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Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

a technology of nucleosides and nucleic acids, applied in the field of modified nucleic acids, can solve the problems of difficult to obtain dna expression in cells, alterations and/or damage to the genomic dna of host cells, etc., and achieve the effect of reducing the innate immune respons

Inactive Publication Date: 2013-05-16
MODERNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are multiple problems with prior methodologies of effecting protein expression.
Introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and / or damage to the host cell genomic DNA.
Further, it is difficult to obtain DNA expression in cells; frequently DNA enters cells but is not expressed or not expressed at reasonable rates or concentrations.
This can be a particular problem when DNA is introduced into cells such as primary cells or modified cell lines.

Method used

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  • Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
  • Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
  • Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

Examples

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example 1

Modified mRNA In Vitro Transcription

A. Materials and Methods

[0826]Modified mRNAs according to the invention are made using standard laboratory methods and materials for in vitro transcription with the exception that the nucleotide mix contains modified nucleotides. The open reading frame (ORF) of the gene of interest is flanked by a 5′ untranslated region (UTR) containing a strong Kozak translational initiation signal and an alpha-globin 3′ UTR terminating with an oligo(dT) sequence for templated addition of a polyA tail for mRNAs not incorporating adenosine analogs. Adenosine-containing mRNAs are synthesized without an oligo (dT) sequence to allow for post-transcription poly (A) polymerase poly-(A) tailing.

[0827]The modified mRNAs may be modified to reduce the cellular innate immune response. The modifications to reduce the cellular response may include pseudouridine (ψ) and 5-methyl-cytidine (5meC, 5 mc or m5C). (See, Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. Mol ...

example 2

Modified mRNA Transfection

A. Reverse Transfection

[0846]For experiments performed in a 24-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 1×105. For experiments performed in a 96-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.5×105. For each modified mRNA to be transfected, modified mRNA: RNAIMAX™ are prepared as described and mixed with the cells in the multi-well plate within 6 hours of cell seeding before cells had adhered to the tissue culture plate.

B. Forward Transfection

[0847]In a 24-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.7×105. For experiments performed in a 96-well collagen-coated tissue culture plate, Keratinocytes are seeded at a cell density of 0.3×105. Keratinocytes are then grown to a confluency of >70% for over 24 hours. For each modified mRNA to be transfected, modified mRNA: RNAIMAX™ are prepared as described and transfected onto the...

example 3

Cellular Innate Immune Response to Modified Nucleic Acids: IFN-Beta ELISA and TNF-Alpha ELISA

[0851]An enzyme-linked immunosorbent assay (ELISA) for Human Tumor Necrosis Factor-α (TNF-α), Human Interferon-β (IFN-β) and Human Granulocyte-Colony Stimulating Factor (G-CSF) secreted from in vitro-transfected Human Keratinocyte cells is tested for the detection of a cellular innate immune response.

[0852]Keratinocytes are grown in EPILIFE® medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen at a confluence of >70%. Keratinocytes are reverse transfected with 0 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of the indicated chemically modified mRNA complexed with RNAIMAX™ from Invitrogen as described in triplicate. Secreted TNF-α in the culture medium is measured 24 hours post-transfection for each of the chemically modified mRNAs using an ELISA kit from Invitrogen according to the manufacturer protocols.

[0853]Secreted IFN-β is measur...

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Abstract

The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using them.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 13 / 644,072, filed Oct. 3, 2012, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof which claims priority to U.S. Provisional Patent Application No. 61 / 542,533, filed Oct. 3, 2011, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof, the contents of each are herein incorporated by reference in their entirety.REFERENCE TO THE SEQUENCE LISTING[0002]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled M009SQLST.txt, was created on Jan. 17, 2013 and is 9,970 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.TECHNICAL FIELD[0003]The present disclosure provides compositions and methods using modified nucleic acids to modulate cellular function. The modified nucleic acids...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/02
CPCC07H21/02C12N15/67A61K38/193A61K48/0066A61P37/04A61P43/00C12N15/11C07K14/535A61K48/0075A61K48/0033A61K9/5123A61K9/0019A61K9/0021
Inventor DE FOUGEROLLES, ANTONINROY, ATANUBANCEL, STEPHANEHATALA, PAUL
Owner MODERNA THERAPEUTICS INC
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