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Method for generating dumbbell-shaped DNA vector

A dumbbell-shaped, vector-based technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc.

Pending Publication Date: 2022-06-10
NAT UNIV OF SINGAPORE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

While viral vectors are costly and often elicit an immune response or pose a risk of genomic vector integration, many nonviral delivery vectors involve non-nucleic acid helper functions that can be 1,2 toxic to cells

Method used

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  • Method for generating dumbbell-shaped DNA vector
  • Method for generating dumbbell-shaped DNA vector
  • Method for generating dumbbell-shaped DNA vector

Examples

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Embodiment

[0328]In the current state-of-the-art protocol, the generation of each new dumbbell vector begins with the individual cloning of the sequence of interest to be implemented into the dumbbell into a plasmid vector. The method of the present invention only needs to prepare a general template, including promoters, enhancers, DNA nuclear localization signals, introns, transcription terminators, RNA nuclear export signals, WPRE and other regulatory sequences. The sequence of interest is then introduced via chemically synthesized PCR primers without further cloning and without the need for endonucleases ( Figure 1-7 ). This generated dumbbell can be used as a molecular bait or an expression vector. Expression vectors can express noncoding RNAs, including shRNA, pre-miRNA, miRNA, aptamers, antisense RNA, and antisense miRNA ( Figure 1-4 , 6, 7) and / or expression of peptide or protein-coding RNA ( Figure 5-7 ). The expression cassettes of hairpin RNAs (such as shRNA and pre-miRN...

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Abstract

The dumbbell-shaped DNA minimum vector represents a genetic vector consisting only of a gene expression cassette of interest and a terminal closed loop structure. The dumbbell vector for small hairpin RNA or microRNA expression is very small in volume, which is advantageous for cell delivery and nuclear diffusion. Conventional strategies for generating dumbbell vectors expressing small RNA entail cloning of corresponding plasmid vectors, which are then used for dumbbell protection. Herein, we provide a novel cloning-free method for generating dumbbell vectors that express small RNA, which do not require any restriction endonuclease. The method comprises performing PCR amplification on a universal DNA template using a primer containing a sense or antisense strand of a sequence of interest, denaturing and refolding the amplification product to form a stem-loop structure, and covalently closing the structure using a DNA ligase to obtain a dumbbell structure.

Description

technical field [0001] The present disclosure relates to a new general template-assisted cloning-free method that allows for the efficient synthesis of dumbbell-shaped DNA vectors at low cost for the delivery of recombinant DNA and RNA into host cells. Background technique [0002] The efficiency of approaches such as gene therapy for inherited and acquired genetic diseases, genetic vaccination, stem cell programming, somatic cell reprogramming, immunotherapy, CRISPR / Cas-mediated genome editing, and in vivo protein expression manipulation relies on the delivery of recombinant DNA to In primary cells ex vivo or in vivo to trigger expression of non-coding RNA or protein. [0003] In primary cells, expression of recombinant exogenous episomal DNA (eg, plasmid) is silenced within 24 hours of delivery, independent of the route of delivery. The mechanisms behind this effect are poorly understood. Only integrating viral delivery vectors, such as retroviruses, lentiviruses, and AA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/64
CPCC12N15/85C12N15/64C12Q2521/325C12Q2521/501C12Q2531/113A61K48/0008C12N2310/531C12N2310/532
Inventor V·帕策尔S·L·西里尔
Owner NAT UNIV OF SINGAPORE
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