EP-4741508-A2 - HYPERACTIVE TRANSPOSONS AND TRANSPOSASES

EP4741508A2EP 4741508 A2EP4741508 A2EP 4741508A2EP-4741508-A2

Abstract

Inventors

SANDIG, VOLKER
KRÜGENER, Sven
ROSE, THOMAS

Assignees

ProBioGen AG

Dates

Publication Date: 20260513
Application Date: 20200717

Claims (15)

A transposable element comprising a piggyBac or piggyBac-like left repeat sequence and left internal repeat sequence, wherein the left internal repeat sequence comprises at least one nucleotide modification, wherein the at least one nucleotide modification increases the homology of the left internal repeat sequence to the left repeat sequence.
The transposable element of claim 1, wherein the at least one nucleotide modification increases binding affinity of a piggyBac or piggyBac-like transposase cysteine-rich domain (CRD) to the left internal repeat sequence.
The transposable element of claims 1 or 2, wherein the transposable element reduces the viability recovery time of transfected cells and/or increases expression performance, including an increased product titer, compared to a corresponding transposable element lacking said nucleotide modification.
The transposable element of any one of claims 1 to 3, wherein the at least one nucleotide modification is selected from the group consisting of a nucleotide substitution, a nucleotide deletion, a nucleotide addition, and a nucleotide insertion, or is a combination thereof.
The transposable element of claim 4, wherein the piggyBac left internal repeat sequence (i) has a nucleotide sequence according to SEQ ID NO: 1 and comprises at least one nucleotide substitution selected from the group consisting of adenosine (A) at nucleotide position 3 is replaced by cytidine (C) (A3C), adenosine (A) at nucleotide position 9 is replaced by thymidine (T) (A9T), adenosine (A) at nucleotide position 10 is replaced by thymidine (T) (A10T), and guanosine (G) at nucleotide position 12 is replaced by thymidine (T) (G12T), or is a variant thereof which is at least 80% identical to said nucleotide sequence, wherein said variant comprises at least one nucleotide substitution selected from the group consisting of adenosine (A) at nucleotide position 3 or at a nucleotide position corresponding thereto is replaced by cytidine (C) (A3C), adenosine (A) at nucleotide position 9 or at a nucleotide position corresponding thereto is replaced by thymidine (T) (A9T), adenosine (A) at nucleotide position 10 or at a nucleotide position corresponding thereto is replaced by thymidine (T) (A10T), and guanosine (G) at nucleotide position 12 or at a nucleotide position corresponding thereto is replaced by thymidine (T) (G12T), (ii) has a nucleotide sequence according to SEQ ID NO: 2 and comprises a nucleotide insertion, wherein adenosine (A) is introduced between nucleotide positions 7 and 8, or is a variant thereof which is at least 80% identical to said nucleotide sequence, wherein said variant comprises a nucleotide insertion, wherein adenosine (A) is introduced between nucleotide positions 7 and 8 or between nucleotide positions corresponding thereto. (iii) has a nucleotide sequence according to SEQ ID NO: 3 and comprises at least one nucleotide substitution selected from the group consisting of guanosine (G) at nucleotide position 7 is replaced by cytidine (C) (G7C), and thymidine (T) at nucleotide position 9 is replaced by cytidine (C) (T9C), or is a variant thereof which is at least 80% identical to said nucleotide sequence, wherein said variant comprises at least one nucleotide substitution selected from the group consisting of guanosine (G) at nucleotide position 7 or at a nucleotide position corresponding thereto is replaced by cytidine (C) (G7C), and thymidine (T) at nucleotide position 9 or at a nucleotide position corresponding thereto is replaced by cytidine (C) (T9C), (iv) has a nucleotide sequence according to SEQ ID NO: 4 and comprises a nucleotide substitution, wherein thymidine (T) at nucleotide position 7 is replaced by adenosine (A) (T7A), or is a variant thereof which is at least 80% identical to said nucleotide sequence, wherein said variant comprises a nucleotide substitution, wherein thymidine (T) at nucleotide position 7 or at a nucleotide position corresponding thereto is replaced by adenosine (A) (T7A), or (v) has a nucleotide sequence according to SEQ ID NO: 5 and comprises at least one nucleotide substitution selected from the group consisting of guanosine (G) at nucleotide position 6 is replaced by thymidine (T) (G6T), and thymidine (T) at nucleotide position 14 is replaced by guanosine (G) (T14G), or is a variant thereof which is at least 80% identical to said nucleotide sequence, wherein said variant comprises at least one nucleotide substitution selected from the group consisting of guanosine (G) at nucleotide position 6 or at a nucleotide position corresponding thereto is replaced by thymidine (T) (G6T), and thymidine (T) at nucleotide position 14 or at a nucleotide position corresponding thereto is replaced by guanosine (G) (T14G).
The transposable element of any one of claims 1 to 5, wherein the left repeat sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 27 or is a variant thereof which is at least 80% identical to said nucleotide sequence.
The transposable element of any one of claims 1 to 6, wherein the left repeat sequence and the left internal repeat sequence are part of a 5'-transposon end sequence.
The transposable element of any one of claims 1 to 7, wherein the transposable element comprises a piggyBac or piggyBac-like 3'-transposon end sequence.
The transposable element of any one of claims 1 to 8, wherein the transposable element comprises at least one polynucleotide of interest, or at least one cloning site for inserting at least one polynucleotide of interest.
The transposable element of claim 9, wherein the at least one polynucleotide of interest is operably linked to the piggyBac or piggyBac-like 5'-transposon end sequence and to the piggyBac or piggyBac-like 3'-transposon end sequence, or the at least one cloning site for inserting the at last one polynucleotide of interest is located between the piggyBac or piggyBac-like 5'-transposon end sequence and the piggyBac or piggyBac-like 3'-transposon end sequence.
The transposable element of claims 9 or 10, wherein the at least one polynucleotide of interest is selected from the group consisting of a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, a polynucleotide encoding a mRNA, a polynucleotide encoding a tag, and a viral polynucleotide.
A transgenic cell comprising a transposable element of any one of claims 1 to 11.
A kit comprising a container and a transposable element of any one of claims 1 to 11.
The kit of claim 13, wherein the kit further comprises a transposase, or a polynucleotide encoding a transposase, or a vector comprising a polynucleotide encoding a transposase.
A method for introducing a polynucleotide of interest into the genome of a cell comprising the steps of: (i) providing a cell, and (ii) introducing a transposable element of any one of claims 1 to 11, and a transposase, or a polynucleotide encoding a transposase, or a vector comprising a polynucleotide encoding a transposase into the cell, thereby integrating the polynucleotide of interest into the genome of the cell.

Description

The present invention relates to a polypeptide comprising a piggyBac transposase or a fragment or a derivative thereof having transposase function comprising at least one amino acid substitution. Further, the present invention relates to a transposable element comprising a piggyBac or piggyBac-like left repeat sequence and left internal repeat sequence, wherein the left internal repeat sequence comprises at least one nucleotide modification. Furthermore, the present invention relates to a kit comprising the above transposase and/or transposable element. In addition, the present invention relates to a targeting system comprising the above transposase and/or transposable element. BACKGROUND OF THE INVENTION Transposons have recently been developed as potent, non-viral gene delivery tools. In particular, the performance of a generated producer cell line can be improved, when the integration of plasmid DNA is supported using a transposon. For instance, a transposon allows the integration of a greater size of heterologous DNA and the integration of a higher number of heterologous DNA copies into each genome. Furthermore, integration via a transposon provides an efficient method for the reduction of plasmid backbone integration and/or the reduction of concatemers. Transposable elements or transposons are DNA-sections, which can move from one locus to another part of the genome. Two classes of transposable elements are distinguished: retrotransposons, which replicate through an RNA intermediate (class 1), and "cut-and-paste" DNA transposons (class 2). Class 2 transposons are characterised by short inverted terminal repeats (ITRs) and element-encoded transposases, enzymes with excision and insertion activity. In the natural configuration, the transposase gene is located between the inverted repeats. A number of class 2 transposons have been shown to facilitate insertion of heterologous DNA into the genome of eukaryotes, for example, a transposon from the moth Trichoplusia ni (piggyBac), a transposon from the bat Myotis lucifugus (piggyBat), a reconstructed transposon from salmon species (Sleeping Beauty), or a transposon from the medaka Oryzias latipes (Tol2). These transposons have many applications in genetic manipulation of a host genome, including transgene delivery and insertional mutagenesis. For instance, the piggyBac (PB) DNA transposon (previously described as IFP2) is used technologically and commercially in genetic engineering by virtue of its property to efficiently transpose between vectors and chromosomes [US6218185 B1]. For these applications the DNA to be integrated is flanked by two PB ITRs in a PB vector. By co-delivery of PB transposase the flanked DNA is excised precisely form the PB vector and integrated into the target genome at TTAA specific sites. To increase transformation efficiencies, more active transposases were developed. These hyperactive transposases yield a greater fraction of cells that integrated a provided transposon and a greater number of transposon integrations per cell compared to wild-type transposases. Different strategies are described in the art: For example, EP2160461 B1 describes hyperactive Sleeping Beauty transposases generated via side directed mutagenesis, US9534234 B2 provides a PB-like transposase derived from the silkworm Bombyx mori and from the frog Xenopus tropicalis fused to a heterologous nuclear localization sequence (NLS), EP1546322 B1 discloses a chimeric integrating enzyme comprising a binding domain recognising a DNA landing pad to drag transposon-transposase complex to the landing pad and promote integration in its vinicity and EP1594972 B1 claims a transposase or a fragment or derivative thereof having transposase function fused to a polypeptide binding domain that can associates with a cellular or engineered polypeptide comprising a DNA targeting domain. Transformation efficiencies can also be increased by using more active transposons. One field of application for transposases is the development of pharmaceutical cell lines. Chinese Hamster Ovary (CHO) cells are the most prevalent mammalian cell factories for producing therapeutic biologics, due to its ability to grow in suspension cultures, its capacity for complex post-translational modifications, and its low susceptibility to human viral infections. One of the main limits for the industrial production of recombinant therapeutic proteins is the time- and labor-intensive process of cell line production and characterisation. The majority of the available methods rely on random transgene integration. Multiple cassettes often integrate in tandem into more or less active sites. Active chromosomal loci are rare and thousands of clones have to be screened to obtain high producers. To reduce the extent of recombinant cell line screening and to enhance the productivity and stability of recombinant CHO cell lines, PB-mediated gene delivery was used [M. Matasci et al., The PiggyBac transposon enhances the fr