HYPERACTIVE TRANSPOSONS AND TRANSPOSASES

Abstract

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.

Claims

1. A polypeptide comprising a piggyBac transposase or a fragment or a derivative thereof having transposase function comprising at least one amino acid substitution selected from the group consisting of isoleucine (I) at amino acid position 30 or at an amino acid position corresponding thereto is replaced by alanine (A) (I30A), glutamine (Q) at amino acid position 118 or at an amino acid position corresponding thereto is replaced by proline (P) (Q118P), methionine (M) at amino acid position 185 or at an amino acid position corresponding thereto is replaced by valine (V) (M185V), methionine (M) at amino acid position 282 or at an amino acid position corresponding thereto is replaced by leucine (L) (M282L), and asparagine (N) at amino acid position 538 or at an amino acid position corresponding thereto is replaced by arginine (R) (N538R).

2. The polypeptide of claim 1, wherein the piggyBac transposase or the fragment or the derivative thereof having transposase function comprises the following amino acid substitutions: isoleucine (I) at amino acid position 30 or at an amino acid position corresponding thereto is replaced by alanine (A) (I30A), methionine (M) at amino acid position 282 or at an amino acid position corresponding thereto is replaced by leucine (L) (M282L), and asparagine (N) at amino acid position 538 or at an amino acid position corresponding thereto is replaced by arginine (R) (N538R).

3. The polypeptide of claim 1, wherein the piggyBac transposase or the fragment or the derivative thereof having transposase function comprises the following amino acid substitutions: isoleucine (I) at amino acid position 30 or at an amino acid position corresponding thereto is replaced by alanine (A) (I30A), glutamine (Q) at amino acid position 118 or at an amino acid position corresponding thereto is replaced by proline (P) (Q118P), methionine (M) at amino acid position 185 or at an amino acid position corresponding thereto is replaced by valine (V) (M185V), methionine (M) at amino acid position 282 or at an amino acid position corresponding thereto is replaced by leucine (L) (M282L), and asparagine (N) at amino acid position 538 or at an amino acid position corresponding thereto is replaced by arginine (R) (N538R).

4. The polypeptide of claim 1, wherein the piggyBac transposase has an amino acid sequence according to SEQ ID NO: 18 and comprises at least one amino acid substitution selected from the group consisting of isoleucine (I) at amino acid position 30 is replaced by alanine (A) (I30A), glutamine (Q) at amino acid position 118 is replaced by proline (P) (Q118P), methionine (M) at amino acid position 185 is replaced by valine (V) (M185V), methionine (M) at amino acid position 282 is replaced by leucine (L) (M282L), and asparagine (N) at amino acid position 538 is replaced by arginine (R) (N538R), or is a variant thereof which is at least 80% identical to said amino acid sequence, wherein said variant comprises at least one amino acid substitution selected from the group consisting of isoleucine (I) at amino acid position 30 or at an amino acid position corresponding thereto is replaced by alanine (A) (I30A), glutamine (Q) at amino acid position 118 or at an amino acid position corresponding thereto is replaced by proline (P) (Q118P), methionine (M) at amino acid position 185 or at an amino acid position corresponding thereto is replaced by valine (V) (M185V), methionine (M) at amino acid position 282 or at an amino acid position corresponding thereto is replaced by leucine (L) (M282L), and asparagine (N) at amino acid position 538 or at an amino acid position corresponding thereto is replaced by arginine (R) (N538R).

5-6. (canceled)

7. The polypeptide of claim 1, wherein the polypeptide comprises at least one heterologous chromatin reader domain (CRD).

8. The polypeptide of claim 7, wherein the at least one heterologous CRD is connected to the transposase or the fragment or the derivative thereof having transposase function, preferably via a linker.

9. The polypeptide of claim 8, wherein the CRD recognises histone methylation degree and/or acetylation state of histones.

10-13. (canceled)

14. A polynucleotide encoding the polypeptide of claim 1.

15. A vector comprising the polynucleotide of claim 14.

16. 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.

17. The transposable element of claim 16, 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.

18. The transposable element of claim 17, wherein the piggyBac left internal repeat sequence 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).

19. The transposable element of claim 17, wherein the piggyBac-like left internal repeat sequence (i) 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, or (ii) 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), or (iii) 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 (iv) 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).

20-22. (canceled)

23. The transposable element of claim 16, 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.

24-25. (canceled)

26. The transposable element of claim 16, 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.

27-30. (canceled)

31. A method for producing a transgenic cell comprising the steps of: (i) providing a cell, and (ii) introducing (iia) a transposable element, and a polypeptide of claim 1, (iib) 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, and a transposase or a fragment or a derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or a derivative thereof having transposase function, or a vector comprising a polynucleotide encoding a transposase or a fragment or a derivative thereof having transposase function, or (iic) 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, and a polypeptide of claim 1, into the cell, thereby producing the transgenic cell.

32-35. (canceled)

36. The method of claim 31, wherein the transposable element of (iia) comprises at least one polynucleotide of interest.

37. The method of claim 36, wherein the at least one polynucleotide of interest is flanked by terminal repeats (TRs).

38-47. (canceled)

48. A kit comprising (i) a transposable element, and a polypeptide of claim 1; or (ii) 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, and a transposase or a fragment or a derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or a derivative thereof having transposase function, or a vector comprising a polynucleotide encoding a transposase or a fragment or a derivative thereof having transposase function; or (iii) 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, and a polypeptide of claim 1.

49-51. (canceled)

52. The kit of claim 48, wherein the transposable element of (i) comprises at least one polynucleotide of interest, or at least one cloning site for inserting at least one polynucleotide of interest.

53-69. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0237] The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0238] FIG. 1: Tested variants of hyperactive piggyBac transposase. PBw: wild-type (wt) PiggyBac transposase, Trichoplusia ni, GenBank accession number #AAA87375.2; haPB1: transposase mutated in 130A, Q118P, M185V, M282L, and N538R compared to wt piggyBac transposase; haPB2: transposase mutated in 130A, M282L, and N538R compared to wt piggyBac transposase; The nucleotide sequences and the corresponding amino acid sequences are listed under SEQ ID NO: 17 and SEQ ID NO: 18 for PBw, SEQ ID NO: 19 and SEQ ID NO: 20 for haPB1 and SEQ ID NO: 21 and SEQ ID NO: 22 for haPB2.

[0239] FIG. 2: Tested variants of transposon end sequences (TES). A: schematic representation of transposon B: Tested transposon variants

[0240] FIG. 3: Maps of transposon expression vectors. Promoter regions are shown as black blocks, Polyadenylation signals (PolyA) are shown as white boxes. Antibiotic resistance genes, selection marker genes and the coding region for the light chain gene or rather the heavy chain gene are shown as arrows: pac=puromycin-N-acetyltransferase; dhfr=dehydrofolate reductase; aph=kanamycin resistance. The location of the tested transposon end sequences (TES) are shown as triangles.

[0241] FIG. 4a: Viability recovery during selection phase. A: circles: PB minimal wild type TES Transposable Element and PBw; triangles: PB wild type TES Transposable Element (5′TES 247 bp) and PBw; squares: PB artificial TES Transposable Element (5′TES 247 bp) and PBw; asterisk: PB artificial TES (5′TES 247 bp) and haPB2 B: Viability at day 5 post selection start of PB wild type TES Transposable Element and PB artificial TES Transposable Element and PBw.

[0242] FIG. 5: Fed Batch IgG antibody titer concentrations of CHO-DG44 clones pools after brief selection. Relative day 14 titers derived from PBw and hyperactive transposase variants.

[0243] FIG. 6: Shows single substitutions and preferred combinations of substitutions of the piggyBac transposase or fragment or derivative thereof. At least one of the following substitutions is present: I30A, Q118P, M185V, M282L, and/or N538R.

EXAMPLES

[0244] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Example 1

Gene Optimization, Synthesis and Construction of the Transposase Expression Plasmids

[0245] The amino acid sequences of piggyBac wt transposase (Trichoplusia ni; GenBank accession number #AAA87375.2; SEQ ID NO: 18 [Virology 172(1) 156-169 1989]) and hyperactive variants thereof (SEQ ID NO: 20 and SEQ ID NO: 22) were reverse translated.
The nucleotide sequences were optimized by knockout of cryptic splice sites and RNA destabilizing sequence elements, optimized for increased RNA stability and adapted to match the requirements of CHO cells (Cricetulus griseus) regarding the codon usage. The nucleotide sequences were synthesized by GeneArt Gene Synthesis (Life technologies) and used to generate the constructs shown in FIG. 1 using standard cloning procedures. The coding sequence (CDS) of PBw is shown under SEQ ID NO: 17, the coding sequence (CDS) of haPB1 is shown under SEQ ID NO: 19 and the coding sequence (CDS) of haPB2 is shown under SEQ ID NO: 21.
The constructs were ligated into an expression vector, which allows transient expression of the transposase variants under control of the CMV promoter. General procedures for constructing expression plasmids are described in Sambrook, J E. F. Fritsch and T. Maniatis: Cloning I/II/III, A Laboratory Manual New York/Cold Spring Harbor Laboratory Press, 1989, Second Edition.

Example 2

Construction of the Transposon Plasmids

[0246] Transposons were created containing variants of natural and artificial PB transposon end sequences and tested for their ability to be recognized by the PB transposase. The tested constructs are shown in FIG. 2b. The nucleotide sequences of the transposon end sequences are listed here under SEQ ID NO: 13 (piggyBac, Trichoplusia ni, 5′-transposon end sequence (wt) (357 bp)), SEQ ID NO: 14 (piggyBac, Trichoplusia ni, 3′-transposon end sequence (wt)), SEQ ID NO: 6 (piggyBac, Trichoplusia ni, 5′-transposon end sequence (wt) (248 bp)), SEQ ID NO: 11 (piggyBac, Trichoplusia ni, minimal 5′-transposon end sequence (wt)), SEQ ID NO: 12 (piggyBac, Trichoplusia ni, minimal 3′-transposon end sequence (wt)), SEQ ID NO: 16 (piggyBac, artificial 5′-transposon end sequence (357 bp)) and SEQ ID NO: 15 (piggyBac, artificial 5′-transposon end sequence (248 bp)). The transposon end sequences were integrated in the empty expression vectors PBGGPE×2.0m and PBGGPE×2.0p in 5′ and 3′ position to the bacterial backbone sequence with bacterial replication origin and antibiotic resistance gene.
Synthetic heavy or rather light chain fragments of an monoclonal antibody assembled with a signal peptide were ligated into the transposon containing empty expression vectors to generate the plasmids shown in FIG. 3. General procedures for constructing expression plasmids are described in Sambrook, J E. F. Fritsch and T. Maniatis: Cloning I/II/III, A Laboratory Manual New York/Cold Spring Harbor Laboratory Press, 1989, Second Edition.

Example 3

Generation and Analysis of Clone Pools

[0247] As starter cell line the dihydrofolate reductase-deficient CHO cell line, CHO/DG44 [Urlaub et al., 1986, Proc Natl Acad Sci USA. 83 (2): 337-341] was used. The cell line was maintained in serum-free medium. Plasmids containing one of the transposon variants and transient expression vectors for expression of one of the transposase variants each were transfected by electroporation according to the manufacturer's instructions (Neon Transfection System, Thermo Fisher Scientific). In each transfection 1.5 μg of circular HC and LC transposon vector DNA and 1.2 μg of circular transposase DNA were used. Transfectants were subjected to selection with puromycin and methotrexate to eliminate untransfected cells, as well as non- and low-producer. Two consecutive series of transfections and selections were performed using the same vector combinations, DNA amounts and selection conditions. After a selection period of two weeks selection pressure was removed and resulting clone pools were subjected to Fed-batch processes under generic conditions with defined seeding cell densities. Fed batch processes were performed in shake flasks (SF125, Corning) with working volumes of 30 mL in chemically defined culture medium. A chemically defined feed was applied every two days following a generic feeding regiment. Viability were measured using a Vi-CELL viability analyzer (Beckman Coulter). Antibody concentrations of cell culture supernatant samples were determined by the Octet® RED96 System (Fortebio) against purified material of the expressed antibody as standard curve.

[0248] FIG. 4 shows viability recovery during selection phase of transfectants generated with wild type PB transposase or hyperactive transposase haPB2 and the transposons of Example 2. A faster recovery of viability was observed compared to the wild type transposons. The faster recovery of viability was observed by using hyperactive transposase in combination with a transposon comprising an artificial TES.

[0249] FIG. 5 shows the fed batch results at day 14 of clone pools derived from wt PB transposase and hyperactive transposase variants by using the PB minimal wild type TES Transposons. An increase in titer was observed compared to the wild type transposase for the hyperactive transposase variants (5 to 6 fold).

Sequence Listing Summary:

[0250] SEQ ID NO: 1 piggyBac, Trichoplusia ni, left internal repeat wild-type (wt) [0251] SEQ ID NO: 2 piggyBac-like, Xenopus tropicalis, left internal repeat (wt) [0252] SEQ ID NO: 3 piggyBac-like, Bombyx mori, left internal repeat (wt) [0253] SEQ ID NO: 4 piggyBac-like, Mytois lucifugus #1, left internal repeat (wt) [0254] SEQ ID NO: 5 piggyBac-like, Mytois lucifugus #2, left internal repeat (wt) [0255] SEQ ID NO: 6 piggyBac, Trichoplusia ni, 5′-transposon end sequence (wt), encompassing SEQ ID NO: 1 (wt) (248 bp) [0256] SEQ ID NO: 7 piggyBac-like, Xenopus tropicalis, 5′-transposon end sequence (wt), encompassing SEQ ID NO: 2 (wt) [0257] SEQ ID NO: 8 piggyBac-like, Bombyx mori, 5′-transposon end sequence (wt), encompassing SEQ ID NO: 3 (wt) [0258] SEQ ID NO: 9 piggyBac-like, Mytois lucifugus #1, 5′-transposon end sequence (wt), encompassing SEQ ID NO: 4 (wt) [0259] SEQ ID NO: 10 piggyBac-like, Mytois lucifugus #2, 5′-transposon end sequence (wt), encompassing SEQ ID NO: 5 (wt) [0260] SEQ ID NO: 11 piggyBac, Trichoplusia ni, minimal 5′-transposon end sequence (wt) [0261] SEQ ID NO: 12 piggyBac, Trichoplusia ni, minimal 3′-transposon end sequence (wt) [0262] SEQ ID NO: 13 piggyBac, Trichoplusia ni, 5′-transposon end sequence (wt) (357 bp) [0263] SEQ ID NO: 14 piggyBac, Trichoplusia ni, 3′-transposon end sequence (wt) [0264] SEQ ID NO: 15 piggyBac, artificial 5′-transposon end sequence (248 bp) [0265] SEQ ID NO: 16 piggyBac, artificial 5′-transposon end sequence (357 bp) [0266] SEQ ID NO: 17 wt piggyBac transposase, Trichoplusia ni, DNA [0267] SEQ ID NO: 18 wt piggyBac transposase, Trichoplusia ni, protein [0268] SEQ ID NO: 19 piggyBac transposase mutant haPB1, DNA [0269] SEQ ID NO: 20 piggyBac transposase mutant haPB1, protein [0270] SEQ ID NO: 21 piggyBac transposase mutant haPB2, DNA [0271] SEQ ID NO: 22 piggyBac transposase mutant haPB2, protein [0272] SEQ ID NO: 23 piggyBac, Trichoplusia ni, left repeat sequence (wt) [0273] SEQ ID NO: 24 piggyBac-like, Xenopus tropicalis, left repeat sequence (wt) [0274] SEQ ID NO: 25 piggyBac-like, Bombyx mori, left repeat sequence (wt) [0275] SEQ ID NO: 26 piggyBac-like, Mytois lucifugus #1, left repeat sequence (wt) [0276] SEQ ID NO: 27 piggyBac-like, Mytois lucifugus #2, left repeat sequence (wt) [0277] SEQ ID NO: 28: piggyBac, artificial left internal repeat