WO2010085699A2 - Mammalian piggybac transposon and methods of use - Google Patents

Mammalian piggybac transposon and methods of use Download PDF

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WO2010085699A2
WO2010085699A2 PCT/US2010/021871 US2010021871W WO2010085699A2 WO 2010085699 A2 WO2010085699 A2 WO 2010085699A2 US 2010021871 W US2010021871 W US 2010021871W WO 2010085699 A2 WO2010085699 A2 WO 2010085699A2
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transposon
seq
nucleic acid
transposase
cell
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WO2010085699A3 (en
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Nancy Lynn Craig
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The Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Definitions

  • Typical methods for introducing DNA into a cell include DNA condensing reagents such as calcium phosphate, polyethylene glycol, and the like, lipid-containing reagents, such as liposomes, multi- lamellar vesicles, and the like, as well as virus-mediated strategies.
  • DNA condensing reagents such as calcium phosphate, polyethylene glycol, and the like
  • lipid-containing reagents such as liposomes, multi- lamellar vesicles, and the like
  • virus-mediated strategies can have limitation.
  • the amount of nucleic acid that can be transfected into a cell is limited in virus strategies.
  • Virus-mediated strategies can be cell- type or tissue-type specific and the use of virus-mediated strategies can create immunologic problems when used in vivo.
  • Transposons include a (short) nucleic acid sequence, with terminal repeat sequences upstream and downstream.
  • Active transposons encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.
  • Transposable elements represent a substantial fraction of many eukaryotic genomes. For example, -50% of the human genome is derived from transposable element sequences, and other genomes, for example plants, may consist of substantially higher proportions of transposable element-derived DNA.
  • Transposable elements are typically divided into two classes, class 1 and class 2.
  • Class 1 is represented by the retrotransposons (LINEs, SINEs, LTRs, and ERVs).
  • Class 2 includes the "cut-and-paste" DNA transposons, which are characterized by terminal inverted repeats (TIRs) and are mobilized by an element-encoded transposase.
  • TIRs terminal inverted repeats
  • 10 superfamilies of cut-and-paste DNA transposons are recognized in eukaryotes (Feschotte and Pritham 2007).
  • the present inventors have identified mammalian piggyBac transposons and transposases.
  • the present inventors have identified hyperactive mammalian piggyBac variants.
  • the mammalian piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell.
  • the gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.
  • the invention features a transposon comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposon activity.
  • the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae. In another embodiment, the mammalian piggyBac nucleic acid sequence is from the genus Myotis. In another further embodiment, the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
  • the transposon comprises a nucleic acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. In further embodiments, the transposon is capable of inserting into the DNA of a cell.
  • the transposon further comprises a marker protein.
  • the transposon is inserted in a plasmid.
  • the transposon further comprises at least a portion of an open reading frame. In another related embodiment, the transposon further comprises at least one expression control region. In a further embodiment, the expression control region is selected from the group consisting of a promoter, an enhancer or a silencer. In another embodiment, the transposon further comprises a promoter operably linked to at least a portion of an open reading frame. In certain exemplary embodiments, the plasmid comprises SEQ ID NO: 7 or SEQ ID
  • the cell is obtained from an animal.
  • the cell is from a vertebrate or an invertebrate.
  • the vertebrate is a mammal.
  • the present invention features a transposase comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposase activity.
  • the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae. In another embodiment, the mammalian piggyBac nucleic acid sequence is from the genus Myotis. In another further embodiment, the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
  • the nucleic acid sequence is selected from SEQ ID NO: 3 and SEQ ID NO: 5.
  • amino acid sequence is selected from SEQ ID NO: 4 and SEQ ID NO: 6. .
  • the present invention features a gene transfer system comprising a transposon and a mammalian piggyBac transposase.
  • the transposon is inserted into the genome of the cell.
  • the cell is obtained from an animal.
  • the cell is from a vertebrate or an invertebrate.
  • the vertebrate is a mammal.
  • the invention features a cell comprising the transposon of the above aspects.
  • the present invention provides a pharmaceutical composition comprising a transposon comprising a mammalian piggyBac nucleic acid sequence and a mammalian piggyBac transposase, together with a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • the present invention features a method for introducing exogenous DNA into a cell comprising contacting the cell with the gene transfer system of claim 26, thereby introducing exogenous DNA into a cell.
  • the cell is a stem cell.
  • the present invention provides a kit comprising a transposon comprising a mammalian piggyBac nucleic acid sequence and instructions for introducing DNA into a cell.
  • the kit further comprises a mammalian piggyBac transposase.
  • the present invention features a hyperactive mammalian piggyBac transposon that has a higher level of transposon excision compared to a wildtype mammalian piggyBac transposon.
  • the wildtype mammalian piggyBac transposon is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the hyperactive transposon comprises a nucleotide change in SEQ ID NO 3 selected from C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A, A687G, G715A, T13C, C23T, G161A, G25A, T1050C, A1356G, A26G, A1033G, A1441G, A32G, A389C, A32G, A389C, A32G, T1572A, G456A, T1641C, Tl 155C, G1280A, T22C, A106G, A29G, and Cl 137T.
  • SEQ ID NO 3 selected from C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A
  • the hyperactive transposon comprises an amino acid change in SEQ ID NO 4 selected from A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, I345V, M481V, EI lG, K130T, G9G, R427H, S8P, S36G, DlOG, S36G and silent.
  • nucleotide or “polynucleotide” is meant to refer to both double- and single-stranded DNA and RNA, and combinations thereof.
  • a polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences.
  • a polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques.
  • a polynucleotide can be linear or circular in topology.
  • a polynucleotide can be, for example, a portion of a vector, or a fragment.
  • a "coding sequence” or a “coding region” is a polynucleotide that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide.
  • the boundaries of a coding region are generally determined by a translational start codon at its 5' end and a translational stop codon at its 3' end.
  • a regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked.
  • Nonlimiting examples of regulatory sequences include promoters, transcriptional initiation sites, translational start sites, translational stop sites, transcriptional terminators (including, for instance, poly-adenylation signals), and intervening sequences (introns).
  • operably linked is meant to refer a nucleotide sequence that is placed in a functional relationship with another nucleotide sequence.
  • a coding sequence is operably linked to a promoter sequence
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary join two protein coding regions, contiguous and in reading frame. Since enhancers may function when separated from the promoter by several kilobases and intron sequences may be of variable lengths, some nucleotide sequences may be operably linked but not contiguous.
  • polypeptide is meant to refer to a polymer of amino acids of any length.
  • peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide.
  • This term also includes post- expression modifications of the polypeptide, for example, glycosylations (e.g., the addition of a saccharide), acetylations, phosphorylations and the like.
  • transposon or “transposable element” is meant to refer to a polynucleotide that is able to excise from a donor polynucleotide, for instance, a vector, and integrate into a target site, for instance, a cell's genomic or extrachromosomal DNA.
  • a transposon includes a polynucleotide that includes a nucleic acid sequence flanked by cis- acting nucleotide sequences on the termini of the transposon.
  • a nucleic acid sequence is "flanked by" cis-acting nucleotide sequences if at least one cis-acting nucleotide sequence is positioned 5' to the nucleic acid sequence, and at least one cis-acting nucleotide sequence is positioned 3' to the nucleic acid sequence.
  • Cis-acting nucleotide sequences include at least one inverted repeat (also referred to herein as an inverted terminal repeat, or ITR) at each end of the transposon, to which a transposase, preferably a member of the mammalian piggyBac family of transposases, binds.
  • the transposon is a mammalian piggyBac transposon.
  • An "isolated" polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized.
  • a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities.
  • transposase is meant to refer to a polypeptide that catalyzes the excision of a transposon from a donor polynucleotide (e.g., a vector) and the subsequent integration of the transposon into the genomic or extrachromosomal DNA of a target cell.
  • a donor polynucleotide e.g., a vector
  • the transposase binds an inverted sequence or a direct repeat.
  • the transposase may be present as a polypeptide.
  • the transposase is present as a polynucleotide that includes a coding sequence encoding a transposase.
  • the polynucleotide can be RNA, for instance an mRNA encoding the transposase, or DNA, for instance a coding sequence encoding the transposase.
  • the coding sequence may be present on the same vector that includes the transposon, i.e., in cis.
  • the transposase coding sequence may be present on a second vector, i.e., in trans.
  • the transposase is a mammalian piggyBac transposon.
  • Figure 1 shows piggyBac transposition in HeLa cells.
  • Figure 2 (A - E) shows the results of phylogenetic analyses.
  • Tc2 transposases (B) Tc2 transposases.
  • C mariner transposases.
  • D TcI transposases.
  • E piggyBac transposases.
  • Figure 3 shows electropherogram results from PCR analysis of a taxonomically diverse panel of bat genomes. Oligonucleotide primer pairs are indicated in white.
  • Figure 4 shows divergence estimates and age ranges for each autonomous and nonautonomous transposon family described.
  • Figure 5 shows filled and intact preintegration site sequences for individual nonautonomous piggyBacl ML elements from M. lucifugus and M. austroriparius, respectively. Target site duplications for each insertion are shaded.
  • (A) Locus 340 3005.
  • Locus primer sequences (5'-3 ? ) for the two loci presented are as follows:
  • Figure 6 is a graph that shows proportion of insertions nested within instances of each
  • Figure 9 is a representation of mammalian bat piggyBac helper plasmid which expresses the bat piggyBac transposase and corresponds to SEQ ID NO: 7.
  • Figure 10 is a representation of mammalian bat piggyBac donor plasmid which encodes bat piggyBac transposon and a drug resistance marker and corresponds to SEQ ID
  • the present inventors have isolated the first piggyBac transposon from a mammal. No active form of this family of transposons has been isolated from a mammal, and very few other transposons that are active in mammalians cells and other metazoans have been isolated.
  • the present invention features mammalian piggyBac transposon and transposases.
  • a transposon, or transposable element that includes a nucleic acid sequence, as described herein, positioned between at least two repeats (for example, inverted repeats (IRs)), at least one repeat on either side of the nucleic acid sequence.
  • the inventive transposon comprises a nucleic acid sequence positioned between at least two repeats, wherein these repeats can bind to a transposase protein as defined herein and wherein the transposon is capable of inserting into DNA of a cell.
  • repeats are preferably sequences that are recognized and bound by the transposase as defined herein.
  • a mammalian piggyBac transposon is bound by an inventive transposase, contains a pair of repeat sequences.
  • the first repeat is typically located upstream to the nucleic acid sequence and the second repeat is typically located downstream of the nucleic acid sequence.
  • the second repeat represents the same sequence as the first repeat, but shows an opposite reading direction as compared with the first repeat (5' and 3' ends of the complementary double strand sequences are exchanged).
  • IRs inverted repeats
  • repeats may occur in a multiple number upstream and downstream of the above mentioned nucleic acid sequence.
  • the number of repeats located upstream and downstream of the above mentioned nucleic acid sequence is identical.
  • the repeats are short, between 10 - 20 base pairs, and preferably 15 base pairs.
  • the repeats (IRs) as described herein preferably flank a nucleic acid sequence which is inserted into the DNA of a cell.
  • the nucleic acid sequence can include all or part of an open reading frame of a gene (i.e., that part of a protein encoding gene), one or more expression control sequences (i.e., regulatory regions in nucleic acid) alone or together with all or part of an open reading frame.
  • Preferred expression control sequences include, but are not limited to promoters, enhancers, border control elements, locus-control regions or silencers.
  • the nucleic acid sequence comprises a promoter operably linked to at least a portion of an open reading frame.
  • transposons of the present invention can preferably occur as a linear transposon (extending from the 5' end to the 3' end, by convention) that can be used as a linear fragment or circularized, for example in a plasmid.
  • the present invention features a transposon comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposon activity.
  • the present invention also features a mammalian piggyBac transposase comprising a nucleic acid sequence and variants, derivatives and fragments thereof that retain transposase activity.
  • the mammalian piggyBac transposase may be present as a polypeptide.
  • the transposase is present as a polynucleotide that includes a coding sequence encoding a transposase.
  • the polynucleotide can be RNA, for instance an mRNA encoding the transposase, or DNA, for instance a coding sequence encoding the transposase.
  • the coding sequence may be present on the same vector that includes the transposon, i.e., in cis.
  • the transposase coding sequence may be present on a second vector, i.e., in trans.
  • the mammalian piggyBac transposon or transposase nucleic acid sequence is from the family Vespertilionidae. In further exemplary embodiments, the mammalian piggyBac transposon or transposase nucleic acid sequence is from the genus Myotis. In other further preferred embodiments, the mammalian piggyBac transposon or transposase nucleic acid sequence is from the species Myotis lucifugus.
  • nucleic acids encoding a mammalian piggyBac transposon or a mammalian piggyBac transposase as defined herein.
  • Nucleic acids according to the present invention typically comprise ribonucleic acids, including mRNA, DNA, cDNA, chromosomal DNA, extrachromosomal DNA, plasmid DNA, viral DNA or RNA.
  • a nucleic acid is preferably selected from any nucleic sequence encoding the same amino acid sequence of a mammalian transposon or tranposase protein due to degeneration of its genetic code.
  • nucleic acid sequences may lead to an improved expression of the encoded fusion protein in a selected host organism.
  • Tables for appropriately adjusting a nucleic acid sequence are known to a skilled person. Preparation and purification of such nucleic acids and/or derivatives are usually carried out by standard procedures (see Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.). Other variants of these native nucleic acids may have one or more codon(s) inserted, deleted and/or substituted as compared to native nucleic acid sequences.
  • nucleic acid sequences of the present invention may code for modified (non-natural) transposon or transposase sequences.
  • promoters or other expression control regions can be operably linked with the nucleic acids encoding the proteins described herein to regulate expression of the protein in a quantitative or in a tissue-specific manner.
  • the nucleic acid sequence encoding an mammalian piggyBac transposon protein comprises a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, shown below.
  • a mammalian piggyBac transposase comprises a nucleic acid sequence corresponding to SEQ ID NO: 3, and the amino acid sequence encoding the protein, SEQ ID NO: 4, shown below:
  • a mammalian piggyBac transposase comprises a nucleic acid sequence corresponding to SEQ ID NO: 5, and the amino acid sequence encoding the protein, SEQ ID NO: 6, shown below:
  • nucleic acid encoding the mammalian piggyBac transposon has a nucleic acid sequence showing at least 75% or 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with a nucleic acid sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.
  • nucleic acid encoding the mammalian piggyBac transposase has a nucleic acid sequence showing at least 75% or 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the nucleic acid sequence corresponding to SEQ ID NO: 3 or SEQ ID NO: 5.
  • nucleic acid encoding the mammalian piggyBac transposon or transposase is selected from a nucleic acid sequence encoding the mammalian piggyBac transposon as defined above and being capable of hybridizing to a complement of a nucleic acid sequence as defined above under stringent conditions.
  • nucleic acid encoding the mammalian piggyBac transposon or transposase is selected from a nucleic acid sequence encoding the mammalian piggyBac transposase as defined above and being capable of hybridizing to a complement of a nucleic acid sequence as defined above under stringent conditions.
  • Stringent conditions are, for example: 30% (v/v) formamide in 0.5*SSC, 0.1% (w/v) SDS at 42 C for 7 hours.
  • a hyperactive mammalian piggyBac transposon of the present invention has a higher level of transposon excision compared to a wildtype mammalian piggyBac transposon.
  • a mammalian piggyBac of the present invention preferably catalyzes the transposition of a transposon at a frequency that is greater than a "baseline" transposase.
  • the mammalian piggyBac nucleic acid sequence is hyperactive compared to a wildtype mammalian piggyBac nucleic acid sequence.
  • the wildtype mammalian piggyBac transposon is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the nucleotide change in SEQ ID NO 3 is selected from the group consisting of: C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A, A687G, G715A, T13C, C23T, G161A, G25A, T1050C, A1356G, A26G, A1033G, A1441G, A32G, A389C, A32G, A389C, A32G, T1572A, G456A, T1641C, and T1155C.
  • the nucleotide change in SEQ ID NO 3 comprises C41T, A1424G, C1472A, G1681A.
  • the nucleotide change in SEQ ID NO 3 comprises T150C, A351G.
  • nucleotide change in SEQ ID NO 3 comprises A279G. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
  • the nucleotide change in SEQ ID NO 3 comprises A898G.
  • the nucleotide change in SEQ ID NO 3 comprises A880G, G1558A. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A687G, G715A.
  • the nucleotide change in SEQ ID NO 3 comprises T13C, C23T. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
  • the nucleotide change in SEQ ID NO 3 comprises G25A, T1050C, A1356G.
  • the nucleotide change in SEQ ID NO 3 comprises A26G, A1033G.
  • the nucleotide change in SEQ ID NO 3 comprises A1441G.
  • nucleotide change in SEQ ID NO 3 comprises A32G, A389C. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
  • the nucleotide change in SEQ ID NO 3 comprises G456A, T 1641C.
  • the nucleotide change in SEQ ID NO 3 comprises T1155C.
  • the nucleotide change in SEQ ID NO 3 comprises G1280A.
  • the nucleotide change in SEQ ID NO 3 comprises T22C. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
  • the nucleotide change in SEQ ID NO 3 comprises A29G, A106G, and Cl 137T.
  • an amino acid change in SEQ ID NO 4 is selected from the group consisting of: A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, I345V, M481V, EI lG, K130T, G9G, and silent.
  • the amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising C41T, A1424G, C1472A, G1681A, comprises A14V, D475G, P491Q, A561T.
  • amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising T150C, A351G, is silent.
  • amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising A279G, is silent.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T1638C, comprises T546T.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A898G comprises T300A.
  • the amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising A880G, G1558A, comprises T294A, A520T.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A687G, G715A, comprises silent, G239S.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T13C, C23T comprises S5P, S8F.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising Gl 6 IA comprises S54N.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising G25A, T 1050C, A1356G comprises D9N, silent, silent.
  • the amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising A26G, A1033G, comprises D9G, I345V.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A 144 IG, comprises M481V.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A32G, T1572A comprises G9G, silent.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising G456A, T 1641C, comprises silent, silent.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising Tl 155C, comprises silent.
  • the amino acid change in SEQ ID NO 4 corresponding to the nucleotide change in SEQ ID NO 3 comprising G 1280A, comprises R427H.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T22C, comprises S8P.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A106G, comprises S36G.
  • the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A29G, A106G, and C1137T comprises DlOG, S36G, and silent.
  • Assays for measuring the excision of a transposon from a vector, the integration of a transposon into the genomic or extrachromosomal DNA of a cell, and the ability of transposase to bind to an inverted repeat are described herein and are known to the art (see, for instance, (Ivies et al.
  • a transposon of the present invention transposes at a frequency that is, in increasing order of preference, at least about 50%, at least about 100%, at least about 200%, most preferably, at least about 300% greater than a baseline transposon.
  • both transposons i.e., the baseline transposon and the transposon being tested
  • the invention also features protein sequence showing at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the protein sequence of SEQ ID NO: 4 that is encoded by the nucleic acid sequence corresponding to SEQ ID NO: 3.
  • the invention also features protein sequence showing at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the protein sequence of SEQ ID NO: 6 that is encoded by the nucleic acid sequence corresponding to SEQ ID NO: 5.
  • identity is understood as the degree of identity between two or more proteins, nucleic acids, etc., which may be determined by comparing these sequences using known methods such as computer based sequence alignments (basic local alignment search tool, S. F. Altschul et al., J. MoI. Biol. 215 (1990), 403-410). Such methods include without being limited thereto the GAG programme, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 287 (1984); Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP or BLASTN, and FASTA (Altschul, S., et al., J. MoI. Biol. 215:403-410) (1999)). Additionally, the Smith Waterman-algorithm may be used to determine the degree of identity between two sequences.
  • Functional derivatives according to the present invention preferably maintain the biological function of the mammalian transposase, i.e. the transposase activity, the excision of the nucleic acid sequence and its insertion activity concerning the excised sequences into specific target sequences.
  • Functional derivatives according to the present invention may comprise one or more amino acid insertion(s), deletion(s) and/or substitution(s) of the transposase as shown by SEQ ID NO: 4 or SEQ ID NO: 6.
  • Amino acid substitutions as described herein are preferably conservative amino acid substitutions, which do not alter the biological activity of the transposon or transposase protein.
  • amino acid sequences may include, for example, amino acid sequences containing conservative changes that do not significantly alter the activity or binding characteristics of the resulting transposase.
  • substitutions for an amino acid sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations are not expected to substantially affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.
  • Particularly preferred conservative substitutions include, but are not limited to, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free NH 2 .
  • Amino acid insertions and substitutions are preferably carried out at those sequence positions of that do not alter the spatial structure or which relate to the catalytic center or binding region of the mammalian piggyBac transposase.
  • a change of a spatial structure by insertion(s) or deletion(s) can be detected readily with the aid of, for example, CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (Ed.), Elsevier, Amsterdam).
  • Suitable methods for generating proteins with amino acid sequences which contain substitutions in comparison with the native sequence(s) are disclosed for example in the publications U.S. Pat. No.
  • the transposon of the present invention may further comprise a marker protein.
  • the nucleic acid sequence can be of any variety of recombinant proteins, e.g. any protein known in the art.
  • the protein encoded by the nucleic acid sequence can be a marker protein such as green fluorescent protein (GFP), the blue fluorescent protein (BFP), the photo activatable-GFP (PA-GFP), the yellow shifted green fluorescent protein (Yellow GFP), the yellow fluorescent protein (YFP), the enhanced yellow fluorescent protein (EYFP), the cyan fluorescent protein (CFP), the enhanced cyan fluorescent protein (ECFP), the monomeric red fluorescent protein (mRFPl), the kindling fluorescent protein (KFPl), aequorin, the auto fluorescent proteins (AFPs), or the fluorescent proteins JRed, TurboGFP, PhiYFP and PhiYFP-m, tHc-Red (HcRed-Tandem), PS-CFP2 and KFP-Red (all available commercially available), or other suitable fluorescent proteins chloramphenicol acetyl
  • GFP green fluorescent protein
  • BFP
  • the protein further may be selected from growth hormones, for example to promote growth in a transgenic animal, or from beta-galactosidase (lacZ), luciferase (LUC), and insulin-like growth factors (IGFs), alpha-anti-trypsin, erythropoietin (EPO), factors VIII and XI of the blood clotting system, LDL-receptor, GATA-I, etc.
  • the nucleic acid sequence further may be a suicide gene encoding e.g. apoptotic or apoptose related enzymes and genes including AlF, Apaf e.g.
  • the mammalian piggyBac transposase preferably in combination a mammalian piggyBac transposon, has several advantages compared to approaches in the prior art, e.g. with respect to viral and retroviral methods.
  • transposon insertions can be (re)mobilized by supplying the transposase activity in trans.
  • transposon insertions can be (re)mobilized by supplying the transposase activity in trans.
  • the mammalian piggyBac transposon and transposase protein as defined above can be transfected into a cell as a protein or as ribonucleic acid, including mRNA, as DNA, e.g. as extrachromosomal DNA including, but not limited to, episomal DNA, as plasmid DNA, or as viral nucleic acid.
  • the nucleic acid encoding the transposase protein can be transfected into a cell as a nucleic acid vector such as a plasmid, or as a gene expression vector, including a viral vector. Therefore, the nucleic acid can be circular or linear.
  • a vector refers to a plasmid, a viral vector or a cosmid that can incorporate nucleic acid encoding the transposase protein or the transposon of this invention.
  • the terms "coding sequence” or “open reading frame” refer to a region of nucleic acid that can be transcribed and/or translated into a polypeptide in vivo when placed under the control of the appropriate regulatory sequences.
  • DNA encoding the transposase protein can be stably inserted into the genome of the cell or into a vector for constitutive or inducible expression.
  • the transposase encoding sequence is preferably operably linked to a promoter.
  • promoters There are a variety of promoters that could be used including, but not limited to, constitutive promoters, tissue-specific promoters, inducible promoters, and the like. Promoters are regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding sequence.
  • a DNA sequence is operably linked to an expression-control sequence, such as a promoter when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • operably linked includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence to yield production of the desired protein product.
  • ATG e.g., ATG
  • nucleic acid sequences encoding the mammalian piggyBac transposon are provided as SEQ ID NO: 1 or SEQ ID NO: 2 or hyperactive variants as described herein.
  • Exemplary nucleic acid sequences encoding the mammalian piggyBac transposase protein are provided as SEQ ID NO: 3 or SEQ ID NO: 5.
  • DNA or RNA sequences encoding the mammalian piggyBac transposase protein there are other DNA or RNA sequences encoding the mammalian piggyBac transposase protein. These DNA or RNA sequences have the same amino acid sequence as a mammalian piggyBac transposase protein, but take advantage of the degeneracy of the three letter codons used to specify a particular amino acid. For example, it is well known in the art that various specific RNA codons (corresponding DNA codons, with a T substituted for a U) can be used interchangeably to code for specific amino acids.
  • the present invention also features a gene transfer system comprising a mammalian piggyBac transposon as described herein and a mammalian piggyBac transposase as described herein.
  • the mammalian piggyBac transposase protein preferably recognizes repeats (e.g. IRs) on the mammalian piggyBac transposon.
  • the gene transfer system of this invention therefore, preferably comprises two components: the transposase as described herein and a transposon as described herein.
  • the transposon has at least two repeats (e.g. IRs). When put together these two components provide active transposon activity and allow the transposon to be relocated.
  • the transposase binds to the repeats and promotes insertion of the intervening nucleic acid sequence into DNA of a cell as defined below.
  • the gene transfer system comprises a mammalian piggyBac transposon as defined above in combination with a mammalian piggyBac transposase protein (or nucleic acid encoding the inventive mammalian piggyBac transposase protein to provide its activity in a cell).
  • a mammalian piggyBac transposase protein or nucleic acid encoding the inventive mammalian piggyBac transposase protein to provide its activity in a cell.
  • This combination preferably results in the insertion of the nucleic acid sequence into the DNA of the cell.
  • the gene transfer system mediates insertion of the mammalian piggyBac transposon into the DNA of a variety of cell types and a variety of species by using the mammalian piggyBac transposase protein.
  • such cells include any cell suitable in the present context, including but not limited to animal cells or cells from bacteria, fungi (e.g., yeast, etc.) or plants.
  • Preferred animal cells can be vertebrate or invertebrate.
  • preferred vertebrate cells include cells from mammals including, but not limited to, rodents, such as rats or mice, ungulates, such as cows or goats, sheep, swine or cells from a human.
  • such cells can be pluripotent (i.e., a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells) and totipotent cells (i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells).
  • pluripotent i.e., a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells
  • totipotent cells i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells.
  • oocytes, eggs, and one or more cells of an embryo may also be considered as targets for stable transfection with the present gene transfer system.
  • the cells are stem cells.
  • Cells receiving the mammalian piggyBac transposon and/or the mammalian piggyBac transposase protein and capable of inserting the transposon into the DNA of that cell also include without being limited thereto, lymphocytes, hepatocytes, neural cells, muscle cells, a variety of blood cells, and a variety of cells of an organism, embryonic stem cells, somatic stem cells e.g. hematopoietic cells, embryos, zygotes, sperm cells (some of which are open to be manipulated by an in vitro setting).
  • the cell DNA that acts as a recipient of the transposon of described herein includes any DNA present in a cell (as mentioned above) to be transfected, if the mammalian piggyBac transposon is in contact with an mammalian piggyBac transposase protein within the cell.
  • the DNA can be part of the cell genome or it can be extrachromosomal, such as an episome, a plasmid, a circular or linear DNA fragment.
  • Typical targets for insertion are e.g. double-stranded DNA.
  • the components of the gene transfer system described herein i.e. the mammalian piggyBac transposase protein (either as a protein or encoded by a nucleic acid as described herein) and the mammalian piggyBac transposon can be transfected into a cell, preferably into a cell as defined above, and more preferably into the same cell. Transfection of these components may furthermore occur in subsequent order or in parallel.
  • the mammalin piggyBac transposase protein or its encoding nucleic acid may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposon.
  • the transposon may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposase protein or its encoding nucleic acid.
  • transfected parallel preferably both components are provided in a separated formulation and/or mixed with each other directly prior to administration in order to avoid transposition prior to transfection.
  • administration of at least one component of the gene transfer system may occur repeatedly, e.g. by administering at least one, two or multiple doses of this component.
  • the gene transfer system may be formulated in a suitable manner as known in the art, or as a pharmaceutical composition or kit as described herein.
  • the components of the gene transfer system may preferably be transfected into one or more cells by techniques such as particle bombardment, electroporation, microinjection, combining the components with lipid-containing vesicles, such as cationic lipid vesicles, DNA condensing reagents (e.g., calcium phosphate, polylysine or polyethyleneimine), and inserting the components (i.e. the nucleic acids thereof into a viral vector and contacting the viral vector with the cell.
  • the viral vector can include any of a variety of viral vectors known in the art including viral vectors selected from the group consisting of a retroviral vector, an adenovirus vector or an adeno- associated viral vector.
  • nucleic acid encoding the mammalian piggyBac transposase protein may be RNA or DNA.
  • nucleic acid encoding the mammalian piggyBac transposase protein or the transposon of this invention can be transfected into the cell as a linear fragment or as a circularized fragment, preferably as a plasmid or as recombinant viral DNA.
  • nucleic acid encoding the mammalian piggyBac transposase protein is thereby preferably stably or transiently inserted into the genome of the cell to facilitate temporary or prolonged expression of the mammalian piggyBac transposase protein in the cell.
  • Non-viral vectors as described herein, are generated largely from synthetic starting materials and are therefore more easily manufactured than viral vectors.
  • Non- viral reagents are less likely to be immunogenic than viral agents making repeat administration possible.
  • Non-viral vectors are more stable than viral vectors and therefore better suited for pharmaceutical formulation and application than are viral vectors.
  • inventive gene transfer system is a non- viral gene transfer system that facilitates insertion into DNA and markedly improves the frequency of stable gene transfer.
  • the present invention further provides an efficient method for producing transgenic animals, including the step of applying the inventive gene transfer system to an animal.
  • Transgenic DNA has not been efficiently inserted into chromosomes. Only about one in a million of the foreign DNA molecules is inserted into the cellular genome, generally several cleavage cycles into development. Consequently, most transgenic animals are mosaic (Hackett et al. (1993). The molecular biology of transgenic fish. In Biochemistry and Molecular Biology of Fishes (Hochachka & Mommsen, eds) Vol. 2, pp. 207-240). As a result, animals raised from embryos into which transgenic DNA has been delivered must be cultured until gametes can be assayed for the presence of inserted foreign DNA. Many transgenic animals fail to express the transgene due to position effects. A simple, reliable procedure that directs early insertion of exogenous DNA into the chromosomes of animals at the one-cell stage is needed. The present system helps to fill this need.
  • the gene transfer system of this invention can readily be used to produce transgenic animals that carry a particular marker or express a particular protein in one or more cells of the animal.
  • methods for producing transgenic animals are known in the art and incorporation of the inventive gene transfer system into these techniques does not require undue experimentation, e.g. there are a variety of methods for producing transgenic animals for research or for protein production including, but not limited to Hackett et al. (1993, supra).
  • Other methods for producing transgenic animals are described in the art (e.g. M. Markkula et al. Rev. Reprod., 1, 97-106 (1996); R. T. Wall et al., J. Dairy ScL, 80, 2213-2224 (1997)), J. C. Dalton, et al. (Adv. Exp. Med. Biol, 411, 419-428 (1997)) and H. Lubon et al. (Transfus. Med. Rev., 10, 131-143 (1996)).
  • transgenic animals may preferably contain a nucleic acid sequence inserted into the genome of the animal by the gene transfer system, thereby enabling the transgenic animal to produce its gene product, e.g. a protein.
  • this protein is preferably a product for isolation from a cell, for example the inventive protein can be produced in quantity in milk, urine, blood or eggs.
  • Promoters can be used that promote expression in milk, urine, blood or eggs and these promoters include, but are not limited to, casein promoter, the mouse urinary protein promoter, beta-globin promoter and the ovalbumin promoter respectively.
  • Recombinant growth hormone, recombinant insulin, and a variety of other recombinant proteins have been produced using other methods for producing protein in a cell.
  • Nucleic acids encoding these or other proteins can be inserted into the transposon of this invention and transfected into a cell. Efficient transfection of the inventive transposon as defined above into the DNA of a cell occurs when mammalian piggyBac transposase protein is present. Where the cell is part of a tissue or part of a transgenic animal, large amounts of recombinant protein can be obtained.
  • Transgenic animals may be selected from vertebrates and invertebrates, e.g. fish, birds, mammals including, but not limited to, rodents, such as rats or mice, ungulates, such as cows or goats, sheep, swine or humans.
  • rodents such as rats or mice
  • ungulates such as cows or goats, sheep, swine or humans.
  • the present invention furthermore provides a method for gene therapy comprising the step of introducing the gene transfer system into cells as described herein.
  • the mammalian piggyBac transposon as described herein preferably comprises a gene to provide a gene therapy to a cell or an organism.
  • the gene is placed under the control of a tissue specific promoter or of a ubiquitous promoter or one or more other expression control regions for the expression of a gene in a cell in need of that gene.
  • genes are being tested for a variety of gene therapies including, but not limited to, the CFTR gene for cystic fibrosis, adenosine deaminase (ADA) for immune system disorders, factor IX and interleukin-2 (IL-2) for blood cell diseases, alpha- 1 -antitrypsin for lung disease, and tumor necrosis factors (INFs) and multiple drug resistance (MDR) proteins for cancer therapies.
  • CFTR gene for cystic fibrosis
  • ADA adenosine deaminase
  • IL-2 interleukin-2
  • INFs tumor necrosis factors
  • MDR multiple drug resistance
  • an advantage of the inventive gene transfer system for gene therapy purposes is that it is not limited to a great extent by the size of the intervening nucleic acid sequence positioned between the repeats.
  • the gene transfer system may be transfected into cells by a variety of methods, e.g. by microinjection, lipid-mediated strategies or by viral-mediated strategies.
  • microinjection lipid-mediated strategies
  • viral-mediated strategies do not have substantial size limitations.
  • other strategies for introducing the gene transfer system into a cell such as viral-mediated strategies could limit the length of the nucleic acid sequence positioned between the repeats.
  • the gene transfer system as described herein can be delivered to cells via viruses, including retroviruses (such as lentiviruses, etc.), adenoviruses, adeno-associated viruses, herpes viruses, and others.
  • viruses including retroviruses (such as lentiviruses, etc.), adenoviruses, adeno-associated viruses, herpes viruses, and others.
  • both the transposon and the transposase gene can be contained together on the same recombinant viral genome; a single infection delivers both parts of the gene transfer system such that expression of the transposase then directs cleavage of the transposon from the recombinant viral genome for subsequent insertion into a cellular chromosome.
  • the transposase and the transposon can be delivered separately by a combination of viruses and/or non- viral systems such as lipid-containing reagents. In these cases either the transposon and/or the transposase gene can be delivered by a recombinant virus.
  • the expressed transposase gene directs liberation of the transposon from its carrier DNA (viral genome) for insertion into chromosomal DNA.
  • mammalian piggyBac transposons may be utilized for insertional mutagenesis, preferably followed by identification of the mutated gene.
  • DNA transposons, particularly the transposons have several advantages compared to approaches in the prior art, e.g. with respect to viral and retroviral methods. For example, unlike proviral insertions, transposon insertions can be remobilized by supplying the transposase activity in trans.
  • transposon insertions at new loci by crossing stocks transgenic for the above mentioned two components of the transposon system, the inventive transposon and the inventive transposase.
  • the gene transfer system is directed to the germline of the experimental animals in order to mutagenize germ cells.
  • transposase expression can be directed to particular tissues or organs by using a variety of specific promoters.
  • remobilization of a mutagenic transposon out of its insertion site can be used to isolate revertants and, if transposon excision is associated with a deletion of flanking DNA, the inventive gene transfer system may be used to generate deletion mutations.
  • transposons are composed of DNA, and can be maintained in simple plasmids, inventive transposons and particularly the use of the inventive gene transfer system is much safer and easier to work with than highly infectious retroviruses.
  • the transposase activity can be supplied in the form of DNA, mRNA or protein as defined above in the desired experimental phase.
  • the present invention also provides an efficient system for gene discovery, e.g. genome mapping, by introducing a mammalian piggyBac transposon as defined above into a gene using a gene transfer system as described in the present invention.
  • the mammalian piggyBac transposon in combination with the mammalian piggyBac transposase protein or a nucleic acid encoding the mammalian piggyBac transposase protein is transfected into a cell.
  • the transposon preferably comprises a nucleic acid sequence positioned between at least two repeats, wherein the repeats bind to the mammalian piggyBac transposase protein and wherein the transposon is inserted into the DNA of the cell in the presence of the mammalian piggyBac transposase protein.
  • the nucleic acid sequence includes a marker protein, such as GFP and a restriction endonuclease recognition site. Following insertion, the cell DNA is isolated and digested with the restriction endonuclease.
  • the cell DNA is cut into about 4000-bp fragments on average.
  • These fragments can be either cloned or linkers can be added to the ends of the digested fragments to provide complementary sequence for PCR primers.
  • linkers are added, PCR reactions are used to amplify fragments using primers from the linkers and primers binding to the direct repeats of the repeats in the transposon. The amplified fragments are then sequenced and the DNA flanking the direct repeats is used to search computer databases such as GenBank.
  • Using the gene transfer system for methods as disclosed above such as gene discovery and/or gene tagging permits, for example, identification, isolation, and characterization of genes involved with growth and development through the use of transposons as insertional mutagens or identification, isolation and characterization of transcriptional regulatory sequences controlling growth and development.
  • the invention provides a method for mobilizing a nucleic acid sequence in a cell.
  • the mammalian piggyBac transposon is inserted into DNA of a cell, as described herein.
  • Mammalian piggyBac protein or nucleic acid encoding the mammalian piggyBac transposase protein is transfected into the cell and the protein is able to mobilize (i.e. move) the transposon from a first position within the DNA of the cell to a second position within the DNA of the cell.
  • the DNA of the cell is preferably genomic DNA or extrachromosomal DNA.
  • the inventive method allows movement of the transposon from one location in the genome to another location in the genome, or for example, from a plasmid in a cell to the genome of that cell.
  • RNA interference is a technique in which exogenous, double-stranded RNAs (dsRNAs), being complementary to mRNA's or genes/gene fragments of the cell, are introduced into this cell to specifically bind to a particular mRNA and/or a gene and thereby diminishing or abolishing gene expression.
  • dsRNAs double-stranded RNAs
  • the technique has proven effective in Drosophila, Caenorhabditis elegans, plants, and recently, in mammalian cell cultures.
  • the inventive transposon preferably contains short hairpin expression cassettes encoding small interfering RNAs (siRNAs), which are complementary to mRNA's and/or genes/gene fragments of the cell.
  • siRNAs small interfering RNAs
  • These siRNAs have preferably a length of 20 to 30 nucleic acids, more preferably a length of 20 to 25 nucleic acids and most preferably a length of 21 to 23 nucleic acids.
  • the siRNA may be directed to any mRNA and/or a gene, that encodes any protein as defined above, e.g. an oncogene.
  • the present invention further refers to pharmaceutical compositions containing either a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, and/or a mammalian piggyBac transposon, or a gene transfer system as described herein comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, in combination with a mammalian piggyBac transposon.
  • the pharmaceutical composition may optionally be provided together with a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • a pharmaceutically acceptable carrier, adjuvant, or vehicle according to the invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the component(s) with which it is formulated.
  • compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the pharmaceutical compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parenterally-acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavouring or colouring agents may also be added.
  • the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the inventive gene transfer system or components thereof with a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and Therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
  • the pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the inventive gene transfer system or components thereof suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the components of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene component, emulsifying wax and water.
  • the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
  • compositions of this invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • the amount of the components of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. It has to be noted that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific component employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a component of the present invention in the composition will also depend upon the particular component(s) in the composition.
  • the pharmaceutical composition is preferably suitable for the treatment of diseases, particular diseases caused by gene defects such as cystic fibrosis, hypercholesterolemia, hemophilia, immune deficiencies including HIV, Huntington disease, .alpha.-anti-Trypsin deficiency, as well as cancer selected from colon cancer, melanomas, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, gliomas, thyroid cancer, mamma carcinomas, prostate tumors, hepatomas, diverse virus-induced tumors such as e.g.
  • diseases particular diseases caused by gene defects such as cystic fibrosis, hypercholesterolemia, hemophilia, immune deficiencies including HIV, Huntington disease, .alpha.-anti-Trypsin deficiency, as well as cancer selected from colon cancer, melanomas, kidney cancer, lymph
  • papilloma virus induced carcinomas e.g. cervix carcinoma
  • adeno carcinomas herpes virus induced tumors (e.g. Burkitt's lymphoma, EBV induced B cell lymphoma), Hepatitis B induced tumors (Hepato cell carcinomas), HTLV-I und HTLV-2 induced lymphoma, lung cancer, pharyngeal cancer, anal carcinoma, glioblastoma, lymphoma, rectum carcinoma, astrocytoma, brain tumors, stomach cancer, retinoblastoma, basalioma, brain metastases, medullo blastoma, vaginal cancer, pancreatic cancer, testis cancer, melanoma, bladder cancer, Hodgkin syndrome, meningeoma, Schneeberger's disease, bronchial carcinoma, pituitary cancer, mycosis fungoides, gullet cancer, breast cancer, neurinoma, spinaliom
  • kits comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, and/or a mammalian piggyBac transposon; or a gene transfer system as described herein comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid as described herein, in combination with a mammalian piggyBac transposon; optionally together with a pharmaceutically acceptable carrier, adjuvant or vehicle, and optionally with instructions for use.
  • Any of the components of the inventive kit may be administered and/or transfected into cells in a subsequent order or in parallel, e.g.
  • the mammalian piggyBac transposase protein or its encoding nucleic acid may be administered and/or transfected into a cell as defined above prior to, simultaneously with or subsequent to administration and/or transfection of the inventive transposon.
  • the mammalian piggyBac transposon may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposase protein or its encoding nucleic acid.
  • transfected parallel preferably both components are provided in a separated formulation and/or mixed with each other directly prior to administration in order to avoid transposition prior to transfection.
  • administration and/or transfection of at least one component of the kit may occur in a time staggered mode, e.g. by administering multiple doses of this component.
  • the piggyBac transposon from insects is becoming an increasingly popular tool for use in transgenesis and insertional mutagenesis in many organisms including mammals and insects.
  • the present inventors have isolated the first piggyBac transposon from a mammal. No active form of this family of transposons has been isolated from a mammal, and very few other transposons that are active in mammalians cells and other metazoans have been isolated.
  • the piggyBac transposon described by the present invention as it is from a mammal, may be better able to escape mammalian transposon repression systems.
  • the present inventors have already established that it is active in the yeast S.cerevisiae and thus have employed yeast to isolate hyperactive bat piggyBacs that could be more active than existing versions of the insect element.
  • yeast There is mounting evidence for recent and substantial DNA transposon activity in the vespertilonid bat Myotis lucifugus.
  • this conclusion was inferred from the presence of minimally diverged nonautonomous hAT transposons, the polymorphic status of some of these transposons in natural Myotis populations, and the discovery of an apparently full-length and potentially functional autonomous hAT transposon (Ray et at. 2007).
  • the present experiments characterize seven new families of DNA transposons from the current whole-genome sequence of M. lucifugus.
  • the present experiments provide additional information on the previously described Myotis hAT transposons (Ray et at. 2007). Together, these elements represent three distinct superfamiles and exhibit clear signs of recent activity in the vespertilonid lineage, that is,within the last 40 - 50 million years (Myr).
  • Myr Myr - 50 million years
  • the present inventors have also identified a ninth transposon lineage that likely expanded prior to the chiropteran divergence.
  • One family represents the youngest DNA transposon family so far recorded in any mammalian species, one that is likely still expanding in the genome.
  • the discovery of this unprecedented level of DNA transposon activity in a mammalian genome represents a dramatic shift in our view of mobile element biology in mammals.
  • the consensus sequence for Myotis hATl was previously described (Ray et at. 2007). To summarize, the entire sequence spans 2921 nucleotides with a single ORF consisting of bases 700-2631 that encodes an apparently intact transposase of 643 amino acids (aa). The present analysis confirms the characteristic target-site duplications (TSDs) for Myotis hAT elements, 8 bp with a central TA dinucleotide, and the typical short TIR (terminal inverted repeats) sequence. Myotis hATl and its nonautonomous derivatives are by far the most abundant family of elements analyzed in this study, with > 96,000 hits of -100 bp in the current WGS data.
  • TSDs target-site duplications
  • hAT2_ML A second family of hAT elements was identified, called hAT2_ML.
  • the TIRs of hAT2_ML are highly similar to Myotis hATl, but the internal sequence is only weakly similar.
  • hAT2_ML is predicted to encode a 428-aa transposase with only minimal sequence similarity and 33% amino acid identity to the Myotis hATl transposase.
  • Phylogenetic comparison to other hAT transposases confirms that Myotis hATl and hAT2_ML belong to the hAT superfamily and are closely related to hATI MD (Repbase Update, volume 12, issue 10), a hAT transposon family recently identified in the opossum Monodelphis domestica (Fig.
  • Eukaryotic Tel/mariner elements are divided into several anciently diverged lineages (Robertson 2002; Feschotte and Pritham 2007). Three distinct Tel/ lineages have been previously identified in mammals and characterized most extensively in human: pogo, Tc2, and D34D mariner. Members of all three groups were identified in M. lucifugus as well as the first mammalian member of the Tel group. The pogo lineage is represented by Tigger elements in mammals. It is known to include both eutherianwide and primate-specific families (Robertson 1996; Smit and Riggs 1996; Pace and Feschotte 2007). Thus, the presence of Tigger elements was somewhat expected in M. lucifugus.
  • Tiggerl ML Remnants of what appears to be an ancient family of Tigger- like elements called Tiggerl ML were identified. A tentative consensus was reconstructed from the alignment of the seven longest copies. The size (2.8 kb) and TIRs are consistent with other Tigger elements and the consensus is similar to consensus sequences in Repbase as Tiggerla CAR and Tiggerla ART, which represent Tiggerl-like familes specific to carnivores and artiodactyls, respectively. Also identified were several highly eroded copies of a related family of Tiggerl transposons in the European hedgehog, Erinaceus europaeus (e.g., accession AANN01830819, position 1599-4234).
  • the Tc2 group is sister to the pogo lineage and was first identified in Caenorhabditis elegans (Ruvolo et at. 1992). There are relatively few Tc2-like elements in human, and their amplification predates the split of eutherian mammals (Pace and Feschotte 2007). In contrast, a distinct and much more recent family of Tc2-like elements was identified in M. lucifugus.
  • the Tc2_l_ML consensus is 1728 bp with 23 bp TIRs similar to known or newly identified Tc2 elements. It contains a single ORF spanning positions 213-1559 and encoding a putative transposase of 448 aa.
  • Tc2_l_ML belongs to the Tc2 group, but does not cluster with either of the mammalian Tc2-like transposases identified in human (Kanga), opossum (Tc2_MD2), and tenrec (Tc2_Et), or with the human transposase- derived proteins POGK and POGZ (Fig. IB).
  • Tc2_l_ML defines a distinct lineage of Tc2-like elements.
  • Tc2_l_ML is the least abundant of the DNA transposon familes described in this study with only 589 hits -100 bp.
  • Tel- like elements represent another distinct lineage of Tel/ mariner elements that are widespread and common in invertebrates and lower vertebrates (fish, frogs), but no Tel- like element has previously been identified in mammals (Avancini et at. 1996; Leaver 2001; Sinzelle et at. 2005). Consistent with this observation, TBLASTN searches with representative Tel-like transposases from fish or nematode return no significant hits in the complete genome of human, mouse, rat, and dog. In contrast, the same query returns hundreds of hits from the M. lucifugus WGS database. A single Tel- like family, TeI l ML, predominates in the genome.
  • the TeI l ML consensus spans 1222 bp with 29 bp TIR with a 5'-CACTG-3' terminal motif typical of many Tel-like elements.
  • the ORF occupies position 150-1190 of the consensus and encodes a putative transposase of 346 residues.
  • Phylogenetic analyses show that the TeI l ML transposase forms a well- supported clade with Tel-like transposases from fish (Fig. 2D). 3788 TeI l ML fragments of 100 bp or larger were detected in the M. lucifugus WGS database.
  • TeI l ML elements are inserted in (TA) dinucleotide repeats, an insertion preference also observed for other Tel elements (Vigdal et at. 2002).
  • TA TA dinucleotide repeat
  • Tel elements Vigdal et at. 2002.
  • TeI l ML The consensus of this subfamily has an intact ORF that represents 2/3 (708 bp; 235 aa) of the complete ORF for TeI l ML. Five hundred copies of this subfamily of Tel are distributed throughout the M. lucifugus genome.
  • piggyBac superfamily piggyBac-like elements have been identified in a wide range of animal species and Entamoeba (Sarkar et at. 2003; Pritham et at. 2005).
  • piggyBac elements have so far only been characterized in the human genome, where they are predominantly represented by two families of nonautonomous elements (MER85 and MER75) and by several stationary "domesticated" transposases (PGBD1-5 genes) (Sarkar et at. 2003). Two familes of piggyBac-like elements in M. lucifugus were identified.
  • piggyBacl ML is defined by a 2626-bp consensus with short TIRs (15 bp) that are very similar to other piggyBac transposons.
  • the consensus contains a 1719-bp ORF (position 587-2305) that likely encodes a transposase of 572 residues.
  • piggyBac2_ML is similar in length at 2639 bp and with a 583-aa transposase (nt 716-2467).
  • piggyBac2_ML elements are currently more numerous, with at least 3869 instances.
  • TcI l ML, Tc2_l_ML, and Mlmarl appear to be limited to Vespertilonidae with the possible exception of Tel- like sequences in Pteronotis parnelli (Family Mormoopidae). All three hAT families are restricted to the genus Myotis.
  • piggyBac2_ML shares a similar distribution to the hAT elements, but positive results were obtained from the lone representative of Miniopteridae, a family that was recently elevated from subfamily status within Vesperdilionidae (Miler-Butterworth et al. 2007). Amplification of the miniopterid and mormoopid representatives suggests the need for additional exploration of these taxa. PCR data using piggyBacl ML-derived oligonucleotides suggests that these elements are even further restricted. Amplification was obtained in Myotis austroriparius, a fellow North American bat, but not the Asian representative, Myotis horsfieldii. These results may not confirm that no class 2 transposons are active in other bat lineages.
  • Example 4 Age estimations of DNA transposon families Table 1 , shown below, and Figure 4 show the estimated ages for each family based on average divergence from the consensus sequence and an estimated neutral mutation rate of -2.366 X 10- 9 (see Methods).
  • CpG to non-CpG mutations densities were calculated for all families (Table 2, shown below).
  • Average CpG dinucleotide mutation densities were approximately eight times higher than non-CpG sites. It is noted that this is higher than the rate observed for primates ( ⁇ 6X; Xing et at.
  • Nested self- insertions are theoretically possible and were expected when the analysis was performed. Indeed, there were some possible instances observed. However, in each of these cases the TSDs were not clearly identifiable. Because TSD identification was one a priori criterion for assessing the presence of any nested insertion (see Methods), these were excluded from Table 3. Finally, one would expect older elements to have accumulated more nested insertions than more recently mobilzed elements. To test this hypothesis, the percent nested insertion content for all instances of each family of elements recovered from the M. lucifugus genome was examined.
  • Figure 6 illustrates a clear pattern of younger elements (i.e., piggyBac-like and hAT-like) having lower relative nested insertion content when compared with older elements from the Tel/mariner superfamily.
  • the one exception to this pattern is TeI l ML, which exhibits a relatively low percentage of nested insertions. It is unclear as to why this family exists as an outlier.
  • DNA transposons are widespread and a diversity of elements have been recently active in the genomes of many eukaryotes, including lower vertebrates (Aparicio et al. 2002; Koga et al. 2006). In contrast, initial analyses of the transposable element landscape in the complete human, mouse, rat, and dog genomes have led to the common belief that mammalian genomes are devoid of recently active DNA transposons (Lander et al. 2001; Waterston et al. 2002; Gibbs et al. 2004; Lindblad-Toh et al. 2005).
  • the elements appear to be almost exclusively limited to Vespertilionidae, and in some cases to selected taxa within the family.
  • the level of sequence divergence among copies (and to their respective consensus sequences) suggests that these families were active from ⁇ 36 Mya to the present.
  • Analysis of nested insertions suggests a hierarchical pattern of insertion consistent with sequence divergence estimates.
  • two potentially full-length and intact piggyBacl ML transposons could be identified in the available genome sequence data, supporting the hypothesis that the expansion of these elements and their nonautonomous relatives is ongoing.
  • piggyBacl ML was verified by recovering "empty" orthologous sites in a closely related Myotis species and by the limited taxonomic distribution of piggyBacl ML to North American Myotis species. Together with the recent study reporting the massive amplification of HeliBats 30-36 Mya in the lineage of M. lucifugus (Pritham and Feschotte 2007), these data demonstrate that the genomes of vespertilonid bats have been subjected to multiple waves of amplification of diverse DNA transposons, from ⁇ 40 Mya to the present. One lineage of piggyBac, piggyBacl ML, is likely still active.
  • Myotis is one of the most species-rich of all mammalian genera, and repeated waves of transposon activity suggest a mechanism for generating the genetic variability necessary to produce its tremendous species diversity.
  • a recent analysis of Myotis phylogeny based on nuclear and mitochondrial DNA sequences becomes more interesting when one considers the data presented here. Stadelmann et at. (2007) found that a burst of Myotis diversification occurred -12-13 Mya. These dates correspond well to the estimated time during which the most active DNA transposon families were expanding in the Myotis genome (Fig. 3).
  • viruses remain the best candidates as potential vectors for the horizontal introduction of DNA transposons and other TEs (Miler and Miler 1982; Fraser et at. 1983; Jehle et at. 1998; Piskurek and Okada 2007).
  • the propensity of bats to tolerate massive and diverse viral infections may have facilitated the recurrent horizontal introduction of DNA transposons and/ or their evolutionary persistence in vespertilonid bats.
  • the data presented herein provides evidence that the previously identified trend toward DNA transposon extinction in mammals is not universal and that a wide diversity of DNA transposons have been active throughout the diversification of the vespertilionid bats, that is, within the last 40 Myr.
  • the genus Myotis is an excellent candidate for studying the impact and influence of mobile elements, especially class 2 elements, on the evolution of genomic, species, and ecological diversity in mammals. Concomitantly, studies of bats may represent a unique opportunity to investigate lie history characteristics that make some organisms more susceptible to transposon activity than others.
  • stem cells The ability to specifically modify the genomes of stem cells would be of great benefit in the treatment of human disease. In diseases that result from the lack of a particular gene product because of a defective gene, addition of an intact copy of the gene to stem cells could lead to the alleviation of disease upon reintroduction. Alternatively it may be useful to supplement stem cells with a gene product from a heterologous gene such that the modified cells would produce an agent that would kill other cells, for example an anti-tumor agent.
  • transposons A powerful method for the introduction of new DNA into cells is via transposons.
  • cut and paste transposons where an element- encoded transposase recognizes and binds to specific sites at the ends of the transposon and excises the transposon from that donor site and inserts it into a new target site.
  • the element Upon integration, the element becomes stably associated with the host genome and can serve as a long-term source of an element-encoded therapeutic product.
  • Such DNA cut and paste elements can be used for the modification of stem cell genomes.
  • a general feature of transposable elements is that they insert in many different target sites. While this is a valuable property when such elements are used as insertional mutagens, such widespread insertion is a hazard in gene therapy.
  • the present inventors have generated "target site-specific transposons" by making chimeric transposases in which a highly target site specific DNA binding domain is fused to the transposase.
  • Figure 9 shows a representation of mammalian bat piggyBac helper plasmid which expresses the bat piggyBac transposase and corresponds to SEQ ID NO: 7.
  • Figure 10 shows a representation of mammalian bat piggyBac donor plasmid which encodes bat piggyBac tranposon and a drug resistance marker and corresponds to SEQ ID NO: 8.
  • An exemplary Mammalian bat piggyBac donor plasmid is shown below, and corresponds to SEQ ID NO: 8.
  • Example 8 Hyperactive mammalian piggyBac variants
  • the following experiments demonstrate bat piggyBac transposition in S. cerevisiae.
  • a simple genetic assay was established for the excision of piggyBAT in yeast (Saccharomyces cerevisiae), using a modified version of the yeast URA3 gene as a transposon donor.
  • the yeast actin intron has been introduced into the URA3 gene to form a URA3::actin intron gene.
  • the actin intron can be efficiently spliced from mRNA of this gene so that a strain carrying the URA3:: actin intron is a uracil prototroph.
  • a transposon donor plasmid contains a mini-piggyBAT transposon composed of 153 bp of the piggyBAT-L end and 208 bp of the piggyBAT-R end flanking a kanamycin resistance gene in the URA3::actin intron cassette.
  • the transposase is supplied by a second helper plasmid containing the piggyBAT transposase gene under the galactose-inducible control of the GALS promoter.
  • This assay can be used for screening for hyperactive piggyBacs by random mutagenesis of the transposase gene and screening for an increased frequency of Ura+ reversion.
  • the WGS data set represented -73% of the unassembled M. lucifugus genome with contig sizes averaging ⁇ 2.4 kb (Pritham and Feschotte 2007).
  • the WGS data was queried using TBLASTN to detect the presence of coding sequences related to all known DNA transposon superfamilies.
  • Age estimation of M. ludfugus transposons Ages for each transposon family were estimated by extracting full or near full-length ORF's based on the coordinates of the repeats drawn from the output files of a locally implemented version of RepeatMasker 3.1.6. Additionally, it was hypothesized that nonautonomous subfamiles were likely mobilized by autonomous elements sharing the same TIRs, and thus were deposited during the same time period as the autonomous elements. Therefore, representatives were collected from a limited number of nonautonomous derivatives for all familes. All extracted elements were aligned with their respective consensus sequence using MUSCLE (Edgar 2004).
  • oligonucleotide primers were designed to survey the presence/absence of 15 individual insertions of a nonautonomous piggyBacl ML subfamily (npiggy_156) in M. austroriparius, which diverged from M. lucifugus -8-12 Mya (Stadelmann et at. 2007). Primers were designed in the genomic regions flanking the elements using sequence data from M. lucifugus and tested on a panel of 10 individuals from natural populations of M. austroriparius. Details on sample collection, DNA extraction, amplification, cloning, and sequencing are as previously described (Ray et at. 2007). Sequences from the two intact preintegration site amplicons shown in Figure 4 have been deposited in GenBank under accession nos. EU177095 and EU177096.
  • BLASTN or TBLASTN were used with nucleotide or amino acid sequence queries corresponding to each family of transposon in separate searches of custom databases representing the following taxa: Chiroptera (taxid:9397, excluding Myotis lucifugus), Xenarthra (taxid:9348), Afrotheria (taxid:311790), Laurasiatheria (taxid:314145), Euarchontoglires (taxid:314146), Mammalia (taxid:40674).
  • Vertebrate DNA transposon as a natural mutator The medaka fish Tol2 element contributes to genetic variation without recognizable traces. MoI. Biol. Evol. 23: 1414-1419.

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Abstract

The present invention provides mammalian piggyBac transposons and transposases. In particular embodiments, the present inventors have identified hyperactive mammalian piggyBac variants. The mammalian piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.

Description

MAMMALIAN PIGGYBAC TRANSPOSON AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 61/146,798, filed on January 23, 2009, the entire contents of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Typical methods for introducing DNA into a cell include DNA condensing reagents such as calcium phosphate, polyethylene glycol, and the like, lipid-containing reagents, such as liposomes, multi- lamellar vesicles, and the like, as well as virus-mediated strategies. However, such methods can have limitation. For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies. Further, the amount of nucleic acid that can be transfected into a cell is limited in virus strategies. Not all methods facilitate insertion of the delivered nucleic acid into cellular nucleic acid and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the insertion of nucleic acid into viral vectors can be labor intensive. Virus-mediated strategies can be cell- type or tissue-type specific and the use of virus-mediated strategies can create immunologic problems when used in vivo.
One suitable tool in order to overcome these problems are transposons. Transposons, or transposable elements, include a (short) nucleic acid sequence, with terminal repeat sequences upstream and downstream. Active transposons encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.
Transposable elements represent a substantial fraction of many eukaryotic genomes. For example, -50% of the human genome is derived from transposable element sequences, and other genomes, for example plants, may consist of substantially higher proportions of transposable element-derived DNA. Transposable elements are typically divided into two classes, class 1 and class 2. Class 1 is represented by the retrotransposons (LINEs, SINEs, LTRs, and ERVs). Class 2 includes the "cut-and-paste" DNA transposons, which are characterized by terminal inverted repeats (TIRs) and are mobilized by an element-encoded transposase. Currently, 10 superfamilies of cut-and-paste DNA transposons are recognized in eukaryotes (Feschotte and Pritham 2007).
While class 2 elements are widespread and active in a variety of eukaryotes, they have been thought to be transpositionally inactive in mammalian genomes. This conclusion was based primarily on the initial analyses of the human and mouse genome sequences. While both species harbor a significant number and a diverse assortment of DNA transposons, they show no signs of recent activity (Lander et at. 2001; Waters ton et at. 2002). For example, there are more than 300,000 DNA elements recognizable in the human genome, which are grouped into 120 families and belong to five superfamiles. A large subset of these elements (40 families; -98,000 copies) were integrated in the last 40-80 million years (Myr), but there remains no evidence for any human DNA transposon familes younger than ~37 Myr (Pace and Feschotte 2007).
The natural process of horizontal gene transfer can be mimicked under laboratory conditions. In plants, transposons of the Ac/Ds and Spm families have been routinely transfected into heterologous species (Osborne and Baker, 1995 Curr. Opin. Cell Biol. 7, 406- 413). In animals, however, a considerable obstacle to the transfer of an active transposon system from one species to another has been that of species-specificity of transposition due to the requirement for factors produced by the natural host.
Both invertebrate and vertebrate transposons hold potential for transgenesis and insertional mutagenesis in model organisms. Particularly, the availability of alternative transposon systems in the same species opens up new possibilities for genetic analyses. There still remains a need for new methods for introducing DNA into a cell, and particularly methods that promote the efficient insertion of transposons of varying sizes into the nucleic acid of a cell or the insertion of DNA into the genome of a cell while allowing more efficient transcription/translation results than constructs as available in the state of the art.
SUMMARY OF THE INVENTION
As described in more detail below, the present inventors have identified mammalian piggyBac transposons and transposases. In particular embodiments, the present inventors have identified hyperactive mammalian piggyBac variants. The mammalian piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery. Accordingly, in a first aspect, the invention features a transposon comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposon activity.
In one embodiment, the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae. In another embodiment, the mammalian piggyBac nucleic acid sequence is from the genus Myotis. In another further embodiment, the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
In certain exemplary embodiments, the transposon comprises a nucleic acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. In further embodiments, the transposon is capable of inserting into the DNA of a cell.
In another embodiment, the transposon further comprises a marker protein.
In another further embodiment, the transposon is inserted in a plasmid.
In a related embodiment, the transposon further comprises at least a portion of an open reading frame. In another related embodiment, the transposon further comprises at least one expression control region. In a further embodiment, the expression control region is selected from the group consisting of a promoter, an enhancer or a silencer. In another embodiment, the transposon further comprises a promoter operably linked to at least a portion of an open reading frame. In certain exemplary embodiments, the plasmid comprises SEQ ID NO: 7 or SEQ ID
NO: 8.
In one embodiment, the cell is obtained from an animal. In another further embodiment, the cell is from a vertebrate or an invertebrate. In still another further embodiment, the vertebrate is a mammal. In another aspect, the present invention features a transposase comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposase activity.
In one embodiment, the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae. In another embodiment, the mammalian piggyBac nucleic acid sequence is from the genus Myotis. In another further embodiment, the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
In certain exemplary embodiments, the nucleic acid sequence is selected from SEQ ID NO: 3 and SEQ ID NO: 5.
In further exemplary embodiment, the amino acid sequence is selected from SEQ ID NO: 4 and SEQ ID NO: 6. .
In another aspect, the present invention features a gene transfer system comprising a transposon and a mammalian piggyBac transposase.
In one embodiment, the transposon is inserted into the genome of the cell. In one embodiment, the cell is obtained from an animal. In another further embodiment, the cell is from a vertebrate or an invertebrate. In still another further embodiment, the vertebrate is a mammal.
In another embodiment, the invention features a cell comprising the transposon of the above aspects.
In another aspect, the present invention provides a pharmaceutical composition comprising a transposon comprising a mammalian piggyBac nucleic acid sequence and a mammalian piggyBac transposase, together with a pharmaceutically acceptable carrier, adjuvant or vehicle. In another further aspect, the present invention features a method for introducing exogenous DNA into a cell comprising contacting the cell with the gene transfer system of claim 26, thereby introducing exogenous DNA into a cell.
In one embodiment, the cell is a stem cell.
In another aspect, the present invention provides a kit comprising a transposon comprising a mammalian piggyBac nucleic acid sequence and instructions for introducing DNA into a cell.
In one embodiment, the kit further comprises a mammalian piggyBac transposase.
In another aspect, the present invention features a hyperactive mammalian piggyBac transposon that has a higher level of transposon excision compared to a wildtype mammalian piggyBac transposon.
In one embodiment, the wildtype mammalian piggyBac transposon is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
In another embodiment, the hyperactive transposon comprises a nucleotide change in SEQ ID NO 3 selected from C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A, A687G, G715A, T13C, C23T, G161A, G25A, T1050C, A1356G, A26G, A1033G, A1441G, A32G, A389C, A32G, A389C, A32G, T1572A, G456A, T1641C, Tl 155C, G1280A, T22C, A106G, A29G, and Cl 137T.
In another embodiment, the hyperactive transposon comprises an amino acid change in SEQ ID NO 4 selected from A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, I345V, M481V, EI lG, K130T, G9G, R427H, S8P, S36G, DlOG, S36G and silent.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims. Definitions
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1- 3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-3,4-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is herein incorporated by reference.
As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
As used herein, the term "nucleotide" or "polynucleotide" is meant to refer to both double- and single-stranded DNA and RNA, and combinations thereof. A polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences, and non-coding sequences such as regulatory sequences. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide can be, for example, a portion of a vector, or a fragment. A "coding sequence" or a "coding region" is a polynucleotide that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translational start codon at its 5' end and a translational stop codon at its 3' end. A regulatory sequence is a nucleotide sequence that regulates expression of a coding region to which it is operably linked.
Nonlimiting examples of regulatory sequences include promoters, transcriptional initiation sites, translational start sites, translational stop sites, transcriptional terminators (including, for instance, poly-adenylation signals), and intervening sequences (introns).
As used herein, the term "operably linked" is meant to refer a nucleotide sequence that is placed in a functional relationship with another nucleotide sequence. For example, if a coding sequence is operably linked to a promoter sequence, this generally means that the promoter may promote transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary join two protein coding regions, contiguous and in reading frame. Since enhancers may function when separated from the promoter by several kilobases and intron sequences may be of variable lengths, some nucleotide sequences may be operably linked but not contiguous..
As used herein, the term "polypeptide" is meant to refer to a polymer of amino acids of any length. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes post- expression modifications of the polypeptide, for example, glycosylations (e.g., the addition of a saccharide), acetylations, phosphorylations and the like.
As used herein, the term "transposon" or "transposable element" is meant to refer to a polynucleotide that is able to excise from a donor polynucleotide, for instance, a vector, and integrate into a target site, for instance, a cell's genomic or extrachromosomal DNA. A transposon includes a polynucleotide that includes a nucleic acid sequence flanked by cis- acting nucleotide sequences on the termini of the transposon. A nucleic acid sequence is "flanked by" cis-acting nucleotide sequences if at least one cis-acting nucleotide sequence is positioned 5' to the nucleic acid sequence, and at least one cis-acting nucleotide sequence is positioned 3' to the nucleic acid sequence. Cis-acting nucleotide sequences include at least one inverted repeat (also referred to herein as an inverted terminal repeat, or ITR) at each end of the transposon, to which a transposase, preferably a member of the mammalian piggyBac family of transposases, binds. In certain preferred embodiments, the transposon is a mammalian piggyBac transposon. An "isolated" polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Preferably, a polypeptide or polynucleotide of this invention is purified, i.e., essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities. As used herein, the term "transposase" is meant to refer to a polypeptide that catalyzes the excision of a transposon from a donor polynucleotide (e.g., a vector) and the subsequent integration of the transposon into the genomic or extrachromosomal DNA of a target cell. Preferably, the transposase binds an inverted sequence or a direct repeat. The transposase may be present as a polypeptide. Alternatively, the transposase is present as a polynucleotide that includes a coding sequence encoding a transposase. The polynucleotide can be RNA, for instance an mRNA encoding the transposase, or DNA, for instance a coding sequence encoding the transposase. When the transposase is present as a coding sequence encoding the transposase, in some aspects of the invention the coding sequence may be present on the same vector that includes the transposon, i.e., in cis. In other aspects of the invention, the transposase coding sequence may be present on a second vector, i.e., in trans. In certain preferred embodiments, the transposase is a mammalian piggyBac transposon.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows piggyBac transposition in HeLa cells. Figure 2 (A - E) shows the results of phylogenetic analyses. (A) hAT transposases.
(B) Tc2 transposases. (C) mariner transposases. (D) TcI transposases. (E) piggyBac transposases.
Figure 3 shows electropherogram results from PCR analysis of a taxonomically diverse panel of bat genomes. Oligonucleotide primer pairs are indicated in white. Figure 4 shows divergence estimates and age ranges for each autonomous and nonautonomous transposon family described.
Figure 5 shows filled and intact preintegration site sequences for individual nonautonomous piggyBacl ML elements from M. lucifugus and M. austroriparius, respectively. Target site duplications for each insertion are shaded. (A) Locus 340 3005. (B)
Locus primer sequences (5'-3?) for the two loci presented are as follows:
Mluc contl .015340 3005 L- TTGTACCAAAAGGTGCCAAA,
Mluc_contl.015340_3005_RTTTCCTCAT ATACCATCCCATTTT; Mluc contl .000939 - 4 755 L-CAAAAT AAGGGAGAAAGGAAACA, M luc contl .000939 - 47
55 RGGGCTGAGAACAAGATCCA. (Mluc) M. lucifugus; (Maus) M. austroriparius.
Figure 6 is a graph that shows proportion of insertions nested within instances of each
DNA transposon family described. Families are arranged along the X-axis in order of age based on genetic divergence estimates in Table 3. Values can be found in Table 3. Figure 7 (A - C) shows representative alignments for three subfamilies recovered during the analysis. (A) a nonautonomous piggyBac l ML element; (B) a nonautonomous hATI ML element; (C) a nonautonomous Tc2 element. For all, the presumed TSD is outlined in black and the TSDs that are characteristic of the family are shaded in gray.
Identical residues below the consensus are indicated by "." And indels are indicated by "-". Figure 8 shows complete alignments used to determine the full-length consensus sequence for each family.
Figure 9 is a representation of mammalian bat piggyBac helper plasmid which expresses the bat piggyBac transposase and corresponds to SEQ ID NO: 7.
Figure 10 is a representation of mammalian bat piggyBac donor plasmid which encodes bat piggyBac transposon and a drug resistance marker and corresponds to SEQ ID
NO: 8.
DETAILED DESCRIPTION
The present inventors have isolated the first piggyBac transposon from a mammal. No active form of this family of transposons has been isolated from a mammal, and very few other transposons that are active in mammalians cells and other metazoans have been isolated.
Accordingly, the present invention features mammalian piggyBac transposon and transposases. Another aspect of this invention refers to a transposon, or transposable element, that includes a nucleic acid sequence, as described herein, positioned between at least two repeats (for example, inverted repeats (IRs)), at least one repeat on either side of the nucleic acid sequence. Preferably, the inventive transposon comprises a nucleic acid sequence positioned between at least two repeats, wherein these repeats can bind to a transposase protein as defined herein and wherein the transposon is capable of inserting into DNA of a cell. Accordingly, repeats are preferably sequences that are recognized and bound by the transposase as defined herein.
According to certain preferred embodiments of the present invention, a mammalian piggyBac transposon is bound by an inventive transposase, contains a pair of repeat sequences. In certain preferred embodiments, the first repeat is typically located upstream to the nucleic acid sequence and the second repeat is typically located downstream of the nucleic acid sequence. Accordingly, the second repeat represents the same sequence as the first repeat, but shows an opposite reading direction as compared with the first repeat (5' and 3' ends of the complementary double strand sequences are exchanged). These repeats are then termed "inverted repeats" (IRs), due to the fact that both repeats are just inversely repeated sequences. In certain embodiments, repeats may occur in a multiple number upstream and downstream of the above mentioned nucleic acid sequence. Preferably, the number of repeats located upstream and downstream of the above mentioned nucleic acid sequence is identical. In certain embodiments, the repeats are short, between 10 - 20 base pairs, and preferably 15 base pairs.
The repeats (IRs) as described herein preferably flank a nucleic acid sequence which is inserted into the DNA of a cell. The nucleic acid sequence can include all or part of an open reading frame of a gene (i.e., that part of a protein encoding gene), one or more expression control sequences (i.e., regulatory regions in nucleic acid) alone or together with all or part of an open reading frame. Preferred expression control sequences include, but are not limited to promoters, enhancers, border control elements, locus-control regions or silencers. In a preferred embodiment, the nucleic acid sequence comprises a promoter operably linked to at least a portion of an open reading frame. According to certain preferred embodiment, transposons of the present invention can preferably occur as a linear transposon (extending from the 5' end to the 3' end, by convention) that can be used as a linear fragment or circularized, for example in a plasmid.
The present invention features a transposon comprising a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposon activity. The present invention also features a mammalian piggyBac transposase comprising a nucleic acid sequence and variants, derivatives and fragments thereof that retain transposase activity.
The mammalian piggyBac transposase may be present as a polypeptide. Alternatively, the transposase is present as a polynucleotide that includes a coding sequence encoding a transposase. The polynucleotide can be RNA, for instance an mRNA encoding the transposase, or DNA, for instance a coding sequence encoding the transposase. When the transposase is present as a coding sequence encoding the transposase, in some aspects of the invention the coding sequence may be present on the same vector that includes the transposon, i.e., in cis. In other aspects of the invention, the transposase coding sequence may be present on a second vector, i.e., in trans.
In certain preferred embodiments, the mammalian piggyBac transposon or transposase nucleic acid sequence is from the family Vespertilionidae. In further exemplary embodiments, the mammalian piggyBac transposon or transposase nucleic acid sequence is from the genus Myotis. In other further preferred embodiments, the mammalian piggyBac transposon or transposase nucleic acid sequence is from the species Myotis lucifugus.
Preferred embodiments of the present invention refer to nucleic acids encoding a mammalian piggyBac transposon or a mammalian piggyBac transposase as defined herein. Nucleic acids according to the present invention typically comprise ribonucleic acids, including mRNA, DNA, cDNA, chromosomal DNA, extrachromosomal DNA, plasmid DNA, viral DNA or RNA. In certain preferred embodiments, a nucleic acid is preferably selected from any nucleic sequence encoding the same amino acid sequence of a mammalian transposon or tranposase protein due to degeneration of its genetic code. These alternative nucleic acid sequences may lead to an improved expression of the encoded fusion protein in a selected host organism. Tables for appropriately adjusting a nucleic acid sequence are known to a skilled person. Preparation and purification of such nucleic acids and/or derivatives are usually carried out by standard procedures (see Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.). Other variants of these native nucleic acids may have one or more codon(s) inserted, deleted and/or substituted as compared to native nucleic acid sequences. These sequence variants preferably lead to mammalian piggyBac transposon or transposase proteins having at least one amino acid substituted, deleted and/or inserted as compared to the native nucleic acid sequences of transposases. Therefore, nucleic acid sequences of the present invention may code for modified (non-natural) transposon or transposase sequences. Further, promoters or other expression control regions can be operably linked with the nucleic acids encoding the proteins described herein to regulate expression of the protein in a quantitative or in a tissue-specific manner. In a particularly preferred embodiment, the nucleic acid sequence encoding an mammalian piggyBac transposon protein comprises a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, shown below.
SEQ ID NO: 1 piggyBac 1_M1 Transposon DNA CACTTGGATTGCGGGAAACGAGTTAAGTCGGCTCGCGTGAATTGCGCGTACTCCGCGGGA GCCGTCTTAACTCGGTTCATATAGATTTGCGGTGGAGTGCGGGAAACGTGTAAACTCGGG CCGATTGTAACTGCGTATTACCAAATATTTGTTTGTTTGCCGTTCACAAAGATACCTACC TGCGTATGCGAACCGTTTCATACCCATTGGGGTAATAGGTATCATAAAAATCCCTTGTAA GTAGAGAACACTTTATGGCGCTGGTCGAGTACAATACCGTTAGGGTACCAATGTATTGTT TTCTCATTTTATAACCGATATCCCTGGATATCTCACCATGCAAAGAGAATAGTTATTGAG AAATACTTCTTGTGTCCGGACGTCTTTAGTTTTGAGTTTTGTGAATATTATTACGTGTTT AAATATTTAAATATATTGTGATAAAGACATTCATATAGAATTTTTTAACCAAATAAAATA AAATCGTTCCGCAAAGTGTTCGCATAATAATTATTTTTTTATATCAGATATCTATCGGAG TACCGCTATTTTCAGTTAGTATTATTTTTATTTGCTTTAACCTAGAATGTCGCAGCATTC AGACTATAGTGATGATGAGTTTTGTGCAGACAAGTTGTCCAATTATTCTTGTGATAGCGA TCTTGAAAATGCGAGTACAAGTGATGAAGATTCTAGTGATGATGAAGTAATGGTGCGTCC CAGGACATTGAGGCGACGAAGAATTTCGAGCTCCAGCTCTGACTCAGAGTCAGATATAGA AGGCGGGAGAGAAGAATGGTCGCATGTTGATAATCCACCGGTCTTAGAAGATTTTTTAGG GCATCAAGGATTAAACACAGATGCTGTTATAAATAATATAGAAGATGCCGTGAAATTATT TATCGGAGATGATTTTTTTGAATTTCTTGTAGAGGAGTCAAACAGGTATTATAATCAAAA TAGGAATAATTTCAAACTTTCAAAAAAAAGCCTAAAGTGGAAAGATATAACCCCTCAAGA GATGAAGAAGTTTTTAGGGTTAATTGTTCTCATGGGACAGGTGCGCAAAGATAGAAGAGA TGACTATTGGACCACGGAGCCATGGACGGAGACGCCATATTTTGGTAAAACGATGACGAG AGACAGGTTCCGACAGATATGGAAAGCTTGGCACTTCAATAATAATGCGGATATCGTAAA TGAATCAGATAGACTTTGCAAAGTGAGACCAGTACTAGATTATTTTGTGCCTAAATTTAT AAATATTTACAAACCTCATCAGCAATTATCACTAGATGAAGGGATCGTACCTTGGAGGGG AAGATTATTCTTTAGGGTATATAATGCTGGCAAGATCGTTAAATATGGAATATTGGTTCG TTTGTTGTGCGAAAGTGATACAGGATATATCTGTAACATGGAAATTTATTGCGGCGAAGG AAAGCGATTATTGGAAACGATACAAACAGTAGTGTCTCCATACACTGATTCGTGGTACCA TATATATATGGACAATTATTATAATAGCGTCGCAAATTGTGAAGCACTTATGAAAAACAA ATTCAGAATATGTGGAACAATCCGGAAAAATCGAGGTATACCTAAAGATTTTCAAACAAT TTCTTTGAAAAAAGGTGAAACAAAATTTATAAGGAAAAATGATATATTGTTACAAGTGTG GCAATCAAAAAAGCCTGTATACCTGATTTCTTCGATTCATTCTGCGGAGATGGAAGAAAG TCAGAATATTGACAGAACATCAAAAAAGAAAATTGTCAAACCGAATGCACTCATTGACTA CAATAAACATATGAAAGGTGTTGACCGGGCCGACCAATACCTTTCATATTATTCGATATT GCGGAGGACGGTCAAATGGACAAAAAGGTTGGCAATGTATATGATAAATTGCGCATTATT TAATTCTTATGCAGTTTACAAATCAGTGAGGCAAAGAAAAATGGGTTTTAAAATGTTTTT GAAACAAACAGCTATCCACTGGTTGACGGATGATATTCCAGAGGACATGGACATTGTTCC AGACCTTCAACCAGTACCGTCTACTTCTGGAATGCGGGCTAAACCACCTACATCTGATCC ACCATGCAGGCTATCGATGGACATGAGAAAGCATACGTTACAGGCAATTGTCGGAAGTGG AAAAAAGAAAAACATTTTGAGAAGGTGTCGCGTATGTTCCGTTCATAAATTGCGCAGTGA GACACGCTACATGTGCAAATTTTGCAATATACCTCTACATAAAGGGGCGTGTTTTGAAAA ATATCATACGCTAAAAAACTATTAATTTCTATTTGTTTTGATTTATTATGAAAATTTTAT TTTAAACTTTAAAAATATGTGTATATTTTTAGGTAATAAGTATTTTTTGGGTTTTTCGTA TTTAGTTTATATATATCGAATTATTTATGTACTGAATAGATAAAAAAATGTCTGTGATTG AATAAATTTTCATTTTTTACACAAGAAACCGAAAATTTCATTTCAATCGAACCCATACTT CAAAAGATATAGGCATTTTAAACTAACTCTGATTTTGCGCGGGAAACCTAAATAATTGCC CGCGCCATCTTATATTTTGGCGGGAAATTCACCCGACACCGTAGTG
SEQ ID NO: 2 piggyBac2_ML Transposon DNA
CACATTGCGTACCGCTCACGAGTTTTCTCGTGTTTCGCGCGCCATCTGTTAAGGACCGCT CACGAGTGTTCTCGTTTTTCACGCGCCATCTGTTATGGACCTTAGATGTCAACACACTGT CTTGTCCACTGTGGGGCGCGGTTACAGTGTTTTGGCCAGGTTCAAGCCTCGGACTAATGA AAGGACAGGGTCCTCTCACTGCCACGTGCAAGTCCCAGCTGGAGGGCAGGGCCCTCCCAG CACAATCATAGCCAACGGCTGTGGTTGTAAGCTTGAACCTATGGTCCGAACACGTAGCCC CACGTGCCTTGTGATAGAGTTCGGGTGCATGTAGTTGAGTAGGGTTGAGACTCACGAGAA TGCTGTAAACAACGTGATCACGCCCCTACTTTGCCTCGTGGCATCTGCTATAAAATAAAG ACACGGCTTGTGGGCGCTGGCGTTGTCTCCTCTTCAGGGAGCAGCGTCCCACCGAGACCC AGCTATTATTCTCTTGTCTGTCTTTCCTTAATCCTTTCACCCCCCCACTCAGAGACACCC TTGGCCGTGCTGGCGCGGCACAGTCCACATCGAATGAAGCGATCTCATTGGTGGAAACCG TGCAGGTCAATCTACGAAAAACTATATAATTGCACGAACCCATAAAGCATTGCAGTTACA TTGTATTTTGGTCATTCGAATAGTCTTCGTCTTCAAGTTCCTGGCGCTTTTAGAAATGCC CTCTCTCAGAAAAAGGAAGGAAACCAACGAAACTGATACACTTCCGGAAGTATTTAACGA TAATTTATCAGATATTCCTAGTGAGATCGAAGATGCGGATGACTGTTTTGACGATTCCGG AGACGATTCTACTGATTCTACTGACAGTGAAATTATTAGACCTGTAAGGAAGCGCAAGGT GGCGGTGCTTTCAAGTGATTCCGACACTGACGAAGCTACTGATAATTGTTGGTCTGAAAT TGACACACCACCACGCTTACAAATGTTTGAAGGTCATGCTGGGGTCACTACATTTCCGTC TCAGTGTGACTCTGTACCCTCTGTGACCAATCTCTTTTTTGGTGATGAATTGTTTGAGAT
GTTGTGCAAAGAGCTGTCCAACTATCACGATCAAACCGCAATGAAACGCAAAACACCATC TAGAACACTAAAGTGGTCTCCGGTTACACAGAAGGACATCAAGAAATTCCTTGGCCTAAT TATTCTGATGGGTCAAACAAGAAAAGATAGCTTGAAAGACTATTGGTCAACAGATCCTTT GATATGTACCCCTATATTTCCACAGACAATGAGTCGCCATAGATTTGAGCAAATATGGAC ATTCTGGCATTTCAATGATAACGCCAAAATGGACAGTCGCTCGGGGAGACTTTTCAAGAT CCAACCTGTGCTGGATTATTTCCTGCATAAATTTCGAACAATATACAAACCAAAGCAACA GTTGTCTTTGGACGAGGGAATGATTCCATGGAGAGGACGTTTCAAATTTCGCACGTACAA CCCAGCGAAAATAACAAAATACGGTTTACTTGTTCGGATGGTGTGCGAGAGTGACACCGG CTATATCTGCAGTATGGAGATATACACTGCTGAAGGAAGGAAATTGCAAGAAACTGTTCT TTCAGTCCTTGGACCCTATCTTGGCATATGGCACCATATTTACCAGGATAATTATTACAA TGCTACATCTACTGCTGAATTGCTGCTACAGAACAAAACTAGAGTCTGTGGGACTATTAG GGAGAGTAGAGGTTTACCGCCAAATTTGGAAATGAAAACATCAAGAATGAAGAAAGGTGA CATAATATTTTCCAGAAAAGGCGATATTCTTCTCCTAGCATGGAAAGACAAGCGGGTTGT CCGAATGATATCAACGATCCATGACACTTCTGTCTCGACAACAGGAAAAAAAAATAGAAA AACGGGAGAGAATATTGTAAAACCTACCTGCATCAAGGAATACAATGCCCACATGAAAGG CGTTGACCGTGCGGATCAATTCCTTTCGTGTTGTTCCATTCTAAGGAAAACGATGAAATG GACAAAAAAAGTAGTGCTGTACCTTATAAACTGTGGACTTTTCAATTCATTTAGAGTGTA CAACGTCCTCAATCCACAAGCAAAAATGAAGTATAAACAGTTTCTGCTATCGGTGGCGAG AGACTGGATAACGGATGACAATAATGAAGGCTCTCCGGAACCAGAGACAAATCTGTCCAG CCCTTCCCCTGGGGGTGCAAGGAGAGCACCTCGTAAAGATCCACCCAAAAGGTTGTCAGG TGATATGAAGCAGCATGAACCTACGTGTATTCCAGCGAGTGGAAAGAAAAAATTTCCTAC GAGAGCCTGCAGAGTTTGTGCCGCCCATGGAAAAAGGAGCGAATCTAGATACTTATGTAA ATTTTGTTTGGTCCCTCTTCATAGAGGAAAATGTTTTACGCAGTACCATACGTTAAAAAA GTACTAGGAACTTTAATTGTTTAATTGTTTTTGTAAATAAAAATGTTATAATTATTGAAA AACAACACCTAAAGTGCATTATGATCTGTAGTTATGATGATTTAAATAACGTGCAGTTTG CCCAAAAACGTGTGGTCCCTGGCGTATGTCTTAGAGATTTCTATGCGGTACGCAATGTG
In other particularly preferred embodiment, a mammalian piggyBac transposase comprises a nucleic acid sequence corresponding to SEQ ID NO: 3, and the amino acid sequence encoding the protein, SEQ ID NO: 4, shown below:
SEQ ID NO: 3 piggyBac 1_ML Transposase DNA
ATGTCGCAGCATTCAGACTATACTCATGATGAGTTTTGTGCAGACAAGTTGTCCAATTATTCTTGTG ATAGCGATCTTGAAAATGCGAGTACAAGTGATGAAGATTCTAGTGATGATGAAGTAATGGTGCGT CCCAGGACATTGAGGCGACGAAGAATTTCGAGCTCCAGCTCTGACTCAGAGTCAGATATAGAAGG CGGGAGAGAAGAATGGTCGCATGTTGATAATCCACCGGTCTTAGAAGATTTTTTAGGGCATCAAG GATTAAACACAGATGCTGTTATAAATAATATAGAAGATGCCGTGAAATTATTTATCGGAGATGATT TTTTTGAATTTCTTGTAGAGGAGTCAAACAGGTATTATAATCAAAATAGGAATAATTTCAAACTTT CAAAAAAAAGCCTAAAGTGGAAAGATATAACCCCTCAAGAGATGAAGAAGTTTTTAGGGTTAATT GTTCTCATGGGACAGGTGCGCAAAGATAGAAGAGATGACTATTGGACCACGGAGCCATGGACGGA GACGCCATATTTTGGTAAAACGATGACGAGAGACAGGTTCCGACAGATATGGAAAGCTTGGCACT TCAATAATAATGCGGATATCGTAAATGAATCAGATAGACTTTGCAAAGTGAGACCAGTACTAGAT TATTTTGTGCCTAAATTTATAAATATTTACAAACCTCATCAGCAATTATCACTAGATGAAGGGATC GTACCTTGGAGGGGAAGATTATTCTTTAGGGTATATAATGCTGGCAAGATCGTTAAATATGGAATA TTGGTTCGTTTGTTGTGCGAAAGTGATACAGGATATATCTGTAACATGGAAATTTATTGCGGCGAA GGAAAGCGATTATTGGAAACGATACAAACAGTAGTGTCTCCATACACTGATTCGTGGTACCATATA TATATGGACAATTATTATAATAGCGTCGCAAATTGTGAAGCACTTATGAAAAACAAATTCAGAATA TGTGGAACAATCCGGAAAAATCGAGGTATACCTAAAGATTTTCAAACAATTTCTTTGAAAAAAGG TGAAACAAAATTTATAAGGAAAAATGATATATTGTTACAAGTGTGGCAATCAAAAAAGCCTGTAT ACCTGATTTCTTCGATTCATTCTGCGGAGATGGAAGAAAGTCAGAATATTGACAGAACATCAAAA AAGAAAATTGTCAAACCGAATGCACTCATTGACTACAATAAACATATGAAAGGTGTTGACCGGGC CGACCAATACCTTTCATATTATTCGATATTGCGGAGGACGGTCAAATGGACAAAAAGGTTGGCAAT GTATATGATAAATTGCGCATTATTTAATTCTTATGCAGTTTACAAATCAGTGAGGCAAAGAAAAAT GGGTTTTAAAATGTTTTTGAAACAAACAGCTATCCACTGGTTGACGGATGATATTCCAGAGGACAT GGACATTGTTCCAGACCTTCAACCAGTACCGTCTACTTCTGGAATGCGGGCTAAACCACCTACATC TGATCCACCATGCAGGCTATCGATGGACATGAGAAAGCATACGTTACAGGCAATTGTCGGAAGTG GAAAAAAGAAAAACATTTTGAGAAGGTGTCGCGTATGTTCCGTTCATAAATTGCGCAGTGAGACA CGCTACATGTGCAAATTTTGCAATATACCTCTACATAAAGGGGCGTGTTTTGAAAAATATCATACG CTAAAAAACTAT
SEQ ID NO: 4 piggyBacl_ML Transposase protein
MSQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDS ESDIEGGREEWSHVDNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNR
YYNQNRNNFKLSKKSLKWKDITPQEMKKFLGLIVLMGQVRKDRRDDYWTTEPWTETPYFG KTMTRDRFRQIWKAWHFNNNADIVNESDRLCKVRPVLDYF VPKFINIYKPHQQLSLDEGI VPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGEGKRLLETIQTWSPYT DSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFIRKNDI LLQVWQSKKPVYLISS1HSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDRADQYLS
YYSILRRWKWTKRLAMYMINCALFNSYAVYKSVRQRKMGFKMFLKQTA1HWLTDDIPED
MDIVPDLQPVPSTSGMRAKPPTSDPPCRLSMDMRKHTLQAIVGSGKKKNILRRCRVCSVH
KLRSETRYMCKFCNIPLHKGACFEKYHTLKN
In other particularly preferred embodiment, a mammalian piggyBac transposase comprises a nucleic acid sequence corresponding to SEQ ID NO: 5, and the amino acid sequence encoding the protein, SEQ ID NO: 6, shown below:
SEQ ID NO: 5 piggyBac2_ML Transposase DNA ATGCCCTCTCTCAGAAAAAGGAAGGAAACCAACGAAACTGATACACTTCCGGAAGTATTTAACGA TAATTTATCAGATATTCCTAGTGAGATCGAAGATGCGGATGACTGTTTTGACGATTCCGGAGACGA TTCTACTGATTCTACTGACAGTGAAATTATTAGACCTGTAAGGAAGCGCAAGGTGGCGGTGCTTTC AAGTGATTCCGACACTGACGAAGCTACTGATAATTGTTGGTCTGAAATTGACACACCACCACGCTT ACAAATGTTTGAAGGTCATGCTGGGGTCACTACATTTCCGTCTCAGTGTGACTCTGTACCCTCTGTG ACCAATCTCTTTTTTGGTGATGAATTGTTTGAGATGTTGTGCAAAGAGCTGTCCAACTATCACGATC AAACCGCAATGAAACGCAAAACACCATCTAGAACACTAAAGTGGTCTCCGGTTACACAGAAGGAC ATCAAGAAATTCCTTGGCCTAATTATTCTGATGGGTCAAACAAGAAAAGATAGCTTGAAAGACTAT TGGTCAACAGATCCTTTGATATGTACCCCTATATTTCCACAGACAATGAGTCGCCATAGATTTGAG CAAATATGGACATTCTGGCATTTCAATGATAACGCCAAAATGGACAGTCGCTCGGGGAGACTTTTC AAGATCCAACCTGTGCTGGATTATTTCCTGCATAAATTTCGAACAATATACAAACCAAAGCAACAG TTGTCTTTGGACGAGGGAATGATTCCATGGAGAGGACGTTTCAAATTTCGCACGTACAACCCAGCG AAAATAACAAAATACGGTTTACTTGTTCGGATGGTGTGCGAGAGTGACACCGGCTATATCTGCAGT ATGGAGATATACACTGCTGAAGGAAGGAAATTGCAAGAAACTGTTCTTTCAGTCCTTGGACCCTAT CTTGGCATATGGCACCATATTTACCAGGATAATTATTACAATGCTACATCTACTGCTGAATTGCTGC TACAGAACAAAACTAGAGTCTGTGGGACTATTAGGGAGAGTAGAGGTTTACCGCCAAATTTGGAA ATGAAAACATCAAGAATGAAGAAAGGTGACATAATATTTTCCAGAAAAGGCGATATTCTTCTCCT AGCATGGAAAGACAAGCGGGTTGTCCGAATGATATCAACGATCCATGACACTTCTGTCTCGACAA CAGGAAAAAAAAATAGAAAAACGGGAGAGAATATTGTAAAACCTACCTGCATCAAGGAATACAA TGCCCACATGAAAGGCGTTGACCGTGCGGATCAATTCCTTTCGTGTTGTTCCATTCTAAGGAAAAC GATGAAATGGACAAAAAAAGTAGTGCTGTACCTTATAAACTGTGGACTTTTCAATTCATTTAGAGT GTACAACGTCCTCAATCCACAAGCAAAAATGAAGTATAAACAGTTTCTGCTATCGGTGGCGAGAG ACTGGATAACGGATGACAATAATGAAGGCTCTCCGGAACCAGAGACAAATCTGTCCAGCCCTTCC CCTGGGGGTGCAAGGAGAGCACCTCGTAAAGATCCACCCAAAAGGTTGTCAGGTGATATGAAGCA GCATGAACCTACGTGTATTCCAGCGAGTGGAAAGAAAAAATTTCCTACGAGAGCCTGCAGAGTTT GTGCCGCCCATGGAAAAAGGAGCGAATCTAGATACTTATGTAAATTTTGTTTGGTCCCTCTTCATA GAGGAAAATGTTTTACGCAGTACCATACGTTAAAAAAGTAC
SEQ ID NO: 6 piggyBac2_ML Transposase protein
MPSLRKRKETNETDTLPEVFNDNLSDIPSEIEDADDCFDDSGDDSTDSTDSEIIRPVRKR KVAVLSSDSDTDEATDNCWSEIDTPPRLQMFEGHAGVTTFPSQCDSVPSVTNLFFGDELF EMLCKELSNYHDQTAMKRKTPSRTLKWSPVTQKDIKKFLGLIILMGQTRKDSLKDYWSTD PLICTPIFPQTMSRHRFEQIWTFWHFNDNAKMDSRSGRLFKIQPVLDYFLHKFRTIYKPK QQLSLDEGMIPWRGRFKFRTYNPAKITKYGLLVRMVCESDTGYICSMEIYTAEGRKLQET VLSVLGPYLGIWHHIYQDNYYNATSTAELLLQNKTRVCGTIRESRGLPPNLEMKTSRMKK GDIIFSRKGDILLLAWKDKRWRMIST1HDTSVSTTGKKNRKTGENIVKPTCIKEYNAHM KGVDRADQFLSCCSILRKTMKWTKKWLYLINCGLFNSFRVYNVLNPQAKMKYKQFLLSV ARD WITDDNNEGSPEPETNLSSPSPGGARRAPRKDPPKRLSGDMKQHEPTCIPASGKKKF PTRACRVCAAHGKRSESRYLCKFCLVPLHRGKCFTQYHT
In a further embodiment of the nucleic acid encoding the mammalian piggyBac transposon has a nucleic acid sequence showing at least 75% or 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with a nucleic acid sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2. In another further embodiment of the nucleic acid encoding the mammalian piggyBac transposase has a nucleic acid sequence showing at least 75% or 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the nucleic acid sequence corresponding to SEQ ID NO: 3 or SEQ ID NO: 5.
In another preferred embodiment the nucleic acid encoding the mammalian piggyBac transposon or transposase is selected from a nucleic acid sequence encoding the mammalian piggyBac transposon as defined above and being capable of hybridizing to a complement of a nucleic acid sequence as defined above under stringent conditions. In another preferred embodiment the nucleic acid encoding the mammalian piggyBac transposon or transposase is selected from a nucleic acid sequence encoding the mammalian piggyBac transposase as defined above and being capable of hybridizing to a complement of a nucleic acid sequence as defined above under stringent conditions. Stringent conditions are, for example: 30% (v/v) formamide in 0.5*SSC, 0.1% (w/v) SDS at 42 C for 7 hours.
In some aspects, a hyperactive mammalian piggyBac transposon of the present invention has a higher level of transposon excision compared to a wildtype mammalian piggyBac transposon.
In some other aspects, a mammalian piggyBac of the present invention preferably catalyzes the transposition of a transposon at a frequency that is greater than a "baseline" transposase. In certain preferred embodiments, the mammalian piggyBac nucleic acid sequence is hyperactive compared to a wildtype mammalian piggyBac nucleic acid sequence. Preferably, the wildtype mammalian piggyBac transposon is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 is selected from the group consisting of: C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A, A687G, G715A, T13C, C23T, G161A, G25A, T1050C, A1356G, A26G, A1033G, A1441G, A32G, A389C, A32G, A389C, A32G, T1572A, G456A, T1641C, and T1155C.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises C41T, A1424G, C1472A, G1681A.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises T150C, A351G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A279G. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
T1638C.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A898G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A880G, G1558A. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A687G, G715A.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises T13C, C23T. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
G161A.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises G25A, T1050C, A1356G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A26G, A1033G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A1441G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A32G, A389C. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
A32G, T1572A.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises G456A, T 1641C.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises T1155C.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises G1280A.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises T22C. In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises
A106G.
In certain exemplary embodiments, the nucleotide change in SEQ ID NO 3 comprises A29G, A106G, and Cl 137T. In other exemplary embodiments, an amino acid change in SEQ ID NO 4 is selected from the group consisting of: A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, I345V, M481V, EI lG, K130T, G9G, and silent. In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising C41T, A1424G, C1472A, G1681A, comprises A14V, D475G, P491Q, A561T.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T150C, A351G, is silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A279G, is silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T1638C, comprises T546T.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A898G, comprises T300A.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A880G, G1558A, comprises T294A, A520T.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A687G, G715A, comprises silent, G239S. In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T13C, C23T, comprises S5P, S8F.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising Gl 6 IA, comprises S54N. In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising G25A, T 1050C, A1356G, comprises D9N, silent, silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A26G, A1033G, comprises D9G, I345V.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A 144 IG, comprises M481V. In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A32G, T1572A, comprises G9G, silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising G456A, T 1641C, comprises silent, silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising Tl 155C, comprises silent.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising G 1280A, comprises R427H.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising T22C, comprises S8P.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A106G, comprises S36G.
In certain exemplary embodiments, the amino acid change in SEQ ID NO 4, corresponding to the nucleotide change in SEQ ID NO 3 comprising A29G, A106G, and C1137T, comprises DlOG, S36G, and silent. Assays for measuring the excision of a transposon from a vector, the integration of a transposon into the genomic or extrachromosomal DNA of a cell, and the ability of transposase to bind to an inverted repeat are described herein and are known to the art (see, for instance, (Ivies et al. Cell, 91, 501-510 (1997); WO 98/40510 (Hackett et al.); WO 99/25817 (Hackett et al.), WO 00/68399 (Mclvor et al.), incorporated by reference in their entireties herein. For purposes of determining the frequency of transposition of a transposon of the present invention, the activity of the baseline transposon is normalized to 100%, and the relative activity of the transposon of the present invention determined. Preferably, a transposon of the present invention transposes at a frequency that is, in increasing order of preference, at least about 50%, at least about 100%, at least about 200%, most preferably, at least about 300% greater than a baseline transposon. Preferably, both transposons (i.e., the baseline transposon and the transposon being tested) are flanked by the same nucleotide sequence in the vector containing the transposons.
The invention also features protein sequence showing at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the protein sequence of SEQ ID NO: 4 that is encoded by the nucleic acid sequence corresponding to SEQ ID NO: 3.
The invention also features protein sequence showing at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98% sequence identity with the protein sequence of SEQ ID NO: 6 that is encoded by the nucleic acid sequence corresponding to SEQ ID NO: 5.
The term "identity" is understood as the degree of identity between two or more proteins, nucleic acids, etc., which may be determined by comparing these sequences using known methods such as computer based sequence alignments (basic local alignment search tool, S. F. Altschul et al., J. MoI. Biol. 215 (1990), 403-410). Such methods include without being limited thereto the GAG programme, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 287 (1984); Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP or BLASTN, and FASTA (Altschul, S., et al., J. MoI. Biol. 215:403-410) (1999)). Additionally, the Smith Waterman-algorithm may be used to determine the degree of identity between two sequences.
Functional derivatives according to the present invention preferably maintain the biological function of the mammalian transposase, i.e. the transposase activity, the excision of the nucleic acid sequence and its insertion activity concerning the excised sequences into specific target sequences. Functional derivatives according to the present invention may comprise one or more amino acid insertion(s), deletion(s) and/or substitution(s) of the transposase as shown by SEQ ID NO: 4 or SEQ ID NO: 6. Amino acid substitutions as described herein are preferably conservative amino acid substitutions, which do not alter the biological activity of the transposon or transposase protein. Conservative amino acid substitutions are characterized in that an amino acid belonging to a group of amino acids having a particular size or characteristic can be substituted for another amino acid, particularly in regions of the inventive protein that are not associated with catalytic activity or DNA binding activity, for example. Other amino acid sequences may include, for example, amino acid sequences containing conservative changes that do not significantly alter the activity or binding characteristics of the resulting transposase. Substitutions for an amino acid sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations are not expected to substantially affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Particularly preferred conservative substitutions include, but are not limited to, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free NH2.
Amino acid insertions and substitutions are preferably carried out at those sequence positions of that do not alter the spatial structure or which relate to the catalytic center or binding region of the mammalian piggyBac transposase. A change of a spatial structure by insertion(s) or deletion(s) can be detected readily with the aid of, for example, CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (Ed.), Elsevier, Amsterdam). Suitable methods for generating proteins with amino acid sequences which contain substitutions in comparison with the native sequence(s) are disclosed for example in the publications U.S. Pat. No. 4,737,462, U.S. Pat. No. 4,588,585, U.S. Pat. No. 4,959,314, U.S. Pat. No. 5,116,943, U.S. Pat. No. 4,879,111 and U.S. Pat. No. 5,017,691, incorporated by reference in their entireties herein. Other functional derivatives may be additionally stabilized in order to avoid physiological degradation. Such stabilization may be obtained by stabilizing the protein backbone by a substitution of by stabilizing the protein backbone by substitution of the amide-type bond, for example also by employing [beta]-amino acids. According to certain preferred embodiments of the present invention, the transposon of the present invention may further comprise a marker protein. For example, in certain preferred embodiments, the nucleic acid sequence can be of any variety of recombinant proteins, e.g. any protein known in the art. e.g. the protein encoded by the nucleic acid sequence can be a marker protein such as green fluorescent protein (GFP), the blue fluorescent protein (BFP), the photo activatable-GFP (PA-GFP), the yellow shifted green fluorescent protein (Yellow GFP), the yellow fluorescent protein (YFP), the enhanced yellow fluorescent protein (EYFP), the cyan fluorescent protein (CFP), the enhanced cyan fluorescent protein (ECFP), the monomeric red fluorescent protein (mRFPl), the kindling fluorescent protein (KFPl), aequorin, the auto fluorescent proteins (AFPs), or the fluorescent proteins JRed, TurboGFP, PhiYFP and PhiYFP-m, tHc-Red (HcRed-Tandem), PS-CFP2 and KFP-Red (all available commercially available), or other suitable fluorescent proteins chloramphenicol acetyltransferase (CAT). The protein further may be selected from growth hormones, for example to promote growth in a transgenic animal, or from beta-galactosidase (lacZ), luciferase (LUC), and insulin-like growth factors (IGFs), alpha-anti-trypsin, erythropoietin (EPO), factors VIII and XI of the blood clotting system, LDL-receptor, GATA-I, etc. The nucleic acid sequence further may be a suicide gene encoding e.g. apoptotic or apoptose related enzymes and genes including AlF, Apaf e.g. Apaf-1, Apaf-2, Apaf-3, or APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik, CAD, Calpain, Caspases e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, or Granzyme B, ced- 3, ced-9, Ceramide, c-Jun, c-Myc, CPP32, crm A, Cytochrome c, D4-GDP-DI, Daxx, CdRl, DcRl, DD, DED, DISC, DNA-PK.sub.CS, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas, Fas-ligand CD95/fas (receptor), FLICE/MACH, FLIP, Fodrin, fos, G-Actin, Gas-2, Gelsolin, glucocorticoid/glucocorticoid receptor, granzyme A/B, hnRNPs C1/C2, ICAD, ICE, JNK, Lamin A/B, MAP, MCL-I, Mdm-2, MEKK-I, MORT-I, NEDD, NF-.sub..kappa.B, NuMa, p53, PAK-2, PARP, Perform, PITSLRE, PKC.delta., pRb, Presenilin, prICE, RAIDD, Ras, RIP, Sphingomyelinase, SREBPs, thymidine kinase from Herpes simplex, TNF-. alpha., TNF- alpha receptor, TRADD, TRAF2, TRAIL-Rl, TRAIL-R2, TRAIL-R3, Transglutaminase, Ul 70 kDa snRNP, YAMA, etc.
The mammalian piggyBac transposase, preferably in combination a mammalian piggyBac transposon, has several advantages compared to approaches in the prior art, e.g. with respect to viral and retroviral methods. For example, unlike proviral insertions, transposon insertions can be (re)mobilized by supplying the transposase activity in trans. Thus, for example, instead of performing time-consuming microinjections, it is possible according to the present invention to generate transposon insertions at new loci.
The mammalian piggyBac transposon and transposase protein as defined above can be transfected into a cell as a protein or as ribonucleic acid, including mRNA, as DNA, e.g. as extrachromosomal DNA including, but not limited to, episomal DNA, as plasmid DNA, or as viral nucleic acid. Furthermore, the nucleic acid encoding the transposase protein can be transfected into a cell as a nucleic acid vector such as a plasmid, or as a gene expression vector, including a viral vector. Therefore, the nucleic acid can be circular or linear. A vector, as used herein, refers to a plasmid, a viral vector or a cosmid that can incorporate nucleic acid encoding the transposase protein or the transposon of this invention. The terms "coding sequence" or "open reading frame" refer to a region of nucleic acid that can be transcribed and/or translated into a polypeptide in vivo when placed under the control of the appropriate regulatory sequences. DNA encoding the transposase protein can be stably inserted into the genome of the cell or into a vector for constitutive or inducible expression. Where the transposase protein is transfected into the cell or inserted into the vector as nucleic acid, the transposase encoding sequence is preferably operably linked to a promoter. There are a variety of promoters that could be used including, but not limited to, constitutive promoters, tissue-specific promoters, inducible promoters, and the like. Promoters are regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding sequence. A DNA sequence is operably linked to an expression-control sequence, such as a promoter when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operably linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence to yield production of the desired protein product. Exemplary nucleic acid sequences encoding the mammalian piggyBac transposon are provided as SEQ ID NO: 1 or SEQ ID NO: 2 or hyperactive variants as described herein. Exemplary nucleic acid sequences encoding the mammalian piggyBac transposase protein are provided as SEQ ID NO: 3 or SEQ ID NO: 5. In addition to the conservative changes discussed above that would necessarily alter the transposase-encoding nucleic acid sequence (all of which are disclosed herein as well), there are other DNA or RNA sequences encoding the mammalian piggyBac transposase protein. These DNA or RNA sequences have the same amino acid sequence as a mammalian piggyBac transposase protein, but take advantage of the degeneracy of the three letter codons used to specify a particular amino acid. For example, it is well known in the art that various specific RNA codons (corresponding DNA codons, with a T substituted for a U) can be used interchangeably to code for specific amino acids.
Methods for manipulating DNA and proteins are known in the art and are explained in detail in the literature such as Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press or Ausubel, R. M., ed. (1994). Current Protocols in Molecular Biology.
Gene Transfer System
The present invention also features a gene transfer system comprising a mammalian piggyBac transposon as described herein and a mammalian piggyBac transposase as described herein. As mentioned above, the mammalian piggyBac transposase protein preferably recognizes repeats (e.g. IRs) on the mammalian piggyBac transposon. The gene transfer system of this invention, therefore, preferably comprises two components: the transposase as described herein and a transposon as described herein. Preferably, in certain embodiments, the transposon has at least two repeats (e.g. IRs). When put together these two components provide active transposon activity and allow the transposon to be relocated. In use, the transposase binds to the repeats and promotes insertion of the intervening nucleic acid sequence into DNA of a cell as defined below.
In further exemplary embodiments, the gene transfer system comprises a mammalian piggyBac transposon as defined above in combination with a mammalian piggyBac transposase protein (or nucleic acid encoding the inventive mammalian piggyBac transposase protein to provide its activity in a cell). This combination preferably results in the insertion of the nucleic acid sequence into the DNA of the cell. Alternatively, it is possible to insert the transposon of the present invention into DNA of a cell through non-homologous recombination through a variety of reproducible mechanisms. In either event the inventive transposon can be used for gene transfer by using this gene transfer system.
In certain preferred embodiments, the gene transfer system mediates insertion of the mammalian piggyBac transposon into the DNA of a variety of cell types and a variety of species by using the mammalian piggyBac transposase protein. Preferably, such cells include any cell suitable in the present context, including but not limited to animal cells or cells from bacteria, fungi (e.g., yeast, etc.) or plants. Preferred animal cells can be vertebrate or invertebrate. For example, preferred vertebrate cells include cells from mammals including, but not limited to, rodents, such as rats or mice, ungulates, such as cows or goats, sheep, swine or cells from a human.
In other further exemplary embodiments, such cells, particularly cells derived from a mammals as defined above, can be pluripotent (i.e., a cell whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells) and totipotent cells (i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells). These cells are advantageously used in order to affirm stable expression of the transposase or to obtain a multiple number of cells already transfected with the components of the inventive gene transfer system. Additionally, cells such as oocytes, eggs, and one or more cells of an embryo may also be considered as targets for stable transfection with the present gene transfer system.
In certain preferred embodiments of the invention, the cells are stem cells.
Cells receiving the mammalian piggyBac transposon and/or the mammalian piggyBac transposase protein and capable of inserting the transposon into the DNA of that cell also include without being limited thereto, lymphocytes, hepatocytes, neural cells, muscle cells, a variety of blood cells, and a variety of cells of an organism, embryonic stem cells, somatic stem cells e.g. hematopoietic cells, embryos, zygotes, sperm cells (some of which are open to be manipulated by an in vitro setting).
In other certain exemplary embodiments, the cell DNA that acts as a recipient of the transposon of described herein includes any DNA present in a cell (as mentioned above) to be transfected, if the mammalian piggyBac transposon is in contact with an mammalian piggyBac transposase protein within the cell. For example, the DNA can be part of the cell genome or it can be extrachromosomal, such as an episome, a plasmid, a circular or linear DNA fragment. Typical targets for insertion are e.g. double-stranded DNA.
The components of the gene transfer system described herein, i.e. the mammalian piggyBac transposase protein (either as a protein or encoded by a nucleic acid as described herein) and the mammalian piggyBac transposon can be transfected into a cell, preferably into a cell as defined above, and more preferably into the same cell. Transfection of these components may furthermore occur in subsequent order or in parallel. E.g. the mammalin piggyBac transposase protein or its encoding nucleic acid may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposon. Alternatively, the transposon may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposase protein or its encoding nucleic acid. If transfected parallel, preferably both components are provided in a separated formulation and/or mixed with each other directly prior to administration in order to avoid transposition prior to transfection. Additionally, administration of at least one component of the gene transfer system may occur repeatedly, e.g. by administering at least one, two or multiple doses of this component.
For any of the above transfection reactions, the gene transfer system may be formulated in a suitable manner as known in the art, or as a pharmaceutical composition or kit as described herein. In further preferred embodiments, the components of the gene transfer system may preferably be transfected into one or more cells by techniques such as particle bombardment, electroporation, microinjection, combining the components with lipid-containing vesicles, such as cationic lipid vesicles, DNA condensing reagents (e.g., calcium phosphate, polylysine or polyethyleneimine), and inserting the components (i.e. the nucleic acids thereof into a viral vector and contacting the viral vector with the cell. Where a viral vector is used, the viral vector can include any of a variety of viral vectors known in the art including viral vectors selected from the group consisting of a retroviral vector, an adenovirus vector or an adeno- associated viral vector.
As already mentioned above the nucleic acid encoding the mammalian piggyBac transposase protein may be RNA or DNA. Similarly, either the nucleic acid encoding the mammalian piggyBac transposase protein or the transposon of this invention can be transfected into the cell as a linear fragment or as a circularized fragment, preferably as a plasmid or as recombinant viral DNA.
Furthermore, the nucleic acid encoding the mammalian piggyBac transposase protein is thereby preferably stably or transiently inserted into the genome of the cell to facilitate temporary or prolonged expression of the mammalian piggyBac transposase protein in the cell.
The gene transfer system as disclosed above represents a considerable refinement of non-viral DNA-mediated gene transfer. For example, adapting viruses as agents for gene therapy restricts genetic design to the constraints of that virus genome in terms of size, structure and regulation of expression. Non- viral vectors, as described herein, are generated largely from synthetic starting materials and are therefore more easily manufactured than viral vectors. Non- viral reagents are less likely to be immunogenic than viral agents making repeat administration possible. Non-viral vectors are more stable than viral vectors and therefore better suited for pharmaceutical formulation and application than are viral vectors. Additionally, the inventive gene transfer system is a non- viral gene transfer system that facilitates insertion into DNA and markedly improves the frequency of stable gene transfer.
The present invention further provides an efficient method for producing transgenic animals, including the step of applying the inventive gene transfer system to an animal. Transgenic DNA has not been efficiently inserted into chromosomes. Only about one in a million of the foreign DNA molecules is inserted into the cellular genome, generally several cleavage cycles into development. Consequently, most transgenic animals are mosaic (Hackett et al. (1993). The molecular biology of transgenic fish. In Biochemistry and Molecular Biology of Fishes (Hochachka & Mommsen, eds) Vol. 2, pp. 207-240). As a result, animals raised from embryos into which transgenic DNA has been delivered must be cultured until gametes can be assayed for the presence of inserted foreign DNA. Many transgenic animals fail to express the transgene due to position effects. A simple, reliable procedure that directs early insertion of exogenous DNA into the chromosomes of animals at the one-cell stage is needed. The present system helps to fill this need.
In certain preferred embodiments, the gene transfer system of this invention can readily be used to produce transgenic animals that carry a particular marker or express a particular protein in one or more cells of the animal. Generally, methods for producing transgenic animals are known in the art and incorporation of the inventive gene transfer system into these techniques does not require undue experimentation, e.g. there are a variety of methods for producing transgenic animals for research or for protein production including, but not limited to Hackett et al. (1993, supra). Other methods for producing transgenic animals are described in the art (e.g. M. Markkula et al. Rev. Reprod., 1, 97-106 (1996); R. T. Wall et al., J. Dairy ScL, 80, 2213-2224 (1997)), J. C. Dalton, et al. (Adv. Exp. Med. Biol, 411, 419-428 (1997)) and H. Lubon et al. (Transfus. Med. Rev., 10, 131-143 (1996)).
In another embodiment, the present invention features a transgenic animal produced by the methods described herein, preferably by using the gene transfer system presently described. For example, transgenic animals may preferably contain a nucleic acid sequence inserted into the genome of the animal by the gene transfer system, thereby enabling the transgenic animal to produce its gene product, e.g. a protein. In transgenic animals this protein is preferably a product for isolation from a cell, for example the inventive protein can be produced in quantity in milk, urine, blood or eggs. Promoters can be used that promote expression in milk, urine, blood or eggs and these promoters include, but are not limited to, casein promoter, the mouse urinary protein promoter, beta-globin promoter and the ovalbumin promoter respectively. Recombinant growth hormone, recombinant insulin, and a variety of other recombinant proteins have been produced using other methods for producing protein in a cell. Nucleic acids encoding these or other proteins can be inserted into the transposon of this invention and transfected into a cell. Efficient transfection of the inventive transposon as defined above into the DNA of a cell occurs when mammalian piggyBac transposase protein is present. Where the cell is part of a tissue or part of a transgenic animal, large amounts of recombinant protein can be obtained.
Transgenic animals may be selected from vertebrates and invertebrates, e.g. fish, birds, mammals including, but not limited to, rodents, such as rats or mice, ungulates, such as cows or goats, sheep, swine or humans.
The present invention furthermore provides a method for gene therapy comprising the step of introducing the gene transfer system into cells as described herein. Therefore, the mammalian piggyBac transposon as described herein preferably comprises a gene to provide a gene therapy to a cell or an organism. Preferably, the gene is placed under the control of a tissue specific promoter or of a ubiquitous promoter or one or more other expression control regions for the expression of a gene in a cell in need of that gene. Presently, a variety of genes are being tested for a variety of gene therapies including, but not limited to, the CFTR gene for cystic fibrosis, adenosine deaminase (ADA) for immune system disorders, factor IX and interleukin-2 (IL-2) for blood cell diseases, alpha- 1 -antitrypsin for lung disease, and tumor necrosis factors (INFs) and multiple drug resistance (MDR) proteins for cancer therapies. These and a variety of human or animal specific gene sequences including gene sequences to encode marker proteins and a variety of recombinant proteins are available in the known gene databases such as GenBank.
An advantage of the inventive gene transfer system for gene therapy purposes is that it is not limited to a great extent by the size of the intervening nucleic acid sequence positioned between the repeats. There is no known limit on the size of the nucleic acid sequence that can be inserted into DNA of a cell using the mammalian piggyBac transposase protein. In particular preferred embodiments, for gene therapy purposes, but also for other inventive purposes the gene transfer system may be transfected into cells by a variety of methods, e.g. by microinjection, lipid-mediated strategies or by viral-mediated strategies. For example, where microinjection is used, there is very little restraint on the size of the intervening sequence of the transposon of this invention. Similarly, lipid-mediated strategies do not have substantial size limitations. However, other strategies for introducing the gene transfer system into a cell, such as viral-mediated strategies could limit the length of the nucleic acid sequence positioned between the repeats.
Accordingly, in certain exemplary embodiments, the gene transfer system as described herein can be delivered to cells via viruses, including retroviruses (such as lentiviruses, etc.), adenoviruses, adeno-associated viruses, herpes viruses, and others. There are several potential combinations of delivery mechanisms that are possible for the mammalian piggyBac transposon portion containing the transgene of interest flanked by the terminal repeats and the gene encoding the transposase. For example, both the transposon and the transposase gene can be contained together on the same recombinant viral genome; a single infection delivers both parts of the gene transfer system such that expression of the transposase then directs cleavage of the transposon from the recombinant viral genome for subsequent insertion into a cellular chromosome. In another example, the transposase and the transposon can be delivered separately by a combination of viruses and/or non- viral systems such as lipid-containing reagents. In these cases either the transposon and/or the transposase gene can be delivered by a recombinant virus. In every case, the expressed transposase gene directs liberation of the transposon from its carrier DNA (viral genome) for insertion into chromosomal DNA. In certain preferred embodiments of the present invention, mammalian piggyBac transposons may be utilized for insertional mutagenesis, preferably followed by identification of the mutated gene. DNA transposons, particularly the transposons, have several advantages compared to approaches in the prior art, e.g. with respect to viral and retroviral methods. For example, unlike proviral insertions, transposon insertions can be remobilized by supplying the transposase activity in trans. Thus, instead of performing time-consuming microinjections, it is possible according to the present invention to generate transposon insertions at new loci by crossing stocks transgenic for the above mentioned two components of the transposon system, the inventive transposon and the inventive transposase. In a preferred embodiment the gene transfer system is directed to the germline of the experimental animals in order to mutagenize germ cells. Alternatively, transposase expression can be directed to particular tissues or organs by using a variety of specific promoters. In addition, remobilization of a mutagenic transposon out of its insertion site can be used to isolate revertants and, if transposon excision is associated with a deletion of flanking DNA, the inventive gene transfer system may be used to generate deletion mutations. Furthermore, since transposons are composed of DNA, and can be maintained in simple plasmids, inventive transposons and particularly the use of the inventive gene transfer system is much safer and easier to work with than highly infectious retroviruses. The transposase activity can be supplied in the form of DNA, mRNA or protein as defined above in the desired experimental phase.
In another embodiment, the present invention also provides an efficient system for gene discovery, e.g. genome mapping, by introducing a mammalian piggyBac transposon as defined above into a gene using a gene transfer system as described in the present invention. In one example, the mammalian piggyBac transposon in combination with the mammalian piggyBac transposase protein or a nucleic acid encoding the mammalian piggyBac transposase protein is transfected into a cell. In certain preferred embodiments, the transposon preferably comprises a nucleic acid sequence positioned between at least two repeats, wherein the repeats bind to the mammalian piggyBac transposase protein and wherein the transposon is inserted into the DNA of the cell in the presence of the mammalian piggyBac transposase protein. In certain preferred embodiments, the nucleic acid sequence includes a marker protein, such as GFP and a restriction endonuclease recognition site. Following insertion, the cell DNA is isolated and digested with the restriction endonuclease. For example, if the endonuclease recognition site is a 6-base recognition site and a restriction endonuclease is used that employs a 6-base recognition sequence, the cell DNA is cut into about 4000-bp fragments on average. These fragments can be either cloned or linkers can be added to the ends of the digested fragments to provide complementary sequence for PCR primers. Where linkers are added, PCR reactions are used to amplify fragments using primers from the linkers and primers binding to the direct repeats of the repeats in the transposon. The amplified fragments are then sequenced and the DNA flanking the direct repeats is used to search computer databases such as GenBank.
Using the gene transfer system for methods as disclosed above such as gene discovery and/or gene tagging, permits, for example, identification, isolation, and characterization of genes involved with growth and development through the use of transposons as insertional mutagens or identification, isolation and characterization of transcriptional regulatory sequences controlling growth and development.
In another exemplary embodiment of the present invention, the invention provides a method for mobilizing a nucleic acid sequence in a cell. According to this method the mammalian piggyBac transposon is inserted into DNA of a cell, as described herein.
Mammalian piggyBac protein or nucleic acid encoding the mammalian piggyBac transposase protein is transfected into the cell and the protein is able to mobilize (i.e. move) the transposon from a first position within the DNA of the cell to a second position within the DNA of the cell. The DNA of the cell is preferably genomic DNA or extrachromosomal DNA. The inventive method allows movement of the transposon from one location in the genome to another location in the genome, or for example, from a plasmid in a cell to the genome of that cell.
In another exemplary embodiments, the inventive gene transfer system can also be used as part of a method involving RNA- interference techniques. RNA interference (RNAi), is a technique in which exogenous, double-stranded RNAs (dsRNAs), being complementary to mRNA's or genes/gene fragments of the cell, are introduced into this cell to specifically bind to a particular mRNA and/or a gene and thereby diminishing or abolishing gene expression. The technique has proven effective in Drosophila, Caenorhabditis elegans, plants, and recently, in mammalian cell cultures. In order to apply this technique in context with the present invention, the inventive transposon preferably contains short hairpin expression cassettes encoding small interfering RNAs (siRNAs), which are complementary to mRNA's and/or genes/gene fragments of the cell. These siRNAs have preferably a length of 20 to 30 nucleic acids, more preferably a length of 20 to 25 nucleic acids and most preferably a length of 21 to 23 nucleic acids. The siRNA may be directed to any mRNA and/or a gene, that encodes any protein as defined above, e.g. an oncogene. This use, particularly the use of mammalian piggyBac transposons for integration of siRNA vectors into the host genome provides a long-term expression of siRNA in vitro or in vivo and thus enables a long-term silencing of specific gene products.
Pharmaceutical Compositions
The present invention further refers to pharmaceutical compositions containing either a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, and/or a mammalian piggyBac transposon, or a gene transfer system as described herein comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, in combination with a mammalian piggyBac transposon.
The pharmaceutical composition may optionally be provided together with a pharmaceutically acceptable carrier, adjuvant or vehicle. In this context, a pharmaceutically acceptable carrier, adjuvant, or vehicle according to the invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the component(s) with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the pharmaceutical compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
The pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavouring or colouring agents may also be added.
Alternatively, the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the inventive gene transfer system or components thereof with a suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and Therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the inventive gene transfer system or components thereof suspended or dissolved in one or more carriers. Carriers for topical administration of the components of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene component, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
The amount of the components of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration. It has to be noted that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific component employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a component of the present invention in the composition will also depend upon the particular component(s) in the composition. The pharmaceutical composition is preferably suitable for the treatment of diseases, particular diseases caused by gene defects such as cystic fibrosis, hypercholesterolemia, hemophilia, immune deficiencies including HIV, Huntington disease, .alpha.-anti-Trypsin deficiency, as well as cancer selected from colon cancer, melanomas, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, gliomas, thyroid cancer, mamma carcinomas, prostate tumors, hepatomas, diverse virus-induced tumors such as e.g. papilloma virus induced carcinomas (e.g. cervix carcinoma), adeno carcinomas, herpes virus induced tumors (e.g. Burkitt's lymphoma, EBV induced B cell lymphoma), Hepatitis B induced tumors (Hepato cell carcinomas), HTLV-I und HTLV-2 induced lymphoma, lung cancer, pharyngeal cancer, anal carcinoma, glioblastoma, lymphoma, rectum carcinoma, astrocytoma, brain tumors, stomach cancer, retinoblastoma, basalioma, brain metastases, medullo blastoma, vaginal cancer, pancreatic cancer, testis cancer, melanoma, bladder cancer, Hodgkin syndrome, meningeoma, Schneeberger's disease, bronchial carcinoma, pituitary cancer, mycosis fungoides, gullet cancer, breast cancer, neurinoma, spinalioma, Burkitt's lymphoma, lyryngeal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin lymphoma, urethra cancer, CUP- syndrome, oligodendroglioma, vulva cancer, intestinal cancer, oesphagus carcinoma, small intestine tumors, craniopharyngeoma, ovarial carcinoma, ovarian cancer, liver cancer, leukemia, or cancers of the skin or the eye; etc.
Kits
The present invention also features kits comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid, and/or a mammalian piggyBac transposon; or a gene transfer system as described herein comprising a mammalian piggyBac transposase as a protein or encoded by a nucleic acid as described herein, in combination with a mammalian piggyBac transposon; optionally together with a pharmaceutically acceptable carrier, adjuvant or vehicle, and optionally with instructions for use. Any of the components of the inventive kit may be administered and/or transfected into cells in a subsequent order or in parallel, e.g. the mammalian piggyBac transposase protein or its encoding nucleic acid may be administered and/or transfected into a cell as defined above prior to, simultaneously with or subsequent to administration and/or transfection of the inventive transposon. Alternatively, the mammalian piggyBac transposon may be transfected into a cell as defined above prior to, simultaneously with or subsequent to transfection of the mammalian piggyBac transposase protein or its encoding nucleic acid. If transfected parallel, preferably both components are provided in a separated formulation and/or mixed with each other directly prior to administration in order to avoid transposition prior to transfection. Additionally, administration and/or transfection of at least one component of the kit may occur in a time staggered mode, e.g. by administering multiple doses of this component.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES The piggyBac transposon from insects is becoming an increasingly popular tool for use in transgenesis and insertional mutagenesis in many organisms including mammals and insects. The present inventors have isolated the first piggyBac transposon from a mammal. No active form of this family of transposons has been isolated from a mammal, and very few other transposons that are active in mammalians cells and other metazoans have been isolated. The piggyBac transposon described by the present invention, as it is from a mammal, may be better able to escape mammalian transposon repression systems. The present inventors have already established that it is active in the yeast S.cerevisiae and thus have employed yeast to isolate hyperactive bat piggyBacs that could be more active than existing versions of the insect element. There is mounting evidence for recent and substantial DNA transposon activity in the vespertilonid bat Myotis lucifugus. In one study, this conclusion was inferred from the presence of minimally diverged nonautonomous hAT transposons, the polymorphic status of some of these transposons in natural Myotis populations, and the discovery of an apparently full-length and potentially functional autonomous hAT transposon (Ray et at. 2007). In a separate report, an extensive and recently amplified population of Helitrons, a distinct subclass of DNA transposons, was discovered in the M. lucifugus genome (Pritham and Feschotte 2007). These surprising observations raised a number of intriguing questions about the activity of DNA transposons and its genomic impact in Myotis and other bats. In particular, the present inventors investigated whether the discovery of relatively recent hAT and Helitron activity in Myotis were merely coincidental or whether it reflected some unique biological or genomic features of bats in general or vespertilonids in particular that has allowed DNA transposons to invade and expand in these genomes, while such occurrences appear to be rare or nonexistent in other mammalian genomes. As a prelude to addressing these questions M. lucifugus was examined for additional traces of DNA transposon activity. Consequently, the diversity, abundance, and evolutionary history of DNA transposons in the M. lucifugus genome was examined.
The present experiments characterize seven new families of DNA transposons from the current whole-genome sequence of M. lucifugus. In addition, the present experiments provide additional information on the previously described Myotis hAT transposons (Ray et at. 2007). Together, these elements represent three distinct superfamiles and exhibit clear signs of recent activity in the vespertilonid lineage, that is,within the last 40 - 50 million years (Myr). The present inventors have also identified a ninth transposon lineage that likely expanded prior to the chiropteran divergence. One family represents the youngest DNA transposon family so far recorded in any mammalian species, one that is likely still expanding in the genome. The discovery of this unprecedented level of DNA transposon activity in a mammalian genome represents a dramatic shift in our view of mobile element biology in mammals.
Example 1. Activity of the piggyBac element
In order to demonstrate that the piggyBac element was active, a plasmid that expressed the transposase and another plasmid with a transposon encoding a transposon carrying a drug resistance gene were transfected into HeLa cells. It was found that the transposon inserted in the host genome as measured by a much high frequency of drug- resistant cells than occurred with a no transposase control. The results of this experiment are shown in Figure 1. As shown in Figure 1 , the blue spots on the Petri dish are cells that live in the presence of drug. To further show that the piggyBac element was active, bat transposon excision was assayed using the ura — > ura+ assay as described in Mitra R, et al. EMBO J. 2008 Apr 9;27(7) : 1097-109. The frequency of bat piggyBac excision was about 1 x 10" .
Example 2. Identification of transposons in the M. lucifugus genome hA T superfamily
The consensus sequence for Myotis hATl was previously described (Ray et at. 2007). To summarize, the entire sequence spans 2921 nucleotides with a single ORF consisting of bases 700-2631 that encodes an apparently intact transposase of 643 amino acids (aa). The present analysis confirms the characteristic target-site duplications (TSDs) for Myotis hAT elements, 8 bp with a central TA dinucleotide, and the typical short TIR (terminal inverted repeats) sequence. Myotis hATl and its nonautonomous derivatives are by far the most abundant family of elements analyzed in this study, with > 96,000 hits of -100 bp in the current WGS data. A second family of hAT elements was identified, called hAT2_ML. The TIRs of hAT2_ML are highly similar to Myotis hATl, but the internal sequence is only weakly similar. hAT2_ML is predicted to encode a 428-aa transposase with only minimal sequence similarity and 33% amino acid identity to the Myotis hATl transposase. Phylogenetic comparison to other hAT transposases confirms that Myotis hATl and hAT2_ML belong to the hAT superfamily and are closely related to hATI MD (Repbase Update, volume 12, issue 10), a hAT transposon family recently identified in the opossum Monodelphis domestica (Fig. 2A). RepeatMasker results using the full consensus as a query suggests that hA T2_ML is not nearly as widespread as Myotis_hATl with only 2004 hits of -100 bp. A third hAT family member, hAT3_ML, was also recovered from the M. lucifugus genome. The consensus sequence contains a large ORF spanning base pairs 1595-3403 and encoding a putative protein of 602 aa, which nests in phylogenetic analysis within a clade encompassing several mammalian hAT transposases (Fig. 2A). With its 3904-bp consensus, it is the longest of all of the elements described in this study and is second to Myotis hATl in terms of its abundance with ) > 12,000 elements of -100 bp identified in the current WGS assembly of M. lucifugus.
Tel/mariner superfamily
Eukaryotic Tel/mariner elements are divided into several anciently diverged lineages (Robertson 2002; Feschotte and Pritham 2007). Three distinct Tel/ lineages have been previously identified in mammals and characterized most extensively in human: pogo, Tc2, and D34D mariner. Members of all three groups were identified in M. lucifugus as well as the first mammalian member of the Tel group. The pogo lineage is represented by Tigger elements in mammals. It is known to include both eutherianwide and primate-specific families (Robertson 1996; Smit and Riggs 1996; Pace and Feschotte 2007). Thus, the presence of Tigger elements was somewhat expected in M. lucifugus. Remnants of what appears to be an ancient family of Tigger- like elements called Tiggerl ML were identified. A tentative consensus was reconstructed from the alignment of the seven longest copies. The size (2.8 kb) and TIRs are consistent with other Tigger elements and the consensus is similar to consensus sequences in Repbase as Tiggerla CAR and Tiggerla ART, which represent Tiggerl-like familes specific to carnivores and artiodactyls, respectively. Also identified were several highly eroded copies of a related family of Tiggerl transposons in the European hedgehog, Erinaceus europaeus (e.g., accession AANN01830819, position 1599-4234). Given that hedgehog, carnivores, artiodactyls, and chiropterans are all part of the well- supported Laurasiatheria superorder, it was hypothesized that Tiggerl ML amplified during the early part of the Laurasiatherian radiation. This is by far the oldest family of DNA transposons that were identified and, as it does not appear to be unique to chiropterans, the details of this family were not focused on in the current study.
The Tc2 group is sister to the pogo lineage and was first identified in Caenorhabditis elegans (Ruvolo et at. 1992). There are relatively few Tc2-like elements in human, and their amplification predates the split of eutherian mammals (Pace and Feschotte 2007). In contrast, a distinct and much more recent family of Tc2-like elements was identified in M. lucifugus. The Tc2_l_ML consensus is 1728 bp with 23 bp TIRs similar to known or newly identified Tc2 elements. It contains a single ORF spanning positions 213-1559 and encoding a putative transposase of 448 aa. Phylogenetic analysis confirms that Tc2_l_ML belongs to the Tc2 group, but does not cluster with either of the mammalian Tc2-like transposases identified in human (Kanga), opossum (Tc2_MD2), and tenrec (Tc2_Et), or with the human transposase- derived proteins POGK and POGZ (Fig. IB). Thus, Tc2_l_ML defines a distinct lineage of Tc2-like elements. Tc2_l_ML is the least abundant of the DNA transposon familes described in this study with only 589 hits -100 bp. Two distinct lineages of canonical D34D mariner are known to occur in mammals: the cecropia subfamily, represented by Hsmarl in anthropoid primates and Mmarl in mouse, and the irritans subfamily, represented by Hsmar2 in primates (Robertson 2002). An abundant (2036 hits >100 bp) family of transposons in M. lucifugus were detected, Mlmarl, that displays all the features of a canonical D34D mariner. The Mlmarl consensus is 1287 bp with 30-bp TIRs and a single ORF, spanning position 174-1184 and predicted to encode a 336-aa transposase. Surprisingly, phylogenetic analyses robustly place the predicted Mlmarl transposase within the mauritiana subfamily of mariner (Fig. 2e), a subfamily typified by the original mosl element active in Drosophila mauritiana (Hartl et at. 1997; Robertson 2002). No members of the irritans or cecropia subfamiles of mariner in M. lucifugus were detected using protein queries representing these lineages, although these two lineages have been identified in a broad range of mammals (Demattei et at. 2000; Robertson 2002; Waters ton et at. 2002; Sinzelle et at. 2006), including other bat species (see below).
Tel- like elements represent another distinct lineage of Tel/ mariner elements that are widespread and common in invertebrates and lower vertebrates (fish, frogs), but no Tel- like element has previously been identified in mammals (Avancini et at. 1996; Leaver 2001; Sinzelle et at. 2005). Consistent with this observation, TBLASTN searches with representative Tel-like transposases from fish or nematode return no significant hits in the complete genome of human, mouse, rat, and dog. In contrast, the same query returns hundreds of hits from the M. lucifugus WGS database. A single Tel- like family, TeI l ML, predominates in the genome. The TeI l ML consensus spans 1222 bp with 29 bp TIR with a 5'-CACTG-3' terminal motif typical of many Tel-like elements. The ORF occupies position 150-1190 of the consensus and encodes a putative transposase of 346 residues. Phylogenetic analyses show that the TeI l ML transposase forms a well- supported clade with Tel-like transposases from fish (Fig. 2D). 3788 TeI l ML fragments of 100 bp or larger were detected in the M. lucifugus WGS database. Many of the TeI l ML elements are inserted in (TA) dinucleotide repeats, an insertion preference also observed for other Tel elements (Vigdal et at. 2002). Of note is one subfamily derived from TeI l ML. The consensus of this subfamily has an intact ORF that represents 2/3 (708 bp; 235 aa) of the complete ORF for TeI l ML. Five hundred copies of this subfamily of Tel are distributed throughout the M. lucifugus genome.
piggyBac superfamily piggyBac-like elements have been identified in a wide range of animal species and Entamoeba (Sarkar et at. 2003; Pritham et at. 2005). Among mammals, piggyBac elements have so far only been characterized in the human genome, where they are predominantly represented by two families of nonautonomous elements (MER85 and MER75) and by several stationary "domesticated" transposases (PGBD1-5 genes) (Sarkar et at. 2003). Two familes of piggyBac-like elements in M. lucifugus were identified. piggyBacl ML is defined by a 2626-bp consensus with short TIRs (15 bp) that are very similar to other piggyBac transposons. The consensus contains a 1719-bp ORF (position 587-2305) that likely encodes a transposase of 572 residues. Two potentially active elements, with intact ORFs and identifiable TSDs, were located in the available genome data. piggyBac2_ML is similar in length at 2639 bp and with a 583-aa transposase (nt 716-2467). Sequence comparison with other piggyBac transposases and phylogenetic analyses indicate that the two families are relatively closely related and form a strong cluster with the sea squirt, Ciona intestinalis (Fig. IE). Myotis piggy-Bac-like elements tend to be flanked by the canonicalS' -TIAA-3' TSD. piggyBacl-ML elements number at least 2056 in the M. lucifugus genomes and are likely still mobilizing (see below). However, piggyBac2_ML elements are currently more numerous, with at least 3869 instances.
In summary, a diverse array of hAT, Tel/mariner, and piggyBac transposons in the M. lucifugus genome have been identified. The Myotis hATl and hAT3_ML elements are considerably abundant compared to the others. As with Myotis hATl elements (Ray et al. 2007), all of the newly discovered transposon families are associated with multiple nonautonomous MITE subfamilies. It is beyond the scope of the present study to describe all of these subfamilies in detail, but it is noted that nonautonomous elements appear to have outnumbered their autonomous partners, as is typically observed for DNA transposon families in humans and most other eukaryotes. (Feschotte et al. 2002; Pace and Feschotte 2007).
Example 3. Taxonomic distribution of DNA transposons identified in Myotis lucifugus
PCR-based analyses of 15 taxa representing nine bat families were performed using two oligonucleotide primer pairs for each transposon family (Fig. 3). Results indicate that the families described have limited taxonomic distributions. TcI l ML, Tc2_l_ML, and Mlmarl appear to be limited to Vespertilonidae with the possible exception of Tel- like sequences in Pteronotis parnelli (Family Mormoopidae). All three hAT families are restricted to the genus Myotis. piggyBac2_ML shares a similar distribution to the hAT elements, but positive results were obtained from the lone representative of Miniopteridae, a family that was recently elevated from subfamily status within Vesperdilionidae (Miler-Butterworth et al. 2007). Amplification of the miniopterid and mormoopid representatives suggests the need for additional exploration of these taxa. PCR data using piggyBacl ML-derived oligonucleotides suggests that these elements are even further restricted. Amplification was obtained in Myotis austroriparius, a fellow North American bat, but not the Asian representative, Myotis horsfieldii. These results may not confirm that no class 2 transposons are active in other bat lineages. Instead, they lend support to the hypothesis that these particular transposons have a limited taxonomic distribution. With the exception of Tiggerl ML, whose amplification likely predates the split of Chiroptera or perhaps even Laurasiatheria, TBLASTN and BLASTN queries of chiropteran taxa with the consensus transposon sequences from M. lucifugus yielded only very few significant hits in other bat species. A handful of hits were obtained with the Mlmarl predicted transposase in the phyllostomid bats Artibeiis jamaicensis and Carolla perspieilata, and in the rhino lophid bat, Rhino lophus femimenquinum. Interestingly, the hits correspond to cecropia-like and the previously described irritans-like mariners (Sinzelle et al. 2006) (Fig. 2C). Thus, it appears that distinct types of mariners have colonized different bat lineages. Mlmarl is most closely related to Mboumarl, a mauritiana- like mariner from the ant Messor bouvieri (see also phylogeny in Fig. 2C). The transposase regions are 75% identical at the nucleotide level and 85% similar at the protein level. This level of sequence similarity is remarkable given the evolutionary distance of their host species, ).800 Myr (Gu 1998; Blair and Hedges 2005). No traces of other mauritiana-like mariner transposases in any of the ~30 additional mammalian species nor any other chordate species represented in the NCBI databases were detected.
Example 4. Age estimations of DNA transposon families Table 1 , shown below, and Figure 4 show the estimated ages for each family based on average divergence from the consensus sequence and an estimated neutral mutation rate of -2.366 X 10-9 (see Methods).
Figure imgf000043_0001
Age analyses of the eight youngest families produce a clear temporal pattern indicative of successive expansions in the genome. The earliest invasions began with Tel- like and mariner-like elements and were followed by the colonization of TcI I ML, hAT- like, and piggyBac-like families. In mammals, CpG dinucleotides within repeats are generally heavily methylated, and thus tend to degrade into TpG or CpA dinucleotides at a substantially higher rate than other base combinations (Coulondre et al. 1978; Razin and Riggs 1980; Xing et al. 2004). Thus, lower divergence estimates were expected and observed when calculated using sequences from which the CpG dinucleotides had been removed. CpG to non-CpG mutations densities were calculated for all families (Table 2, shown below). The youngest subfamiles, derived from piggyBacl ML, produced consistently lower ratios than any other subfamily. This is most likely explained by their very recent deposition in the genome (see below) and because they have not resided long enough to accumulate many mutations of any kind. This hypothesis, however, does not explain the relatively low ratios observed for the nhATl subfamilies, which are believed to have been mobilized as much as 10 Myr earlier. Average CpG dinucleotide mutation densities were approximately eight times higher than non-CpG sites. It is noted that this is higher than the rate observed for primates (~6X; Xing et at. 2004), bringing to mind the hypothesis that increased methylation, and consequently, high CpG:non-CpG mutation ratios may serve as a genomic defense mechanism against large bursts of transposable element activity (Schmid 1998; Xing et at. 2004).
Figure imgf000044_0001
Example 5. Current activity by piggyBacl-ML
Both genetic distance and CpG analyses above provide evidence for very recent and likely ongoing piggyBacl ML activity. Indeed, a large fraction of the nonautonomous piggyBael elements identified in M. lucifugus are strictly identical (65.7% of the elements in the npiggy_156 subfamily and 30.7% in the npiggy_239 subfamily), which strongly indicates that they have been inserted very recently. As seen in Figure 4, most of the DNA transposon families analyzed in this study appear to have reached an amplification peak and then entered a period of reduced activity and extinction. In contrast, the npiggy_156 subfamily appears to be nearing its amplification peak, suggesting that piggyBacl_ML may still be active in the Myotis genome. The identification of two full-length elements with intact ORFs in the current genome sequence data further support this hypothesis. Further evidence for recent piggyBacl ML activity is he observation that it was possible to experimentally isolate intact preintegration sites for npiggy_156 elements in a related species, M. austroriparius (Fig. 5). Finally, experimental results from a diverse panel of bat species indicate that piggyBacl ML is present in only a subset of species in the genus (Fig. 3). Thus, this particular subfamily was mobilzed within the last -8-12 Myr (Stadelmann et at. 2007). Example 6. Nested insertion analysis
As a second, independent method to estimate relative ages of the elements, he number and identity of nested insertions were compared within individual transposon copies. Assuming that DNA transposon insertions occur fairly randomly throughout the genome, older elements should have higher proportions of nested insertions than younger elements. Furthermore, while it is possible for representatives from older transposon families to become nested within younger families if their periods of activity overlapped, these instances should be relatively rare. RepeatMasker hits for each family of transposons were subjected to a second RepeatMasker analysis using a custom library that included each of the new transposable elements described here and Ves, a SINE found in the suborder Yangochiroptera (Borodulina and Kramerov 1999, 2005). The studies were limited to these elements because their sequences have been well characterized here or elsewhere (Borodulina and Kramerov 1999; Ray et at. 2007) and their target site duplications are easily identified.
Analysis of nested insertions supports the age hierarchy suggested by divergence estimates. Among the hAT and piggyBac elements examined, only two nested DNA transposon insertions were recovered-two piggyBacl insertions inside a Myotis hATl element (Table 3, shown below)-lending support to the hypothesis that these families are the most recently active DNA transposon families in the M. lucifugus genome. In contrast, the older elements represented by the Tel, Tel2, and mariner families have been subjected to numerous interruptions by the more recently active elements. Mlmarl elements have suffered nested insertions from all other families except Te2_l_ML, which is likely older. It is interesting that the Te2 elements have only accumulated two nested insertions by their fellow DNA transposons. This could be explained by the low occurrence of these elements (n = 593) and the relatively small size of these insertions (-560 bp). Mariner elements are present in larger numbers ( >2000) and larger average size (778 bp), and thus present approximately five times the number of potential insertion targets during an only slightly shorter time frame. Analyses of Ves SINE nested insertions present a similar picture. The four most recently active elements, piggyBacl_ML, piggyBac2_ML, hAT2_ML, and hAT3_ML have been subjected only to Ves insertions. Nested self- insertions are theoretically possible and were expected when the analysis was performed. Indeed, there were some possible instances observed. However, in each of these cases the TSDs were not clearly identifiable. Because TSD identification was one a priori criterion for assessing the presence of any nested insertion (see Methods), these were excluded from Table 3. Finally, one would expect older elements to have accumulated more nested insertions than more recently mobilzed elements. To test this hypothesis, the percent nested insertion content for all instances of each family of elements recovered from the M. lucifugus genome was examined. Figure 6 illustrates a clear pattern of younger elements (i.e., piggyBac-like and hAT-like) having lower relative nested insertion content when compared with older elements from the Tel/mariner superfamily. The one exception to this pattern is TeI l ML, which exhibits a relatively low percentage of nested insertions. It is unclear as to why this family exists as an outlier.
Figure imgf000046_0001
DNA transposons are widespread and a diversity of elements have been recently active in the genomes of many eukaryotes, including lower vertebrates (Aparicio et al. 2002; Koga et al. 2006). In contrast, initial analyses of the transposable element landscape in the complete human, mouse, rat, and dog genomes have led to the common belief that mammalian genomes are devoid of recently active DNA transposons (Lander et al. 2001; Waterston et al. 2002; Gibbs et al. 2004; Lindblad-Toh et al. 2005). Supporting this idea, a detailed investigation of the evolutionary history of human DNA transposons revealed that many transposons were intensively active during early primate evolution (-80-40 million years ago (Mya), but this activity ceased approximately ~40 Mya (Pace and Feschotte 2007). Analyses of the rodent and carnivore lineages reveal a similar trend, with no DNA transposon family identified in either mouse or dog that is significantly younger than 50 Myr (Waterston et al. 2002; J. Pace and C. Feschotte, unpubl). The present studies report the identification of seven new families of DNA transposons in the M. lucifugus genome which, in contrast to observations made for other mammalian genomes, have apparently been active within the last 40 Myr. Several lines of evidence support this conclusion. First, the elements appear to be almost exclusively limited to Vespertilionidae, and in some cases to selected taxa within the family. Second, the level of sequence divergence among copies (and to their respective consensus sequences) suggests that these families were active from ~36 Mya to the present. Analysis of nested insertions suggests a hierarchical pattern of insertion consistent with sequence divergence estimates. Third, two potentially full-length and intact piggyBacl ML transposons could be identified in the available genome sequence data, supporting the hypothesis that the expansion of these elements and their nonautonomous relatives is ongoing. Finally, recent activity of piggyBacl ML was verified by recovering "empty" orthologous sites in a closely related Myotis species and by the limited taxonomic distribution of piggyBacl ML to North American Myotis species. Together with the recent study reporting the massive amplification of HeliBats 30-36 Mya in the lineage of M. lucifugus (Pritham and Feschotte 2007), these data demonstrate that the genomes of vespertilonid bats have been subjected to multiple waves of amplification of diverse DNA transposons, from ~40 Mya to the present. One lineage of piggyBac, piggyBacl ML, is likely still active. This is the first documented evidence of the recent activity of a diverse population of DNA transposons in any mammal. At least 3.5% of the available genome data is made up of sequence derived from the transposons described herein. It should be noted, however, that this number is very likely an underestimate. The previous study of hATl elements in M. lucifugus revealed an abundance of nonautonomous nbAT families, which have similar TIRs, but little internal sequence similarity with each other or with larger autonomous elements (Ray et at. 2007). Because of this lack of similarity, RepeatMasker fails to identify a majority of the previously identified nhAT elements when the consensus for Myotis hA Tl is used as the part of the database. Indeed, masking the genome using the Myotis hA Tl sequence produces only -21,000 hits greater than 100 bp. Creating a database that contains the full-length consensus and the nhAT elements described by Ray et at. (2007) as a single repeat database increases the number of hits to >96,000. It is believed that the other transposon families described herein will follow a similar pattern, and therefore, the estimate of the amount of DNA derived from DNA transposons may be too low. Add the impact of HeliBats, which accounts for at least an additional 3% of the M. lucifugus genome (Pritham and Feschotte 2007), and the as yet uncharacterized influence of retrotransposons suggests an extremely dynamic genomic landscape over the past 40 Myr of vespertilionid bat evolution.
Myotis is one of the most species-rich of all mammalian genera, and repeated waves of transposon activity suggest a mechanism for generating the genetic variability necessary to produce its tremendous species diversity. Interestingly, a recent analysis of Myotis phylogeny based on nuclear and mitochondrial DNA sequences becomes more intriguing when one considers the data presented here. Stadelmann et at. (2007) found that a burst of Myotis diversification occurred -12-13 Mya. These dates correspond well to the estimated time during which the most active DNA transposon families were expanding in the Myotis genome (Fig. 3).
The young age and narrow taxonomic distribution of all (but one) of the transposon families identified in M. lucifugus raises the puzzling question of their evolutionary origin. Based on the data presented here and elsewhere (Pace and Feschotte 2007; Ray et at. 2007), a favored hypothesis is that most of these families hail from repeated episodes of horizontal introduction of founder autonomous transposons from yet unknown source(s) occurring at different evolutionary time points in the lineage of M. lucifugus. The only alternative hypothesis would be that each family arose from active transposons that were vertically inherited during chordate evolution. This hypothesis may be less likely, because the lack of closely related elements in any of the 30+ other mammalian species (including three nonvespertilionid bats) for which complete or large amounts of genome sequence data are currently available would entail that these elements have persisted in the lineage of vespertilonid bats, while being systematically lost in all other lineages.
Under a scenario dominated by horizontal introduction, it appears that mariner, Tel, and Tc2-like elements colonized the ancestral vespertilonid genome prior to diversification of the family. Later invasions of the hA T-like and piggyBac-like elements may have occurred prior to diversification of Myotis and, in the case of piggyBacl_ML, New World Myotis. Such a scenario of repeated horizontal transfer is consistent with the well-known propensity and the apparent necessity of class 2 elements to undergo horizontal transfers (Hartl et at. 1997; Robertson 2002). A more thorough analysis of the distribution of these elements in bats and other vertebrates will shed additional light onto their origin.
These data also raised the question as to why bats (and Vespertilonidae in particular) exhibit such a high level of recent transposon activity, while these types of elements have seemingly gone extinct in other mammalian lineages examined. Bats exhibit a variety of life history traits unique among mammals (flight, high population sizes and densities, torpor) that provide potential avenues for future research. One of the most intriguing of these characteristics is the fact that bats are notorious viral reservoirs (Calis her et at. 2006). Like viruses, DNA transposons are examples of genomic parasites, and the ability of bats to safely harbor large loads of a variety of viruses may suggest similar genetic tolerance with regard to DNA transposons. On the other hand, viruses remain the best candidates as potential vectors for the horizontal introduction of DNA transposons and other TEs (Miler and Miler 1982; Fraser et at. 1983; Jehle et at. 1998; Piskurek and Okada 2007). Thus, the propensity of bats to tolerate massive and diverse viral infections may have facilitated the recurrent horizontal introduction of DNA transposons and/ or their evolutionary persistence in vespertilonid bats. In conclusion, the data presented herein provides evidence that the previously identified trend toward DNA transposon extinction in mammals is not universal and that a wide diversity of DNA transposons have been active throughout the diversification of the vespertilionid bats, that is, within the last 40 Myr. Furthermore, it is proposed that the genus Myotis is an excellent candidate for studying the impact and influence of mobile elements, especially class 2 elements, on the evolution of genomic, species, and ecological diversity in mammals. Concomitantly, studies of bats may represent a unique opportunity to investigate lie history characteristics that make some organisms more susceptible to transposon activity than others.
Example 7. Modification of stem cell genomes
The ability to specifically modify the genomes of stem cells would be of great benefit in the treatment of human disease. In diseases that result from the lack of a particular gene product because of a defective gene, addition of an intact copy of the gene to stem cells could lead to the alleviation of disease upon reintroduction. Alternatively it may be useful to supplement stem cells with a gene product from a heterologous gene such that the modified cells would produce an agent that would kill other cells, for example an anti-tumor agent.
A powerful method for the introduction of new DNA into cells is via transposons. In particular, on class of such elements are cut and paste transposons: where an element- encoded transposase recognizes and binds to specific sites at the ends of the transposon and excises the transposon from that donor site and inserts it into a new target site. Upon integration, the element becomes stably associated with the host genome and can serve as a long-term source of an element-encoded therapeutic product. Such DNA cut and paste elements can be used for the modification of stem cell genomes. A general feature of transposable elements is that they insert in many different target sites. While this is a valuable property when such elements are used as insertional mutagens, such widespread insertion is a hazard in gene therapy. Insertion of even a therapeutic gene may inactivate a host gene, leading to a deleterious mutation. Such a consequence has already been realized: in a gene therapy trial for a SCID defect in France using retroviral vectors, although cured of the SCID disease, several subjects acquired leukemia because of retroviral insertion next to a cellular oncogenes. Thus, avoiding random mutation, i.e. having selective insertion of an element, in highly desirable.
The present inventors have generated "target site-specific transposons" by making chimeric transposases in which a highly target site specific DNA binding domain is fused to the transposase.
Figure 9 shows a representation of mammalian bat piggyBac helper plasmid which expresses the bat piggyBac transposase and corresponds to SEQ ID NO: 7.
Figure 10 shows a representation of mammalian bat piggyBac donor plasmid which encodes bat piggyBac tranposon and a drug resistance marker and corresponds to SEQ ID NO: 8.
An exemplary Mammalian bat piggyBac helper plasmid is shown below, and corresponds to SEQ ID NO: 7.
Figure imgf000051_0001
Figure imgf000052_0001
An exemplary Mammalian bat piggyBac donor plasmid is shown below, and corresponds to SEQ ID NO: 8.
Figure imgf000053_0001
Example 8. Hyperactive mammalian piggyBac variants The following experiments demonstrate bat piggyBac transposition in S. cerevisiae. A simple genetic assay was established for the excision of piggyBAT in yeast (Saccharomyces cerevisiae), using a modified version of the yeast URA3 gene as a transposon donor. In this modified URA3 gene, the yeast actin intron has been introduced into the URA3 gene to form a URA3::actin intron gene. The actin intron can be efficiently spliced from mRNA of this gene so that a strain carrying the URA3:: actin intron is a uracil prototroph. However, if a large DNA segment such as a several kb transposon is introduced into the actin intron, the resulting intron is too large to be spliced from mRNA, making the strain a uracil auxotroph. Thus excision of the transposon and restoration of the donor site to the parental URA3:: actin intron configuration can be followed by assaying for reversion of uracil auxotrophy to uracil prototrophy. The restoration of the gapped donor site in yeast could occur by end-joining or gene conversion using the chromosomal actin gene intron as a template.
In the yeast two plasmid piggyBAT system, a transposon donor plasmid contains a mini-piggyBAT transposon composed of 153 bp of the piggyBAT-L end and 208 bp of the piggyBAT-R end flanking a kanamycin resistance gene in the URA3::actin intron cassette. The transposase is supplied by a second helper plasmid containing the piggyBAT transposase gene under the galactose-inducible control of the GALS promoter.
In the absence of transposase, the frequency of URA3 reversion was very low, about 10-7 . Upon galactose induction of the piggyBAT transposase gene, however, the frequency of URA3 reversion was very much higher, about 10-3, indicating a high level of transposon excision. Considerable excision, i.e. uracil prototrophy, was observed even without galactose induction, indicating that the low level of transposase present because of leakiness of the GALS promoter can promote transposition.
This assay can be used for screening for hyperactive piggyBacs by random mutagenesis of the transposase gene and screening for an increased frequency of Ura+ reversion. Table 6, below, shows a list of nucleotide changes and the corresponding amino acid changes in 20 hyperactive piggyBacs identified using the present methods.
Figure imgf000054_0001
Mutant 2 T150C SILENT A351G SILENT
Mutant 3 A279G SILENT
Mutant 4 T1638C T546T
Mutant 5 A898G T300A
Mutant 6 A880G T294A G1558A A520T
Mutant 7 A687G SILENT G715A G239S
Mutant 8 T13C S5P C23T S8F
Mutant 9 G161A S54N
Mutant 10 G25A D9N
T1050C SILENT
A1356G SILENT
Mutant 11 A26G D9G A1033G I345V
Mutant 12 A1441G M481V Mutant 13 A32G EI lG
A389C K130T
Mutant 14 A32G EI lG
T1572A SILENT
Mutant 15 G456A SILENT
T1641C SILENT
Mutant 16 T1155C SILENT
Mutant 17 G1280^ R427H
Mutant 18 T22C S8P
Mutant 19 A106G S36G
Mutant 20 A29G DlOG
A106G S36G
C1137T SILENT
Methods The experiments described herein were performed with, but not limited to, the following methods and materials.
Transposon identification and consensus reconstruction and classification Initial computational searches were carried out from January through April 2007 against the M. lucifugus WGS data deposited at NCBI (http://www.ncbLnlm.nih.gov/,
GenBank accession no.AAPEOOOOOOOO; K. Lindblad-Toh, pers. comm.). At that time, the WGS data set represented -73% of the unassembled M. lucifugus genome with contig sizes averaging ~2.4 kb (Pritham and Feschotte 2007). The WGS data was queried using TBLASTN to detect the presence of coding sequences related to all known DNA transposon superfamilies. When significant hits were obtained (generally Evalue «10~5), complete sequence entries spanning the top 10-50 subject hits (depending on the overall abundance and sequence heterogeneity of the hits) were retrieved from the database and aligned using a local installation of MUSCLE (Edgar 2004) or CLUSTAL (Thompson et at. 1994). Sequences were trimmed to eliminate unalignable flanks and leave only TSDs, non coding regions, and the presumed coding regions of the elements. Complete alignments used to determine the full-length consensus sequence for each family are presented in Figure 8. Consensus sequences were derived from each multiple alignment based on a majority rule. For each consensus, coding sequences were predicted using ORF Finder (on the world wide web at ncbLnlm.nih.gov/projects/gorf/) and, if necessary, refined manually at ambiguous positions in the consensus using multiple alignments of protein sequences from individual copies. Unless previous nomenclature had been established, families and subfamiles were disclosed according to the standard principles of TE classification (Wicker et at. 2007). hAT3_ML was discovered independently using methods described in Arensburger et at. (2005). After its initial characterization, it was subjected to the same methods as all other elements described herein. All transposon consensus sequences used in the experiments described herein have been deposited in Repbase (on the world wide web at girinst.org/repbase/index.html).
Age estimation of M. ludfugus transposons Ages for each transposon family were estimated by extracting full or near full-length ORF's based on the coordinates of the repeats drawn from the output files of a locally implemented version of RepeatMasker 3.1.6. Additionally, it was hypothesized that nonautonomous subfamiles were likely mobilized by autonomous elements sharing the same TIRs, and thus were deposited during the same time period as the autonomous elements. Therefore, representatives were collected from a limited number of nonautonomous derivatives for all familes. All extracted elements were aligned with their respective consensus sequence using MUSCLE (Edgar 2004). The most appropriate model for estimating nucleotide divergence from each consensus was determined using MODELTEST v3.7 (Posada and Crandall 1998) and genetic distances were calculated in PAUP* v4.0bl0 (Swofford 2002). Specific neutral mutation rate estimates for bats are not currently available. Thus, published sequences from representative vespertilionid (Nycticeinops schlieffeni, Myotis tricolor, Scotophilus dinganii, Eptesicus hottentotus, Cistugo lesueuri, and Cistugo seabrai) and miniopterid (Minioptems natalensis, M. inflatus, M. fraterculus, M. australis, and M. macrocneme) taxa were used to obtain an estimate. Specifically, sequence data generated by Eick et al. (2005) was obtained for four introns (AJ865401-5, AJ865438-43, AJ865646-50, AJ865683-88, AJ866297-301, AJ866329-34, AJ866349-53, AJ866383-87). A concatenated alignment was constructed using MUSCLE (Edgar 2004) and the most appropriate evolutionary Primer model for the data was determined using MODELTEST v3.7 (Posada and Crandall 1998), TIM+G (r = 1.2597). The average genetic distance between families Miniopteridae and Vespertilionidae as defined by Eick et at. (2005) was determined using PAUP* v4.0bl0 (Swofford 2002). By incorporating the estimated divergence time between these two families, ~45 Mya (Eick et at. 2005; Miler- Butterworth et at. 2007), an estimated mutation rate of 2.366 X 10-9 was determined, which was then used to determine likely periods of activity. Representative alignments for several nonautonomous subfamilies are presented in Figure 8. Ages for each subfamily of elements were estimated using sequences as recovered from the genome and sequences from which the CpG dinucleotides had been removed (see Table 1). The relative ratio of CpG to non-CpG mutations were also estimated for each family (Table 2). This was accomplished using a modified Perl script originally designed to estimate CpG dinucleotide mutation rates in primate AIu elements (Xing et at. 2004) and modified for this work.
Nested insertion analysis
All RepeatMasker hits for each family of transposons were extracted and each was subjected to a second RepeatMasker analysis using a custom library that included each of the new transposable elements described here. Each nested insertion was validated manually by checking for the presence of the expected target-site duplications.
Identification of intact preintegration sites in M. austroriparius
To test the hypothesis that piggyBacl_ML has been recently active in Myotis, oligonucleotide primers were designed to survey the presence/absence of 15 individual insertions of a nonautonomous piggyBacl ML subfamily (npiggy_156) in M. austroriparius, which diverged from M. lucifugus -8-12 Mya (Stadelmann et at. 2007). Primers were designed in the genomic regions flanking the elements using sequence data from M. lucifugus and tested on a panel of 10 individuals from natural populations of M. austroriparius. Details on sample collection, DNA extraction, amplification, cloning, and sequencing are as previously described (Ray et at. 2007). Sequences from the two intact preintegration site amplicons shown in Figure 4 have been deposited in GenBank under accession nos. EU177095 and EU177096.
Figure imgf000059_0001
Taxonomic distribution of M. ludfugus transposons
To investigate the presence of related transposon families in other chiropteran or mammalian lineages, BLASTN or TBLASTN were used with nucleotide or amino acid sequence queries corresponding to each family of transposon in separate searches of custom databases representing the following taxa: Chiroptera (taxid:9397, excluding Myotis lucifugus), Xenarthra (taxid:9348), Afrotheria (taxid:311790), Laurasiatheria (taxid:314145), Euarchontoglires (taxid:314146), Mammalia (taxid:40674). The trace data from the sequencing efforts for the flying fox Pteropus vampyms (7,782,409 sequences averaging 970 bp) was also queried. Oligonucleotide primers complementary to portions of the coding region of each autonomous element (Table 4) were used to survey a taxonomically diverse panel of bat genomic DNAs (Table 5) via PCR. Reaction conditions were as described previously (Ray et al. 2007) and annealing temperatures for each primer pair are found in Table 4.
Figure imgf000060_0001
Phylogenetic analyses
Amino acid sequences for known transposases from each superfamily were obtained from database searches and aligned using MUSCLE (Edgar 2004). Phylogenetic analyses were conducted using MEGA 4 (Kumar et al. 2004). Neighbor-joining trees were constructed using the equal input model with 5000 bootstrap replicates.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. References:
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Pace II, J.K. and Feschotte, C 2007. The evolutionary history of human DNA transposons: Evidence for intense activity in the primate lineage. Genome Res. 17: 422-432.

Claims

What is claimed is:
1. A transposon comprising: a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposon activity.
2. The transposon of claim 1 , wherein the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae.
3. The transposon of claim 1 , wherein the mammalian piggyBac nucleic acid sequence is from the genus Myotis.
4. The transposon of claim 1 , wherein the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
5. The transposon of claim 1 , comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2.
6. The transposon of claim 1 , wherein the transposon is capable of inserting into the DNA of a cell.
7. The transposon of claim 1 , wherein the transposon further comprises a marker protein.
8. The transposon of claim 1 , wherein the transposon is inserted in a plasmid.
9. The transposon of claim 8 wherein the transposon further comprises at least a portion of an open reading frame.
10. The transposon of claim 8 wherein the transposon further comprises at least one expression control region.
11. The transposon of claim 8 wherein the expression control region is selected from the group consisting of a promoter, an enhancer or a silencer.
12. The transposon of claim 8 wherein the transposon further comprises a promoter operably linked to at least a portion of an open reading frame.
13. The transposon of claim 8, wherein the plasmid comprises SEQ ID NO: 7 or SEQ ID NO: 8.
14. The transposon of claim 5 wherein the cell is obtained from an animal.
15. The transposon of claim 17 wherein the cell is from a vertebrate or an invertebrate.
16. The transposon of claim 18 wherein the vertebrate is a mammal.
17. A transposase comprising: a mammalian piggyBac nucleic acid sequence and variants, derivatives and fragments thereof that retain transposase activity.
18. The transposase of claim 17, wherein the mammalian piggyBac nucleic acid sequence is from the family Vespertilionidae.
19. The transposase of claim 17, wherein the mammalian piggyBac nucleic acid sequence is from the genus Myotis.
20. The transposase of claim 17, wherein the mammalian piggyBac nucleic acid sequence is from the species Myotis lucifugus.
21. The transposase of claim 17, comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 5.
22. An amino acid sequence corresponding to the nucleic acid sequence of claim 21, wherein the amino acid sequence is selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 6.
23. A gene transfer system comprising: a transposon according to claim 1; and a mammalian piggyBac transposase according to claim 17.
24. The gene transfer system of claim 23 wherein the transposon is inserted into the genome of the cell.
25. The gene transfer system of claim 24, wherein the cell is obtained from an animal.
26. The gene transfer system of claim 24, wherein the cell is from a vertebrate or an invertebrate.
27. The gene transfer system of claim 24, wherein the vertebrate is a mammal.
28. A cell comprising the transposon of claim 1.
29. A pharmaceutical composition comprising: a transposon comprising a mammalian piggyBac nucleic acid sequence and a mammalian piggyBac transposase, together with a pharmaceutically acceptable carrier, adjuvant or vehicle.
30. A method for introducing exogenous DNA into a cell comprising:
contacting the cell with the gene transfer system of claim 26, thereby introducing exogenous DNA into a cell.
31. The method of claim 33, wherein the cell is a stem cell.
32. A kit comprising: a transposon comprising a mammalian piggyBac nucleic acid sequence and instructions for introducing DNA into a cell.
33. The kit of claim 32, further comprising a mammalian piggyBac transposase.
34. A hyperactive mammalian piggyBac transposon that has a higher level of transposon excision compared to a wildtype mammalian piggyBac transposon.
35. The mammalian piggyBac of claim 34, wherein the wildtype mammalian piggyBac transposon is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
36. The hyperactive transposon of claim 34, comprising a one or more nucleotide changes in SEQ ID NO 3 selected from the group consisting of: C41T, A1424G, C1472A, G1681A, T150C, A351G, A279G, T1638C, A898G, A880G, G1558A, A687G, G715A, T13C, C23T, G161A, G25A, T1050C, A1356G, A26G, A1033G, A1441G, A32G, A389C, A32G, A389C, A32G, T1572A, G456A, T1641C, Tl 155C, G1280A, T22C, A106G, A29G, and Cl 137T.
37. The hyperactive transposon of claim 34, comprising one or more amino acid changes in SEQ ID NO 4 selected from the group consisting of: A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, 1345 V, M481V, EI lG, K130T, G9G, R427H, S8P, S36G, DlOG, S36G, and silent.
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