WO2014117050A2 - Modified mirna as a scaffold for shrna - Google Patents

Abstract

What is described is a modified miRNA molecule for producing an artificial siRNA/mature small RNA molecule that inhibits the expression of a target transcript of a host cell, comprising a stem region modified to comprise a sequence encoding the artificial siRNA molecule, consisting of a guide and a passenger strand; a conserved region having specific sequences; and a nonconserved region modified to include a recognition site for a restriction enzyme while preserving the native secondary structure of the miRNA. The modified miRNA molecule produced with these elements substantially inhibits the expression of the target transcript when expressed from an endogenous or exogenous promoter in the host cell.

Description

MODIFIED MIRNA AS A SCAFFOLD FOR SHRNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/757,104, filed January 26, 2013, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] RNA interference, gene knockdown, regulation of gene expression, genetic engineering, RNA biology, small RNA biology, genetics, genetic manipulation.

[0003] Application fields: Molecular biology, cancer biology, study of disease biology, biomedical sciences, life sciences, therapeutic studies, drug target research, drug target development.

BACKGROUND

[0004] RNA interference (RNAi) is an endogenous pathway that fine tunes gene expression (among others). This pathway provides a powerful tool to suppress the expression of a specific gene at will, for a specific period of time, reversibly. It can be used to uncover gene function and biochemical pathways and/or to test new therapeutic strategies. To implement RNAi as a tool, various triggers can be used, including siRNAs, stem-loop shRNAs, and miRNA-embedded shRNAs. MicroRNA-embedded shRNAs that use a miRNA backbone resemble most closely the endogenous triggers, and are least toxic and thus preferable. However, potent shRNA sequences must be identified among hundreds to thousands of possibilities; thus they are rare and hard to identify.

[0005] Conventional stem loop shRNA structures provide a simple si-based design and in many cases potent knockdown. However, these shRNA molecules are often toxic, lack a robust regulatable system, and are more prone to off-targets effects, e.g. due to imprecision in Dicer processing or saturation of Exportin-5 export.

[0006] Target transcript knockdown efficiency is a measure of potent, clearly controlled knockdown. Achieving knockdown efficiency depends on i) delivery and expression of the precursor of the RNAi trigger, e.g., a vector coding for a miRNA-embedded shRNA, ii) the processing efficiency of the precursor molecule (biogenesis, thus its ability to become a mature small RNA that can trigger an RNAi response), and iii) the propensity of the chosen shRNA sequence itself to elicit a strong target knockdown through mature RISC complexes. There is a need to generate microRNA (miRNA) backbones that lead to efficient/better processing of the synthetic miRNA precursor molecules, to increase the overall percentage of sequences that lead to potent target knockdown.

SUMMARY

[0007] One aspect of the description is a modified miRNA molecule for producing an artificial siRNA (or shRNA) molecule that inhibits the expression of a target transcript, comprising

• a stem region modified to comprise a sequence encoding the artificial siRNA

molecule, consisting of a guide and a passenger strand;

• a conserved region comprising a sequence consisting of 5'-DC*NNC-3', wherein N consists of A, G, U, or C, D consists of A, G or U, and wherein the C* is located at position 16-20 of the 3' arm;

wherein the modified miRNA molecule substantially inhibits the target transcript when integrated into a host cell genome and expressed by the host cell. The conserved region preferably comprises the sequence 5'-ACUUCAA-3', 5'-GCUUCGA-3', or 5'-UCUUCUG-3'.

[0008] One embodiment of the description is when the modified miRNA further comprises a region consisting of a recognition site for a restriction enzyme. Another embodiment is a modified miRNA wherein the native secondary structure is conserved. Another embodiment is when a modified miRNA molecule is derived from miR-30, preferably in which the region comprises the sequence 5'-CUUUAG-3'. In another embodiment, the recognition site consists of a sequences recognized by EcoRI or Xhol. When the restriction enzyme consists of EcoRI, the modified sequence may consist of 5'-GACUUC-3' and the partially complementary sequence containing the EcoRI restriction sites consists of 5'-GAAUUC-3'. The modified miR-30 molecule may further comprise a modified loop region. A bulge in the guide strand may be absent from stem region of the modified miR-30 molecule.

[0009] In another embodiment, the miRNA molecule is derived from a miRNA selected from miR-30, miR-22, miR-15, miR-16, miR-103, and miR-107. In another embodiment the sequence of the conserved region is modified from the sequence 5'- ACUUCAA-3' to a sequence selected from the group consisting of 5'- ACGUCAA-3', 5'- ACCUCAA-3', and 5'- ACAUCAA- 3'.

[0010] In another embodiment, the modified miRNA molecule may be derived from miR-22. In this connection, the conserved region may comprise the sequence 5'-GCUUCGA-3'. [0011] In another embodiment, the modified miRNA molecule is derived from miR-

15/16.

[0012] In another embodiment, the modified miRNA molecule consists of a precursor miRNA molecule, preferably a pri-miRNA molecule. The modified pri-miRNA molecule may be efficiently processed by Drosha into a pre-miRNA in the nucleus of a host cell expressing the modified miRNA. The pre-miRNA molecule may be subsequently processed by Dicer in the cytoplasm of the host cell.

[0013] In another aspect of the description, a nucleic acid construct encodes the modified miRNA. In one embodiment, the Pol II promoter may be a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, and/or a developmental stage- specific promoter. In another embodiment, the Pol II promoter may be a CMV-derived promoter. The nucleic acid construct may further comprise at least one selectable marker. The nucleic acid construct may further comprise a reporter transcript. The nucleic acid construct may further comprise a Pol III promoter upstream of the coding sequence for expressing the precursor molecule.

[0014] In another embodiment of the description, a cell may comprise the nucleic acid construct encoding the modified miRNA. In another embodiment the cell may be a mammalian cell, or another vertebrate cell (e.g. a chicken cell). The cell may be cultured in vitro (e.g. in a plastic dish), or be part of an organism, including a mammal or other vertebrate. In this respect, another embodiment of the description herein is in the design of a therapeutic for use in treating a disease, or in the design of a therapeutic.

[0015] Another embodiment of the description is a method for inhibiting the expression of a target transcript of interest in a cell-free system, or in a cell, comprising introducing a nucleic acid construct encoding the modified miRNA into the cell, wherein the siRNA molecule derived from the modified miRNA molecule is specific for the target transcript. In another embodiment, the method further comprises inhibiting at least one additional target transcript(s) of interest in the cell by introducing at least one additional nucleic acid construct encoding another modified miRNA molecule into the cell, wherein each of the siRNA molecules derived from the modified miRNA molecule is specific for the additional target transcripts, respectively.

[0016] Another embodiment of the description is a method for treating a transcript- mediated disease, comprising introducing into an individual having the disease a nucleic acid construct encoding the modified miRNA molecule, where the siRNA derived from the modified miRNA molecule is specific for the transcript mediating the disease, or specific for an auxiliary transcript necessary for disease progression or maintenance. [0017] Another embodiment of the description is a method of validating a transcript as a potential target for treating a disease, comprising: introducing a nucleic acid construct encoding the modified miRNA into a cell associated with the disease, wherein the siRNA molecule derived from the modified miRNA is specific for the transcript; and assessing the effect of inhibiting the expression of the transcript or gene surrogate on one or more disease-associated phenotypes; wherein a positive effect on at least one disease-associated phenotype is indicative that the transcript is a potential target for treating the disease.

[0018] Another embodiment of the description is a method for producing a modified miRNA molecule for inhibiting the expression of a target transcript by preparing a nucleic acid construct encoding the modified miRNA comprising the steps of

• modifying the stem sequence of the miRNA molecule to substitute a nucleic acid sequence encoding a siRNA molecule;

• substituting a conserved region comprising a sequence consisting of 5'- DC*NNC-3', wherein N consists of A, G, U, or C, D consists of A, G or U, and wherein the C* is located at position 16-20 of the 3' arm; wherein the modified miRNA molecule substantially inhibits the target transcript when the nucleic acid construct is integrated into a host cell genome. The conserved region preferably comprises the sequence 5'-ACUUCAA-3', 5'-GCUUCGA-3', or 5'-UCUUCUG-3'.

[0019] An embodiment comprises modifying a region to include a recognition site for a restriction enzyme. Another embodiment is to preserve the miRNA secondary structure. Another embodiment is the method wherein a miRNA molecule is derived from miR-30 and the region comprises the sequence 5'-CUUUAG-3'. Further, the restriction enzyme preferably consists of EcoRI or Xhol, more preferably wherein the restriction enzyme consists of EcoRI, the modified sequence consists of GACUUC and the partially complementary sequence containing the EcoRI site consists of GAATTC. Alternatively, the miRNA molecule may be derived from miR-22 and the conserved region comprises the sequence 5'-GCUUCGA-3' or may be derived from miR- 15/16 and the conserved region comprises the sequence 5'-UCUUCUG3'.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Fig. 1 shows a comparison of core sequence of endogenous miR-30 (human MIR30A, above) and synthetic miR-30 (below). The regions of endogenous miR-30 in red in the upper sequence show completely conserved nucleotides. The regions in synthetic miR-30 that are altered in the conserved region are shown in red in the lower sequence. A synthetic sequence dement (Eco I site) in a 3' conserved region, highlighted in blue art the Sower, synthetic sequence, contains mutations in one highly conserved nucleotide.

[00211 ¾■ 2 shows a representations of mi -30 vs. ms -30A (Fig. 2A), msR-30 vs. miR~E (Fig, 2B)_; core structures of endogenous human miR-30 A, synthetic miR-30, and miR-E (red arrow, SNP (C>A); green circle, relocated EcoRJ site; blue circle, bulge in stem loop; orange circle, loop variation; miR-30 and miR-E are shown with sh.Yapl .8 1 E) (Fig. 2C), miR- 22 (Fig. 2D), and mlR-15/16 (Fig. 2E},

]§022] Fig. 3 shows the results of modifying the conserved (lank region of rniR~30. Single point mutations were generated always exchanging one nucleotide. All constructs had the repositioned EeoRI site and 5' arm adaptations. Ail were tested in NIH3T3 cells at single copy. Nucleotide substitutions are highlighted in green. The conserved nucleotide (C) thai was changed irs the synthetic miR-30 (indicated with an asterisk) is highlighted in purple,

J9Q23] Fig. 4 shows the results of modifying the stem loop region of miR-30, The miR- 30 molecule without and with bulge was compared to mi - 155 (BLOC -iT₅ invitrogen), which has a guide bulge. Two validated LMG shraiR molecules were tested, shmir,Lue, l 309_s originally validated in miR-30; and shmir.Lnc,20 i, a control shR A from invitrogen. All were cloned in pL N and infected at identical single-copy conditions, Fig, 4A shows the sensor shRNA reporter assay system. Fig. 4B shows results of testing expression at day 1 (left panel) and day 6 (right panel). Fig. 4C summarizes the results for shmir.Luc.201 {left panel) and shmir.Luc.1309 (right panel). Percentage knockdown (vertical axis) relative to each shRNA^" is shown for 2 days (dark brown) and for 6 days (light brown).

[0024] Fig. 5 shows the results of reversing the guide and passenger strands. Fig. 5A shows the sequence variants of the PtenJ 524 shRNA (04-18) along with the Ren.713 (02) and Pten,! 523 (03) control s RNAs. Human MIR30A (01) is shown for reference.

Eridogeitotis sequences:

01 ) Human MiSUOA

Experimental sequences with single features:

02) Ren. 713 (standard miR-30 design)

03) Fieri. 1323 {si3fidard miR-30 design)

04) Piers. 1524 (standard raiR-30 design}

05) Ptest. 1524 L (endogenous loop)

06) Fieri. 1524 B S (bulge 1. contains a 2 nt ulge in the guide sirand)

0?) Ptert, 1524 B2 (bulge 2, contests a 2 ni bulge in the guide strand, positioned closer la the loep}

OS) Piers. 1524 L+Bl (endogenous loop and bulge 1)

09) Fieri. 1524 L+B2 (endogenous loop and bulge 2)

SO) Plen. 1524 Ri (reverse 1, swapped guide passenger position, with a longer overall stem)

! 1 ) Plert. 1524 R2 (reverse 2, swapped guide/passengir position)

12) Plen. 1524 RiB l (reverse 1 and bulge 1}

13) Plen. 1524 R2B 1 (reverse 2 and bulge I)

14) Fieri. 1524 R2B2 (reverse 2 and bulge 2) 15} Pten, 1524 E (endogenous V (lank at Eco I site, repositioned EcoRi site, adapted 5' complement region

Experimental requracsis with combined features, unitin sirsgie attributes that strosgiy enhance knockdown potency:

16} Pies, 1524 E+L {miR-E and endogenous op}

17} Pten. 1524 E+R!BI (miR-E and reverse 1 with ulge J

18} Ptets, 1524 E+R2S2 (miR-E and reverse 2 with bulge 2}

Structural microRNA regions are labeled and highlighted by shaded boxes. Guide strands are shown in green, and alterations highlighted In red. The Xhol/EsoR! restric i n sites are underlined; the repositioned ("new") EeoRI site and 5' complement region are marked in blue. Fig. 5B compares the knockdown for various miRNA sequences,

[0025] Fig, 6 shows the results of using msR~£ as backbone to express shRNAs. miR-E expression of shRNAs targeting Ften (mi RE Piers J 523 and mi RE Pten.1524) is compared to the same shRNA sequences as synthetic mlR~30 (sh.Ptm l 523 and sh.Pten.1524) and miR-G5 (miRGS PtenJ 523 and miRGS Pten , 1524) constructs,

10826] Fig, 7 shows the results of using miR-E to express a variety of shRNAs targeting different transcripts, and compares it to miR-30 (labeled as s ,X). Fig, 7 A shows the results of expressing various Pten shRNA. Fig. 7B shows the resu lts of expressing various shRNA For knockdown of the Bel2 (B-eell CLL/lymphoma 2} gene. Fig, 7C shows the results of expressing various shRNA for knockdown of the Mel I (Myeloid eel! leukemia sequence I ) gene,

[0027] Fig, 8 shows the results of testing the knockdown of a panel of shRNAs integrated at single copy, MiR-30 (blue) is compared to miR-E (red) for Dnmi,¾ shRNAs (left panel) and Asxil shRNAs (right panel). The percentage knockdown compared to shRNA negative cells is shown,

]M2S| Fig. 9 shows the rescue of viral packaging efficiency (viral titers) for vectors coding for & miRNA-embedded shRNA + its target site, upon suppression of the RNAs pathway in a packaging cell line using stem-loop shRNA targeting components of the Drosha complex (specifically DGCR8), Fig. 9A shows the sensor vector with the target site (labeled as "Sensor"), Fig, 9B shows percentage infected ceils, proportional to viral titer, resulting from packaging under a variety of conditions in PlatGP packaging cells, Suppression of the RNAs pathway using the stem loop shRNA shDGCRS.C folly rescued packaging efficiency of Sensor vectors containing a target sites, as compared to Sensor vectors without target site.

Fig, 10 shows generation and validation of retro- and lenttvira! vectors featuring miR-E. Fig. 1 QA shows reporter-based validation of new miR-E shRNAs expression vectors. Reporter cells (e.g. NIH3T3s) are infected with a reporter construct constitutive ly expressing a transcript coding for Ametrme and harboring multiple target sites of established control shRNAs, Sorted Ametrine-poskive cells are then infected with one of several retroviral expression vectors to be tested, expressing another fluorescent protein coupled to mIR-E, After 6 days of culture (in presence of doxycyelme for Tet-regulated vectors), levels of the Amet ine-reporier are quantified by flow cytometry in both shRNA~ and shRNA^" cells. Fig, J OB shows a schematic representation of tested retroviruses. While the vectors LEPG and LENG eonsiitutivefy express the contained shRNA (here en.713), the vectors RTREYIR and RT3GEP1R introduce rtTA3. allowing for doxycyc!ine-regulated expression of the contained shRNA (here Ren.713). Flow cytometry blots show Ametrine levels at day 6 post-infection (constitutive vectors) or after 6 days of doxycycline treatment (inducible vectors). Bar graphs show the corresponding quantification of Ametrine levels.

Fig, 1 1 A shows schematic maps of validated constitutive (pMSCV) and Tet-reguiated (pQCXIX) retroviral msR~E expression vectors, featuring different drug selection and fluorescent markers.

Fig. ! I B shows schematic maps of validated constitutive and Tet-regulated lentiviral ntiR-E expression vectors, featuring different drug selection and fluorescent markers,

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

]Θ029] What is described herein is engineering of a new variant of an endogenous miRNA ck o e_j with drastically increased general knockdown efficiency, The increased knockdown efficiency results from the discovery of a conserved structure/sequence element in the 3" flank of the miR-30 backbone, a sequence that had previously been unknown and mutated. What is further described is a means of engineering of a cloning site (e.g., EcoRI) in a non- conserved part of the backbone, and in a way that conserves the general secondary structure,

[0030} What is described is engineering of a new backbone of miRNA molecules that leads to better target knockdown through generally improved primary miRNA processing. This involved discovery of conserved sequence/structure elements required for efficient pri-miRNA processing, Implementation of conserved structures In an engineered backbone for efficient processing of synthetic RNAi triggers, leading to highly improved target knockdown,

Specifically, what Is described is an evolutionary conserved region of miR-30 that is altered in synthetic miR-30 currently used as a scaffold for producing selected shRNA. This highly conserved region lies in the 3' flank to the stem loop (Fig, ! and Fig. 2A), A matching conserved region in miR-22 and mir- 15/16 is also described herein (Fig. 2B and Fig. 2C_S respectively). j 031] What is further described is discovery and implementation of sequence/structure elements (as part of a miRNA backbone) that enhance primary miRN A processing. What is described herein Is a combination of these conserved elements with strategies to insert cloning sites that do not affect knockdown efficiency, and assist easy and fast generation of synthetic sh NA vectors and libraries thereof

[0032] What is described is a means of utilizing a mi ~30~based system to overcome the limitations of simple stem loop systems. The modified miR-30 scaffold as a means of producing shRNA is less toxic in-vitro / ιη-νίνο, is readily adaptable to a simple reportable Tet- system, and promises educed off-target effects. The only downside thai remains is that design of potent shRNAs is harder.

[0033] The issue is likely suboptimal pri-miRNA processing, because potent mlR30- shRNAs are harder to find than potent stem-loop shRNAs, even Sensor-optimized shRNAs show huge variability in pri-miRNA processing, as evidenced in the Sensor deep-seq dataset and {indirectly} by variations in packaging efficacy and expression levels of spacer reporters, and even when expressed from strong promoters (e.g. TRE), mature small RNAs of synthetic miR30~ shRNAs are less abundant than many endogenous miRs.

[6034| A technical Issue Is whether the synthetic mil 30 structure can be further optimized to improve primary msRNA processing. What is described herein is a means of improving msRNA processing of miR-30 and thereby increasing knockdown efficiency,

[00351 ί¾ Α interference (RNAs) is normally triggered by double stranded R A (dsRNA) or endogenous mieroRNA precursors (pri-mlRNAs/pre-miRNAs). Since its discovery, RNAi has emerged as a powerful genetic tool for suppressing gene expression in mammalian cells. Stable gene knockdown can be achieved by expression of synthetic short hairpin RNAs (shRNAs),

[0036] The current modified miRNA can be produced from one single stably integrated expression construct The single expression construct may be stably transfected/infected into a target cell, or may be a germ line transgene. Transgenic animals with the subject RNAi constructs, which may be regulated to express miRNA-embedded shRNA in an inducible, reversible, constitutive and/or tissue-specific manner, can be used to establish valuable animal models for certain disease, such as those associated with loss-of-flinciion of certain target genes, or to assess target genes for their effect on disease or normal physiology upon knockdown. The ability to control both the timing (e.g., at certain developmental stages) and location (e.g.. tissue- specific) of target gene knock-down, including the ability to reverse the course of

snduction inactivation, renders the subject system a powerful tool to study gene function and disease progression. Such animal models or cells thereof may also be used for drug and drug target screening or validation. [CS037J In certain embodiments, Pol Π promoters control the transcription of the subject miRNA shRNA coding sequence. In general, any Pol II compatible promoters may be used for the instant description. In certain embodiments, various inducible Pol it promoters may be used to direct precursor miRNA shRNA expression. Exemplar)' inducible Pol 11 promoters include the tightly reguiatabie Tet system (either Te On or TetQFF), and a number of other inducible expression systems known in the art and/or described herein. The tet systems allows incremental and reversible induction of precursor miRNA shRNA expression in vitro and in vivo, with no or minimal leaklness in precursor miRNA/shRNA expression. Such inducible system has advantages over the existing unidirectional Cre-lox strategies. Other systems of inducible expression may also be used with the instant constructs and methods.

j0038] In certain embodiments, expression of the subject miRNA/shRNA may be under the control of a tissue specific promoter, such as & promoter that is specific for a variety of tissues including liver, pancreas (exocrine or endocrine portions), spleen, esophagus, stomach, large or small intestine, colon, Gl tract heart, lung, kidney, thymus, parathyroid, pineal gland, pituitary gland, mammary gland, salivary gland, ovary, ytems, cervix (e.g., neck portion), prostate, testis, germ cell, ear, eye, brain, retina, cerebellum, cerebrum. FNS or CN5, placenta, adrenal cortex or medulla, skin, lymph node, muscle, fat, bone, cartilage, synovium, bone marrow, epithelial, endothelial, vascular, and nervous tissues. The tissue specific promoter may also be specific for certain disease tissues, such as cancers. Any tissue s ecific promoters may be used in the connection with the nucleic acid constructs described herein.

[ S3 ] In certain embodiments, artificial raiRNA constructs based on, for example, rs iR30 (microRNA 30), may be used to express precursor miRNA/shRNA from single/low copy stable integration in cells in vivo, or through germiirse transmission in transgenic animals, in certain embodiments, even a single copy of stably integrated precursor miRNA/shRNA construct results in effective knockdown of a target gene. In certain embodiments, the inducible Tet system, coupled with the low-copy integration feature of description, allows more flexible screening applications, such as in screening for potentially lethal shRNAs or synthetic lethal shRNAs,

|0©40j In certain embodiments, the subject precursor miRNA cassette may be inserted within a gene encoded by the subject yector. For example, the subject precursor miRNA coding sequence may be inserted with art intron, or in the 5'- or 3 -LTR of a reporter gene such as GFP.

|D04I] In certain embodiments, cultured cells, such as wild type mouse fibroblasts or primary ee!ls can be switched from proliferative to senescent states simply through regulated knockdown of p53 using the subject constructs and methods, [00421 The constructs and methods of the description are advantageous in several respects. In one respect, stable precursor miRNA/shRNA expression may be effected through retroviral or lentivira! delivery of the ms NA/shRNAs, which is shown to be effective at single copy per cell. This allows very effective stable gene expression regu ation at extremely low copy number per cell {e.g. one per cell), thus vastly advantageous over systems requiring the introduction of a large copy number of constructs into the target ceil by, for example, transient iransfection, High copy numbers can be achieved also through other means, e.g. viral transduction. In some instances single-copy is required, as for example pooled shRNA screens that require single-copy for the deconvoiution of screening results, or in transgenic animals that may require single-copy for site specific integration of the trarrsgene (expressing the shRNA) at a given locus.

[0043] Compared to transection where there are multiple copies (such as multiple episomal copies) of the shRNA construct, and the LTR is active, the instant system is preferable for stable expression of the shRNA. A system described herein allows for stable expression, but could also be used In a transfection case. Even when infected and stably integrated into a target cell, the LTR promoter might be used (as Is the case for example in the LMP vector, where the LTR promoter drives expression of the shRNA cassette). Another useful feature of the modified m RNA described herein is that it is compatible with an established miR30 mIRNA/sbRNA library, which contains designed miRNA/shRNA constructs targeting almost all human and mouse genes (e.g., Silva et ah, 2G05, Nature Genetics 37: 1281 -1288). Any specific member of the library can be readily cloned (such as by PCR) into the vectors of the instant description for Pol It-driven regulated and stable expression.

[3044] Expression in some vector designs with different promoters depends on position of transcriptional start and stop sites. The subject method/system described herein, particularly using pel II promoters, has no such stringent requirements.

[ S45] Another aspect of the description provides a method for drug target validation. The outcome of inhibiting the function of a gene, especially the associated effect in vivo, is usually hard to predict Gene knock-out experiments offer valuable data for this purpose, but is expensive, time consuming, and potentially non-Informative since many genes are required for normal development, such that loss-of-function mutation in such genes causes embryonic lethality. Using the methods of the instant description, especially the inducible expression regulation system of the description, any potential drug target/candidate gene for therapeutic intervention may be tested first by selectively up- and/or down-regulating their expression in vitro, ex vivo, or In vivo, and determining the effect of such regulated expression, especially in vivo effects on an organism. If disruption of the norma! expression pattern of a candidate gene shows desired phenotypes in vitro and/or in vivo, the candidate gene is chosen as a target for therapeutic intervention. Various candidate compounds cars then he screened to identify inhibitors or activators of such validated targets,

[0046| Another aspect of the description provides a method to determine the effect of coordinated expression regulation of two or more genes. For example, miRNA/shR A constructs for two more target genes may be introduced into a target eel! (e,g, by stable integration) or an organism (e.g. by viral vector infection or transgenic techniques), and their expression may be individually or coordinated regulated using the inducible, or constitutive, and/or ubiquitous, or tissue specific or developmental specific promoters according to the instant description, Since different inducible promoters are available, the expression of the two or more target genes may be reguiated either in the s me or opposite direction (e.g., both up- or down- regulating, or one up one down, etc,). Such experiments can provide useful information regarding, inter alia, genetic interaction between related genes.

]0047] In certain embodiments, the nucleic acid constructs encode for modified miRNA that allows highly efficient knockdown of a target gene from a single (retroviral) integration event, thus providing a hig ly efficient means for certain screening applications. For example, the instant system and methods may be used to test potentially lethal miRNA/shR As or synthetic lethal miRNA/shRNAs,

[00481 Herein described also is a method to treat certain cancer, especially those cancer overexpressing Ras pathway genes (e.g., Ras itself) and having impaired p53 function, comprising introducing into such cells an active p53 gene or gene product to induce senescence and/or apoptosis, thereby killing the cancer ceils, or at least inhibit cancer progression and/or g owth.

MicroRJNA and RNAi Design

[00491 vectors thai express perfect complementary short hairpins RNAs

(sh NAs) are commonly used to generate functional siR As/maiure small RNA triggers of RNAi. (As used herein, shRNA, SJRNA and mature small RNA are a unified group of molecules that all function to knockdown expression of target genes, and are used interchangeably in that context.) The efficacy of gene silencing mediated by different shRNA-derived siRNAs may be inconsistent, and a number of short-hairpin RNA expression vectors can trigger an anti-viral interferon response. Moreover, shRNAs are typically processed symmetrically, In that both the functional si RNA strand and its complement strand are incorporated into the RISC complex. E ry of both strands Into the RISC can decrease the efficiency of the desired egu at on and increase the number of off-target mRNAs that are influenced, In com a ison_s endogenous microRNA {miR A} processing and maturation is a fairly efficient process that is not expected to trigger an anti-viral interferon response. This process involves sequential steps thai are specified by the information contained in miRNA hairpin and its flanking sequences.

|ΘΘ5Θ] Mature microRNAs (miRNAs) are endogeno sly encoded ~22~nt~long RNAs that are generally expressed in a highly tissue- or deveioprnental-stage-speeific fashion and that post-transcript ioral I y regulate target genes, More than -800 {mammals} distinct miRNAs have been identified in plants and animals. These small regulator)' RNAs are believed to serve important biological functions by three prevailing modes of action: (I) by repressing the translation of target mR As, (2) through deadenylation of transcripts, and (3) through cleavage and degradation of m NAs. In the latter case, miRNAs function analogously to small Interfering RNAs {si R As), in that they lead to a direct cleavage of the transcript rather than a non-cleavage mediated repression of gene expression, Importantly; niRNAs are expressed irs a highly tissue- specific or developmental^ regulated manner and this regulation is likely key to their predicted roles in eukaryotic development and differentiation. Analysis of the normal role of miRNAs will be facilitated by techniques that allow the regulated over-expression or inappropriate expression of authentic miRNAs in vivo, whereas the ability to regulate the expression of si NAs will greatly increase their utility both in cultured cells arid In vivo. Thus one can design and express artificial microRNAs based on the features of existing microRNA genes, such as the gene encoding the human miR~3G microRNA, These miR3G~based shRNAs have complex folds, and, compared with simpler stein/loop style shRNAs, are less toxic at inhibiting gene expression in transient and long-term assays.

[0051] Expression requires the insertion of the entire or core elements of the predicted miRNA precursor stem-loop structure into the expression vector at an arbitrary location. Because the actual extent of the precursor stem loop can sometimes be difficult to accurately predict It is generally appropriate to include ^"20-150 bp of flanking sequence on each side of the predicted ^"60~8G~ni miRNA stem-loop precursor to be sure that all ess-acting sequences necessary for accurate and efficient Drosha processing are included.

[0052] Genome wide libraries of shRNAs based on the miR.30 precursor RNA have also been generated. Each member of such libraries target specific human or mouse genes (or other species, including rat, dog, cat, monkey, cow, ...), and may be readily converted to the vectors/expression systems of the instant description. The following section describes the design of such libraries. ]QGS3J MicroRNAs (including the siRNA/maiure small RNA products and artificial microRNAs as well as endogenous m croRNAs) have potential for use as therapeutics as well as research tools, e.g. analyzing gene function. As a general method, the mature micro NA (miR) of the description,, especially those non-miR-30 based microRNA constructs of the description may also be produced according to the following description,

jOG54f In certain em odiments, the methods for efficient expression of microRNAs involve the use of a precursor microRNA molecule having a microRNA sequence in the contex of microRNA flanking sequences. The precursor microRNA is composed of any type of nucleic acid based molecule capable of accommodating the microRNA flanking sequences and the microRNA sequence, Examples of precursor microRNAs and the individual components of the precursor (flanking sequences and microRNA sequence) are provided herein, The description, however, is not limited to the examples provided. The description s based, at least in part, on the discover)' of an important component of precursor microRNAs, that is, the microRNA flanking sequences. The nucleotide sequence of the precursor and its components may vary widely.

[0055] In one aspect a precursor microRNA molecule is an isolated nucleic acid including microRNA flanking sequences and having a stem-loop structure with a microRNA sequence incorporated therein, An "isolated molecule" is a molecule that Is free of other substances with which it is ordinarily ound in nature or in vivo systems to an extent practical and appropriate for its intended use. In particular, the molecular species are sufficiently free from other biological constituents of host cells or if they are expressed in host cells they are free of the form or context in which they are ordinarily found in nature. For instance, a nucleic acid encoding a precursor microRNA having homologous microRNA sequences and flanking sequences may ordinarily be found in a host cell In the context of the host cell genomic DNA, An isolated nucleic acid encoding a microRNA precursor may be delivered to a host cell, but is not found in the same context of the host genomic DNA as the natural system, Alternatively, an isolated nucleic acid is removed from the host ceil or present in a host ceil that does not ordinarily have such a nucleic acid sequence. Because an isolated molecular species of the description may be admixed with a pharmaceutical !y~aeceptab|e carrier in a pharmaceutical preparation or delivered to a host cell, the molecular species may comprise only a small percentage by weight of the preparation or cell. The molecular species is nonetheless isolated in that it has been substantially separated from the substances with which it may be associated in living systems.

[0Θ561 An "isolated precursor microRNA molecule" is one which Is produced from a vector having a nucleic acid encoding the precursor microRNA. Thus, the precursor microRNA produced from the vector may be in a host cell or removed from a host cell The isolated precursor microRNA ma be found within a host cell that is capable of expressing the same precursor. Since the isolated precursor miRMA is a synthetic construct produced from a vector, it may be isolated from the host cell by ordinary methods of the art.

[0057] The term "nucleic acid" is used to mean multiple nucleotides (i.e. molecules comprising a sugar (e.g. nhose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidlne (e.g. cytosine (€), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G}), The term shall also include po!ynucleosides (i.e. a polynucleotide minus the phosphate} and any other organic base containing polymer. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, inosme, S-meihyScytosioe, 2-aminopurine_t 2-arrsino-6-ch ropurine_} 2,6- diaminopurine, b poxanthine, and other naturally and non-natural iy occurring nucleobases. substituted and unsubsiituied aromatic moieties. Other such modifications are well known to those of skill in the art. Thus, the term nucleic acid also encompasses nucleic acids with substitutions or modifications, such as in the bases and/or sugars, e.g. locked nucleic acids

(LNAs).

[005S] "MicroRNA flarsksrig sequence" as used herein refers to nucleotide sequences including microRNA processing elements. MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from precursor microRNA. Often these elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure. In some instances the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.

[00591 Thus, in some embodiments the flanking sequences are 3 to 4,000 nt In length. As a result, the length of the precursor molecule may be, in some instances at least about 50 nt or about 100 nt in length. The total length of the precursor molecule, however, may be greater or less than these values. In other embodiments the minimal length of the microRNA Hanking sequence is 10, 20, 30, 40, 50, 60, 70, 8Θ, 90, 100, 150, 200 and any integer there between, in other embodiments the maximal length of the microRNA flanking sequence is 2,000, 2 J 00, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3, 100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900 4,000 and any integer there between.

]006i| The microRNA flanking sequences may be native microRNA Hanking sequences or artificial microRNA flanking sequences, A native microRNA flanking sequence is a nucleotide sequence that Is ordinarily associated in naturally existing systems with microRNA sequences, i.e., these sequences are found within the genomic sequences surrounding the minima! microRNA hairpin in vivo. Artificial microRNA flanking sequences are nucleotides sequences that are not found to be flanking to microRNA sequences in naturally existing systems, The artificial microRNA flanking sequences may be flanking sequences found naturally in the context of other microRNA sequences. Alternatively they may be composed of minima! microRNA processing elements which are found within naturally occurring flanking sequences and inserted into other random nucleic acid sequences that do not naturally occur as flanking sequences or only partially occur as natural flanking sequences.

[006!] The microRNA flanking sequences within the precursor microRNA molecule may flank one or both sides of the stem-loop structure encompassing the microRNA sequence. Thus, one end (i,e,_s 5^f) of the stem-loop structure may be adjacent to a single flanking sequence and the other end (i.e., 3') of the ste n-loop structure may not be adjacent to a flanking sequence. Preferred structures have flanking sequences on both ends of the stem-loop structure. The flanking sequences may be directly adjacent to one or both ends of the stem-loop structure or may be connected to the stem-loop structure through a linker, additions! nucleotides or other molecules.

[90621 A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion), In some cases, the loop may also be very short and thereby not be recognized by Dicer, leading to Dicer-independent shR As (comparable to the endogenous miR0431 ). The terms "hairpin" and "fold-back" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art, The actual primary sequence of nucleotides within the stem-loop structure is not critical to the practice of the description as long as the secondary structure is present. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem may include one or mo e base mismatches. Alternatively, the base-pairing may be exact, i.e. not include any mismatches.

[0063] In some instances the precursor microRNA molecule may include more than one stem-loop structure. The multiple stem-loop structures may be linked to one another through a linker, such as, for example, a nucleic acid linker or by a microRNA flanking sequence or other molecule or some combination thereof.

[806 [ In an alternative embodiment, useful interfering RNAs can be designed with a number of software programs, e.g., the OlsgoEngine siRNA design tool available at wwv.oHoengine.com. The siRNAs of this description may range about, e.g., 1 -29 basepa!rs in length for the double-stranded portion. In some embodiments, the siRNAs are hairpin RNAs having an about 1 -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 1 -29 bp fragment complementary to a target gene mRNA thai has at least one, preferably at least two or three, bp mismatch with a nonterget gene-related sequence. In some embodiments, the preferred siR As do not bind to RNAs having more than 3 mismatches with the target region.

Expression Vectors and Host Cells

[00651 The description also Includes vectors for producing precursor microRNA molecules. Generally these vectors include a sequence encoding a primary microRNA and (in vivo) expression elements. The expression elements include at least one promoter, such as a Pol 11 promoter, which may direct the expression of the operably linked microRNA precursor (e.g. the shRNA encoding sequence). The vector or primary transcript is first processed to produce the stem-loop precursor molecule, The stem-loop precursor is then processed to produce the mature microRNA.

| 66] R A polymerase II (Pol 11} transcription units (e.g., units containing a CMV promoter) is preferred for use with inducible expression. It will be appreciated that in the vectors of the description, the subject shRNA encoding sequence may be operably linked to a variety of other promoters. In some embodiments, the promoter is a type II tRNA promoter such as the tRNAVa promoter and the tRNAmet promoter. These promoters may also be modified to increase promoter activity. In addition, enhancers can be placed near the promoter to enhance promoter activity. Pot IS enhancer may also be used for Pol Hi promoters. For example, an enhancer from the CMV promoter can be placed near the U6 promoter to enhance U6 promoter. In certain embodiments, the subject Pol II promoters are inducible promoters. Exemplary inducible Pol IS systems are available from commercial sources. Inducible promoters include Tet-resporssive promoters, macrolide responsive promoters and the iae operator system.

[0067] The interfering RNA can be mdudbly expressed in a tissue-specific manner dictated by a tissue-speciilc promoter driving expression of a iet-irarssaciivator protein

(tTA;TET-off or rtTA;TET-on). In certain embodiments, tissue-specificity can be obtained by coupling tissue-specific promoters to the Cre-LoxP system. In general, a triple transgenic can be produced consisting of ( 1 ) TRE-Lox-Stop-Lox(LSL)-GFP~miR30/E, (2) CAG-riTA, and( 3) tissue specific promoter-CRE; or (1) TRE-GFP-miRSO E, (2) CAG-LSL-rtTA, and (3) tissue specific promoter-CRE, Tissue-speciilc 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 Vi!Hn or FABP promoter in Ihe case of Intestinal cells and/or cancers; a RIP promoter m the case of pancreatic beta cells; a Keratin promoter 1st the case of keratisioevtes; a Probasin promoter in the ease of prostatic epithelium; a Nesttn or GFAP promoter in the ease of CNS cells and/or cancers; a Tyrosine Hydroxylase, S 00 promoter or neurofilament promoter in the case of neurons; the pancreas- specific promoter; a Clara cell secretory protein promoter in the ease of lung cancer; and an Alpha myosin promoter in the ease of cardiac cells,

[0068] Cre and/or teMransactivator expression also can be controlled in a temporal manner, e,g,_s by using an inducible promoter, or a promoter that is temporally restricted during development such as Pax3 or Protein O (neural crest), Hoxal (f!oorplate and notoehord), Hoxb6 {extraembryonic mesoderm, lateral plate and limb mesoderm and midbrain-hindbraiii 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 (E ) 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 (Gfl). Natural G Hgands include cordcosterone, Cortisol, and aldosterone, Mutant versions of the GR receptor, which respond to, e.g,, dexamethasone, triamcinolone aoetomde, and/or RU3 486, may also be fused to the Cre protein.

[0069] In certain embodiments, additional transcription units may be present 3^f to the shR A portion. For example, an internal ribosomal entry site (IRES) may be positioned downstream of the shRNA insert, the transcription of which is under the control of a second promoter, such as the PGK promoter. The IRES sequence may be used to direct the expression of an operably linked second gene, such as a reporter gene (e.g,, a fluorescent protein such as GFP, BFP, YFP, etc, an enzyme such as fuciferase (Promega), etc.). The reporter gene may serve as an indication of mfection/iransfeclion. and the efficiency and/or amount of m A transcription of the shRNA-I ES-reporter cassette/insert. Optionally, one or more selectable markers (such as puromycin resistance gene, neomycin resistance gene, hygromycin resistance gene, zeocsn resistance gene, etc.) may aiso be present on the same vector, and are under the transcriptional control of the second promoter. Such markers may be useful for selecting stable integration of the vector into a host cell genome.

[ΘΟ70] Certain exemplary vectors useful for expressing the precursor microR As are shown in the examples. Thus the description encompasses the nucleotide sequence of such vectors as well as variants thereof.

[11071] In general, variants typically w ll share at least 40% nucleotide identity with any of the described vectors, In some instances, will share at least 50% nucleotide identity; and in still other instances, will share at least 60% nucleotide identify, The preferred variants have at least 70% sequence homology. More preferably the preferred variants have at least 80% and, most preferably, at least 90% sequence homology to the described sequences. (If the common element of the shown vector(s) and the variant vector is only the m!R-E backbone of the precursor miRNAs described herein, then the sequenee/n eieotide identity will only be ~-3QGnt of an ~6G0S-9000nt vector, which is about 3-5%.)

When using the molecules described herein to produce variants from libraries, variants with high percentage sequence homology can be identified, for example, using stringent hybridization conditions, The term "stringent conditions", as used herein, refers to parameters with which the art is familiar. More specifically, stringent conditions, as used herein, refe to hybridization at 65° C, in hybridization buffer (SJxSSC, 0,02% Fsco , 0.02% polyvinyl pyrolidone, 0,02% bovine serum albumin, 2.5 mM NaH2P04 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.I 5M sodium chloride/0. ISM sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetie acid. After hybridization, the membrane to which the DNA is transferred is washed at 2* SSC at room temperature and then at 0.1 ^SSC Q. S xSDS at 65° C. There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency. Such variants may be further subject to functional testing such that variants that substantially preserve the desired/relevant function of the original vectors are selected/identified.

[0073] The "in vivo expression elements" are any regulatory nucleotide sequence,, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient expression of the nucleic acid to produce the precursor mlcroRNA. The In vivo expression element may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter or a tissue specific promoter. Constitutive mammalian promoters include, but are not limited to, polymerase II promoters as well as the promoters for the following genes:

h poxanthine phosphorihosyi transferase (HPTR), adenosine deaminase, pyruvate kinase, and β- aetsn. Exemplary viral promoters which function const itutiveiy in eukaryotic ceils include, for example, promoters from the simian virus, papilloma virus, adenovirus, human immunodeficiency vims (HIV), Rous sarcoma virus, cytomegalovirus, the long Sermi saS repeats (LTR) of mo!oney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as in vivo expression element of the description also include inducible promoters, inducible promoters are expressed in the presence of an inducing agent, For example, the nietallotbionein promoter Is induced to promote transcription in the presence of certain metal ions, Other inducible promoters are known to those of ordinary skill in the art,

[0074] Vectors include, but are not limited to, p!asmids, pbagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for producing the precursor microR^' A, and free nucleic acid fragments which can be attached to these nucleic acid sequences. Viral and retroviral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses; retroviruses, such as: Moloney murine leukemia virus; Murine stem cell virus, Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcoma virus; adenovirus; adeno-assodated virus; SV4G-fype viruses; polyoma viruses;

Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses;

!enitviruses; and RNA viruses such as any retrovirus. One can readily employ other unnamed vectors known in the art,

[0075] Viral vectors are generally based on nors-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleic acid sequence of interest, Hon- cytopathie viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent provirai integration into host cellular DMA. Retroviruses have been approved for human gene therapy trials. Genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of nucleic acids in vivo. Standard protocols tor producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasm id, transection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M._f "Gene Transfer and Expression, A Laboratory Manual," W,H. Freeman Co,₅ New York (1 90) and Murry, E, J. Ed. "Methods in Molecular Biology," vol 7, Humana Press, Inc., Clifton, NJ. (1991).

[δ 7β] The description also encompasses host cells trarssiecied with the subject vectors, especially host cell lines with stably integrated shR A constructs. In certain embodiments, the subject host eel! contains only a single copy of the integrated construct expressing the desired sh NA (optionally under She control of an inducible and/or tissue specific promoter). Host cells include for instance, ceils (such as primary ceils) and cell lines, e.g. prokaryotic (e.g., E. colt), and eukaryotic (e.g., dendritic ceils, CHO ceils, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells, etc.). Exemplary cells include: N1H3T3 cells, MEFs, HE&293 or HE 293T cells, CHO cells, DPI cells, hematopoietic stem/progenitor cells, cancer cells, etc.

Methods of Using

[H077J In certain aspects, methods of the description comprise contacting and introducing into a target cell with a subject vector capable of expressing a precursor microRNA as described herein, to regulate the expression of a target gene in the cell. The vector produces the microRNA transcript, which is then processed into precursor microRNA in the cell, which is thers processed to produce the mature functional microRNA which is capable of altering accumulation of a target protein in the target cell. Accumulation of the protein may be effected in a number of different ways. For instance the mkro NA may directly or indirectly affect translation or may result in cleavage of the mRNA transcript or even effect stability of the protein being translated from the target mRNA, MicroRNA may function through a number of different mechanisms. The methods and products of the description are not limited to any one mechanism, The method may be performed in vitro, e.g., for studying gene function, ex vivo or in vivo, e.g. for therapeutic purposes.

[SS78] An "ex vivo" method as used herein is a method which involves isolation of a cell from a subject, manipulation of the cell outside of the body, and reimplantation of the manipulated ceil into the subject. The ex vivo procedure may be used on autologous or heterologous cells, hut is preferably used on autologous cells, in preferred embodiments, the ex vivo method is performed on cells that are isolated from bodily fluids such as peripheral blood or bone marrow, but may be isolated from any source of cells. When returned to the subject the manipulated ceil will he programmed for ceil death or division, depending on the treatment to which it was exposed. Ex vivo manipulation of cells has been described in several references in the art. The ex vivo activation of cells of the description may be performed by routine ex vivo manipulation steps known in the art. In vivo methods are also well known in the art. The description thus is useful for therapeutic purposes and also is useful for research purposes such as testing in animal or in vitro models of medical, physiological or metabolic pathways or conditions. [0079] The ex vivo and In vivo methods are performed on a subject. A "subject" shall mean a human or non-human mammal, including but not limited to, a dog, cat horse, cow, pig, sheep, goat, primate, rat, and mouse, etc. In other instances, the "subject" may also be a non- mammal vertebrate, including but not limited to chicken, frog, fish, etc.

[0080] in some Instances the mature microRNA is expressed at a level sufficient to cause at least a 2-fold, or in some instances, a 10 fold reduction in accumulation of the target protein, The level of accumulation of a target protein may be assessed using routine methods known to those of skill in the art. For instance, protein may be isolated from a target cell and quantitated using Western blot analysis or other comparable methodologies, optionally in comparison to a control. Protein levels may also be assessed using reporter systems or fkorescerttly labeled antibodies, !n other em odiments, the mature mtcrorlNA is expressed at a level sufficient to cause at least a 2, 5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 5, 70, 75, or 100 fold reduction in accumulation of the target protein. The "fold reduction" may be assessed using any parameter for assessing a quantitative value of protein expression. For instance, a quantitative value can be determined using a label i.e. fluorescent, radioactive linked to an antibody. The value is a relative value that is compared to a control or a known value,

[0081] Different microRNA sequences have different levels of expression of mature microRNA and thus have different effects on target mRNA and/or protein expression. For instance, in some eases a microRNA may be expressed at a high level and may be very efficient such that the accumulation of the target protein is completely or near completely blocked. In other instances the accumulation of ihs target protein may be only reduced slightly over the level that would ordinarily be expressed in that cell at that time under those conditions in the absence of the mature microRNA. Complete inhibition of the accumulation of the target protein is not essential, for example, for therapeutic purposes. In many cases partial or low inhibition of accumulation may produce a preferred phenotype. The actual amount thai is useful will depend on the particular cell type, the stage of differentiation, conditions to which the cell is exposed, the modulation of other target proteins, etc.

[0082 [ The microRNAs may be used to knock down gene expression in vertebrate cells for gene-function studies, including target-validation studies during the development of new pharmaceuticals, as well as the development of human disease models and therapies, and ultimately, human gene therapies.

[0083] The methods of the description are useful for developing therapies For any type of "disease", "disorder" or "condition" ire which It is desirable to reduce the expression or accumulation of a particular target protein(s). Diseases include, for instance, but are not limited to, cancer, infectious disease, cystic fibrosis, blood disorders, including leukemia and lymphoma, spinal muscular dystrophy, early-onset Parkinsonism (Waisman syndrome) and X-lmked mental retardation (M x3).

[0 S4] Cancers include but are not limited to biliary tract cancer; bladder cancer; breast cancer; brain cancer including glioblastomas and meduiloblastomas; cervica! cancer;

choriocarcinoma; colon cancer including colorectal carcinomas; endometrial cancer; esophageal cancer; gastric cancer; head and neck cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia, multiple myeloma, AlDS-assoclated leukemias and adult T-eell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Pagers disease; liver cancer; lung cancer including small cell lung cancer and non-small cell lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; osteosarcomas; ovarian cancer Including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,

HpGsarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skin cancer including melanomas, Kaposi's sarcoma, basocelhilar cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stroma! tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; transitional cancer and renal cancer including adenocarcinoma and Wilms tumor.

[0085] An infectious disease, as used herein, is a disease arising from the presence of a foreign microorganism in the body. A microbial antigen, as used herein, is an antigen of a microorganism. Microorganisms include but are not limited to, infectious virus, infectious bacteria, and infectious fungi,

|00S6] Examples of Infectious virus include but are not limited to: Retroviridae (e.g. human immunodeficiency viruses, such as HlV-1 (also referred to as HTLV-Ui, LAV or HTLV- ili/LAV, or HJV-IIF); and other isolates, such as H1V-LP); Picomavirldae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinovi ruses, echoviruses);

Calcivtridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronavi uses); R abdoviradae (e,g. vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g. coronavi ruses); Hhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Fiioviridae (e.g. eboia viruses); Paramyxovlridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthoniyxoviridae (e.g. influenza viruses); Bungavirldae (e.g. Hantaan viruses, buuga viruses, ph!eboviruses and airo viruses) Arena viridae (hemorrhagic fever viruses): Reovirldae (e.g. reovir ses, orbiviurses and rotaviruses); Birnaviridae; Hepadnavlridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenovsridae (most adenoviruses);

Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster vims, cytomegalovirus (C V), herpes virus); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and indov ridae (e.g. African swine fever virus); and unclassified viruses (e.g, the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (tlioughi to be a defective satellite of hepatitis B virus), the agents of nors-A, non-B hepatitis (class intema!Iy transmitted; class 2^!«parenteraIIy transmitted (Le. Hepatites C); orwalk and related viruses, and astroviruses).

[0087] Examples of infectious bacteria include but are not limited to; Helicobacter pyloriSj Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M tuberculosis, ML avium, M. intraceilulare, M kansail, M. gordooae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agaJactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.).

Streptococcus pneumoniae, pathogenic Campylobacter sp._» Enierococcus sp„ Haemophilus influenzae, Bacillus anirseis, corynebseterium diphtheriae, corynebseterium sp,, Er sipelothnx rhusiopathiae, Clostridium perfringers, Clostridium ietani, Enterobacter aerogerses, Klebsiella pneumoniae, Pasturefia rnuHocida, Bacteroides sp.₃ Fusobacterium nueloatum, Streptobaci!lus monilifonrsts, Treponema palladium, Treponema perienue, Leptospira, Rickettsia, and

Actinomyces israelii,

[θθ§8[ Examples of infectious fungi include: Cryptococcus neoformans, Hisiopiasma eapsulaium, Coccidioides immiiis, Blastomyces dermatitidts, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii.

[ΘΘ89] The vectors of this description can be delivered into host ceils via a variety of methods, including but not limited to, liposome fusion, infection by viral vectors, and routine nucleic acid irasisfection methods such as electroporation, calcium phosphate precipitation and microinjection. In some embodiments, the vectors are integrated into the genome of a transgenic animal (e.g., a mouse, a rabbit, a hamster, or a nojihuman primate). Diseased or disease-prone ceils containing these vectors can be used as a model system to study the development, maintenance, or progression of a disease that is affected by ihe presence or absence of the interfering RNA,

[0090 | Expression of the mlRNA/sIR A introduced into a target cell may be confirmed by art-recognized techniques, such as Northern blotting using a nucleic acid probe. For cell lines that are more difficult to transfect, more extracted RNA can be used for analyses, optionally coupled with exposing the film longer. Once expression of the miR A/si& A is confirmed, the DNA construct can then be tested for NAt efficacy against a cotransfected construct encoding the target protein or directly against an endogenous target In the latter case, one preferably should have a clear idea of transfection efficiency and of the half-!ife of the target protein before performing the experiment.

Pharmaceutical Use and Methods of Administration

[0091] In one aspect, the description provides a method of administering any of the compositions described herein to a subject. When administered, the compositions are applied in a therapeutically effective, pharmaceutically acceptable amount as a pharmaceutically acceptable formulation, As used herein, the term "pharmaceutically acceptable" is given its ordinary meaning. Pharmaceutically acceptable compounds are generally compatible with other materials of the formulation and are not generally deleterious to the subject. Any of the compositions of the present description may be administered to the subject in a therapeutically effective dose. A "therapeutically effective'" or ars "effective" as used herein means that amount necessary to delay the onset of, inhibit the progression of, halt altogether the onset or progression of, diagnose a particular condition being treated, or otherwise achieve a medically desirable result, i.e., that amount which is capable of at least partially preventing, reversing, reducing, decreasing, ameliorating, or otherwise suppressing the particular condition being treated, A therapeutically effective amount can be determined on an individual basis and will be based, at least In part, on consideration of the species of mammal, the mammal's age, sex. size, and health; the compound and/or composition used, the type of delivery system used; the time of administration relative to the severity of the disease; and whether a single, multiple, or contro!fed-re!ease dose regiment is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

|S092| The terms "treat" "treated," "treating," and the like, when used herein, refer to administration of the systems and methods of the description to a subject, which may, for example, increase the resistance of the subject to development or further development of cancers, to administration of the composition in order to eliminate or at least control a cancer or an infectious disease, and/or to reduce the severity of the cancer or infectious disease, or symptoms thereof. Such terms also include prevention of disease/condition n, for example,

subjects/individuals predisposed to such diseases/conditions, or at high risk of developing such diseases conditions.

0©93| When administered to a subject, effective amounts will depend on the particular condition being treated and the desired outcome. A therapeutically effective dose may be determined by those of ordinary skill in the art, for instance, employing factors such as those further described below and using no more than routine experimentation.

Exemplary Uses

Drug Target Validation

[0894] Good drugs are potent and specific; that is, ideally, they must have strong effects on a specific biological pathway or tissue (such as the disease tissue), while having minimal effects on all other pathways or all other tissues (e.g., healthy tissues). Confirmation that a compound inhibits the intended target (drug target validation) and the identification of undesirable secondary effects are among the main challenges In developing new drugs, Drag target validation also includes determining whether the inhibition of the target under

consideration has a therapeutic benefit,

[0095 [ Modern drug screening typically requires tremendous amounts of time and financial resources. Ideally, before even committing to such an extensive drug development program to identify a drug, one would like to know whether the intended drug target would even make a good target for treating a disease. That is, whether antagonizing the function of the intended target (such as a disease-associated oncogene or survival gene), would be

sufficient effective to treat the disease, and whether such treatment would bear an acceptable risk or side effect. For example, if a cancer is determined to be caused by an activating mutation in the Ras pathway, or caused by abnormal activity of a survival gerte such as Bci-2, the subject system can be used to generate animal models for drug target validation. Specifically, one can generate a transgenic mouse with the subject tet-responsive iRNA-embedded shRNA expression, with the miRNA-embedded shRNA targeting a gene that is a potential drug target {i.e., Ras or Be!-2 in this example). Tumors with various initiating lesions can then be made in the mouse, and the miRNA-embedded shRNA can be switched on in the tumor (if, for example, a tet-ON regulator is used), Such miR A -embedded shRNA expression mimics the action of a (yet to be identified) drug that would interfere with that target, if knocking down the target gene is effective to reverse or stall the course of the disease, the target gene is a valid target. [0096] Optionally, the miRNA-embedded shRNA transgene can be switched on in a number of tissues or organs, or even in the whole organism, in order to verify the potential side effects of the (yet to be identified) drug on other healthy tissues/organs.

[0097] Thus another aspect of the description provides an animal useful for drug target validation, comprising a germline transgene encompassing the subject artificial nucleic acid, which transcription is driven by a Pol II promoter. The expression of the encoded precursor molecule (such as one based on the miR30-design) leads to an siRNA/mature small RNA trigger of RNAi that targets a (yet to be identified) candidate drug target. Optionally, the precursor molecule is expressed in an inducible, reversible, and/or tissue-specific manner.

[0098] In a related aspect, the description provides a method for drug target validation, comprising antagonizing the function of a candidate drug target (gene) using a subject cell or animal (e.g., a transgenic animal) encompassing the subject artificial nucleic acid, either in vitro or in vivo, and assessing the ability of the encoded precursor molecule to reverse or stall the disease progress or a particular phenotype associated with a pathological condition. Optionally, the method further comprises assessing any side effects of inhibiting the function of the target gene on one or more healthy organs/tissues.

Animal Disease Model

[0099] The subject nucleic acid constructs enables one to switch on or off a target gene or certain target genes (e.g., by using crossing different lines of transgenic animals to generate multiple-transgenic animals) inducibly, reversibly, and/or in a tissue-specific manner. This would facilitate conditional knock-out/knock-down or turning-on of any target gene(s) in a tissue-specific manner, and/or during a specific developmental stage (e.g., embryonic, fetal, neonatal, postnatal, adult, etc.). Animals bearing such transgenes may be treated, such as by providing a tet analog in drinking water, to turn on or off certain genes to allow certain diseases to develop/manifest. Such system and methods are particularly useful, for example, to analyze the role of any known or suspected oncogene (or tumor suppressor genes) in the maintenance of immortalized or transformed states, and in continued tumor growth in vivo.

[0100] In certain embodiments, the extent of gene knock-down may be controlled to achieve a desired level of gene expression. Such animals or cell (healthy or diseased) may be used to study disease progress, response to certain treatment, and/or screening for drug leads.

[0101] The ability of the subject system to use a single genomic copy of the Pol II promoter-driven miRNA-embedded shRNA cassette to control gene expression is particularly valuable for complex library screening. [0102] The subject gene knock-down by expression of shRNA-mirs may be very similar to overexpression of protein-coding cDNAs. Thus any expression systems allowing targeted, regulated and tissue-specific expression, which have traditionally be limited to gene overexpression studies, may now be adapted for loss-of-function studies, especially when combined with the available genome-wide, sequence-verified banks of miR-30-based shRNAs targeting model organisms, such as human and mouse.

EXAMPLES

Example 1, Conserved Region

[0103] Comparison of miR-30 sequence across several species shows an evolutionary conserved region (Fig. 1). The alignment shows a highly conserved region extends from reference position 239 to 340. A comparison of complete sequence of endogenous and synthetic miR-30 shows that the guide/passenger positioning is reversed in synthetic miR-30, and that a bulge in position 12-13 of the guide is missing. Importantly, a synthetic loop in a 3' conserved region contains mutations in one highly conserved nucleotide. This is because a EcoRI site is placed there, and alters a highly conserved nucleotide, i.e., a C is changed to A.

[0104] Modifications were made in the conserved region by modifying the vector sequence encoding the miR-30 sequence 5'-GAAUUCAA-3' to determine the effect on the efficiency of knockdown. The following sequence was used in the vector sh.Yapl.891, noting that that vector contains the DNA sequence 5'-GAATTCAA-3' where underscored.

TGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTG GAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGA GAAGGTATATTGCTGTTGACAGTGAGCGCTGGAGAAGTTTACTACATAAA T AG T GAAG CCACAGATGTATTTATGTAG TAAACT TCTCCATTGCCTACTG CCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAA CTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAA TGGTATAAATTAAATCACTTT

[0105] The following sequence was used in the vector miR-E sh.Yapl.891, noting that that vector contains the conserved DNA sequence 5'-GACTTCAA-3' where underscored.

TGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTG GAAACACTTGCTGGGATTACTTCGACTTCTTAACCCAACAGAAGGCTCGA GAAGGTATATTGCTGTTGACAGTGAGCGCTGGAGAAGTTTACTACATAAA T AG T GAAG CCACAGATGTATTTATGTAG TAAACT TCTCCATTGCCTACTG CCTCGGACTTCAAGGGGCTAGAATrCGAGCAATTATCTTGTTTACTAAAA CTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAA TGGTATAAATTAAATCACTTT

[0106] The sequence contains a shifted EcoRI site (GAATTC) in a nonconserved region and modifications to preserve the secondary structure in the region predicted to hybridize with the EcoRI site in the miRNA secondary structure.

[0107] The results (Fig. 3) show that single nucleotide substitutions to produce the miRNA sequences 5' -UACUUCAA-3', and 5'-GACGUCAA-3' produced the knockdown equivalent to the endogenous human miR-30 sequence (5'-GACUUCAA-3'). These effects are due to the presence of the original conserved 3' flank, not the synthetic EcoRI site of miR-E.

[0108] The results show that there are critical nucleotides in this region. The perfect configuration was found to be 5'-NACNTCAA-3'. These results show that the modified miRNA- backbone must contain the native sequence 5'-GACUUCAA-3' or a modified sequence 5'- UACUUCAA-3', or 5'-GACGUCAA-3' to maximize knockdown of the embedded shRNA.

Example 2, Stem Loop Region

[0109] Modifications were made in the stem loop region of miR-30. The miR-30 molecule without and with a bulge was compared to miR-155 (BLOCK-iT, Invitrogen), which has a guide bulge. Two validated Luc shmiR molecules were tested, shmir.Luc.1309, originally validated in miR-30; and shmir.Luc.201, a control shRNA from Invitrogen. All were cloned in pLMN and infected at identical single-copy conditions. A sensor shRNA reporter assay system was used according to Fellmann et al., 2011, Mol Cell 41:733-46, hereby incorporated by reference, with some modifications. The experimental design and results are shown in Fig. 4. Expression at day 2 and day 6 for each nucleic acid construct were measured and compared. The results show that a bulge in miR-30 guide strand at position 12-13 reduces knockdown.

Example 3, Alternative miR-30 loops

[0110] Modifications were made in the miR-30 loops. Modifying the loop during testing of the original guide/passenger configuration did not affect knockdown potential. Seven out of seven alternative synthetic loops turned out to be worse than the miR-E/miR-30 loop. According to Gu et al., 2012, Cell 151:900-11, hereby incorporated by reference, the current configuration perfectly supports precise DICER positioning and minimizes off-target effects. The results show that the currently used miR-30-loop leads to efficient processing in the nucleus and by Dicer.

Example 4, Reversing Guide and Passenger Strands in the Stem Loop region

[0111] Modifications were made in the stem loop region to determine whether reversing the guide and passenger strands affects the degree of knockdown achieved. A miRNA- embedded shRNA for knocking down expression of the phosphatase and tensin homolog gene (Pten) was used (Fig. 5A). miR-30 sh.Pten.1523 contained the guide on the 3' strand connected via a 19 nucleotide loop to the passenger strand on the 5' loop, as follows.

CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGACCAGCTAAAGGTGAA GATATATAGTGAAGCCACAGATGTATATATCTTCACCTTTAGCTGGCTG CCTACTGCCTCGGAATTC

[0112] miR-30 sh.Pten.1524 contained a different guide and passenger strand, as follows.

CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGACAGCTAAAGGTGAAG ATATATTAGTGAAGCCACAGATGTAATATATCTTCACCTTTAGCTGGTG CCTACTGCCTCGGAATTC

[0113] miR-G5 sh.Pten.1523 and sh.Pten.1524 both contained the guide on the 5' strand connected via a loop to the passenger strand on the 3' loop. The sequence of miR-G5 sh.Yapl.891 is as follows.

TGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTT GGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTC GAGAAGGTATATTGCTGTTGACAGTGAGCGACTTTATGTAGTAAACTTC TCCATGTGAAGCCACAGATGATGGAGAAGTTTACTACATAAAGCTGCCT ACTGCCTCGGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTAC TAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAA T T AAAAT G GT AT AAAT T AAAT C AC T T T

[0114] The core (XhoI/EcoRI) sequence of miR-G5 sh.Pten.1523 is as follows.

CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGACTATATCTTCACCTT TAGCTGGCGTGAAGCCACAGATGGCCAGCTAAAGGTGAAGATATAGCTG CCTACTGCCTCGGAATTC [0115] The core (XhoI/EcoRI) sequence of miR-G5 sh.Pten.1524 is as follows.

CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGACATATATCTTCACCT TTAGCTGGGTGAAGCCACAGATGCCAGCTAAAGGTGAAGATATATGCTG CCTACTGCCTCGGAATTC

[0116] The sequence encoding modified miR-30 (miR-G5) with these shRNAs (sh.Pten.2634.sh.Pten.1524) was inserted into a LMT vector (LMP with turbo-GFP instead of GFP). A small portion of the sequence of LMP miR-30, around the miRNA-embedded shRNA is as follows.

CCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGT ACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCC CTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCC TCACTCCTTCTCTAGGCGCCGGAATTAGATCTCTCGACTAGGGATAACA GGGTAATTGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCTGC ACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAG AAGGCTCGAGCAACCAGAATTCAAGGGGCTACTTTAGGAGCAATTATCT TGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAA AG C T G AAT T AAAAT G G T AT AAAT T AAAT C AC T T T T T T C AAT T G AC G C G T GATCTAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGA GCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCC TCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCC GT

[0117] NIH3T3 full cell lysates in single copy infection were used, and the

transfectants were selected by puromycin. The results showed (Fig. 5B) that reversing the guide and passenger strands had no major effects on knockdown potency of miR30-shRNAs.

Example 5, Compiling modifications to produce miR-E

[0118] A modified miR-30 molecule was prepared by correcting the synthetic molecule to restore the endogenous human MIR30A sequence, by including a EcoRI site in a

nonconserved region. The resulting nucleic acid sequence of the new molecule, miR-E (with a stem sequence coding for an shRNA targeting the Yapl gene), is as follows (restriction sites for Xhol 5'-CTCGAG-3' and EcoRI 5'-GAATTC-3' are shown in italic):

TGTTTGAATGAGGCTTCAGTACTTTACAGAATCGTTGCCT GCACATCTTGGAAACACTTGCTGGGATTACTTCGACTTCT T AAC C C AAC AG AAGGCTCGA GAAG GTATATTGCTGTTGAC AGTGAGCGCTGGAGAAGTTTACTACATAAATAGTGAAGCC ACAGATGTATTTATGTAG TAAAC TTCTCCATTGCCTACTG CCTCGGACTTCAAGGGGCTAGAATTCGAGCAATTATCTTG TTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATT

TTTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTT

T

[0119] This miR-E sequence was modified and included shRNA directed to target Pten mRNA. The sequences of these shRNAs (XhoI/EcoRI fragments) are shown in Table 1.

[0120] The miR-E encoding vector was transduced into NIH3T3 cells at single copy infection, and the transfectants were selected by puromycin. The results showed (Fig. 6) that the miR-E configuration substantially enhanced Pten knockdown, particularly for intermediate shRNAs.

[0121] The structure of miR-E was compared to endogenous miR-30 (MIR30A) and synthetic miR-30, as folded with the mfold algorithm (Fig. 7). The conserved region is shown with an arrow, and the non-conserved region circled. The EcoRI site in a conserved region of miR-30 is moved to a nonconserved region in miR-E. The structure shows that the secondary structure of miR-E in the nonconserved region containing the EcoRI site is preserved compared to synthetic and endogenous miR-30.

Example 6, General Utility of miR-E

[0122] Synthetic miR-30 was compared to miR-E for expression of a variety of siRNA/mature small RNA molecules. Systematic single-copy knockdown testing of a panel of shRNAs (designed based on Sensor rules) in miR-E vs. miR-30 configurations using western blot and/or Sensor-reporter assays (details see above reference to Fellmann et al., 2011, Mol Cell 41:733-46). Primers used to clone shRNAs into miR-E were miR30-Xho-fw (miR30-XhoI-short- fw; 24bp, Tm 63 °C, GC 45%)

5'-AGAAGGCTCGAGAAGGTATATTGC-3'

and miRE-Eco-rev (30bp, Tm 72°C, GC 56%)

5 ' -GCTCGAATTCTAGCCCCTTGAAGTCCGAGG-3 '

[0123] The sequences used in comparison are shown in Tables 1 and 2.

[0124] The results (Figs. 8 and 9) show that the miR-E configuration dramatically enhances knockdown for almost every shRNA. Only known super-shRNAs (e.g. p53.1224, Ren.713) are not further improved, likely because the knockdown is already saturated, while many intermediate shRNAs in miR-E trigger knockdown in the range of super-potent shRNAs. The results show that combining Sensor-based design rules and miR-E configuration produces potent shRNAs in >70%. Example 7, Production of miR-E virions

[0125] PlatGP packaging cells were first stably transduced with stem-loop DGCR8 shRNAs (A-G) and selected. Two shRNAs were lethal. Others were then transfected with the Sensor vector (Fig. 10A). In the presence of the target site (TS) for the miRNA-embedded shRNA expressed from the same plasmid, the Sensor vector dramatically reduces its own viral titers, which can be partially rescued with DGCR8 siRNA cotransfection. One of the five stable DGCR8 knockdown packaging lines shows full restoration of packaging efficacies indicating that pri-miRNA processing is sufficiently blocked (Fig. 10B). These packaging cell lines will not only be valuable for even packaging of Sensor constructs or miR-E based shRNAs, but also to correct shRNA-processing induced biases in complex shRNA pools.

Table 1, shRNA sequences

mi -E sh.Pten.2049 ctcgagaaggtatattgctgttgaca gtgagcgaaagatcagcattcacaaa ttatagtgaagccacagatgtataat ttgtgaatgctgatcttctgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Bcl2.783 ctcgagaaggtatattgctgttgaca gtgagcgattatacaaggagacttct gaatagtgaagccacagatgtattca gaagtctccttgtataagtgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Bcl2.851 ctcgagaaggtatattgctgttgaca gtgagcgcaagggtaaacttgacaga agatagtgaagccacagatgtatctt ctgtcaagtttacccttttgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Bcl2.906 ctcgagaaggtatattgctgttgaca gtgagcgcgcacaggaattttgttta atatagtgaagccacagatgtatatt aaacaaaattcctgtgcatgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Bcl2.1132 ctcgagaaggtatattgctgttgaca gtgagcgcgactgatattaacaaagc ttatagtgaagccacagatgtataag ctttgttaatatcagtcttgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Bcl2.1241 ctcgagaaggtatattgctgttgaca gtgagcgccaagtgttcggtgtaact aaatagtgaagccacagatgtattta gttacaccgaacacttgatgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Mcll.772 ctcgagaaggtatattgctgttgaca gtgagcgactccggaaactggacatt aaatagtgaagccacagatgtattta atgtccagtttccggagctgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Mcll.866 ctcgagaaggtatattgctgttgaca gtgagcgcggattgtgactcttattt ctttagtgaagccacagatgtaaaga aataagagtcacaatccttgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Mcll.1334 ctcgagaaggtatattgctgttgaca gtgagcgaaagagtcactgtctgaat gaatagtgaagccacagatgtattca ttcagacagtgactcttctgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Mcll.1792 ctcgagaaggtatattgctgttgaca gtgagcgaaacagcctcgatttttaa gaatagtgaagccacagatgtattct taaaaatcgaggctgttctgcctact gcctcggacttcaaggggctagaatt c

miR-E sh.Mcll.2018 ctcgagaaggtatattgctgttgaca gtgagcgcggactggttatagattta taatagtgaagccacagatgtattat aaatctataaccagtccatgcctact gcctcggacttcaaggggctagaatt c

Table 2, Additional sh NA sequences

B 04.6 2 {haswan)

TGCTG1 GACAGTGAGCGACAGGACTTCAACACTATGTTTTAGTGAAGCCACAGATGTAAAACATAGT OTTQAAGTCCTGOTGCCTACTGCCTCGGA

BRD4J 81? <bMmffls)

TGCTGTTGACAGTOAGCOCAAGGAGAAAGACAAGAA JAATAGTGAAGCCACAGATGTA'rrCCTTCT TGTCTTTCTCCT TTGCCTACTGCC CGGA

&Rm,m (hum )

TGCTGTTGACAGTGAGCGCAAGGAGAAAGACAAGAAGGAATAGTGAAGCCACAGATGTATTCCTTCT TGTCTTTCTCCTTTTGCCTACTGCCTCGGA

TGCTGTTGACAGTGAGCGACAAGGTAATTGCAGTAATGAATAGTGAAGCCACAGATGTATTCATTACT GCAATTACCTTGGTGCCTACTGCCTCGGA

ί>»ϋΐι ίί.733 {jusjiase)

TOCl^'GTrOACAGTGAGCGACAGAAAGAGCACAACAGAGAATAGTGAAGCCACACATGTATTCTCTGTT GTGCTCTTTCTGGTGCCTACTGCCTCGGA

TGCTGl ¾ACAGTGAGCGCCCAGATGl ^"C iOCCAATAATAGTGAAGCCACAGATGTATTATTGGCA AAGAACATCTGGATGCCTACTGCCTCGGA

Daj»e3a,l 177 (mease)

TGCTGTTGACAGTGAGCGCCCAGATGTTCTTTGCCAATAATAGTGAAGC:CACAGA'i'GTATi^'ATrGCiCA AAGAACATCTriGATGCCTACTGCCTCGGA

amt3iiA42S (mosise)

TGCTG^'n^'GACAGTGAGCGAGCAGCGTCACACAGAAGCATATAGTGAAGCCACAGATGTATATGCT CT^' GTGTGACGCTGCGTGCCTACTGCCTCGGA

TGCI'GTTGACAG'rOAGCJGACCGCCTC'ri'CTTTGAGTTCTATAGTGAAGCCACAGATGTATAGAACTCAA AGAAGAGGCGGCTGCCTACTGCCTCGGA

TGCTGTTGACAGTGAGCGCGGCATCCACTG^' GAAl^'GATAATAGTGAAGCCACAGATGTATTATCATTC ACAGTGGATGCCATGCXrrACTGCCTCGGA

Bnm&aAW {mmss)

TGGTGTTGACAGTGAGCGCGGCATCCAC'rG'i^'GAATGA'rAATAGTGAAGCCACAGATGTATTATCATTC ACAGTGGATGCCATGCCTACTGCCTCGGA

TGCTGITGACAGTGAGCGACTGGTGCTAATTCCTCTCATATAGTGAAGCCACAGATGTATATGAGAGG AATTAGCACCAGCTGCCTACTGCCTCGQA

0ijss 3se,4!73 (msuse)

TGCTGTTGACAGTGAGCGCTAGCTGWaAAGAAATATTAATAGTGAAGCCACAGATG^'E^'ATrAATATTT CTTCAACAGCTATTGCCTACTGCCTCGGA

Diiii 3a,4575 (mts s)

TGCTGTTGACAGTGAGCGCAAGGTGAAGTTGTCTCGTTTATAGTGAAGCCACAGA^'iOTA^'[^'AAACGAGA CAACTTCACCTTATGCCTACTGCCTCGGA LiBci.i309 {Firefly Liiesferaise, «Οίϊίϊ¾

TGCTGrrGACAOTCAOCGCCCGCCTGAAGTCTCTGATTAATAGTGAAGCCACAGATGTATTAATCAGA GACTTCAGGCGGTTGCCTACTGCCTCGGA

MVB.721 {buwa!s)

TGCTGTTGACAGTGAGCGACI'GGACGAACTGATAATGCTATAGTGAAGCCACAGATGTATAGCATTAT CAGTTCGTCCAGGTGCCTACTGCCTCGGA

MYEJ4S? { mm}

TGCTGTrGACAGTGAGCGCAAi^'sAAAC^'rrGGTGTTAGGTAATAGTGAAGCCACAGATGTATTACCTAAC ACCAAGTTTCTTTTGCCTACTGCCTCGGA Y S834 {l¾atsa«5 & taeesse)

TGCTGTTGACAGTGAGCGCACGACGAGAACAGTTGAAACA1'ACJ! AAfJCCACA(lATGTAT^'G'!'f'{'CAAC TGlTCTGG'f'CG'il'f^'GCCTACTGCCTCGGA

RPA114U { m)

TGCnOTTGACAGTG CGACCACCATCTTGTGTACATCTTTAGTGAAGCCACAGATGTAAAGA'rGTAC ACAAGATGGTGGCTGCCTACTGCCTCGGA

Claims

What is Claimed:

1. A modified miRNA molecule for producing an artificial RNA molecule that inhibits the expression of a target transcript of a host cell, comprising

• a stem region modified to comprise a sequence encoding the artificial RNA molecule, wherein the artificial RNA molecule consists of a guide and a passenger strand; and

• a conserved region comprising a sequence consisting of 5'-DC*NNC-3', wherein N consists of A, G, U, or C, D consists of A, G or U, and wherein the C* is located at position 16-20 of the 3' arm;

wherein the modified miRNA molecule substantially inhibits the target transcript when expressed in the host cell.

2. The modified miRNA molecule of claim 1, wherein the conserved region comprises the sequence 5'-ACUUCAA-3', 5'-GCUUCGA-3', or 5'-UCUUCUG-3'.

3. The modified miRNA molecule of claim 1, further comprising an exogenous sequence encoding a recognition site for a restriction enzyme.

4. The modified miRNA molecule of claim 3, wherein the recognition site consists of a sequences recognized by EcoRI or Xhol.

5. The modified miRNA molecule according to any of claims 3 or 4, wherein the modified sequence consists of 5'-GAAUUC-3' and the complementary strand consists of the sequence 5'-GACUUC-3'.

6. The modified miRNA molecule according to any of claims 1-5, wherein the modified miRNA preserves the secondary structure of the native miRNA molecule.

7. The modified miRNA molecule according to any of claims 1-6, wherein the stem region does not include a bulge or a mismatch between the guide and passenger strand.

8. The modified miRNA molecule according to any of claims 1-7, wherein the miRNA molecule is derived from miR-30.

9. The modified miRNA molecule of claim 1, wherein the miRNA molecule is derived from miR-15 or miR-16 and the conserved region comprises the sequence 5'- UCUUCUG-3'.

10. The modified miRNA molecule according to any of claims 1-9, consisting of a pre- miRNA molecule.

11. The modified miRNA molecule of claim 10, consisting of a pri-miRNA molecule.

12. The modified miRNA molecule of claim 11, wherein the pri-miRNA molecule is efficiently processed to a pre-miRNA molecule in the nucleus.

13. The modified miRNA molecule according to any of claims 10-12, wherein the pre- miRNA molecule is efficiently processed by Dicer.

14. A nucleic acid construct encoding the modified miRNA molecule of any of claims 1-13.

15. The nucleic acid construct of claim 14 comprising a Pol II promoter.

16. The nucleic acid construct of claim 15, wherein the Pol II promoter is a constitutive

promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, a cell-type-specific promoter, and/or a developmental stage-specific promoter.

17. The nucleic acid construct of claim 16, wherein the Pol II promoter is a CMV-derived promoter.

18. The nucleic acid construct of claim 14, further comprising at least one selectable marker.

19. The nucleic acid construct of claim 14, further comprising a reporter gene.

20. The nucleic acid construct of claim 14, further comprising a Pol III promoter upstream of the coding sequence for expressing the precursor molecule.

21. A cell comprising the nucleic acid construct according to any of claims 14-20.

22. The cell of claim 21, which is a cell from a vertebrate organism.

23. A mammal comprising the cell according to claim 21.

24. A method for inhibiting the expression of a target transcript of interest in a cell,

comprising introducing a nucleic acid construct according to any of claims 14-20 into the cell, wherein the siRNA/mature small RNA molecule derived from the modified miRNA molecule is specific for the target transcript.

25. A method for treating a gene-mediated disease, comprising introducing into an individual having the disease a nucleic acid construct according to any of claims 14-20, wherein the nucleic acid construct encodes a plurality of siRNA/mature small RNA molecules that are specific for the a plurality of target transcripts.

26. A method for treating a gene-mediated disease, comprising introducing into an individual having the disease a nucleic acid construct according to any of claims 14-20, where the nucleic acid construction encodes an siRNA/mature small RNA that is specific for the transcript mediating the disease or specific for an auxiliary transcript required for disease progression or maintenance.

27. A method of validating a transcript as a potential target for treating a disease, comprising: • introducing a nucleic acid construct according to any of claims 12-18 into a cell associated with the disease, wherein the nucleic acid construct encodes an siRNA/mature small RNA molecule that is specific for the transcript; and

• assessing the effect of inhibiting the expression of the transcript on one or more

disease-associated phenotype; wherein a positive effect on at least one disease- associated phenotype is indicative that the transcript is a potential target for treating the disease.

28. A method for producing a non-naturally occurring, modified miRNA molecule for

inhibiting the expression of a target transcript by preparing a nucleic acid construct encoding the modified miRNA comprising the steps of

• modifying the stem sequence of the miRNA molecule to substitute it with a nucleic acid sequence encoding a siRNA molecule; and

• substituting a part of the backbone sequence of the miRNA molecule with a conserved region comprising a sequence consisting of 5'-DC*NNC-3', wherein N consists of A, G, U, or C, D consists of A, G or U, and wherein the C* is located at position 16-20 of the 3' arm;

wherein the nucleic acid construct is integrated into a host cell genome, and

wherein the modified miRNA molecule substantially inhibits the target transcript.

29. The method of claim 28, wherein the conserved region comprises the sequence 5'- ACUUCAA-3', 5'-GCUUCGA-3', or 5'-UCUUCUG-3'.

30. The method of claim 28, further comprising introducing an exogenous sequence

encoding a recognition site for a restriction enzyme.

31. The method of claim 30, wherein the recognition site consists of a sequence recognized by EcoRI or Xhol.

32. The method according to any of claims 30-31, wherein the modified miRNA sequence consists of GAAUUC and the complementary sequence consists of GACUUC.

33. The method according to any of claims 30-32, further comprising a modified loop region.

34. The method according to any of claims 30-33, wherein the stem region does not include a bulge or a mismatch between the guide and passenger strand.

35. The method of claim 28, wherein the miRNA molecule is derived from miR-155.

36. The method of claim 28, wherein the miRNA molecule is derived from miR-22 and the conserved region comprises the sequence 5'-GCUUCGA-3'.

37. The method of claim 28, wherein the modified miRNA molecule is derived from a miRNA selected from miR-30, miR-22, miR-15, miR-16, miR-103, and miR-107.

38. The method according to any of claims 28-37, wherein the nucleic acid construct encodes for a pri-miRNA molecule,

39. The method of claim 38, wherein the pri-miRNA molecule is processed to a pre-miRNA molecule in the nucleus of the host cell.