WO1997030064A1 - Oligonucleotides contenant de l'hexitol et leur utilisation dans des strategies antisens - Google Patents

Oligonucleotides contenant de l'hexitol et leur utilisation dans des strategies antisens Download PDF

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Publication number
WO1997030064A1
WO1997030064A1 PCT/EP1997/000762 EP9700762W WO9730064A1 WO 1997030064 A1 WO1997030064 A1 WO 1997030064A1 EP 9700762 W EP9700762 W EP 9700762W WO 9730064 A1 WO9730064 A1 WO 9730064A1
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Prior art keywords
oligomer
complex
anhydro
heterocyclic ring
derived
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PCT/EP1997/000762
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English (en)
Inventor
Piet André Maurits Maria HERDEWIJN
Arthur Albert Edgard Van Aerschot
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Stichting Rega Vzw
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Publication date
Application filed by Stichting Rega Vzw filed Critical Stichting Rega Vzw
Priority to AU18743/97A priority Critical patent/AU1874397A/en
Publication of WO1997030064A1 publication Critical patent/WO1997030064A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure

Definitions

  • This invention relates to oligomers comprising or containing in part 1,5-anhydrohexitol nucleotide analogues which exhibit sequence-specific binding to complementary sequences of natural oligonucleotides.
  • This invention further relates to the chemical synthesis of these oligomers and their use in antisense strategies which comprise diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification, and therapeutics
  • a European Patent Application n° 94202342.5 discloses the synthesis of phosphoramidite building blocks of hexitol nucleoside analogues, their assembly into oligonucleotides and the hybridization potential of this new class of oligonucleotide constructs, made up of or containing in part 1,5-anhydro-2,3-dideoxyhexitol nucleoside analogues, and phosphorylated at their 4'-end.
  • the synthesis and antiviral activity of the afore mentioned nucleoside analogues themselves is subject of a European Patent Application n°92.201803.1, dd. 18 juni 1992.
  • the present invention enlarges the diversity of possible constructs which can be envisaged for strong and selective hybridization with a complementary sequence as disclosed in the afore mentioned Patent Application n° 94202342.5, and further simplifies the synthesis of these constructs by elimination of the need for a 4'-terminal phosphate moiety.
  • oligomers comprising or containing in part 1,5-anhydro-2,3- dideoxy-D -arabino-hexitol nucleotide analogues as the monomer units A, wherein the hexitol is coupled via its 2-position to the heterocyclic ring of a pyrimidine or purine base, provide such improved stability and binding affinity.
  • the monomer units A are represented by the formula I:
  • B is a heterocyclic ring which is derived from a pyrimidine or purine base and X represents oxygen or sulfur.
  • monomer units A are combined with 2'-deoxynucleotides (monomer units B), the latter represented by formula II:
  • B is a heterocyclic ring which is derived from a pyrimidine or purine base and X represents oxygen or sulfur.
  • the monomers are connected to each other through a phosphodiester bridge with formula III representing the structure of these oligomers.
  • n and p are integers ;
  • each B independently is a heterocyclic ring which is derived from a pyrimidine or purine base ;
  • each X independently represents oxygen or sulfur ;
  • the 5'-end, respectively the 6'-end, of the oligonucleotide may optionally be dephosphorylated and the 3'-end, respectively the 4'-end, may optionally be phosphorylated;
  • the oligomers may be comprised solely of monomer units A (formula I) as represented by formula IV.
  • q is an integer with q ⁇ 2 ;
  • each B independently is a heterocyclic ring which is derived from a pyrimidine or purine base ;
  • each X independently represents oxygen or sulfur ;
  • the 6'-end of the oligonucleotide may optionally be dephosphorylated
  • oligomers of formula III and IV are new compounds. They display a certain similarity with oligonucleotides consisting of the natural 2'-deoxynucleosides represented by formula V
  • k is an integer and wherein each B independently is a heterocyclic ring which is derived from a pyrimidine or purine base, and wherein the 5'-end of the oligonucleotide may optionally be dephosphorylated.
  • the oligomers of formula III and IV and their respective salts exhibit sequence specific binding to natural oligonucleotides represented by formula V or its 5'-dephosphorylated congener.
  • a new class of hybridons or sequence- specific binding polymers has therefore been found.
  • Oligomers of formula III or IV may form a complex with another oligomer of formula III or IV, or they may form a complex with a complementary single-stranded or double-stranded natural oligonucleotide. Each strand may be of the same or different length.
  • the compounds according to the invention are therefore oligomers comprising or containing partially nucleotide analogues wherein a 1,5-anhydro-2,3-dideoxy-D-hexitol is coupled via its 2-position according to an arabino-configuration to the heterocyclic ring of a pyrimidine or purine base.
  • the oligomers consist of the above mentioned nucleotide analogues connected to each other as phosphate diesters or thiophosphate diesters.
  • the oligomers can be represented either by the formula lu or IV, wherein m, n, p, q, B and X have the above stated designations.
  • the oligomers can be exclusively composed of the hexitol nucleotide analogues of the formula I (yielding oligomers of formula IV) or can have natural 2'-deoxynucleotides interspersed or at the end of the molecule (yielding oligomers of formula III).
  • the hexitol has the (D)-configuration and the stereochemistry of both substituents is according to an (S) configuration.
  • group B When group B is derived from a pyrimidine base it can be either cytosine, 5-methylcytosine, uracil or thymine When B is derived from a purine base it can be either adenine, guanine, 2,6-diaminopurine, hypoxanthine or xanthine.
  • the invention also relates to the chemical synthesis of these oligomers and their salts and to their use in antisense strategies which comprise diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification and therapeutics. Chemical synthesis
  • nucleoside analogues, monomer components of the present invention can be prepared in different ways and one of the preparation methods is subject of European patent application n° 92.201 803. 1. These syntheses have been described likewise ( 15). Assembly of the monomers into an oligomer follows the classical schemes and can be done either by standard phosphoramidite chemistry (compare ref. 16) or by H-phosphonate chemistry (compare ref. 17) All procedures are conveniently carried out on an automated DNA synthesizer as for standard oligonucleotide synthesis. For these standard conditions also compare e.g. ref. 18.
  • the preferred method is the phosphoramidite method making use of the phosphoramidites of the hexitol nucleoside analogues as the incoming building blocks for assembly in the "6'-directien".
  • the method of preparation for these phosphoramidite building units is subject of European patent application n° 94202342.5.
  • Synthesis of the afore mentioned oligonucleotide constructs of formula III makes use of standard solid supports (e.g. controlled pore glass) functionalized with an appropriately protected 2'-deoxynucleoside.
  • standard solid supports e.g. controlled pore glass
  • phosphoramidite monomers of 2'-deoxynucleosides on one hand and phosphoramidite building units of 1,5-anhydro-2,3-dideoxyhexitol analogues as subject of European patent application n° 94202342.5 on the other hand
  • assembly of any composition of formula III can be obtained.
  • the primary hydroxyl group optionally can be phosphorylated by well-known commercially available reagents (e.g. phosphorylation reagent of Glen Research) or can be left intact.
  • Synthesis of the afore mentioned oligonucleotide constructs of formula IV can be done starting from solid supports functionalized with an appropriately protected 1,5-anhydro-2,3-dideoxyhexitol analogue.
  • the synthesis of such supports is outlined below. Following standard solid phase procedures and using the afore mentioned phosphoramidite building units of 1,5-anhydro-2,3-dideoxyhexitol analogues, constructs of any composition of formula IV can be obtained.
  • the primary hydroxyl group optionally can be phosphorylated by well-known commercially available reagents (e.g phosphorylation reagent of Glen Research) or can be left intact.
  • Solid supports containing a 1,5-anhydrohexitol analogue can be prepared by succinylation of the compounds la-d yielding 2a-d, which can be coupled to the amino function of either long chain alkylamino controlled pore glass (CCAA-CPG) or a suitable amino functionalized polystyrene (e g. Tentagel R - RAPP Polymere) making use of a carbodiimide and yielding 3a-d (for functionalization of supports compare ref. 19).
  • CCAA-CPG long chain alkylamino controlled pore glass
  • a suitable amino functionalized polystyrene e g. Tentagel R - RAPP Polymere
  • the obtained oligonucleotides are cleaved from the support and deprotected by ammonia treatment for 16 h at 55°C.
  • Purification of the obtained oligomers of the above stated formula's III or IV can be accomplished in several ways (compare e.g. ref. 20).
  • the preferred method is purification by anion-exchange FPLC at a basic pH of 12 to disrupt all possible secondary structures (compare e.g. ref 13). Desalting is done by simple gel filtration techniques followed by lyophilization. All acceptable salts can be prepared in a conventional manner.
  • LSIMS liquid secondary ion mass spectrometry Thgly thioglycerol, TEA triethylamine, TEAB triethylammonium carbonate, TEAA triethylammonium acetate.
  • the obtained foam was treated with 40 mL of a 80% aqueous acetic acid solution for 1 h at 60°C Evaporation and coevaporation with toluene left an oil which was purified by flash chromatography on 20 g of silica gel (gradient of CH 2 Cl 2 to CH 2 Cl 2 -MeOH 94:6). Besides some 4'-O, N 3 -diben-zoylated product, 108 mg (0.3 mmol, 71%) of the title product 5a was isolated as a white foam.
  • the mixture was diluted with 100 mL of CH 2 Cl 2 and washed with 50 mL of 10% aqueous Na 2 S 2 O 3 and with 50 mL of 1 M TEAB .
  • the organic phase was dried, evaporated and purified by column chromatography on 20 g of silica gel (gradient of CH 2 Cl 2 -TEA 99: 1 to CH 2 Cl 2 -TEA-MeOH 93: 1 :6) affording 200 mg (0.17 mmol, 95%) of the fully protected dimer.
  • the white foam obtained was dissolved in 32 mL of a 3 : 1 mixture of cone ammonia and ethanol and heated overnight at 40°C. The mixture was evaporated and coevaporated with 1,4-dioxane. The residue was dissolved in 20 mL of 80% aqueous acetic acid and heated at 60 °C for 1 h. After evaporation, the residue was partitioned between 10 mL of 10 mM TEAA solution and 10 mL of ether.
  • the aqueous phase was washed 3 times more with 10 mL of diethylether and concentrated and purified by reverse phase HPLC on a polystyrene-divinyl-benzene support (PLRP-S, 250 ⁇ 9 mm) with an acetonitrile gradient in 0.1 M TEAA.
  • Product containing fractions were pooled and the TEAA salt was exchanged for the sodium salt by chromatography on a Dowex 50X8-200 cation exchange resin under its sodium form. Lyophylization afforded 52 mg (S5 ⁇ mol, 47% overall) as a white voluminous powder.

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Abstract

L'invention concerne de nouveaux oligomères comprenant des analogues du nucléotide 1,5-anhydro-2,3-didésoxyhexitol et des nucléotides de désoxyribose. Elle concerne également un complexe constitué du nouvel oligomère et d'un oligonucléotide complémentaire à simple ou double brin. Elle concerne enfin des compositions pharmaceutiques comprenant l'oligomère et/ou le complexe, ainsi que leur utilisation tant en biologie moléculaire qu'en génie génétique pour l'hybridation, l'isolement d'acides nucléiques, la modification d'ADN spécifique de site et la cartographie, par exemple.
PCT/EP1997/000762 1996-02-16 1997-02-14 Oligonucleotides contenant de l'hexitol et leur utilisation dans des strategies antisens WO1997030064A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18743/97A AU1874397A (en) 1996-02-16 1997-02-14 Hexitol containing oligonucleotides and their use in antisense strategies

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP96200403.2 1996-02-16
EP96200403 1996-02-16
EP96201089 1996-04-23
EP96201089.8 1996-04-23

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1578765A2 (fr) * 2002-11-05 2005-09-28 Isis Pharmaceuticals, Inc. Composes oligomeres contenant un substitut de sucre et compositions a utiliser dans la modulation genique
WO2006047842A2 (fr) * 2004-11-08 2006-05-11 K.U. Leuven Research And Development Nucleosides modifies pour interference arn
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7790691B2 (en) 2003-06-20 2010-09-07 Isis Pharmaceuticals, Inc. Double stranded compositions comprising a 3′-endo modified strand for use in gene modulation
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
US20110244025A1 (en) * 2005-04-26 2011-10-06 Eugen Uhlmann Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
WO2015034928A1 (fr) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Polynucléotides chimériques
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
WO2015051214A1 (fr) 2013-10-03 2015-04-09 Moderna Therapeutics, Inc. Polynucléotides codant pour un récepteur de lipoprotéines de faible densité
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
WO2016014846A1 (fr) 2014-07-23 2016-01-28 Moderna Therapeutics, Inc. Polynucléotides modifiés destinés à la production d'anticorps intracellulaires
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
EP3533873A1 (fr) 2011-09-14 2019-09-04 Translate Bio MA, Inc. Composés d'oligonucléotides multimères
US10815291B2 (en) 2013-09-30 2020-10-27 Modernatx, Inc. Polynucleotides encoding immune modulating polypeptides
EP4159741A1 (fr) 2014-07-16 2023-04-05 ModernaTX, Inc. Procédé de production d'un polynucléotide chimérique pour coder un polypeptide ayant une liaison internucléotidique contenant un triazole
US11744801B2 (en) 2017-08-31 2023-09-05 Modernatx, Inc. Methods of making lipid nanoparticles
US11786607B2 (en) 2017-06-15 2023-10-17 Modernatx, Inc. RNA formulations

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Cited By (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9096636B2 (en) 1996-06-06 2015-08-04 Isis Pharmaceuticals, Inc. Chimeric oligomeric compounds and their use in gene modulation
US7695902B2 (en) 1996-06-06 2010-04-13 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US7812149B2 (en) 1996-06-06 2010-10-12 Isis Pharmaceuticals, Inc. 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
EP1578765A2 (fr) * 2002-11-05 2005-09-28 Isis Pharmaceuticals, Inc. Composes oligomeres contenant un substitut de sucre et compositions a utiliser dans la modulation genique
US8604183B2 (en) 2002-11-05 2013-12-10 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
EP1578765A4 (fr) * 2002-11-05 2008-04-23 Isis Pharmaceuticals Inc Composes oligomeres contenant un substitut de sucre et compositions a utiliser dans la modulation genique
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
US7790691B2 (en) 2003-06-20 2010-09-07 Isis Pharmaceuticals, Inc. Double stranded compositions comprising a 3′-endo modified strand for use in gene modulation
US8569474B2 (en) 2004-03-09 2013-10-29 Isis Pharmaceuticals, Inc. Double stranded constructs comprising one or more short strands hybridized to a longer strand
US8394947B2 (en) 2004-06-03 2013-03-12 Isis Pharmaceuticals, Inc. Positionally modified siRNA constructs
US7884086B2 (en) 2004-09-08 2011-02-08 Isis Pharmaceuticals, Inc. Conjugates for use in hepatocyte free uptake assays
WO2006047842A3 (fr) * 2004-11-08 2006-09-28 Leuven K U Res & Dev Nucleosides modifies pour interference arn
WO2006047842A2 (fr) * 2004-11-08 2006-05-11 K.U. Leuven Research And Development Nucleosides modifies pour interference arn
US20110244025A1 (en) * 2005-04-26 2011-10-06 Eugen Uhlmann Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9701965B2 (en) 2010-10-01 2017-07-11 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10751386B2 (en) 2011-09-12 2020-08-25 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
EP3533873A1 (fr) 2011-09-14 2019-09-04 Translate Bio MA, Inc. Composés d'oligonucléotides multimères
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
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