Modified polynucleotides for the production of factor ix

a technology of factor ix and modified polynucleotides, which is applied in the field of delivery methods, can solve the problems of affecting the expression of dna by cells, each step represents an opportunity for error and damage, and is difficult to obtain dna expression in cells, so as to increase the level of a polypeptide, increase the level, and increase the effect of the level

Inactive Publication Date: 2013-09-19
MODERNATX 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 deliverying pharmaceutical compositions in order to achieve effective protein expression both for therapeutics and bioprocessing applications.
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 frequently DNA 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.
Assuming the proper management of the foregoing, effective delivery and achievement of therapeutically relevant levels of proteins for a time sufficient to product clinical outcomes remains a significant hurdle.

Method used

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  • Modified polynucleotides for the production of factor ix
  • Modified polynucleotides for the production of factor ix
  • Modified polynucleotides for the production of factor ix

Examples

Experimental program
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example 1

Modified mRNA Production

[0349]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); herein incorporated by reference).

[0350]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 limited to, DNA2.0 (Menlo Park, Calif.)...

example 2

PCR for cDNA Production

[0366]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.

[0367]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.

[0368]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)

[0369]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.

[0370]A typical in vitro transcription reaction includes the following:

1.Template cDNA1.0μg2.10x transcription buffer (400 mM Tris-HCl2.0μlpH 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 20.0μl. and7.Incubation at 37° C. for 3 hr-5 hrs.

[0371]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 electro...

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Abstract

Provided are formulations, compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the delivery of biological moieties, and are useful for production of proteins.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 13 / 437,034, filed Apr. 2, 2012, entitled Delivery and Formulation of Engineered Nucleic Acids which claims priority to U.S. Provisional Patent Application No. 61 / 470,451, filed Mar. 31, 2011, entitled Delivery and Formulation of Engineered Nucleic Acids the contents, the contents of each is incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING[0002]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled M003USSQLST.txt created on May 17, 2013 which is 17,058 bytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0003]The invention relates to delivery methods. These methods are specifically useful in therapeutic delivery of modified nucleic acids such as modified mRNA (mmR...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/535C12N9/64
CPCA61K31/7088C12N15/87C07K14/535C12N9/644A61K31/7115A61K48/00A61K38/4846A61K47/18A61K47/22A61K47/28A61K38/193C12N15/67C12N2310/335A61P1/00A61P11/00A61P13/00A61P17/00A61P19/00A61P21/00A61P25/00A61P29/00A61P3/00A61P31/00A61P31/12A61P35/00A61P37/02A61P43/00A61P5/00A61P7/00A61P9/00
Inventor DE FOUGEROLLES, ANTONINELBASHIR, SAYDA M.SCHRUM, JASON P.
Owner MODERNATX INC
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