# Patent

## Title
Sequence-specific MRNA interferase and uses thereof

## Abstract

The present invention provides an improved and specific mRNA interferase and related methods of protein-based mRNA interference.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority of U.S. Provisional Application No. 61/515,049, filed on Aug. 4, 2011. The content of the application is incorporated herein by reference in its entirety.


STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under grant 1RO1GM081567, awarded by the National Institute of Health. Accordingly, the U.S. Government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to a novel sequence-specific interferase and an improved method of achieving protein-based mRNA interference comparable to RNA-based mRNA interference, which is more sequence-specific.
BACKGROUND OF THE INVENTION
RNA-based mRNA interference has been well documented including the initial finding of natural antisense RNA regulation in E. coli and its application to regulate specific gene expression and phage infection and later the discovery of miRNA and siRNA. A common mechanism for all these systems is use of an RNA sequence complementary to a target mRNA. However, despite the existing technologies surrounding RNA-based mRNA interference, there are no functional technologies to inhibit the function of a specific mRNA by protein-based mRNA interference.
SUMMARY OF THE INVENTION
The present invention provides a novel mRNA interferase, which can mediate protein-based mRNA interference. Shown below are the polypeptide and related DNA sequences for an exemplary novel mRNA interferase, MazF-hw.







(SEQ ID NO: 1)


VTPRCRYVQVRRGDIVIVDLSPTKGSEQQGTNRPCVVIQNDVGNRNSPT


 


TIIAPFTKQYNPDNTYPFEVEVLASNTSLNQDSVADLSQIRVVDINKGV


 


KTNIGSVPSARMAKIDTAIKTSLGL


 


(SEQ ID NO: 2)


CATATGACTCCGCGTTGTCGTTACGTGCAAGTACGCCGCGGCGATATCG


 


TCATTGTTGACTTGAGTCCGACGAAGGGTAGCGAGCAGCAGGGTACCAA


 


CCGCCCTTGTGTAGTTATCCAAAATGATGTGGGCAACCGTAACTCCCCG


 


ACCACGATCATCGCTCCGTTCACGAAGCAGTATAACCCGGATAATACGT


 


ACCCGTTCGAAGTAGAGGTACTGGCATCGAATACCTCGCTGAATCAGGA


 


TTCGGTGGCAGACCTGAGTCAAATCCGCGTAGTGGATATTAATAAGGGC


 


GTGAAGACCAATATCGGCTCAGTTCCTTCCGCTCGCATGGCAAAAATCG


 


ATACCGCGATTAAGACGAGTCTGGGTCTGTGA






Accordingly, one aspect of this invention provides an isolated polypeptide (i) comprising an amino acid sequence that is at least 70% (e.g., 80, 85, 90, 95, or 99%) identical to the sequence of SEQ ID NO: 1 and (ii) having an mRNA interferase activity. In one embodiment, the polypeptide comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 1 as shown below. In a preferred example, the polypeptide has an activity of cleaving an RNA sequence having the target sequence of UUACUCA (SEQ ID NO: 3).
Another aspect of this invention provides an isolated nucleic acid comprising a sequence that encodes the above-mentioned polypeptide. The nucleic acid can contain a sequence that is at least 70% (e.g., 80, 85, 90, 95, or 99%) identical to SEQ ID NO: 2. The invention also features a vector, such as an expression vector, comprising the nucleic acid and a host cell comprising the nucleic acid.
The afore-mentioned nucleic acid, vector, and host cell can be used for producing a polypeptide of this invention. Accordingly, this invention also provides a method for producing the polypeptide. The method includes culturing the host cell in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell in the medium of the cell.
In a third aspect, the invention provides a composition containing the aforementioned polypeptide or a nucleic acid encoding the polypeptide; and a carrier. As disclosed herein, this composition is useful for protein-based mRNA interference.
In a fourth aspect, the invention provides a method for detecting whether RNA molecules in a test sample have the target sequence of UUACUCA. The method includes incubating the test sample with the aforementioned polypeptide under conditions permitting cleaving an RNA sequence by the polypeptide to generated a treated sample; and examining the treated sample to identify any change in molecular weight or size of the RNA molecules. The change indicates that the RNA molecules have the target sequence. In one embodiment, the examining step is conducted by comparing the treated sample with a molecular weight maker sample or with a control sample that is identical to the test sample.
In a fifth aspect, the invention provides a method for decreasing the level of RNA in a cell. The method includes introducing to the cell the above-mentioned polypeptide, nucleic acid, or vector. In one example, the RNA contains the sequence of UUACUCA. The cell can be a prokaryotic cell (e.g., a bacterial cell such as an E. coli, cell) or a eukaryotic cell (e.g., a yeast cell, a plant cell, an insect cell, or a mammalian cell).
In a sixth aspect, the invention provides an isolated nucleic acid comprising (i) a sequence encoding a biological active agent and (ii) a restrictive sequence encoding UUACUCA. This nucleic acid allows one to generate various constructs where the expression level of the agent in a cell can be regulated by the polypeptide of this invention.
In a seventh aspect, the invention provides a kit comprising the polypeptide mentioned above and a buffer. The kit allows one to detect whether RNA molecules in a test sample have a specific target sequence, such as UUACUCA, or to protein-based mRNA interference.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. la-c show the characteristics of the MazF homologue from H. walsbyi. a, Amino acid sequence alignment of H. walsbyi MazF-hw (SEQ ID NO: 1) with 17 other MazF homologues (SEQ ID NOs: 7-23) from B. subtilis 168 (MazF-bs), C. perfigens 13 (MazF-cp), S. Aureus COL (MazF-sa), Nostoc PCC7120 (MazF-no), Synechocystis PCC6803(MazF-sy), M. tuberculosis H37Rv (MazF-mt1 to -mt7), ChpBK in E. coli (ChpBK-ec), PemK in E. coli (PemK-ec), M. xanthus (MazF-mx) and MazF from E. coli K12 (MazF-ec). Identical amino acid residues are shown in black shades and conservative substitutions in gray shades. S represents β sheet strands and H represents α helices. b, Location of the mazF-hw gene on the H. walsbyi chromosome obtained from TIGR. c, Toxicity of H. walsbyi MazF-hw. E. coli BL21 cells were transformed with pColdIIImazF-hw and spread on M9-glycerol-casamino acids plates with and without IPTG (1 mM). The plates were incubated at 37° C. for 18 h. d, Effect of MazF-hw induction on cell growth. Cell growth was measured by a Klett meter. E. coli BL21 cells harboring pColdIII-mazF-hw were cultured in M9-glycerol-casamino acids medium at 37° C. until cell density reached 30 Klett (equivalent to 3×107 cells/ml). Then, the culture was divided into two; one was incubated at 37° C. with (open circles) and the other without IPTG (1 mM; closed circles). The cultures were diluted 10 fold at 2 h and 5 h time points, respectively.
FIGS. 2a-f show the endoribonuclease activity of MazF-hw in vitro. Cleavage of MS2phage RNA (a), E. coli total RNA (b), and yeast total RNA (c) by MazF-hw. Purified MazF-hw was incubated with E. coli total RNA, yeast total RNA and MS2 phage RNA at 37° C. for 30 min with or without purified CspA protein (lanes 2 and 3). The reaction mixture (20 μl) consisted of each RNA, 0.5 μg MazF-hw, 120 μg CspA, 0.1 M EDTA, 40 mM NaC1 and 0.5 μl of RNase inhibitor (Roche) in 20 mM Tris-HC1 (pH8.0)12. The reaction products were analyzed on a 1.2% agarose gel. The products are indicated by arrows. d, Analysis of MazF-hw cleavage sites in MS2 phage RNA by in vitro primer extension. Lane 1 represents a control reaction in which no protein was added; lane 2, MS2 RNA was incubated with MazF-hw. The reactions were analyzed on a 6% polyacrylamide gel containing 8 M urea and the products were visualized by autoradiography. The cleavage site is indicated by an arrow on the RNA sequence (SEQ ID NO: 6) and determined using the RNA ladder shown on the left. e and f, Synthesized 13-base RNA (5′-A1A2G3U4U5A6C7U8C9A10A11A12G13-3′, SEQ ID NO: 3) in which the U4 and A10 residues were replaced with A, G, and C or with G, C, and U residues, respectively, used as substrates. The substrates labeled at the 5′-end with 32P were incubated with MazF-hw for 0, 5 and 30 min (lanes 1-3, respectively) or without MazF-hw for 30 min (lane 4). The reaction products were analyzed on a 20% polyacrylamide gel containing 8 M urea and the products were visualized by autoradiography.
FIGS. 3a-e demonstrate that four essential genes containing hepta-sequence are essential for MazF-hw toxicity. Total RNA was extracted from E. coli BL21(DE3) cells harboring pColdII mazF-hw (a) and pColdII (b) at various time points (0, 5, 10, 30 and 60 min) after the addition of 0.1 mM IPTG and subjected to reverse transcriptase PCR (RT-PCR). RT-PCR was performed using the protocol for the Transcriptor first strand cDNA synthesis kit (Roche) and the primers for four genes were designed to amplify the fragment containing hepta-sequence cutting site. e, The four UUACUCA-less genes were cloned into pACYC-Duet and pCOLA-Duet. E. coli BL21 (DE3) cells harboring these plasmids and pColdII-mazF-hw were streaked on M9 plates with or without 0.1 mM IPTG and incubated at 30° C. for 20 h.



DETAILED DESCRIPTION OF INVENTION
RNA-based mRNA interference by antisense RNA and RNAi has been well documented and used to suppress a specific gene expression. More recently, protein-based mRNA interference by sequence-specific endoribonulceases (i.e., mRNA interferases) has been implicated by earlier findings of three or five-base sequence-specific mRNA interferases in bacteria. However, these enzymes were not specific enough to regulate expression of a specific gene(s) in the cells. To achieve protein-based mRNA interference comparable to RNA-based mRNA interference, more sequence-specific mRNA interferases are required.
As disclosed herein, an mRNA interferase was identified from an extreme halophilic archaeon, which recognizes a specific seven-base sequence cleaving only a specific group of genes required for ATP production. This archaeon, Haloquadra walsbyi, isolated from a hypersaline pool on the Sinai Peninsula contains a gene encoding a protein (MazF-hw) homologous to Escherichia coli MazF-ec, an ACA-specific mRNA interferase. The induction of MazF-hw in E. coli resulted in complete cell growth arrest only after three generations in contrast to MazF-ec causing almost immediate growth arrest. Purified MazF-hw cleaved only at a single site in 3.5-kb MS2 phage RNA, but could not cleave E. coli 16S and 23S rRNAs or yeast 18S and 28S rRNAs. Determination of the cleavage site in MS2 RNA and assays with synthetic oligoribonucleotides revealed that MazF-hw cleaves RNA specifically at UU^ACUCA, (cleaved at ^). This sequence was found to be unusually abundant in the mRNAs for rhodopsin transcription activator and some membrane proteins of the archaeon. E. coli contains four essential genes having the hepta sequence. When all the cleavage sites in these genes were eliminated, E. coli was no longer sensitive to MazF-hw, demonstrating that cell growth can be regulated by a sequence-specific mRNA interferase. These findings suggest that, in addition to antisense RNA, protein-based mRNA interference is another effective way to silence specific gene expression in cells.
The present invention provides an enzyme isolated from a super halophilic archaeon isolated from a hypersaline pool on the Sinai Peninsula, which functions as an endoribonuclease or an mRNA interferase, termed MazF-hw. It recognizes a specific 7-base RNA sequence. Theoretically, a specific 7-base sequence exists once in every 16,384-base sequence, but interestingly the hepta RNA sequence is overrepresented in the rhodopsin transcription activator gene and a few membrane protein genes of this archaeon, indicating that protein-based mRNA interference occurs in this organism to silence specific gene expression and regulate cell growth. When MazF-hw is induced in E. coli, cell growth is arrested after three generations, indicating that there are a few essential genes containing the hepta sequence. In fact, four essential genes on the E. coli genome were found to contain one hepta sequence each. To verify if protein-based mRNA interference can regulate cell growth, all four hepta sequences were altered to uncleavable sequences without changing the amino acid sequences. It was found that when these hepta sequence-less genes are induced, the cells become resistant to MazF-hw induction. Thus, this is the first demonstration that cell growth can be regulated by protein-based mRNA interference targeting specific genes. This result further demonstrates that protein-based mRNA interference by sequence-specific mRNA interferases may be widely applicable for regulation of specific gene expression and thus cell growth from bacteria to human.
As used herein, the percent identity of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic. Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
An “isolated polypeptide” refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitute at least 10% (i.e., any percentage between 10% and 100% inclusive, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide of the invention can be purified from a natural source, produced by recombinant DNA techniques, or by chemical methods.
A “recombinant” peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired peptide. A “synthetic” peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis. The term “recombinant” when used with reference e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
Within the scope of this invention are fusion proteins containing one or more of the afore-mentioned sequences and a heterologous sequence. A heterologous polypeptide, nucleic acid, or gene is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form. Two fused domains or sequences are heterologous to each other if they are not adjacent to each other in a naturally occurring protein or nucleic acid.
The amino acid composition of the above-mentioned mRNA interferase peptide/polypeptide/protein may vary without disrupting the ability to recognize a specific RNA sequence and cleaved it. For example, it can contain one or more conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in SEQ ID NO: 1 is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for the ability to bind to the respective receptor and trigger the respective cellular response to identify mutants that retain the activity as descried below in the examples.
A functional equivalent of a peptide, polypeptide, or protein of this invention refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having ore or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity to of the above-mentioned mRNA interferase. The isolated polypeptide can contain SEQ ID NO: 1 or a functional fragment thereof. In general, the functional equivalent is at least 70% (e.g., any number between 70% and 100%, inclusive, e.g., 70%, 80%, 85%, 90%, 95%, and 99%) identical to SEQ ID NO: 1.
A polypeptide described in this invention can be obtained as a recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., glutathione-s-transferase (GST), 6x-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention.
Alternatively, the peptides/polypeptides/proteins of the invention can be chemically synthesized (see e.g., Creighton, “Proteins: Structures and Molecular Principles,” W. H. Freeman & Co. NY, 1983), or produced by recombinant DNA technology as described herein. For additional guidance, skilled artisans may consult Ausubel et al. (supra), Sambrook et al. (“Molecular Cloning, A Laboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), and, particularly for examples of chemical synthesis Gait, M. J. Ed. (“Oligonucleotide Synthesis,” IRL Press, Oxford, 1984).
As an mRNA interferase functions intracellularly, the above-disclosed polypeptide can be associated with, e.g., conjugated or fused to, one or more of an amino acid sequence comprising a cell-penetrating peptide (CPP) sequence and the like. In this manner, as composition of the invention as discussed below can include a transport enhancer. For example, the composition may include a penetration enhancing agent, such as MSM, for the delivery of the mRNA interferase or related gene-silencing polypeptides to a cell and/or through the cell membrane and into the cytosol or nucleus of the cell. The mRNA interferase or related therapeutic polypeptides then function to down-regulate the mRNA level of a target gene, thereby resulting in a desired cell status and phenotype. As indicated above, the mRNA interferase or related gene-silencing polypeptides may be delivered by itself or as a fusion with one or more of an NLS, CPP, and/or other domains. See, e.g., Tachikawa et al. PNAS (2004) vol. 101, no. 42:15225-15230.
A cell-penetrating peptide (CPP) generally consists of less than 30 amino acids and has a net positive charge. CPPs internalize in living animal cells in vitro and in vivo in endocytotic or receptor/energy-independent manner. There are several classes of CPPs with various origins, from totally protein-derived CPPs via chimeric CPPs to completely synthetic CPPs. Examples of CPPs are known in the art. See, e.g., U.S. Application Nos. 20090099066 and 20100279918. It is know that CPPs can delivery an exogenous protein to a specific cell.
Although the mRNA interferase or related gene-silencing polypeptides to be delivered may be fusion proteins including a CPP, in certain instances, the interferase does not include a CPP as the aforementioned transport enhancer may serve the function of delivering the biologically active interferase directly to the cell, and/or through the cell membrane into the cytoplasm of the cell and/or into the nucleus of the cell as desired. For instance, in certain instances, it may be desirable to deliver a biologically active protein to the cell wherein the protein is not conjugated or fused to another molecule. In such an instance, any biologically active protein may be delivered directly in conjunction with the transport enhancer.
All of naturally occurring mRNA interferase, genetic engineered mRNA interferase, and chemically synthesized mRNA interferase can be used to practice the invention disclosed therein. mRNA interferase polypeptides obtained by recombinant DNA technology may have the same amino acid sequence as naturally a occurring mRNA interferase or an functionally equivalent thereof. They also include chemically modified versions. Examples of chemically modified polypeptides include polypeptides subjected to conformational change, addition or deletion of a side chain, and polypeptides to which a compound such as polyethylene glycol has been bound. Once purified and tested by standard methods or according to the method described in the examples below, an mRNA interferase polypeptide can be included in pharmaceutical composition for silencing a gene.
The present invention also provides a nucleic acid that encodes any of the polypeptides mentioned above. Preferably, the nucleotide sequences are isolated and/or purified. A nucleic acid refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA), an RNA molecule (for example, but not limited to, an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded. An “isolated nucleic acid” is at nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
The present invention also provides recombinant constructs or vectors having one or more of the nucleotide sequences described herein. Example of the constructs include a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred embodiment, the construct further includes regulatory sequences, including a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).
A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector of this invention includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
Examples of expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of or Simian virus 40 (SV40), bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, as nucleic acid sequence encoding one of the polypeptides described above can be inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are within the scope of those skilled in the art.
The nucleic acid sequence in the aforementioned expression vector is preferably operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: the retroviral long terminal (LTR) or SV40 promoter, the E. coli lac or tip promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or viruses. In a preferred embodiment, the promoter is a tissue specific promoter that drives mRNA synthesis in a cell or tissue of interest.
The expression vector can also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may include appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli. 
The vector containing the appropriate nucleic acid sequences as described above, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host to permit the host to express the polypeptides described above (e.g., SEQ ID NO: 1). Such vectors can be used in gene therapy. Examples of suitable expression hosts include bacterial cells (e.g., E. coli, Streptomyces, Salmonella typhimurium), fungal cells (yeast), insect cells (e.g., Drosophila and Spodoptera frugiperda (Sf9)), animal cells (e.g., CHO, COS, and HEX 293), adenoviruses, and plant cells. The selection of an appropriate host is within the scope of those skilled in the art. In some embodiments, the present invention provides methods for producing the above mentioned polypeptides by transfecting a host cell with an expression vector having a nucleotide sequence that encodes one of the polypeptides. The host cells are then cultured under a suitable condition, which allows for the expression of the polypeptide.
The present invention further provides gene therapy using nucleic acids encoding one or more of the polypeptides mentioned above or an analog or homolog thereof. Targeted gene therapy involves the use of vectors (e.g., organ-homing peptide) that are targeted to specific organs or tissues after systemic administration.
In certain embodiments, the present invention provides gene therapy for the in vivo production of the above-mentioned polypeptides. Such therapy would achieve its therapeutic effect by introduction of the nucleic acid sequences into cells or tissues of a human or a non-human animal in need of deceasing in or silencing of a target gene. Delivery of the nucleic acid sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Preferred for therapeutic delivery of the nucleic acid sequences is the use of targeted liposomes.
Various viral vectors which can be utilized for gene therapy disclosed herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney marine leukemia virus (MoMuLV), Harvey marine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using a tissues- or cell-specific antibody or hormone that has a receptor in a cell. Those of skill in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector.
Another targeted system for delivery of nucleic acids is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based system including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Methods for efficient gene transfer using a liposome vehicle, are known in the art. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art. A nucleic acid sequence of this invention can be a DNA or a RNA. The terms “RNA,” “RNA molecule,” and “ribonucleic acid molecule” are used interchangeably herein, and refer to a polymer of ribonucleotides. The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA also can be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively).
Compositions
This invention also provides a composition that contains a suitable carrier and one or more of the agents described above. The composition can be a pharmaceutical composition that contains a pharmaceutically acceptable carrier. The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
The above-described composition, in any of the forms described above, can be used for modulating the mRNA level of a gene. An effective amount refers to the amount of an active compound/agent that is required to confer a therapeutic effect on a treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of conditions treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.
A pharmaceutical composition of this invention can be administered to a subject parenterally, orally, nasally, rectally, topically, or buccally. The term “parenteral” as used herein refers to, but not limited to, subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, or intraarterial injection, as well as any suitable infusion technique. A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, polyoxyethylated versions thereof. These oil solutions or suspensions also can contain a long chain alcohol diluent or dispersant such as, but not limited to, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as, but not limited to, TWEENS or SPANS or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms also can be used for the purpose of formulation. As used herein, “administering” does not include microinjection of a fertilized oocyte and intergenerational transmission via germ cells.
As used herein, a “subject” refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human mammals, non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, and non-mammals, such as birds, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model. “Treating” or “treatment” refers to administration of a compound or agent to a subject who has a disorder with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder. An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
Kits
The invention provides a kit for determining whether RNA molecules in a test sample have a specific target sequence, such as UUACUCA, or for protein-based mRNA interference. To that end, a wide variety of kits may be prepared according to present invention. For example, a kit may include the above-motioned polypeptide, buffers, and instructional materials for RNA restriction enzymatic reaction. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The kits of the present invention may further include one or more of the following components or reagents: a reverse transcriptase, an RNase inhibitor, an enzyme for attaching a 3′ oligodeoxynucleotide tail onto DNA molecules (e.g., terminal deoxynucleotidyl transferase), an enzyme for degrading RNA in RNA/DNA duplexes (e.g., RNase H); and one or more RNA polymerases (e.g., T7, T3 or SP6 RNA polymerase). Additionally, the kits may include buffers, primers (e.g., oligodT primers, random primers), nucleotides, labeled nucleotides, an RNase inhibitor, polyA polymerase, RNase-free water, containers, vials, reaction tubes, and the like compatible with the analysis of RNA molecules according to the methods of the present invention. The components and reagents may be provided in containers with suitable storage media.
EXAMPLE 1
Materials and Methods
This example describes general materials and methods used in EXAMPLES 2-4 below.
Bacterial Strains and Plasmids
E. coli BL21(DE3) and DH5a were used. The mazF-hw gene was synthesized (Genscript) and cloned into pColdII (Takara Bio) to express the MazFhw. The four UUACUCA-less genes (rpoB, lolD, rplC and rpmD) were cloned into pACYCDuet and pCOLA-Duet (Novagen), respectively.
Poem Purification
To purify N-terminal His-tagged MazF-hw, pColdII-mazF-hw was introduced into E. coli BL21(DE3). The expression of MazF-hw was induced with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 15° C. for 3 h. The MazF-hw was purified with Ni-NTA agarose (Qiagen) following the manufacturer's protocol.
mRNA Interferase Activity of MazF-hw
Purified MazF-hw was incubated with E. coli total RNA, yeast total RNA and MS2 phage RNA at 37° C. for 30 min with or without purified CspA protein, an RNA chaperone. The reaction mixture (20 μl) consisted of each RNA, 0.5 μg MazF-hw, 120 μg CspA, 0.1 mM EDTA, 400 mM NaCl and 0.5 μl of RNase inhibitor (Roche) in 20 mM Tris-HCl (pH8.0). After denaturation in urea, the products were separated on an 1.2% agarose gel.
Primer Extension Analysis in Vitro
MS2 RNA was incubated with or without purified MazF-hw as described above and the products were analyzed as described previously.
Cleavage of Synthetic RNA by MazF-hw
Synthesized 13-base RNA (5′-A1A2G3U4U5A6C7U8C9A10A11A12G13-3′) (SEQ ID NO.: 3) in which the U4 and A10 residues were replaced with A, G, and C or G, C, and U residues, respectively, were used as substrate. The labeled substrates were incubated with MazF-hw for 0, 5 and 30 min or without MazF-hw for 30 min at 20° C. in a reaction mixture containing 20 mM Tris-HCl (pH8.0), 1 mM EDTA, 400 mM NaCl and 0.5 μl of RNase inhibitor. The reaction products were analyzed as described previously
EXAMPLE 2
Identification of a MazF Homologue in H. Walsbyi 
In this example, assays were carried out to identify a MazF homologue Haloquadra walsbyi. 
Haloquadra walsbyi was isolated from a hypersaline pool on Sinai Peninsula. The cells were extremely thin and square, measuring 2-5 μm wide but less than 0.2 μm thick. Using blast search with E. coli MazF, the gene HQ2202A was identified. It encodes a 124-residue protein. This protein has 31% identity and 46% similarity to E. coli MazF (111 residues) (FIG. 1a). The gene for MazF-hw appears to be co-translated with the gene for an upstream ORF, which overlaps with MazF ORF in a manner similar to that of the E. coli mazE-mazF operon (FIG. 1b). However the upstream ORF (termed MazY) does not show homology to E. coli MazE, the antitoxin of MazF.
In order to examine MazF-hw toxicity in E. coli, the mazF-hw gene was synthesized and cloned into pColdII vector. As shown in FIG. 1c, the induction of MazF-hw inhibited colony formation on an agar plate in the presence of 1 mM isopropyl-β-D-1-thiogalactoside (IPTG). The gene for MazY-hw was also synthesized and cloned it into pET28a. As shown in FIG. 1c, co-induction of MazY-hw neutralized the toxicity of MazF-hw, suggesting that MazY is the antitoxin for MazF-hw. The toxicity of MazF-hw in was also examined a liquid culture (FIG. 1d). When MazF-hw was induced by the addition of 1 mM IPTG, cell growth was completely inhibited only after 5 hr or three generations. This slow growth inhibition is in a sharp contrast to that observed with E. coli MazF, which inhibits cell growth within 15 min after induction.
EXAMPLE 3
MazF-hw 1s an mRNA Interferase
It was possible that this slow inhibitory effect of MazF-hw was likely due to its RNA cleavage specificity. Therefore, in this example, MazF-hw protein was purified to determine its cleavage specificity.
First, 3.5-kb MS2 phage RNA was used as substrate and found that MazF-hw cleaves this mRNA only at one site (FIG. 2a). It is important to note that preincubation of MazF-hw with MazY-hw completely inhibited its endoribonuclease activity, suggesting that the observed cleavage is caused by MazF-hw (FIG. 2a; lane 4). The specific cleavage site in MS2 RNA between—ACUUU and ACUCA—was detected by primer extension (FIG. 2d). The MazF-hw MS2 RNA cleavage activity was completely inhibited by addition of 10 mM MgCl2 in the reaction as found with E. coli MazF8 and required NaCl (10-50 mM).
Next, assays were carried out using 165 and 23S rRNAs from E. coli and 18S and 28S rRNAs from yeast as substrate. Surprisingly, none of these RNAs were cleaved by MazF-hw (FIGS. 2b and e, respectively). Using Perl programming language for computational analysis, these RNAs were analyzed for the presence of different sequences (all possible four-, five- and six-base combinations) around the sequence of the sole cleavage site found in MS2 RNA. The results from these analyses are presented in Table 1 below and demonstrate that there are two possible six-base RNA cleavage sequences for MazF-hw, UUACUC and UACUCA, neither of which exists in E. coli 16S and 23S rRNAs or in yeast 18S and 28S rRNAs (the common bases in the sequences are underlined).
The results presented in FIG. 2 show that there is one cleavage site in MS2 RNA for MazF-hw and no cleavage sites in the E. coli 16S and 23S RNAs and the yeast 18S and 28S RNAs. Using Perl programming language for computational analysis, these RNAs were analyzed for the presence of all possible four-, five- and six-base combinations around the sequence of the sole cleavage site found in MS2 RNA. Results from these analyses are presented in Table 1.







TABLE 1







Putative MazF-hw cleavage sites in


MS2 RNA, 23S, 16S, 28S and 18S rRNA













MS2RNA
23S rRNA
16S rRNA
28S rRNA
18S rRNA
















CUUU
>10






UUUA
>10


UUAC
>10


UACU
>10


ACUC
>10


UUUAC
9
1
0
2
2


UUACU
3
2
1
2
2


UACUC
1
3
0
1
0


ACUCA
2
1
2
1
2


CUCAG
5
1
2
1
2


UUUACU
1
1
0
1
2


UUACUC
1
0
0
0
0


UACUCA
1
0
0
0
0


ACUCAG
1
1
0
0
0


CUCAGU
1
1
0
0
0









EXAMPLE 4
MazF-hw Cleaves RNA at a Specific Seven-Base Sequence
In order to determine which of the two sequences was the actual cleavage site, two 13-base oligonucleotides, 5′-AAGUUACUCCAGG-3′ (SEQ ID NO.: 4) and 5′-AAGCUACUCAAGG-3′ (SEQ ID NO.: 5) were synthesized The underlined sequences are from MS2 RNA including the common UACUC sequence. However, they were not cleaved by the MazF-hw (data not shown).
Subsequently, seven more 13-base oligoribonucleotides were synthesized. These oligoribonucleotides have various bases on both sides of the six-base sequences (FIGS. 2e and 2f). Surprisingly, it was found that MazF-hw cleaved only one of these substrates, containing UUACUCA sequence, which is consistent with the cleavage sequence found in MS2 RNA. Thus, the results demonstrate that MazF-hw recognizes the seven-base sequence, UU^ACUA and cleaves between the second (U) and the third residue (A)(^ indicates the cleavage site).
Since a specific seven-base sequence can be found on average only once every 16,384-base RNA sequence, it is possible that MazF-hw cleaves only a specific group of mRNAs. Out of 2610 ORFs on the H. walsbyi genome, only 183 ORFs have the heptad sequence, of which one has three heptad sequences, twelve have two (Table 2), and the remaining 170 have only one. Since one can assume that the mRNA sensitivity to MazF-hw is proportional to the number of the heptad cleavage sites present, the boa gene for a putative transcription activator for rhodopsin (bacterio-opsin) having three heptad sequences may be the most sensitive, suggesting that upon induction of MazF-hw the expression of the light-driven proton pump may be turned off.







TABLE 2







ORFs in Haloquadra walsbyi, which are most sensitive to MazF-hw



















Halofex




Length
Number of


Function


Gene ID
Gene
(bp)
UUACUCA
Protein Name
COGs
Class
















HQ1739A
boa
5418
3
bacterio-opsin activator-like
COG2202T,
SIG






transcription regulator
COG2203T,








COG3413R



HQ3529A
—
402
2
probable sulfatase
COG3119P
MIS


HQ2658A
—
717
2
conserved hypothetical protein
COG2220R
CHY


HQ2731A
glnP
855
2
ABC-type glutamine/glutamate/polar
COG0765E
TP






amino acids transport system,








permease protein




HQ2726A
trmB
1062
2
probable sugar-specific
COG1378K
REG






trancriptional regulator TrmB




HQ1250A
gtl3
1065
2
probable glycosyltransferase, type 2
COG0463M
GEN


HQ3464A
aslA
1605
2
probable arylsulfatase; probable
COG3119P
MIS






choline-sulfatase




HQ1036A
cstA
1806
2
carbon starvation protein A
COG1966T
SIG


HQ1786A
—
1938
2
ABC-type transport system ATP-
COG1132V
TP






binding/permease protein




HQ2295A
chlID
2289
2
magnesium chelatase (protoporphyrin
COG1239H,
COM






IX magnesium-chelatase)
COG1240H



HQ2542A

2367
2
conserved hypothetical protein
—
CHY


HQ2550A
glcD
3111
2
oxidoreductase (glycolate oxidase
COG0247C,
GEN






iron-sulfur subunit)
COG0277C



HQ3461A
polA2
6870
2
DNA-directed DNA polymerase large
COG1372L,
RRR






subunit (family D) (archaeal DNA
COG1933L







polymerase II)









EXAMPLE 5
Regulation of Specific Gene By MazF-hw in E. Coli 
In E. coli, out of 233 ORFs containing the heptad sequence, only four (lolD, rplC, rpmD and rpoB,) are essential for cell growth. Upon MazF-hw induction, mRNAs from all four of these genes were degraded (FIG. 3a), assays were carried out to test whether MazFhw was no longer toxic in E. coli if all the cleavage sites in the four genes were eliminated.
As shown in FIG. 3c, cell growth was recovered when the cleavage sites were removed from the four genes. Importantly, when one of the four genes was left intact, cell growth was blocked even in the presence of IPTG, demonstrating that cell growth can be regulated by a sequence-specific mRNA interferase. The data also confirmed that MazF-hw cleaves mRNA at UUACUCA sequences. Notably, the MazF-hw activity was strongly inhibited in the presence of 10 mM MgCl2 or 500 mM NaCl, while H. walsbyi requires 3 M NaCl for cell growth and is resistant to 2 M MgCl2 suggesting that MazF-hw is not active under normal growth conditions. This archaeon lives on the surface of saturated salt water, effectively utilizing light for the production of ATP. However, it is assumed that upon hypo-osmotic stress in nature such as rain or influx of water from a river lowering specific gravity of water, the cells cannot float on the surface of water, reducing ATP production. As a result, the cellular salt concentration decreases to activate MazF-hw, which then degrades the mRNA for transcription activator for the rhodopsin gene. Most of the genes containing two heptad sequences such as ABC-type transporters, cAMP-dependent carbon starvation protein A, sulfatase and FAD-linked oxidase (Table 2) also seem to be involved in hypo-osmotic stress.
While sequence-specific DNA restriction enzymes have been known for many years, sequence-specific endoribonucleases have just been recently discovered. To date, a number of MazF homologues are found in bacteria, having a wide range of cleavage specificities from three to five bases. It has been shown that pathogenic bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus contain mRNA interferases that recognize specific pentad RNA sequences, which are either overpresented or underpresented in genes associated with their pathogenicity. In Myxococcus xanthus, a pentad sequence-specific mRNA interferase has been shown to be required for programmed cell death during fruiting body formation. The present discovery of a MazF homologue, specific to a heptad RNA sequence raises an intriguingly possibility that there may be many other MazF homologues which target only a specific group of cellular mRNAs to regulate cellular physiology. Notably, in addition of antisense RNA and siRNA technology, mRNA interferases will open a new avenue to interfere with expression of a specific mRNA or a specific group of mRNAs, preventing viral infection and harmful gene expression from bacteria to human.
The genomic ORF sequences of H. walsbyi DSM 16790 from NCBI RefSeq (Accession NC_008212) were retrieved and the number of UUACUCA sequences was estimated by using Perl script. For function clustering of each gene, two clusters (COGs and HaloLex Function class) were used from NCBI and HaloLex database, respectively. Clusters of Orthologous Groups of proteins (COGs) were created by comparing protein sequences encoded in complete genomes, representing major phylogenetic lineages. Each COG consists of individual proteins or groups of paralogs from at least 3 lineages and thus corresponds to an ancient conserved domain. HaloLex is a comprehensive genome information system for archaea, and the function classes of HaloLex are used to analyze all MazF-hw sensitive genes.
The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated herein in their entireties.

## Claims

1. An isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1.

2. The polypeptide of claim 1, having an mRNA interferase activity.

3. The polypeptide of claim 1, wherein the amino acid sequence is at least 99% identical to the sequence of SEQ ID NO: 1.

4. The polypeptide of claim 3, wherein the amino acid sequence comprises the sequence of SEQ ID NO: 1.

5. The polypeptide of claim 1, wherein the polypeptide has an activity of cleaving an RNA sequence having the target sequence of UUACUCA.

6. A composition comprising the polypeptide of claim 1 and a carrier.

7. A kit comprising the polypeptide of claim 1 and a buffer.