WO2004056964A2

WO2004056964A2 - Vectors for inducible rna interference

Info

Publication number: WO2004056964A2
Application number: PCT/US2003/040548
Authority: WO
Inventors: William Winston; Ronan C. O'hagan; Joerg Heyer; William Rideout; Bijan Etemad-Moghadam
Original assignee: Genpath Pharmaceuticals, Incorporated
Priority date: 2002-12-18
Filing date: 2003-12-18
Publication date: 2004-07-08
Also published as: WO2004056964A3; AU2003299732A1; AU2003299732A8

Abstract

Recombinant vectors for inducibly expressing double-stranded RNA molecules that interfere with the expression of a target gene.

Description

VECTORS FORINDUCIBLE RNA INTERFERENCE

BACKGROUND OF THE INVENTION

RNA interference (RNAi) has been used to silence the expression of a target gene. RNAi is a sequence-specific posttranscriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA). It causes degradation of mRNAs homologous in sequence to the dsRNA. The mediators of the degradation are 21- to 23 -nucleotide small interfering RNAs (siRNAs) generated by cleavage of longer dsRNAs (including hairpin RNAs) by DICER, a ribonuclease Ill-like protein. Molecules of siRNA typically have 2- to 3-nucleotide 3' overhanging ends resembling the RNAse III processing products of long dsRNAs that normally initiate RNAi. When introduced into a cell, they assemble with an endonuclease complex (RNA-induced silencing complex), which then guides target mRNA cleavage. As a consequence of degradation of the targeted mRNA, cells with a specific phenotype of the suppression of the corresponding protein product are obtained (e.g., reduction of tumor size, metastasis, angiogenesis, and growth rates). The small size of siRNAs, compared with traditional antisense molecules, prevents activation of the dsRNA-inducible interferon system present in mammalian cells. This helps avoid the nonspecific phenotypes normally produced by dsRNA larger than 30 base pairs in somatic cells. See, e.g., Elbashir et al, Methods 26:199-213 (2002); McManus and Sharp, Nature Reviews 3:737-747 (2002); Hannon, Nature 418:244-251 (2002); Brummelkamp et al., Science 296:550-553 (2002); Tuschl, Nature Biotechnology 20:446-448 (2002); U.S. Application US2002/0086356 Al; WO 99/32619; WO 01/36646; and WO 01/68836.

SUMMARY OF THE INVENTION This invention features recombinant vectors for expressing double- stranded RNA (dsRNA) molecules in a controllable mam er and cells and animals comprising the vectors. These dsRNA molecules interfere with (i.e., inhibit) the expression of a target gene, particularly a disease-related gene such as an oncogene or a tumor suppressor gene. In another aspect, the invention provides a modified nucleic acid molecule encoding a tetracycline repressor (TetR), for use in mammalian cells .

The vectors of this invention can be used to express the interfering dsRNAs at a desired time point to study the biological functions of a target gene in vitro or in vivo. Inducible expression of RNAi also has potential uses where RNAi is used as a therapeutic intervention to control the point of initiation and duration of expression of the therapeutic RNAi. The vectors can also be used to assess the toxicity of the dsRNAs in vivo, i.e., to determine whether expression of a given dsRNA has side effects in nontargeted cells and tissues and/or whether inhibition of a target gene causes undesired physiological problems.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this specification, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers .

Other features and advantages of this invention will be apparent from the description below. BRLEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a drawing illustrating the construction of a vector system (constructs J-P, infra) that contains a tetracycline-inducible system for expressing an RNAi sequence, ⁽'χχχχχ" denotes a stretch of nucleotide T that serves as a transcription tenninator sequence.

Fig. 2 A is a drawing illustrating several constructs (constructs A-E, infra) that contain an IPTG-inducible system for expressing an RNAi sequence. "TTTTT" denotes a stretch of nucleotide T that serves as a transcription terminator sequence. Fig. 2B is a bar graph showing that modified human U6 promoters containing two Lac operator sequences (constructs C-E) are more strongly repressed by Lad than modified human U6 promoters containing only one Lac operator sequence (constructs A and B). "FFl" denotes an RNAi-encoding sequence that targets luciferase gene expression. Fig. 2C is a bar graph showing that constructs A-E retain wildtype

Lac operator function.

Fig. 3 A is a diagram illustrating constructs F, G, H, and I (infra) of this invention.

Fig. 3B is a bar graph showing that removal of the LoxP-Stop-LoxP cassette in constructs F-I allows efficient transcription of the FFl coding sequence. Figs. 3 C-E are drawings illustrating another construct that contains a Cre-inducible system for expressing an RNAi sequence. Upon activation of the Cre recombinase, the LoxP sites recombine, deleting the intervening STOPPER sequence. Fig. 4 A is a bar graph showing that constructs J-P (infra) of this invention retain wildtype Tet operator function.

Fig. 4B is a bar graph showing that modified human U6 promoters containing a TetO are repressed by TetR.

Fig. 5 A is a bar graph showing that a modified human U6 promoter containing a TetO is repressed in a stable setting by a codon optimized TetR.

Fig. 5B is a line graph showing that RNAi of luciferase in xenograft tumors expressing (1) luciferase, (2) a luciferase shRNA from a modified human U6 promoter TetO, and (3) a codon optimized TetR can be regulated by doxycycline.

Fig. 6 is a diagram illustrating a combined system of this invention, in which the LoxP system is combined with the TetO or LacO system. Fig. 7 is a diagram illustrating the use of the Cre-Lox system to switch expression from one RNAi-encoding sequence (shRNA) to a second.

DETAILED DESCRIPTION OF THE INVENTION This invention features recombinant vectors containing inducible systems for expressing dsRNA molecules that interfere with expression of target genes, including disease-related genes (e.g., cancer-related genes such as oncogenes and tumor suppressor genes). These vectors can be based on plasmids or viruses such as retroviruses (e.g., Moloney amphotropic murine virus), adenoviruses, and lentiviruses.

The vectors of this invention can be delivered into host cells via a variety of methods, including but not limited to, liposome fusion (transposomes), infection by viral vectors, and routine nucleic acid transfection methods such as electroporation, calcium phosphate precipitation and microinjection. Host cells include cultured cells and cells in an animal. In some embodiments, the vectors are integrated into the genome of a transgenic animal (e.g., a mouse, a rabbit, a hamster, or a nonhuman primate). Diseased or disease-prone cells containing these vectors can be used as a model system to study the development, maintenance, or progression of a disease that is affected by the presence or absence of the interfering RNA.

This model system can also be used to identify other disease-related elements. For instance, a detailed expression profile of gene expression in tumors undergoing regression or regrowth due to the expression or nonexpression of the interfering RNA can be established. Techniques used to establish the profile include the use of suppression subtraction (in cell culture), differential display, proteomic analysis, serial analysis of gene expression (SAGE), and expression/transcription profiling using cDNA and/or oligonucleotide microarrays. Then, comparisons of expression profiles at different stages of cancer development can be performed to identify genes whose expression patterns are altered. Animals harboring the vectors of this invention can also be used to identify surrogate biomarkers for diagnosis or for following disease progression in patients. The biomarkers can be identified based on the differences between the expression profiles of the "on" and "off" states in the animal model. Blood or urine samples from the animal can be tested with ELISAs or other assays to determine which biomarkers are released from the diseased tissue (e.g., tumor) into circulation during genesis, maintenance, or regression of the disease. These biomarkers are particularly useful clinically in following disease progression post RNAi therapy or post-drug therapy which targets the same gene as the RNAi. These biomarkers can also be used clinically to assess the toxicity of any such therapy.

I. DESIGN OF VECTOR INSERTS

Useful interfering RNAs can be designed with a number of software programs, e.g., the OligoEngine siRNA design tool available at http://www.oligoengme.com. The siRNAs of this invention may range about, e.g., 19-29 basepairs in length for the double-stranded portion. In some embodiments, the siRNAs are hairpin RNAs having an about 19-29 bp stem and an about 4-34 nucleotide loop. Preferred siRNAs are highly specific for a region of the target gene and may comprise any about 19-29 bp fragment of a target gene mRNA that has at least one , preferably at least two or three, bp mismatch with a nontarget gene-related sequence. In some embodiments, the preferred siRNAs do not bind to RNAs having more than 3 mismatches with the target region.

Intracellular transcription of dsRNAs can be achieved by cloning the dsRNA-encoding sequences into RNA polymerase III (Pol III) transcription units, which normally encode the small nuclear RNA U6 or the human RNAse P RNA HI, or into RNA polymerase I (Pol I) or II (Pol II) transcription units (e.g., units containing a CMV promoter). However, it will be appreciated that in the vectors of the invention, the dsRNA-encoding sequences may be operably linked to a variety of other promoters. In some embodiments, the promoter is a convergent RNA polymerase III prompter, e.g., a convergent U6 snRNA promoter (Tran et al, BMC Biotechnology 3:21(2003)); a type II tRNA promoter such as the tRNA^va promoter and the tRNA^met promoter. These promoters may also be modified to increase promoter activity. In addition, enhancers can be placed near the promoter to enhance promoter activity. For example, an enhancer from the CMV promoter can be placed near the U6 promoter to enhance U6 promoter activity (Xia et al., Nuc Acids Res 31 (2003)). Exemplary inducible Pol II systems are available from Invitrogen, e.g., the GeneSwitch™ and T-Rex™ systems.

Two approaches can be used for expressing dsRNA: (1) sense and antisense strands constituting the dsRNA duplex are transcribed by individual promoters; or (2) dsRNAs are expressed as fold-back stem-loop structures (hairpms) that give rise to dsRNAs after intracellular processing. Inducible transcription-regulatory elements are inserted into the promoter region for controlled expression of the dsRNAs. See, e.g., discussions below on the tetracycline-inducible, LPTG-inducible, and Cre-inducible Pol Ill-based transcription units. For Pol I- or Pol LI-based transcription units, well-established inducible systems such as tetracycline transactivator systems, reverse tetracycline transactivator systems, and ecdysone systems can be used. However, it will be appreciated that for controlled expression of the dsRNAs, other operators that are controlled by a small molecule are useful in the vectors of the invention.

An exemplary human U6 transcription unit has the following sequence:

LOCUS SP6-U6 genomic 860 bp DNA FEATURES Location/Qualifiers snRNA 529..635

/label=U6 transcript enhancer 286..317

/label=Distal Sequence Element misc_binding 63..482

/label=Proximal Sequence Element TATA_signal 498..506 /label=TATA Box terminator 631..635

/label=Transcriptional Termination Signal promoter 1..18

/label=SP6 promoter misc_feature 51..528

/label=U6 promoter misc feature 529..529 /label=Start of Transcription misc_feature 51..860

/label=U6 genomic fragment misc_feature 457.-462 /label=NdeI

BASE COUNT 227 a 195 c 205 g 233 t ORIGIN

1 atttaggtga cactatagaa tacaagcttg gctgcaggtc gacggatccc cccgagtcca 61 acacccgtgg gaatcccatg ggcaccatgg cccctcgctc caaaaatgct ttcgcgtcgc

121 gcagacactg ctcggtagtt tcggggatca gcgtttgagt aagagcccgc gtctgaaccc

181 tccgcgccgc cccggcccca gtggaaagac gcgcaggcaa aacgcaccac gtgacggagc

241 gtgaccgcgc gccgagcgcg cgccaaggtc gggcaggaag agggcctatt tcccatgatt

301 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact 361 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag

421 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag

481 tatttcgatt tcttggc|ttt atatat|cttg tggaaaggac gaaacaccgt gctcgcttcg

541 gcagcacata tactaaaatt ggaacgatac agagaagatt agcatggccc ctgcgcaagg

601 atgacacgca aattcgtgaa gcgttccata tttttacatc aggttgtttt tctgttttta 661 catcaggttg tttttctgtt tggttttttt tttacaccac gtttatacgc cggtgcacgg

721 tttaccactg aaaacacctt tcatctacag gtgatatctt ttaacacaaa taaaatgtag

781 tagtcctagg agacggaata gaaggaggtg gggcctaggc agattcatct ctgcggtgca

841 ttttgcctct ggccctcggg (SEQ ID Nθ:l)

In this sequence, the U6-transcript sequence is underlined, with the PSE italicized and the transcription initiation site G double-underlined. The promoter region spans from nucleotide 51 to the nucleotide immediately preceding the initiating G. The TATA box in the promoter region is boxed. Luukkonen et al., RNA 4:231 -8 (1998). To construct an RNAi vector, the U6-transcri.pt sequence can be replaced in part or in its entirety by a sequence encoding an interfering dsRNA. In some embodiments, it may be preferred that the spacing between the PSE and the TATA box and the spacing between the TATA box and the GTG site are maintained for proper transcription. The cytosine (C) immediately preceding the GTG site and the purine immediately following this site may also be preserved for proper start of transcription. Goomer et al, Nucleic Acids Research 20:4903-12 (1992).

II. A TETRACYCYLINE-INDUCIBLE SYSTEM

Fig. 1 illustrates one vector system of this invention (see Working Examples). To construct this vector, a Tet operator sequence (TetOp) is inserted into the promoter region of the vector. TetOp is preferably inserted between the PSE and the transcription initiation site, upstream or downstream from the TATA box. hi some embodiments, the TetOp is immediately adjacent to the TATA box. The expression of the RNAi molecule is thus under the control of tetracycline (or doxycycline, or any other tetracycline analogue). Addition of tetracycline relieves repression of the promoter by a tetracycline repressor that the host cells are also engineered to express. Since the tetracycline repressor is derived from bacteria, its coding sequence may be optionally modified to adapt to the codon usage by mammalian transcriptional systems and to prevent methylation. In some embodiments, the host cells comprise (i) a first expression construct containing a gene encoding a tetracycline repressor operably linked to a first promoter, such as any tissue or cell type-specific promoter or any general promoter, and (ii) a second expression construct containing the dsRNA-coding sequence operably linked to a second promoter that is regulated by the tetracycline repressor and tetracycline. Administration of tetracycline or an analogue thereof (e.g., doxycycline) results in expression of the dsRNA in a manner dictated by the tissue specificity of the first promoter.

in. A LAC OPERATOR SYSTEM

Fig. 2 A illustrates yet another vector system of this invention (see Working Examples). To construct this vector, a Lac operator sequence (LacO) is inserted into the promoter region. The LacO is preferably inserted between the PSE and the transcription initiation site, upstream or downstream of the TATA box. In some embodiments, the LacO is immediately adjacent to the TATA box. The expression of the RNAi molecule is thus under the control of IPTG (or any analogue thereof). Addition of IPTG relieves repression of the promoter by a Lac repressor (i.e., the Lad protein) that the host cells are also engineered to express. Since the Lac repressor is derived from bacteria, its coding sequence may be optionally modified to adapt to the codon usage by mammalian transcriptional systems and to prevent methylation. In some embodiments, the host cells comprise (i) a first expression construct containing a gene encoding a Lac repressor operably linked to a first promoter, such as any tissue or cell type- specific promoter or any general promoter, and (ii) a second expression construct containing the dsRNA-coding sequence operably linked to a second promoter that is regulated by the Lac repressor and IPTG. Administration of JJPTG results in expression of dsRNA in a manner dictated by the tissue specificity of the first promoter.

IV. A LOXP-STOP-LOXP SYSTEM

Figs. 3 A-E illustrate yet another vector system of this invention. The RNAi vector of this system contains a LoxP-Stop-LoxP cassette before the hairpin (Fig. 3 A) or within the loop of the hairpin (Figs. 3 C-E). Any suitable stop sequence for the promoter can be used in the cassette. One version of the LoxP- Stop-LoxP system for Pol LI is described in, e.g., Wagner et al, Nucleic Acids Research 25:4323-4330 (1997). The "Stop" sequences (such as the one described in Wagner, supra, or a run of five or more T nucleotides) in the cassette prevent the RNA polymerase III from extending an RNA transcript beyond the cassette. Upon introduction of a Cre recombinase, however, the LoxP sites in the cassette recombine, removing the Stop sequences and leaving a single LoxP site. Removal of the Stop sequences allows transcription to proceed through the hairpin sequence, producing a transcript that can be efficiently processed into an open-ended, interfering dsRNA. Thus, expression of the RNAi molecule is induced by addition of Cre. In some embodiments, the host cells contain a Cre-encoding transgene under the control of a constitutive, tissue-specific promoter. As a result, the interfering RNA can only be inducibly expressed in a tissue-specific manner dictated by that promoter. Tissue-specific promoters that can be used include, without limitation: a tyrosinase promoter or a TRP2 promoter in the case of melanoma cells and melanocytes; an MMTV or WAP promoter in the case of breast cells and/or cancers; a Villin or FABP promoter in the case of intestinal cells and/or cancers; a RLP promoter in the case of pancreatic beta cells; a Keratin promoter in the case of keratinocytes; a Probasin promoter in the case of prostatic epithelium; a Nestin or GFAP promoter in the case of CNS cells and/or cancers; a Tyrosine Hydroxylase, SI 00 promoter or neurofilament promoter in the case of neurons; the pancreas-specific promoter described in Edlund et al. Science 230:912-916 (1985); a Clara cell secretory protein promoter in the case of lung cancer; and an Alpha myosin promoter in the case of cardiac cells. Cre expression also can be controlled in a temporal manner, e.g., by using an inducible promoter, or a promoter that is temporally restricted during development such as Pax3 or Protein O (neural crest), Hoxal (floorplate and notochord), Hoxb6 (extraembryonic mesoderm, lateral plate and limb mesoderm and midbrain-hindbrain junction), Nestin (neuronal lineage), GFAP (astrocyte lineage), Lck (immature thymocytes). Temporal control also can be achieved by using an inducible form of Cre. For example, one can use a small molecule controllable Cre fusion, for example a fusion of the Cre protein and the estrogen receptor (ER) or with the progesterone receptor (PR). Tamoxifen or RU486 allow the Cre-ER or Cre-PR fusion, respectively, to enter the nucleus and recombine the LoxP sites, removing the LoxP Stop cassette. Mutated versions of either receptor may also be used. For example, a mutant Cre-PR fusion protein may bind RU486 but not progesterone. Other exemplary Cre fusions are a fusion of the Cre protein and the glucocorticoid receptor (GR). Natural GR ligands inlcude corticosterone, cortisol, and aldosterone. Mutant versions of the GR receptor, which respond to, e.g., dexamethasone, triamcinolone acetonide, and or RU38486, mya also be fused to the Cre protein. V. CELLS AND ANIMALS

This invention also provides nonhuman transgenic animals whose somatic and germ cells contain an inducible RNAi contsruct of this invention (including both heterozygotes and homozygotes). Such animals can be used to study the effect of the RNAi coding sequence on tumorigenicity and tumor development, to study the role of the targeted gene in normal tissue development and differentiation, and to screen for and establish toxicity profiles of anti-cancer drugs. Also included are chimeric animals that can be used to generate the transgenic animals. The non-human animal is preferably a mammal, more preferably a cow, goat, sheep, or rodent such as a rat or mouse.. As used herein, a "chimeric animal" is one in which one or more of the cells of the animal includes a transgene. In other embodiments, the transgenic or chimeric animals can be non- human primates, dogs, chickens, amphibians, etc.

VI. WORKING EXAMPLES

The following examples are meant to illustrate the methods and materials of the present invention. Suitable modifications and adaptations of the described conditions and parameters normally encountered in the art which are obvious to those skilled in the art are within the spirit and scope of the present invention.

A. IPTG-Inducible U6 Promoters

The following describes several IPTG-inducible U6 promoters that were made. All of these constructs encoded a small interfering RNA molecule that inhibited expression of a luciferase gene. Four versions of the E. coli LacO sequence were used. Short and long versions of the natural LacO sequence were used, 5'-aattgtgagcggataacaatt-3(SEQ ID NO:2) and 5'- tgtgtggaattgtgagcggataacaatttcacaca-3' (SEQ ID NO:3), respectively. Also, short and long versions of a synthetic LacO sequence (a perfect palindrome of the 5' half of the natural LacO) were used, 5'-gaattgtgagcgctcacaattc-3' (SEQ ID NO:4) and 5'-tgtggaattgtgagcgctcacaattccaca-3' (SEQ ID NO:5), respectively. In construct A (also called U6 LOM FFl), a short version of the natural E. coli LacO sequence (SEQ ID NO:2; supra) was inserted between the TATA box and the transcription initiation site, replacing a native sequence of the same length (Fig. 2A). Construct A has the following sequence in the promoter region and the siRNA-coding region:

LOCUS U6 LOM FFl 636 bp DNA

FEATURES Location/Qualifiers precursor_RNA 563..625

/label=FFl shRNA misc_feature 536..562

/label=U6 leader sequence terminator 626..630

/label=Termination Signal misc_feature 58..535

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc_feature 536..536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 470..489

/label=Proximal Sequence Element tRNA 505..513

/label=TATA Box misc_binding 514..534

/label=Natural Lac Operator misc_feature 1..6

/label=XhoI misc_feature 631..636

/label=EcoRI misc feature 464..469

/label=NdeI

BASE COUNT 166 a 150 c 158 g 162 t ORIGIN

1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

241 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc 301 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttacσj'taac

481 ttgaaagrtat ttcgatttct tgg ttata tat|AATTGTG AGCGGATAAC AATTcgtgct

541 cgcttcggca gcacatatac taggattcca attcagcggg agccacctga tttggatcgg

601 gtggctctcg ctgagttgga atccattttt gaattc (SEQ ID NO: 6)

In this sequence, the PSE is italicized; the TATA box is boxed; the LacO sequence is in uppercase; and the G transcription initiation site is double-underlined.

In construct B (also U6 LOP FFl), a short synthetic LacO sequence (SEQ ID NO: 4, supra) was inserted into the U6 promoter region in the same fashion. Construct B has the following sequence:

LOCUS U6 LOP FFl 636 bp DNA FEATURES Location/Qualifiers ρrecursor_RNA 563..625

/label=FFl shRNA misc_feature 536..562

/label=U6 leader sequence terminator 626..630

/label=Termination Signal misc feature 58..513

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc feature 536..536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc feature 470..489

/label=Proximal Sequence Element tRNA 505..513 /label=TATA Box misc_binding 514..535

/label=Synthetic Lac Operator misc feature 1..6

/label=XhoI misc feature 631..636 /label=EcoRI misc_feature 464.. 69

/label=NdeI BASE COUNT 164 a 152 c 158 g 162 t ORIGIN 1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccg'taac

481 ttgaaagtat ttcgatέtct tgg ttata tat|GAATTGT GAGCGCTCAC AATTCgtgct

541 cgcttcggca gcacatatac taggattcca attcagcggg agccacctga tttggatcgg 601 gtggctctcg ctgagttgga atccattttt gaattc (SEQ ID NO: 7)

In the above sequence, the markings are the same as in construct A's sequence.

Constructs A and B both showed wild-type transcriptional activity when Lad was not present, and showed repressed transcription activity when Lad was present. To take advantage of Lad's ability to cooperatively bind two LacO sites, three more constructs, constructs C-E, were made, each containing two LacO sites. Construct C (also called U6 LO Ndel S LOM FFl) had the long synthetic LacO sequence inserted at the Ndel site and the short natural LacO sequence inserted between the TATA box and the G initiation site. It has the following sequence:

LOCUS LO Ndel S LOM FFl 670 bp DNA FEATURES Location/Qualifiers precursor_RNA 597..659

/label=FFl shRNA misc feature 570..596 /label=U6 leader sequence terminator 660..664

/label=Termination Signal misc_feature 58..569 /label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc_feature 570..570

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 504..523

/label=Proximal Sequence Element tRNA • 539..547 /label=TATA Box misc_binding 548..568

/label=Natural Lac Operator misc_binding 469..498

/label=Synthetic Lac Operator (LO Ndel S) insertion_seq 466..499

/label=L0 Ndel S insert misc_feature 1..6

/label=XhoI misc_feature 665..670 /label=EcoRI

BASE COUNT 176 a 157 c 165 g 172 t ORIGIN

1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc 61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

241 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc

301 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa 361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatattg tggaattgtg 481 agcgctcaca attccacaat atgcttaccg taacttgaaa gtatttcgat ttcttggcjttj

541 |tatatat|AAT TGTGAGCGGA TAACAATTcg tgctcgcttc ggcagcacat atactaggat

601 tccaattcag cgggagccac ctgatttgga tcgggtggct ctcgctgagt tggaatccat

661 ttttgaattc (SEQ ID NO: 8)

In the above sequence, the long synthetic LacO sequence is in boldface; and the short natural LacO sequence is in uppercase.

In construct D (also called U6 LO Ndel N LOM FFl), the long natural LacO was inserted at the Ndel site, and the short natural LacO was inserted between the TATA box and the G initiation site. This construct has the following sequence:

LOCUS L LOO NNddeell NN LLOOMM FFFFll 667755 bbpp DNA FEATURES Location/Qualifiers precursor_RNA 602..664

/label=FFl s RNA misc_feature 575..601

/label=U6 leader sequence terminator 665..669

/label=Termination Signal misc_feature 58..574

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc_feature 575..575

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 509..528

/label=Proximal Sequence Element tRNA 544..552

/label=TATA Box misc_binding 553..573

/label=Natural Lac Operator insertion_seq 466..504

/label=LO Ndel N insert misc_binding 469..503

/label=Natural Lac Operator (LO Ndel N) misc feature 670..675

/label=EcoRI misc_feature 1..6

/label=XhoI BASE COUNT 179 a 155 c 167 g 174 t ORIGIN 1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatattg tgtggaattg

481 tgagcggata acaatttcac acaatatgct taccgtaact tgaaagtatt tcgatttctt

541 ggcjtttatat at[AATTGTGA GCGGATAACA ATTcgtgctc gcttcggcag cacatatact 601 aggattccaa ttcagcggga gccacctgat ttggatcggg tggctctcgc tgagttggaa

661 tccatttttg aattc (SEQ ID NO: 9)

In the above sequence, the long natural LacO sequences is in boldface, and the short natural LacO sequence is in uppercase. hi construct E (also called U6 LO Ndel S LOP FFl), the long synthetic LacO was inserted at the Ndel site, and the short synthetic LacO was inserted between the TATA box and the G initiation site. This construct has the following sequence:

LOCUS LO Ndel S LOP 670 bp DNA FEATURES Location/Qualifiers precursor_RNA 597..659

/label=FFl shRNA misc_feature 570..596

/label=U6 leader sequence terminator 660..664

/label=Termination Signal misc feature 58..547 /label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc_feature 570..570 /label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 504..523

/label=Proximal Sequence Element tRNA 539.-547

/label=TATA Box misc_binding 548..569

/label=Synthetic Lac Operator misc_binding 469.. 98 /label=Synthetic Lac Operator (LO Ndel S) insertion_seq 466..499

/label=LO Ndel S insert misc__feature 1..6

/label=XhoI misc_feature 665..670

/label=EcoRI BASE COUNT 174 a 159 c 165 g 172 t ORIGIN

1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc 181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

241 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc

301 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatattg tggaattgtg / 481 agcgctcaca attccacaat atgcttaccg- taacttgaaa gtatttcgat ttcttggc[tt|

541 [tatatatjGAA TTGTGAGCGC TCACAATTCg tgctcgcttc ggcagcacat atactaggat 601 tccaattcag cgggagccac ctgatttgga tcgggtggct ctcgctgagt tggaatccat

661 ttttgaattc (SEQ ID NO: 10)

In the above sequences, the long synthetic LacO sequence is in boldface, and the short synthetic LacO sequence is in uppercase.

In all of constructs A-E, the spacing was not changed between the TATA box and the G initiation site when the LacO sequence was inserted therein. That is, the LacO sequence replaced a native sequence of the same length. The following shows an alignment of the promoter sequences of wildtype U6 and constructs A-E. The numbers above the sequences denote nucleotide positions, where position "1" corresponds to nucleotide 444 in SEQ ID NO: 1. The following sequences are assigned SEQ ID NOs:l 1-16, respectively.

1 65

U6 WT taaaatggactatca tatgcttaccg A taaaatggactatca tatgct taccg

B taaaatggactatca tatgcttaccg

C taaaatggactatcatat--tgtggaattgtgagc-gctcacaattccaca--atatgrc taccg

D taaaatggactatcatattgtgtggaattgtgagcggataacaatttcacacaatatgcttaccg

E taaaatggactatcatat~~tgtggaattgtgagc~gctcacaattccaca--atatg-cttaccg 66 127 U6 WT taacttgaaagtatttcgatttcttggc|tttatatat|cttgtggaaaggacgaaacaccg

A taacttgaaagtatttcgatttcttggc[tttatatat|AATTGTGAGCGGATAACAATTcg

B taacttgaaagtatttcgatttcttggcltttatatatlGAATTGTGAGCGCTCACAATTCg

C taacttgaaagtatttcgatttcttggc|tttatatat|AATTGTGAGCGGATAACAATTcg D taacttgaaagtatttcgatttcttggcjtttatatat[ARTTGTGAGCGGATAACAATTcg

E taacttgaaagtatttcgatttcttggc|tttatatat|GAATTGTGAGCGCTCACAATTCg

Also, in all of constructs A-E, the transcription termination site is "xxxxx" following the G initiation site and the short-hairpin sequence. The siRNA coding sequence encodes a short-hairpin sequence (FFl) targeting Luciferase expressed from the pGL3 vector (Promega).

Constructs A-E were constructed as described below. For construct A, to insert the LacO sequence into the promoter region, the following primer sets were used in two separate polymerase chain reactions (PCR): (1) a first external primer (5'-ggccctcgaggatttaggtgacactatag-3'; SEQ ID NO: 17) that targeted a vector region 5' to the U6 transcription unit, and a first internal primer having the sequence of 5 '-agcacgaattgttatccgctcacaattatatataaagccaagaaatcgaaatact-3 ' (SEQ ID NO:18); and (2) a second internal primer having the sequence of 5'- tggctttatatataattgtgagcggataacaattcgtgctcgcttcggcagcacatatac-3' (SEQ ID NO: 19), and a second external primer that targeted a vector region 3' to the U6 transcription unit. The PCR products from these two reactions were mixed and subjected to PCR again using the two external primers. This PCR reaction generated a complete, modified U6 transcription unit having the LacO sequence. To construct an RNAi vector, the U6-coding sequence was then replaced in part or in its entirety by a sequence encoding an interfering dsRNA, using routine recombinant techniques (e.g., PCR). For construct B, to insert the LacO sequence into the promoter region, the following primer sets were used in two PCRs: (1) a first external primer (SEQ ID NO: 17, supra) that targeted a vector region 5' to the U6 transcription unit, and a first internal primer having the sequence of 5'- agcacgaattgtgagcgctcacaattcatatataaagccaagaaatcgaaatact-3' (SEQ ID NO: 20); and (2) a second internal primer having the sequence of 5 '- tggctttatatatgaattgtgagcgctcacaattcgtgctcgcttcggcagcacatatac-3' (SEQ ID NO:21), and a second external primer that targeted a vector region 3' to the U6 transcription unit. The PCR products from these two reactions were mixed and subjected to PCR again using the two external primers. This PCR reaction generated a complete, modified U6 transcription unit having the LacO sequence. To construct an RNAi vector, the U6-coding sequence was replaced in part or in its entirety by a sequence encoding an interfering dsRNA, using routine recombinant techniques (e.g., PCR).

For constructs A and B, the FFl sequence with an EcoRI site was introduced into the human U6 promoter DNA sequence using PCR with an Xhol- containing forward primer (SEQ ID NO: 17, supra) and the FFl -containing reverse primer 5'- ggaattcaaaaatggattccaactcagcgagagccacccgatccaaatcaggtggctcccgctgaattggaatcctagt atatgtgctgccgaagc-3' (SEQ ID NO:22). U6 promoter DNA fragments containing the FFl sequence were digested with EcoRI and Xhol and inserted into pENTRl 1 (Invitrogen) digested with EcoRI and Xhol. Construct C was made by digesting construct A with Ndel and inserting the annealed self-complementary oligonucleotide 5'- tattgtggaattgtgagcgctcacaattccacaa-3' (SEQ ID NO:23), introducing the long synthetic LacO sequence. Construct D was made by digesting construct A with Ndel and inserting the annealed oligonucleotides 5'-tattgtgtggaattgtgagcggataacaatttcacacaa- 3' (SEQ ID NO:24) and 5'-tattgtgtgaaattgttatccgctcacaattccagaca-3' (SEQ ID NO:25), introducing the long natural LacO sequence.

Construct E was made by digesting construct B with Ndel and inserting the annealed self-complementary oligonucleotide SEQ ID NO:23 (supra), introducing the long synthetic LacO sequence.

The constructs were then cotransfected into NTH 3T3 cells with pGL3 Control (for directing luciferase expression; Promega), pCMV La (for direction Lad expression; Stratagene), and pSEAP2 Control (for directing SEAP expression; as a control for co-transfection; BD Biosciences). Transfected cells were treated with 5 mM JPTG and were compared to untreated cells.

Forty-eight hours after the start of transfection, 10 μl of supernatant from the cells was removed and used for a SEAP luminescence assay (Great ESCAPE SEAP Chemiluminescence Assay, BD Biosciences). The cells were subjected to a luciferase luminescence assay (STEADY-GLO, Promega). Data were normalized for transfection efficiency by dividing luciferase assay values with SEAP assay values. The comparison between transfection with the empty vector (pENTRl 1) and transfection with U6 FFl vectors shows the degree of inhibition of luciferase expression from pGL3. As shown in Fig. 2B, U6 constructs containing two LacO sequences

(i.e., constructs C-E), when co-transfected with pCMV Lad, exhibited stronger (approximately 15% more) repression by Lad in the absence of IPTG (i.e., thus less expression of the FFl transcript and less inhibition of luciferase expression), compared to U6 constructs containing only one LacO sequence (e.g., constructs A- B). Fig. 2C shows that constructs A-E inhibited luciferase expression significantly in the absence of Lad expression. The extents of the inhibition among the constructs were comparable. Constructs A and B had a combined average inhibition of 91%, and constructs C-E had a combined average inhibition of 83%. In conclusion, U6 constructs containing two LacO sequences retained wild type promoter activity, while having tighter inducibility control by Lad. B. Cre-LoxP Systems The Cre-LoxP system was used to create U6 promoter constructs for tissue-specific expression of short-hairpin RNAs (shRNAs) that target the expression of a luciferase gene. A LoxP-Stop-LoxP cassette was inserted between the G transcriptional start site of the U6 promoter and the FFl shRNA-coding sequence (Fig. 3A). The approximately 400 base pair LoxP Stop cassette consisted of two LoxP sites in the same orientation bracketing six RNA Polymerase III transcriptional termination sites (stretches of four or more Ts from a luciferase gene fragment and a U6 RNA transcriptional termination fragment). The LoxP Stop cassette prevented transcription from proceeding through to the shRNA- coding sequence. Cre-mediated recombination would remove the intervening Stop sequence between the two LoxP sites, leaving only one LoxP site. Transcription could then proceed through to the shRNA-coding sequence.

Four U6 LoxP Stop constructs were made, termed constructs F, G, H, and I. The differences among these constructs were the position of the transcriptional start site relative to the first LoxP site and the sequence used as a spacer between the second LoxP site and the FFl sequence. In construct F (also U6 LoxP Stop 1A FFl), the initiation start site was placed directly 5' to the first LoxP site, while the spacer between the second LoxP site and the FFl sequence was 5'-CGACGAGGC-3'. Construct G (also U6 LoxP Stop IB FFl) was identical to construct F except that it had a spacer sequence of 5'-CGACCTCCC-3 '. In construct H (also U6 LoxP Stop 2 A FFl), the transcription initiation start site was placed within the first LoxP site, maintaining the U6 transcriptional nucleotide as G, preceded by C as in the wild-type promoter. This construct had the same spacer sequence as construct F. Construct I (also U6 LoxP Stop 2B FFl) was the same as construct H except that the former had the spacer sequence of construct G. In order to compare the U6 LoxP constructs to their recombined form (i.e., with the STOPPER sequence removed), constructs F, G, H, and I were each recombined in vitro using the Cre recombinase. The ability of the cloned, recombined construct to silence Luciferase was then assayed and compared to a wild-type U6 promoter expressing the FFl shRNA targeting Luciferase. The promoter sequence of construct F is shown below.

LOCUS U6 Lox Stop Lox 1A FFl 1021 bp DNA

FEATURES Location/Qualifiers misc_feature 58..535

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter mmiisscc__ffeeaattuurree 536.-536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 470.. 89 /label=Proximal Sequence Element tRNA 505..513

/label=TATA Box misc_recomb 537..570

/label=LoxP misc_recomb 905..938

/label=LoxP terminator 620..625

/label=Transcriptional Termination 1 misc feature 571..739

/label=pGL3 Luciferase fragment terminator 753..904

/label=U6 Transcriptional Termination precursor_RNA 948..1010

/label=FFl shRNA terminator 1011..1015

/label=Transcriptional Termination 7 misc feature 1016..1021

/label=EcoRI misc_feature 1..6 /label=XhoI terminator 789..793

/label=Transcriptional Termination 2 terminator 805..809

/label=Transcriptional Termination 3 misc_feature 813..817

/label=Transcriptional Termination 4 terminator 829..833

/label=Transcriptional Termination 5 terminator 842..851

/label=Transcriptional Termination 6 BASE COUNT 265 a 223 c 253 g 280 t ORIGIN 1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac

481 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa

541 cttcgtatag catacattat acgaagttat tacacccgag ggggatgata aaccgggcgc 601 ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac

661 gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta

721 tgtaaacaat ccggaagcgc cgcggccgct aggcaaggat gacacgcaaa ttcgtgaagc

781 gttccatatt tttacatcag gttgtttttc tgtttttaca tcaggttgtt tttctgtttg

841 gttttttttt tacaccacgt ttatacgccg gtgcacggtt taccactgaa aacacctttc 901 atctataact tcgtatagca tacattatac gaagttatcg acgaggcgga ttccaattca

961 gcgggagcca cctgatttgg atcgggtggc tctcgctgag ttggaatcca tttttgaatt

1021 c (SEQ ID NO: 26) In the above sequence, the PSE is italicized; the LoxP sites are in boldface; and the initiation start site is double-underlined. After recombination, only one LoxP site remains and the sequence is the same as if the G nucleotide at position 536 is joined immediately 5' to the A nucleotide at position 905.

The promoter sequence of construct G is shown below.

LOCUS U6 Lox Stop Lox IB FFl 1021 bp DNA

FEATURES Location/Qualifiers misc_feature 58..535

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter mmiisscc_ffeeaattuurree 536..536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc__feature 470..489 /label=Proximal Sequence Element tRNA 505..513

/label=TATA Box misc_recomb 537..570

/label=LoxP misc_recomb 905..938

/label=LoxP terminator 620..625

/label=Transcriptional Termination 1 misc feature 571..739

/label=pGL3 Luciferase fragment terminator 753..904

/label=U6 Transcriptional Termination precursor_RNA 948..1010

/label=FFl shRNA terminator 1011..1015

/label=Transcriptional Termination 7 misc feature 1016..1021

/label=EcoRI misc feature 1..6 /label=XhoI terminator 789..793

/label=Transcriptional Termination 2 terminator 805..809

/label=Transcriptional Termination 3 misc_feature 813..817

/label=Transcriptional Termination 4 terminator 829..833

/label=Transcriptional Termination 5 terminator 842..851

/label=Transcriptional Termination 6 BASE COUNT 264 a 226 c 250 g 281 t ORIGIN 1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac

481 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa

661 gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta

721 tgtaaacaat ccggaagcgc cgcggccgct aggcaaggat gacacgcaaa ttcgtgaagc

781 gttccatatt tttacatcag gttgtttttc tgtttttaca tcaggttgtt tttctgtttg

841 gttttttttt tacaccacgt ttatacgccg gtgcacggtt taccactgaa aacacctttc 901 atctataact tcgtatagca tacattatac gaagttatcg acctcccgga ttccaattca

961 gcgggagcca cctgatttgg atcgggtggc tctcgctgag ttggaatcca tttttgaatt

1021 C (SEQ ID NO:27) The PSE, the LoxP sites, and the transcription start site are marked as in the sequence shown for construct F. After recombination, only one LoxP site remains and the sequence is the same as if the G nucleotide at position 536 is joined immediately 5' to the A nucleotide at position 905.

The promoter sequence of construct H is shown below.

LOCUS U6 Lox Stop Lox 2A FFl 1012 bp DNA FEATURES Location/Qualifiers misc_feature 58..513

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter mmiisscc__ffeeaattuurree 536.-536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 470..489 /label=Proximal Sequence Element tRNA 505..513

/label=TATA Box misc_recomb 528..561

/label=LoxP misc_recomb 896..929

/label=LoxP terminator 611..616

/label=Transcriptional Termination 1 misc feature 562..730

/label=pGL3 Luciferase fragment terminator 744..895

/label=U6 Transcriptional Termination precursor_JRNA 939..1001

/label=FFl shRNA terminator 1002..1006

/label=Transcriptional Termination 7 misc feature 1007..1012

/label=EcoRI misc feature 1..6 /label=XhoI terminator 780..784

/label=Transcriptional Termination 2 terminator 796..800

/label=Transcriptional Termination 3 misc_feature 804..808

/label=Transcriptional Termination 4 terminator 820..824

/label=Transcriptional Termination 5 terminator 833..842

/label=Transcriptional Termination 6 BASE COUNT 261 a 220 c 251 g 280 t ORIGIN 1 ctcgaggatt taggtgacac tatagaatac aagcttggct gcaggtcgac ggatcccccc

61 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

361 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac

481 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacata acttcgtata

541 gcatacatta tacgaagtta ttacacccga gggggatgat aaaccgggcg cggtcggtaa 601 agttgttcca ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt

661 taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt atgtccggtt atgtaaacaa

721 tccggaagcg ccgcggccgc taggcaagga tgacacgcaa attcgtgaag cgttccatat

781 ttttacatca ggttgttttt ctgtttttac atcaggttgt ttttctgttt ggtttttttt

841 ttacaccacg tttatacgcc ggtgcacggt ttaccactga aaacaccttt catctataac 901 ttcgtatagc atacattata cgaagttatc gacgaggcgg attccaattc agcgggagcc

961 acctgatttg gatcgggtgg ctctcgctga gttggaatcc atttttgaat tc (SEQ ID NO:28)

The PSE, the LoxP sites, and the transcription start site are marked as in the sequence shown for construct F. After recombination, the one of the LoxP site remains and the sequence is the same as if the T nucleotide at position 561 is joined immediately 5' to the C nucleotide at position 930. The promoter sequence of construct I is shown below.

LOCUS U6 Lox Stop Lox 2B 1012 bp DNA FEATURES Location/Qualifiers misc_feature 58..513

/label=U6 Promoter promoter 8..25

/label=SP6 Promoter misc_feature 536..536

/label=Start of Transription enhancer 293..324

/label=Distal Sequence Element misc_feature 470..489

/label= roximal Sequence Element tRNA 505..513 /label=TATA Box misc recomb 528..561 /label=LoxP misc recomb 896..929 /label=LoxP terminator 611..616

/label=Transcriptional Termination 1 misc feature 562..730

/label=pGL3 Luciferase fragment terminator 744..895

/label=U6 Transcriptional Termination precursor_RNA 939..1001 /label=FFl shRNA terminator 1002..1006

/label=Transcriptional Termination 7 misc feature 1007..1012 /label=EcoRI misc feature 1..6

/label=XhoI terminator 780..784

/label=Transcriptional Termination 2 terminator 796..800

/label=Transcriptional Termination 3 misc feature 804..808

/label=Transcriptional Termination 4 terminator 820..824

/label=Transcriptional Termination 5 terminator 833..842 /label=Transcriptional Termination 6 BASE COUNT 260 a 223 c 248 g 281 t ORIGIN

121 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc

181 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg

241 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc

421 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaσcgtaac

481 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacata acttcgtata

541 gcatacatta tacgaagtta ttacacccga gggggatgat aaaccgggcg cggtcggtaa

601 agttgttcca ttttttgaag cgaaggttgt ggatctggat accgggaaaa cgctgggcgt 661 taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt atgtccggtt atgtaaacaa

721 tccggaagcg ccgcggccgc taggcaagga tgacacgcaa attcgtgaag cgttccatat

781 ttttacatca ggttgttttt ctgtttttac atcaggttgt ttttctgttt ggtttttttt

841 ttacaccacg tttatacgcc ggtgcacggt ttaccactga aaacaccttt catctataac ^•

901 ttcgtatagc atacattata cgaagttatc gacctcccgg attccaattc agcgggagcc 961 acctgatttg gatcgggtgg ctctcgctga gttggaatcc atttttgaat tc (SEQ

ID NO: 29)

The PSE, the LoxP sites, and the transcription start site are marked as in the sequence shown for construct F. After recombination, one of the LoxP sites remains and the sequence is the same as if the T nucleotide at position 561 is joined immediately 5' to the C nucleotide at position 930.

Constructs F-I were made by introducing the FFl sequence and the LoxP Stop cassette into the human U6 promoter DNA sequence using PCR. The U6 promoter DNA fragments were then digested with EcoRI and Xhol and inserted into pENTRl 1 (Invitrogen) digested with these two enzymes. The constructs were cotransfected into NTH 3T3 cells with pGL3 and pSEAP2. Forty- eight hours after the start of transfection, 10 μl supernatant from the cells was removed and, used for a SEAP luminescence assay (see above). The cells were subjected to a Luciferase luminescence assay (Steady-Glo, Promega). Data were normalized by dividing Luciferase assay values with SEAP assay values. The comparison between the empty vector (pENTRl 1) and U6 FFl vectors shows the degree of inhibition of Luciferase expression from pGL3. Fig. 3B shows that the recombined forms of construct F-I effectively silenced Luciferase expression. The non-recombined forms of these constructs, in contrast, showed no silencing of Luciferase. h conclusion, Cre-mediated removal of the LoxP Stop cassette allows effective expression of shRNAs from the U6 promoter. These shRNAs are capable of efficient gene-specific silencing even though the LoxP sequence is also present in the transcript. These vectors will allow one to temporally and spatially restrict the expression of shRNAs through the use of spatially and/or temporally- restricted promoters. Additionally, the U6 LoxP Stop vector design can be combined with IP TG or Tetracycline inducible U6 promoters (below) to allow inducible control after Cre-mediated removal of the LoxP Stop. This allows the advantages of inducible expression of shRNAs while restricting expression only to specific tissues, thus preventing possible systemic effects of expression of shRNAs in non-target tissues.

C. Tetracycline-Inducible Promoters The Tet operon contains two different operator sequences, designated TO1 (5'-ACTGTATCATTGATAGAGT-3'; SEQ ID NO:30) and TO2 (5'-TCCCTATCAGTGATAGAGA-3'; SEQ ID NO:31), respectively. Both TO1 and TO2 bind the tetracycline repressor (TetR) protein. TO1 and TO2 sequences were inserted between the TATA and the transcription initiation site in the human U6 promoter at various positions while maintaining the spacing between the TATA and the initiation site and preserving the C preceding the G at the initiation site. These constructs are designated J, K, L, M, N, O, and P (also referred to as U6TO1A, U6TO1B, U6TO1C, U6TO2A, U6TO2B, U6TO2C, and U6TO2D, respectively). Their sequence alignment (from the PSE to the transcriptional start) is shown below (SEQ ID NOs:32-38).

J cttaccgtaacttgaaag-tatttcgatttcttggc|Ettatatat|ACTCTATCATTGATAGAGTaccg

hi the above alignment, the PSE is italicized; the TATA box is shown in a box; the TO1 and TO2 sequences are capitilized; and the first G of the transcription initiation site is double-underlined. Constructs J-P were constructed as described below:

To insert the TetOp sequence into the promoter region, the following primer sets were used in two PCRs: (1) a first external primer SEQ ID NO:17 (supra), which targeted a vector region 5' to the U6 transcription unit, and a first internal primer having the following sequence for each construct of: J: 5'-agcacggtactctatcaatgatagagtatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:39),

K: 5'-agcacggactctatcaatgatagagttatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:40),

L: 5'-agcacgactctatcaatgatagagtttatatataaagccaagaaatcgaaatact-3' (SEQ ID NO:41,

M: 5'-agcacggtgtctctatcactgatagggatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:42),

N: 5'-agcacggttctctatcactgatagggaatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:43), O: 5'-agcacggtctctatcactgatagggagatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:44), and

P: 5'-agcacgtctctatcactgatagggaagatatataaagccaagaaatcgaaatact-3' (SEQ ID

NO:45); and (2) a second internal primer having following sequence for each construct of: J: 5 '-tggctttatatatactctatcattgatagagtaccgtgctcgcttcggcagcacatatac-3 ' (SEQ ID NO:46),

K: 5 '-tggctttatatataactctatcattgatagagtccgtgctcgcttcggcagcacatatac-3 ' (SEQ ID NO:47),

L: 5 ' -tggctttatatataaactctatcattgatagagtcgtgctcgcttcggcagcacatatac-3 ' (SEQ JO NO:48),

M: 5 ' -tggctttatatatccctatcagtgatagagacaccgtgctcgcttcggcagcacatatac-3 ' (SEQ ID NO:49), N: 5'-tggctttatatattccctatcagtgatagagaaccgtgctcgcttcggcagcacatatac-3' (SEQ

ID NO:50),

O: 5 '-tggctttatatatctccctatcagtgatagagaccgtgctcgcttcggcagcacatatac-3 ' (SEQ ID NO:51), and

P: 5 '-tggctttatatatcttccctatcagtgatagagacgtgctcgcttcggcagcacatatac-3 ' (SEQ ID NO:52); and a second external primer that targeted a vector region 3' to the U6 transcription unit. The PCR products from these two reactions were mixed and subjected to PCR again using the two external primers. This PCR reaction generated a complete, modified U6 transcription unit having the TetOp sequence. To construct an RNAi vector, the U6-coding sequence was then replaced in part or in its entirety by a sequence encoding an interfering dsRNA, using routine recombinant techniques (e.g., PCR).

U6 promoter DNA fragments containing the FFl sequence were digested with EcoRI and Xhol and inserted into pENTRl 1 (Invitrogen) digested with the same enzymes. U6 promoter constructs were cotransfected with pGL3 Control (Luciferase expression, Promega), pcDNA6/TR (for directing TetR expression, Invitrogen), and pSEAP2 Control (SEAP expression used as a cotransfection control, BD Biosciences) into NTH 3T3 cells. Cells were treated with tetracycline and were compared to untreated cells. Forty-eight hours after the start of transfection, 10 μl supernatant from the cells was removed and used for a SEAP luminescence assay. The cells were subjected to a Luciferase luminescence assay (Steady-Glo, Promega). Data were normalized by dividing Luciferase assay values with SEAP assay values. These values were then normalized to an empty vector control.

As shown in Fig. 4A, the TetO constructs maintained wildtype transcriptional activity in the absence of TetR. When TetR and tetracycline were present, the constructs significantly inhibited Luciferase expression, but this inhibition was significantly repressed by TetR in the absence of tetracycline (Fig. ^• 4B). Construct J consistently appeared to be the most strongly repressed U6 TO1 promoter, and construct N consistently appeared to be the most strongly repressed U6 TO2 promoter. Notably, the positions of the TetOp in constructs J and N were the same. In conclusion, when inserting the TetOp sequence into the U6 promoter, it is important to maintain the spacing between the TATA box and the transcription initiation site, and to preserve the C nucleotide immediately 5' to the G initiation site.

Because the TetR gene is a prokaryotic gene, we designed a version of the gene that is codon-optimized for expression in mammalian cells. This version was designated gpTetR. This sequence was also designed to prevent possible GpC methylation, which would prevent silencing of the gene in mammals. An S V40 nuclear localization signal was added to the C-terminal end to allow localization of the protein product to the nucleus, allowing gpTetR to bind the TetOp more efficiently.

The gpTetR sequence is as follows.

LOCUS gpTetR 648 bp DNA FEATURES Location/Qualifiers misc_signal 622..645

/label=SV40 Nuclear Localization Signal BASE COUNT 190 a 149 c 164 g 145 t

1 atgagcaggc ttgacaaatc aaaagtgatt aactcagctc ttgaattgct caatgaagtg

61 gggatcgagg gtctaactac acgaaagctg gcacagaagc taggggtgga acagccaacc

121 ctgtattggc atgtgaaaaa caaaagagcc ctgcttgatg cactggctat tgagatgcta 181 gacagacacc atacccactt ctgtcctctg gaaggggaga gctggcagga cttcctgaga

241 aacaatgcga agtctttccg ttgtgcactc ctgagccatc gcgatggagc caaagttcat 301 ttagggactc ggcccacaga aaagcaatac gagacactag agaatcagct cgcctttctg

361 tgccagcaag gctttagtct ggaaaatgca ctctatgctc tcagcgccgt gggacacttt

421 accttgggat gtgtccttga agatcaagag catcaggttg caaaggaaga gagagaaact

481 ccaactacag acagtatgcc cccattgctg aggcaggcta tagaattatt cgaccaccag

541 ggcgcagaac ctgcctttct ctttggtctg gagctgatta tttgtggctt agagaaacaa 601 ctcaaatgtg aatcaggctc tccacctaag aagaaacgga aggtttaa (SEQ ID

NO: 53)

The inducible tetracycline system was further validated using stable cell lines and gpTetR. Human colorectal carcinoma cells, HCT116, were infected with virus containing a luciferase expression construct containing the Hygromycin resistance gene. After selection with Hygromycin, the cells were subsequently transfected with gpTetR, under the control of the PGK promoter. The gpTetR expression construct contains a Zeocin resistance gene. The stably transfected cells were selected with Zeocin and subcloned. Single clones were tested for adequate gpTetR expression, using real time RT-PCR, and subsequently infected with lenti viral vectors containing either the luciferase-directed FFl shRNA under the control of the inducible promoter U6TO2B (promoter construct N), or empty vector. As a control to test the effect of the hairpin construct in the absence of the gpTetR repressor, HCT116 cells were infected with virus containing the luciferase expression construct and then infected with the empty virus or U6TO2B-FF1 lentivirus. 1 ug/ml Doxycyline was added to the cells and luciferase activity was measured after 72 hours. The luciferase values were determined by treating cells with Steady Glo reagent (Promega) and measuring luminescence with a plate reader (Bio-Tek). Luciferase values were averaged from 4 replicates for each sample. The standard deviation was then calculated from those replicates. Values and standard deviations were rescaled with empty vector equal to 1. Figure 5 A shows that in cells lacking gpTetR, FFl -driven RNAi of luciferase occurs with or without Doxycycline. On the other hand, cells containing gpTetR show repression of RNAi in the absence of Doxycyclin, and FFl-driven RNAi of luciferase in the presence of Doxycycline.

The system was then tested in vivo. Cell were produced as described above. Nude mice were injected sub-cutaneously with 10⁶ cells per injection site, at three separate sites, on both flanks. Doxycyline (2 μg/ml)was admininstered orally in drinking water (changed twice a week). Tumor size and luciferase activity were measured over a period of 14 days, using a caliper and the NightOWL imaging system (Berthold Technolgies GmbH & Co KG), respectively. The luciferase values for each tumor at each time point were determined by injecting (interperitoneal) mice with luciferin (Molecular Probes) and measuring luminescence with the NightOwl imaging system. Each tumor luminescence value was divided by its size (area), and then these values were averaged per sample type. Average values were rescaled so that the average value for tumors not containing U6TO2B FFl (thus containing empty virus control, not shown in Figure 5B) is equal to one for each time point. HCT116 Luciferase PGKgpTetR (empty virus control) is revalued to one when comparing to HCT116 Luciferase PGKgpTetR U6TO2B FFl (minus doxycycline samples are compared to each other and plus doxycycline samples are compared to each other). HCT116 Luciferase (empty virus control) is revalued to one when comparing to HCT116 U6TO2B FFl (both plus and minus doxcycline samples are grouped together for each tumor type). The results show that the down-regulation, repression, and de- repression phenotypes seen in vitro are also observed in vivo (Fig. 5B). In summary, gpTetR effectively represses the U6TO2B promoter (construct N) in the absence of Doxycycline, and the presence of Doxycycline allows effective expression from U6TO2B, allowing RNAi to function.

D. Combined Promoter Systems and an Alternative Use for the Cre-Lox System

This example describes a promoter system combining a small molecule inducible system (e.g., the tetracycline-inducible system or the IPTG- inducible system) with the Cre-Lox system (Fig. 6). By combining the two systems, one can control RNAi both spatially and temporally. First, a small molecule inducible U6 promoter, containing a LacO or TetOp sequence, is fused to a LoxP-Stop-LoxP shRNA cassette. The LoxP-Stop-LoxP cassette prevents transcription of the shRNA, preventing RNAi of a target gene. Upon Cre

Recombinase-mediated recombination, the Stop cassette is removed, leaving only one LoxP site (Fig. 6). Tissue-specific expression of Cre Recombinase allows spatial control of shRNA expression. Temporal control can be achieved by a promoter that is temporally restricted during development or through an inducible form of Cre. An example of inducible Cre is a fusion between Cre and the

Estrogen Receptor (ER). Tamoxifen allows the Cre-ER fusion to enter the nucleus and recombine the LoxP sites, removing the LoxP Stop cassette. Temporal control of Cre can also be accomplished by the use of an inducible promoter for Cre. After Cre-mediated removal of the LoxP-Stop-LoxP cassette, expression of the shRNA from the U6 promoter can further be controlled by the use of U6 promoters containing Lac Operator or Tet Operator sequences (Fig. 6). When Lad is also expressed in the cell, expression from the U6 Lac Operator promoter will only occur in the presence of IPTG. When TetR is also expressed in cells, expression from the U6 Tet Operator promoter will only occur in the presence of tetracycline or doxycycline. The addition of the Lac Operator or Tet Operator controlled system to the Cre-Lox controlled system allows one to control the amount of expression from the U6 promoter in specific tissues and at desired times. The Cre-Lox system alone allows one to turn on shRNA expression at desired times in desired tissues. However, once the LoxP Stop LoxP cassette is removed, transcription of the shRNA is continuous. The addition of the Lac

Operator or Tet Operator system allows one to turn on and off expression of the shRNA at desired times in desired tissues. Additionally, the level of shRNA expression can be controlled by using different doses of the small molecule inducer. An alternative use of the Cre-Lox system is to use Cre-mediated recombination to switch from expression of one shRNA to another (Fig. 7). Instead of using a Stop sequence between two LoxP sites, a shRNA is placed immediately after the first LoxP sequence and is followed by a stretch of four or more Ts. The second LoxP sequence (in the same orientation as the first), is followed by a second shRNA. Before Cre-mediated recombination, the first shRNA will be expressed and transcription terminates before the second one. After Cre-mediated recombination, only the second shRNA is present and expressed.

Claims

What is claimed is:

1. A nucleic acid construct comprising a coding sequence for a small interfering RNA molecule, said coding sequence linked operably to a mammalian or viral promoter, wherein a part of the nucleotide region between the PSE and the transcription initiation site of the promoter has been replaced with an operator sequence, wherein said operator sequence is controlled by a small molecule, and wherein said operator sequence has the same length as said part, or is no more than two nucleotides longer than said part.

2. The nucleic acid construct of claim 1, wherein said promoter is an RNA pol I, pol II, or pol III promoter.

3. The nucleic acid construct of claim 2, wherein said promoter is a U6, HI or CMV promoter.

4. The nucleic acid construct of claim 1, wherein said operator sequence is between the TATA box and the transcription initiation site of the promoter.

5. The nucleic acid construct of claim 1, wherein said operator sequence is a Lac operator sequence or a tetracycline operator sequence.

6. The nucleic acid construct of claim 1 , wherein said Lac operator sequence is SEQ ID NO:2 or 4, or said sequence with up to 4 terminal deletions from either end of the sequence.

7. The nucleic acid construct of claim 5, selected from SEQ ID NO: 6 or 7.

8. The nucleic acid construct of claim 1, wherein a part of the nucleotide region between the TATA box and the transcription initiation site of the promoter has been replaced with a first Lac operator sequence, and wherein a part of the nucleotide region between the distal element sequence and the proximal element sequence has been replaced with an additional Lac operator sequence.

9. The nucleic acid construct according to claim 8, wherein the first LacO sequence and the additional LacO sequence are selected from the group consisting of SEQ ID NOS: 2-5.

10. The nucleic acid construct according to claim 9, selected from the group consisting of: SEQ ID NOS: 8-10.

11. The nucleic acid construct of claim 5, wherein said tetracycline operator sequence is SEQ ID NO:30 or 31.

12. The nucleic acid construct according to claim 11, selected from the group consisting of: SEQ ID NOS: 32-38.

13. The nucleic acid construct of claim 5, wherein said tetracycline operator sequence is placed immediately downstream from the TATA box.

14. The nucleic acid construct of claim 1, wherein said small interfering RNA molecule inhibits expression of a disease-related gene.

15. The nucleic acid construct of claim 14, wherein the disease is cancer.

16. A nucleic acid construct comprising a coding sequence for a small interfering RNA molecule linked operably to a mammalian or viral promoter, wherein the transcription initiation site of the promoter and said coding sequence are separated by a LoxP-Stop-LoxP cassette consisting of a transcription termination sequence flanked by two LoxP sites, wherein upon recombination between said two LoxP sites in the presence of a cre recombinase, said transcription termination sequence is removed, allowing transcription to proceed through said coding sequence.

17. The nucleic acid construct of claim 16, wherein said promoter is an RNA pol I, pol II, or pol III promoter.

18. The nucleic acid construct of claim 17, wherein said promoter is a U6, HI or CMV promoter.

19. The nucleic acid construct according to claim 18, selected from the group consisting of SEQ ID NOS: 26-29.

20. The nucleic acid construct of claim 16, wherein said small interfering RNA molecule inhibits expression of a disease-related gene.

21. The nucleic acid construct of claim 20, wherein the disease is cancer.

22. A nucleic acid construct comprising a coding sequence for a first small interfering RNA molecule linked operably to a mammalian RNA polymerase III promoter, wherein the first small interfering RNA molecule is placed between a LoxP site and a stretch of at least four thymines, wherein the construct further comprises a second LoxP site followed by a coding sequence for a second small interfering RNA molecule, wherein before recombination between said two LoxP sites in the presence of cre recombinase, the first small interfering RNA molecule is expressed; and wherein upon recombination between said two LoxP sites in the presence of a cre recombinase, the first small interfering RNA molecule is no longer expressed and the second small interfering RNA molecule is expressed.

23. The nucleic acid construct of claim 16 or claim 22, further comprising an operator sequence, wherein said operator sequence is controlled by a small molecule, and wherein said operator sequence replaces a portion of nucleic acid sequence of a selected from: a portion between the TATA box and the transcription initiation site, a portion between the PSE and the TATA box, a portion between the distal element sequence and the proximal element sequence and both a portion between the TATA box and the transcription initiation site and a portion between the distal element sequence and the proximal element sequence.

24. A mammalian cell comprising the nucleic acid construct of any one of claims 1 to 15 and claim 23.

25. The mammalian cell according to claim 24, further comprising an additional construct for the expression of a repressor of the operator sequence.

26. The mammalian cell according to claim 25 , wherein the additional construct encodes Lad or TetR.

27. A mammalian cell comprising the nucleic acid construct according to any one of claim 16 to 22.

28. The mammalian cell according to claim 27, further comprising a construct that encodes Cre recombinase.

29. The cell according to any one of claims 25 or 26, wherein expression of the repressor is under the control of a promoter selected from the group consisting of: an inducible promoter, a stage-specific promoter and a tissue- specific promoter.

30. The cell according to claim 28, wherein Cre recombinase expression is under the control of a promoter selected from the group consisting of: an inducible promoter, a stage-specific promoter and a tissue-specific promoter.

31. A non-human mammal comprising the cell according to any one of claims 24 to 30.

32. The non-human mammal according to claim 31, which is a chimeric mammal some of whose somatic or germ cells are a cell according to any one of claims 24 to 30.

33. The non-human mammal according to claim 31, which is a transgenic mammal all of whose somatic or germ cells are cells according to any one of claims 24 to 30.

34. A method for making a chimeric non-human mammal according to claim 32 comprising the step of introducing a construct according to any one of claims 1-23 into an embryonic stem (ES) cell and generating a chimeric mammal from the ES cell.

35. A method for making a transgenic non-human mammal according to claim 33 comprising the step of mating a chimeric non-human mammal according to claim 34 with another animal from the same species.

36. A tetracycline repressor encoded by SEQ ID NO:53 or a degenerate variant thereof.

37. A method for inhibiting the expression of a gene of interest in a cell comprising the step of introducing a construct according to any one of claims 1 to 23 into the cell, wherein the small interfering RNA molecule is specific for the gene of interest.

38. A method for treating a gene-mediated disease comprising introducing into a cell a construct according to any one of claims 1 to 23 where the small interfering RNA molecule is specific for the gene mediating the disease.