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(12) Patent Application Publication (10) Pub. No.: US 2007/0136827 A1 Collins Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2007/0136827 A1 Collins Et Al US 2007013 6827A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0136827 A1 Collins et al. (43) Pub. Date: Jun. 14, 2007 (54) CSFTRANS RIBOREGULATORS Publication Classification (51) Int. Cl. (75) Inventors: James J. Collins, Newton, MA (US); AOIK 67/027 (2006.01) Farren J. Isaacs, Brookline, MA (US); C7H 2L/04 (2006.01) Charles R. Cantor, Del Mar, CA (US); CI2N 15/09 (2006.01) Daniel J. Dwyer, Brookline, MA (US) CI2N 5/06 (2006.01) (52) U.S. Cl. ............................ 800/14; 435/325; 435/455; 536/23.2 Correspondence Address: CHOATE, HALL & STEWART LLP (57) ABSTRACT TWO INTERNATIONAL PLACE BOSTON, MA 02110 (US) The present invention provides nucleic acid molecules, DNA constructs, plasmids, and methods for post-transcrip tional regulation of gene expression using RNA molecules to (73) Assignee: TRUSTEES OF BOSTON UNIVER both repress and activate translation of an open reading SITY, Boston, MA (US) frame. Repression of gene expression is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5' untranslated (21) Appl. No.: 10/535,128 region (5' UTR) of an mRNA molecule. The nucleic acid element forms a hairpin (stem/loop) structure through complementary base pairing. The hairpin blocks access to (22) PCT Fed: Nov. 14, 2003 the mRNA transcript by the ribosome, thereby preventing translation. In particular, in embodiments of the invention PCT No.: PCT/USO3A36506 designed to operate in prokaryotic cells, the stem of the (86) hairpin secondary structure sequesters the ribosome binding S 371(c)(1), site (RBS). In embodiments of the invention designed to (2), (4) Date: Apr. 4, 2006 operate in eukaryotic cells, the stem of the hairpin is positioned upstream of the start codon, anywhere within the 5' UTR of an mRNA. A small RNA (trans-activating RNA, Related U.S. Application Data or taRNA), expressed in trans, interacts with the crRNA and alters the hairpin structure. This alteration allows the ribo Some to gain access to the region of the transcript upstream (60) Provisional application No. 60/426,891, filed on Nov. of the start codon, thereby activating transcription from its 15, 2002. previously repressed state. Patent Application Publication Jun. 14, 2007 Sheet 1 of 12 US 2007/0136827 A1 A. 2. Artificial Riboswitch System DNA s' ...A.Sassass RNA ''Ris Translation cis-Repression Protein &S m r me - - - - - -n - I - - - - - - - - - trans-Activation ulcars TrvashgraseracterstarsagesAUG C RBS Gule Patent Application Publication Jun. 14, 2007 Sheet 2 of 12 US 2007/O136827 A1 R a'yaay wicuccucuuuccaucuu "' ...IEEEEEEEEEEGU an C (Seo ID Jo: sa)er crR7 "Y "Yff freffy." G.gu' A aaasniAdil AAA Aug ( Seq \D No! S3) cR10 " i "iff"fify"2 G a "ErikCa a disceau (set d No. 54) C CrPB 'uuluu("Yi" AucucutAAShikigasaucCau (StG ) NO SS)er 4. CA UUA AA gif d CAGGy C heatedCU i. 'CCAusACUA5 (SEQ D WJO SSC) Figure 2a Patent Application Publication Jun. 14, 2007 Sheet 3 of 12 US 2007/0136827 A1 150 10 Fluorescence (Arbitrary Units) Figure 2b Patent Application Publication Jun. 14, 2007 Sheet 4 of 12 US 2007/0136827 A1 - 10 10 Fluorescence (Arbitrary Units) Figure 2c F - ta1 Ocr10 he ta12cr12 O.B 2. 8 0.6 0.4 90.2 O O o o 16 % Arabinose G a. Arabinose (FL) - Arabinose (RNA) +Arabinose (FL) +Arabinose (RNA) s - - Fluorescence (Arbitrary Units) F. ( u(26 3A-3G Patent Application Publication Jun. 14, 2007 Sheet 6 of 12 US 2007/O136827 A1 JAAAccA. RS Crr2 GA UseYUNR AUUAAGAggAGAAAG Guaccaus. (S Q D Nos.) Gu(22 3-H Patent Application Publication Jun. 14, 2007 Sheet 7 of 12 US 2007/O136827 A1 a III) M&A loop NS IILL Patent Application Publication Jun. 14, 2007 Sheet 8 of 12 US 2007/O136827 A1 taRNA T ampf 1 GURC C Patent Application Publication Jun. 14, 2007 Sheet 9 of 12 US 2007/0136827 A1 taR10-cirrit taR12-cirr taR10-cr-0 taR7-cirr10 taR12-Cr 10 taR2-crr2 taR7-cr2 taR10-cr-2 8 do 20 140 160 BO 200 220 24g Patent Application Publication Jun. 14, 2007 Sheet 10 of 12 US 2007/013.6827 A1 s--- D ---- -1N--/ 00 20 140 60 O 220 240 60 --2 Patent Application Publication Jun. 14, 2007 Sheet 11 of 12 US 2007/013.6827 A1 0.6 0.5 0.4 0.3 0.2 0.1 0.1 0.2 0.3 04 0.5 0.6 0.7 x (1-x) FIGule 8 Patent Application Publication Jun. 14, 2007 Sheet 12 of 12 US 2007/O136827 A1 S. t V / CD - a 9 it 3. as S. s N. o & - CO \\D Sl 3 e 3 A <A. Ch. s A: CSs CD f o CD c tre s CN n O Ol US 2007/013.6827 A1 Jun. 14, 2007 CIS/TRANS RIBOREGULATORS 0005 Although the approaches described above have proven extremely valuable, they have a variety of features RELATED APPLICATION INFORMATION that limit their usefulness. For example, methods that 0001) This application claims the benefit of U.S. Provi involve alterations in RNA transcription may have slower sional Application Ser. No. 60/426,891, filed Nov. 15, 2002, response times than methods that are based on post-tran which is hereby incorporated by reference. Scriptional regulation. Techniques involving modulation of transcription factors are generally limited to well-character ized transcription factors. Antisense, ribozyme, and RNAi GOVERNMENT SUPPORT based approaches typically require sequence-specific 0002 This invention was made with Government Sup design. It is evident that a need exists in the art for additional port under Grant Number F30602-01-2-0579 awarded by the systems and methods for the control of gene expression. In Air Force Research Laboratory and Grant Number EIA particular, there exists a need for modular systems that 0130331 awarded by the National Science Foundation. The function with a wide variety of genes and that can be Government has certain rights in the invention. integrated into biological networks. Furthermore, there exists a need in the art for systems that would afford the BACKGROUND OF THE INVENTION ability to artificially control gene expression within cells in 0003) Virtually all forms of life exhibit the ability to response to external stimuli. control gene expression, e.g., in response to environmental conditions or as part of the developmental process, and a SUMMARY OF THE INVENTION myriad of different mechanisms for controlling gene expres 0006 The present invention addresses these needs, sion exist in nature. These mechanisms permit cells to among others, by providing systems and methods for the express particular Subsets of genes and allow them to adjust post-transcriptional control of gene expression in prokary the level of particular gene products as required. For otic or eukaryotic cells. The invention provides an artificial example, bacteria and eukaryotic cells are often able to RNA-based system that enables precise control through adjust the expression of enzymes in synthetic or metabolic highly specific RNA-RNA interactions. According to the pathways depending on the availability of Substrates or end invention effective repression is achieved by engineering an products. Similarly, many cells are able to induce synthesis RNA molecule (or template for the RNA molecule), so that of protective molecules such as heat shock proteins in the engineered RNA forms a secondary structure that pre response to environmental stress. Inherited or acquired vents the ribosome from gaining access to the RNA at an defects in mechanisms for control of gene expression are appropriate location to begin translation. Repression of gene believed to play a significant role in human diseases (e.g., expression is achieved through the presence of a regulatory cancer), and targeted disruption of important regulatory nucleic acid element (the cis-repressive RNA or crRNA) molecules in mice frequently results in severe phenotypic within the 5' untranslated region (5' UTR) of an mRNA defects. molecule. The nucleic acid element forms a hairpin (stem/ 0004. A number of approaches have been developed in loop) structure through complementary base pairing. The order to artificially control levels of gene expression, many hairpin blocks access to the mRNA transcript by the ribo of which are modeled on naturally occurring regulatory Some, thereby preventing translation. A Small RNA (trans systems. In general, gene expression can be controlled at the activating RNA, or taRNA), expressed in trans, interacts level of RNA transcription or post-transcriptionally, e.g., by with the crRNA and alters the hairpin structure. This alter controlling the processing or degradation of mRNA mol ation allows the ribosome to gain access to the region of the ecules, or by controlling their translation. For example, transcript upstream of the start codon, thereby activating modulating the activity of transcription factors (e.g., by transcription from its previously repressed state. administration of small molecule activators or inhibitors) is being pursued as a method of controlling mRNA levels (see, 0007. In one aspect, the invention provides an engineered e.g., Nyanguile O, Uesugi M., Austin DJ, Verdine G L. Proc nucleic acid molecule comprising: (i) a first stem-forming Natl Acad Sci USA. 1997, 94(25): 13402-6. A nonnatural portion; (ii) a second stem-forming portion, wherein the two transcriptional coactivator.). Antisense strategies for gene stem-forming portions are complementary or Substantially silencing, in which an antisense RNA or DNA binds to a complementary, and (iii) a non-stem-forming portion that target RNA and results in inactivation, are also being forms a loop connecting the 3' end of the first stem-forming actively pursued for applications ranging from functional portion and the 5' end of the second stem-forming portion, genomics to therapeutics (Giles RV, “Antisense oligonucle wherein the engineered nucleic acid molecule forms a otide technology: from EST to therapeutics'Curr Opin Mol stem-loop structure that represses translation when posi Ther: 2000, 203):238-52).
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