US 12,473,571 B2
Expression cassette for expressing gene comprising overlapping open reading frames in insect cell and application thereof
He Xiao, Hubei (CN); Xiaobin He, Hubei (CN); Gang Huang, Hubei (CN); Ying Hu, Hubei (CN); Xing Pan, Hubei (CN); He Huang, Hubei (CN); Liang Du, Hubei (CN); and Mengdie Wang, Hubei (CN)
Assigned to GENVEOYAGER (WUHAN) CO., LTD., Hubei (CN)
Appl. No. 18/016,866
Filed by GENEVOYAGER (WUHAN) CO., LTD., Hubei (CN)
PCT Filed Sep. 27, 2021, PCT No. PCT/CN2021/120828
§ 371(c)(1), (2) Date Jan. 19, 2023,
PCT Pub. No. WO2023/039936, PCT Pub. Date Mar. 23, 2023.
Claims priority of application No. 202111105263.5 (CN), filed on Sep. 18, 2021.
Prior Publication US 2024/0247282 A1, Jul. 25, 2024
Int. Cl. C12N 15/86 (2006.01); C12N 7/00 (2006.01)
CPC C12N 15/86 (2013.01) [C12N 7/00 (2013.01); C12N 2710/14043 (2013.01); C12N 2750/14122 (2013.01); C12N 2750/14152 (2013.01); C12N 2800/105 (2013.01); C12N 2830/42 (2013.01)] 20 Claims
 
1. An expression cassette for expressing a gene comprising overlapping open reading frames in an insect cell, comprising from 5′ to 3′ and operably linked:
a promoter capable of driving transcription in the insect cell;
an artificially constructed sequence comprising a 5′ part of a first open reading frame;
a 3′ part of the first open reading frame comprising at least one additional open reading frame;
wherein the artificially constructed sequence comprises a native or engineered intron with splicing activity in the insect cell, the intron comprises a translation start codon ATG of the first open reading frame or the intron is located between any two adjacent nucleotides in ATG, so that the 5′ part of the first open reading frame lacks a translation start codon, and the intron does not comprise a promoter capable of driving transcription in the insect cell; the at least one additional open reading frame comprises at least one additional translation start codon;
the expression cassette can only transcribe a precursor mRNA in the insect cell, and -during a post-transcriptional processing process, a translation start codon AUG in the artificially constructed sequence is retained or deleted or a translation start codon AUG is formed in the artificially constructed sequence only through an alternative splicing function of the intron, thereby implementing translation and expression of different overlapping open reading frames.