Method for producing dumbbell-shaped DNA vectors

A PCR-based, cloning-free method synthesizes dumbbell-shaped DNA vectors efficiently and cost-effectively, addressing production costs and safety risks associated with existing methods, enhancing cellular delivery and reducing transgene silencing.

JP7751885B2Active Publication Date: 2025-10-09NATIONAL UNIVERSITY OF SINGAPORE
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Patent Information

Application Number
JP2022511226
Authority / Receiving Office
JP · JP
Patent Type
Patents
Current Assignee / Owner
Priority Date
2019-08-22
Filing Date
2020-08-21
Publication Date
2025-10-09
Estimated Expiration
2040-08-21

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Abstract

A dumbbell-shaped DNA minimal vector is a gene vector consisting only of a gene expression cassette of interest and a closed-end loop structure. Dumbbell vectors for expressing small hairpin RNAs or microRNAs have an extremely small size, which is advantageous for cellular delivery and nuclear spread. Conventional strategies for creating small RNA-expressing dumbbell vectors require cloning of each plasmid vector, which is then used for dumbbell protection. Herein, the inventors present a novel cloning-free method for creating small RNA-expressing dumbbell vectors, which also does not require any restriction endonucleases. The method involves PCR amplification of a universal DNA template using primers containing the sense or antisense strand of the target sequence, denaturing and refolding the amplified product to form a stem-loop structure, and covalently closing the structure using DNA ligase to obtain a dumbbell structure.
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Citation Information

Patent Citations

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    CN103045726A

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