US 2024/0035037 A1Published application

Transposon-based vectors and uses

AssigneeJust-Evotec Biologics, Inc.

InventorsJeffrey T. McGrew, James Joyce McDermott, Sherman Ku

Life of this patent

Estimating the term…

Loading patent…

Abstract

Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence or a human cytomegalovirus (huCMV) intron A sequence. Some of the vectors are useful for inducible expression. Some of the vectors contain a 5′ PiggyBac ITR and a 3′ PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and methods of producing a protein of interest, in vitro, involving the mammalian host cell.

Claims19

Dependent claims

A recombinant expression vector, comprising:

(a) a 5′ PiggyBac ITR comprising the nucleotide sequence of SEQ ID NO:45;
(b) a first expression cassette, comprising:
(i) a control sequence comprising:
(A) a murine cytomegalovirus (mCMV) enhancer sequence, comprising a mCMV enhancer element (mCMV-E) and a CMV promoter (CMV-P) sequence at its 3′ end, operably linked 5′ to a human cytomegalovirus (huCMV) intron A sequence, and optionally, comprising one or more TetO sequences inserted within the CMV-P sequence;
(B) an intervening first leader sequence operably linked, 3′ to the CMV-P sequence of the mCMV enhancer sequence, and 5′ to the huCMV intron A sequence;
and
(C) a second leader sequence operably linked 3′ to the huCMV intron A sequence;
(ii) an open reading frame encoding a protein of interest operably linked to the control sequence;
and
(iv) a polyadenylation site operably linked 3′ to the open reading frame;
(c) a second expression cassette, comprising:
(iii) a weak constitutive promoter, operably linked to an open reading frame encoding a selectable marker;
and
(iv) a polyadenylation site operably linked 3′ to the open reading frame;
and
(d) a 3′ PiggyBac ITR comprising the nucleotide sequence of SEQ ID NO:47.

13 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/217,863Filed 3 Jul 2023
Priority: 27 Jan 2016
Published: 1 Feb 2024
Term ends: Not estimatedThe source records don’t support an estimate.

Classification14 codes

C12N 15/52
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: genes encoding for enzymes or proenzymes
C12N 15/113
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: non-coding nucleic acids modulating the…
C12N 15/65
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: using markers
C12N 15/67
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for enhancing…

Family

Application62/388,391Us provisional application · filed 27 Jan 2016
Application16/072,180Continuation in part · filed 24 Jul 2018
Application16/286,551Division · filed 26 Feb 2019
Application17/190,954Continuation in part · filed 3 Mar 2021
Application18/217,863Continuation in part
Application17/190,954Division
Application16/286,551Continuation in part
US 11,028,410 B2Related patent
US 11,098,310 B2Related patent
US 11,692,193 B2Related patent
US 2019/0055580 A1Published application
US 2019/0185863 A1Published application
US 2021/0189403 A1Published application
US 2024/0035037 A1This patent

From the US filing history. Foreign family members aren’t listed.