# Patent

## Title
Synthetic mammalian retrotransposon gene

## Abstract

The invention relates to synthetic transposon and retrotransposon genes that exhibit higher levels of expression relative to natural transposon and retrotransposon genes. The invention further relates to transposons and retrotransposons comprising such synthetic genes.

## Background
This application claims the benefit of U.S. Provisional Application No. 60/473,658 filed on May 28, 2003, which is incorporated herein by reference in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 12, 2009, is named 61241716.txt, and is 127,114 bytes in size.
BACKGROUND OF THE INVENTION
Transposons are discrete mobile DNA segments that are common constituents of plasmid, virus, and bacterial chromosomes. These elements are detected by their ability to transpose self-encoded phenotypic traits from one replicon to another, or to transpose to a known gene and inactivate it. There are two types of transposons, ranging in size from about 750 to greater than 50,000 nucleotide base pairs. One type, known as the small insertion sequence or IS element, does not encode any known phenotypic traits. The other type encompasses relatively large units that do encode phenotypic traits such as antibiotic resistance (Plasmids and Transposons Environmental Effects and Maintenance Mechanisms; Edited by C. Stuttard and K. Rozee; Academic Press, New York; Pages 165-205). Transposons or transposable elements include a piece of nucleic acid bounded by repeat sequences. Active transposons encode enzymes that facilitate the insertion of the nucleic acid into DNA sequences.
In vertebrates, the discovery of DNA transposons, mobile elements that move via a DNA intermediate, is relatively recent (Radice, A. D., et al., 1994. Mol. Gen. Genet. 244, 606-612). Since then, inactive, highly mutated members of the Tc1/mariner as well as the hAT (hobo/Ac/Tam) superfamilies of eukaryotic transposons have been isolated from different fish species, Xenopus and human genomes (Oosumi et al., 1995. Nature 378, 873; Ivics et al. 1995. Mol. Gen. Genet. 247, 312-322; Koga et al., 1996. Nature 383, 30; Lam et al., 1996. J. Mol. Biol. 257, 359-366 and Lam, W. L., et al. Proc. Natl. Acad Sci. USA 93, 10870-10875).
Retrotransposons are naturally occurring DNA elements which are found in cells from almost all species of animals, plants and bacteria which have been examined to date. They are capable of being expressed in cells, can be reverse transcribed into an extrachromosomal element and reintegrate into another site in the same genome from which they originated.
Retrotransposons may be grouped into two classes, the retrovirus-like LTR retrotransposons, and the non-LTR elements such as human L1 elements, Neurospora TAD elements (Kinsey, 1990, Genetics 126:317-326), I factors from Drosophila (Bucheton et al., 1984, Cell 38:153-163), and R2Bm from Bombyx mori (Luan et al., 1993, Cell 72: 595-605). These two types of retrotransposon are structurally different and also retrotranspose using radically different mechanisms.
Unlike the LTR retrotransposons, non-LTR elements (also called polyA elements) lack LTRs and instead end with polyA or A-rich sequences. The LTR retrotransposition mechanism is relatively well-understood; in contrast, the mechanism of retrotransposition by non-LTR retrotransposons has just begun to be elucidated (Luan and Eickbush, 1995, Mol. Cell. Biol. 15:3882-3891; Luan et al., 1993, Cell 72:595-605). non-LTR retrotransposons can be subdivided into sequence-specific and non-sequence-specific types. L1 is of the latter type being found to be inserted in a scattered manner in all human, mouse and other mammalian chromosomes.
The L1 element (also known as a LINE) has been extremely successful at colonizing the human genome. Early approximations estimated that L1s are present at 100,000 copies in the human genome and comprise 5% of nuclear DNA (Fanning and Singer, 1987, Biochim Biophys Acta 910:203-121). However, recent studies suggest that as many as 850,000 L1s may exist in the human genome (Smit et al., 1996, Current Opinion in Genetics and Development). Most of these copies are truncated at the 5′ end and are presumed to be defective. Similar to full-length elements, the 5′ truncated copies are often flanked by short target site duplications (TSDs).
A 6.1 kb full-length L1 consensus sequence reveals the following conserved organization: a 5′ untranslated leader region (UTR) with an internal promoter; two non-overlapping reading frames (ORF1 and ORF2); a 200 bp 3′ UTR and a 3′ poly A tail. ORF1 encodes a 40 kd protein and may serve a packaging function for the RNA (Martin, 1991, Mol. Cell Biol. 11:4804-4807; Hohjoh et al., 1996, EMBO J. 15:630-639), while ORF2 encodes a reverse transcriptase (Mathias et al., 1991, Science 254:1808-1810). ORF1 and possibly ORF2 proteins associate with L1 RNA, forming a ribonucleoprotein particle. Reverse transcription by ORF2 protein may occur, resulting in L1 cDNAs, which are integrated into the genome (Martin, 1991, Curr. Opin. Genet. Dev. 1:505-508). Additionally, L1 elements are usually flanked by TSD's ranging from 7 to 20 bp. The full L1 and other non-LTR retrotransposons lack recognizable homologs of retroviral integrase, protease and RNase H. This group of elements employs a fundamentally different mechanism for transposition than the LTR-retrotransposons.
Some human L1 elements can retrotranspose (express, cleave their target site, and reverse transcribe their own RNA using the cleaved target site as a primer) into new sites in the human genome, leading to genetic disorders. For example, germ line L1 insertions into the factor VIII and dystrophin gene give rise to hemophilia A and muscular dystrophy, respectively (Kazazian et al., 1988, Nature 332:164-166; Narita et al., 1993, J. Clinical Invest. 91:1862-1867; Holmes et al., 1994, Nature Genetics 7:143-148), while somatic cell L1 insertions into the c-myc and APC tumor suppressor gene are implicated in rare cases of breast and colon cancer, respectively (Morse et al., Nature 333:87-90; Miki et al., 1992, Cancer Research 52:643-645). L1 retrotransposons account, directly or indirectly, for more than 30% of mammalian genomes by mass (Lander et al., 2001, Nature 409:860-921), by means of self-mobilization and trans-mobilization of Alu elements (Dewannieux et al., 2003, Nature Genet. 35:41-48). A full-length (about 6-kilobase) L1 consists of two open reading frames, ORF1 and ORF2, encode proteins for retrotransposition (Feng et al., 1996, Cell 87:905-916; Moran et al., 1996, Cell 87:917-927).
Thus, a highly active L1 element would be potentially useful as a tool for mammalian genetics.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the invention relates to a synthetic transposon gene. In another embodiment, the invention relates to a synthetic mammalian transposon gene.
We have now found that synthetic transposon and retrotransposon genes may be useful for gene therapy applications and for other genetic applications described herein.
In a further embodiment, the invention relates to a synthetic retrotransposon gene. An additional embodiment of the invention provides a synthetic mammalian retrotransposon gene.
In one embodiment, the invention relates to a synthetic ORF2 gene. In another embodiment, the invention relates to a synthetic mammalian ORF2 gene. The synthetic gene exhibits a higher level of expression relative to natural L1 retrotransposon ORF2 gene.
In one embodiment, the invention relates to a synthetic ORF1 gene. In another embodiment, the invention relates to a synthetic mammalian ORF1 gene. The synthetic gene exhibits a higher level of expression relative to natural L1 retrotransposon ORF1 gene.
In further embodiments, the invention relates to a synthetic ORF2 or ORF1 gene comprising human-associated codons. In additional embodiments of the invention, the synthetic ORF2 or ORF1 gene comprise only human-associated codons. In these embodiments, human-associated codons refer to those codons most frequently used in highly expressed mammalian genes. Of note, there are often two or more such codons encoding each amino acid, thus, many possible synthetic elements are possible.
In another embodiment, the invention provides a synthetic mammalian ORF2 gene comprising SEQ ID NO:1.
In another embodiment, the invention provides a synthetic mammalian ORF1 gene comprising SEQ ID NO:2.
In another embodiment, the invention provides a synthetic mammalian ORF2 gene comprising SEQ ID NO:3.
In another embodiment, the invention provides a synthetic mammalian ORF1 gene comprising SEQ ID NO:4.
In one embodiment, the invention relates to a transposon comprising a synthetic transposon gene. In another embodiment, the invention relates to a retrotransposon comprising a synthetic retrotransposon gene. In additional embodiments, the invention relates to an L1 retrotransposon comprising a synthetic ORF2 or ORF1 gene. In another embodiment, the invention relates to an L1 retrotransposon comprising a synthetic ORF2 and a synthetic ORF1 gene. Additional embodiments of the invention provide a mammalian L1 retrotransposon comprising a synthetic mammalian ORF2 gene, a synthetic mammalian ORF1 gene, or both a synthetic mammalian ORF2 and a synthetic mammalian ORF1 gene. The retrotransposon retrotransposes at least as efficiently as a native L1 retrotransposon. In a further embodiment of the invention, the retrotransposon retrotransposes more efficiently than a native L1 retrotransposon.
In another embodiment, the invention provides recombinant vector constructs comprising a synthetic ORF2 gene. In another embodiment, the invention provides recombinant vector constructs comprising a synthetic ORF1 gene. In another embodiment, the invention provides recombinant vector constructs comprising a synthetic ORF2 gene and a synthetic ORF1 gene. Eukaryotic cells transfected, transformed, or infected with such constructs are likewise provided.
In additional embodiments, the invention provides methods for preparing a synthetic ORF2 or ORF1 gene.
In another embodiment, the invention relates to a method of delivering a desired gene, or a biologically active fragment thereof, to the cells of a mammal. The method comprises the administration of a synthetic ORF2 or ORF1 gene, or a combination thereof and the desired gene to said mammal.
A further embodiment of the invention provides compositions comprising a cassette comprising a synthetic ORF2 or ORF1 gene, or a combination thereof, and a desired gene and a pharmaceutically acceptable carrier. In another embodiment, the invention relates to a method of treating a genetic disorder in a mammal. The method comprises administering a composition comprising a synthetic ORF2 or ORF1 gene or a combination thereof and a desired gene and a pharmaceutically acceptable carrier to a mammal having the genetic disorder in question.
In another embodiment, the invention relates to a method of identifying an uncharacterized gene, or a biologically active fragment thereof, in cells. The method comprises the administration of a synthetic ORF2 or ORF1 gene, or a combination thereof, and a detectable tag sequence, the identification of those cells expressing the tag sequence, and the isolation and characterization of DNA flanking the tag sequence.
Other embodiments of the invention are disclosed infra.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a schematically depicts the L1 structure. TSD refers to target site duplication; UTR refers to untranslated region.
FIG. 1b provides a schematic overview of the synthesis of the mammalian ORF2 gene. Oligonucleotides encoding each fragment are mixed in a PCR assembly reaction and subsequently used as template for amplification. Amplification products are cloned and ligated together with unique restriction sites (labelled A to J).
FIG. 1c shows a plasmid structure. The test sequences (lacZ, mORF2 or smORF2) are fused, in frame, downstream of the GFP ORF. An independent neo transcript is used to monitor transfection efficiency and loading. The short lines extending along parts of the plasmid represent probes used in FIG. 1d. 
FIG. 1d provides an analysis of smORF2 expression. Top: RNA expression of GFPlacZ, GFPmORF2 and GFPsmORF2. Middle: RNA expression of loading control. Bottom: protein expression of GFPlacZ, GFPmORF2 and GFPsmORF2.
FIG. 2a provides a schematic representation of a retrotransposition assay. The L1 element contains an intron-interrupted neo reporter in the 3′ untranslated region with its own promoter and polyadenylation signal. Wen neo is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become G418-resistant. The short lines extending along parts of the plasmid represent probes for RNA analysis. SD refers to the splice donor; SA refers to the splice acceptor.
FIG. 2b shows the results obtained when retrotransposition was assayed in HeLa cells (N=3). pTN201 contains only wild-type native mouse L1 sequence, and pTN203 contains wild-type native mouse L1 sequence with a D709Y reverse transcriptase point mutation. The average absolute number of colonies for pTN201 was 440 events per 106 transfected cells.
FIG. 3a shows the results of inverse polymerase chain reaction to amplify each complete insertion and flanking sequence. Primers flanking each insertion were used for amplification from G418-resistant clones. Odd-numbered lanes show that, for each primer pair, parental HeLa cells produced only empty site products. Even-numbered lanes show that the respective G418-resistant clones produced both empty site and filled smL1 insertion products of predicted sizes.
FIG. 3b provides characteristics of cloned insertions. TSD refers to target site duplication.
FIG. 3c depicts the structure and flanking sequence of cloned insertions schematically (nucleotides disclosed as SEQ ID NOS 412, 414, 416-418, 422-424, 428, and 430 respectively in order of appearance on the left side of the figure and nucleotides disclosed as 413, 415, 419-421, 425-427, 429, & 431 respectively in order of appearance on the right side of the figure). Insertion no. 8 contained an additional 7 bp (highlighted in blue) not found in the human genome sequence. Insertion no. 10 contained one untemplated base pair relative to the human genome sequence database followed by a 10-bp deletion (indicated in blue) immediately upstream of the L1 insertion. TSDs are highlighted in red, and presumptive endonuclease cleavage sites are underlined.
FIG. 4 depicts high-frequency retrotransposition in mouse cells: total RNA analysis of snL1 expression. Expression of native, partly synthetic, and completely synthetic mL1 was compared in HeLa cells.
FIG. 5a schematically depicts the conditionally activated mouse retrotransposon which only retrotransposes when exposed to the cre recombinase protein.
FIG. 5b shows results in tissue culture cells.
FIG. 6 lists SEQ ID NO:1 (synthetic murine ORF2).
FIG. 7 lists SEQ ID NO:2 (synthetic murine ORF1).
FIG. 8 lists SEQ ID NO:3 (synthetic human ORF2).
FIG. 9 lists SEQ ID NO:4 (synthetic human ORF1).
FIG. 10 lists the sequence of oligonucleotides used (SEQ ID NO:5-209) for the murine experiments.
FIG. 11 lists the sequence of oligonucleotides used (SEQ ID NO:210-411) for the human experiments.
FIG. 12 provides a sequence alignment of native mouse L1 with synthetic mouse L1. MacVector 6.5.3 (Oxford Molecular) was used to create a nucleic acid ClustalW alignment of native mouse L1 (SEQ ID NO: 432) and synthetic mouse L1 (SEQ ID NO: 433), starting at the ATG of ORF1 and ending at the stop codon of ORF2. For these sequences, the base composition of L1 spa is 41% A, 20% T, 21% C, 18% G. The base composition of smL1 is 27% A, 14% T, 33% C, 26% G. L1spa (Genbank accession #AF016099) was used as the sequence for native mouse L1. Identities are shaded in grey.



DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is a synthetic transposon gene. Further disclosed herein is a synthetic mammalian transposon gene. Additionally disclosed herein is a synthetic retrotransposon gene. Further disclosed herein is a synthetic mammalian retrotransposon gene.
A synthetic retrotransposon may retrotranspose at least as efficiently as a native retrotransposons in one embodiment. In another embodiment, the synthetic retrotransposon retrotransposes more efficiently than a native retrotransposon. In yet another embodiment, the synthetic retrotransposon of the invention retrotransposes about 2-fold more efficiently than a native retrotransposon. In yet another embodiment, the retrotransposon of the invention retrotransposes about 25-fold more efficiently than a native retrotransposon. In yet another embodiment, the retrotransposon of the invention retrotransposes about 100 to 200 fold more efficiently than a native retrotransposon.
A synthetic transposon may transpose at least as efficiently as a native transposons in one embodiment. In another embodiment, the synthetic transposon transposes more efficiently than a native retrotransposon. In yet another embodiment, the synthetic transposon of the invention transposes about 2-fold more efficiently than a native transposon. In yet another embodiment, the transposon of the invention transposes about 25-fold more efficiently than a native transposon. In yet another embodiment, the transposon of the invention transposes about 100 to 200 fold more efficiently than a native transposon.
Disclosed herein is a synthetic ORF2 gene. In one embodiment, the synthetic ORF2 gene of the invention is vertebrate, including fish, reptiles, amphibians, birds, insects, or the like. In one embodiment, the synthetic ORF2 gene of the invention is mammalian, including murine, bovine, equine or the like. One embodiment of the synthetic gene not only uses codons optimal for humans, but also destroys a nucleotide sequence bias that may be responsible for its poor expression.
In one embodiment, the synthetic ORF2 gene exhibits a higher level of expression relative to natural L1 retrotransposon ORF2 gene.
In another embodiment of the invention, the synthetic ORF2 gene encodes the same protein as does the native gene, yet comprises human-associated codons. In another embodiment of the invention, the synthetic ORF2 gene comprises only human-associated codons. “Human-associated codons” refer to those codons most frequently used in highly expressed mammalian genes. One of skill in the art would be able to determine which codons were most frequently associated with a particular gene. Of note, there are often two or more such codons encoding each amino acid, thus, many possible synthetic elements are possible. For example, human-associated codons for each amino acid may include: alanine GCC, arginine CGC or CGG, asparagine AAC, aspartate GAC, cysteine TGC, glutamine CAG, glutamate GAG, glycine GGC or GGG, histidine CAC, isoleucine ATC, leucine CTG or CTC, lysine AAG, methionine ATG, proline CCC, phenylalanine TTC, serine AGC or TCC, threonine ACC, tryptophan TGG, tyrosine TAC, and valine GTG or GTC.
In one embodiment, the synthetic ORF2 gene has a DNA sequence that has less than 100% identity with that of the natural L1 retrotransposon ORF2 gene. In another embodiment, the synthetic ORF2 gene has a DNA sequence that has no more than about 90% identity with that of the natural L1 retrotransposon ORF2 gene. In another embodiment, the synthetic ORF2 gene has a DNA sequence that has no more than about 80% identity with that of the natural L1 retrotransposon ORF2 gene.
In another embodiment, the synthetic mammalian ORF2 gene has a DNA sequence that has at least about 70% identity with SEQ ID NO:1. In yet another embodiment, the synthetic mammalian ORF2 gene of the invention comprises SEQ ID NO:1.
Further disclosed herein is a synthetic ORF1 gene. In another embodiment of the invention, the synthetic ORF1 gene of the invention is vertebrate, including fish, reptiles, amphibians, birds, insects, or the like. In another embodiment of the invention, the synthetic ORF1 gene of the invention is mammalian, including murine, bovine, equine or the like. In a further embodiment of the invention, the synthetic ORF1 gene exhibits a higher level of expression relative to natural L1 retrotransposon ORF1 gene.
In another embodiment of the invention, the synthetic ORF1 gene encodes the same protein as does the native gene, yet comprises human-associated codons. In another embodiment of the invention, the synthetic ORF1 gene comprises only human-associated codons.
In one embodiment, the synthetic ORF1 gene of the invention has a DNA sequence that has less than 100% identity with that of the natural L1 retrotransposon ORF1 gene. In another embodiment, the synthetic ORF1 gene of the invention has a DNA sequence that has no more than about 90% identity with that of the natural L1 retrotransposon ORF1 gene. In another embodiment, the synthetic ORF1 gene of the invention has a DNA sequence that has no more than about 80% identity with that of the natural L1 retrotransposon ORF1 gene.
In another embodiment, the synthetic mammalian ORF1 gene of the invention has a DNA sequence that has at least about 70% identity with SEQ ID NO:2. In yet another embodiment, the synthetic mammalian ORF1 gene of the invention comprises SEQ ID NO:2.
In another embodiment, the synthetic mammalian ORF2 gene of the invention has a DNA sequence that has at least about 70% identity with SEQ ID NO:3. In yet another embodiment, the synthetic mammalian ORF2 gene of the invention comprises SEQ ID NO:3.
In another embodiment, the synthetic mammalian ORF1 gene of the invention has a DNA sequence that has at least about 70% identity with SEQ ID NO:4. In yet another embodiment, the synthetic mammalian ORF1 gene of the invention comprises SEQ ID NO:4.
In one embodiment, the synthetic mammalian ORF2 gene of the invention is human. In another embodiment of the invention, the synthetic mammalian ORF1 gene of the invention is human.
A transposon comprising a synthetic transposon gene of the invention is provided in one embodiment. A retrotransposon comprising a synthetic retrotransposon gene of the invention is provided in another embodiment.
An L1 retrotransposon comprising a synthetic ORF2 gene of the invention is provided in an additional embodiment. A further embodiment provides an L1 retrotransposon comprising a synthetic ORF1 gene of the invention. Yet another embodiment provides an L1 retrotransposon comprising both a synthetic ORF2 gene of the invention and a synthetic ORF1 gene of the invention. The transposon or retrotransposon transposes or retrotransposes, respectively, at least as efficiently as a native transposon or retrotransposon in one embodiment. In another embodiment, the transposon or retrotransposon transposes or retrotransposes, respectively, more efficiently than a native transposon or retrotransposon.
A mammalian L1 retrotransposon comprising a synthetic mammalian ORF2 gene of the invention is provided in an additional embodiment. A further embodiment provides a mammalian L1 retrotransposon comprising a synthetic mammalian ORF1 gene of the invention. Yet another embodiment provides a mammalian L1 retrotransposon comprising both a synthetic mammalian ORF2 gene of the invention and a synthetic mammalian ORF1 gene of the invention. The mammalian retrotransposon retrotransposes at least as efficiently as a native L1 retrotransposon in one embodiment. In another embodiment, the mammalian retrotransposon retrotransposes more efficiently than a native L1 retrotransposon. In yet another embodiment, the retrotansposon of the invention retrotransposes about 2-fold more efficiently than a native L1 retrotransposon. In yet another embodiment, the retrotransposon of the invention retrotransposes about 25-fold more efficiently than a native L1 retrotransposon. In yet another embodiment, the retrotransposon of the invention retrotransposes about 100 to 200 fold more efficiently than a native L1 retrotransposon.
Recombinant vector constructs comprising a synthetic ORF2 gene of the invention are provided in an additional embodiment. Recombinant vector constructs comprising a synthetic ORF1 gene of the invention are provided in an additional embodiment. Recombinant vector constructs comprising both a synthetic ORF2 gene of the invention and a synthetic ORF1 gene of the invention are provided in an additional embodiment. The recombinant vector may be selected, without limitation, from chemical conjugates, viral (DNA or RNA) vectors, such as Epstein Barr virus (EBV), polyoma-based virus, adeno-associated virus, lentivirus, parvovirus, herpes simplex virus, retroviruses, poxviruses, and the like, fusion proteins, plasmids, and phage. It is not necessary that the vector sequences be limited to naturally occurring eukaryotic viral elements. Artificial chromosomes are also contemplated in the invention, including mammalian artificial chromosomes.
In one embodiment, the subject plasmid is pCEP4, with the native mORF2 sequence replaced with the DNA sequence of the synthetic mammalian ORF2 or ORF1 gene of the invention.
A recombinant vector construct according to the invention may exhibit a retrotransposition frequency that is more active than that observed for the native vector. In one embodiment, the construct of the invention exhibits a retrotransposition frequency that may be between about 2 to about 100 times more active than that observed for the native vector.
Eukaryotic cells transfected, transformed, or infected with recombinant vector constructs according to the invention are provided in another embodiment.
A method of preparing a synthetic ORF2 or ORF1 gene of the invention is provided. In one embodiment, the method comprises preparing a synthetic ORF2 or ORF1 gene of the invention that comprises “human-associated codons”. In another embodiment, the method comprises preparing a synthetic ORF2 or ORF1 gene of the invention that comprises only “human-associated codons”. In another embodiment, the method further comprises the step of incorporating unique restriction enzyme sites at intervals of between about 400 to about 600 base pairs. In yet another embodiment, the method further comprises the step of resynthesizing the 3′ untranslated region of the nucleotide sequence with more than one unique restriction enzyme sites, or using the 3′ UTR of a cellular gene. In yet another embodiment, the method further comprises the step of designing oligonucleotides of between about 30 base pairs to about 90 base pairs in length to cover both the sense and complementary strands of the sequence. In yet another embodiment, the method further comprises the step of employing the oligonucleotides to synthesize between about 6 to about 12 fragments of DNA, which are between about 400 to about 600 base pairs in length each. In yet another embodiment, the method further comprises the step of performing PCR employing approximately equimolar mixtures of the sense and antisense oligonucleotides corresponding to each of said fragments. In yet another embodiment, the method further comprises the step of subcloning the PCR-resultant fragments together using the unique restriction enzyme sites to generate the complete gene.
A synthetic ORF2 or ORF1 gene may be prepared comprising “human-associated codons.” The resulting nucleotide sequence may further be altered to incorporate unique restriction enzyme sites at approximately 500 base pair intervals. In addition, the 3′ untranslated region may be resynthesized with multiple unique restriction enzyme sites to facilitate subcloning. Oligonucleotides may be designed to completely cover both strands of this new sequence, for example oligonucleotides of between about 30 to about 90 bpase pairs may be designed. Hairpins in these oligonucleotides (as predicted, for example, by the computer program Oligo 5.0) with a melting temperature greater than approximately 65° C. may be destroyed with mutations that conserved the amino acid sequence. These mutations may change the optimal codon to a less optimal alternative.
The resulting optimized ORF2 or ORF1 nucleotide sequence may then be subjected to a modified Polymerase Chain Reaction (Stemmer et al., 1995, Gene 164:49-53). For example, oligonucleotides from between about 40 base pairs to about 80 base pairs, which cover the entire sense strand may be synthesized. The complementary strand may also be synthesized with oligonucleotides from between about 40 base pairs to about 80 base pairs, with approximately a 30 base pair stagger with respect to the sense strand oligonucleotides. Oligonucleotides may have between about 20 to about 50 base pairs of hybridization with two oligonucleotides from the complementary strand. Using these oligonucleotides, the ORF2 or ORF1 nucleotide sequence may be synthesized in between about 6 to about 12 fragments of between about 400 to about 600 base pairs each. The sense and antisense oligonucleotides corresponding to each fragment may be mixed in approximately equimolar amounts, and a PCR reaction may be performed. For example, a 25-cycle assembly PCR may be performed. Other PCR cycles may also be employed and those of skill in the art having the benefit of this disclosure would be able to determine the optimal number of cycles for a particular purpose. This assembly reaction may be diluted into the PCR reactions containing, for example, the outer-most oligonucleotides for the fragment as amplification primers.
The resulting fragments may be cloned, for example separately, into a plasmid, and approximately 20-30 clones may be sequenced for each. By standard subcloning procedures, mutations introduced during the oligosynthesis, assembly, or PCR may be removed. The resulting fragments may be subcloned together using the unique restriction enzymes sites previously engineered into the sequence, generating a complete optimized ORF2 or ORF1 nucleotide sequence in a plasmid.
A method of delivering a desired gene, or a biologically active fragment thereof, to the cells of a mammal, is likewise provided in an embodiment of the invention. In one embodiment of the invention, the desired gene is a therapeutic gene. The method comprises the administration of a synthetic ORF2 and/or ORF1 gene according to the invention and the desired gene to a mammal. Gene therapy methods are also contemplated according to embodiments of the invention. Genes that may be delivered via gene therapy by retrotransposition include, without limitation, Factor VIII, Factor IX, tyrosine hydroxylase, aromatic amino acid decarboxylase, apoptotic protease activating factor-1-dominant negative inhibitor (Apaf-1-DN), alpha-galactosidase A (AGA). Disorders and/or diseases that could be targeted via gene therapy by retrotransposition include, without limitation, hemophilia, Parkinson's disease, Fabry's disease, familial hypercholesterolemia, Gaucher's disease, Cystic Fibrosis, and adrenoleukodystrophy, adenosine deaminase deficiency (SCID), alpha-antitrypsin deficiency, Duchenne muscular dystrophy, pheylketouria, sickle cell anemia, Tay-Sachs disease, and the thalessemias.
Another embodiment provides a composition comprising a cassette comprising a synthetic ORF2 and/or ORF1 gene of the invention and a desired gene and a pharmaceutically acceptable carrier. In one embodiment, the cassette may comprise a heterologous or homologous DNA (non-L1 DNA) which may be inserted into the cell genome. This DNA may be positioned within the 3′ UTR sequences, or between the 3′ UTR and the polyA signal, and oriented such that expression of the DNA is under the control of a promoter. The type of DNA that may be inserted includes, without limitation, DNA which functions as a marker for identification of the site of insertion, for example, the neomycin (neo) resistance gene or other drug resistance genes (e.g., zeo, hygro, gpt), the green fluorescence protein (GFP) gene, lacZ, the herpes simplex virus (HSV) thymidine kinase gene, and even cell surface receptor genes such as, but not limited to, T cell receptor genes.
The route of administration of a composition according to the invention may also vary depending upon the disorder to be treated. The compositions may be administered to a subject in one of the traditional modes (e.g., orally, parenterally, transdermally or transmucosally), in a sustained release formulation using a biodegradable biocompatible polymer, or by on-site delivery using micelles, gels and liposomes, or rectally (e.g., by suppository or enema). In one embodiment, for treatment of patients having lung infection, the route of administration may be for example intranasal delivery by aerosol or via the blood. The appropriate pharmaceutically acceptable carrier will be evident to those skilled in the art and will depend in large part upon the route of administration.
In one embodiment of the invention, the promoter may be selected, without limitation, from RNA polymerase II promoters such as housekeeping promoters—actin, PGK, DNA polII or a ubiquitin promoter; tissue-specific promoters—the albumin, globin, ovalbumin promoter sequences, skin-specific promoters—K12 or K14, inducible promoters—steroid-inducible promoters, tetracycline-inducible promoters, and the like, and viral promoters—the SV40 early promoter, the Rous sarcoma virus (RSV) promoter and the cytomegalovirus immediate early promoter (CMV), as well as other retroviral LTRs and RNA polymerase III promoters such as an L1 element promoter, tRNA promoters and the 5S RNA promoter. The type of promoter which provides optimal expression of the desired DNA will depend on the desired result and will be apparent to the person of ordinary skill in the art.
To effect retrotransposition in a cell and, therefore, insertion of a desired DNA into the genome of a cell, a cassette comprising a synthetic ORF2 and/or ORF1 gene of the invention and a desired gene may be added to a population of cells in a composition suitable to effect uptake by the cells of the DNA. For example, for transfection of cells in vitro when the recombinant vector is in plasmid form, the cassette may be added to the cells in any number of formulations, including, but not limited to, a calcium phosphate transfection mixture, a liposome transfection formulation, and the like. Such types of transfection procedures are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.). The cassette may also be added to cells in the form of a virus which has been manipulated using recombinant DNA technology to accommodate the cassette, which is suitable for delivery of the cassette to the desired cells.
To direct insertion of DNA into inactive regions of host cell DNA, it may be possible to alter the ORF2 and/or ORF1 protein such that it cleaves host cell DNA at innocuous sequences, for example, in ribosomal RNA gene sequences. Alternatively, it may be possible to replace the endonuclease domain of ORF2 or ORF1 with the domain of another enzyme that specifically cleaves DNA at innocuous sequences. Such enzymes include, but are not limited to, the rep gene of adeno-associated virus and certain group I intron-encoded, site-specific endonucleases, e.g., the universal code equivalent of the yeast mitochondrial SCE1 gene.
In another embodiment, the DNA comprised in the cassette may be useful for the correction of a genetic defect in the cell into which the insertion is made. DNAs which can be used to effect correction of such genetic defects may be derived from, or comprise wild-type forms of genes which are mutated in the cell, thereby giving rise to the genetic defect. Such DNAs include, without limitation, any known or unknown DNA which can be used to correct a genetic defect in cells having such a defect. Examples of such DNA include cDNAs encoding the cystic fibrosis transmembrane conductance regulator (CFTR), cDNA encoding beta-globin, cDNA encoding blood clotting proteins, cDNA encoding enzymes such as, but not limited to adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase (HGPRT) and the like, cDNAs which play a role in cancer, such as, but not limited to, tumor suppressor genes, p53, p21, p16, retinoblastoma, Wilm's tumor, and the like, and also cytokines, interleukins and genes which encode therapeutic peptides, and the like.
Accordingly, in one embodiment, the invention provides a method of treating a genetic disorder in a mammal, wherein the method comprises administering a composition comprising a cassette comprising a synthetic ORF2 and/or ORF1 gene of the invention and a desired gene and a pharmaceutically acceptable carrier to the mammal having said genetic disorder.
In another embodiment, the invention relates to the correction of genetic defects in animals. Mammals and, even more preferably, humans, are contemplated in further related embodiments. To correct a genetic defect in an animal, the above-mentioned method of gene delivery is included in the invention that may be based upon delivery of a desired gene, or a biologically active fragment thereof, by retrotransposition, to the cells of an animal having the genetic defect. To effect retrotransposition in a cell in vivo in an animal for treatment of the animal, the cassette comprising a synthetic ORF2 and/or ORF1 gene of the invention, wherein the desired gene is administered to the animal using technology known in the art and described, for example, in WO 94 28938 and U.S. Pat. No. 5,240,846, each of which is hereby incorporated herein by reference. Upon administration to the animal in the compositions described herein, the cassette, which may be taken up by the target cells subsequently undergoes retrotransposition.
Genetic defects which may be corrected accordingly using retrotransposition include, without limitation, cystic fibrosis, mutations in the dystrophin gene, genetic defects associated with blood clotting, and any other either known or as yet unknown genetic defect (e.g., lysosomal storage diseases and other metabolic diseases).
In an additional embodiment, a method of identifying an uncharacterized gene, or a biologically active fragment thereof, in cells is provided, comprising the administration of a synthetic ORF2 and/or ORF1 gene according to the invention and a detectable tag sequence, the identification of those cells expressing the tag sequence, and the isolation and characterization of DNA flanking the tag sequence. In another embodiment, the method may include cloning genes that may be heretofore unknown or unclonable. The “tag” DNA may be selected, without limitation, from neoR, the GFP gene, lacZ, and the like.
Following retrotransposition of the tag DNA into cells, cells having the tag sequence inserted in the genome may be identified using any number of techniques that are well known in the art. For example, hybridization may be used wherein a probe comprising the tag DNA is used to identify cells having the tag DNA or RNA. Where the tag DNA is expressed as a protein, any number of immunological techniques may be used to identify cells expressing the tag protein. Such technology is well known in the art and is described, for example, in Sambrook et al. (supra). The DNA flanking tag DNA may then be isolated and cloned using ordinary technology described in, for example, Sambrook (supra), thereby effecting isolation and characterization of genes and regions of DNA which may be heretofore unknown.
“Retrotransposition” as used herein, includes the process of integration of a sequence into a genome, expression of that sequence in the genome, reverse transcription of the integrated sequence to generate an extrachromosomal copy of the sequence and reintegration of the sequence into the genome.
“Gene” as used herein, includes an actual gene including both the exons and introns of the gene.
“Heterologous DNA” as used herein, includes DNA which may not naturally be found in the cell into which it is inserted. For example, when mouse or bacterial DNA is inserted into the genome of a human cell, such DNA is referred to herein as “heterologous DNA.” In contrast, the term “homologous DNA” as used herein, denotes DNA that is found naturally in the cell into which it is inserted. For example, the insertion of mouse DNA into the genome of a mouse cell constitutes insertion of “homologous DNA” into that cell. In the latter case, it is not necessary that the homologous DNA be inserted into a site in the cell genome in which it is naturally found; rather, homologous DNA may be inserted at sites other than where it is naturally found, thereby creating a genetic alteration (a mutation) in the inserted site.
“Non-L1 DNA” as used herein, includes DNA which does not naturally occur in an L1 element.
It will be appreciated that embodiments of the invention should not be construed to be limited in any way to the precise DNA sequences which are disclosed herein. Homologous DNA sequences having substantially the same function as the disclosed DNA sequences are also considered to be included in the invention. Furthermore, it is recognized that gene sequences among closely related structures (such as retrotransposon ORFs, which have essentially the same function) may vary considerably, and that such sequences are likewise considered to be included in the invention.
As used herein, the term “homology” refers to the subunit sequence identity or similarity between two polymeric molecules e.g., between two nucleic acid molecules, e.g., between two DNA molecules, or two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two polypeptide molecules is occupied by phenylalanine, then they are identical at that position. The homology between two sequences, most clearly defined as the % identity, is a direct function of the number of identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length) of the positions in two polypeptide sequences are identical then the two sequences are 50% identical; if 70% of the positions, e.g., 7 out of 10, are matched or homologous, the two sequences share 70% identity. By way of example, the polypeptide sequences ACDEFG and ACDHIK share 50% identity and the nucleotide sequences CAATCG and CAAGAC share 50% identity.
“Complementary,” as used herein, refers to the subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. For example, when a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
As discussed herein, certain embodiments of the invention provide DNA encoding a protein product which may be used in gene therapy to correct a genetic defect in a cell. It should be understood that such a protein may comprise native polypeptide sequences, or may comprise modifications which render the protein in general more suitable as a gene therapy agent and more stable in a cell.
An additional embodiment of the invention provides for analogs of proteins or peptides encoded by a DNA sequence to be inserted into the genome of a cell. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups, but are not limited to these groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. In the latter instance, this is most applicable if reconstituted nucleic acid/protein particles are used for delivery. It is essentially and in vitro modification followed by an in vivo delivery.
Also included are polypeptides which have been modified using ordinary molecular biological techniques to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as therapeutic agents. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
The term “correction of a genetic defect” as used herein, includes the expression of a wild type gene product in a cell in an amount to restore normal function to the cell, which function was considered to be abnormal due to the genetic defect. The term also applies to situations wherein the genetic defect in the animal is corrected by delivering a wild type copy of the defective gene to a cell type other than the actual cell expressing the defective protein. Expression of the wild type copy of the gene in the other cells, and secretion of the wild type protein expressed therein may also serve to correct a genetic defect in the animal.
EXAMPLES
It should be appreciated that the invention should not be construed to be limited to the examples which are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
Example 1
smORF2 and ORF1 sequences were created by replacing each codon in the mouse L1 ORFs with the favored codons in highly expressed human genes (Haas et al., 1996, Curr. Biol. 6:315-324). The sequence was further altered with silent mutations introducing unique cleavage sites and eliminating potential hairpins that might have inhibited gene assembly. 60-mer oligonucleotides collectively encoding both strands of smORF2 were ordered from Qiagen, and gene synthesis (Stemmer et al., 1995, Gene 164:49-53) was performed on each ‘500-bp segment, as shown in FIG. 1b. 
Assembly reactions contained each primer at 30 nM and 1× ExTaq mix (Takara) in a total of 25 μl. Amplification reactions contained each outer primer at 0.5 μM, 2.5 μl assembly reaction, and 1× ExTaq mix in a total volume of 25 μl. PCR conditions were 94° C. for 4 min, 25 cycles of 94 ′C. for 30 s, 65 ′C. for 30 s, and 72 ′C. for 30 s, followed by 72 ′C. for 7 min. PCR products were cloned into pCRII with the TOPO-TA cloning kit (Invitrogen). A total of 24-48 clones were sequenced for each fragment, and mutations were removed by standard cloning techniques. Finally, synthesized fragments were ligated together in pBluescriptKS−. Oligonucleotide sequences used are shown in FIG. 10.
Fusion vectors were prepared (as shown in FIG. 1c), wherein the test sequences (lacZ, mORF2 or smORF2) are fused, in frame, downstream of the GFP ORF. An independent neo transcript is also inserted.
The expression of smORF2 was subsequently analyzed via immunoblot analysis. Cells were harvested in 5% SDS/PBS; this was followed by sonication. Total lysates were subjected to 7.5% SDS-polyacrylamide-gel electrophoresis and transferred to poly(vinylidene difluoride) (Amersham). Antibody incubations were performed in PBS containing 0.05% Tween-20 and 5% milk. Washes were performed in PBS, 0.1% Tween-20. Anti-GFP(FL) antibody (Santa Cruz) was used at 1:250 dilution. Anti-rabbit IgG (Amersham) was used at 1:5,000 dilution. Blots were developed with ECL-plus (Amersham).
In both human and mouse cells, transfection of GFPsmORF2 led to a massive increase in RNA compared with wild-type GFPmORF2 (FIG. 1d, top panel, lanes 3 and 4). The introduction of two mutations that abolish the endonuclease and reverse transcriptase activities of mORF2 provided a further slight increase in smORF2 RNA levels (FIG. 1d, top panel, lanes 5). Probing for the vector-encoded neo transcript showed that these increases in RNA were not due to differences in transfection efficiency or loading (FIG. 1d, middle panel). Immunoblotting these samples with anti-GFP (FIG. 1d, bottom panel) showed that protein levels were correlated with RNA increase, marking the first instance of the reproducible expression of detectable amounts of recombinant full-length ORF2 protein in a mammalian system.
Example 2
In order to determine whether the increased RNA levels led to altered retrotransposition efficiency, an established tissue culture assay, the retrotransposition assay (FIG. 2a), was used to measure relative retrotransposition frequencies in HeLa cells. The standard retrotransposition assay in HeLa cells was performed essentially as described in Moran et al., 1996, Cell 87:917-927. Transfected cells were selected with 200 μg ml−1 hygromycin for 10-12 days, then counted and seeded in 600 μg ml−1 G418 for 10 days. Colonies were stained with 0.4% Giemsa in PBS.
mORF2 was replaced with smORF2 in a fill-length mouse L1 to make a partly synthetic mouse L1 (psmL1). Because we were concerned that recoded mORF2 might lack potentially important cis-acting sequences required for retrotransposition (for example, an internal ribosomal entry site), we also constructed a partly synthetic version of ORF2 (psmL1-2) in which the first roughly 500 bp of mORF2 consisted of wild-type L1 sequence and the remainder was synthetic. In HeLa cells, both psmL1 and psmL1-2 were about 20-25-fold more active than wild-type mL1 (FIG. 2b). Synthesis and incorporation of a synthetic mORF1 (smORF1) and partly synthetic mORF1 variants led to further increases in retrotransposition, reaching a maximum of more than 200-fold increase over wild type (FIG. 2b) in the element with two fully synthetic ORFs.
The transient retrotransposition assays in HeLa, 3T3 and L cells were performed essentially as described in Wei et al., 2000, Anal. Biochem. 284:435-438. Each transposition construct was cotransfected with the GFP-expressing plasmid pTracerEF (Invitrogen) to normalize for transfection efficiency. At 24 h after transfection, cells were split 1:2, 1:20 and 1:200 into 100-mm dishes. At 36 h after transfection, the diluted cells were selected with G418 and the remaining cells were analyzed for GFP expression by flow cytometry to normalize for transfection efficiency. 3T3 cells were selected in 1 mg ml−1 G418; L cells were selected in 400 μg ml−1 G418. Colonies were stained with 0.4% Giemsa or 0.5% Coomassie brilliant blue.
With the use of the transient assay, synthetic mouse L1 (pCEPsmL1) retrotransposition frequency was compared with that of wild-type native human L1 and wild-type native mouse L1 (N=3). The average absolute numbers of colonies of pJM101L1rp (colonies per 106 transfected cells) for HeLa, 3T3 and L cells were 2,904, 108 and 1,568, respectively.







TABLE 1







high-frequency retrotransposition in mouse cells









Relative transposition



frequency










Plasmid
HeLa
3T3
L













pCEP4 (empty vector)
0
0
0


pTN201 (native mouse wild-type)
<0.005
<0.002
<0.002


pTN203 (native mouse mutant)
0
0
0


pJM101L1 (native human wild-type)
0.13
0.017
0.07


pCEPsmL1 (synthetic mouse wild-type)
1
1
1


pCEPsml1mut2 (synthetic mouse mutant)
0
0
<0.002









Example 3
To verify that the smL1 G418-resistant colonies resulted from authentic L1 retrotransposition, we characterized six smL1 insertions. The mutant loci were identified by inverse polymerase chain reaction (PCR), enabling the amplification of each complete insertion and flanking sequence. For each primer pair, parental HeLa cells produced only empty site products (FIG. 3a, odd-numbered lanes), whereas the respective G418-resistant clones produced both empty site and filled smL1 insertion products of predicted sizes (FIG. 3a, even-numbered lanes).
Amplicons were cloned and sequenced to determine their general structures and genomic flanks. Integration sites were determined by inverted PCR essentially as described in Morrish et al., 2002, Nature Genet. 31:159-165. Genomic DNA (5 μg) from each clone was digested with EcoRI, inactivated by heat, diluted to 1 ml and ligated overnight, precipitated with ethanol, resuspended in 30 μl water and subjected to two rounds of inverted PCR with oligonucleotides JB6466/JB6467 (round 1) and JB6468/JB6469 (round 2). Sequencing with JB3529, JB3530 and JB3531 identified the 30 flanking sequences. Primers based on flanking sequence were used to amplify intact smL1 insertions, which were subsequently sequenced.
As summarized in FIG. 3b, all amplicons showed a properly spliced neo gene, a poly(A) tail, and most (five of six) had target site duplications 5-108 bp long. Insertion no. 10 had a 10-bp target deletion and insertion no. 18 had a 5′ L1 inversion, features commonly found in L1 insertions.
In addition, various chromosomes served as targets, and the endonuclease cleavage sites inferred from target site duplications matched the previously reported degenerate consensus (5′-TTTT/AA-3′ on the bottom strand) (FIG. 3c).
Example 4
The activity of the synthetic mouse L1 retrotransposons was compared with wild-type human and mouse L1 in mouse cells. Because episomal plasmids used to introduce marked retrotransposons do not replicate efficiently in mouse cells, we used a transient retrotransposition assay in 3T3 and L cells. We also performed the transient assay in HeLa cells, verifying the relative retrotransposition frequencies obtained with the standard assay (compare pTN201 and pCEPsmL1 from FIG. 2b and table 1). The synthetic mouse L1 (pCEPsmL1) underwent retrotransposition at much higher frequencies (more than 200-fold) than its wild-type counterpart in mouse cells.
In addition, we compared smL1 with a human L1 (pJM101L1rp), because L1rp has previously been used to generate transgenic mouse lines and, thus, serves as a benchmark for retrotransposition frequencies in mice. smL1 was significantly more active than L1rp in all cell types tested, making it the most active L1 element known so far. Introducing catalytic mutations into smL1 to produce smL1mut2 essentially abolished retrotransposition.
Example 5
Northern blot analysis of wild-type full-length mL1 and its synthetic counterparts was performed. Total RNA was isolated with TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Total RNA (6 μg) from each sample was treated with 10 units of DNase I for 15 min at 37 ′C., then run on a 0.8% agarose/formaldehyde gel, blotted overnight to a Genescreen plus nylon membrane (NEN) in 10×SSC, and crosslinked by ultraviolet radiation. Prehybridizations and hybridizations were both performed in ULTRAhyb (Ambion) at 42° C. The following [′y-32P]ATP end-labeled oligonucleotides were used as probes: GFP probe, JB4057; GFP plasmid neo probe, JB4059; transposition plasmid neo probe, JB4541; hyg probe, JB6341. Washes were performed in 2×SSC, 0.1% SDS and in 0.2×SSC, 0.1% SDS. Radioactive signal was detected with Fuji imaging plates and a Fuji scanner (BAS-1500). For subsequent reprobing, membranes were stripped with three 10-min washes in boiling 0.1×SSC, 1% SDS.
The Northern blot analysis of wild-type full-length mL1 and its synthetic counterparts revealed that increasing lengths of synthetic L1 sequence led to increasing full-length L1 RNA levels (FIG. 4). pCEPsmL1mut2 was used in place of pCEPsmL1, because pCEPsmL1 was difficult to maintain episomally, as determined by the hygro transfection/loading control (data not shown). The intact pCEPsmL1 plasmid is not maintained in transfected cells for long periods.
Example 6
A conditionally activated version synthetic mouse retrotransposon which only retrotransposes when exposed to the cre recombinase protein was constructed (FIG. 5a). A construct was created in which the synthetic retrotransposon is inactivated by an intervening reporter gene (such as β-GEO), operationally joined to sequences leading lead to premature RNA truncation. This cassette is flanked by the loxP recombination sited. When this DNA segment is in the presence of cre protein (provided in vitro, via co-transfection, or via cross-breeding to a cre-expressing transgenic mouse), cre will effect recombination at the loxP sites, deleting β-geo and activating the synthetic retrotransposon.
The reporter for retrotransposition is a UV-excited green fluorescent protein (gfp), which allows the tracking of new mutations visually by simply shining ultraviolet light on them. This is shown in FIG. 5b. The inactivated and activated DNA constructs (depicted in FIG. 5a) were transfected into HELA cells, and the phenotypes were assessed by X-gal staining for β-geo expression and fluorescence microscopy for transposition (gfp activity).
Example 7
A human version of a synthetic L1 element was synthesized using the same methods as described above. This element was based on the amino acid sequence of a consensus human L1 element (Brouha et al., 2003, Proc Natl Acad Sci USA 100:5280-5285). ORF1 and ORF2 were recoded, as well as the interORF region and 3′UTR, generating a completely synthetic human L1.
Retrotransposition activity was measured and revealed a several-fold increase in activity in comparison to native human L1.
The disclosures of each and every patent, patent application and publication cited herein are hereby incorporated herein by reference in their entirety.
While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

## Claims

1. A synthetic transposon gene comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

2. A transposon comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.