Inducible expression systems

Abstract

The invention provides inducible promoter systems and their components incorporating components of a tetracycline operon, cumate or lactose. By coordinating expression of different transcriptional units in these systems as a result of selection of promoters and / or linking the units into the same DNA molecule, these systems can achieve higher levels of expression of coding segments of interest, increased differential levels of expression between on- and off-states, and / or greater responsiveness to inducing agents than conventional systems.

Description

[0001] INDUCIBLE EXPRESSION SYSTEMS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] The application claims the benefit of US 63 / 656,026, filed June 4, 2024, US 63 / 623,741 filed January 22, 2024, and US 63 / 585,365 filed September 28, 2023, each incorporated by reference in its entirety for all purposes.

[0004] REFERENCE TO A SEQUENCE LISTING

[0005]

[0002] The application refers to sequences disclosed in a file named 618882SEQLST.XML of 613,400 bytes created September 18, 2024, incorporated by reference in its entirety for all purposes.

[0006] BACKGROUND

[0007]

[0003] The tet operon confers tetracycline resistance in bacteria. The operon includes tet operator sites, a promoter, and a bicistronic transcriptional unit encoding a tet repressor and a tetracyclineresistance protein. In the absence of tetracycline, the tet repressor binds to the tet operator sites inhibiting expression from the promoter of both the tet repressor and tetracycline-resistance protein. When tetracycline is present it binds to the tet repressor inhibiting its own binding to the tet operators. Tetracycline-resistance protein is then expressed as is more tet repressor, which again shuts downs expression of the tet operon when the tetracycline has been exhausted.

[0008]

[0004] The components of the tet operon have been incorporated into various inducible expression systems for regulating expression of a coding segment of interest (see, e.g., US Nos. 5814618, 5654168, 5650298 and 5,464,758 and 9181556). Generally, such systems have two components. A first construct includes one or more tet operators, a promoter and a coding segment of interest. A second construct includes a promoter and a tet repressor (or modified form thereof), sometimes fused to a transcriptional activation domain. The presence of tetracycline controls binding of the tet repressor to the tet operators, and consequently expression of the coding segment.

[0009]

[0005] Pseudomonas putida Fl utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate. Upstream of the cym operon is a regulatory gene, cymR. cymR binds to an operator repressing expression of both cym and cmt operons. Repression is relieved by cumate binding to the repressor.

[0006] The lactose (Lac) operon is an operon required for transport and metabolism of lactose in E. coli and other bacteria. The operon includes three structure genes, LacZ, LacY and LacA, a promoter, terminator, regulator and operator. LacZ encodes bet-galactosidase an enzyme that cleaves lactose into glucose and galactose. LacY encodes beta-galactoside permease a transporter that pumps lactose into a cell, and LacA encodes a beta-galactoside transacetylase, which transfers an acetyl group from acetyl- CoA to thioacetamide.

[0010]

[0007] A first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called the lactose repressor to hinder production of P-galactosidase in the absence of lactose. The Lacl gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive). If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the lac operator. The repressor binding to the operator interferes with binding of RNA polymerase to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels. When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose, made from lactose by the product of the lacZ gene, binds to the repressor, causing an allosteric shift. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.

[0011]

[0008] The second control mechanism is a response to glucose, which uses the catabolite activator protein (CAP) homodimer to greatly increase production of p-galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence about 60 bp upstream of the transcription start site), which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of P-galactosidase, enabling the cell to hydrolyze lactose and release galactose and glucose.

[0012]

[0009] Related application W02022 / 204067 is incorporated by reference in its entirety for all purposes.

[0013] SUMMARY OF THE CLAIMED INVENTION

[0014]

[0010] The invention provides a dual inducible expression system comprising:

[0015] (a) a minimal promoter in operable linkage with first and second operator segments, and a heterologous coding segment; (b) a first promoter operably linked to a segment encoding a modified repressor linked to a transcriptional activation domain, which binds to the first operator segment in the presence of a first inducer inducing expression of the heterologous coding segment; and

[0016] (c) a second promoter operably linked to a segment encoding a repressor, which binds to the second operator segment in the absence a second inducer repressing transcription of the heterologous coding segment; wherein in the presence of the first and second inducer expression of the heterologous coding segment is induced.

[0017] [Oil] Optionally, (a), (b) and (c) are separate transcriptional units. Optionally, the first operator segment is 5' to the minimal promoter. Optionally, the second operator segment is about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

[0018]

[0012] Optionally, the first and / or segment operator segment comprises an array of operators separated by spacers.

[0019]

[0013] Optionally, the first and second inducers can be the same or different.

[0020]

[0014] Optionally, the first and second promoter are the same type of promoter.

[0021]

[0015] Optionally, the repressor is any of a tet repressor, cumate repressor or lac repressor and the modified repressor is any of a modified tet repressor, modified cumate repressor or modified lac repressor.

[0022]

[0016] The invention further provides a contiguous nucleic acid comprising a dual inducible expression system as described above.

[0023]

[0017] Optionally, the contiguous nucleic acid is a transposon in which the dual inducible expression system is flanked by inverted repeats of the transposon.

[0024]

[0018] The invention further provides a cell comprising the contiguous nucleic acid.

[0025]

[0019] The invention further provides a method of inducing expression of a heterologous coding segment, comprising culturing a cell comprising the dual expression system as described above; and supplying the first and second inducers to the culture, wherein expression is induced.

[0026]

[0020] The invention further provides a transposon comprising a first transcriptional unit comprising a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least two tet operators, and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a tet repressor effective to bind a tet operator in the absence of tetracycline or doxycycline or a modified-tet repressor effective to bind a tet operator in the presence of tetracycline or doxycycline, wherein the tet repressor or the modified tet repressor is fused to a transcriptional activation domain, and the first and second transcriptional units are flanked by inverted repeats of the transposon.

[0027]

[0021] The invention further provides a transposon comprising a first transcriptional unit comprising a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least one cumate operator and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a cumate repressor effective to bind a cumate operator in the absence of cumate or encoding a modified cumate repressor effective to bind a cumate operator in the presence of cumate, wherein the cumate repressor or the modified cumate repressor is fused to a transcriptional activation domain and the first and second transcriptional units are flanked by inverted repeats of the transposon.

[0028]

[0022] Optionally, the second transcriptional unit further comprises a polyadenylation sequence. Optionally, the tet repressor has an amino acid sequence comprising SEQ ID NO:5. Optionally, the modified tet repressor has an amino acid sequence comprising SEQ ID NO:6.

[0029]

[0023] Optionally, the cumate repressor has an amino acid sequence comprising SEQ ID NO:170. Optionally, the modified cumate repressor linked to the transcriptional activation domain has an amino acid sequence comprising SEQ ID NO:172.

[0030]

[0024] Optionally, the minimal chimpanzee promoter is operably linked to two or more heterologous coding segments separated by an IRES sequence or sequence encoding a CHYSEL peptide. Optionally, the two or more heterologous coding segments encode two or more proteins of a viral packaging system.

[0031]

[0025] Optionally, the two or more heterologous coding segments encode each required protein of a viral packaging system. Optionally, the transposon is a piggyBac or piggyBac-like transposon.

[0032]

[0026] The invention further provides a cell transformed with a transposon.

[0033]

[0027] Optionally, the cell is mammalian.

[0034]

[0028] The invention further provides a non-human animal transformed with a transposon.

[0035]

[0029] Optionally the animal is transgenic.

[0036]

[0030] The invention further provides a cell or nonhuman transgenic animal having a genome comprising a transposon comprising (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least two tet operators and a heterologous coding segment encoding a protein of viral packaging system, and (b) a promoter operably linked to a tet repressor fused to a transcriptional activation domain or modified tet repressor fused to transcriptional activation domain, wherein expression of the heterologous coding segment can be regulated by supplying tetracycline or doxycycline, or other tetracycline analog to the cell or nonhuman transgenic animal, wherein (a) and (b) are flanked by inverted repeats of the transposon.

[0031] The invention further provides a cell or nonhuman transgenic animal having a genome comprising a transposon comprising (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least one cumate operator and a heterologous coding segment encoding a protein of a viral packaging system, and (b) a promoter operably linked to a segment encoding cumate repressor linked to a transcriptional activation domain or a modified cumate repressor fused to transcriptional activation domain, wherein expression of the heterologous coding segment can be regulated by supplying cumate to the cell or nonhuman transgenic animal, wherein (a) and (b) are flanked by inverted repeats of the transposon.

[0037]

[0032] The invention further provides a method for inducible expression of a heterologous coding segment comprising; providing a first transcriptional unit comprising in operable linkage with a least two tet operators, a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a tet repressor fused to a transcriptional activation domain or a modified tet repressor fused to a transcriptional activation domain, wherein the tet repressor is expressed and in the absence of tetracycline or doxycycline, the tet repressor fused to the transcriptional activation domain binds to the at least two tet operators and expression of the heterologous coding segment is increased, or the modified tet repressor fused to the transcriptional activation domain is expressed and in the presence of tetracycline or doxycycline, the modified tet repressor fused to the transcriptional activation domain binds to the at least two tet operators and expression of the heterologous coding segment is increased, wherein the first and second transcriptional units are flanked by inverted repeats of the transposon.

[0033] The invention further provides a method for inducible expression of a heterologous coding segment comprising; providing a first transcriptional unit comprising in operable linkage with a least one cumate operator, a promoter having a nucleotide sequence comprising SEQ ID NO:24 and a heterologous coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a cumate repressor fused to a transcriptional activation domain or a modified cumate repressor fused to a transcriptional activation domain, wherein the cumate repressor fused to the transcriptional activation domain is expressed and in the absence of cumate the cumate repressor binds to the at least one cumate operator and expression of the heterologous coding segment is increased, or the modified cumate repressor fused to the transcriptional activation domain is expressed and in the presence of tetracycline or doxycycline the modified cumate repressor binds to the at least one cumate operator and expression of the heterologous coding segment is increased, wherein the first and second transcriptional units are flanked by inverted repeats of the transposon.

[0038]

[0034] Optionally in such methods, the first and second transcriptional units are components of the same contiguous DNA molecule.

[0039]

[0035] Optionally, the transposon is a piggyBac or piggyBac-like transposon.

[0040]

[0036] Optionally, the methods further comprise introducing the transposon into a cell.

[0041]

[0037] Optionally, the cell is a mammalian cell. Optionally, the first and second transcriptional units integrate into the genome of the cell.

[0042]

[0038] Optionally, the method further comprises culturing the cell. Optionally, the method further comprises supplying tetracycline or doxycycline or other tetracycline analog to culture media of the cell.

[0039] Optionally, the method further comprises supplying cumate or an analog to culture media of the cell.

[0043]

[0040] The invention further provides a system for regulating expression, comprising:

[0044] (a) a first transcriptional unit comprising a minimal promoter operably linked to at least two lac operators and a heterologous coding segment and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a modified lac repressor fused to a transcriptional activator, wherein in the presence of lactose or an analog thereof, the modified la - repressor fused to a transcriptional activator binds to the at least two lac operators and induces expression of the heterologous coding segment.

[0045]

[0041] Optionally, the promoter has a sequence comprising SEQ ID NO:24 and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with SEQ ID NO:24 is naturally associated.

[0042] Optionally, the promoter lacks a 5' flanking sequence from a natural chimpanzee CMV promoter with which it is naturally associated. Optionally, the first and second lac operators are 5' to the promoter.

[0046]

[0043] Optionally, the first transcriptional unit comprises three, six or eight lac operators 5' to the promoter.

[0047]

[0044] Optionally, the three, six or eight lac operators are separated by spacers of 10-25 nucleotides.

[0048]

[0045] Optionally, at least some of the spacers differ from one another.

[0049]

[0046] Optionally, the first transcriptional unit further comprises a segment encoding a 5' UTR.

[0050]

[0047] Optionally, the segment encoding the 5' UTR has a sequence comprising SEQ ID NO:29.

[0051]

[0048] Optionally, the second transcriptional unit further comprises a polyadenylation sequence.

[0052]

[0049] Optionally, the heterologous coding segment encodes a protein.

[0053]

[0050] Optionally, the protein is a protein of a viral packaging system.

[0054]

[0051] Optionally, the minimal promoter is operably linked to two or more heterologous coding segments separated by an IRES sequence or sequence encoding a CHYSEL peptide.

[0055]

[0052] Optionally, the two or more heterologous coding segments encode two or more proteins of a viral packaging system.

[0056]

[0053] Optionally, the two or more heterologous coding segments encode each required protein of a viral packaging system.

[0057]

[0054] Optionally, the protein is a therapeutic protein or a membrane protein.

[0058]

[0055] Optionally, the first and second transcriptional units are with the same contiguous nucleic acid. The invention further provides a transposon comprising a transcriptional unit as described above flanked by inverted repeats of the transposon.

[0059]

[0056] Optionally, the transposon further comprises target sites flanking the inverted repeats. Optionally, the transposon is a piggyBac or piggyBac-like transposon.

[0060]

[0057] The invention further provides a cell transformed with an expression system or transposon as described above.

[0061]

[0058] Optionally, the cell is mammalian. The invention further provides a non-human animal transformed with an expression system or transposon as described above.

[0062]

[0059] Optionally, the non-human animal is transgenic.

[0063]

[0060] The invention further provides a cell or nonhuman transgenic animal having a genome comprising (a) a minimal promoter operably linked to at least two lac operators and a heterologous coding segment, and (b) a promoter operably linked to modified lac repressor fused to transcriptional activator, wherein expression of the coding segment can be induced by supplying lactose or an analog thereof to the cell or nonhuman transgenic animal.

[0064]

[0061] The invention further provides a method for inducible expression of a heterologous coding segment comprising providing a first transcriptional unit comprising a minimal promoter operably linked to a least two lac operators, and a heterologous coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding modified lac repressor fused to a transcriptional activator, wherein the modified lac repressor fused to the transcriptional activator is expressed and in the presence of lactose or an analog thereof, the modified lac repressor fused to the transcriptional activator binds to the at least two lac operators and expression of the heterologous coding segment is increased.

[0065]

[0062] Optionally, in the above methods, the first and second transcriptional units are components of the same contiguous DNA molecule.

[0066]

[0063] Optionally, the first and second transcriptional units are components of a transposon. Optionally, the transposon is a piggyBac or piggyBac-like transposon.

[0067]

[0064] Optionally, the method further comprises introducing the contiguous DNA molecule into a cell.

[0068]

[0065] Optionally, the cell is a mammalian cell.

[0069]

[0066] Optionally, the first and second transcriptional units integrate into the genome of the cell.

[0070]

[0067] Optionally, the method further comprises culturing the cell.

[0071]

[0068] Optionally, the method further comprises supplying lactose or an analog to culture media of the cell.

[0072]

[0069] The invention further provides a nucleic acid for expression of adeno-associated virus proteins VP1, VP2 and VP3 comprising overlapping coding segments for VP1, VP2 and VP3 operably linked to a promoter, wherein the initiation codon for the segment encoding VPl is CUG.

[0073] BRIEF DESCRIPTION OF THE FIGURES

[0074]

[0070] Fig. 1 shows alignments of mouse (SEQ. ID NO:16) , human (SEQ ID NO:13) and hybrid (SEQ ID NO:12) CMV promoters. The transcriptional start site is indicated as the underlined G labelled +1. The methylation-sensitive CG dinucleotide in the human sequence is underlined and labelled CpG -179. The TATA boxes of each promoter is underlined.

[0071] Figs. 2 and 3 show first and second transcriptional units respectively for a system for tet inducible expression of an open reading frame ("tet off").

[0075]

[0072] Fig. 4 shows a first transcriptional unit of a different system for tet inducible expression of an open reading frame ("tet on"}.

[0076]

[0073] Figs. 5 and 6 show first and second transcriptional units respectively for a system for cumate inducible expression of an open reading frame ("cumate off").

[0077]

[0074] Fig. 7 shows a first transcriptional unit of a different system for cumate inducible expression of an open reading frame ("cumate on").

[0078]

[0075] Figs. 8A, B show a lac / cumate dual expressible system in (A) absence and (B) presence of lactate / cumate.

[0079]

[0076] Figs. 9A, B show a lac / tet dual expressible system in (A) absence and (B) presence of lactate and tetracycline.

[0080]

[0077] Figs. 10A, B show a lac / lac dual expressible system in (A) absence and (B) presence of lactose.

[0081]

[0078] Figs. 11A, B show a cumate / lac dual expressible system in (A) absence and (B) presence of cumate / lactose.

[0082]

[0079] Figs. 12A, B show a cumate / tet dual expressible system in (A) absence and (B) presence of cumate / tetracycline.

[0083]

[0080] Figs. 13A, B show a cumate / cumate dual expressible system in (A) absence and (B) presence of cumate.

[0084]

[0081] Figs. 14A, B show a tet / lac dual expressible system in (A) absence and (B) presence of tetracycline / lactose.

[0085]

[0082] Figs. 15A, B show a tet / cumate dual expressible system in (A) absence and (B) presence of tetracycline / cumate.

[0086]

[0083] Figs. 16A, B show a tet / tet dual expressible system in (A) absence and (B) presence of tetracycline.

[0087]

[0084] Fig. 17 shows an alignment of minimal CMV promoters. Bold text indicates bases that may be removed to fuse the minimal promoter to an operator sequence to impose a dual control.

[0088]

[0085] Figs. 18A, B show a tet / tet dual expressible system with an additional tet operator placed between the promoter and its operably linked modified repressor-transcriptional activator fusion in (A) absence and (B) presence of tetracycline.

[0086] Figs. 19A, B show Western blots of AAV proteins expressed from IPTG and doxycycline inducible systems.

[0089]

[0087] Figs. 20A, B shows a tet single control expressible system in (A) absence and (B) presence of tetracycline.

[0090]

[0088] Fig. 21 shows expression of AAV2 capsid proteins VP1, VP2 and VP3.

[0091] DEFINITIONS

[0092]

[0089] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of nucleic acids, reference to "a substrate" includes a plurality of such substrates, reference to "a variant" includes a plurality of variants, and the like.

[0093]

[0090] Terms such as "connected," "attached," "linked," and "conjugated" are used interchangeably to encompass direct as well as indirect connection, attachment, linkage or conjugation unless the context indicates otherwise.

[0094]

[0091] When a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. When a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. When a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention. When a combination is disclosed, each sub combination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, when different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. When any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0095]

[0092] Unless defined otherwise, all technical and scientific terms used have their ordinary meaning. Singleton, et. al., Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY, 1991 provide guidance as to ordinary meaning.

[0096]

[0093] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0097]

[0094] If a DNA sequence is provided, the specification should be understood as additional disclosing the sequence of the RNA, which will be the same with the exception that thymine (T) is replaced with uracil (U), and vice versa.

[0098]

[0095] Nucleic acids are preferably provided with codon preferences for a cell in which expression is intended. The term "codon usage" or "codon bias" refers to the relative frequencies with which different synonymous codons are used to encode an amino acid within an open reading frame. A nucleic acid sequence having codon preferences for a particular target cell has a balance of synonymous codon choices that result in efficient translation in that cell type. This balance is often not calculable from observed genomic codon frequencies, but must be empirically determined, for example as described in US patents 7,561,972 and 7,561,973 and 8,401,798 and in Welch et. al. (2009) "Design Parameters to Control Synthetic Gene Expression in Escherichia coli". PLoS ONE 4(9): e7002. https: / / doi.org / 10.1371 / journal.pone.0007002. A nucleic acid originally isolated from one cell type to be introduced into a target cell of another type can undergo selection of codon preferences for the target site cell such that at least 1 and sometimes, 5, 20, 15, 20, 50, 100 or more choices among synonymous codons differ between the nucleic acid introduced into the target cell from the original nucleic acid.

[0099]

[0096] Two nucleic acids are "complementary" if the bases of one hydrogen bond to the bases of the other. For perfect complementarity, adenine (A) in the first nucleic acid must correspond with thymine (T) (or uracil for RNA) in the second (and vice versa), and cytosine (C) in the first nucleic acid must correspond with guanine (G) in the second (and vice versa). The two nucleic acid s must also be antiparallel. If two nucleic acid are complementary, one may be described as the "reverse complement" of the other to indicate that their bases are complementary when one is in the 5' to 3' direction and the other is in the 3' to 5' direction. When one nucleic acid sequence is described as complementary to another, it is intended to indicate that the sequences are antiparallel and able to base-pair with one another.

[0100]

[0097] The "configuration" of a nucleic acid refers to the presence, order and direction of functional segments with the nucleic acid.

[0098] A 'transposase' is a polypeptide that catalyzes the excision of a corresponding transposon from a donor nucleic acid, for example a vector, and (providing the transposase is not integrationdeficient) the subsequent integration of the transposon into a target nucleic acid.

[0101]

[0099] "Transposition" refers to action of a transposase in excising a transposon from one nucleic acid and then integrating it, either into a different site in the same nucleic acid, or into a second nucleic acid.

[0102]

[0100] A "transposon" means a nucleic acid that can be excised from a first nucleic acid, for instance, a vector, and be integrated into a second position in the same nucleic acid, or into a second nucleic acid, for instance, the genomic or extrachromosomal DNA of a cell, by the action of a corresponding trans-acting transposase. A transposon comprises a first transposon end and a second transposon end, which are nucleic acid sequences recognized by and transposed by a transposase. The first and second transposon ends include inverted terminal repeats. Two copies of a transposon target site are usually present on the outside of the transposon ends (one on each side). A transposon usually further comprises a nucleic acid between the two transposon ends, which along with the two transposon ends is transposed by the action of the transposase. In natural transposons, the nucleic acid between the transposon ends is typically a corresponding transposase. Transposons of the present invention are "synthetic transposons" comprising a heterologous nucleic acid, which is transposable by virtue of its juxtaposition between two transposon ends. Synthetic transposons may or may not further comprise flanking nucleic acid sequence(s) outside the transposon ends, such as a sequence encoding a transposase, a vector sequence or sequence encoding a selectable marker.

[0103]

[0101] A "transposon end" means the cis-acting nucleotide sequences that are sufficient for recognition by and transposition by a corresponding transposase. Transposon ends of piggyBac-like transposons comprise perfect or imperfect repeats such that the respective repeats in the two transposon ends are reverse complements of each other. These are referred to as inverted terminal repeats (ITR) or terminal inverted repeats (TIR). A transposon end may or may not include additional sequence proximal to the ITR that promotes or augments transposition.

[0104]

[0102] The terms "corresponding transposon" and "corresponding transposase" are used to indicate an activity relationship between a transposase and a transposon. A transposase transposes its corresponding transposon. Many transposases correspond with a single transposon, and many transposons correspond with a single transposase. The term "orthogonal" refers to a lack of interaction between two systems. A first transposon and its corresponding first transposase and a second transposon and its corresponding second transposase are orthogonal if the first transposase does not excise or transpose the second transposon and the second transposase does not excise or transpose the first transposon.

[0105]

[0103] A "target site" for a transposase is a site or sequence in a molecule into which a transposon can be inserted by a transposase. The piggyBac transposase from Trichoplusia ni inserts its transposon predominantly into the target sequence 5'-TTAA-3'. Other useable target sequences for piggyBac transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'-AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'- TTGA-3', 5'-TTTA-3', 5’-TTAC-3', 5’-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-GTAA-3', 5'-AGGT-3', 5'-ATCA -3', , 5'- CTCC-3', 5'- T AAA-3', 5'-TCTC -3', 5'-TGAA -3', 5'- AAAT-3', 5'- AATC-3', 5'-ACAA -3', 5'- ACAT-3', 5'- ACTC -3', 5'-AGTG -3', 5'-ATAG -3', 5'- CAAA-3', 5'-CACA -3', 5'-CATA -3', 5'-CCAG -3', 5'-CCCA -3', 5'-CGTA -3', 5'-CTGA -3', 5'- GTCC-3', 5'- TAAG-3', 5'-TCTA -3', 5'-TGAG -3', 5'-TGTT -3', 5'-TTCA -3', 5'- TTCT-3' and 5'-TTTT -3' (Li et al., 2013. Proc. Natl. Acad. Sci vol. 110, no. 6, E478-487) and 5'-TTAT. PiggyBac-like transposases transpose their transposons using a cut-and-paste mechanism, which results in duplication of their 4 base pair target sequence on insertion into a DNA molecule. The target sequence is thus found on each side of an integrated piggyBac-like transposon.

[0106]

[0104] A "coupling element" or "translational coupling element" means a DNA sequence that allows the expression of a first polypeptide to be linked to the expression of a second polypeptide. Internal ribosome entry site elements (IRES elements) and cis-acting hydrolase elements (CHYSEL elements) are examples of coupling elements.

[0107]

[0105] A DNA sequence, segment of DNA, RNA sequence or RNA sequence means a contiguous nucleic acid sequence, which can be an oligonucleotide of 2 to 20 nucleotides in length to a full-length genomic sequence of thousands or hundreds of thousands of base pairs.

[0108]

[0106] A vector is a nucleic acid that facilitates any of transfection, integration, replication or expression of a coding segment incorporated into the vector. An expression vector is a vector comprising a promoter which has been or can be operably linked to a coding segment to be expressed. Transfection of the expression vector into a cell allows the cell to express the coding segment. An expression vector can be a genetically engineered plasmid, virus, recombinant virus, or an artificial chromosome derived from, for example, a bacteriophage, adenovirus, adeno-associated virus, retrovirus, lentivirus, poxvirus, or herpesvirus. Such expression vectors can include sequences from bacteria, viruses or phages. Such vectors include chromosomal, episomal and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids.

[0107] A "gene" refers to a transcriptional unit including a promoter and sequence to be expressed from it as an RNA or polypeptide. The sequence to be expressed can be genomic or cDNA or one or more non-coding RNAs including siRNAs or microRNAs among other possibilities. Other elements, such as introns, and other regulatory sequences may or may not be present.

[0109]

[0108] A gene transfer system refers to an expression vector and optionally one or more other features to facilitate gene transfer. For example, a gene transfer system may comprise an expression vector and a lipid or viral packaging mix for enabling a first nucleic acid to enter a cell, or it may comprise a nucleic acid that includes a transposon and a second nucleic acid encoding a corresponding transposase for genomic integration of the transposon. A transposase and transposon of a gene transfer system may be on the same nucleic acid molecule or on different nucleic acid molecules.

[0110]

[0109] Two elements are "heterologous" to one another if not naturally associated. For example, a heterologous coding segment linked to a promoter (or equivalently a heterologous promoter linked to a coding segment) means a promoter other than that which naturally drives expression of the coding segment. A heterologous nucleic acid flanked by transposon ends or ITRs means a heterologous nucleic acid not naturally flanked by those transposon ends or ITRs, such as a nucleic acid encoding a polypeptide other than a transposase, including an antibody heavy or light chain. A nucleic acid is heterologous to a cell if not naturally found in the cell or if naturally found in the cell but in a different location (e.g., episomal or different genomic location) than the location described.

[0111]

[0110] An expression system refers to a nucleic acid for expressing a coding segment. As well as the coding segment, the expression system can include a promoter and one or more regulatory sequences, such as an operator, as further described herein. An expression system may or may not be a component of a vector. Also an expression system may or may not be a component of a transposon.

[0112]

[0111] A "hyperactive" transposase is a transposase that is more active than the naturally occurring transposase from which it is derived. "Hyperactive" transposases are thus not naturally occurring sequences.

[0113]

[0112] An "IRES" or "internal ribosome entry site" means a specialized sequence that directly promotes ribosome binding, independent of a cap structure.

[0114]

[0113] An 'isolated' object, such as a polypeptide or nucleic acid, means the object has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Objects can also be purified, that is, provided at least 90%, 95% or 99% free w / w of other materials with which they are naturally associated or are used in their production or purification. The terms isolated and purified do not exclude presence of other components not naturally associated with the object that facilitate its use, such as a heterologous promoter for a coding segment, or pharmaceutical excipient.

[0115]

[0114] Unless otherwise apparent from the context, the terms "nucleoside" and "nucleotide" include those moieties which contain not only the standard purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or is functionalized as ethers, amines, or the like. The term "nucleotidic unit" encompasses nucleosides and nucleotides.

[0116]

[0115] An "Open Reading Frame" or "ORF" means a portion of a nucleic acid that, when translated into amino acids, contains no stop codons. An open reading frame presumptively encodes a polypeptide. The genetic code reads DNA sequences in groups of three base pairs, which means that a double-stranded DNA molecule can read in any of six possible reading frames-three in the forward direction and three in the reverse. An ORF typically also includes an initiation codon at which translation may start.

[0117]

[0116] The term "operably linked" refers to functional linkage between two sequences such that one sequence operationally modifies the behavior of the other. For example, a promoter is operably linked to a coding segment when the promoter can initiate transcription of the coding segment, optionally with subsequent translation of the transcript. A promoter is operably linked to one or more operators, when initiation of transcription by the promoter can be regulated by binding of a repressor or modified repressor to the operon. A mature polypeptide and signal peptide are operably linked when the signal peptide regulates secretion or subcellular location of the mature polypeptide.

[0118]

[0117] The term "overhang" or "DNA overhang" means the single-stranded portion at the end of a double-stranded DNA molecule. Complementary overhangs are those which will base-pair with each other.

[0119]

[0118] A "piggyBac-like transposase" means a transposase with at least 20% amino acid sequence identity as identified using the TBLASTP algorithm to the piggyBac transposase from Trichoplusia ni (SEQ. ID NO:116), and as more fully described in Sakar, A. et. al., 2003. Mol. Gen. Genomics 270: 173-180.

[0120] "Molecular evolutionary analysis of the widespread piggyBac transposon family and related 'domesticated' species", and further characterized by a DDE-like DDD motif, with aspartate residues at positions corresponding to D268, D346, and D447 of Trichoplusia ni piggyBac transposase on maximal alignment. PiggyBac-like transposases are also characterized by their ability to excise their transposons precisely with a high frequency. A "piggyBac-like transposon" means a transposon having transposon ends which are the same or at least 80% and preferably at least 90, 95, 96, 97, 98, 99% or 100% identical to the nucleotide sequences of the transposon ends of a naturally occurring transposon that encodes a piggyBac-like transposase. A piggyBac-like transposon includes an inverted terminal repeat (ITR) sequence of approximately 12-16 bases at each end. These repeats may be identical at the two ends, or the repeats at the two ends may differ at 1 or 2 or 3 or 4 positions in the two ITRs. The transposon is flanked on each side by a 4 base sequence corresponding to the integration target sequence which is duplicated on transposon integration (the Target Site Duplication or Target Sequence Duplication or TSD). PiggyBac-like transposons and transposases occur naturally in a wide range of organisms including Argyrogramma agnate (GU477713), Anopheles gambiae (XP_312615; XP_320414; XP_310729), Aphis gossypii (GU329918), Acyrthosiphon pisum (XP_001948139), Agrotis ypsilon (GU477714), Bombyx mori (BAD11135), Ciona intestinalis (XP_002123602), Chilo suppressalis (JX294476), Drosophila melanogaster (AAL39784), Daphnia pulicaria (AAM76342), Helicoverpa armigera (ABS18391), Homo sapiens (NP_689808), Heliothis virescens (ABD76335), Macdunnoughia crassisigna (EU287451), Macaca fascicularis (AB179012), Mus musculus (NP_741958), Pectinophora gossypiella (GU270322), Rattus norvegicus (XP_220453), Tribolium castaneum (XP_001814566) and Trichoplusia ni (AAA87375) and Xenopus tropicalis (BAF82026), although transposition activity has been described for almost none of these.

[0121]

[0119] A regulatory element such as promoter is active in a specified target cell, such as a mammalian cell, means a regulatory element configurable to result in a level of expression of at least 1 transcript and optionally at least ten, 100 or 1000 transcripts per cell in a mammalian cell into which the regulatory element has been introduced.

[0122]

[0120] Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated the window of comparison between two sequences is defined by the longer of (a) entire length of the shorter of the two sequences being compared, or ( b) at least 25 contiguous nucleotides. Matched positions in maximally aligned sequences can be referred to as corresponding to one another.

[0123]

[0121] Specific binding between two entities refers to binding detectably higher in magnitude and distinguishable from non-specific binding of each of the entities to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Exemplary specific binding affinity can be at least 107, 108, 109, or 1010 M-l.

[0124]

[0122] Preferential binding between two entities refers to a substantial difference in binding affinity between two different conditions, such as presence or absence of tetracycline. For example, the affinities can differ by a factor of a least 5, 10, 25, 50 or 100. Preferential expression in one condition compared with another likewise refers to variation in expression by a factor of at least 5, 10, 25, 50 or 100 between the conditions.

[0125]

[0123] A polypeptide refers to any polymer of amino acids natural or synthetic regardless of length and thus includes full length proteins, fragments thereof, and peptides.

[0126]

[0124] A promoter can be represented by a single-stranded sequence of nucleotides present on a transcribed strand or a double-stranded sequence of nucleotide formed of the single-stranded sequence of nucleotides just described duplexed with its complement. Depending on the context, reference to a promoter may refer to either single- or double-stranded forms or both.

[0127]

[0125] Transfection is used generically for any process for introducing a nucleic into cells.

[0128]

[0126] The term "comprising" indicates that other features besides those recited may or may not be present. Thus, for example, reference to a nucleic acid comprising a tet operator comprising SEQ ID NO:5 means that additional flanking residues can be present on either or both sides of SEQ ID NO:5.

[0129] The term "consisting essentially of" is used in accordance with convention to refer to the basic and novel features of an invention.

[0130] DETAILED DESCRIPTION

[0131] I. General

[0132]

[0127] The invention provides inducible promoter systems and their components incorporating components of a tetracycline, cumate or lactose operon. By coordinating expression of different transcriptional units in these systems as a result of selection of promoters and / or linking the units into the same DNA molecule, these systems can achieve higher levels of expression of coding segments of interest, increased differential levels of expression between on- and off-states, and / or greater responsiveness to inducing agents than conventional systems.

[0133] II. Promoters

[0134]

[0128] The invention provides a hybrid mouse-human CMV promoter. The sequences of mouse and human CMV promoters are set out aligned in Fig. 1 with the transcriptional start site (first transcribed nucleotide) indicated as the underlined G annotated as +1. The human CMV promoter is a constitutive promoter often used for expression in mammalian cells. However, this promoter contains several CG dinucleotide motifs, which may reduce transcriptional efficiency, particularly a CG motif occupying positions -178 and -179 counted from the transcriptional start site of human CMV promoter (corresponding to positions 42 and 43 of SEQ ID NO:13, or corresponding positions of any mouse-human chimeric promoter sequence maximally aligned with SEQ ID NO:13). CG motifs can be eliminated by replacing sequence from the human CMV promoter with aligned sequence from the mouse CMV promoter lacking one or more of the CG motifs, particularly the CG motif occupying positions -178 and - 179 of the human CMV promoter sequence (SEQ ID NO:13). Among other possibilities, replacement can be effected by combining an upstream segment of a mouse CMV promoter of SEQ ID NO:16 and a downstream segment of a human CMV promoter of SEQ ID NO:13 or 14. The junction between the segments is preferably within a segment of nucleotides ACGTCAATGGGA, which is common to the human and mouse CMV promoter sequences. One hybrid comprises 149 bp of the mouse CMV sequence of SEQ ID NO:16 upstream of 114 bp of the human CMV sequence of SEQ ID NO:13. Preferred promoters have sequences comprising, consisting of or consisting essentially of the sequence of SEQ ID N0:10. SEQ ID NQ:10 lacks a 13-nucleotide sequence (SEQ ID NO:35) immediately upstream of the human CMV transcription initiation site. The omitted sequence can be replaced by regulatory elements, such as tet, cumate or lactose operators as further described below. Another preferred promoter sequence comprises, consists essentially of or consists of SEQ ID NO:12. SEQ ID NO:12 includes the 13- nucleotide sequence of SEQ ID NO:35 omitted in SEQ ID N0:10. Although exemplified for a combination of human and mouse CMV promoters, the same principles can be applied in forming a hybrid promoter between a human CMV promoter and CMV promoters of other species, particularly rodent species, such as rat CMV. Hybrid promoters, as described above, preferably confer enhanced transcription compared with either or both of the component promoters forming the hybrid.

[0135]

[0129] The invention also provides a minimal chimpanzee CMV promoter. A minimal promoter is a promoter that by itself shows no or minimal transcription (e.g., a mean of < 1, 5 or 10 transcripts per cell), but which can show substantially enhanced transcription (e.g., at least 10-fold, 50-fold or 100-fold), when combined with an upstream regulatory sequence, which serves to recruit a polymerase and transcriptional factors. A minimal promoter sequence is typically a contiguous sequence of nucleotides starting at or near the first nucleotide upstream of the transcriptional initiation site and including a TATA box. An exemplary minimal chimpanzee CMV promoter has a sequence comprising, consisting of or consisting essentially of SEQ ID NO:24. Optionally, up to 1, 2, 3, 4 or 5 nucleotides can be deleted from either end. Optionally, a few , e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the chimpanzee CMV promoter can be included upstream from the minimal promoter sequence, but inclusion of additional nucleotides is not preferred because this may reduce the differential levels of expression in the presence and absence of upstream activation. An exemplary minimal promoter comprises SEQ ID NO:24 and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with SEQ ID NO:24 is naturally associated. Corresponding minimal promoters can be obtained from human or mouse CMV or other organisms, (i.e., sequences with greatest identify to chimp sequences when maximally aligned). Other exemplary minimal promoters have sequences comprising, consisting of or consisting essentially of any of SEQ ID NOS:22, 23 and 179. Other minimal promoters have sequences comprising, consisting of or consisting essentially of any one of SEQ ID NOS:307-309. Preferably such minimal promoters have none, or no more than 1, 2, 3, 4, 5, 20,

[0136] 20, 25, 50 or 60 contiguous nucleotides on the 5' side of the selected SEQ ID NO. with which the selected SEQ ID NO. is naturally associated.

[0137]

[0130] The invention also uses intermediate strength promoters. Such promoters have reduced transcriptional activity relative to the hybrid promoters described above or their component promoters but greater activity than a minimal promoter. For example, the transcriptional activity can be reduced by 2-20 fold relative to the hybrid promoters described above or their components. Examples of such promoters have sequences comprising, consisting of or consisting essentially of any of SEQ ID NOS:17-

[0138] 21.

[0139]

[0131] The invention can also make use of other strong promoters besides the hybrid promoters and their components described above. Such promoters are preferably active in eukaryotic cells, more preferably in mammalian cells. Examples of such strong promoters are CMV, EFla (human elongation factor 1-alpha), SV40, PGK1 (phosphoglycerate kinase), human ubiquitin C, and human beta actin.

[0140]

[0132] The promoters described above can be incorporated into transcriptional units, which in addition to the promoter include a coding segment, and sometimes other regulatory sequences, such as tet, cumate or lactose operators as described further below or an enhancer, among other components. The heterologous coding segment can encode a polypeptide or RNA and can include a 5'UTR and / or a 3' UTR and a polyadenylation sequence among other components. Such transcriptional units can be transformed into cells for expression.

[0141]

[0133] Promoters can be compared by forming otherwise identical transcriptional units between promoters to be compared, transforming into the same cell type, e.g., CHO, HeLa, 293, COS, U20S, 3T3, or other mammalian cell and comparing transcription levels.

[0142] III. Components of Tet Regulatory Systems

[0143]

[0134] Some of the components of the present systems can be the same as those of conventional tet dependent regulatory systems. Such components include tet operators, tet repressors, which bind tet operators in the absence of tetracycline or other analogs thereof, modified tet repressors, which bind tet operators in the presence of tetracycline or other analogs thereof, and inducers, such as tetracycline and analogs thereof.

[0144]

[0135] An exemplary tet operator comprises, consists of, or consists essentially of the sequence of any of SEQ ID NO:1 and SEQ ID NOS:192-195 Other tet operators can have up to 1, 2, 3 or 4 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to the exemplified sequence and retain the ability of the exemplified sequence for preferential binding of a tet repressor in the absence of tetracycline and preferential binding of a modified tet repressor in the presence of tetracycline. Examples of other tet operator sequences include 5'- TCGCTATCAGTGATAGAGA-3' and 5'-ACTCTATCATTGATAGAGT-3' (Wissmann et al, 1986, NucL Acids Res. 14: 4253-4266) Other examples of tet operators comprise, consist of or consist essentially of the sequence of any of SEQ ID NOS:192-195. Tet operators are typically used in tandem arrays (i.e., including at least two tet operator sequences). Some arrays include 2 to 10 tet operator sequences. Some such arrays include 2, 3, 4, 5, 6, 7, 8, 9 or 10 operator sequences. Some arrays have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 operator sequences. Some arrays have more than 10 operator sequences. The multiple operator sequences in such a tandem array are typically the same sequence. The operators are typically separated by spacers of e.g., 1-25 nucleotides. The identity and length of spacers can vary between different operators in an array. The number of operators and length of spacer can vary depending on the location of the operators relative to other components of a transcriptional unit as further described below. Exemplary tet arrays are provided in SEQ ID NQS:201-204. Exemplary tet operator arrays having sequences linked to a minimal promoter have sequences selected from any of SEQ ID NOS:26 and 202-204.

[0136] An exemplary tet repressor polypeptide comprises or consists of the amino acid sequence

[0145] SEQ ID NO:5. Other tet repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to such a sequence. Examples of variants include those described in Gossen and Bujard, Proc. Natl. Acad. Sci. USA Vol. 89, pp. 5547-5551, 1992 and a T40A substitution described by Altschmied et aL, EMBO J. 7:4011-4017, (1988). Substitutions are numbered according to position in SEQ ID NO:5 or if present in a sequence with a different number of residues than SEQ ID NO:5, the position of SEQ ID NO:5 corresponding to the substitution when the sequences are maximally aligned. A tet repressor polypeptide has the property of specifically binding to a tet operator or an array thereof in the absence of tetracycline. A tet repressor shows at least preferential binding to a tet operator or array thereof in the absence of tetracycline compared with the presence of tetracycline.

[0146]

[0137] An exemplary modified tet repressor polypeptide comprises or consists of an amino acid sequence selected from SEQ ID NOS:6, 314 or 322-328. Other modified tet repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to this sequence. Examples of variants include those described in Das et al. Current Gene Therapy, 2016, 16, 156-167, including tTA (E71K D95N L101S G102D), S2 (E19G A56P D148E H179R), M2 (S12G E19G A56P D148E H179R), 2S-S2 (E19G A56P D148E H179R), 2S-M2 (S12G E19G A56P D148E H179R), VI (E19G A56P F86Y D148E H179R), rtTA3 (S12G E19G A56P F86Y D148E H179R), V10 (E19G A56P F67S F86Y D148E R171K H179R), V16 (V9I E19G A56P F67S F86Y D148E R171K H179R). Substitutions are numbered according to position in SEQ ID NO:6 or if present in a sequence with a different number of residues than SEQ ID NO:6, the position of SEQ ID NO:6 corresponding to the substitution when the sequences are maximally aligned. In contrast to a tet repressor polypeptide, a modified tet repressor polypeptide has the property of specifically binding to a tet operator or an array thereof in the presence of tetracycline. An exemplified modified tet repressor fused to a transcriptional activation domain has an amino acid sequence comprising, consisting of or consisting essentially of SEQ ID NO:8 or 9. A modified tet repressor shows at least preferential binding to a tet operator or array thereof in the presence of tetracycline compared with the absence of tetracycline.

[0147]

[0138] Inducers of expression used in the present system include tetracycline itself, doxycycline and other tetracycline analogs. Unless otherwise apparent from the context, reference to tetracycline should be understood as alternatively disclosing that tetracycline analogs can be used. A tetracycline analog is a compound structurally related to tetracycline and which specifically binds to a tet repressor or modified tet repressor as described herein. Examples of tetracycline analogs are anhydrotetracycline (ate), chlorotetracycline, oxytetracycline, or deoxytetracycline and minocycline. Further analogs are disclosed by Hlavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, R. K. Blackwood et al. (eds.), Springer-Verlag, Berlin, N.Y., 1985; Mitscher, "The Chemistry of the Tetracycline Antibiotics", Medicinal Research 9, Dekker, N.Y., 1978; Noyee Development Corporation, "Tetracycline Manufacturing Processes" Chemical Process Reviews, Park Ridge, N.J., 2 volumes, 1969; Evans, "The Technology of the Tetracyclines," Biochemical Reference Series 1, Quadrangle Press, New York, 1968; and Dowling, "Tetracycline," Antibiotic Monographs, no. 3, Medical Encyclopedia, New York, 1955 and W02007 / 133797 and WO2007 / 133798.

[0148] IV. Components of Cumate Regulatory Systems

[0149]

[0139] A cumulate regulatory system has analogous compounds and mode of operation to a tetracycline regulatory system. These include cumate operators, cumate repressors, which bind a cumate operator in the absence of cumate and modified cumate repressors, which bind cumate operators in the presence of cumate. The description in relation to tetracycline regulatory sequences applies mutatis mutandis to cumate operator systems.

[0150]

[0140] An exemplary cumate operator comprises, consists of, or consists essentially of the sequence of any of SEQ ID NOS:156-160 or 191. Other cumate operators can have up to 1, 2, 3 or 4 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to the exemplified sequence and retain the ability of the exemplified sequence for preferential binding of a cumate repressor in the absence of cumate and preferential binding of a modified cumate repressor in the presence of cumate. Cumate operators can be used individually or in tandem arrays (i.e., including at least two cumate operators). Some arrays include 2 to 10 cumate operators. Some such arrays include 2, 3, 4, 5, 6, 7, 8, 9 or 10 operator sequences. Some arrays have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 operator sequences. Some arrays have more than 10 operator sequences. The multiple operators in such a tandem array can have the same or different sequences. The operators may be separated by spacers of e.g., 1-25 nucleotides. The identity and length of spacers can vary between different operators in an array. The number of operators and length of spacer can vary depending on the location of the operators relative to other components of a transcriptional unit as further described below.

[0151] Some exemplary arrays of cumate operators comprise, consist of or consist essentially of sequences of any of SEQ ID NQS:196-200.

[0152]

[0141] An exemplary cumate repressor polypeptide comprises or consists of the amino acid sequence SEQ ID NQ:170. Other cumate repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to this sequence. A cumate repressor polypeptide has the property of specifically binding to a cumate operator or an array thereof in the absence of cumate. A cumate repressor shows at least preferential binding to a cumate operator or array thereof in the absence of cumate compared with the presence of cumate.

[0153]

[0142] An exemplary modified cumate repressor polypeptide comprises or consists of the amino acid sequence SEQ ID NO:171. Other modified cumate repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to this sequence. SEQ ID NO:171 includes three mutations relative to SEQ ID NQ:170: E142G , I144M and V125A An exemplary modified cumate repressor fused to a transcriptional activation domain has an amino acid sequence comprising, consisting of or consisting essentially of SEQ ID NO:172 or 200. In contrast to a cumate repressor polypeptide, a modified cumate repressor polypeptide has the property of specifically binding to a cumate operator or an array thereof in the presence of cumate. A modified cumate repressor shows at least preferential binding to a cumate operator or array thereof in the presence of cumate compared with the absence of cumate.

[0154]

[0143] Inducers of expression used in the present system include cumate itself and analogs. An analog is a compound structurally related to cumate and which specifically binds to a cumate repressor or modified cumate repressor as described herein. Examples of cumate analogs include di-methyl p- aminobenzoic acid (DM PABA), trimethyl cumate, and ethylbenzoate, or a salt thereof, mainly para- or 4-substituted benzoate consisting of a bulky group of heteroatom, such as those selected from the group consisting of 3,4-dimethylbenzoate, 4-ethylbenzoate, 4-t-butylbenzoate, 4-phenylbenzoate, 4- benzylbenzoate, 4-ethoxybenzoate, 4-propyloxybenzoate, 4-n-butyloxybenzoate, 4-chlorobenzoate, 4- bromobenzoate, 4-iodobenzoate, 4-bromomethylbenzoate, 3,4-dichlorobenzoate, 4- trifluoromethylbenzoate, 4-ethyl-m-xylene, 4-vinyltoluene, 4-n-propyltoluene, 4-allytoluene, 4-fluoro-p- toluate, 3-chloro-p-toluate, and 4-bromo-m-toluate, an analogue of cumate such as Benzoic acid, p- methylbenzoic acid, p-ethylbenzoic acid, p-Propylbenzoic acid, cumic acid, p-isobutylbenzoic acid, p-tert- butylbenzoic acid, ibuprofen, p-aminobenzoic acid, p-N-methylaminobenzoic acid, p-N- dimethylaminobenzoic acid, p-N-methyl-N-ethylaminobenzoic acid and p-N-ethylaminobenzoic acid.

[0155] V. Components of lactose regulatory system

[0156]

[0144] Some of the components of the present systems can be the same as those of conventional lactose-dependent regulatory systems. Such components include lac operators, a lac repressor, which binds operators in the absence of lactose or an analog thereof, and a modified lac repressor, which binds lac operators in the presence of lactose or other analogs thereof

[0157]

[0145] An exemplary lac operator comprises, consists of, or consists essentially of the sequence of any of SEQ ID NOS:173-177. Other lac-operators can have up to 1, 2, 3 or 4 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to the exemplified sequence and retain the ability of the exemplified sequence for preferential binding of a lac repressor in the absence of lactose and preferential binding of the modified lactose repressor in the present of lactose. Lac operators are typically used in tandem arrays (i.e., including at least two lac operators). Some arrays include 2 to 10 lac operators. Some such arrays include 2, 3, 4, 5, 6, 7, 8, 9 or 10 operators. Some arrays have at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 operators. Some arrays have more than 10 operators. The multiple operator sequences in such a tandem array are typically the same sequence. The operators are typically separated by spacers of e.g., 1-25 nucleotides. The identity and length of spacers can vary between different operators in an array. The number of operators and length of spacer can vary depending on the location of the operators relative to other components of a transcriptional unit as further described below. Exemplary arrays of six lac operators comprises, consist of or consists essentially of a nucleotide sequence selected from SEQ ID NO:178 and 317.

[0158]

[0146] An exemplary lac repressor polypeptide comprises or consists of the amino acid sequence SEQ ID NO:184. Other modified lac repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to this sequence. Substitutions are numbered according to position in SEQ ID NO:184 or if present in a sequence with a different number of residues than SEQ ID NO:184, the position of SEQ ID NO:184 corresponding to the substitution when the sequences are maximally aligned. A lac repressor specifically binding to a lac operator or an array thereof in the absence of lactose. A lac repressor shows at least preferential binding to a lac operator or array thereof in the absence of lactose compared with the presence of lactose.

[0159]

[0147] An exemplary modified lac repressor polypeptide comprises or consists of the amino acid sequence SEQ ID NO:185. Other modified lac repressor polypeptides can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or additions and / or have at least 90, 95, 97, 98 or 99% identity relative to this sequence. Substitutions are numbered according to position in SEQ ID NO:185 or if present in a sequence with a different number of residues than SEQ ID NO:185, the position of SEQ ID NO:185 corresponding to the substitution when the sequences are maximally aligned. An exemplary lactose repressor comprises 3 mutations: S97P, R207L and T258A, relative to SEQ ID NO:184. A second exemplary modified lac repressor comprises 5 mutations: S69Y, S97P, Q131P, M242I and L346stop, relative to SEQ ID NO:184. A third exemplary modified lac repressor comprises 3 mutations: S97P, G315D and P339H, relative to SEQ ID NO:184. A fourth exemplary modified lac repressor comprises 8 mutations: A72V, A92V, C107S, S120R, L295M, V301A, T334M and L346stop, relative to SEQ ID NO:184. A fifth exemplary modified lac repressor comprises 4 mutations: S97P, K108N, E235D and Q352E, relative to SEQ ID NO:184. A sixth exemplary modified lac repressor comprises 6 mutations: K84I, S97P, R2O7L, T258A, L307H and L349P, relative to SEQ ID NO:184. Modified lac repressors containing other permutations of the mutations indicated above can also be constructed.

[0160]

[0148] In contrast to a lac repressor, a modified lac repressor polypeptide has the property of specifically binding to a lac operator or an array thereof in the presence of lactose. A modified lac repressor shows at least preferential binding to a lac operator or array thereof in the presence of lactose compared with the absence of lactose. A modified lac repressor can be linked to a transcriptional activation domain, e.g., having a sequence comprising, consisting of or consisting essential of any of SEQ ID NQS:40 and 186-188. An exemplary lactose / IPTG inducible transcriptional activator that comprises a modified E. coli lac repressor has amino acid sequence SEQ ID NO:189. A lactose / IPTG inducible transcriptional activator may further comprise a eukaryotic nuclear localization signal. An exemplary lactose / IPTG inducible transcriptional activator that comprises a modified E. coli lac repressor and a nuclear localization signal has amino acid sequence SEQ ID N0:190.

[0161]

[0149] Inducers of expression used in the present system include lactose and analogs isopropyl 0-d- 1-thiogalactopyranoside (IPTG), phenyl-d-galactopyranoside, thiomethyl galactoside and allolactose. Unless otherwise apparent from the context, reference to lactose should be understood as alternatively disclosing that lactose analogs can be used. A lactose analog is a compound structurally related to lactose and which specifically binds to a modified lac repressor as described herein.

[0162] VI. Transcriptional Activation Domains

[0163]

[0150] Some of the present systems include a transcriptional activation domain. Such a domain can be expressed as a fusion protein with a repressor polypeptide or modified repressor polypeptide. Fusion is preferably between the C-terminus of the repressor or modified repressor and the N-terminus of the transcriptional activation domain. More than one transcriptional domain can be included as a tandem array of such domains in a fusion protein. Examples of transcriptional activations domains include the HSV VP16 activation domain (see, e.g., Hagmann et aL, J. Virol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et aL, Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11, 4961-4968 (1992)). Other transcriptional activation domains include VP64, p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol. 23:255- 275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim. PoL 46:77-89; McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al (1999) Curr. Opin. Genet. Dev. 9:499-504. Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, API, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419- 429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353. Exemplary VP16 activation domains comprise or consist of a sequence selected from SEQ ID NOS:7 or 40. Exemplary fusions of a modified tet repressor to a VP16 activation domain comprises or consists of a sequence selected from SEQ ID NOS:8 or 9.

[0164] VII. Coding Segments

[0165]

[0151] Coding segments can encode any polypeptide or RNA of interest. Coding segments can include an open reading frame encoding a polypeptide. Coding segments can also include a segment encoding a 5' UTR, 3' UTR or polyadenylation region. Examples of polypeptides includes therapeutic proteins, proteins associated with a disease phenotype, enzymes, proteins used as selection markers or counter-selection markers, or suicide proteins. Examples of RNA include mRNA, tRNA, rRNA as well as various RNA molecules used for interference of expression, such as anti-sense, siRNA, shRNA or micro- RNA including artificial micro-RNA. Some examples of therapeutic proteins include antibodies or their component heavy and light chains, or heavy and light chain fused to one another as a single-chain antibody, antibodies engineered to produce heteromeric multi-chain molecules capable of binding more than one target protein (e.g. bispecific or multi-specific antibodies), T cell engagers, chimeric antigen receptors combining an antigen binding region with transmembrane and T cell signaling domains, Fc fusion proteins, antigens from pathogens (e.g. for use in vaccines), anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factor hormones, interferons, interleukins and thrombolytics. Proteins associated with disease are often mutated forms of human proteins. Some examples of such proteins are huntingtin, cystic fibrosis trans-membrane regulator, hemoglobin, alpha-1 antitrypsin, phenylalanine hydroxylase, beta-hexosaminidase, amyloid precursor protein, alpha-synuclein prion protein, transthyretin, crystallin and p53. It is particularly useful to be able to control the timing of expression of coding segments whose expression can be toxic for the cell that expresses them, for example so that the growth phase of an expression host can be decoupled from the expression phase. Examples of potentially toxic coding segments include open reading frames encoding membrane proteins such as ion channels, G-protein coupled receptors (GPCRs) and viral membrane proteins (such as the coronavirus spike proteins). Other examples of potentially toxic coding segments include open reading frames encoding proteins normally targeted to the lysozome, kinases and cytokines.

[0166]

[0152] A selection marker is a nucleic acid or expression product that allows for selection of a molecule or cell containing the marker often under particular conditions. These markers can encode an activity, such as, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions. Examples of selectable markers include: (1) DNA segments that encode products which provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) DNA segments that encode products which suppress the activity of a gene product; (4) DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as beta-galactosidase, green fluorescent protein (GFP), and cell surface proteins); (5) DNA segments that bind products which are otherwise detrimental to cell survival and / or function; (6) DNA segments that otherwise inhibit the activity of any of the DNA segments described in Nos. 1-5 above (e.g., antisense oligonucleotides); (7) DNA segments that bind products that modify a substrate (e.g. restriction endonucleases); (8) DNA segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) DNA segments that encode a specific nucleotide sequence which can be otherwise non-functional (e.g., for PCR amplification of subpopulations of molecules); and / or (10) DNA segments, which when absent, directly or indirectly confer sensitivity to particular compounds. Some examples of selection markers include glutamine synthetase, dihydrofolate reductase, blasticidin-resistance, neo- resistance, hygromycin-resistance, puromycin- resistance and zeocin-resistance.

[0167]

[0153] The term "counter-selectable marker" means a polynucleotide sequence that confers a selective disadvantage on a host cell. Examples of counter-selectable markers include sacB, rpsL, tetAR, pheS, thyA, gata-1, ccdB, kid and barnase (Bernard, 1995, Journal / Gene, 162: 159-160; Bernard et. al., 1994. Journal / Gene, 148: 71-74; Gabant et. al., 1997, Journal / Biotechniques, 23: 938-941; Gababt et. al., 1998, Journal / Gene, 207: 87-92; Gababt et. aL, 2000, Journal / Biotechniques, 28: 784-788; Galvao and de Lorenzo, 2005, Journal / Appl Environ Microbiol, 71: 883-892; Hartzog et. al., 2005, Journal / Yeat, 22:789-798; Knipfer et. aL, 1997, Journal / Plasmid, 37: 129-140; Reyrat et. al., 1998, Journal / lnfect Immun, 66: 4011-4017; Soderholm et. al., 2001, Journal / Biotechniques, 31: 306-310, 312; Tamura et. al., 2005, Journal / Appl Environ Microbiol, 71: 587-590; Yazynin et. al, 1999, Journal / FEBS Lett, 452: 351- 354).

[0168]

[0154] A suicide gene is a gene that causes a cell to kill itself e.g., by inducing apoptosis or by metabolizing a pro-drug into a drug that is toxic to a cell, e.g., caspase-9 (see Yagyu et aL, Mol. Ther. 23: 1475-85 (2015)). The present systems are particularly useful for regulated expression of coding segments whose expression is detrimental or lethal to a cell.

[0169]

[0155] Coding segments also include one or more proteins of a viral packaging system, such as lentivirus, adenovirus or adenoassociated virus (see US2023 / 0051793); Lee et aL, (doi. / org / 10.1021 / ascsynbio.2c00207). Lentivirus packaging components include VSV-G (e.g., Addgene 12259), rev response element (RRE) (e.g., Acc# P69718), gag (Addgene 12251), pol (e.g., Acc# B9W4K0)

[0170]

[0156] Production of AAV by mammalian cells can be accomplished by expression of a subset of adenoviral genes as an alternative to co-infection with an adenovirus helper virus (Matsushita et al, 1998. Gene Therapy 5, 938-945. The adenoviral genes required are a subset of the following genes: (i) the E2A protein (for example sequences SEQ ID NOS:268-276); (ii) the E4 ORF 6 protein (for example sequences SEQ ID NOS:259-267); (iii) the RNA genes VAI (for example sequences SEQ ID NQS:250-252) and VA2 (for example sequences SEQ ID NOS:253-255); (iv) the El region comprising E1A (for example sequences SEQ ID NOS:277-282), E1B 19K (for example sequences SEQ ID NOS:283-286) and E1B 55K (for example sequences SEQ ID NQS:287-290).

[0171]

[0157] Exemplary sequences comprising the RNA genes VAI and VA2 are SEQ ID NOS:253-255. Exemplary sequences comprising the E2A protein are SEQ ID NOS:273-275. Exemplary sequences comprising the E4 orf 6 protein are SEQ ID NOS:261-263.

[0172]

[0158] The AAV genome comprises two genes: Rep and Cap. Each of these genes comprises multiple open reading frames (Xiao et al, 1998. J Virol. 72:2224-32). Proteins encoded by the Rep gene comprise Rep 78 (for example sequences SEQ ID NOS:213-215), Rep 68 (for example sequences SEQ ID NOS:216-218), Rep 52 (for example sequences SEQ ID NQS:219-220) and Rep 40 (for example sequences SEQ ID NOS:221-222). Proteins encoded by the Cap gene comprise VP1 (for example sequences SEQ ID NOS:224-228), VP2 (for example sequences SEQ ID NOS: 231-232, 242-243), VP3 (for example sequence SEQ ID NOS:235, 244-245), assembly protein 1 (for example sequences SEQ ID NQS:305-306) and X protein (for example sequence SEQ ID NO:304).

[0173]

[0159] It is common to use AAV vectors that comprise the terminal repeats for AAV2. These are optimally replicated by the AAV2 Rep gene product but can be packaged into viral particles that use Cap genes from other serotypes. Caps from different serotypes may preferentially infect different cell types. It is therefore useful to use not only the Rep and Cap genes from the AAV2 genome (for example SEQ ID NOS: repcap3-4), but also chimeric genomes comprising the AAV2 Rep gene and the Cap gene from a different serotype. Exemplary sequences are AAV2 Rep, AAV1 Cap (SEQ ID NOS:292-293), AAV2 Rep, AAV3 Cap (SEQ ID NOS:296-297), AAV2 Rep, AAV4 Cap (SEQ ID NOS:298-299), AAV2 Rep, AAV5 Cap (SEQ ID NQS:300-301), AAV2 Rep, AAV6 Cap (SEQ ID NQS:302-303).

[0174]

[0160] Two or more coding segments can be placed under control of the same inducible promoter by separating the segments with IRES elements or by sequence encoding peptides such as CHYSEL (2a). Linkage of two or more coding segments to the same inducible promoter can be used to coordinate expression of multiple proteins performing interrelated functions, for examples, a plurality of proteins, which together operate as a viral packaging system. Such proteins can include one or more capsid proteins, and / or nonstructural proteins required for packaging, such as a nuclease to cut viral DNA and a polymerase. Each required protein of a viral packaging system can be included under the control of the same inducible promoter. Alternatively, proteins of a viral packaging system can be expressed individually from an inducible promoter system. If multiple proteins are expressed from the same type of inducible promoter system, their expression can be coordinately induced by supplying or withdrawing the same molecule, e.g., tetracycline, cumate or lactose. If expressed form different types of inducible promoter system, their expression can be controlled to follow a specified order. For example, AAV rep68 / rep78, and adenovirus E2a and E4orf6 can be expressed first to begin amplifying the AAV genome, before expressing the AAV capsid (VP1, VP2, VP3) and assembly proteins (including Rep 40 / 52).

[0175]

[0161] AAV VP1, VP2 and VP3 are expressed from overlapping nucleic acid segments in the same open reading frame from different translational initiation sites. When expressing an open reading frame including overlapping segments encoding AAV VP1, VP2 and VP3, the ratio of VP1 to VP2and / or VP3 can be increased by replacing the normal ACG initiation codon for VP1 with CUG and preferably further replacing nucleotides at the -1 and -2 position to C. This selection of initiation codon and / or preceding two nucleotides can be accommodated in any of the inducible and / or transposon-based expression systems described herein. VIII. Gene Transfer Systems

[0176]

[0162] The present transcription units can be incorporated into one or more gene transfer systems. For inducible control systems including two transcriptional units, both are preferably included in the same gene transfer system as part of the same contiguous nucleic acid.

[0177]

[0163] A gene transfer system comprises a nucleic acid to be transferred into a host cell and one or more other elements to facilitate uptake, integration, expression or election of the nucleic acid. A gene transfer system can include a transposon and corresponding transposase. Although transposons are preferred gene transfer systems because of their large cargo sizes and because multiple different coding segments with all of their associated regulatory elements can be incorporated without compromising packaging and delivery of the gene transfer system, other genes transfer systems including a lentiviral system, an adenoviral system or an adeno-associated viral system, or other expression vector can be used.

[0178]

[0164] A gene transfer system or one or more if its components can 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 (example, calcium phosphate, polyline or polyethyleneimine)

[0179]

[0165] After transfection, introduced nucleic acids can remain the cytoplasm (e.g., as an episome) or can be integrated into the genome. Integration of a nucleic acid into the genome of a host cell generally makes it stably heritable, by subjecting it to the same mechanisms that ensure the replication and division of genomic DNA. Such stable heritability is desirable for achieving good and consistent expression over long growth periods.

[0180]

[0166] Nucleic acid can be efficiently integrated into nucleic acids, such as a genome, by a transposase system. The nucleic acid into which a transposed nucleic acid is integrated is sometimes referred to as a target nucleic acid. A transposase system includes a transposon and a corresponding transposase. The transposon includes a heterologous nucleic acid to be transposed flanked by inverted transposon repeats (ITRs). The ITR on one side of the heterologous nucleic acid is a perfect or substantial (e.g., one or two mismatched nucleotides) reverse complement of the ITR on the other side. Between the ITRs and heterologous nucleic acids, additional transposon sequences may be present. Outside the ITRs (distal to the heterologous nucleic acid) are two copies, one for each side, of a target site, typically of four nucleotides. A benefit of a transposon is that the entire nucleic acid between transposon inverted terminal repeats (ITRs) can be integrated. There are several different classes of transposon. piggyBac and piggyBac-like transposons include the piggyBac transposon from the looper moth Trichoplusia ni, Xenopus piggyBac-like transposons, Bombyx piggyBac-like transposons, Heliothis piggyBac-like transposons, Helicoverpa piggyBac-like transposons, Agrotis piggyBac-like transposons, Amyelois piggyBac-like transposons, piggyBat piggyBac-like transposons and Oryzias piggyBac-like transposons. hAT transposons include TcBuster. Mariner transposons include Sleeping Beauty. Each of these transposons can be integrated into the genome of a mammalian cell by a corresponding transposase. A transposase can be provided as a protein or encoded by a nucleic acid. A transposase can include or be operably linked to a nuclear localization signal (NLS). The NLS can be internal or fused to either end of the transposase. The NLS can be naturally associated with the transposase or heterologous to it. A transposon comprising a heterologous nucleic acid and its corresponding transposase can be transfected into a cell at the same time, or sequentially. For example, a transposase protein or its encoding nucleic acid may be transfected into a cell prior to, simultaneously with or subsequently to transfection of a corresponding transposon. Additionally, administration of either component of the gene transfer system may occur repeatedly, for example, by administering at least two doses of this component.

[0181]

[0167] A nucleic acid encoding a transposase protein can be transfected into a cell as a nucleic acid vector such as a plasmid, a viral vector or as an mRNA molecule. The nucleic acid can be circular or linear. The nucleic acid encoding the transposase protein can be stably inserted into the genome of the cell or can remain in the cytoplasm. The transposase can be expressed constitutively or from an inducible system. DNA encoding a transposase is preferably linked to a promoter. A variety of promoters can be used including constitutive promoters, tissue-specific promoters, inducible promoters, species-specific promoters, cell-type specific promoters and the like. Alternatively, a transposase can be introduced into a cell directly as protein, for example using cell-penetrating peptides (e.g. as described in Ramsey and Flynn, 2015. Pharmacol. Ther. 154: 78-86"Cell-penetrating peptides transport therapeutics into cells"); using small molecules including salt plus propane betaine (e.g. as described in Astolfo et. al., 2015. Cell 161: 674-690); or electroporation (e.g. as described in Morgan and Day, 1995. Methods in Molecular Biology 48: 63-71"The introduction of proteins into mammalian cells by electroporation").

[0182]

[0168] Transposase proteins can be introduced into cells encoded as an mRNA molecule. RNA molecules can include substitutions to reduce toxicity effects on the cell, for example substitution of uridine with pseudouridine, and substitution of cytosine with 5-methyl cytosine. mRNA encoding the transposase can be prepared with a 5'-cap structure to improve expression in a target cell. Exemplary cap structures are a cap analog (G(5')ppp(5')G), an anti-reverse cap analog (3'-0-Me- m.sup.7G(5')ppp(5')G, a clean cap(m7G(5')ppp(5')(2'OI\ / leA)pG), an mCap (m7G(5')ppp(5)G). mRNA encoding the transposase may be prepared such that some bases are partially or fully substituted, for example uridine may be substituted with pseudo-uridine, cytosine may be substituted with 5-methyl- cytosine. Any combinations of these caps and substitutions may be made. Similarly, a nucleic acid encoding a transposase protein or the transposon of this invention can be transfected into the cell as a linear fragment or as a circularized fragment, either as a plasmid or as recombinant viral DNA. If the transposase is introduced as a DNA sequence encoding the transposase, then the coding segment encoding the transposase is preferably operably linked to a promoter suitable for use in the intended target cell.

[0183]

[0169] An exemplary piggyBac-like transposon for modifying the genome of a mammalian cell is a Xenopus transposon which comprises an ITR with the with sequence given by SEQ ID NO:41, a heterologous nucleic acid to be transposed and a second ITR with sequence given by SEQ ID NO:42. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95%or 100% identical to SEQ ID NO:43 or 44 on one side of the heterologous nucleic acid, preferably the left side, and a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:45 or 46 on the other side of the heterologous nucleic acid, preferably the right side. This transposon may be transposed by a corresponding Xenopus transposase comprising a sequence at least 90% or 100% identical to the sequence given by SEQ ID NO:47 or 48, for example, any of SEQ ID NOS:47-79. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:47: Y6L, Y6H, Y6V, Y6I,Y6C, Y6G, Y6A, Y6S, Y6F, Y6R, Y6P, Y6D, Y6N, S7G, S7V, S7D, E9W, E9D,E9E, M16E, M16N, M16D, M16S, M16Q, M16T, M16A, M16L, M16H, M16F, M16I,S18C, 518Y, S18M, S18L, S18Q, S18G, S18P, S18A, S18W, S18H, S18K, S18I,S18V, S19C, S19V, S19L, S19F, S19K, S19E, S19D, S19G, S19N, S19A, S19M,S19P, 519Y, S19R, S19T, S19Q, S20G, 520M, S20L, S20V, S20H, S20W, S20A,S20C, S20Q, S20D, S20F, S20N, S20R, E21N, E21W, E21G, E21Q, E21L, E21D,E21A, E21P, E21T, E21S, E21Y, E21V, E21F, E21M, E22C, E22H, E22R, E22L,E22K, E22S, E22G, E22M, E22V, E22Q, E22A, E22Y, E22W, E22D, E22T, F23Q,F23A, F23D, F23W, F23K, F23T, F23V, F23M, F23N, F23P, F23H, F23E, F23QF23R, F23Y, S24L, S24W, 524H, S24V, S24P, S24I, S24F, S24K, 524Y, S24D,S24C, 524N, 524G, S24A, S26F, S26H, S26V, S26Q, S26Y, S26W, S28K, S28Y,S28C, S28M, S28L, S28H, S28T, S28Q, V31L, V31T, V31I, V31Q, V31K, A34L,A34E, L67A, L67T, L67M, L67V, L67C, L67H, L67E, L67Y, G73H, G73N, G73K,G73F, G73V, G73D, G735, G73W, G73L, A76L, A76R, A76E, A76I, A76V, D77N,D77Q, D77Y, D77L, D77T, P88A, P88E, P88N, P88H, P88D, P88L, N91D, N91R,N91A, N91L, N91H, N91V, Y141I, Y141M, Y141Q, Y141S, Y141E, Y141W, Y141V,Y141F, Y141A, Y141C, Y141K, Y141L, Y141H, Y141R, N145C, N145M, N145A,N145Q, N145I, N145F, N145G, N145D, N145E, N145V, N145H, N145W, N145Y,N145L, N145R, N145S, P146V, P146T, P146W, P146C, P146Q, P146L, P146Y,P146K, P146N, P146F, P146E, P148M, P148R, P148V, P148F, P148T, P148C,P148Q, P148H, Y150W, Y150A, Y150F, Y150H, Y150S, Y150V, Y150C, Y150M,Y150N, Y150D, Y150E, Y150Q, Y150K, H157Y, H157F, H157T, H157S, H157W,A162L, A162V, A162C, A162K, A162T, A162G, A162M, A162S, A162I, A162Y,A162Q, A179T, A179K, A179S, A179V, A179R, L182V, L182I, L182Q, L182T,L182W, L182R, L182S, T189C, T189N, T189L, T189K, T189Q, T189V, T189AJ189W, T189Y, T189G, T189F, T189S, T189H, L192V, L192C, L192H, L192M,L192I, S193P, S193T, S193R, S193K, S193G, S193D, S193N, S193F, S193H,S193Q, S193Y, V196L, V196S, V196W, V196A, V196F, V196M, V196I, S198G,S198R, S198A, S198K, T200C, T200I, T200M, T200L, T200N, T200W, T200VJ200Q, T200Y, T200H, T200R, S202A, S202P, L210H, L210A, F212Y, F212N,F212M, F212C, F212A, N218V, N218R, N218T, N218C, N218G, N218I, N218P,N218D, N218E, A248S, A248L, A248H, A248C, A248N, A248I, A248Q, A248Y,A248M, A248D, L263V, L263A, L263M, L263R, L263D, Q270V, Q270K, Q270A,Q270C, Q270P, Q270L, Q270I, Q270E, Q270G, Q270Y, Q270N, Q270T, Q.270W,Q.270H, S294R, S294N, S294G, S294T, S294C, T297C, T297P, T297V, T297MJ297L, T297D, E304D, E304H, E304S, E304Q, E304C, S308R, S308G, L310R,L310l, L310V, L333M, L333W, L333F, Q336Y, Q336N, Q336M, Q336A, Q336T,Q336L, Q336I, Q336G, Q336F, Q336E, Q336V, Q336C, Q336H, A354V, A354W,A354D, A354C, A354R, A354E, A354K, A354H, A354G, C357Q, C357H, C357W,C357N, C357I, C357V, C357M, C357R, C357F, C357D, L358A, L358F, L358E,L358R, L358Q, L358V, L358H, L358C, L358M, L358Y, L358K, L358N, L358I,D359N, D359A, D359L, D359H, D359R, D359S, D359Q, D359E, D359M, L377V,L377I, V423N, V423P, V423T, V423F, V423H, V423C, V423S, V423G, V423A,V423R, V423L, P426L, P426K, P426Y, P426F, P426T, P426W, P426V, P426C,P426S, P426Q, P426H, P426N, K428R, K428Q, K428N, K428T, K428F, S434A,S434T, S438Q, S438A, S438M, T447S, T447A, T447C, T447Q, T447N, T447G,L450M, L450V, L450A, L450I, L450E, A462M, A462T, A462Y, A462F, A462K,A462R, A462Q, A462H, A462E, A462N, A462C, V467T, V467C, V467A, V467K, 1469V, I469N, 1472V, I472L, 1472W, I472M, I472F, L476I, L476V, L476N,L476F, L476M, L476C, L476Q, P488E, P488H, P488K, P488Q, P488F, P488M,P488L, P488N, P488D, Q498V, Q498L, Q498G, Q498H, Q498T, Q498C, Q498E,Q498M, L502I, L502M, L502V, L502G, L502F, E517M, E517V, E517A, E517K,E517L, E517G, E517S, E517I, P520W, P520R, P520M, P520F, P520Q, P520V,P520G, P520D, P520K, P520Y, P520E, P520L, P520T, S521A, S521H, S521C,S521V, S521W, S521T, S521K, S521F, S521G, N523W, N523A, N523G, N523S,N523P, N523M, N523Q, N523L, N523K, N523D, N523H, N523F, N523C, I533M, 1533V, I533T, I533S, I533F, I533G, 1533E, D534E, D534Q, D534L, D534R,D534V, D534C, D534M, D534N, D534A, D534G, D534F, D534T, D534H, D534K,D534S, F576L, F576K, F576V, F576D, F576W, F576M, F576C, F576R, F576Q,F576A, F576Y, F576N, F576G, F576I, F576E, K577L, K577G, K577D, K577R,K577H, K577Y, K577I, K577E, K577V, K577N, 1582V, 1582K, I582R, I582M,I582G, I582N, 1582E, I582A, I582Q, Y583L, Y583C, Y583F, Y583D, Y583Q,L587F, L587D, L587R, L587I, L587P, L587N, L587E, L587S, L587Y, L587M,L587Q, L587G, L587W, L587K or L587T.

[0184]

[0170] Another exemplary piggyBac-like transposon for modifying the genome of a cell is a Bombyx transposon which comprises an ITR with the sequence of SEQ ID N0:80, a heterologous nucleic acid to be transposed and a second ITR with the sequence of SEQ ID NO:81. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:82 on one side of the heterologous nucleic acid, preferably the left side, and a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:83 on the other side of the heterologous nucleic acid, preferably the right side. This transposon may be transposed by a corresponding Bombyx transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:84, for example, any of SEQ ID NQS:84-106.

[0185] Preferably the transposase is a hyperactive variant of a naturally occurring transposase of SEQ ID NO:84 including one or more of the following mutations: Q92A, Q92P, Q92N, Q92I, Q92Y, Q92H, Q92F, Q92R, Q92D, Q92M, Q92W, Q92C, Q92G, Q92L, Q92V, Q92T, V93P, V93K, V93M, V93F, V93W, V93L, V93A, V93I, V93Q, P96A, P96T, P96M, P96R, P96G, P96V, P96E, P96Q, P96C, F97Q, F97K, F97H, F97T, F97C, F97W, F97V, F97E, F97P, F97D, F97A, F97R, F97G, F97N, F97Y, H165E, H165G, H165Q, H165T, H165M, H165V, H165L, H165C, H165N, H165D, H165K, H165W, H165A, E178S, E178H, E178Y, E178F, E178C, E178A, E178Q, E178G, E178V, E178D, E178L, E178P, E178W, C189D, C189Y, C189I, C189W, C189T, C189K, C189M, C189F, C189P, C189Q, C189V, A196G, L200I, L200F, L200C, L200M, L200Y, A201Q, A201L, A201M, L203V, L203D, L203G, L203E, L203C, L203T, L203M, L203A, L203Y, N207G, N207A, L211G, L211M, L211C, L211T, L211V, L211A, W215Y, T217V, T217A, T217I, T217P, T217C, T217Q, T217M, T217F, T217D, T217K, G2195, G219A, G219C, G219H, G219Q, Q235C, Q235N, Q235H, Q235G, Q235W, Q235Y, Q235A, Q235T, Q235E, Q235M, Q235F, Q238C, Q238M, Q238H, Q238V, Q238L, Q238T, Q238I, R242Q, K246I, K253V, M258V, F261L, S263K, C271S, N303C, N303R, N303G, N303A, N303D, N303S, N303H, N303E, N303R, N303K, N303L, N303Q, I312F, I312C, I312A, I312L, I312T, 1312V, I312G, I312M, F321H, F321R, F321N, F321Y, F321W, F321D, F321G, F321E, F321M, F321K, F321A, F321Q, V323I, V323L, V323T, V323M, V323A, V324N, V324A, V324C, V324I, V324L, V324T, V324K, V324Y, V324H, V324F, V324S, V324Q, V324M, V324G, A330K, A330V, A330P, A330S, A330C, A330T, A330L, Q333P, Q333T, Q333M, Q333H, Q333S, P337W, P337E, P337H, P337I, P337A, P337M, P337N, P337D, P337K, P337Q, P337G, P337S, P337C, P337L, P337V, F368Y, L373C, L373V, L373I, L373S, L373T, V389I, V389M, V389T, V389L, V389A, R394H, R394K, R394T, R394P, R394M, R394A, Q395P, Q395F, Q395E, Q395C, Q395V, Q395A, Q395H, Q395S, Q395Y, S399N, S399E, S399K, S399H, S399D, S399Y, S399G, S399Q, S399R, S399T, S399A, S399V, S399M, R402Y, R402K, R402D, R402F, R402G, R402N, R402E, R402M, R402S, R402Q, R402T, R402C, R402L, R402V, T403W, T403A, T403V, T403F, T403L, T403Y, T403N, T403G, T403C, T403I, T403S, T403M, T403Q, T403K, T403E, D404I, D404S, D404E, D404N, D404H, D404C, D404M, D404G, D404A, D404Q, D404L, D404P, D404V, D404W, D404F, N408F, N408I, N408A, N408E, N408M, N408S, N408D, N408Y, N408H, N408C, N408Q, N408V, N408W, N408L, N408P, N408K, S409H, S409Y, S409N, S409I, S409D, S409F, S409T, S409C, S409Q, N441F, N441R, N441M, N441G, N441C, N441D, N441L, N441A, N441V, N441W, G448W, G448Y, G448H, G448C, G448T, G448V, G448N, G448Q, E449A, E449P, E449T, E449L, E449H, E449G, E449C, E449I, V469T, V469A, V469H, V469C, V469L, L472K, L472Q, L472M, C473G, C473Q, C473T, C473I, C473M, R484H, R484K, T507R, T507D, T507S, T507G, T507K, T507I, T507M, T507E, T507C, T507L, T507V, G523Q, G523T, G523A, G523M, G523S, G523C, G523I, G523L, I527M, 1527V, Y528N, Y528W, Y528M, Y528Q, Y528K, Y528V, Y528I, Y528G, Y528D, Y528A, Y528E, Y528R, Y543C, Y543W, Y543I, Y543M, Y543Q, Y543A, Y543R, Y543H, E549K, E549C, E549I, E549Q, E549A, E549H, E549C, E549M, E549S, E549F, E549L, K550R, K550M, K550Q, S556G, S556V, S556I, P557W, P557T, P557S, P557A, P557Q, P557K, P557D, P557G, P557N, P557L, P557V, H559K, H559S, H559C, H559I, H559W, V560F, V560P, V560I, V560H, V560Y, V560K, N561P, N561Q, N561G, N561A, V562Y, V562I, V562S, V562M, V567I, V567H, V567N, S583M, E601V, E601F, E601Q, E601W, E605R, E605W, E605K, E605M, E605P, E605Y, E605C, E605H, E605A, E605Q E605S, E605V, E605I, E605G, D607V, D607Y, D607C, D607N, D607W, D607T, D607A, D607H, D607Q, D607E, D607L, D607K, D607G, S609R, S609W, S609H, S609V, S609Q, S609G, S609T, S609K, S609N, S609Y, L610T, L610I, L610K, L610G, L610A, L610W, L610D, L610Q, L610S, L610F or L610N .

[0186]

[0171] Another exemplary piggyBac-like transposon for modifying the genome of a cell is a piggyBat transposon which comprises an ITR with the sequence of SEQ ID N0:107, a heterologous nucleic acid to be transposed and a second ITR with the sequence of SEQ ID N0:108. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:109 on one side of the heterologous nucleic acid, preferably the left side, and a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:110 on the other side of the heterologous nucleic acid, preferably the right side. This transposon may be transposed by a corresponding piggyBat transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:111. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:111: A14V, D475G, P491Q, A561T, T546T, T300A,T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, 1345V. M481V, EI1G, K130T,G9G, R427H, S8P, S36G, D1OG, S36G.

[0187]

[0172] Another exemplary piggyBac-like transposon for modifying the genome of a cell comprises an ITR with the sequence of SEQ ID NO:112, a heterologous nucleic acid to be transposed and a second ITR with the sequence of SEQ ID NO:113. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:114 on one side of the heterologous nucleic acid, preferably the left side, and a sequence immediately adjacent to the ITR and proximal to the heterologous nucleic acid that is at least 95% or 100% identical to SEQ ID NO:115 on the other side of the heterologous nucleic acid preferably the right side. This transposon may be transposed by a corresponding piggyBac transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:116. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:116: G2C, Q40R, 130V, G1655, T43A, S61R,S103P, S103T, M194V, R281G, M282V, G316E, 1426V, Q497L, N505D, Q573L,S509G, N570S, N538K, Q591P, Q591R, F594L, M194V, 130V, S103P, G165S,M282V, S509G, N538K, N571S, 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, C137T,A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, SSP, SBF,S54N, D9N, D9G, 1345V, M481V, E11G, K130T, G9G, R427H, S8P, S36G, DI0G,S36G, A51T, C153A, C277T, G201A, G202A, T236A, A103T, A104C, T140C,G138T, T118A, C74T, A179C, S3N, 130V, A46S, A46T, I82W, S103P, R119P,C125A, C125L, G165S, Y177K, Y177H, F180L, F180I, F180V, M185L, A187G,F200W, V207P, V209F, M226F, L235R, V240K, F241L, P243K, N258S, M282Q,L296W, L296Y, L296F, M298V, M298A, M298L, P311V, P311I, R315K, T319G,Y327R, Y328V, C340G, C340L, D421H, V436I, M456Y, L470F, S486K, M503l,M503L, V552K, A570T, Q591P, Q591R, R65A, R65E, R95A, R95E, R97A, R97E,R135A, R135E, R161A, R161E, R192A, R192E, R208A, R208E, K176A, K176E,K195A, K195E, S171E, M14V, D270N, 130V, G165S, M282L, M282I, M282V orM282A.

[0188]

[0173] Another example of a piggyBac-like transposon for modifying the genome of a cell is an Amyelois transposon comprising an ITR with the sequence of SEQ ID NO:117, a heterologous nucleic acid, and a second ITR with the sequence of SEQ ID NO:118. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence that is at least 95% or 100% identical to SEQ ID NO:119 on one side of the heterologous nucleic acid, and a sequence that is at least 95% or 100%identical to SEQ ID N0:120 on the other side of the heterologous nucleic acid. This transposon may be transposed by a corresponding Amyelois transposase comprising a sequence at least 90% identical to SEQ ID NO:121. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:121: P65E, P65D, R95S, R95T, V100I, V100L,V100M, L115D, L115E, E116P, H121Q, H121N, K139E, K139D, T159N, T159Q,V166F, V166Y, V166W, G179N, G179Q, W187F, W187Y, P198R, P198K, L203R,L203K, I209L, 1209V, 1209M, N211R, N211K, E238D, L273I, L273V, L273M,D304K, D304R, I323L, I323M, 1323V, Q329G, Q329R, Q329K, T345L, T345I,T345V, T345M, K362R, T366R, T366K, T380S, L408M, L408I, L408V, E413S,E413T, S416E, S416D, I426M, I426L, 1426V, S435G, L458M, L458I, L458V,A472S, A472T, V475I, V475L, V475M, N483K, N483R, I491M, 1491V, I491L,A529P, K540R, S560K, S560R, T562K, T562R, S563K, S563R.

[0189]

[0174] Another exemplary piggyBac-like transposon for modifying the genome of a cell is a Heliothis transposon comprising an ITR with the sequence of SEQ ID NO:122, a heterologous nucleic acid and a second ITR with the sequence of SEQ ID NO:123. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence that is at least 95% or 100% identical to SEQ ID NO:124 on one side of the heterologous nucleic acid, and a sequence that is at least 95% or 100% identical to SEQ ID NO:125 on the other side of the heterologous nucleic acid. This transposon may be transposed by a corresponding Heliothis transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:126. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:126: S41V, 5411, S41L, L43S, L43T, V81E,V81D, D83S, D83T, V85L, V85I, V85M, P125S, P125T, Q126S, Q126T, Q131R,Q131K, Q131T, Q131S, S136V, S136I, S136L, S136M, E140C, E140A, N151Q,K169E, K169D, N212S, I239L, 1239V, I239M, H241N, H241Q, T268D, T268E,T297C, M300R, M300K, M305N, M305Q, L312I, C316A, C316M, L321V, L321M,N322T, N322S, P351G, H357R, H357K, H357D, H357E, K360Q, K360N, E379P,K397S, K397T, Y421F, Y421W, V450I, V450L, V450M, Y495F, Y495W, A447N,A447D, A449S, A449V, K476L, V492A, 1500M, L585K and T595K.

[0190]

[0175] An advantageous piggyBac-like transposon for modifying the genome of a cultured mammalian cell is an Oryzias transposon comprising an ITR with the sequence of SEQ ID NO:127, a heterologous nucleic acid and a second ITR with the sequence of SEQ ID NO:128. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence that is at least 95% or 100% identical to SEQ ID NO:129 on one side of the heterologous nucleic acid, and a sequence that is at least 95% or 100% identical to SEQ ID N0:130 on the other side of the heterologous nucleic acid. This transposon may be transposed by a corresponding Oryzias transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:131. Preferably the transposase is a hyperactive variant of a naturally occurring transposase. Preferably the hyperactive variant transposase comprises one or more of the following amino acid changes, relative to the sequence of SEQ ID NO:131: E22D, A124C, Q131D, Q131E, L138V, L138I,L138M, D160E, Y164F, Y164W, I167L, 1167V, I167M, T202R, T202K, I206L, 1206V, I206M, I210L, 1210V, I210M, N214D, N214E, V253I, V253L, V253M,V258L, V258I, V258M, A284L, A284I, A284M, A284V, V386I, V386M, V386L,M400L, M400I, M400V, S408E, S408D, L409I, L409V, L409M, V458L, V458M,V458I, V467I, V467M, V467L, L468I, L468V, L468M, A514R, A514K, V515I,V515M, V515L, R548K, D549K, D549R, D550R, D550K, S551K and S551R

[0191]

[0176] Another exemplary piggyBac-like transposon for modifying the genome of a cell is an Agrotis transposon comprising an ITR with the sequence of SEQ ID NO:132, a heterologous nucleic acid, and a second ITR with the sequence of SEQ ID NO:133. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence that is at least 95% or 100% identical to SEQ ID NO:134 on one side of the heterologous nucleic acid, and a sequence that is at least 95% or 100% identical to SEQ ID NO:135 on the other side of the heterologous nucleic acid. This transposon may be transposed by a corresponding Agrotis transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:136. Preferably the transposase is a hyperactive variant of a naturally occurring transposase.

[0177] Another piggyBac-like transposon for modifying the genome of a cell is a Helicoverpa transposon comprising an ITR with the sequence of SEQ ID NO:137, a heterologous nucleic acid and a second ITR with the sequence of SEQ ID NO:138. The transposon may further be flanked by a copy of the tetranucleotide 5'-TTAA-3' on each side, immediately adjacent to the ITRs and distal to the heterologous nucleic acid. The transposon may further comprise a sequence that is at least 95% or 100% identical to SEQ ID NO:139 on one side of the heterologous nucleic acid, and a sequence that is at least 95% or 100% identical to SEQ ID NQ:140 on the other side of the heterologous nucleic acid. This transposon may be transposed by a corresponding Helicoverpa transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:141. Preferably the transposase is a hyperactive variant of a naturally occurring transposase.

[0192]

[0178] Another exemplary transposon for modifying the genome of cell is a Sleeping Beauty transposon from the Mariner family of transposons, for example one that comprises an ITR with the sequence of SEQ ID NO:142, a heterologous nucleic acid and a second ITR with the sequence of SEQ ID NO:143. Such a transposon can comprise a first transposon end with at least 90% or 100% sequence identity to SEQ ID NO:144, and a second transposon end with at least 90% or 100% sequence identity to SEQ ID NO:145. This transposon may be transposed by a corresponding Sleeping Beauty transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:146, including hyperactive variants thereof.

[0193]

[0179] Another example transposon for modifying the genome of a mammalian cell is a TcBuster transposon, from the hAT family of transposons, for example one that comprises an ITR with the sequence of SEQ ID NO:147, a heterologous nucleic acid and a second ITR with the sequence of SEQ ID NO:148. Such a transposon can comprise a first transposon end with at least 90% or 100% sequence identity to SEQ ID NO:149, and a second transposon end with at least 90% or 100% sequence identity to SEQ ID NO:150. This transposon may be transposed by a corresponding TcBuster transposase comprising a sequence at least 90% or 100% identical to SEQ ID NO:151, including hyperactive variants thereof.

[0194] IX. Cells and Transgenic Animals

[0195]

[0180] Nucleic acids comprising one or more transcriptional units as further described herein and gene transfer systems including the nucleic acids can be introduced into various cells and transgenic non-human animals. Cells into which nucleic acids are introduced are sometimes referred to as host cells or target cells or target host cells. Cells can be prokaryotic or eukaryotic. Mammalian cells, such as human, primate, or rodent are preferred. Insect cells can also be used. Cells can be a cell line of substantially identical cells obtained by expansion of a single cell or a mixed population of cells. Cells can be an immortal cell line or cells of finite life span. Some exemplary cell lines include CHO cell lines, various COS cell lines, HeLa cells, COS cells, 293 cells, U20S, HEK293 cells, L cells, and non-antibody- producing myelomas including Sp2 / 0 and NSO. Examples of cell types include hepatocytes, neural cells, muscle cells, blood cells, lymphocytes (B cells, natural killer cells and T cells), embryonic stem cells, somatic stem cells, hematopoietic cells, embryos, zygotes and sperm cells (some of which are open to be manipulated in an in vitro setting). Cells can be totipotent (i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells), pluripotent (cells whose descendants can differentiate into several restricted cell types, such as hematopoietic stem cells or other stem cells). Nucleic acid introduced into cells can also be used to generate transgenic nonhuman animals, e.g., rodents, such as mice, rats and rabbits, insects, fish, farm animals, such as goats, sheep, pigs, and cattle and non-human primates. Such transgenic animals typically incorporate an introduced heterologous nucleic acid into the genome of their germline and other cells. Such cells and transgenic animals can be used for production of proteins, e.g., therapeutic proteins or enzymes. Cells and transgenic animals can also be used for analyzing phenotypes conferred by expressed proteins, e.g., disease-associated phenotypes, and screening compounds for activity against such disease. Introduced nucleic acids can also be used to modify expression of genes of cells and transgenic animals, as for example, when the introduced nucleic acid encodes an inhibitor RNA to suppress expression of an endogenous gene. Suitable cells into which a nucleic acid has been introduced, particularly stem cells, can also be used for gene therapy. In some such application, the introduced nucleic acid includes a coding segment under inducible control, such that the coding segment encodes a polypeptide, which when expressed is lethal to the cell. Such a system allows an introduced cell to be eliminated by supplying an inducer of expression should the cell start undergoing inappropriate growth (e.g., becoming cancerous). After introduction of transcriptional units into cells, cells can be cultured and tetracycline or an analog, cumate or analog, or lactose or analog can be introduced into the culture medium to turn on or off expression of a coding segment. Likewise animals into which transcriptional units have been introduced can be contacted with tetracycline or an analog, cumate or analog or lactose or analog, when a developmental stage is reached at which induction or suppression of expression of the coding segment on the first transcriptional unit is desired. Contacting can be by any conventional route including intravenous, intraperitoneal, subcutaneous, oral, transdermal, and intramuscular.

[0181] Nucleic acid encoding one or more transcriptional units as described below can also be used in a coupled in vitro transcription and translation systems kits for which are commercially available from e.g., Thermo Fisher Scientific or New England Biolabs.

[0196] X. Inducible Systems

[0197]

[0182] The invention provides several expression systems for placing a coding segment of interest under inducible control depending on the presence or absence of tetracycline, doxycycline or other analog. Such systems typically include two transcriptional units, one for expressing a coding segment of interest, the other for expressing a tet repressor or modified tet repressor to make expression of the first transcriptional unit dependent on the presence or absence of tetracycline or an analog thereof.

[0198]

[0183] One expression system has a first transcriptional unit including in operable linkage and in order from 5' to 3', a promoter, one or more tet operator sites, and a coding segment to be expressed. An exemplary transcriptional unit is shown in Fig. 2, which includes a promoter with a TATA box, and adjacent segment ending at a position upstream from the transcriptional start site, first and second tet operators separated by a spacer, and a coding segment encoding a 5' UTR and an open reading frame to be expressed. The second tet operator can be immediately adjacent to the first nucleotide of the 5' UTR as shown. When the first and second tet operators replace a segment of the promoter immediately upstream from the transcription initiation site, transcription may initiate downstream of the remaining promoter within the first or second tet operator or the 5' UTR. As shown, the promoter includes DNA between the TATA box and a position upstream of the promoter's normal transcriptional start site (the first transcribed nucleotide). It is preferred that the first tet operator sequence replaces a contiguous segment of the promoter including at least the first base pair upstream of the transcriptional start site. More preferably the first tet operator sequence replaces a contiguous segment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 bp 5' of the promoter starting at the nucleotide immediately upstream of the transcriptional start site. Most preferably the first tet operator sequence replaces at least 6-bp 5' of the promoter starting at the nucleotide immediately upstream of the transcriptional start site. A preferred 5' UTR comprises the Xenopus globin UTR (SEQ. ID NO:29).

[0199]

[0184] A second transcriptional unit includes a promoter operably linked to a segment encoding a tet repressor. An exemplary transcriptional unit is shown in Fig. 3. In the system shown a promoter operable in a mammalian cell is operably linked to a coding segment including an open reading frame encoding a tet repressor followed by a segment encoding a polyA tail. In the absence of tetracycline, doxycycline or other analog, expressed tet repressor binds to the operator sites in the first transcriptional unit, hindering initiation of transcription and thereby inhibiting or eliminating expression of the coding segment. The operators are preferably located within 20 bases upstream and 50 bases downstream of the transcriptional start site of the promoter. In the presence of tetracycline, doxycycline or other analog, the tet repressor binds to the tetracycline or analog, inhibiting or eliminating binding of the tet repressor to tet operator(s) in the first transcriptional unit and removing inhibition of the coding segment. Thus, the coding segment is expressed in the presence of tetracycline or analog and not in the absence or at least expression in the presence is much greater than in the absence (e.g., by a factor of at least 2, 5, 10, 20, 50 or 100).

[0200]

[0185] The invention further provides several systems for placing a coding segment of interest under inducible control depending on the presence or absence of cumate or other analog. Such systems typically include two transcriptional units, one for expressing a coding segment of interest, the other for expressing a cumate repressor or modified cumate repressor to make expression of the first transcriptional unit dependent on the presence or absence of cumate or an analog thereof.

[0201]

[0186] One expression system has a first transcriptional unit including in operable linkage and in order from 5' to 3', a promoter, one or more cumate operators, and a coding segment to be expressed. An exemplary transcriptional unit is shown in Fig. 5, which includes a promoter with a TATA box at least one cumate operator a coding segment encoding a 5' UTR and an open reading frame to be expressed. When the cumate operator(s) replace a segment of the promoter immediately upstream from the transcription initiation site, transcription may initiate downstream of the remaining promoter within the first or second cumate operator or the 5' UTR. Sequence of exemplary promoter-cumate operator fusions are provided as SEQ ID NOS:161-163. A preferred 5' UTR comprises the Xenopus globin UTR (SEQ ID NO:29).

[0202]

[0187] A second transcriptional unit includes a promoter operably linked to a segment encoding a cumate repressor. An exemplary transcriptional unit is shown in Fig. 6. In the system shown a promoter operable in a mammalian cell is operably linked to a coding segment including an open reading frame encoding a cumate repressor followed by a segment encoding a polyA tail. In the absence of cumate or other analog, expressed cumate repressor binds to the operator sites in the first transcriptional unit, hindering initiation of transcription and thereby inhibiting or eliminating expression of the coding segment. In the presence of cumate or analog, the cumate repressor binds to the cumate or analog, inhibiting or eliminating binding of the cumate repressor to cumate operator(s) in the first transcriptional unit and removing inhibition of the coding segment. Thus, the coding segment is expressed in the presence of cumate or analog and not in the absence or at least expression in the presence is much greater than in the absence (e.g., by a factor of at least 2, 5, 10, 20, 50 or 100).

[0203]

[0188] The promoter of the first transcriptional unit (tet and cumate systems) is preferably a strong promoter to increase expression of the coding sequence after induction. The promoter is preferably a chimeric mouse-human CMV promoter as described above. Such a promoter is a preferred promoter to a human CMV promoter, which has been used for tetracycline-inducible expression previously. This is because elimination of one or more CpG sites from the human CMV promoter reduces the silencing effects of CpG methylation.

[0204]

[0189] The promoter for the second transcriptional unit (tet and cumate systems) is preferably a promoter of reduced strength compared with the chimeric mouse-human CMV promoter described above or even with the human CMV promoter used in some conventional systems. Use of a reduced strength promoter reduces the expression of the tet repressor and thus allows induction of expression from the first transcriptional unit at lower levels of tetracycline or analog than when the same or similar strength promoters are used for both transcriptional units.

[0205]

[0190] The first and second transcriptional units of this system (tet and cumate systems) are preferably combined on the same contiguous nucleic acid for introduction into cells. The nucleic acid can then be part of a transposon or other vector or gene delivery system as described above.

[0206] Incorporating both transcriptional units on the same nucleic acid facilitates introduction of both units into cells because only one transfection and identification of transformed cells is necessary, but also contributes to cells integrating the same number of copies of each of the transcriptional units. The presence of equal numbers of both transcriptional units allows reproducible control of the ratio of tet repressor to the number of copies of the first transcriptional unit. This reproducible control, preferably in combination with appropriate promoter selection as described above, can result in any of high levels of inducible expression, greater differentiation between levels of expression in presence and absence of inducer and inducible expression using lower levels of inducer.

[0207]

[0191] A second tet system for inducible expression has a first transcriptional unit (i.e., a nucleic acid) comprising in operable linkage and in order from 5' to 3', one or more tet operators, a minimal promoter and a coding segment to be expressed.

[0208]

[0192] Fig. 4 shows an exemplary form of this transcriptional unit with an array of tet operators separated by spacers upstream of a minimal promoter, operably linked to a coding segment encoding a 5' UTR and open reading frame to be expressed. The 5' UTR preferably comprises Xenopus globin UTR of SEQ ID NO:29. Preferably 2-10 or 3-8, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 operators are present in the array separated by spacers of 10-25 bp, optionally 15-20 bp. A second transcriptional unit includes in operable linkage a promoter and a segment encoding either a tet repressor or a modified tet repressor fused to a transcriptional activation domain, such as VP16. When the tet repressor fused to the transcriptional activation domain is expressed from the second transcriptional unit, it binds to the array of tet operators in the absence of tetracycline or analog. The transcriptional activation domain recruits polymerase and other transcription factors resulting in transcription from the minimal promoter and expression of the coding segment. In the presence of tetracycline or analog, the tet repressor can no longer bind the tet operator array or does so to a much-reduced extent, inhibiting or eliminating expression of the coding segment. Thus, the coding segment is placed under tetracycline or analog inducible control being expressed in the absence of tetracycline or analog.

[0209]

[0193] The reverse form of induction occurs when the tet repressor is replaced with a modified form of tet repressor, which binds to tet operators in the presence of tetracycline or analog. Here, in the absence of tetracycline or analog, the modified tet repressor linked to transcriptional activation domain is expressed but does not bind significantly if at all to the tet operators resulting in little or no recruitment of polymerase and other transcription factors, and little if any expression from the minimal promoter. This arrangement is shown in Fig. 20A. When tetracycline or analog is supplied, the tetracycline or analog binds to the modified tet repressor, which in turn binds to the tet operators. The linked transcriptional activation domain then recruits polymerase and other transcriptional factor resulting in transcription of the coding segment from the minimal promoter. This arrangement is shown in Fig. 20B. The coding segment is thus placed under inducible control of tetracycline or analog, being expressed in the presence of tetracycline and not expressed or expressed at much lower levels in the absence of tetracycline (e.g., least 2-, 5-, 10-, 20-, 50- or 100-fold increased expression on inductions).

[0210]

[0194] Exemplary tet operator arrays can have a nucleotide sequence selected from SEQ ID NOS:8 and 9. An exemplary minimal human CMV promoter has SEQ ID NO:22. Exemplary 5' UTRs comprising the Xenopus globin 5' UTR can have a nucleotide sequence selected from SEQ ID NOS:29, 30 and 31. The exemplary arrays are linked in the order from 5' to 3': tet operator array, minimal human CMV promoter, 5' UTR. Any of the exemplified tet operator arrays, can be used with any exemplified minimal human CMV promoter, and any exemplified 5' UTR in the order specified. An exemplary tet operator array fused to the 5' end of a chimpanzee minimal CMV promoter has a nucleotide sequence SEQ ID NO:26. Preferably the 3' end of the minimal chimpanzee CMV promoter is joined to a 5' UTR comprising the Xenopus globin 5' UTR with nucleotide sequence SEQ ID NO:29. An exemplary sequence comprising the minimal chimpanzee CMV promoter joined to a 5' UTR comprising the Xenopus globin 5' UTR has nucleotide sequence SEQ ID NO:25. An exemplary sequence comprising a tet operator array fused to the minimal chimpanzee CMV promoter joined to a 5' UTR comprising the Xenopus globin 5' UTR has nucleotide sequence SEQ ID NO:27.

[0211]

[0195] A second cumate system for inducible expression has a first transcriptional unit (i.e., a nucleic acid) comprising in operable linkage and in order from 5' to 3' , one or more cumate operators, a minimal promoter and a coding segment to be expressed. Fig. 7 shows an exemplary form of this transcriptional unit with an array of cumate operators separated by spacers upstream of a minimal promoter, operably linked to a coding segment encoding a 5' UTR and open reading frame to be expressed. The 5' UTR preferably comprises Xenopus globin UTR of SEQ ID NO:29. Preferably 2-10 or 3- 8, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 operators are present in the array, optionally separated by spacers of 1-25 bp. A second transcriptional unit includes in operable linkage a promoter and a segment encoding either a cumate repressor or a modified cumate repressor fused to a transcriptional activation domain, such as VP16. When the cumate repressor fused to the transcriptional activation domain is expressed from the second transcriptional unit, it binds to the array of cumate operators in the absence of cumate or analog. The transcriptional activation domain recruits polymerase and other transcription factors resulting in transcription from the minimal promoter and expression of the coding segment. In the presence of cumate or analog, the cumate repressor can no longer bind the cumate operator array or does so to a much-reduced extent, inhibiting or eliminating expression of the coding segment. Thus, the coding segment is placed under cumate or analog inducible control being expressed in the absence of cumate or analog.

[0212]

[0196] The reverse form of induction occurs when the cumate repressor is replaced with a modified form of cumate repressor, which binds to cumate operators in the presence of cumate or analog. Here, in the absence of cumate or analog, the modified cumate repressor linked to transcriptional activation domain is expressed but does not bind significantly if at all to the cumate operators resulting in little or no recruitment of polymerase and other transcription factors, and little if any expression from the minimal promoter. When cumate or analog is supplied, the cumate or analog binds to the modified cumate repressor, which in turn binds to the cumate operators. The linked transcriptional activation domain then recruits polymerase and other transcriptional factor resulting in transcription of the coding segment from the minimal promoter. The coding segment is thus placed under inducible control of cumate or analog, being expressed in the presence of cumate and not expressed or expressed at much lower levels in the absence of cumate (e.g., least 2-, 5-, 10-, 20-, 50- or 100-fold increased expression on inductions).

[0197] Exemplary cumate operator array-promoter fusions are SEQ ID NOS:164-166 and 196-199. Exemplary cumate operator promoter fusions including a 5' UTR are SEQ ID NOS:167-169.

[0213]

[0198] Another system for inducible expression has a first transcriptional unit (i.e., a nucleic acid) comprising in operable linkage, one or more lac operators, a minimal promoter and a coding segment to be expressed. The components preferably are preferably in the order stated from 5' to 3'. An exemplary form of this transcriptional unit with an array of lac operators separated by spacers upstream of a minimal promoter, operably linked to a coding segment encoding a 5' UTR and open reading frame to be expressed. The 5' UTR preferably comprises Xenopus globin UTR of SEQ ID NO:29. Preferably 2-10 or 3-8, e.g., 2, 3, 4, 5, 6, 7, 9, or 10 operators are present in the array separated by spacers of 10-25 bp, optionally 15-20 bp. A second transcriptional unit includes in operable linkage a promoter and a segment encoding a modified lac repressor fused to a transcriptional activation domain, such as VP16. When the modified lac repressor fused to the transcriptional activation domain is expressed from the second transcriptional unit, it binds to the array of lac operators in the absence of lactose or analog. The transcriptional activation domain recruits polymerase and other transcription factors resulting in transcription from the minimal promoter and expression of the coding segment. In the presence of lactose or analog, the modified lac repressor can no longer bind the lac-operator array or does so to a much-reduced extent, inhibiting or eliminating expression of the coding segment. Thus, the coding segment is placed under lactose or analog inducible control being expressed in the absence of lactose or analog.

[0214]

[0199] In the absence of lactose or analog, the modified lac repressor linked to a transcriptional activation domain is expressed but does not bind significantly if at all to the lac operators resulting in little or no recruitment of polymerase and other transcription factors, and little if any expression from the minimal promoter. When lactose or analog is supplied, the lactose or analog binds to the modified lac repressor, which in turn binds to the lac operators. The linked transcriptional activation domain then recruits polymerase and other transcriptional factor resulting in transcription of the coding segment from the minimal promoter. The coding segment is thus placed under inducible control of lactose or analog, being expressed in the presence of lactose and not expressed or expressed at much lower levels in the absence of lactose (e.g., least 2-, 5-, 10-, 20-, 50- or 100-fold increased expression on inductions).

[0215]

[0200] Exemplary lac operator arrays can have a nucleotide sequence selected from SEQ ID

[0216] NOS:173-177. An exemplary minimal human CMV promoter has the sequence of SEQ ID NO:22-24 or

[0217] 79. Exemplary 5' UTRs comprising the Xenopus globin 5' UTR can have a nucleotide sequence selected from SEQ ID NOS:29, 30 and 31. The exemplary arrays can be linked in the order from 5' to 3': lac operator array, minimal human CMV promoter, 5' UTR. Any of the exemplified lac operator arrays, can be used with any exemplified minimal promoter, and any exemplified 5' UTR in the order specified. An exemplary lac operator array fused to the 5' end of a chimpanzee minimal CMV promoter has a nucleotide sequence of any of SEQ ID NOS180-183: Preferably the 3' end of the minimal chimpanzee CMV promoter is joined to a 5' UTR comprising the Xenopus globin 5' UTR with nucleotide sequence SEQ ID NO:29. An exemplary sequence comprising the minimal chimpanzee CMV promoter joined to a 5' UTR comprising the Xenopus globin 5' UTR has nucleotide sequence SEQ ID NO:25.

[0218]

[0201] Lac inducible systems can also be constructed in which a first transcriptional unit has a minimal promoter operably linked to one or more lac operators and a heterologous coding segment, and a second transcriptional unit in which a promoter is operably linked to a segment encoding a lac repressor. An unexemplary unmodified lac repressor comprises, consists of or consists essentially of the sequence of SEQ ID NO:184.

[0219]

[0202] Selection of promoters for the first and second transcriptional units and combination of the units into the same contiguous molecule can improve the efficiency and reproducibility of inducible control. As already mentioned, the promoter for the first transcriptional unit is a minimal promoter. A preferred promoter is the minimal chimp CMV promoter described above, which results in higher level expression of a coding segment in the present systems than a minimal human CMV promoter used in conventional systems. The minimal chimp promoter, when activated by binding of a transcriptional activation domain as described above, is more active than a minimal human CMV promoter used in conventional lactose-inducible vectors. Preferred promoters for the second transcriptional unit are the intermediate strength promoters described above, although other promoters can also be used. Again, incorporation of both transcriptional units on the same nucleic acid for introduction into cells is advantageous for obtaining a cell that has integrated both transcription units and coordinating expression of the second transcriptional unit with the number of copies of the first transcriptional unit to be regulated. This reproducible control preferably in combination with appropriate promoter selection as described above can result in any of high levels of inducible expression, greater differentiation between levels of expression in presence and absence of inducer and inducible expression using lower levels of inducer.

[0220]

[0203] Again (tet, cumate and lactose systems), selection of promoters for the first and second transcriptional units and combination of the units into the same contiguous molecule can improve the efficiency and reproducibility of inducible control. As already mentioned, the promoter for the first transcriptional unit is a minimal promoter. A preferred promoter is the minimal chimp CMV promoter described above, which results in higher level expression of a coding segment in the present systems than a minimal human CMV promoter used in conventional systems. The minimal chimp promoter, when activated by binding of a transcriptional activation domain as described above, is more active than a minimal human CMV promoter used in conventional tetracycline-inducible vectors. Preferred promoters for the second transcriptional unit are the intermediate strength promoters described above, although other promoters can also be used. Again, incorporation of both transcriptional units on the same nucleic acid for introduction into cells is advantageous for obtaining a cell that has integrated both transcription units and coordinating expression of the second transcriptional unit with the number of copies of the first transcriptional unit to be regulated. This reproducible control preferably in combination with appropriate promoter selection as described above can result in any of high levels of inducible expression, greater differentiation between levels of expression in presence and absence of inducer and inducible expression using lower levels of inducer.

[0221]

[0204] One of the nucleic acids described in the above systems includes from 5' to 3', an array of at least one operator, a minimal mammalian promoter, a heterologous coding segment and a polyadenylation signal. The array can include for example, at least two lac operators, at least one cumate operators or at least two tet operators. The minimal promoter can compromise, consist of or consisting essentially of any one of SEQ ID NOS:307-309 among other minimal promoters disclosed herein. If any nucleotides naturally associated with the selected SEQ ID NO. are present, there are preferably no more than 1, 2,3, 4, 5, 10, 15, 20, 25, 50 or 60 such contiguous nucleotides naturally associated with the selected ID NO. on its 5' side. The heterologous coding segment can encode for example a protein of a viral packing system as has been further described herein. The nucleic acid can include a further operator between the minimal promoter and heterologous coding segment e.g., within a segment starting about 20 bases upstream and ending about 40 bases downstream of the transcriptional start site of the minimal promoter. Some such nucleic acids include at least 2, 3, 6 or 8 lac operators 5' of the minimal promoter and at least one lac operator between the minimal promoter and heterologous coding segment. Some such nucleic acids includes 2-20 cumate operators 5' of the minimal promoter and at least one cumate operator between the minimal promoter and heterologous coding segment. Some such nucleic acid include an array of 2-20 tet operators 5' of the minimal promoter and at least one tet operator between the minimal promoter and the heterologous coding segment. XI. Dual inducible expression systems

[0222]

[0205] In some instances, it is desirable to express within a cell a protein whose expression is toxic to that cell. In some instances, a protein may be sufficiently toxic that even a very low level of expression of that protein is detrimental to the cell. For example, some viral proteins required for the replication and packaging of parvoviruses or retroviruses are toxic to mammalian cells. Examples include adenoviral Ela, Elb, E2a, E4orf6 proteins, adeno-associated virus Rep78, Rep68, Rep52, Rep40, VP1, VP2, VP3, AAP and DBP proteins, HIVl gag, pol and rev proteins and vesicular stomatitis virus G protein. The toxicity of some of these proteins has been a barrier to the development of stable mammalian cell lines (for example stable human cell lines, including HEK293 cell lines and amniocyte-derived cell lines) to produce modified viruses for cell and gene therapy applications. In some instances, the basal expression from an inducible promoter, even in the uninduced state, results in the production of sufficient toxic protein to exert a negative selective pressure upon the host cell. Negative selective pressure favors cells that are able to silence expression of the toxic protein, and the ability to express the toxic protein is lost from the cell population. There is thus a need to further reduce basal expression in inducible expression systems, including for development of stable mammalian viral producer and packaging cell lines.

[0223]

[0206] Natural bacterial tetracycline, cumate and lactose repressors bind to their respective operators in the absence of their ligands. A first type of inducible system comprises (a) a first promoter, constitutively active in the host , (b) an operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first promoter and (d) an open reading frame encoding a repressor sequence operably linked to regulatory sequences such that it is expressible in the host cell. In this type of inducible expression system, the repressor is expressed within the host cell and binds to repress transcription of the open reading frame encoding the target conditionally expressed protein. Addition of the ligand for the repressor converts the repressor to a form that no longer binds to the operator, relieving inhibition of transcription and thereby leading to expression of the open reading frame encoding the target conditionally expressed protein. Exemplary cumate operator sequences have a nucleic acid sequence selected from SEQ ID NOS:156-160 or 191. An exemplary cumate repressor has amino acid sequence SEQ ID NO:170. Exemplary tet operator sequences have a nucleic acid sequence selected from SEQ ID NOS:1 or 192-195. An exemplary tet repressor has amino acid sequence SEQ ID

[0224] NO:56.

[0207] Modified (i.e., reversed) versions of the tetracycline, cumate and lactose repressors have been engineered to bind to their operators in the presence of their ligands. A second type of inducible system comprises (a) an array of 2 or more operators operably linked to a first minimal promoter, (b) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter and (c) an open reading frame encoding a modified repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell. In this type of inducible expression system the repressor-transcriptional activation domain fusion is expressed within the host cell but only binds to induce transcription of the open reading frame encoding the target conditionally expressed protein after addition of the ligand for the modified repressor converts the repressor to a form that binds to the operator array; thereby leading to expression of the open reading frame encoding the target conditionally expressed protein. Exemplary cumate operator arrays operably linked to a minimal promoter have a nucleic acid sequence selected from SEQ ID NOs: 164-166 or 196-199. An exemplary modified cumate repressor fused with a transcriptional activation domain has an amino acid sequence selected from SEQ ID NO:172 or 200. Exemplary tet operator arrays operably linked to a minimal promoter have a nucleic acid sequence selected from SEQ ID NOS:26 or 202-204. An exemplary modified tet repressor fused with a transcriptional activation domain repressor has an amino acid sequence selected from SEQ ID NOS:8-9.

[0225]

[0208] A dual inducible expression system may be construed by combining two modes of induction, particularly a repressor and a modified repressor. A dual inducible system includes at least three elements, (a), (b) and (c). Element (a) comprises a minimal promoter in operable linkage with first and second operator segments and a heterologous coding segment to be expressed. The first and second operator segments are bound by a modified repressor and a repressor respectively. The first operator segment is preferably 5' to the promoter. The second operator segment is preferably between 20 bases upstream and 50 bases downstream of the transcriptional start point of the minimal promoter. Either or both of the operator segments can include multiple operators, e.g., an operator array in which operators are separated by spacers. Fig. 17 show a Clustal alignment of 3 minimal CMV promoters (from human, mouse and chimp CMV respectively), the SEQ ID NOs of the corresponding nucleic acid sequences are shown in brackets after the name. The TATA box for each promoter is shown underlined, and the transcriptional start site (which corresponds to a G in all 3 sequences) is indicated in bold and underlined with +1 annotation above. Some or all of the bold nucleotides may be replaced, for example with nucleotides corresponding to the beginning of an operator sequence. The nucleotide sequences of these truncated minimal promoters are SEQ ID NOS:307-309. Element (b) comprises a promoter operably linked to a segment encoding a modified repressor fused to a transcriptional activation domain (modified repressor fusion). The modified repressor fusion binds to the first operator segment in the presence of a first inducer. Element (c) comprises a promoter operably linked to a segment encoding a repressor. The repressor binds to the second operator segment in the absence of a second inducer. In the absence of first and second inducers, the repressor binds to its operator segment repressing expression of the coding segment, and the modified repressor does not bind to its operator segment thereby not inducing transcription. In the presence of both inducers, the repressor no longer binds to its operator segment relieving inhibition by the repressor and the modified repressor now binds to its operator inducing expression. As a result of the dual control using two repressors and two inducers a greater differential between transcriptional level in the absence of both inducers to their presence can be obtained than either of the corresponding single inducible expression systems using the repressor or modified repressor and corresponding inducer by itself.

[0226]

[0209] In a variation of the above scheme, element (b) includes a third operator segment operably linked to the first promoter and the segment encoding the modified repressor fused to a transcriptional activation domain to make expression of the modified repressor fusion dependent on the presence of the second inducer. When the repressor binds the third operator segment in the absence of the second inducer, expression of the modified repressor fusion is repressed by the repressor binding to the third operator segment inhibiting transcription of mRNA encoding the modified repressor fusion. When the second inducer is present it binds to the repressor inhibiting it from binding to the third operator segment, and resulting in expression of the modified repressor fusion. When the first inducer is present, the modified repressor fusion domain binds to the first inducer and induces expression of the heterologous coding segment of element (a). The third operator segment can be located between the promoter and open reading frame of element (b). Thus, for examples, the third operator segment can occur within about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the first promoter on element (b). As for the first and second operator segments, the third operator can include a single operator or an array of operators. Because the second and third operator segments are both for binding to the same repressor, they typically include the same type and number of operators and may have the same sequence. This type of system further increases the differential expression between on states, when both inducers are present and off states when they are both absent. The type of system is particularly useful when the first and second inducer are the same, as in the lac-lac, cumate- cumate or tet-tet dual expression systems described below in that the same inducer can contribute to differential expression at three levels by affecting binding of a repressor or modified repressor to the first, second or third operator segments.

[0227]

[0210] Figs. 18A, B illustrate off and on states of such a system. Fig. 18A upper shows the configuration of elements (a), (c) and (b) from top to bottom. The arrangement is the same as Fig. 16A except that element (b) includes a tet operator between the promoter and open reading frame encoding the modified repressor transcriptional activator fusion. In the absence of tetracycline (or analog), the tet repressor expressed from element (c) binds the second tet operator in element (a) and third tet operator in element (b) inhibiting transcription of the modified repressor fusion from element (b) and coding segment to be expressed from element (a). Fig. 18B shows the same elements in the presence of tetracycline or analog. Now the tetracycline binds the tet repressor inhibiting it from binding to the second and third operator segments. The modified repressor fusion is now expressed and binds to the first operator segment inducing expression of the heterologous coding segment to be expressed.

[0228]

[0211] The repressor and modified repressor can come from the same system (e.g., tet repressor and modified tet repressor, or cumate repressor and modified cumate repressor, or lactate repressor and modified lactate repressor, or can be a repressor and modified repressor from different systems (e.g., tet repressor with modified cumate repressor or modified lactate repressor, cumate repressor with modified tet or modified lactate repressor or lactate repressor with modified tet or modified cumate repressor. If repressor and modified repressor are from the same system, their inducers can be the same, e.g., tetracycline or analog, cumate or analog, or lactate or an analog. If repressor and modified repressor are from different systems, the inducers are different (e.g., tetracycline or analog and cumate or analog).

[0229]

[0212] The three elements (a), (b) and (c) can be three transcriptional units, or two or more of the transcriptional units can be combined with IRES elements or linking peptides, such as CHYSEL. As with other expression systems, the three elements can be combined on the same contiguous nucleic acid, which can be a transposon, in which case the three elements are flanked by inverted repeats, and typically target sites of the transposon.

[0230]

[0213] A lac / cumate dual expressible system (Figs. 8A, B)

[0231]

[0214] (a) an array of 2 or more lac operators, the operator array operably linked to a first minimal promoter, (b) a cumate operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified lac repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified lac repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a cumate repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the cumate repressor bids to the cumate operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the cumate repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both lactose / IPTG and cumate.

[0232]

[0215] A lac / tet dual expressible system (Figs. 9A, B)

[0233]

[0216] (a) an array of 2 or more lac operators, the operator array operably linked to a first minimal promoter, (b) a tet operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified lac repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified lac repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a tet repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the tet repressor bids to the tet operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the tet repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both lactose / IPTG and tetracycline / doxycycline.

[0234]

[0217] A lac / lac dual expressible system (Figs. IDA, B)

[0235]

[0218] (a) an array of 2 or more lac operators, the operator array operably linked to a first minimal promoter, (b) a lac operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified lac repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified lac repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a lac repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the lac repressor bids to the lac operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the lac repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of lactose / IPTG which simultaneously activates transcription and relieves a block to transcription.

[0236]

[0219] A cumate / lac dual expressible system (Figs. 11A, B)

[0237]

[0220] (a) an array of 2 or more cumate operators, the operator array operably linked to a first minimal promoter, (b) a lac operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified cumate repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified cumate repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a lac repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the lac repressor bids to the lac operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the lac repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both lactose / IPTG and cumate.

[0238]

[0221] A cumate / tet dual expressible system (Figs. 12A, B)

[0239]

[0222] (a) an array of 2 or more cumate operators, the operator array operably linked to a first minimal promoter, (b) a tet operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified cumate repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified cumate repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a tet repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the tet repressor bids to the tet operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the tet repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both tetracycline / doxycycline and cumate.

[0240]

[0223] A cumate / cumate dual expressible system (Figs. 13A, B)

[0241]

[0224] (a) an array of 2 or more cumate operators, the operator array operably linked to a first minimal promoter, (b) a cumate operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified cumate repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified cumate repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a cumate repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the cumate repressor bids to the cumate operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the cumate repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of cumate which simultaneously activates transcription and relieves a block to transcription.

[0242]

[0225] A tet / lac dual expressible system (Figs. 14A, B)

[0243]

[0226] (a) an array of 2 or more tet operators, the operator array operably linked to a first minimal promoter, (b) a lac operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified tet repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified tet repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a lac repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the lac repressor bids to the lac operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the lac repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both lactose / IPTG and tetracycline / doxycycline.

[0244]

[0227] A tet / cumate dual expressible system (Figs. 15A, B)

[0245]

[0228] (a) an array of 2 or more tet operators, the operator array operably linked to a first minimal promoter, (b) a cumate operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified tet repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified lac repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a cumate repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the cumate repressor bids to the cumate operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the cumate repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of both tetracycline / doxycycline and cumate.

[0246]

[0229] A tet / tet dual expressible system (Figs. 16A, B)

[0247]

[0230] (a) an array of 2 or more tet operators, the operator array operably linked to a first minimal promoter, (b) a tet operator sequence placed between about 20 bases upstream of the transcriptional initiation start site and about 50 bases downstream of the transcriptional start site of the minimal promoter, (c) an open reading frame encoding a target conditionally expressed protein, the open reading frame operably linked to the first minimal promoter, (d) an open reading frame encoding a modified tet repressor sequence fused to a transcriptional activation domain, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the modified tet repressor bids to the array of operators operably linked to the first minimal promoter in the presence of its ligand and e) an open reading frame encoding a tet repressor sequence, the open reading frame operably linked to regulatory sequences such that it is expressible in the host cell, wherein the tet repressor bids to the tet operator in the absence of its ligand, thereby inhibiting transcription from the first minimal promoter until the ligand for the tet repressor is added. Expression of the open reading frame encoding a target conditionally expressed protein thus requires the addition of tetracycline / doxycycline which simultaneously activates transcription and relieves a block to transcription.

[0248] XII. EXAMPLES

[0249] 1. Inducible promoters comprising tet binding sites between the promoter and transcription start site in transiently transfected HEK cells.

[0250]

[0231] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline after transient transfection into (human) HEK 293 cells. Two promoters were constructed in which the 12 bp to the 5' of the transcriptional start site were removed and replaced by a pair of tet operators with nucleotide sequence SEQ ID NO:2. The first promoter was constructed by modification of the human CMV promoter with nucleotide sequence SEQ ID NO:14. Removal of the 12 bp to the 5' of the transcriptional start site produced nucleotide sequence SEQ ID NO:36. Addition of 2x tet operators to this truncated promoter produced nucleotide sequence SEQ ID NO:15. The second promoter constructed by modification of a chimeric murine-human CMV promoter comprising the first 161 bp of the murine CMV promoter with nucleotide sequence SEQ ID NO:16 fused to the last 127 bp of the human CMV promoter with nucleotide sequence SEQ ID NO:13: the junction between the two promoters comprised a 12 bp sequence common to both: 5'-ACGTCAATGGGA-3', and the sequence of the resulting chimeric promoter had nucleotide sequence SEQ ID NO:12. Removal of the 12 bp to the 5' of the transcriptional start site produced nucleotide sequence SEQ ID NQ:10.

[0251] Addition of 2x tet operators to this truncated promoter produced nucleotide sequence SEQ ID NO:11.

[0252]

[0232] The 3' end of the second tet operator in each of the two promoters described above, was fused to a 5' UTR with nucleotide sequence SEQ ID NO:31, which comprised a Xenopus globin 5'UTR modified to include a Kozak sequence (the modified Xenopus globin 5'UTR has nucleotide sequence SEQ ID NO:29). The UTR was fused to an open reading frame encoding Dasher GFP with amino acid sequence of SEQ ID NO:32, such that transcription from the promoter would result in expression of the GFP.

[0253]

[0233] Each of the two different tet inducible promoter-GFP transcriptional units was cloned onto a plasmid comprising a second transcriptional unit. The second transcriptional unit comprised an open reading frame encoding a tet repressor with amino acid sequence of SEQ ID NO:5. The open reading frame encoding the repressor was linked to a promoter operable in mammalian cells. Promoters selected were a PGK promoter with nucleotide sequence SEQ ID NO:18, an EEF2 promoter with nucleotide sequence SEQ ID NO:17, a murine CMV promoter with nucleotide sequence SEQ ID NO:16, and a human CMV promoter with nucleotide sequence SEQ ID NO:13.

[0254]

[0234] HEK293 suspension cells were grown in flasks in Expi293 media at 37°C with 8% CO2, in an incubator with 25mm throw at 125RPM. The cells were passaged at a density of 2.5x106 cells / ml 24 hours prior to transfection. Cells were diluted to 3x106 cells / ml, and 700ul were aliquoted into 96 deep well plates. Each DNA construct was transfected three independent times using ExpiFectamine™ 293 Transfection Reagent at a 1:2.7 DNA:Transfection reagent ratio. After transfection, the cells were placed in an incubator at 37°C with 8% CO2, with a 3mm throw shaking at 1,000 RPM. 24 hours posttransfection, the cultures were fed with ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2. After this feeding, the cultures were induced by the addition of doxycycline at either 10, 30, or 100 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. QD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24, 48, and 72 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 1.

[0255]

[0235] Row 6 of Table 1 shows the behavior of a construct in which the human CMV promoter was modified by addition of tet operators to serve as the inducible promoter, and where the human CMV promoter was also used as the promoter from which the tet repressor was transcribed. Expression from this construct was induced only at the highest concentration of doxycycline tested (100 ng / ml). Similar performance was observed when the human-murine chimeric CMV promoter was modified by addition of tet operators to serve as the inducible promoter, and where the murine CMV promoter was also used as the promoter from which the tet repressor was transcribed (Table 1 row 5), except that the maximum inducible expression was about twice as high as obtained using the human CMV promoter. When weaker promoters EEF2 or PGK were used to drive expression of the tet repressor, induction of expression was seen at lower levels of doxycycline (rows 4 and 3 respectively). This shows that the threshold for doxycycline induction of expression can be modulated by choice of promoter used to express the tet repressor: weaker promoters express lower levels of tet repressor, so less doxycycline is required to bind to the tet repressor to prevent repressor binding to the operators and remove the block to transcriptional initiation.

[0256] 2. Inducible promoters comprising tet binding sites between the promoter and transcription start site in transiently transfected CHO-S cells.

[0257]

[0236] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline after transient transfection into CHO cells. The same DNA constructs were used as described in Example 1.

[0258]

[0237] CHO-s cells were grown in flasks in ExpiCHO media at 37°C with 5% CO2, in an incubator with 25mm throw at 125RPM. The cells were passaged at a density of 4.5x106 cells / ml 24 hours prior to transfection. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Each DNA construct was transfected three independent times using ExpiFectamine™ CHO Transfection Reagent at a 1:4 DNA:Transfection reagent ratio. After transfection, the cells were placed in an incubator at 37°C with 5% CO2, with a 3mm throw shaking at 1,000 RPM. 24 hours post-transfection, the cultures were fed with ExpiFectamine™ CHO Enhancer and ExpiCHO Feed. After feeding the cells, the cultures were induced by the addition of doxycycline at either 10, 30, or 100 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. OD600 was measured in a flat- bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24, 48, and 72 hours post- induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 2.

[0259]

[0238] Table 2 shows a similar pattern to inducible expression in transient HEK cells, except that the human and murine CMV constructs shown in rows 5 and 6 have reversed places. Expression from the construct in which the murine CMV promoter was used as the promoter from which the tet repressor was transcribed (Table2 row 5) was induced only at the higher concentrations of doxycycline tested (a little at 30 ng / ml, more at 100 ng / ml). In contrast, higher expression at lower doxycycline concentrations was observed when the human CMV promoter was used as the promoter from which the tet repressor was transcribed (Table 2 row 6). This is consistent with the murine CMV promoter being stronger than the human CMV promoter in rodent cells and the human CMV promoter being stronger than the murine CMV promoter human cells. Again, when weaker promoters EEF2 or PGK were used to drive expression of the tet repressor, induction of expression was seen at lower levels of doxycycline (rows 4 and 3 respectively). Inducible expression was higher from the chimeric human murine CMV promoter than from the human CMV promoter. Again the threshold for doxycycline induction of expression can be modulated by choice of promoter used to express the tet repressor: weaker promoters express lower levels of tet repressor, so less doxycycline is required to bind to the tet repressor to prevent repressor binding to the operators and remove the block to transcriptional initiation.

[0260] 3. Inducible promoters comprising tet binding sites between the promoter and transcription start site in stably transfected CHO-K1 cells.

[0261]

[0239] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline when stably integrated into CHO-K1 cells. DNA constructs were used as described in Example 1. Each construct further comprised a third transcriptional unit expressing glutamine synthetase. The three transcriptional units were flanked by a pair of insulators: an HS4 insulator on one side and a D4Z4 core insulator on the other side. The three transcriptional units and the insulators were all placed into a Xenopus piggyBac-like transposon. On one side of the transposon was a 5'-TTAA'3' target integration sequence, immediately followed by a first ITR with nucleotide sequence SEQ ID NO:318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45), immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence.

[0262]

[0240] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO cell line with no functional glutamine synthetase gene. Cells were grown in the absence of glutamine added to the media until their viability reached 95%. Three cultures for each selected cell pool were then grown in EX-CELL® Advanced™ CHO Fed-batch Medium media at 37°C with 5% CO2, in an incubator with 25mm throw at 120 RPM. The cells were passaged at a density of 2x106 cells / ml 24 hours prior to induction. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Cultures were induced by the addition of doxycycline at either 10, 30, 100, 300, 1000 or 3000 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. QD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24 and 48 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 3.

[0263]

[0241] Table 3 shows a similar pattern to inducible expression in transiently transfected CHO cells, except that the overall induction levels were substantially lower. Lowest inducible expression was seen where the tet repressor was transcribed from the very active human or murine CMV promoters (Table 3 rows 5 and 6). The constructs in which tet repressor was transcribed from the more weakly active promoters showed expression that was induced at lower levels of doxycycline (expression started at 30 ng / ml when the tet repressor was transcribed from EEF2 or PGK promoters, while 300 ng / ml doxycycline) and reached 1.7-fold higher levels at high concentrations of doxycycline than constructs where the tet repressor was transcribed from a CMV promoter.

[0264] 4. Inducible promoters comprising tet binding sites between the promoter and transcription start site in stably transfected CHO-K1 cells.

[0265]

[0242] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline when stably integrated into CHO-K1 cells. DNA transposon constructs were similar to those described in Example 3, except that additional versions were made with other promoters driving expression of the tet repressor. Transposon compositions are indicated in Table 4.

[0266]

[0243] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO cell line with no functional glutamine synthetase gene. Cells were grown in the absence of glutamine added to the media until their viability reached 95%. Three cultures of each stably selected cell pool were then grown in EX-CELL® Advanced™ CHO Fed-batch Medium media at 37°C with 5% CO2, in an incubator with 25mm throw at 120 RPM. The cells were passaged at a density of 2x106 cells / m I 24 hours prior to induction. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Cultures were induced by the addition of doxycycline at either 10, 30, 100, 300 or 1000 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. OD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm.

[0267] Sampling was repeated 24 and 48 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 4.

[0244] Table 4 shows a similar pattern to the inducible expression in stably transfected CHO cells shown in Table 3. Most notably, two constructs whose inducible expression is shown in Table 4 differed only in the 6 nucleotides preceding the tet repressor open reading frame (Table 4 rows 6 and 7). The construct shown in row 6 had an optimal Kozak sequence (5'-GCCGCCACC-3'J, while the construct shown in row 7 had a de-optimized Kozak (5'-GCCT I I I l T-3'). The de-optimized Kozak results in reduced translational initiation and thus in less tet repressor being present. The consequence of this was that this inducible promoter was induced at only 10 ng / ml doxycycline, and was fully induced at 30 ng / ml doxycycline, whereas the otherwise identical construct with the optimal Kozak initiating translation of the tet repressor did not begin induction until 100 ng / ml doxycycline, and only became fully induced by 1,000 ng / ml doxycycline.

[0268]

[0245] Tables 1, 2, 3 and 4 show that expression levels obtained from tet inducible promoters, and the amount of inducer required to induce expression, differ depending on the level of expression of the tet repressor. The benefit of combining the transcriptional unit expressing the tet repressor onto the same transposon as the inducible transcriptional unit, is that it minimizes the potential variation in relative expression of tet repressor compared with the number of copies of the inducible promoter in the cell. This improves the predictability of the performance of the system.

[0269] 5. Inducible promoters comprising tet binding sites 5' of the promoter in transiently transfected HEK 293 cells.

[0270]

[0246] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline after transient transfection into HEK 293 cells.

[0271]

[0247] Three different minimal CMV promoters, from human, mouse and chimpanzee CMV (with nucleotide sequences SEQ ID NO:22, 23 and 24 respectively) were each fused, at their 5' ends, to an array of 8 tet operators with nucleotide sequence SEQ ID NO:3, to create 3 tet inducible promoters. The 3' end of each minimal promoter was fused to a 5'UTR with nucleotide sequence SEQ ID NQ:30. A control inducible promoter was also constructed by fusing a previously described array of 7 tet operators with nucleotide sequence SEQ ID NO:4 to the 5' end of a previously described modified minimal human CMV promoter with nucleotide sequence SEQ ID NO:38. The 3' end of this control minimal promoter was fused to a 5'UTR with nucleotide sequence SEQ ID NO:39. The 3' end of each 5'UTR was fused to an open reading frame encoding Dasher GFP with amino acid sequence SEQ ID NO:32.

[0272]

[0248] Each of the different tet inducible promoter-GFP transcriptional units was cloned onto a plasmid comprising a second transcriptional unit. The second transcriptional unit comprised an open reading frame encoding a transcriptional activator comprising a modified tet repressor with amino acid sequence of SEQ ID NO:6, fused to a VP16 transcriptional activation domain with amino acid sequence SEQ ID NO:7. In addition the control tet inducible promoter GFP transcriptional unit was cloned onto a plasmid comprising a second transcriptional unit comprising an open reading frame encoding a transcriptional activator comprising a modified tet repressor with amino acid sequence of SEQ ID NO:6, fused to an alternative VP16 transcriptional activation domain with amino acid sequence SEQ ID NQ:40. In each case the open reading frame encoding the transcriptional activator was linked to an SV40 promoter with nucleotide sequence SEQ ID NO:28, which promoter is operable in mammalian cells.

[0273]

[0249] HEK293 suspension cells were grown in flasks in Expi293 media at 37°C with 8% CO2, in an incubator with 25mm throw at 125RPM. The cells were passaged at a density of 2.5x106 cells / ml 24 hours prior to transfection. Cells were diluted to 3x106 cells / ml, and 700ul were aliquoted into 96 deep well plates. Each DNA construct was transfected three independent times using ExpiFectamine™ 293 Transfection Reagent at a 1:2.7 DNA:Transfection reagent ratio. After transfection, the cells were placed in an incubator at 37°C with 8% CO2, with a 3mm throw shaking at 1,000 RPM. 24 hours posttransfection, the cultures were fed with ExpiFectamine™ 293 Transfection Enhancer 1 and ExpiFectamine™ 293 Transfection Enhancer 2. After this feeding, the cultures were induced by the addition of doxycycline at either 10, 30, or 100 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. QD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24, 48, and 72 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 5.

[0274]

[0250] Table 5 (rows 4 and 5) shows that the control tet inducible minimal human CMV promoter resulted in similar induction profiles using either of the different transcriptional activators (with amino acid sequences SEQ ID NOS:8 and 9, which comprise the VP16-derived activation domains with amino acid sequences SEQ ID NOS:7 and 40 respectively). Table 5 also shows that the array of 8 tet operators joined to minimal human CMV promoter with nucleotide sequence SEQ ID NO:22 performs very comparably to the control tet inducible minimal human CMV promoter (compare Table 5 row 6 with row 5). The minimal murine CMV promoter showed substantially lower levels of induced gene expression than the human minimal CMV promoter sequences (Table 5 row 7). However the inducible promoter comprising the chimpanzee minimal CMV promoter resulted in consistently higher levels of expression than any of the other inducible promoters (Table 5 row 8).

[0275]

[0251] Thus a tet operator array fused to the 5' end of a chimpanzee minimal CMV promoter with nucleotide sequence SEQ ID NO:24 produces a highly effective tet inducible promoter.

[0276] 6. Inducible promoters comprising tet binding sites 5' of the promoter in transiently transfected CHO-S cells.

[0277]

[0252] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline after transient transfection into CHO-S cells.

[0278]

[0253] Constructs were as described in Example 5. CHO-s cells were grown in flasks in ExpiCHO media at 37°C with 5% CO2, in an incubator with 25mm throw at 125RPM. The cells were passaged at a density of 4.5x106 cells / ml 24 hours prior to transfection. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Each DNA construct was transfected three independent times using ExpiFectamine™ CHO Transfection Reagent at a 1:4 DNA:Transfection reagent ratio. After transfection, the cells were placed in an incubator at 37°C with 5% CO2, with a 3mm throw shaking at 1,000 RPM. 24 hours post-transfection, the cultures were fed with ExpiFectamine™ CHO Enhancer and ExpiCHO Feed. After feeding the cells, the cultures were induced by the addition of doxycycline at either 10, 30, or 100 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. OD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24, 48, and 72 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 6.

[0279]

[0254] Table 6 shows that the tet inducible promoters performed similarly in transiently transfected CHO cells as they did in transiently transfected HEK 293 cells. The inducible promoter comprising the chimpanzee minimal CMV promoter resulted in consistently higher levels of expression than any of the other inducible promoters (Table 6 row 8).

[0280] 7. Inducible promoters comprising tet binding sites 5' of the promoter in stably transfected CHO- K1 cells.

[0281]

[0255] We tested several embodiments of tet inducible promoters for their ability to respond to increasing concentrations of doxycycline after stable integration into CHO-K1 cells.

[0256] DNA constructs were used as described in Example 5. Each construct further comprised a third transcriptional unit expressing glutamine synthetase. The three transcriptional units were flanked by a pair of insulators: an HS4 insulator on one side and a D4Z4 core insulator on the other side. The three transcriptional units and the insulators were all placed into a Xenopus piggyBac-like transposon. On one side of the transposon was a 5'-TTAA'3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41), immediately followed by a left transposon end with nucleotide sequence SEQ ID NO:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence.

[0282]

[0257] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO cell line with no functional glutamine synthetase gene. Cells were grown in the absence of glutamine added to the media until their viability reached 95%. Three cultures for each selected cell pool were then grown in EX-CELL® Advanced™ CHO Fed-batch Medium media at 37°C with 5% CO2, in an incubator with 25mm throw at 120 RPM. The cells were passaged at a density of 2x106 cells / ml 24 hours prior to induction. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Cultures were induced by the addition of doxycycline at either 10, 30 or 100 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. QD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24 and 48 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 7. Table 7 shows that the minimal chimpanzee CMV tet inducible promoter shows very good inducible expression when stably integrated into the genome of a host cell.

[0283] 8. Inducible promoters comprising different numbers of tet binding sites 5' of the promoter in stably transfected CHO-K1 cells.

[0284]

[0258] We tested three embodiments of tet inducible promoters with different numbers of tet operators for their ability to respond to increasing concentrations of doxycycline after stable integration into CHO-K1 cells.

[0259] A minimal CMV promoter from chimpanzee CMV (with nucleotide sequence SEQ ID NO:24) was fused at its 5' end, to an array of 3, 6 or 8 tet operators (with nucleotide sequences SEQ ID NOS: 153, 152 and 3 respectively), to create 3 tet inducible promoters. The 3' end of each minimal promoter was fused to a 5'UTR with nucleotide sequence SEQ ID NQ:30. The 3' end of each 5'UTR was fused to an open reading frame encoding Dasher GFP with amino acid sequence SEQ ID NO:32.

[0285]

[0260] Each of the different tet inducible promoter-GFP transcriptional units was cloned onto a plasmid comprising a second transcriptional unit. The second transcriptional unit comprised an open reading frame encoding a transcriptional activator comprising a modified tet repressor with amino acid sequence of SEQ ID NO:6, fused to a VP16 transcriptional activation domain with amino acid sequence SEQ ID NO:7. In each case the open reading frame encoding the transcriptional activator was linked to an SV40 promoter with nucleotide sequence SEQ ID NO:28, which promoter is operable in mammalian cells. Each construct further comprised a third transcriptional unit expressing glutamine synthetase. The three transcriptional units were flanked by a pair of insulators: an HS4 insulator on one side and a D4Z4 core insulator on the other side. The three transcriptional units and the insulators were all placed into a Xenopus piggyBac-like transposon. On one side of the transposon was a 5'-TTAA'3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41), immediately followed by a left transposon end with nucleotide sequence SEQ ID NQ:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence.

[0286]

[0261] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO cell line with no functional glutamine synthetase gene. Cells were grown in the absence of glutamine added to the media until their viability reached 95%. Three cultures for each selected cell pool were then grown in EX-CELL® Advanced™ CHO Fed-batch Medium media at 37°C with 5% CO2, in an incubator with 25mm throw at 120 RPM. The cells were passaged at a density of 2x106 cells / ml 24 hours prior to induction. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Cultures were induced by the addition of doxycycline at either 10, 30, 100 or 300 ng / ml, with a control to which no doxycycline was added. After doxycycline addition, 100ul of culture was taken and diluted with 100ul of PBS. Expression was measured using a SpectroMax M2. QD600 was measured in a flat-bottom, clear-well plate. Green fluorescent protein expression was measured in a black-well plate, with excitation at 485nm and emission at 525nm. Sampling was repeated 24, 48 and 72 hours post-induction. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number. Results are shown in Table 8. Table 8 shows that the minimal chimpanzee CMV tet inducible promoter, operably linked to 3, 6 or 8 repeats of the tet operator, shows very good inducible expression when stably integrated into the genome of a host cell. It also shows that a smaller number of repeats resulted in a higher level of expression in the absence of doxycycline (compare Table 8 columns B, G, L and Q for row 3 with 8 tet operator repeats, row 4 with 6 tet operator repeats, and row 5 with 3 tet operator repeats. As a control, Table 8 row 6 shows the average fluorescence in cells with no integrated inducible transposon).

[0287] 9. A hybrid human-mouse CMV promoter is advantageous for expression in stably transfected mammalian cells.

[0288]

[0262] We tested a hybrid human-mouse promoter comprising a segment of a mouse CMV promoter of SEQ ID NO:16 upstream from a segment of a human CMV promoter of SEQ ID NO:13, wherein the hybrid promoter lacks a CG motif at positions corresponding to positions 42 and 43 of SEQ ID NO:13. The junction between contiguous segments is within the sequence ACGTCAATGGGA, which is common to the mouse and human CMV promoter sequences. The promoter has nucleotide sequence SEQ ID NO:12, which comprises SEQ ID NQ:10 plus 13 bases (5'-GTTTAGTGAACCG-3') immediately 5' of the transcriptional start site.

[0289]

[0263] The hybrid promoter was operably linked to an open reading frame encoding an antibody heavy chain with amino acid sequence SEQ ID NO:154. The construct further comprised a mouse CMV promoter with nucleotide sequence SEQ ID NO:16 operably linked to an open reading frame with amino acid sequence SEQ ID NO:155. The construct further comprised a third transcriptional unit expressing glutamine synthetase. The three transcriptional units were flanked by a pair of insulators: an HS4 insulator on one side and a D4Z4 core insulator on the other side. The three transcriptional units and the insulators were all placed into a Xenopus piggyBac-like transposon. On one side of the transposon was a 5'-TTAA'3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41), immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence.

[0264] Two additional transposon constructs were prepared, in which the hybrid promoter operably linked to the antibody heavy chain was replaced by either a murine CMV promoter with nucleotide sequence SEQ ID NO:16, or by a human CMV promoter with nucleotide sequence SEQ ID NO:13.

[0290]

[0265] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO cell line with no functional glutamine synthetase gene. Cells were grown in the absence of glutamine added to the media until their viability reached 95%. Recovered pools were then grown in a 7-day fed-batch using Sigma Advanced Fed Batch media. Antibody titers were measured in culture supernatant using an Octet. Table 9 shows the titers measured at day 7, and the specific productivities (calculated as the amount of antibody produced per cell per day).

[0291]

[0266] As shown in Table 9, the volumetric (Table 9 column D) and specific (Table 9 column C) productivities obtained using the hybrid promoter with nucleotide sequence comprising SEQ ID NQ:10 operably linked to the heavy chain open reading frame (Table 9 row 1) were higher than those obtained using either of the promoters from which the hybrid promoter was derived (Table 9 rows 2 and 3). We conclude that the hybrid human / murine CMV promoter comprising SEQ ID NQ:10 is advantageous for expression of heterologous proteins in CHO cells.

[0292] 10. Inducible promoters comprising tet binding sites 5' and 3' of the promoter in stably transfected Jurkat cells.

[0293]

[0267] We synthesized a series of fourteen transposons to test several configurations for a doxycycline-based dual inducible expression system, with the goal of reducing uninduced expression levels, while maintaining induced expression levels. Reduced basal expression is particularly important for inducible expression of toxic proteins such as AAV proteins, particularly Rep78, Rep68, Rep 52 and Rep 40, and lentiviral proteins such as gag / pol, and envelope proteins such as VSVg. The basic scheme for these construct designs are shown in Figs. 16A and B. Each Xenopus piggyBac-like transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'- TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a transcriptional unit comprising an HSV-TK promoter, an open reading frame encoding puromycin acetyl transferase, and an SV40 polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome. Each transposon also comprised an open reading frame encoding luciferase from Cypridina noctiluca, operably linked to an inducible promoter. Each transposon further comprised an expression system as shown schematically in Figs. 16 or 18, with components shown in Table 10.

[0294]

[0268] Transposons were co-transfected with mRNA encoding a corresponding transposase with amino acid sequence SEQ ID NO:54 into a Jurkat cell line. Cells were grown in the presence of 600 ng / ml puromycin added to the media until their viability reached 95%. Cultures were induced in a 96-well plate by the addition of doxycycline to 400 ng / ml, with a control culture for each cell pool to which no doxycycline was added. After doxycycline addition, cells were incubated for 22 hours at 37°C, when 20 pl of supernatant was removed from each well and added to 50 pl of VLAR buffer containing Vargulin substrate (from Targeting Systems). Luciferase activity was measured in a luminometer. The number of cells in each well of the plate were counted using flow cytometry and counting beads. This was used to adjust the relative light units measured in the luminometer to obtain relative light units per cell. Data is shown in Table 10.

[0295]

[0269] The expression systems shown in rows 1-4 of Table 10 comprised no tet operator between the minimal CMV promoter and the open reading frame encoding luciferase. These systems all showed similar levels of luciferase expression per cell after induction with 400 ng / ml doxycycline (Table 10 column M), as well as similar levels of luciferase expression per cell in the absence of doxycycline (Table 10 column L). In contrast, luciferase expression per cell in the absence of doxycycline induction was significantly reduced in several expression systems in which a tet operator was introduced to the 5' of the transcriptional initiation site of the minimal CMV promoter, by removing the 13 bases indicated in bold in Figs. 17A-B and fusing that truncated promoter sequence (with nucleotide sequence SEQ ID N0:309) with tet operator(s) (a single tet operator with nucleotide sequence SEQ ID NO:1 for the systems shown in rows 5-8, a pair of tet operators with nucleotide sequence SEQ ID NO:313 for the systems shown in rows 9-12).

[0296]

[0270] The reduction in uninduced expression was influenced by the other components of the expression system. Comparing the systems shown in rows 6-8 of Table 10, which all comprise a single tet operator between the minimal CMV promoter and the luciferase open reading frame, system 540120 has uninduced luciferase expression levels per cell that are around 10% of the levels for the otherwise identical system without the tet operator between the minimal CMV promoter and the luciferase open reading frame (system 540116): compare Table 10 column L row 2 with row 6. However the luciferase expression levels per cell induced by 400 ng / ml doxycycline are very similar for these two systems (compare Table 10 column M row 2 with row 6). Thus by reducing uninduced expression, the fold induction level is increased from around 10 to over 100.

[0297]

[0271] The uninduced luciferase expression levels per cell for systems 540121 and 540122 are between 5 and 7-fold higher than for system 540120 (compare Table 10 column L rows 7 and 8 with row 6). The primary difference between these is the promoter that is operably linked to the open reading frame encoding the tet repressor (shown in column G). The promoter with nucleotide sequence SEQ ID NO:312 is a stronger promoter than the promoter with nucleotide sequence SEQ ID NO:21. As a consequence, higher levels of tet repressor are expressed in systems with the promoter with nucleotide sequence SEQ ID NO:312. More tet repressor causes better occupancy of the expression-blocking tet operator between the minimal CMV promoter and the luciferase open reading frame and thus results in better repression of uninduced expression in system 540120 than systems 540121 and 540122.

[0298]

[0272] Uninduced expression levels were lower in system 540119 than in 540120 (compare Table 10 column L rows 5 and 6). The difference between these two systems is that while system 540120 resembles the one shown schematically in Figs. 16A-B, system 540119 has an additional blocking tet operator placed between the promoter and the operably linked modified tet repressor fused to a transcriptional activator, as shown in Figs. 18A-B. In this system, in addition to directly blocking transcription of the luciferase, the tet repressor blocks expression of the transcriptional activator, and the result is further reduction in uninduced expression. This feature of the expression systems is shown in Table 10 in column D: the unmodified promoter operably linked to the transcriptional activator has nucleotide sequence SEQ ID NO:17, while the promoter modified by addition of a tet operator has nucleotide sequence SEQ ID NQ:310.

[0299]

[0273] A similar pattern to that described above was seen for systems comprising two blocking tet operators between the minimal CMV promoter and the luciferase open reading frame (see Table 10 rows 9-12 and compare with rows 5-8 respectively). The lowest uninduced expression was again seen in the system with the additional blocking tet operator placed between the promoter and the operably linked modified tet repressor fused to a transcriptional activator, as shown in Figs. 18A, B; system 540123, Table 10 row 9. Two blocking tet operators between the minimal CMV promoter and the luciferase open reading frame reduced uninduced expression compared with a single blocking tet operators between the minimal CMV promoter and the luciferase open reading frame (compare Table 10 column L row 9 with row 5 and row 10 with row 6). However, in these systems, the expression induced by 400 ng / ml doxycycline were also about 2-fold lower than in the otherwise identical systems with only a single blocking tet operator between the minimal CMV promoter and the luciferase open reading frame (compare Table 10 column M row 9 with row 5 and row 10 with row 6). The performance of an equivalent system (540241) without any blocking tet operators, and without a third transcriptional unit expressing the tet repressor is shown in Table 10 row 13. This system has a 10-fold higher per cell expression level in the absence of doxycycline than systems 540119, 540120, 540123 and 540124 with blocking tet operators, with expression levels induced by 400 ng / ml doxycycline only as high as the most tightly repressed systems (540123 and 540124). Thus incorporation of one or two blocking operators between the minimal promoter and the open reading frame to be expressed, and of a blocking operator between the promoter and the operably linked modified repressor fused to a transcriptional activator reduced uninduced expression and improved induction ratios for gene expression.

[0300] 11. Inducible systems comprising tet binding sites 5' and 3' of the minimal promoter in stably transfected Jurkat cells.

[0301]

[0274] The Jurkat cells stably transfected with doxycycline-inducible expression systems described in Example 10 were tested for luciferase expression over a range of doxycycline concentrations as described in Example 10 except that doxycycline concentrations were varied. The relative luminescence divided by cell density is shown in Table 11. Results are consistent with those discussed in Example 10. The primary difference is that the maximal expression was obtained at doxycycline concentrations lower than 400 ng / ml. Thus the maximal expression for systems 540119 and 540120 were observed at 166 ng / ml with induction ratios of 194 and 126 respectively, the maximal expression for 540123 was at 55 ng / ml, and for 540124 at 18 ng / ml with induction ratios of 209 and 102 respectively.

[0302]

[0275] Examples 10 and 11 show the improved performance (lower uninduced expression) of a dual inducible system compared with a single inducible system. The dual inducible systems described in these examples included at least three elements, (a), (b) and (c). Element (a) comprised a minimal CMV promoter in operable linkage with first and second operator segments and a coding segment to be expressed (in these examples luciferase). The first operator segment (in these examples an array of 8 tet operators) was 5' to the promoter. The second operator segment (either 1 or 2 tet operators) was 13 bases upstream of the transcriptional start point of the minimal promoter. Element (b) comprised a promoter operably linked to an open reading frame encoding a modified tet repressor linked to a transcriptional activation domain. The modified tet repressor linked to the transcriptional activation domain bound to the first operator segment in the presence of a first inducer (in this example doxycycline). Element (c) comprised a promoter operably linked to an open reading frame encoding a tet repressor. The tet repressor bound to the operator segments in the absence of a second inducer (in these examples also doxycycline). In the absence of inducer (doxycycline), the repressor bound to its operator segments, blocking transcription of the coding segment by any basal activity of the minimal promoter, while the modified repressor did not bind to its operator segment thereby not inducing transcription. In the presence of doxycycline inducer, the tet repressor no longer bound to its operator segment relieving inhibition by the repressor. Doxycycline also caused the modified repressor to bind to its operator, thereby inducing expression. As a result of the dual control using two repressors even with only a single inducer, a greater differential between transcriptional level in the absence of inducer to its presence was obtained than the corresponding single inducible expression systems using the modified repressor and corresponding inducer by itself.

[0303]

[0276] Examples 10 and 11 also show that uninduced expression can be further reduced when element (b) comprised a promoter operably linked to an open reading frame encoding a modified tet repressor linked to a transcriptional activation domain, and wherein a tet operator was placed between the promoter and the open reading frame encoding a modified tet repressor linked to a transcriptional activation domain. In this case the repressor bound to this tet operator, blocking transcription of the open reading frame encoding a modified tet repressor linked to a transcriptional activation domain, and thereby reducing induced expression of the coding segment in (a).

[0304] 12. Inducible systems comprising a modified lac repressor.

[0305]

[0277] Three transposons were constructed to conditionally express GFP when induced by IPTG. Each Xenopus piggyBac-like transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID NO:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a first transcriptional unit comprising a PGK promoter, a first open reading frame encoding puromycin acetyl transferase, and an SV40 polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome. Each transposon also comprised a second transcriptional unit comprising an inducible promoter operably linked to a second open reading frame, encoding Dasher GFP (with amino acid sequence SEQ ID NO:32) and a bovine growth hormone polyadenylation signal suitable for expressing the second open reading frame in a mammalian cell. Each transposon further comprised a third transcriptional unit comprising a constitutive mammalian promoter operably linked to a third open reading frame encoding either a lac repressor or a modified lac repressor fused to a VP16 transcriptional activation domain, and a polyadenylation signal suitable for expressing the third open reading frame in a mammalian cell.

[0306]

[0278] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a CHO-K1 cell line. Cells were grown in the presence of 10 pg / ml puromycin until their viability reached 95%. Three cultures for each selected cell pool were then grown in EX-CELL® Advanced™ CHO Fed-batch Medium media at 37°C with 5% CO2, in an incubator with 25mm throw at 120 RPM. The cells were passaged at a density of 2x106 cells / ml 24 hours prior to induction. Cells were diluted to 6x106 cells / ml, and 800ul were aliquoted into 96 deep well plates. Cultures were induced by the addition of IPTG (Isopropyl R-D-l-thiogalactopyranoside) at either 0.1 or 1 mM, with a control to which no IPTG was added. Green fluorescent protein expression was measured by flow cytometry 24, 48 and 72 hours post-induction. The mean fluorescent intensity for each sample is shown in Table 12.

[0307]

[0279] In one transposon, 542059, the inducible promoter of the second transcriptional unit comprised a constitutive CMV promoter with nucleotide sequence SEQ ID N0:10 (which lacks the 12 bases upstream of the transcriptional initiation site) fused to an array of two lac operators, the array having nucleotide sequence SEQ ID NO:315. The third transcriptional unit comprised an EEF2 promoter operably linked to a lac repressor with amino acid sequence SEQ ID NO:184, fused at its C-terminus to a nuclear localization signal with amino acid sequence SEQ ID NO:316. In this transposon the lac repressor was constitutively expressed and bound to the operators in the second transcriptional unit, blocking transcription until IPTG addition. Table 12 row 4 shows that the uninduced expression of the GFP was low, and reached a maximum of 3,524 in cells induced for 72 hours at 1 mM IPTG.

[0308]

[0280] In a second transposon, 539433, the inducible promoter of the second transcriptional unit comprised an array of 6 lac operators with nucleotide sequence SEQ ID NO:317 fused to the 5' end of a minimal CMV promoter with nucleotide sequence SEQ ID NO:24. The third transcriptional unit comprised an EFla promoter operably linked to a modified lac repressor with amino acid sequence SEQ ID NO:185, fused at its C-terminus to a transcriptional activation domain with amino acid sequence SEQ ID NQ:40. In this transposon the modified lac repressor was constitutively expressed, and bound to the operators in the first transcriptional unit in the presence of IPTG. Table 12 row 5 shows that the uninduced expression of the GFP was higher than for transposon 542059, but the induced expression was also twice as high as for transposon 542059, reaching a maximum of 8,542 in cells induced for 48 hours at 1 mM IPTG.

[0309]

[0281] In a third transposon, 539432, the inducible promoter of the second transcriptional unit comprised an array of 6 lac operators with nucleotide sequence SEQ ID NO:317 fused to the 5' end of a minimal CMV promoter with nucleotide sequence SEQ ID NO:24. The third transcriptional unit comprised an EFla promoter operably linked to a modified lac repressor with amino acid sequence SEQ ID NO:185, fused at its C-terminus to a transcriptional activation domain with amino acid sequence SEQ ID NQ:40. The modified lac repressor in transposon 539432 was also fused at its N-terminus to a nuclear localization signal with amino acid sequence SEQ ID NO:316. In this transposon the modified lac repressor was constitutively expressed and bound to the operators in the first transcriptional unit in the presence of IPTG. Table 12 row 6 shows that the uninduced expression of the GFP was higher than for transposons 542059 and 539433, but the induced expression was also significantly higher than either of the others, reaching a maximum of 15,592 in cells induced for 72 hours at 1 mM IPTG.

[0310]

[0282] The data shown in Table 12 shows that an array of lac operators upstream of a minimal promoter may be used as part of an inducible system in combination with a modified lac repressor that has been "reversed" such that it binds to its operators in the presence of its ligand (in this case IPTG). The data in Example 12 shows that in the embodiment shown, uninduced expression is high. However by reducing expression of the modified repressor, and / or by implementing dual inducible strategies such as those described in Examples 10 and 11 and shown in Figs. 8A-B, 9A-B and 10A-B, we expect that uninduced expression can be substantially reduced without greatly reducing induced expression levels.

[0311] 13. Inducible systems comprising a modified lac repressor for expression of viral proteins.

[0312]

[0283] Transposons were constructed to conditionally express capsid proteins for adeno-associated virus serotype 2 (AAV2) when induced by either doxycycline or IPTG. Each Xenopus piggyBac-like transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'- TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a first transcriptional unit comprising a promoter, a first open reading frame encoding puromycin acetyl transferase, and a polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome. Promoter and polyadenylation signals were different in different transposons, details are shown in Table 13. Each transposon also comprised a second transcriptional unit comprising an inducible promoter operably linked to a polynucleotide comprising open reading frames encoding AAV2 capsid proteins VP1, 2 and 3 as a single open-reading frame with 3 alternative start sites (with nucleotide sequence SEQ ID NO:321) operably linked to a bovine growth hormone polyadenylation signal suitable for expressing the second open reading frame in a mammalian cell. This capsid open reading frame has the natural initiating codon AUG mutated to a threonine codon. Some transposons also included a fourth open reading frame encoding AAV2 replication protein Rep52, as indicated in Table 13. Each transposon further comprised a third transcriptional unit comprising a constitutive mammalian promoter operably linked to a third open reading frame encoding either a modified tet repressor or a modified lac repressor, in each case fused to a VP16 transcriptional activation domain, and a polyadenylation signal suitable for expressing the third open reading frame in a mammalian cell; details of differences in these features are shown in Table 13.

[0313]

[0284] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a suspension-adapted HEK cell line. Cells were grown in the presence of 1.5 pg / ml puromycin until their viability reached 95%. Cultures (20 ml) for each selected cell pool were then inoculated at 1 million cells per ml in Expi-293 Medium (ThermoFisher) at 37°C with 8% CO2, in an incubator with 25mm throw at 125 RPM. Cultures were induced by the addition of IPTG (Isopropyl R-D- 1-thiogalactopyranoside) to 1 mM, or doxycycline to 50 ng / ml as appropriate. Each pool was also grown in the absence of inducer as a control. Samples corresponding to 1 million cells were harvested 72 hours post-induction, pelleted and frozen at -80°C. Pellets of approximately 1 million cells were pelleted in a centrifuge and resuspended in 100 pl of water. An equal volume of 2x SDS sample buffer was added, the sample mixed thoroughly and then boiled at 100°C for 10 minutes. Samples were then centrifuged at 18,000 ref for 40 minutes at 4°C. Supernatant containing soluble cell lysate was carefully removed and 5 pl samples (corresponding to approximately 25,000 cells) were run on SDS polyacrylamide gels. Proteins were transferred from gels to nitrocellulose and detected using antibodies and horse radish peroxidase (HRP)-linked secondary antibodies. The Western shown in Fig. 19A was probed with Anti-AAV VP1 / VP2 / VP3 rabbit polyclonal (VP51), serum, PROGEN, Cat. N0:61084 as the primary and HRP Goat Anti- Mouse, Jackson, 115-035-003 as the secondary. The Western shown in Fig. 19B was probed with Monoclonal Mouse anti-Adeno-Associated Virus AAV2 Rep78 Antibody, Lifespan Bio, LS-C507794 as the primary and HRP Goat Anti- Mouse, Jackson, 115-035-003 as the secondary. Western blots are shown in Figs. 19A and B. Various dilutions of the soluble cell lysate from induced cells were tested for AAV capsid assembly using a commercial AAV2 titer capsid ELISA kit (Genscript, cat no L00942) according to the manufacturer's directions. Titers were measured at 3 different dilutions, and titer calculated by reference to a standard curve. Titer measurements are shown in column I of Table 13 (as assembled capsids per ml of original culture), and in column J (as assembled capsids per cell).

[0314]

[0285] Expression of AAV2 capsid proteins VP1, VP2 and VP3 are shown in Fig. 19A. Lanes 1 and 17 contained protein molecular weight markers, and the viral proteins are indicated with arrows. Lane 16 contained a standard made from AAV2 empty capsid. Lane 15 contained proteins made by cotransfection of a standard commercial plasmid carrying AAV2 rep cap genes with a standard commercial plasmid carrying adenoviral helper genes. Lane 14 contained untransfected HEK cells as a negative control. A summary of which lane in Fig. 19A corresponds to expression of which transposon is shown in Table 13, column I. Lanes 2-9 in Fig. 19A contained protein samples expressed from transposons wherein the inducible promoter of the second transcriptional unit comprised an array of 6 lacO operators with nucleotide sequence SEQ ID NO:178, operably linked to a minimal chimp CMV promoter with nucleotide sequence SEQ ID NO:24; the nucleotide sequence of the promoter comprising these elements was SEQ ID N0:180. Lanes 2 and 3 contained proteins expressed from transposon 546296. In this transposon the third transcriptional unit comprised an EFl promoter operably linked to an open reading frame encoding a modified lac repressor with amino acid sequence SEQ ID NO:185, fused to a VP16 transcriptional activator with amino acid sequence SEQ ID N0:40. The second transcriptional unit in transposon 546296 comprised an inducible promoter operably linked to a polynucleotide comprising open reading frames encoding AAV2 capsid proteins VP1, 2 and 3 fused to an internal ribosome entry site (IRES) with nucleotide sequence SEQ ID NO:329 which was operably linked to an open reading frame encoding AAV replication protein Rep52 (with amino acid sequence SEQ ID N0:220). The cells whose extracts were run in Fig. 19A lane 2 were grown in the absence of IPTG, the cells whose extracts were run in Fig. 19A lane 3 were grown in the presence of 1 mM IPTG. In lane 2, very little viral capsid protein expression is seen, just a faint band corresponding to the most highly expressed VP3 protein and a band corresponding to the background band seen in untransfected HEK cells (compare Fig. 19A lanes 2 and 14). In contrast, when these same cells were grown in the presence of 1 mM IPTG, strong expression of all 3 AAV viral capsid proteins was seen (Fig. 19A, lane 3); the ELISA titer assay showed that these cells produced 5.77 x 1012 AAV2 capsids per ml of culture +(Table 13 row 1 column I). A similar result was seen in Fig. 19B, a Western showing AAV Rep52 expression. Fig. 19B lanes 2 and 3 show the same samples as in Fig. 19A, again strong expression of the viral protein is seen in lane 3 (with IPTG induction) but not in lane 2 (cells grown without IPTG). This shows that a system for regulating expression comprising: a first transcriptional unit comprising a minimal promoter operably linked to lac operators and a heterologous coding segment and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a modified lac repressor fused to a transcriptional activator is inducible by IPTG, lactose or other lactose analog for the expression of AAV replication and capsid proteins from the heterologous coding segment.

[0315]

[0286] Fig. 19A lanes 6 and 7 contained proteins expressed from transposon 546299. Transposon 546299 comprised the same sequence elements as transposon 546296, except that this transposon lacked the IRES operably linked to the open reading frame encoding Rep52 (see Table 13, row 3). As with transposon 546296, cells stably transfected with transposon 546299 expressed very low levels of AAV capsid proteins in the absence of IPTG (Fig. 19A lane 6), but strongly expressed the viral capsid proteins when cells were induced with 1 mM IPTG (Fig. 19A lane 7) ; the ELISA titer assay showed that these cells produced 2.04 x 1012 AAV2 capsids per ml of culture (Table 13 row 3 column I).

[0316]

[0287] Fig. 19A lanes 4 and 5 contained proteins expressed from transposon 546297. Transposon 546297 comprised the same sequence elements as transposon 546296, except that the third transcriptional unit in transposon 546297 comprised an EEF2 promoter operably linked to an open reading frame encoding a nuclear localization signal with amino acid sequence SEQ ID NO:316, fused to a modified lac repressor with amino acid sequence SEQ ID NO:185, fused to a VP16 transcriptional activator with amino acid sequence SEQ ID NO:7. As with transposon 546296, cells stably transfected with transposon 546297 expressed very low levels of AAV capsid proteins in the absence of IPTG (Fig. 19A lane 4), but strongly expressed the viral capsid proteins when cells were induced with 1 mM IPTG (Fig. 19A lane 5); the ELISA titer assay showed that these cells produced 1.69 x 1012 AAV2 capsids per ml of culture (Table 13 row 2 column I). Also as with transposon 546296, cells stably transfected with transposon 546297 expressed very low levels of AAV replication protein Rep52 in the absence of IPTG (Fig. 19B lane 4), but strongly expressed Rep52 when cells were induced with 1 mM IPTG (Fig. 19B lane 5). Induced expression levels of viral proteins for transposon 546297 were slightly lower than for transposon 546296 (compare Fig. 19A lane 3 with lane 5, and Fig. 19B lane 3 with lane 5. This is likely because the EEF2 promoter operably linked to the modified lac repressor / transcriptional activator fusion in transposon 546297 is weaker than the EFl promoter used in transposon 546296.

[0288] Fig. 19A lanes 8 and 9 contained proteins expressed from transposon 546300. Transposon 546300 comprised the same sequence elements as transposon 546299, except that this transposon lacked the IRES operably linked to the open reading frame encoding Rep52 (see Table 13, row 4). As with transposon 546299, cells stably transfected with transposon 546300 expressed very low levels of AAV capsid proteins in the absence of IPTG (Fig. 19A lane 8), but strongly expressed the viral capsid proteins when cells were induced with 1 mM IPTG (Fig. 19A lane 9); the ELISA titer assay showed that these cells produced 6.99 x 1011 AAV2 capsids per ml of culture (Table 13 row 4 column I).

[0317]

[0289] Lanes 10-13 in Fig. 19A contained protein samples expressed from transposons wherein the inducible promoter of the second transcriptional unit comprised an array of 8 tet operators with nucleotide sequence SEQ ID NO:3, operably linked to a minimal chimp CMV promoter with nucleotide sequence SEQ ID NO:24; the nucleotide sequence of the promoter comprising these elements was SEQ ID NO:26. Lanes 10 and 11 contained proteins expressed from transposon 548582. In this transposon the third transcriptional unit comprised an EEF2 promoter operably linked to an open reading frame encoding a modified tet repressor with amino acid sequence SEQ ID NO:6, fused to a VP16 transcriptional activator with amino acid sequence SEQ ID NO:7. The second transcriptional unit in transposon 548582 comprised an inducible promoter operably linked to a polynucleotide comprising open reading frames encoding AAV2 capsid proteins VP1, 2 and 3. The cells whose extracts were run in Fig. 19A lane 10 were grown in the absence of doxycycline, the cells whose extracts were run in Fig. 19A lane 11 were grown in the presence of 50 ng / ml doxycycline. In lane 10, essentially no viral capsid protein expression is seen, just a faint band corresponding to the background band seen in untransfected HEK cells (compare Fig. 19A lanes 10 and 14). In contrast, when these same cells were grown in the presence of 50 ng / ml doxycycline, good expression of all 3 AAV viral capsid proteins was seen (Fig. 19A, lane 11) ; the ELISA titer assay showed that these cells produced 2.41 x 1012 AAV2 capsids per ml of culture (Table 13 row 5 column I).

[0318]

[0290] Fig. 19A lanes 12 and 13 contained proteins expressed from transposon 548584. Transposon 548584 comprised the same elements as transposon 548582, but it also comprised a fourth transcriptional unit comprising a tet-inducible promoter with nucleotide sequence SEQ ID NO:26 operably linked to an open reading frame encoding AAV replication protein Rep52 and a polyadenylation signal sequence. As with transposon 548582, cells stably transfected with transposon 548584 expressed very low levels of AAV capsid proteins in the absence of doxycycline (Fig. 19A lane 12), but strongly expressed the viral capsid proteins when cells were induced with 50 ng / ml doxycycline (Fig. 19A lane 13) ; the ELISA titer assay showed that these cells produced 6.03 x 1011 AAV2 capsids per ml of culture (Table 13 row 2 column I). Fig. 19B shows the Rep52 levels expressed from transposon 548584. In lane 6 of Fig. 19B, essentially no Rep52 protein expression is seen. In contrast, when these same cells were grown in the presence of 50 ng / ml doxycycline, good expression of Rep52 was seen (Fig. 19B, lane 7).

[0319]

[0291] The data shown here demonstrate that a system for regulating expression comprising: a first transcriptional unit comprising a minimal promoter operably linked to lac operators and a heterologous coding segment and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a modified lac repressor fused to a transcriptional activator is inducible by IPTG to yield comparable or greater levels of AAV viral capsid and replication proteins under the appropriate induction conditions as a doxycycline-based inducible system. The most productive IPTG-inducible transposon tested produced about 3 times as many viral capsids as the most productive doxycycline- inducible transposon tested. A conventional high titer transient AAV production system produces approximately 100,000 AAV capsids per ml. The lac-inducible systems shown here can produce up to 27- fold higher capsid levels.

[0320] 14. Amplification of AAV genomic DNA using inducible promoters comprising tet binding sites 5' and 3' of the promoter in stably transfected HEK cells.

[0321]

[0292] The DNA replicating proteins required to amplify the AAV genome are toxic to the host cells in which they are expressed. It is therefore important to repress their expression in the host cell prior to induction for viral production. In traditional AAV production, this is solved by introducing all genes needed for viral synthesis and assembly by transient transfection, so the host cells do not need to retain genes encoding the DNA replicating genes. In contrast, to build a stable producer cell line, the AAV replicating genes must be thoroughly repressed during cell growth and propagation, and then highly induced at the appropriate time. To test the suitability of the inducible systems of the present invention for this purpose we performed the work described in this example.

[0322]

[0293] A first transposon was constructed to introduce a synthetic AAV genome into the genome of a human cell (HEK 293). The first transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 329, (which is an embodiment of SEQ ID NO:117) immediately followed by a left transposon end with nucleotide sequence SEQ ID NO:119. On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NQ:120, immediately followed by a second ITR with nucleotide sequence SEQ ID NQ:330 (which is an embodiment of SEQ ID NO:118), immediately followed by a 5'-TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 insulator adjacent to the right transposon end. The first transposon further comprised a first transcriptional unit comprising a promoter, a first open reading frame encoding blasticidin-S-deaminase, and a polyadenylation signal sequence, such that constitutive expression of the blasticidin-S-deaminase confers resistance to a mammalian cell that has the transposon integrated into its genome. The first transposon further comprised a synthetic AAV genome. The synthetic AAV genome comprised a first AAV2 ITR with nucleotide sequence SEQ ID NO:332, followed by a first transcriptional unit comprising an open reading frame encoding GFP operably linked to a human CMV promoter and a bovine growth hormone polyadenylation sequence, followed by a stuffer sequence and a second AAV2 ITR with nucleotide sequence SEQ ID NO:333 such that the size of the AAV synthetic genome, including both ITRs was approximately 4.3 kb. The first transposon was co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:331, which is at least 90% identical to SEQ ID NO:121, into a suspension-adapted HEK cell line. Cells were grown in the presence of 2 pg / ml blasticidin until their viability reached 95%. Cells whose genome comprised the first transposon (the "synthetic AAV genome host" were frozen in liquid nitrogen and thawed for use as needed.

[0323]

[0294] A second and third transposon were constructed to conditionally express replication proteins for adeno-associated virus when induced by doxycycline. Each of these additional transposons was a Xenopus piggyBac-like transposon comprising, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID NO:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a first transcriptional unit comprising a promoter, a first open reading frame encoding puromycin acetyl transferase, and a polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome.

[0324]

[0295] The second transposon (539237) comprised a second transcriptional unit comprising a first doxycycline-inducible promoter comprising a first array of 8 tet operators operably linked to a first minimal chimp CMV promoter. The first doxycycline-inducible promoter of transposon 539237 was operably linked to a second open reading frame encoding a polypeptide comprising adenoviral protein E4ORF6 (with amino acid sequence SEQ ID NO:263) fused to a CHYSEL sequence with amino acid sequence SEQ ID NO:335 fused to adenoviral protein E2a with amino acid sequence SEQ ID NO:268. The second open reading frame was also operably linked to an SV40 polyadenylation signal. The second transposon (539237) further comprised a third transcriptional unit comprising a second doxycycline promoter comprising a second array of 8 tet operators operably linked to a second minimal chimp CMV promoter. The second doxycycline-inducible promoter of transposon 539237 was operably linked to a third open reading frame encoding AAV2 replication protein Rep68 with amino acid sequence SEQ ID NO:218. The third open reading frame was also operably linked to an SV40 polyadenylation signal. Because it is particularly toxic to cells when expressed, Rep68 expression was further attenuated by inserting 2 bases (5'-AT-3') between the Kozak sequence (5'-GCCACC-3') immediately preceding the Rep68 open reading frame and the open reading frame. This 2 base insertion typically reduces protein expression approximately 20-fold compared to expression of an otherwise identical construct in which the Kozak sequence is immediately juxtaposed with the initiating ATG of the open reading frame. This attenuation will thus reduce both uninduced and induced expression of Rep68 20-fold. Transposon 539237 further comprised a fourth transcriptional unit, comprising an open reading frame encoding a modified tet repressor fused to a transcriptional activator, operably linked to an EEF2 promoter and a beta globin polyadenylation signal such that the modified repressor was constitutively expressible in a mammalian cell. Transposon 539237 was thus configured as shown in Fig. 4 and Figs. 20A-Bs.

[0325]

[0296] The third transposon (546311) comprised a second transcriptional unit identical to that of 539237. The third transposon (546311) further comprised a third transcriptional unit comprising a second doxycycline promoter comprising an array of 8 tet operators operably linked to a second minimal chimp CMV promoter, wherein the second minimal chimp CMV promoter was truncated by removal of the bold bases as shown in Figs. 17A-B, and the truncated promoter was fused at its 3' end to a pair of tet operators with nucleotide sequence SEQ ID NO:313 (which is an embodiment of SEQ ID NO:2). The second doxycycline-inducible promoter of transposon 546311 was operably linked to a third open reading frame encoding AAV2 replication protein Rep68 with amino acid sequence SEQ ID NO:218. The third open reading frame was also operably linked to an SV40 polyadenylation signal. Because this third transposon (546311) comprised additional repression elements in the form of tet operators between the minimal promoter and the Rep68 open reading frame, Rep68 expression was further attenuated by inserting only a single base (A) between the Kozak sequence (5'-GCCACC-3') immediately preceding the Rep68 open reading frame and the open reading frame. This 1 base insertion typically reduces protein expression approximately 3-fold compared to expression of an otherwise identical construct in which the Kozak sequence is immediately juxtaposed with the initiating ATG of the open reading frame. This attenuation will thus reduce both uninduced and induced expression of Rep68 3-fold, so expression will be approximately 7-fold higher than from transposon 539237. Transposon 546311 further comprised a fourth transcriptional unit, comprising an open reading frame encoding a modified tet repressor fused to a transcriptional activator, operably linked to an EEF2 promoter and a beta globin polyadenylation signal such that the modified repressor was constitutively expressible in a mammalian cell. The fourth transcriptional unit further comprised a tet operator juxtaposed between the EEF2 promoter and the open reading frame. Transposon 546311 further comprised a fifth transcriptional unit, comprising an open reading frame encoding a tet repressor, operably linked to a PGK promoter and a beta globin polyadenylation signal such that the modified repressor was constitutively expressible in a mammalian cell. The configuration of the third transposon (546311) for expression of Rep68 was essentially as shown in Figs. 18A-B.

[0326]

[0297] The second and third transposons were each separately transfected into pools of cells whose genome comprised the first transposon. The second and third transposons were each cotransfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into synthetic AAV genome host. Cells were grown in the presence of 1.5 pg / ml puromycin until their viability reached 95%. Cultures for each selected cell pool were then inoculated at 1 million cells per ml in Expi-293 Medium (ThermoFisher) at 37°C with 8% CO2, in an incubator with 25mm throw at 125 RPM. Cultures were induced by the addition of doxycycline to 50 ng / ml or 300 ng / ml. Each pool was also grown in the absence of inducer as a control. Samples corresponding to 1 million cells were harvested 72 hours postinduction, pelleted and frozen at -80°C. Frozen pellets were thawed on ice and total DNA was extracted using the Monarch Genomic DNA Purification Kit from NEB according to manufacturer's protocol. Copy number of the GFP gene target sequence was determined by ddPCR using a primer and fluorescent probe set specific to that sequence. A primer and probe set specific to GAPDH was used as the genomic reference gene. Total DNA extracted from each sample was lOx serially diluted five times into gDNA from untransfected HEK cells. Droplet generation was carried out on a Biorad Automated Droplet Generator, PCR was completed on a DNA Engine thermocycler using the Supermix for ddPCR kit (BioRad), and droplets were read on a BioRad QX200 Droplet Reader. Values within the range of droplet quantification (typically 2-3 for each sample) were used to calculate the total copy number of target sequence per reference by linear regression analysis (e.g. LINEST function in Excel). The number of copies of AAV genome per cell is shown in Table 14.

[0298] Cells whose genomes comprised the first and second transposons, wherein the second transposon (539237) was configured as shown in Fig. 4 and Figs. 20A-B, resulted in a 615-fold AAV genome amplification after 72 hours when cells were treated with 50 ng / ml doxycycline (Table 14.

[0327] Column D row 2). When 300 ng / ml doxycycline was used, a slightly higher amplification (672-fold) was achieved. In contrast, cells whose genomes comprised the first and third transposons, wherein the third transposon (546311) was configured as shown in Figs. 18A-B, resulted in a 478-fold AAV genome amplification after 72 hours when cells were treated with 50 ng / ml doxycycline (Table 14. Column D row 5). When 300 ng / ml doxycycline was used, a substantially higher amplification (1,492-fold) was achieved. This effect is consistent with the results shown in Example 11, where systems 540241 and 540242 configured as in Fig. 4 gave maximal induction at lower doxycycline concentrations, while systems 540119 and 540123 configured as in Figs. 18A-B gave maximal induction at higher doxycycline concentrations (compare Table 11 rows 13 and 14 with Table 11 rows 5 and 9). In this Example 14, the benefit is seen as allowing lower attenuation of the Rep68 expression in the Figs. 18A-B configuration compared with the Fig. 4 / Figs. 20A-B configuration. The purpose of the attenuation is to prevent toxicity of Rep68 to the host cell under uninduced conditions. However greater general attenuation results in lower induced expression. As can be seen in Table 14, induction with 300 ng / ml doxycycline led to more than twice the genome amplification in the Figs. 18A-B configuration (transposon 546311) compared with the Fig. 4 / Figs. 20A-B configuration (transposon 539237), but the uninduced cells showed no differences in growth or viability, with no evidence of Rep68-mediated toxicity in either case.

[0328] 15. Inducible promoters comprising tet binding sites 5' and 3' of the promoter combined with different transactivator sequences in stably transfected Jurkat cells.

[0329]

[0299] We synthesized a series of thirteen transposons to test two of the configurations described in Examples 10 and 11 with different transactivator sequences. One set had a configuration as shown schematically in Figs. 20A-b (a single modified tet repressor fused to a viral transcriptional activator serving as a transactivator). The other set had a configuration as shown schematically in Figs. 16A-B (a dual tet repressor configuration). Each Xenopus piggyBac-like transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a transcriptional unit comprising a PGK promoter, an open reading frame encoding puromycin acetyl transferase, and an SV40 polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome. Each transposon also comprised an open reading frame encoding luciferase from Cypridina noctiluca, operably linked to an inducible promoter.

[0330]

[0300] Transposons were co-transfected with mRNA encoding a corresponding transposase with amino acid sequence SEQ ID NO:54 into a Jurkat cell line. Cells were grown in the presence of 600 ng / ml puromycin added to the media until their viability reached 95%. Cultures were induced in a 96-well plate by the addition of different concentrations of doxycycline, with a control culture for each cell pool to which no doxycycline was added. After doxycycline addition, cells were incubated for 22 hours at 37°C, when 20 pl of supernatant was removed from each well and added to 50 pl of VLAR buffer containing Vargulin substrate (from Targeting Systems). Luciferase activity was measured in a luminometer. The number of cells in each well of the plate were counted using flow cytometry and counting beads. This was used to adjust the relative light units measured in the luminometer to obtain relative light units per cell. Data is shown in Table 15.

[0331]

[0301] The expression systems shown in rows 1-7 of Table 15 comprised no tet operator between the minimal CMV promoter and the open reading frame encoding luciferase. With the exception of system 550310 these systems all showed similar levels of luciferase expression per cell after induction with 220 ng / ml doxycycline (Table 15 column L), as well as similar levels of luciferase expression per cell in the absence of doxycycline (Table 15 column U). We conclude that modified repressors with amino acid sequences corresponding to any of SEQ ID NOS:322 to 327 may be fused to a transcriptional activation domain (e.g., of VP16 with amino acid sequence SEQ ID NO:7) as part of a doxycycline- inducible expression system.

[0332]

[0302] As seen in Examples 10 and 11, luciferase expression per cell in the absence of doxycycline induction was significantly reduced in expression systems which further comprised a transcriptional unit expressing an unmodified tet repressor, and in which a tet operator was introduced to the 5' of the transcriptional initiation site of the minimal CMV promoter, by removing the 13 bases indicated in bold in Figs. 17A-B and fusing that truncated promoter sequence (with nucleotide sequence SEQ ID NQ:309) with a tet operator with nucleotide sequence SEQ ID NO:1; these are shown in Table 15 rows 8-12. Compare row 12 with row 3; row 11 with row 6; row 9 with row 4 and row 10 with row 5. In all cases, uninduced expression was reduced more than 2-fold, and higher concentrations of doxycycline were required for maximal induction. However maximal induction levels were comparable with levels seen in the absence of the additional tet operator and unmodified tet repressor, so the induction level was substantially improved (Table 15 column X). We conclude that modified repressors with amino acid sequences corresponding to any of SEQ ID NOS:322 to 327 may be used as part of a doxycycline- inducible expression system, for example as schematically represented in Figs. 18A-B.

[0333] 16. Modification of translation initiation context in an inducible system comprising a modified lac repressor for expression of viral proteins.

[0334]

[0303] AAV capsid formation typically requires a higher expression of the smallest protein VP3 than the other two proteins. Expression of VP1 is also critical for viral infectivity. VP1 and VP2 are not required for capsid assembly but the VP3 sequence alone is necessary to form the vector required to transport the therapeutic gene, although an increase in the expression of VP1 and VP2 has been described to generate a higher vector yield. VP1 however, is important for transduction based on the presence of the NLS and PLA2 domains and insufficient VP1 will lead to a reduced rate in vector transduction. Super-expression of VP1 at a ratio of 1.9:0.1:8 (VP1:VP2:VP3, respectively) led to the production of morphologically similar capsids as those composed from standard triple plasmid infection methods. The super VP1 capsids also show higher transduction than its wild-type AAV counterpart (Worner, T.P. et. al. Adeno-associated virus capsid assembly is divergent and stochastic. Nat Commun 12, 1642 (2021)). Naturally AAV2 balances expression of VP1 with VP2 and VP3 by differential splicing, with the minor intron acceptor occurring immediately before and the major intron acceptor occurring shortly after the VP1 initiation codon. In the constructs described in Example 13, the AAV2 intron donor was removed and instead the VP1 initiation codon was changed to a non-canonical ACG start, which is the same codon used naturally by AAV2 to initiate VP2. The 9 bases upstream of the ACG codon at the start of VP2 (with nucleotide sequence SEQ ID NO:337) were duplicated to provide a similar initiation context for VP1. As can be seen in Fig. 19A, however, VP1 expression is significantly lower than VP2 expression. We therefore tested modifications of the nucleotide sequence around the VP1 initiation codon to increase expression of VP1 relative to VP2.

[0335]

[0304] Transposons were constructed to conditionally express capsid proteins for adeno-associated virus serotype 2 (AAV2) when induced by IPTG. Each Xenopus piggyBac-like transposon comprised, on one side of the transposon a 5'-TTAA'-3' target integration sequence, immediately followed by a first ITR with nucleotide sequence 318, (which is an embodiment of SEQ ID NO:41) immediately followed by a left transposon end with nucleotide sequence SEQ ID N0:320 (which is at least 95% identical to SEQ ID NO:44). On the other side of the transposon was a right transposon end with nucleotide sequence SEQ ID NO:45, immediately followed by a second ITR with nucleotide sequence SEQ ID NO:319 (which is an embodiment of SEQ ID NO:42), immediately followed by a 5'-TTAA'3' target integration sequence. Each transposon comprised an HS4 insulator adjacent to the left transposon end and a D4Z4 core insulator adjacent to the right transposon end. Each transposon further comprised a first transcriptional unit comprising a promoter, a first open reading frame encoding puromycin acetyl transferase, and a polyadenylation signal sequence, such that constitutive expression of the puromycin acetyl transferase confers resistance to a mammalian cell that has the transposon integrated into its genome. Each transposon further comprised a second transcriptional unit comprising a constitutive mammalian promoter operably linked to a third open reading frame encoding a modified lac repressor (with amino acid sequence SEQ ID NO:185) fused to a VP16 transcriptional activation domain, and a polyadenylation signal suitable for expressing the third open reading frame in a mammalian cell.

[0336]

[0305] Each transposon also comprised a third transcriptional unit comprising an inducible promoter (with nucleotide sequence SEQ ID NQ:180) operably linked to a polynucleotide comprising open reading frames encoding AAV2 capsid proteins VP1, 2 and 3 operably linked to a bovine growth hormone polyadenylation signal suitable for expressing the third open reading frame in a mammalian cell. This capsid protein-encoding sequence comprised the following silent mutations: codon 4 (aspartate) changed from GAT to GAC to remove an out-of-frame methionine, codon 7 (leucine) changed from CTT to CTG to reduce the polypyrimidine tract upstream of the normal capsid major intron splice acceptor, codon 8 (proline) changed from CCA to CCT to remove the natural capsid major intron splice acceptor; thus the open reading frame encoding codons 2-8 of VPl had nucleotide sequence SEQ ID NO:338 instead of the natural nucleotide sequence SEQ ID NO:337. In this context we tested four different initiation contexts: transposon 552133 comprised 12 bases repeated from the 9 bases upstream of plus the ACG initiation codon of VP2, with nucleotide sequence SEQ ID NO:340; transposon 552218 comprised the same 12 bases, but with a G to C substitution immediately before the ACG (i.e. at position -1), so the initiation context comprised nucleotide sequence SEQ ID NO:341; transposon 552219 comprised a G to C substitution immediately before the ACG (i.e. at position -1) and an A to C substitution at position -2, so the initiation context comprised nucleotide sequence SEQ ID NO:342; finally transposon 552226 comprised the same 12 bases as transposon 552218, but with the initiation codon changed from ACG to CTG, so the initiation context comprised nucleotide sequence SEQ

[0337] ID NO:343.

[0306] Transposons were co-transfected with mRNA encoding transposase with amino acid sequence SEQ ID NO:54 into a suspension-adapted HEK cell line. Cells were grown in the presence of 1.5 pg / ml puromycin until their viability reached 95%. Cultures (20 ml) for each selected cell pool were then inoculated at 1 million cells per ml in Expi-293 Medium (ThermoFisher) at 37°C with 8% CO2, in an incubator with 25mm throw at 125 RPM. Cultures were induced by the addition of IPTG (Isopropyl β-D- 1-thiogalactopyranoside) to 1 mM. Each pool was also grown in the absence of inducer as a control. Samples corresponding to 1 million cells were harvested 72 hours post-induction, pelleted and frozen at -80°C. Pellets of approximately 1 million cells were pelleted in a centrifuge and resuspended in 100 μl of water. An equal volume of 2x SDS sample buffer was added, the sample mixed thoroughly and then boiled at 100°C for 10 minutes. Samples were then centrifuged at 18,000 ref for 40 minutes at 4°C. Supernatant containing soluble cell lysate was carefully removed and 5 pl samples (corresponding to approximately 25,000 cells) were run on SDS polyacrylamide gels. Proteins were transferred from gels to nitrocellulose and detected using antibodies and horse radish peroxidase (HRP)-linked secondary antibodies. The Western shown in Fig. 21 was probed with Anti-AAV VP1 / VP2 / VP3 rabbit polyclonal (VP51), serum, PROGEN, Cat. N0:61084 as the primary and HRP Goat Anti- Mouse, Jackson, 115-035-003 as the secondary. Western bands corresponding to VP1 and VP2 were quantified by comparison with purchased standard VP1 and VP2 proteins, the concentrations of viral proteins in the cell extract are shown in Table 16 columns C and D respectively.

[0338]

[0307] Expression of AAV2 capsid proteins VP1, VP2 and VP3 are shown in Fig. 21. Lane 1 contained protein molecular weight markers, and the viral proteins are indicated with arrows. A summary of which lane in Fig. 21 corresponds to expression of which transposon is shown in Table 16, column F. By manipulating the nucleotide sequence immediately surrounding the VP1 initiation codon we were able to change the expression balance from one favoring VP2 (Table 16 rows 1-3 and Fig. 21 lanes 2-4) to one favoring VP1 (Table 16 row 4 and Fig. 21 lane 5). The main factor that led to this switch involved changing from an ACG to a CUG initiation codon. However552219 in which both the -1 and -2 positions were changed to C increased the ratio of VP1 expression relative to VP2 from 0.83 to 0.91 (compare Table 16 rows 1 and 3), so we expect that these effects will be additive and that using an initiation context with nucleotide sequence selected from SEQ ID NOS:344-347 will further increase the ratio of expression in favor of VP1. XIII. TABLES DESCRIPTION

[0339]

[0308] Table 1. DNA constructs comprising two transcriptional units were prepared as described in Example 3. Each construct comprised a first transcriptional unit comprising an inducible promoter whose name is given in column A, operably linked to an open reading frame encoding a green fluorescent protein. The SEQ ID NO giving the nucleotide sequence of the inducible promoter, lacking the 12 bp before the transcriptional start site is shown in column B, and the SEQ ID NO giving the nucleotide sequence of the inducible promoter including the pair of tet operators is shown in column C. Each construct further comprised a second transcriptional unit comprising a constitutive promoter operable in a mammalian cell, operably linked to an open reading frame encoding a tet repressor with amino acid sequence SEQ ID NO:5. This promoter name is shown in column D, and the SEQ ID NO giving the nucleotide sequence of the promoter linked to the tet repressor is shown in column E. Each construct was transfected into HEK 293 cells in 3 independent transfections, induced with doxycycline and cell numbers and fluorescence were measured as described in Example 1. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns F to U. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2.

[0340]

[0309] Table 2. DNA constructs comprising two transcriptional units were prepared as described in Example 1. Each construct comprised a first transcriptional unit comprising an inducible promoter whose name is given in column A, operably linked to an open reading frame encoding a green fluorescent protein. The SEQ ID NO giving the nucleotide sequence of the inducible promoter, lacking the 12 bp before the transcriptional start site is shown in column B, and the SEQ ID NO giving the nucleotide sequence of the inducible promoter including the pair of tet operators is shown in column C. Each construct further comprised a second transcriptional unit comprising a constitutive promoter operable in a mammalian cell, operably linked to an open reading frame encoding a tet repressor with amino acid sequence SEQ ID NO:5. This promoter name is shown in column D, and the SEQ ID NO giving the nucleotide sequence of the promoter linked to the tet repressor is shown in column E. Each construct was transfected into CHO-s cells in 3 independent transfections, induced with doxycycline and cell numbers and fluorescence were measured as described in Example 2. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns F to U. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2.

[0341]

[0310] Table 3. DNA transposons comprising two transcriptional units were prepared as described in Example 1. Each transposon comprised a first transcriptional unit comprising an inducible promoter whose name is given in column A, operably linked to an open reading frame encoding a green fluorescent protein. The SEQ ID NO giving the nucleotide sequence of the inducible promoter, lacking the 12 bp before the transcriptional start site is shown in column B, and the SEQ ID NO giving the nucleotide sequence of the inducible promoter including the pair of tet operators is shown in column C. Each transposon further comprised a second transcriptional unit comprising a constitutive promoter operable in a mammalian cell, operably linked to an open reading frame encoding a tet repressor with amino acid sequence SEQ ID NO:5. This promoter name is shown in column D, and the SEQ ID NO giving the nucleotide sequence of the promoter linked to the tet repressor is shown in column E. Transposons were stably transfected into CHO cells and selected, after recovery three separate cultures for each stable cell line were induced with doxycycline and cell numbers and fluorescence were measured as described in Example 3. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number shown in columns F to Z. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2.

[0342]

[0311] Table 4. DNA transposons comprising two transcriptional units were prepared as described in Example 4. Each transposon comprised a first transcriptional unit comprising an inducible promoter whose name is given in column A, operably linked to an open reading frame encoding a green fluorescent protein. The SEQ ID NO giving the nucleotide sequence of the inducible promoter, lacking the 12 bp before the transcriptional start site is shown in column B, and the SEQ ID NO giving the nucleotide sequence of the inducible promoter including the pair of tet operators is shown in column C. Each transposon further comprised a second transcriptional unit comprising a constitutive promoter operable in a mammalian cell, operably linked to an open reading frame encoding a tet repressor with amino acid sequence SEQ ID NO:5. This promoter name is shown in column D, and the SEQ ID NO giving the nucleotide sequence of the promoter linked to the tet repressor is shown in column E. The promoter sequences indicated in column E in rows 6 and 7 also include the 5'UTR including the Kozak sequence immediately before the translational initiation site for the tet repressor. Transposons were stably transfected into CHO cells and selected, after recovery three separate cultures for each stable cell line were induced with doxycycline and cell numbers and fluorescence were measured as described in Example 4. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number and is shown in columns F to W. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2.

[0343]

[0312] Table 5. DNA constructs comprising two transcriptional units were prepared as described in Example 5. Each construct comprised a first transcriptional unit comprising a minimal promoter whose name is given in column A, with nucleotide sequence given by the SEQ ID NO shown in column C. The 5' of the minimal promoter was fused to an array of tet operators with nucleotide sequence given by the SEQ ID NO shown in column B. The 3' of the minimal promoter was fused to a 5' UTR with nucleotide sequence given by the SEQ ID NO shown in column D. The 5'UTR was joined to an open reading frame encoding Dasher GFP, with amino acid sequence SEQ ID NO:32. Each construct further comprised a second transcriptional unit comprising a constitutive SV40 promoter operable in a mammalian cell, operably linked to an open reading frame encoding a modified tet repressor fused to a VP16 transcriptional activation domain, the amino acid sequence of the open reading frame given by the SEQ ID NO in column E. Each construct was transfected into HEK 293 cells in 3 independent transfections, induced with doxycycline and cell numbers and fluorescence were measured as described in Example 5. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns F to U. Days postinduction are shown in row 1, doxycycline concentrations are shown in row 2.

[0344]

[0313] Table 6. DNA constructs comprising two transcriptional units were prepared as described in Example 5. Each construct comprised a first transcriptional unit comprising a minimal promoter whose name is given in column A, with nucleotide sequence given by the SEQ ID NO shown in column C. The 5' of the minimal promoter was fused to an array of tet operators with nucleotide sequence given by the SEQ ID NO shown in column B. The 3' of the minimal promoter was fused to a 5' UTR with nucleotide sequence given by the SEQ ID NO shown in column D. The 5'UTR was joined to an open reading frame encoding Dasher GFP, with amino acid sequence SEQ ID NO:32. Each construct further comprised a second transcriptional unit comprising a constitutive SV40 promoter operable in a mammalian cell, operably linked to an open reading frame encoding a modified tet repressor fused to a VP16 transcriptional activation domain, the amino acid sequence of the open reading frame given by the SEQ ID NO in column E. Each construct was transfected into CHO-S cells in 3 independent transfections, induced with doxycycline and cell numbers and fluorescence were measured as described in Example 6. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns F to Q. Days postinduction are shown in row 1, doxycycline concentrations are shown in row 2.

[0345]

[0314] Table 7. DNA transposons comprising two transcriptional units were prepared as described in Example 7. Each transposon comprised a first transcriptional unit comprising a minimal promoter whose name is given in column A, with nucleotide sequence given by the SEQ ID NO shown in column C. The 5' of the minimal promoter was fused to an array of tet operators with nucleotide sequence given by the SEQ ID NO shown in column B. The 3' of the minimal promoter was fused to a 5' UTR with nucleotide sequence given by the SEQ ID NO shown in column D. The 5'UTR was joined to an open reading frame encoding Dasher GFP, with amino acid sequence SEQ ID NO:32. Each construct further comprised a second transcriptional unit comprising a constitutive SV40 promoter operable in a mammalian cell, operably linked to an open reading frame encoding a modified tet repressor fused to a VP16 transcriptional activation domain, the amino acid sequence of the open reading frame given by the SEQ ID NO in column E. Transposons were stably transfected into CHO cells and selected, after recovery three separate cultures for each stable cell line were induced with doxycycline and cell numbers and fluorescence were measured as described in Example 7. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns F to Q. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2.

[0346]

[0315] Table 8. DNA transposons comprising two transcriptional units were prepared as described in Example 8. Each transposon comprised a first transcriptional unit comprising a minimal chimpanzee promoter whose 5' end was fused to an array of tet operators with nucleotide sequence given by the SEQ ID NO shown in column A. The promoter was operably linked to an open reading frame encoding Dasher GFP, with amino acid sequence SEQ ID NO:32. Each construct further comprised a second transcriptional unit comprising a constitutive SV40 promoter operable in a mammalian cell, operably linked to an open reading frame encoding a modified tet repressor fused to a VP16 transcriptional activation domain, the amino acid sequence of the open reading frame given by the SEQ ID NO in column E. Transposons were stably transfected into CHO cells and selected, after recovery three separate cultures for each stable cell line were induced with doxycycline and cell numbers and fluorescence were measured as described in Example 8. Fluorescence was measured at 525 nm and cell density was measured as optical density at 600 nm, with each condition measured for 3 independent transfections. The average fluorescence at 525 nm for each set of 3 replicates was divided by optical density at 600 nm for each set of 3 replicates to provide an average measure of fluorescence normalized by cell number, shown in columns B to U. Days post-induction are shown in row 1, doxycycline concentrations are shown in row 2. Control cells with no integrated transposon are shown in row 6.

[0347]

[0316] Table 9. DNA transposons comprising two transcriptional units were prepared as described in Example 9. Each transposon comprised a first transcriptional unit comprising a mouse CMV promoter operably linked to an open reading frame encoding an antibody light chain. Each transposon further comprised a second transcriptional unit comprising a promoter named in column A and with nucleotide given by the SEQ ID NO in column B, operably linked to an open reading frame encoding an antibody heavy chain. Transposons were stably transfected into CHO cells and selected as described in Example 9. The specific productivity of each CHO cell pool is shown in column C, the volumetric productivity is shown in column D.

[0348]

[0317] Table 10. DNA transposons were prepared as described in Example 10. Each transposon comprised a first transcriptional unit comprising a tet operator array with nucleotide sequence SEQ ID NO:3 fused to the 5' end of a minimal chimp CMV promoter operably linked to an open reading frame encoding Cypridina noctoluca luciferase and followed by a bovine growth hormone polyadenylation signal sequence. The nucleotide sequence of the minimal chimp CMV promoter is given by the SEQ ID NO. shown in column B. In some transposons this promoter was fused at its 3' end to a tet operator or a tet operator array with nucleotide sequence given by the SEQ ID NO. in column C. Each transposon further comprised a second transcriptional unit comprising a promoter with nucleotide sequence given by the SEQ ID NO in column D, operably linked to an open reading frame encoding a modified tet repressor with amino acid sequence given by the SEQ ID NO. in column E. The modified repressor was fused at its C-terminus to a VP16 transcriptional activator with amino acid sequence given by the SEQ ID NO. in column F. The open reading frame encoding the modified repressor / transcriptional activator was followed by a rabbit beta globin polyadenylation signal sequence. Some transposons further comprised a third transcriptional unit comprising an open reading frame encoding a tet repressor with amino acid sequence given by SEQ ID NO in column H. The open reading frame encoding the tet repressor was operably linked to a promoter and 5' UTR with nucleotide sequence given by the SEQ ID NO. in column G. Transposons were stably transfected into Jurkat cells, selected and induced with 400 ng / ml doxycycline as described in Example 10. The luminescence (in relative light units) of each uninduced cell pool is shown in column I, the luminescence of each cell pool after induction with doxycycline for 22 hours is shown in column J. The number of Jurkat cells per microliter of culture is shown in column K. Column L shows the relative light units per cell for the uninduced Jurkat pools (obtained by dividing column I by column K); column M shows the relative light units per cell for the induced Jurkat pools (obtained by dividing column J by column K). Column N shows the fold-induction obtained, that is the relative light units from the induced culture divided by the relative light units from the uninduced culture. Column A shows the transposon / expression system identifier.

[0349]

[0318] Table 11. DNA transposons were prepared as described in Example 10 and as shown in Table 10. Column A shows the transposon / expression system identifier, Sequence components are as for Table 10. Transposons were stably transfected into Jurkat cells, selected and induced with doxycycline concentrations from 8 to 500,000 pg / ml. The luminescence (in relative light units) of each cell pool was determined after 22 hours of induction. The luminescence divided by the number of cells per pl in that pool, is shown in columns B-M. The doxycycline concentrations (in pg / ml) are shown in row 15. The minimum luminescence divided by cells per pl is shown in column O. The maximum luminescence divided by cells per pl is shown in column N. The induction ratio (column N divided by column O) is shown in column P.

[0350]

[0319] Table 12. DNA transposons were prepared as described in Example 12. Transposon name is shown in column A. The lac operator array to the 5' of the inducible promoter had nucleotide sequence of the SEQ ID NO. given in column B. The inducible promoter had nucleotide sequence of the SEQ ID NO. given in column C. The lac operator array to the 3' of the inducible promoter had nucleotide sequence of the SEQ ID NO. given in column D. The lac repressor or modified lac repressor of the third transcriptional unit had amino acid sequence of the SEQ ID NO. in column F. The lac repressor or modified lac repressor was fused to a nuclear localization signal with amino acid sequence of the SEQ ID NO. in column E. The modified lac repressor was fused to a transcriptional activator with amino acid sequence of the SEQ ID NO. in column G. Mean fluorescent intensity (MFI) of FACS analyzed cells is shown in columns H-P. The IPTG concentration used is shown in row 2. The time post-induction is shown in row 1. Row 3 shows the MFI of untransfected CHO-K1 cells.

[0320] Table 13. DNA transposons were prepared as described in Example 13. Transposon name is shown in column A. The type of inducible promoter is shown in column B, and the nucleotide sequence of the inducible promoter (operator array fused to the minimal promoter) is given by the SEQ ID NO. shown in column C. The promoter operably linked to the third open reading frame (encoding the modified repressor-transcriptional activator fusion) is shown in column D. The modified repressor- transcriptional activator fusion encoded in the transposon had the amino acid sequence obtained by concatenating the amino acid sequences for the SEQ ID NOS. shown in column E. Some transposons further comprised an open reading frame encoding AAV2 protein Rep52, as shown in column F. The promoter and polyadenylation sequences operably linked to the puromycin-acetyl transferase-encoding open reading frame are shown in columns G and H respectively. The capsid titer measured by ELISA is shown in column I (in capsids per ml of culture), capsid titer in capsids per cell is shown in column J. The corresponding lanes showing expressed proteins from the transposons in Figs. 19A and 19B are indicated in columns K and L respectively.

[0351]

[0321] Table 14 DNA transposons were prepared as described in Example 14, transfected into a host HEK cell whose genome comprised a transposon comprising an AAV genome, and selected on puromycin. The transposon name is shown in column A. Following recovery from selection, cell [pools were induced with the doxycycline concentration shown in column B. After 72 hours, cell pellets were collected, and the AAV genome copy# per cell was measured in each sample, the AAV genome copy numbers are shown in column C. Column D shows the amplification, calculated as the final copy number per cell divided by the initial copy number per cell, this is shown in column D.

[0352]

[0322] Table 15. DNA transposons were prepared as described in Example 15. Each transposon comprised a first transcriptional unit comprising a tet operator array with nucleotide sequence SEQ ID NO:3 fused to the 5' end of a minimal chimp CMV promoter operably linked to an open reading frame encoding Cypridina noctoluca luciferase and followed by a bovine growth hormone polyadenylation signal sequence. The nucleotide sequence of the minimal chimp CMV promoter is given by the SEQ ID NO. shown in column C. In some transposons this promoter was fused at its 3' end to a tet operator with nucleotide sequence given by the SEQ ID NO. in column D. Each transposon further comprised a second transcriptional unit comprising a promoter with nucleotide sequence given by the SEQ ID NO in column E, operably linked to an open reading frame encoding a modified tet repressor with amino acid sequence given by the SEQ ID NO. in column F. The modified repressor was fused at its C-terminus to a VP16 transcriptional activator with amino acid sequence given by the SEQ ID NO. in column G. The open reading frame encoding the modified repressor / transcriptional activator was followed by a rabbit beta globin polyadenylation signal sequence. Some transposons further comprised a third transcriptional unit comprising an open reading frame encoding a tet repressor with amino acid sequence given by SEQ ID NO in column I. The open reading frame encoding the tet repressor was operably linked to a promoter and 5' UTR with nucleotide sequence given by the SEQ ID NO. in column H. Transposons were stably transfected into Jurkat cells, selected and induced with the concentration of doxycycline indicated in row 14. The luminescence (in relative light units) divided by the number of cells per pl in that pool, is shown in columns J-U. The minimum luminescence divided by cells per pl is shown in column W. The maximum luminescence divided by cells per pl is shown in column V. The induction ratio (column W divided by column V) is shown in column X. Column A shows the transposon / expression system identifier. Column B shows the Fig. in which the induction system is schematically shown.

[0353]

[0323] Table 16. DNA transposons were prepared as described in Example 16. Transposon name is shown in column A. The SEQ ID NO corresponding to the 12 bases of the initiation context: that is the 9 bases immediately to the 5' of the initiation codon plus the initiation codon, is shown in column B. The calculated concentration of VPl in the cell extract is shown in column C; the calculated concentration of VP1 in the cell extract is shown in column D; the ratio of these concentrations is shown in column E. The corresponding lanes showing expressed proteins from the transposons in Fig. 21 is indicated in column

[0354] F.

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369] TABLE 14

[0370]

[0371]

[0372] TABLE 16

[0373]

[0324] All publications, patents and patent applications, accession numbers, websites and the like mentioned in this specification are incorporated by reference to the same extent as if each individual publication, patent or patent application was so individually denoted. To the extent different content is associated with an accession number or other reference at different times, the content in effect as of the effective filing date of this application is meant. The effective filing date is the date of the earliest priority application disclosing the accession number in question. Unless otherwise apparent from the context any element, embodiment, step, feature or aspect of the invention can be performed in combination with any other.

[0374] Ill

Claims

What is claimed is:Dual expression system1. A dual inducible expression system comprising: a minimal promoter in operable linkage with first and second operator segments, and a heterologous coding segment; a first promoter operably linked to a segment encoding a modified repressor linked to a transcriptional activation domain (modified repressor fusion), which binds to the first operator segment in the presence of a first inducer inducing expression of the heterologous coding segment; and a second promoter operably linked to a segment encoding a repressor, which binds to the second operator segment in the absence a second inducer repressing transcription of the heterologous coding segment; wherein in the presence of the first and second inducer expression of the heterologous coding segment is induced.

2. The dual inducible expression system of claim 1, further comprising a third operator segment operably linked to the first promoter and the segment encoding the modified repressor fusion, wherein in the absence of the second inducer, the repressor represses expression of the modified repressor fusion by binding to the third operator segment, and in the presence of the second inducer expression of the modified repressor fusion is expressed, which in the presence of the first inducer, induces expression of the heterologous coding segment.

3. The dual inducible expression system of claim 1, wherein (a), (b) and (c) are separate transcriptional units.

4. The dual inducible expression system of any preceding claim, wherein the first operator segment is 5' to the minimal promoter.

5. The dual inducible expression system of any one of claims 1-4, wherein the second operator segment is about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

6. The dual inducible expression system of any one of claims 1-5, wherein the third operator segment is between about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the first promoter.

7. The dual inducible expression system of any one of claims 1-6, wherein the second and third operator segments have the same sequence.

8. The dual inducible expression system of any preceding claim, wherein the first and / or second and / or third operator segment comprises an array of operators separated by spacers.

9. The dual inducible expression system of any preceding claim, wherein the first and second inducers are different.

10. The dual inducible expression system of any one of claims 1-8, wherein the first and second inducers are the same.

11. The dual inducible expression system of any preceding claim, wherein the first and second promoter are the same type of promoter.

12. The dual expression system of any preceding claim, wherein the repressor is any of a tet repressor, cumate repressor or lac repressor and the modified repressor is any of a modified tet repressor, modified cumate repressor or modified lac repressor.

13. A contiguous nucleic acid comprising the dual inducible expression system of any preceding claim.

14. The contiguous nucleic acid of claim 10, which is a transposon in which the dual inducible expression system is flanked by inverted repeats of the transposon.

15. A cell comprising the contiguous nucleic acid of claim 13 or 14.

16. A method of inducing expression of a heterologous coding segment, comprising culturing a cell comprising the dual expression system of any one of claims 1-9; and supplying the first and second inducers to the culture, wherein expression is induced.Minimal chimpanzee promoter, tet repressor or modified tet repressor linked to transcriptional activation domain, activation in presence of tet17. A transposon comprising a first transcriptional unit comprising a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, operably linked to at least two tet operators, and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a tet repressor effective to bind a tet operator in the absence of tetracycline or doxycycline or a modified-tet repressor effective to bind a tet operator in the presence of tetracycline or doxycycline, wherein the tet repressor or the modified tet repressor is fused to atranscriptional activation domain, and the first and second transcriptional units are flanked by inverted repeats of the transposon.Minimal chimpanzee promoter, cumate repressor or modified cumate repressor linked to transcriptional activation domain, activation in presence of cumate18. A transposon comprising a first transcriptional unit comprising a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated, operably linked to at least one cumate operator and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a cumate repressor effective to bind a cumate operator in the absence of cumate or encoding a modified cumate repressor effective to bind a cumate operator in the presence of cumate, wherein the cumate repressor or the modified cumate repressor is fused to a transcriptional activation domain and the first and second transcriptional units are flanked by inverted repeats of the transposon.

19. The transposon of claim 17 or 18, wherein the second transcriptional unit further comprises a polyadenylation sequence.

20. The transposon of claim 17, wherein the tet repressor has an amino acid sequence comprising SEQ ID NO:

521. The transposon of claim 17, wherein the modified tet repressor has an amino acid sequence comprising SEQ ID NO:6.

22. The transposon of claim 18, wherein the cumate repressor has an amino acid sequence comprising SEQ ID NQ:170.

23. The transposon of claim 18, wherein the modified cumate repressor linked to the transcriptional activation domain has an amino acid sequence comprising SEQ ID NO:172.

24. The transposon of claim 17 or 18, wherein the minimal chimpanzee promoter is operably linked to two or more heterologous coding segments separated by an IRES sequence or sequence encoding a CHYSEL peptide.

25. The transposon of claim 24, wherein the two or more heterologous coding segments encode two or more proteins of a viral packaging system.

26. The transposon of claim 25, wherein the two or more heterologous coding segments encode each required protein of a viral packaging system.

27. The transposon of any one of claims 17-26, which is a piggyBac or piggyBac-like transposon.

28. A cell transformed with the transposon of any one of claims 17-27.

29. The cell of claim 28, which is mammalian.

30. A non-human animal transformed with the transposon of any one of claims 17- 27.

31. The non-human animal of claim 30, which is transgenic.

32. A cell or nonhuman transgenic animal having a genome comprising a transposon comprising (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least two tet operators and a heterologous coding segment encoding a protein of viral packaging system, and (b) a promoter operably linked to a tet repressor fused to a transcriptional activation domain or modified tet repressor fused to transcriptional activation domain, wherein expression of the heterologous coding segment can be regulated by supplying tetracycline or doxycycline, or other tetracycline analog to the cell or nonhuman transgenic animal, wherein (a) and (b) are flanked by inverted repeats of the transposon.

33. A cell or nonhuman transgenic animal having a genome comprising a transposon comprising (a) a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated operably linked to at least one cumate operator and a heterologous coding segment encoding a protein of a viral packaging system, and (b) a promoter operably linked to a segment encoding cumate repressor linked to a transcriptional activation domain or a modified cumate repressor fused to transcriptional activation domain, wherein expression of the heterologous coding segment can be regulated by supplying cumate to the cell or nonhuman transgenic animal, wherein (a) and (b) are flanked by inverted repeats of the transposon.

34. A method for inducible expression of a heterologous coding segment comprising; providing a first transcriptional unit comprising in operable linkage with a least two tet operators, a promoter having a sequence comprising SEQ ID NO:24, and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with which SEQ ID NO:24 is naturally associated and a heterologous coding segment encoding a protein of a viral packaging system, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a tet repressor fused to a transcriptional activation domain or a modified tetrepressor fused to a transcriptional activation domain, wherein the tet repressor is expressed and in the absence of tetracycline or doxycycline, the tet repressor fused to the transcriptional activation domain binds to the at least two tet operators and expression of the heterologous coding segment is increased, or the modified tet repressor fused to the transcriptional activation domain is expressed and in the presence of tetracycline or doxycycline, the modified tet repressor fused to the transcriptional activation domain binds to the at least two tet operators and expression of the heterologous coding segment is increased, wherein the first and second transcriptional units are flanked by inverted repeats of the transposon.

35. A method for inducible expression of a heterologous coding segment comprising; providing a first transcriptional unit comprising in operable linkage with a least one cumate operator, a promoter having a nucleotide sequence comprising SEQ ID NO:24 and a heterologous coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding a cumate repressor fused to a transcriptional activation domain or a modified cumate repressor fused to a transcriptional activation domain, wherein the cumate repressor fused to the transcriptional activation domain is expressed and in the absence of cumate the cumate repressor binds to the at least one cumate operator and expression of the heterologous coding segment is increased, or the modified cumate repressor fused to the transcriptional activation domain is expressed and in the presence of tetracycline or doxycycline the modified cumate repressor binds to the at least one cumate operator and expression of the heterologous coding segment is increased, wherein the first and second transcriptional units are flanked by inverted repeats of the transposon.

36. The method of claim 34 or 35, wherein the first and second transcriptional units are components of the same contiguous DNA molecule.

37. The method of claim 36, wherein the transposon is a piggyBac or piggyBac-like transposon.

38. The method of any one of claims 34-37, further comprising introducing the transposon into a cell.

39. The method of claim 38, wherein the cell is a mammalian cell.

40. The method of any one of claims 34-39, wherein the first and second transcriptional units integrate into the genome of the cell.

41. The method of claim any one of claims 34-40, further comprising culturing the cell.

42. The method of claim 41, further comprising supplying tetracycline or doxycycline or other tetracycline analog to culture media of the cell.

43. The method of claim 41, further comprising supplying cumate or an analog to culture media of the cell.Modified lac repressor, minimal promoter, activation in presence of lactose44. A system for regulating expression, comprising: a first transcriptional unit comprising a minimal promoter operably linked to at least two lac operators and a heterologous coding segment and a second transcriptional unit comprising a second promoter operably linked to a segment encoding a modified lac repressor fused to a transcriptional activator, wherein in the presence of lactose or an analog thereof, the modified lac repressor fused to a transcriptional activator binds to the at least two lac operators and induces expression of the heterologous coding segment.

45. The system of claim 44, wherein the promoter has a sequence comprising SEQ ID NO:24 and no more than ten contiguous nucleotides on the 5' side of SEQ ID NO:24 from a natural chimpanzee CMV promoter with SEQ ID NO:24 is naturally associated.

46. The system of claim 44, wherein the promoter lacks a 5' flanking sequence from a natural chimpanzee CMV promoter with which it is naturally associated.

47. The system of any one of claims 44-46, wherein the first and second lac operators are 5' to the promoter.

48. The system of any one of claims 44-47, wherein the first transcriptional unit comprises three, six or eight lac operators 5' to the promoter.

49. The system of claim 48, wherein the three, six or eight lac operators are separated by spacers of 10-25 nucleotides.

50. The system of claim 49, wherein at least some of the spacers differ from one another.

51. The system of any one of claims 44-50, wherein the first transcriptional unit further comprises a segment encoding a 5' UTR.

52. The system of claim 51, wherein the segment encoding the 5' UTR has a sequence comprising SEQ ID NO:29.

53. The system of any one of claims 44-52, wherein the second transcriptional unit further comprises a polyadenylation sequence.

54. The system of any one of claims 44-53, wherein the heterologous coding segment encodes a protein.

55. The system of claim 54, wherein the protein is a protein of a viral packaging system.

56. The system of any one of claims 44-55, wherein the minimal promoter is operably linked to two or more heterologous coding segments separated by an IRES sequence or sequence encoding a CHYSEL peptide.

57. The system of claim 56, wherein the two or more heterologous coding segments encode two or more proteins of a viral packaging system.

58. The system of claim 57, wherein the two or more heterologous coding segments encode each required protein of a viral packaging system.

59. The system of claim 44, wherein the protein is a therapeutic protein or a membrane protein.

60. The system of any one of claims 44-59, wherein the first and second transcriptional units are with the same contiguous nucleic acid.

61. A transposon comprising the system of any one of claims 44-60, flanked by inverted repeats of the transposon.

62. The transposon of claim 61, further comprising target sites flanking the inverted repeats.

63. The transposon of claim 61 or 62, which is a piggyBac or piggyBac-like transposon.

64. A cell transformed with the system or transposon of any one of claims 44-63.

65. The cell of claim 64, which is mammalian.

66. A non-human animal transformed with the transposon of any one of claims 61- 64.

67. The non-human animal of claim 66, which is transgenic.

68. A cell or nonhuman transgenic animal having a genome comprising (a) a minimal promoter operably linked to at least two lac operators and a heterologous coding segment, and (b) a promoter operably linked to modified lac repressor fused to transcriptional activator, wherein expression of the coding segment can be induced by supplying lactose or an analog thereof to the cell or nonhuman transgenic animal.

69. A method for inducible expression of a heterologous coding segment comprising providing a first transcriptional unit comprising a minimal promoter operably linked to a least two lac operators, and a heterologous coding segment, and a second transcriptional unit comprising in operable linkage a promoter, and a segment encoding modified lac repressor fused to a transcriptional activator, wherein the modified lac repressor fused to the transcriptional activator is expressed and in the presence of lactose or an analog thereof, the modified lac repressor fused to the transcriptional activator binds to the at least two lac operators and expression of the heterologous coding segment is increased.

70. The method of claim 69, wherein the first and second transcriptional units are components of the same contiguous DNA molecule.

71. The method of claim 70, wherein the first and second transcriptional units are components of a transposon.

72. The method of claim 71, wherein the transposon is a piggyBac or piggyBac-like transposon.

73. The method of any one of claims 69-72, further comprising introducing the contiguous DNA molecule into a cell.

74. The method of claim 73, wherein the cell is a mammalian cell.

75. The method of any one of claims 69-74, wherein the first and second transcriptional units integrate into the genome of the cell.

76. The method of claim 75, further comprising culturing the cell.

77. The method of claim 76, further comprising supplying lactose or an analog to culture media of the cell.

78. A nucleic acid comprising, from 5' to 3', (i) an array of at least 2 lac operators, at least one cumate operator or at least two tet operators, (ii) a minimal mammalian promoter having a sequence selected to comprise any of SEQ ID NOS:307-309 and no more than 60 contiguous nucleotides on the 5' side of the selected SEQ ID NO. with which the selected SEQ ID NO. is naturally associated, (iii) a heterologous coding segment and (iv) a polyadenylation signal.

79. The nucleic acid of claim 78, wherein the minimal mammalian promoter comprises the selected SEQ ID NO. and no more than 25 or 10 contiguous nucleotides on the 5' side of the selected SEQ ID NO. with which the selected SEQ ID NO. is naturally associated.

80. The nucleic acid of claim 78, wherein the minimal mammalian promoter lacks any contiguous sequence on the 5' side of the selected SEQ ID NO. with which the selected SEQ ID NO. is naturally associated.

81. The nucleic acid of claim 78, wherein the heterologous coding segment encodes a protein of a viral packaging system.

82. The nucleic acid of claim 78, further comprising at least one lac operator, at least one cumate operator or at least one tet operator between about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

83. The nucleic acid of claim 78, wherein element (i) is an array of at least three, six or eight lac operators, and the nucleic acid further comprises at least one lac operator between about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

84. The nucleic acid of claim 78, wherein element (i) is an array of 2-10 cumate operators, and the nucleic acid further comprises at least one cumate operator between about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

85. The nucleic acid of claim 78, wherein element (i) is an array of 2-10 tet operators, and the nucleic acid further comprises at least one tet operator between about 20 bases upstream to about 50 bases downstream of the transcriptional start site of the minimal promoter.

86. A nucleic acid for expression of adeno-associated virus proteins VP1, VP2 and VP3 comprising overlapping coding segments for VP1, VP2 and VP3 operably linked to a promoter, wherein the initiation codon for the segment encoding VPl is CUG.

87. The nucleic acid of claim 87, wherein -1 and -2 positions to the initiation codon are occupied by C.

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