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Modified polynucleotides for the production of secreted proteins

a technology of modified polynucleotides and secreted proteins, which is applied in the direction of peptide/protein ingredients, peptide delivery, vectors, etc., can solve the problems of affecting the dna expression of host cells, and each step represents an opportunity for error and damage to the cell, and it is difficult to obtain dna expression in cells

Active Publication Date: 2016-12-29
MODERNATX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes methods and compositions for creating modified mRNA molecules. These modifications can be made to improve the function of the mRNA in the body, for example, by adding new information or changing the way the mRNA is translated. This invention can have various technical effects, including improving the efficiency or accuracy of mRNA therapy, which is a promising treatment method for certain genetic or degenerative diseases.

Problems solved by technology

There are multiple problems with prior methodologies of effecting protein expression.
For example, introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and / or damage to the host cell genomic DNA.
Not only do the multiple processing steps from administered DNA to protein create lag times before the generation of the functional protein, each step represents an opportunity for error and damage to the cell.
Further, it is known to be difficult to obtain DNA expression in cells as DNA frequently enters a cell but is not expressed or not expressed at reasonable rates or concentrations.
This can be a particular problem when DNA is introduced into primary cells or modified cell lines.
However, the low levels of translation and the immunogenicity of the molecules hampered the development of mRNA as a therapeutic and efforts have since focused on alternative applications that could instead exploit these pitfalls, i.e. immunization with mRNAs coding for cancer antigens.

Method used

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  • Modified polynucleotides for the production of secreted proteins
  • Modified polynucleotides for the production of secreted proteins
  • Modified polynucleotides for the production of secreted proteins

Examples

Experimental program
Comparison scheme
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example 1

Modified mRNA Production

[1137]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, 5mc 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).

[1138]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, ...

example 2

PCR for cDNA Production

[1155]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 dH20 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.

[1156]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.

[1157]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 (IVT)

[1158]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.

[1159]A typical in vitro transcription reaction includes the following:

1Template cDNA1.0μg210x transcription buffer (400 mM2.0μlTris-HCl pH 8.0, 190 mM MgCl2,50 mM DTT, 10 mM Spermidine)3Custom NTPs (25 mM each)7.2μl4RNase Inhibitor20U5T7 RNA polymerase3000U6dH20Up to 20.0 μl. and7Incubation at 37° C. for 3 hr-5 hrs.

[1160]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 electrophores...

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Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

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 M304_USSQLST.txt, was created on Mar. 9, 2013 and is 31.429.071 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.REFERENCE TO LENGTHY TABLE[0002]The specification includes a lengthy Table 6. Lengthy Table 6 has been submitted via EFS-Web in electronic format as follows: File name: M304TBL.txt, Date created: Mar. 9, 2013; File size: 205,684 Bytes and is incorporated herein by reference in its entirety. Please refer to the end of the specification for access instructions.CROSS REFERENCE TO RELATED APPLICATIONS[0003]This application claims priority to U.S. Provisional Patent Application No. 61 / 681,742, filed, Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides, U.S. Provisional Patent Applicat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/10A61K38/17A61K38/19A61K38/18A61K9/14A61K38/48
CPCA61K47/10A61K9/14A61K38/4846A61K38/193A61K38/1816A61K38/1767C12N15/88A61K9/0019A61K9/1271A61K9/1272A61K9/1277A61K9/5031C07K14/47C12N2840/00A61K47/54A61K48/0066A61K38/191A61K38/212A61K38/215A61K38/36A61K38/363A61K38/44A61K38/4833C07K14/505C07K14/535C12N9/0069C12N9/644C12Y113/12007C12Y304/21005C12Y304/21022A61K31/7088C12N15/85A61K38/1866A61K47/542A61K48/0033A61K48/00C07K19/00A61K39/3955A61K48/0075C07K14/475C07K14/525C07K14/56C07K14/565C07K14/745C07K14/75C07K16/2887C07K16/32
Inventor BANCEL, STEPHANECHAKRABORTY, TIRTHADE FOUGEROLLES, ANTONINELBASHIR, SAYDA M.JOHN, MATTHIASROY, ATANUWHORISKEY, SUSANWOOD, KRISTY M.HATALA, PAULSCHRUM, JASON P.EJEBE, KENECHIELLSWORTH, JEFF LYNNGUILD, JUSTIN
Owner MODERNATX INC
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