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Method for producing a protein of interest at the site of a clot

a clot site and protein technology, applied in the direction of peptide/protein ingredients, immunological disorders, metabolism disorders, etc., can solve the problems of cytokine and interferon production and ultimately cell death, and achieve rapid elimination of lipid nanoparticles, increase hemoglobin, and therapeutic

Inactive Publication Date: 2013-09-12
MODERNA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0090]It has been discovered that unique poly-A tail lengths provide certain advantages to the modified mRNA of the present invention.

Problems solved by technology

In general, exogenous unmodified nucleic acid molecules, particularly viral nucleic acids, introduced into the cell induce an innate immune response which results in cytokine and interferon (IFN) production and ultimately cell death.

Method used

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  • Method for producing a protein of interest at the site of a clot
  • Method for producing a protein of interest at the site of a clot
  • Method for producing a protein of interest at the site of a clot

Examples

Experimental program
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Effect test

example 1

Modified mRNA Production

[0900]Modified mRNAs (mmRNA) according to the invention may be made using standard laboratory methods and materials. The open reading frame (ORF) of the gene of interest may be flanked by a 5′ untranslated region (UTR) which may contain a strong Kozak translational initiation signal and / or an alpha-globin 3′ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 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 or m5C). (see, Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson B R et al. NAR (2010); each of which are herein incorporated by reference in their entireties).

[0901]The ORF may also include various upstream or downstream additions (such as, but not limited to, β-globin, tags, etc.) may be ordered from an optimization service such as, but l...

example 2

PCR for cDNA Production

[0918]PCR procedures for the preparation of cDNA are performed using 2×KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This system includes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl; Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH2O diluted to 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min. then 4° C. to termination.

[0919]The reverse primer of the instant invention incorporates a poly-T120 for a poly-A120 in the mRNA. Other reverse primers with longer or shorter poly(T) tracts can be used to adjust the length of the poly(A) tail in the mRNA.

[0920]The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Larger reactions will require a cleanup using a product with a larger capacity. Following the cleanup, the cDNA is quantified...

example 3

In Vitro Transcription

[0921]The in vitro transcription reaction generates mRNA containing modified nucleotides or modified RNA. The input nucleotide triphosphate (NTP) mix is made in-house using natural and un-natural NTPs.

[0922]A typical in vitro transcription reaction includes the following:

1.Template cDNA1.0μg2.10x transcription buffer (400 mM Tris-2.0μlHCl pH 8.0, 190 mM MgCl2, 50 mM DTT,10 mM Spermidine)3.Custom NTPs (25 mM each)7.2μl4.RNase Inhibitor20U5.T7 RNA polymerase3000U6.dH20Up to 20.0 μl. and7.Incubation at 37° C. for 3 hr-5 hrs.

[0923]The crude IVT mix may be stored at 4° C. overnight for cleanup the next day. 1 U of RNase-free DNase is then used to digest the original template. After 15 minutes of incubation at 37° C., the mRNA is purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following the manufacturer's instructions. This kit can purify up to 500 μg of RNA. Following the cleanup, the RNA is quantified using the NanoDrop and analyzed by agarose gel electrophor...

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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.

Description

REFERENCE TO SEQUENCE LISTING[0001]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled M11CON5SQLST.txt, was created on May 17, 2013 and is 25,618 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS[0002]This application is a continuation of U.S. patent application Ser. No. 13 / 714,458, filed Dec. 14, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions which claims priority to U.S. Provisional Patent Application No. 61 / 576,705, filed Dec. 16, 2011, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61 / 618,957, filed Apr. 2, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S. Provisional Patent Application No. 61 / 648,244, filed May 17, 2012, entitled Modified Nucleoside, Nucleoti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7088
CPCA61K48/00A61K9/1647A61K9/16A61K9/1271A61K38/4833C12P21/00A61K48/0041A61K48/0033A61K48/005A61K48/0066C12N15/88A61K31/7105C12N15/117C07K2/00A61K31/7088A61K9/0019A61K9/0024A61K9/0048A61K9/1272A61K9/14C12N15/00A61K38/193A61P25/28A61P3/00A61P35/00A61P37/02A61P9/00A61P9/10A61P3/10A61K31/7115C12N15/67C12P21/02C07K14/535
Inventor DE FOUGEROLLES, ANTONINWOOD, KRISTY M.
Owner MODERNA THERAPEUTICS INC
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