Supercharge Your Innovation With Domain-Expert AI Agents!

Stabilized nucleic acids encoding messenger ribonucleic acid (MRNA)

A coding, nucleotide technology, applied in the field of DNA, which can solve problems such as change

Pending Publication Date: 2020-05-29
INTELLIA THERAPEUTICS INC
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When poly-A tails are encoded on plasmids, the poly-A tails may become shorter (i.e., lose adenine nucleotides) during cycles of plasmid DNA replication, which can lead to dramatic changes in the resulting DNA and subsequent mRNA populations

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Stabilized nucleic acids encoding messenger ribonucleic acid (MRNA)
  • Stabilized nucleic acids encoding messenger ribonucleic acid (MRNA)
  • Stabilized nucleic acids encoding messenger ribonucleic acid (MRNA)

Examples

Experimental program
Comparison scheme
Effect test

example

[0187] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

example 1

[0188] Example 1 - Design and stability of polyA-encoded stable plasmids

[0189] Design poly-A tails containing non-adenine nucleotides. The stability of plasmids encoding these poly-A tails with contiguous adenine nucleotides and non-adenine nucleotides (eg interrupter sequences) was compared to the stability of poly-A tails consisting only of adenine nucleotides.

[0190] The problem of missing adenosine numbers in mRNA poly-A tails consisting only of adenosine is highlighted in Table 2. The sequence of the poly-A tail containing 96 adenosines was inserted into the pUC57 plasmid (Genscript) and transformed into E. coli. Cells were plated on LB-Amp plates and incubated overnight at 30°C or 37°C. Eight colonies were picked and inoculated into 96-well plates with LB-Amp medium and grown overnight at 30°C or 37°C (day 1). Samples from day 1 cultures were added to fresh LB-Amp medium and grown for two additional days at 30°C or 37°C (day 2). DNA was purified from day 1 and d...

example 2

[0198] Example 2 - Activity of constructs with poly A tails comprising non-adenine nucleotides

[0199] Experiments were performed to determine whether there was a difference in the potency of mRNAs with poly-A tails containing non-adenine nucleotides (interrupting sequences) versus those with poly-A tails containing only adenosine. A model system was used in which mRNA encoding the Cas9 protein was transfected into HEK-293 cells by electroporation together with a reporter plasmid encoding secreted embryonic alkaline phosphatase (SEAP) and a guide RNA targeting SEAP. Successful expression of Cas9 protein from mRNA results in cleavage of the SEAP target sequence, resulting in a color change reflecting reduced SEAP production. SEAP HEK-Blue reporter reagent was obtained from Invivogen. The sequence (SEQ ID NO:6) containing the T7 promoter and encoding the Cas9 mRNA with an adenosine-only poly-A tail (designed to have 100 adenosine nucleotides but was shown to have 97 adenosine ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

This disclosure relates to the field of poly-adenylated (poly-A) tails. In some embodiments, a DNA encodes a poly-A tail located 3' to nucleotides encoding a protein of interest, wherein the poly-A tail comprises one or more non-adenine nucleotide.

Description

technical field [0001] The present disclosure relates to the field of stable messenger ribonucleic acid (mRNA) and DNA encoding stable mRNA. Background technique [0002] Polyadenylation is the process of adding multiple adenine nucleotides to the 3' end of messenger RNA (mRNA), thereby forming a polyA tail. The poly-A tail consists of multiple repeats of adenine nucleotides (eg, adenosine monophosphate) without other base interruptions. The poly A tail is critical for nuclear export, translation and stability of mRNA. In nature, when mRNA is produced from DNA, terminal transferase adds an adenine nucleotide to the 3' end of the mRNA. This enzymatic process can be applied when mRNA is produced ex vivo, but is difficult to control and produces poly-A tails of varying lengths. By encoding the poly-A tail in the plasmid, it is possible to reduce the heterogeneity in the poly-A tail. However, this process does not eliminate heterogeneity and has other disadvantages, such as ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11
CPCC12N15/11C12N9/22C12N15/102C12N15/113C12N2310/20C07H21/02C07K14/475C07K14/521C12N15/63
Inventor C·东布罗夫斯基
Owner INTELLIA THERAPEUTICS INC
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com