0041 the non-active part being polymeric
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61K 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61K 9/14 Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles microspheres A61K9/16; microcapsules A61K9/50; nanocapsules, nanoparticles of the matrix type A61K9/51
• CPC Class Codes: A61K 9/16 Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction A61K9/20 takes precedence if the final form is a tablet; microspheres with drug-free outer coating, microcapsules A61K9/50; mixture of different granules, microcapsules, (coated) microparticles A61K9/5084; nanoparticles A61K9/51
• CPC Class Codes: A61K 9/1647 Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/535 : Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 2/00 : Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C12N 15/00 : Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/117 : Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 15/67 : General methods for enhancing the expression
• CPC Class Codes: C12N 15/88 : using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 : having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The present disclosure provides, inter alia, formulation compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation composition may further include a modified nucleic acid molecule and a delivery agent. The present invention further provides nucleic acids useful for encoding polypeptides capable of modulating a cell'"'"'s function and/or activity.

Claims

1. A method of producing a protein of interest in a cell comprising contacting the cell with a PLGA microsphere particle consisting essentially of PLGA and having no amine-containing polymers or amino acids, said PLGA microsphere particle comprising a modified mRNA greater than 30 nucleotides-in length encoding said protein of interest, wherein said modified mRNA consists of nucleoside modifications where 25% of uridine residues are replaced with the nucleoside modification 2-thiouridine and 25% of cytosine residues are replaced with the nucleoside modification 5-methylcytosine and wherein said modified mRNA is formulated in the PLGA microsphere particle such that the PLGA microsphere has a modified mRNA percent encapsulation efficiency of at least 68.1%, a particle size of at least 25 μ

m and an actual RNA loading of at least 0.31 weight percent.

2. The method of claim 1, wherein the PLGA microsphere comprising the modified mRNA releases less than 50% of the modified mRNA in a 48 hour time period.

3. The method of claim 1, wherein the PLGA microsphere comprising the modified mRNA is stable in serum.

4. The method of claim 1, wherein contacting said mammalian cells or tissues occurs via a route of administration selected from the group consisting of intravenous, intramuscular, intravitreal, intrathecal, intratumoral, pulmonary, and subcutaneous.

5. The method of claim 4, wherein the stability is determined relative to unformulated modified mRNA in 90% serum.

6. The method of claim 1, wherein the PLGA microsphere further comprises a second modified mRNA.

7. The method of claim 6, wherein the PLGA microsphere further comprises a third modified mRNA.

8. The method of claim 1, wherein the modified mRNA comprises at least one 5′

terminal cap selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′

fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

9. The method of claim 8, wherein the 5′

terminal cap is Cap1.