CN1276083C - Altering gene expression with ssDNA produced in vivo - Google Patents

Abstract

An expression vector for altering expression of a target nucleic acid sequence in a host cell by production of single-stranded cDNA (ss-cDNA) in the host cell in vivo. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3' to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and may be transfected into the host cell. Transcription of the cassette by the host cell produces an RNA template which is reverse transcribed with the product of the RT coding gene to produce ssDNA of a specified sequence. The ssDNA is modified to remove flanking vector sequences by taking advantage of the ''stem-loop'' structure of the ssDNA, which forms as a result of the inverted tandem repeat that allows the ssDNA to fold back on itself, forming a double stranded DNA stem. The double-stranded stem may contain one or more restriction endonuclease recognition sites and the loop, which remains as ssDNA, can be any desired nucleotide sequence. This design allows the double-stranded stem of the stem-loop intermediate to be cleaved by the desired corresponding restriction endonuclease(s) and the loop portion is then released as a linearized, single-stranded piece of DNA. The resulting ssDNA binds to an endogenous target nucleic acid sequence to alter the expression of that sequence for such therapeutic purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

Description

Altering gene expression with ssDNA produced in vivo

The present invention relates to the alteration of gene expression using a stable DNA construct, commonly referred to as a cassette, into which a nucleic acid sequence is inserted to serve as a template for the subsequent production of the sequence in a prokaryotic or eukaryotic host cell, and to A system for expressing this sequence in a eukaryotic host cell with no (or minimal) flanking sequences. The construct or cassette includes inverted tandem repeats forming a stem-loop intermediate stem whose function in vivo is to cause expression of a sequence known as the sequence of interest as a single-stranded DNA (ssDNA) sequence that binds to the target gene or An interaction occurs to alter the expression of that gene. The expression system of the present invention removes most or all of the adjacent plasmid (or other vector) sequences from the ssDNA via stem-loop formation followed by stem termination by the reverse transcription reaction or cleavage of the stem-loop intermediate. The ssDNA produced in this way is designed to be complementary to and/or bind to any endogenous nucleic acid sequence target, thereby targeting any gene of interest.

Antisense gene therapy has been successfully used in various applications to downregulate gene function. Jain, KK, Handbook of Gene Therapy, New York: Hofgrefe & Huber Publishing (1998). However, the therapy to date suffers from a number of deficiencies and limitations that have severely reduced the utility of such therapies to some extent, including short half-lives of antisense molecules, nonspecific effects, uncertainty about the mode of action of antisense sequences, and the Potential toxic effects in the trial. For example, antisense oligonucleotides (ODN) and their analogs must be administered intravenously, which creates problems with cellular uptake and distribution (Cossum, PA, et al., ^14C -labeled Trends in phosphorothioate oligonucleotides ISIS 2105, J. Pharmacol. Exp. Ther., 267, 1181-1190 (1993), Sands, H., et al., Biodistribution of internal ³ H-labeled oligonucleotides and metabolism. II. 3', 5'-blocked oligonucleotides, Molecular Pharmacology, 47, 636-646 (1995)) and toxicity problems arising from high blood concentrations (Henry, SP, et al., a sulfur Toxicity assessment of the phosphorothioate oligonucleotide ISIS 2302 in experimental 4-week CD-1 mice, Antisense Nucleic Acid Drug Development, 7, 473-481 (1997), Henry, SP, et al., Phosphorothioate Oligonucleotides Comparison of the toxicity profile of the nucleotide ISIS 1082 and ISIS 2105 after subacute intradermal administration in Sprague-Dawley rats, Toxicology, 116, 77-88 (1997)).

To date the most used antisense ODN analogs in antisense therapy are phosphorothioate or methylphosphonate. However, phosphorothioate ODN tends to bind nonspecifically to serum and intracellular proteins (Crooke, S.T., et al., Pharmacokinetic characterization of some novel oligonucleotide analogues in mice, J.Pharmacol.Exp.Ther., 227, 923-937 (1996), Gao, W.Y., et al., Phosphorothioate oligonucleotides are inhibitors of human DNA polymerase and RNase H: Implications of antisense technology, Molecular Pharmacology, 41, 223-229( 1992)), and inhibit the activity of RNase H at higher concentrations (Crooke, S.T., etc., the kinetic characteristics of Escherichia coli RNase H: the cleavage of various antisense oligonucleotides-RNA double strands, Biochemical Journal, 312 , 599-608 (1995)). Phosphorothioate ODN has a lower Tm for RNA than DNA (average 0.5°C per base pair) (Crooke, S.T. and B. LeBleu, Antisense Research and Applications, Boca Raton: CRC Press (1993)). This lower Tm requires that phosphorothioate ODNs are generally longer than phosphodiester DNA oligonucleotides for efficient binding. However, an increase in the length of the ODN can cause a loss of hybridization specificity (Toulme, J.J., et al., Antisense Technology: A Practical Study, see C. Lichtenstein and W. Nellen (eds.), New York: IRL Publishing, pp. 39-74 ( 1997)). In addition, methylphosphonate ODN does not activate the enzymatic activity of RNase H (Maher, L.J, et al., Oligonucleotide-directed triple-helix formation inhibits DNA-binding proteins, Science, 245, 725-730 (1989), Miller, P.S. , Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, see J.S. Cohen (ed.), Boca Raton: CRC Publishing, p. 79 (1989)) and are rapidly cleared (Chen, T.L., et al., in mice Trends and metabolism of oligodeoxynucleotide methylphosphonates following a single intravenous injection in Trends in Drug Metabolism, 18, 815-818 (1990)).

Another approach to gene therapy is the administration of molecules that are catalytically active on the gene and/or the transcription product of the gene. Ribozymes consist only of RNA molecules that catalyze the cleavage of specific mRNA sequences and are thought to be more efficient than antisense ODNs due to their catalytic ability (Woolf, T.M., Cleave or Not: Ribozymes and Antisense, Advances in Antisense Research, 5, 227-232 (1995)). Ribozymes have been used as inhibitors of gene expression and viral replication (Jain, supra (1998)). Unlike antisense ODNs, ribozymes can be delivered endogenously, eg, through the use of viral vectors, or exogenously. However, ribozymes have limited stability due to degradation by RNases in vivo (Jain, supra (1998)).

Using in vitro selection, it has recently been demonstrated that some small single-stranded DNAs catalyze the cleavage of RNA (Breaker, RR, Catalytic DNA: Training and Finding Work, Nature Biotechnology, 17, 422-423 (1999)), thus providing the ability to target specific Gene targeting activity guaranteed. Patent and scientific literature describe many of these short deoxynucleic acid sequences that have been shown to have catalytic activity (see, Breaker, RR and GF Joyce, Chemical Biology, 1, 223-229 (1994); Cuenoud, B. and JWSzostak, Nature, 375, 611-613 (1995); Santoro, SW and GF Joyce, Proceedings of the National Academy of Sciences, 94, 4262-4266 (1997); Faulhammer and M. Famulok, Journal of Molecular Biology, 269, 188-203 (1997); Carmi , N, et al., Proceedings of the National Academy of Sciences, 95, (1998); Li, Y. and RRBreaker, Proceedings of the National Academy of Sciences, 96, 2746-2751 (1999) and U.S. Patent Nos. 5,807,718 and 5,910,408), including the term "10 -23 DNase" and other ssDNA sequences that act as, for example, copper-dependent DNA ligases and calcium-dependent DNA kinases. The catalytic potency of this sequence has been demonstrated by cleaving mRNA targets at ¹⁰⁹ m ^-1 /min- ¹ in the presence of divalent magnesium, thereby providing an opportunity for directed destruction of substrate molecules (see, e.g., RRBreaker, supra (1999 )). Although the art appears to recognize the potential of this enzymatic activity for therapeutic purposes, to the best of our knowledge, no existing system exists for generating these target-specific enzymatic nucleic acid sequences to produce a therapeutic effect in vivo.

Therefore, there are no existing systems for altering gene expression that take advantage of the potential advantage of the efficient catalytic activity of these enzymatic nucleic acid sequences. Accordingly, it is an object of the present invention to provide a DNA construct that directs the synthesis of ssDNA containing a sequence that specifically cleaves an mRNA target in vivo to alter the expression of the gene producing that target mRNA.

Since secondary structural folding may be critical to the catalytic function of the ssDNA enzymatic sequence, another object of the present invention is to provide methods and DNA constructs for producing ssDNA in eukaryotic cells comprising nuclear Such DNase sequences of nucleotide sequences without unwanted intervening or flanking nucleotide bases in order to protect the enzymatic function of the ssDNA against the target nucleic acid are used to alter the expression of a gene comprising the target nucleic acid.

Another object of the present invention is to provide a method and a DNA construct for use in the method for producing ssDNA containing a DNase sequence in a eukaryotic cell for overcoming the problem of treatment using standard oligonucleotide delivery methods Obvious problems encountered.

Another object of the present invention is to provide a method and a DNA construct for in vivo production of ssDNA of any nucleotide sequence useful as (but not limited to) inhibitory nucleic acid, for example in antisense to mRNA Mode binding downregulates a gene product of interest or a viral gene product, or, for example, binds and inhibits a specific cellular function through a protein that binds to a recognition nucleic acid sequence.

Another object of the present invention is to provide a method and a DNA construct for producing ssDNA designed to preferentially bind to duplexes (natural DNA) to form triplex structures that can interfere with normal gene transcription and regulation.

Another object of the present invention is the production of ssDNA in eukaryotic cells for the purpose of disrupting one or more cellular functions.

Yet another object of the present invention is to provide a method and a DNA construct for producing a ssDNA wherein the secondary structure is designed to enable the ssDNA oligonucleotide to bind and/or inhibit or activate protein-dependent The catalysis or various cellular functions of nucleic acid-protein interactions such as transcription, translation, and DNA replication.

Another object of the present invention is to provide a method and a DNA construct for in vivo generation of ssDNA for site-directed mutagenesis or gene knockout for therapeutic applications.

Another object of the present invention is to provide a method and a DNA construct for producing ssDNA of precisely defined nucleotide composition, which facilitates specific insertion at genomic sites for therapeutic purposes.

Yet another object of the present invention is to provide a method and a DNA construct for generating ssDNA complementary to any endogenous nucleic acid target for altering the expression of a gene comprising the nucleic acid sequence target.

Another object of the present invention is to provide a method and DNA expression used in the method for in vivo production of ssDNA comprising sequences exhibiting catalytic activity against mRNA targets for transfection into eukaryotic cells for overcoming the following Lipofection, direct cellular uptake, and/or microinjection delivery barriers for direct administration of ssDNA.

Another object of the present invention is to provide within a single or binary plasmid expression system all the enzymatic functions necessary for in vivo production of ssDNA containing sequences enzymatically active against selected target mRNAs.

Another object of the present invention is to provide methods and pharmaceutically acceptable compositions for delivering an inhibitory nucleic acid sequence comprising a sequence enzymatically active on a target cell in a therapeutically effective manner.

The list of objects of the invention is not intended to list all of the objects of the invention. There are many other cellular functions mediated by the cellular genome that are not mentioned here for the sake of brevity and utility, and which are readily regulated by ssDNA produced in vivo. For example, exonucleases digest ssDNA much more readily than double-stranded DNA (dsDNA). It is therefore another object of the present invention to provide an ssDNA construct and a method for producing the same in vivo, which is not susceptible to degradation by natural exonucleases in cells like dsDNA. From this example it can be seen that the list of some objects of the invention is provided as an example and not intended to limit the scope of the invention.

These objectives are served by a method of altering the expression of an endogenous nucleic acid target sequence in a target cell, the method comprising introducing into the target a cassette consisting of a sequence of interest flanked by inverted tandem repeats and a 3' primer binding site (PBS). Cells and mRNA transcripts of the cassette were reversed starting from PBS to release single-stranded cDNA transcripts in the cells. The nucleic acid sequence contained in the sequence of interest produces a nucleic acid sequence that, when reverse transcribed, binds to an endogenous target nucleic acid sequence to alter expression of the target sequence.

Some embodiments of the present invention are illustrated in the accompanying drawings, wherein Figure 1 is a schematic diagram of the production of ss-cDNA in a host cell according to the present invention.

Fig. 2 is a schematic diagram of a stem-loop intermediate formed by the method shown in Fig. 1 .

Fig. 3 is a schematic diagram of a pssXA plasmid containing the first component of the first embodiment of the expression system of the present invention. To make pssXA, the reverse transcriptase (RT) and MboII genes were subcloned into the mammalian expression vector pBK-RSV (Stratagene) and expressed as a single polypeptide. The RT and MboII domains are separated by a histidine-rich linker.

4A and 4B are schematic diagrams (Fig. 4A) of the pssXB plasmid containing the second component of the first embodiment of the expression system of the present invention, which includes the sequence of interest and contains (1) the MoMuLV reverse transcriptase promoter region, (2) Two NotI sites, one PacI site and one BamHI site for subcloning the DNA sequence of interest, and (3) the sequence of the tandem inverted repeats IR-L and IR-R and the pssXB plasmid insert region (Figure 4B ).

Figure 5A is a schematic diagram of a pssXC plasmid containing a second embodiment of the expression system of the present invention and including the 10-23 DNase sequence shown in Figure 5B.

Figures 6A and 6B represent a schematic diagram of a pssXD plasmid (Figure 6A) and an enlarged portion of a pssXD plasmid (Figure 6B) containing a third embodiment of the expression system of the present invention.

Figure 7 shows the results of PCR assay of RT activity in lysates of pssXA transfected cells. Lanes 1 and 2: A549 cells transiently transfected with pBK-RSV vector; Lanes 3 and 4: A549 cells transiently transfected with pssXA; Lanes 5 and 6: A549 cells stably transfected with pssXA (E10). Before PCR amplification, reverse transcription reactions were performed at 37°C for 10 minutes (lanes 1, 3, and 5) or 30 minutes (lanes 2, 4, and 6), respectively.

Figure 8 shows the assay results of PCR analysis for detection of ssDNA. Total RNA was isolated from E10 cells transiently transfected with pssXB vector, pssXB-I or pssXB-II. Before PCR amplification, total RNA was pretreated with S1 nuclease (lanes 1 and 3) or RNase (lanes 2, 4, and 5) at 37°C for 30 min. Lanes 1 and 2: pssXB-I; Lanes 3 and 4: pssXB-II; Lane 5: pssXB vector.

Figure 9 shows the results of dot blot analysis for detection of ssDNA. 1: E10 cells transfected with pssXB-I; 2. E10 cells transfected with pssXB-II; 3: E10 cells; 4: A549 cells.

Figure 10 shows a histogram of Northern blot quantification of the antisense sequence ssDNA generative vector constructed according to the present invention capable of producing antisense sequence against c-raf kinase. Lanes 1-3: cells harvested 24 hours after transfection; lanes 4-6: cells harvested 48 hours after transfection. Lane 1: E10 cells transfected with pssXB vector; Lanes 2 and 5: E10 cells transfected with pssXB-1; Lanes 3 and 6: E10 cells transfected with pssXB-II.

Figure 11 shows the results of dot blot analysis for the detection of ssDNA in A549 cells transfected with control pssXD-I or pssXD-II containing the c-raf DNase sequence. No detectable signal could be produced in the presence of S1 nuclease due to the specific degradation of ssDNase by S1 nuclease.

Figure 12 shows the results of quantitative RT-PCR to determine whether ssDNA expressed in A549 cells transfected with pssXD-II alters c-raf mRNA levels. Lane 1: control pssXD-I; lane 2: pssXD-II.

Figure 13 shows the Western blot results of inhibition of c-raf protein expression in A549 cells transfected with pssXD-I or pssXD-II. Lane 1: pssXD-II; Lane 2: control pssXD-I; Lane 3: untransfected cells.

Figure 14 shows the results of Western blot of genomic DNA cleavage activity induced apoptosis by inhibition of c-raf gene expression. Lane 1: pssXD-II; Lane 2: control pssXD-I; Lane 3: untransfected cells.

Figure 15 shows the results of Western blot of PARP cleavage activity that induces apoptosis through inhibition of c-raf gene expression. Lane 1: pssXD-II; Lane 2: control pssXD-I; Lane 3: untransfected cells.

In the specification of the present invention, the in vivo production of almost any predetermined or desired nucleotide base composition, with or without flanking nucleotide sequences, in yeast, prokaryotic cells, and/or eukaryotic cells is described. Methods of deoxyribonucleic acid (ss-cDNA) oligonucleotides and nucleic acid constructs are used to alter the expression of target genes. Methods and constructs are described that use biological synthesis instead of in vitro, or artificial synthesis of ss-cDNA with a desired nucleotide base composition. Since biological reactions, ie, enzymatic reactions, are used in these methods, they can be applied to any in vivo system.

In one embodiment, the expression system of the present invention comprises a vector designed to produce any sequence of interest in a mammalian cell, which is an ss-cDNA molecule, preferably free of most adjacent vector sequences ss-cDNA molecules (the term "vector" as used herein refers to plasmids or modified viral or non-viral recombinant biological constructs for the delivery and manipulation of synthetic and/or naturally occurring nucleic acid sequences). This vector system contains all the necessary enzymatic functions and signaling instructions for the production of ss-cDNA in host cells. Host cells transferred with the vectors of the invention produce RNA transcripts driven by eukaryotic promoters (FIG. 1), which are used as templates to direct the synthesis of any desired single-stranded DNA sequence ("sequence of interest").

More specifically, described herein is a first expression system in which the vector contains two plasmids that are co-transfected into a suitable host cell, which may be yeast or any prokaryotic or eukaryotic cell, so that in the cell Generate ssDNA sequences of interest for altering gene expression. Also described is a secondary expression system comprising a single plasmid carrying the sequence of interest which is transfected into a suitable host cell in order to produce the ssDNA sequence of interest in the cell for altered gene expression.

ssDNA produced in vivo using the expression systems described herein can be inhibitory nucleic acids. The inhibitory nucleic acid can be ssDNA synthesized from the mRNA template or the mRNA template itself, which can specifically bind a complementary nucleic acid sequence. Upon binding to an appropriate target nucleic acid sequence, an RNA-RNA, DNA-DNA, or RNA-DNA duplex or triplex is formed. More commonly, these nucleic acids are often called "antisense" because they are usually complementary to the sense or coding strand of a gene, but the "sense" sequence is also used in cells for therapeutic purposes. Thus, the term "inhibitory nucleic acid" as used herein refers to both "sense" and "antisense" nucleic acids.

Binding of an inhibitory nucleic acid to a target nucleic acid alters the function of the target nucleic acid. This change (usually an inhibitory effect) is caused by, for example, blocking DNA transcription, processing or adding poly(A) to mRNA, DNA replication, translation, or initiating the cell's inhibitory mechanisms (such as initiating RNA degradation) produce. Therefore, the inhibitory nucleic acid approach encompasses many different approaches to alter gene expression. These different types of inhibitory nucleic acid techniques are described in Helene, C. and J. Toulme, Acta Biochem. Biophys. 1049, pp. 99-125, (1990), hereinafter referred to as "Helene and Toulme", and are identified by This specific document is cited in its entirety herein.

Briefly, inhibitory nucleic acid therapeutic regimens can be divided into (1) regimens targeting DNA sequences, (2) regimens targeting RNA sequences (including precursor-mRNA and mRNA), and (3) regimens targeting proteins ( sense strand protocol), and (4) protocols that cause cleavage or chemical modification of target nucleic acids such as ssDNA enzymes, including herein referred to as "10-23 enzymes." The first scheme contains several types. Nucleic acids are designed to bind to the major groove of double-stranded DNA to form a triple helix or "triplex" structure. Alternatively, inhibitory nucleic acids are designed to bind to regions of single-stranded DNA that result from the opening of double-stranded DNA during replication or transcription. More commonly, inhibitory nucleic acids are designed to bind to mRNA or pre-mRNA. Inhibitory nucleic acids can also be used to prevent maturation of the pre-mRNA. Inhibitory nucleic acids can be designed to interfere with RNA processing, splicing or translation. In the second approach, the inhibitory nucleic acid targets mRNA. In this approach, inhibitory nucleic acids are designed to specifically inhibit translation of encoded proteins. Using the second approach, inhibitory nucleic acids are used to selectively inhibit certain cellular functions by inhibiting the translation of mRNAs encoding key proteins. An example of such an inhibitory nucleic acid is a sequence complementary to the c-myc mRNA region, which inhibits c-myc protein expression in the human promyelocytic leukemia cell line HL60, which overexpresses the c-myc proto-oncogene (Wickstrom E.L., et al. Proceedings of the National Academy of Sciences, 85, pp. 1028-1032, (1988) and Harel-Bellan, A., et al., Experimental Medicine, 168, pp. 2309-2318, (1988)). As described in Helene and Toulme, inhibitory nucleic acids targeting mRNA have been shown to act by several different mechanisms that inhibit the translation of encoded proteins.

Inhibitory nucleic acids can also use a third approach, which designs the "sense" strand of a gene or mRNA to trap or compete with enzymes or bind proteins involved in mRNA translation as described in Helene and Toulme. Finally, inhibitory nucleic acids can be used to induce chemical inactivation or cleavage of target genes or mRNAs. For example, chemical inactivation occurs by inducing cross-linking between the inhibitory nucleic acid and the target nucleic acid in the cell and by the methods contained herein (i.e., cleavage of the target nucleic acid by an enzymatically active sequence inserted into the cassette of the invention) .

Briefly, in a first aspect, the invention comprises a set of genetic elements suitable for delivery into cells for the production of ssDNA in vitro or in vivo for altering gene expression, expression systems comprising the set of genetic elements, and sets of genetic elements comprising the set One or more elements stably transfected cells. The set of genetic elements is inserted into an expression system for delivery into cells and it includes

(A) an RNA-dependent DNA polymerase (reverse transcriptase) gene, and

(B) a cassette comprising (1) an inverted tandem repeat (IR), (2) between (a) inverted repeats (IR), (b) the 3' end of IR, or ( c) one or more sequences of interest both between the IRs and at the 3' end of the IR and (3) a reverse transcriptase primer binding site (PBS) located at the 3' end of the IR as shown in Figure 2 .

Although not required, the expression system preferably also includes functional and signaling directions for in vivo transcription of these components and for translation of the reverse transcriptase (RT) gene. Other elements optionally included in the set of genetic elements of the present invention may include one or more RNAse genes, usually associated with the RT gene, a restriction endonuclease (RE) gene (for purposes described below) ), a downstream polyadenylation signal sequence for expression in eukaryotic cells so that the mRNA produced by the sequence of interest contains a poly(A) tail (see Figure 1), and a linearized ssDNA when folded into the corresponding A DNA sequence that is enzymatically active in its secondary configuration. In one embodiment of this set of genetic elements, the DNA enzymatic sequence is located within the sequence of interest, whether the sequence of interest is located between inverted repeats (IRs) or between the 3' end of the IR and the PBS, but The present invention is not limited thereto.

In a first embodiment of the expression system described herein, a vector system is provided containing two plasmids, the above-mentioned set of genetic elements suitable for delivery into cells for in vivo production of ssDNA comprising RNA-dependent DNA polymerase (retro transcriptase) gene and additionally contains an RNAse H gene linked to a restriction endonuclease gene with a histidine-proline linker. These genes are constructed and inserted into plasmid vectors containing the necessary control elements for transcription and translation, as well as polyadenylation tailing sequences. This plasmid is referred to herein as the "A" plasmid, pssXA, and is shown in FIG. 3 . A second plasmid, the "B" plasmid, was constructed which, in the embodiments described herein, contained the three elements of the cassette described above, namely, a primer binding sequence (PBS) matching the reverse transcriptase (RT), Sequence of Interest (SOI), and Inverted Repeat (IR). In the second type of plasmid, taking the plasmid pssXB shown in Figure 4 as an example, the SOI is located between the inverted tandem repeats, or at the 5' position (relative to the mRNA transcript) of the PBS, which is located at the end of the mRNA transcript. 3' end. In other words, the SOI is located (1) between the IR, (2) between the IR and the PBS, and/or (3) both between the IR and the IR and the PBS, as described below. B plasmids, one (pssXB-I) between IR (e.g., NotI site) and containing SOI, and one (pssXB-II) between IR and PBS and containing SOI (e.g., cloned in PacI/ this in the BamHI site). Like plasmid A, plasmid B also includes a set of transcriptional control elements. However, in another preferred embodiment herein, the B plasmid does not include (or require) translational control elements, since no protein product is produced from this construct.

In another embodiment described herein, the expression system of the present invention contains a single plasmid vector as shown in Figures 5 and 6, and named plasmids pssXC and pssXD, respectively, into which the set of genetic elements described above is inserted. Components of the B plasmid described above, such as PBS, SOI, and IR, are located in the 3' untranslated portion of the RT polyprotein in the C plasmid. In other words, when the RT-RNAse H component of the C plasmid is transcribed under the control of an appropriate promoter (in the embodiments described herein, the RSV promoter is used), the resulting mRNA transcript contains the RT-RNAse H polynucleotide. The coding region of the protein, and when translation ends at the stop signal, the other mRNA transcript contains (at the 3' end of the translated protein) elements from the B plasmid, as well as a polyadenylation signal for the 3' downstream signaling event, which Maintained relative to the integrity of the RT-RNAse H component.

The specific single plasmid expression system described herein does not contain a restriction endonuclease (RE) gene and thus does not digest the stem of the stem-loop intermediate formed by the inverted repeat. Therefore, SOI (including DNase) inserts into the C or D plasmid only at the 3′ position of the IR, because the transcript encounters the relatively stable stem of the stem-loop intermediate and cannot continue to transcribe ss-cDNA from the mRNA transcript, making ss - Pre-mature truncation of cDNA products to remove unwanted vector sequences. More specifically, each SOI was inserted only within the PacI/BamHI restriction site of the pssXC and pssXD plasmids, as will be apparent from the description below.

As apparent from the description of the B, C and D plasmids below, the plasmids contain a cloning site for insertion of the SOI. In a preferred embodiment of the B plasmid described herein a NotI site (located between IR) and a PacI/BamHI (3' end of IR, eg, between IR and PBS) sites are provided. The C and D plasmids described herein include only PacI/BamHI sites for this purpose. However, those skilled in the art having the benefit of this description will recognize that these particular cloning sites can be selected for the particular system described herein and that other cloning sites are equally useful for this purpose. The two plasmid vector systems described herein containing the A plasmid are not intended to include SOIs, but those skilled in the art will also recognize that if a two plasmid vector system is used, elements of the set of genetic elements of the invention, and in particular It is SOI and can be easily inserted into any plasmid.

A nucleic acid sequence, referred to herein as a cassette, provides a template for the synthesis of ss-cDNA in a target cell. It is a component including SOI, IR, and PBS. The same is true for other elements in the group of genetic elements of the invention, preferably conditioned by a suitable broad-spectrum or tissue-specific promoter/enhancer, such as the CMV promoter, or a promoter/enhancer located upstream of the genetic element combination of adjustments. As is the case with other genetic elements, the promoter/enhancer may be a constitutive or inducible promoter. Those skilled in the art who have the benefit of this description will recognize that many other eukaryotic promoters can be used to facilitate control including SV-40, RSV (non-cell type specific) or tissue-specific glial fibrillary acidic protein (GFAP) Expression of the SOI.

A primer binding site (PBS) for initiation of cDNA synthesis is located between the 3'IR and the polyadenylation signal. PBS is a sequence complementary to transfer RNA (tRNA) in eukaryotic target cells. In the case of mouse Moloney reverse transcriptase (MoMULV RT) described herein used in conjunction with the present invention, PBS utilizes proline tRNA. The PBS used in connection with the presently preferred embodiments of the invention described herein is taken from the actual 18 nucleotide sequence region of mouse Moloney virus. Shinnick, T.M., et al., Nucleotide sequence of Moloney murine leukemia virus, Nature, 293, 543-548 (1981). For the RT gene from human immunodeficiency virus which was also tested as described below, the PBS used was taken from the nucleotide sequence of HIV. Y. Li, et al., Journal of Virology, 66, 6587-6600 (1992). Briefly, any PBS matched to a specific RT was used for this purpose. PBS is specifically recognized by the target cell endogenous primer tRNA. Each tRNA has the ability to recognize a particular sequence (ie, a codon) encoding an amino acid on an mRNA transcript, and has the ability to covalently link to a particular amino acid (ie, the tRNA becomes "loaded" when bound to a particular amino acid). However, primer tRNA can be used to initiate ssDNA synthesis by RT when bound to mRNA transcript PBS and not covalently linked to amino acids (ie, "unloaded"). For example, the MoMULV RT used in the Examples described herein recognizes and uses an unloaded lysine tRNA, which in turn recognizes and binds its unique sequence in PBS. Therefore, each PBS inserted into the expression system of the present invention must contain a unique sequence recognized by the primer tRNA, and the primer tRNA must be a primer tRNA recognized by the specific RT used.

Other retroviral RT/RNAse H genes can be used according to the invention, preferably the RT/RNase H gene is an RT/RNase regulated by a suitable upstream eukaryotic promoter/enhancer expressed in human cells such as the CMV or RSV promoter H gene. RNA-dependent DNA polymerase/RT genes suitable for use in the present invention include elements from retroviruses, hepatitis B and C virus strains, bacterial retrotranscription elements, and retroviruses isolated from various yeast and bacterial species. those genes. As found in nature, these RNA-dependent DNA polymerases often have associated RNase H components within the same coding transcript. However, the present invention does not require a naturally occurring RNase H gene for a particular RT. In other words, those skilled in the art will recognize from this specification that various combinations of RT and RNase H genes can be spliced together for use in the present invention to achieve this function and function in the desired manner to those skilled in the art Improved and/or hybrid versions of these two enzyme systems are available and/or known. Those skilled in the art will also recognize that the target cell itself has sufficient endogenous RNase H to carry out this function. Likewise, those skilled in the art will recognize that the target cell itself may have sufficient endogenous RT activity, eg, derived from a previous retroviral infection, to carry out this function.

The RT/RNase H gene also preferably includes a downstream polyadenylation signal sequence such that mRNA produced from the RT/RNase H gene includes a 3' poly(A) tail that stabilizes the mRNA. As known to those skilled in the art, multiple poly(A) tails are available and routinely used to generate expressed eukaryotic genes.

Those skilled in the art will also recognize that many tissue-specific or broad-spectrum promoters/enhancers, or combinations of promoters/enhancers, not listed above can also be used to help regulate the RT/RNAse H gene, the RE gene ( if used), and the destination sequence. Although it is not necessary to list all available promoters/enhancers to exemplify the invention, as noted above, promoters/enhancers may be constitutive or inducible and may include the CMV or RSV (non-cell type specific) listed here. ) or GFAP (tissue specific) promoter/enhancer and many other viral or mammalian promoters. Representative promoters/enhancers suitable for cassettes of the invention may include, but are not limited to, HSVtk (S.L.McKnight, et al., Science, 217, p. 316 (1982)), the human β-globin promoter (R .Breathnach, et al., Annals of Biochemistry, 50, p. 349 (1981)), (-actin (T. Kawamoto, et al., Molecular Cell Biology, 8, p. 267 (1988)), Rat Growth Hormone (P.R.Larsen, et al., Proceedings of the National Academy of Sciences of the United States of America, 83, p. 8283 (1986)), MMTV (A.L. Huang, et al., Cell, 27, p. 245 (1981)), adenovirus 5 E2 (M.J. Imperiale, et al., Molecular Cell Biology, 4, p. 875, (1984)), SV40 (P. Angel, et al., Cell, 49, p. 729 (1987)), (-2-macroglobulin (D. Kunz, et al., Nucleic Acids Research, 17, p. 1121 (1989)), MHC class I gene H-2kb (M.A. Blanar et al., EMBOJ., 8, p. 1139 (1989)), and thyroid-stimulating hormone (V.K. Chatterjee, et al., U.S. National Proceedings of the Academy of Sciences, 86, p. 9114 (1989)).

RTs produced in cells synthesize complementary DNA (cDNA) using the SOI-containing genetic elements described below as templates. The RNase H activity of RT degrades the mRNA template component of RNA/cDNA hybrids to generate ss-cDNA in vivo.

The gene encoding the RE (used in the two-plasmid expression system and not a necessary component of the system) can be any of several genes encoding REs, and is preferably regulated by one or more constitutive or inducible genes such as those listed above. Those genes controlled by broad-spectrum and/or tissue-specific promoters/enhancers. The specific REs tested were MboII and FokI, but those skilled in the art having the benefit of this description will recognize that any RE (type I, II, IIS, or III) site may be included in the IR. These enzymes "cut" or digest the stem of the stem-loop intermediate described below to linearize the SOI into single-stranded DNA.

Although the expression of the RE gene can be regulated by a suitable constitutive or inducible promoter/enhancer, such as the CMV or RSV promoter for expression in human cells, located upstream of the restriction endonuclease gene, in the plasmid pssXA , RE gene (MboII) is connected with RT-RNAse H polypeptide. The RE gene preferably also includes a downstream polyadenylation signal sequence so that mRNA transcripts from the RE gene have a 3' poly(A) tail.

The cassettes of the invention also include inverted tandem repeats (IR). After digesting the mRNA of the mRNA-cDNA heteroduplex with RNAse H and releasing the ss-cDNA, IR causes the ss-cDNA to fold itself in the manner described in U.S. Pat. The antiparallel DNA composition of the strands, after the sequence cassette is transcribed in the cell and the RT/RNase H generated by gene transcription produces the target ss-cDNA sequence from the mRNA transcript in the cell, as shown in Figure 2. One or more RE sites (in the case of those plasmids containing the RE gene) that are cleaved by the RE produced by the RE gene can be engineered into a double-stranded portion, the IR, that forms the stem of the stem-loop intermediate. Transcription produces ss-cDNA with 5' and 3' coding regions (consisting of IR) flanking the stem and a loop containing the SOI. The stem is then cleaved (also referred to as digested or cleaved) by any of a number of RE enzymes that recognize a cleavage site designed into the stem (note that even though the RE gene is not included in the vector system of the present invention, An endonuclease recognition site can also be designed in the stem) to release the ss-cDNA circle (see Figure 1). The portion of the ss-cDNA loop that does not form any apparent duplex DNA is not affected by RE activity because REs only recognize double-stranded DNA as a target substrate.

As noted above, those skilled in the art will recognize that if it is desired to generate ssDNA from an SOI located between PBS and IR, the RE site does not have to be designed to form the middle of the stem-loop since the transcription of the cassette terminates at the stem formed by the IR. The IR of the body stem. Another option is to design the IR to contain DNA-binding sites for eukaryotic, prokaryotic, or viral proteins, which are used to competitively titrate out selected cellular proteins. Depending on the base composition of the IR chosen, a combination of restriction sites or other sequence-specific elements may be included in the IR to produce a linearized or precisely digested stem-loop intermediate form of ssDNA. The use of synthetically constructed sequence-specific elements in IR is generally preferred, since naturally occurring inverted repeat sequences are unlikely to have properly arranged restriction sites.

As stated above, a cassette containing one of the elements of the set of genetic elements of the invention also includes a catalytically active DNA sequence. Due to the inclusion of the so-called "DNase" in the sequence cassette (and in the embodiments described herein, the DNase is located within the sequence of interest), the present invention is useful for the synthesis of inhibitory nucleic acids (antisense sequences) when the sequence of interest is used as an inhibitory nucleic acid (antisense sequence). This is especially advantageous when it comes to templates. Thus, the examples set forth herein describe the production of an antisense SOI shown in Figure 5B comprising sequences enzymatically active on mRNA including sequences designed to specifically bind c c-raf lyase of the 3' untranslated region of raf mRNA (Monia, B.P., et al., Nat. Medicine, 2, 668-675 (1996), the specific reference of which is hereby incorporated in its entirety into this specification). The two 9bp target-specific binding arms are flanked by a 15bp catalytic domain (Santoro, S.W. and G.F.Joyce, Mechanism and utility of DNA enzymes that cleave RNA, Biochemistry, 37, 13330-13342 (1998), also the Specific documents are incorporated in this specification in their entirety). Appropriate restriction sites were added to the DNase oligonucleotides so that they could be inserted into the NotI site or the PacI and BamHI sites, and the resulting plasmids were designated pssXB-I and pss-XB-II, respectively.

Those skilled in the art will recognize that the present invention is not limited to antisense sequences, which need not contain catalytically active nucleic acid sequences, and that the inhibitory nucleic acid sequence can also be any other form of inhibitory nucleic acid sequence described above. The above SOI was chosen for demonstration of the present invention because the c-raf kinase in the A549 lung cancer cell system is well characterized (Monia, et al., supra (1996)). Raf proteins are serine/threonine protein kinases that have been shown to act as direct downstream effectors of ras proteins within the MAP kinase signaling pathway for the activation of downstream MEK1/MEK2 and the subsequent activation of ERK1 and ERK2 (Daum, G., et al., Daum, G., et al. The Inside and Out of Mouse Kinases, Current Biological Sciences, 19, 474-480 (1994)). Many solid tumors and leukemias have been shown to contain mutations in ras or upregulation of MAP kinase signaling. Signal transduction pathways such as craf-associated tumors are attractive targets for oncology therapy and the above-mentioned phosphorothioate ODN ISIS 5132 has been shown to be a potent antisense inhibitor (Monia, et al., supra (1996)). In addition, ISIS 5132 has been shown to induce apoptosis (Lau, Q.C., et al., Oncogene, 16, 1899-1902 (1998), which specific document is also incorporated in this specification in its entirety) and appears to represent a promising response to this tumor. potentially effective treatment. This antisense ODN has recently entered phase I clinical trials (O'Dwyer, P.J., et al., C-raf-1 exclusion and Tumor Response, Clinical Cancer Research, 5, 3977-3982 (1999)), and demonstrated usefulness in the treatment of c-raf-associated tumors. Other SOIs that have been cloned into plasmids for expression using the expression system of the present invention include h-ras with the 10-23 DNase sequence (Santoro and Joyce, supra (1997)) inserted between the 5' and 3' complementary sequences The partial sequence of the 23rd codon of the antisense binding sequence, the partial sequence of the antisense binding sequence of the pleiotropin (pleiotropin) with 10-23 DNase sequence inserted between the 5' and 3' complementary sequences, and the insertion Partial sequence of SIV sequence tat antisense binding region with 10-23 DNase sequence between 5' and 3' complementary sequences. Although each of these sequences includes DNase sequences, those skilled in the art will recognize from this specification that DNase sequences need not include these, or any other SOIs.

The enzymatically active nucleic acid sequence used in the methods of altering gene expression described herein is 10-23 DNase (Santoro and Joyce, supra (1997)). The enzyme sequence is inserted into the cassette in one or both of two locations, e.g., (a) between the IR and the internal SOI (at the NotI site) or (b) the second 3' of the IR and 5' of the PBS. Inside the SOI (at the PacI/BamHI site). In each way, the resulting aptamer is specific for the SOI target and is thus used to target other DNA sequences, mRNA sequences, and any other suitable substrates to inhibit or alter the machinery of DNA or mRNA splicing, or even to Directly alter the cellular genome in a specific manner.

Those skilled in the art will recognize from this specification that any DNA sequence having enzymatic activity will function for the desired purpose when inserted into a cassette of the invention. Numerous nucleic acid sequences having enzymatic activity have been reported in the literature, including:

Sequences with RNAse activity, such as the so-called "10-23" and "8-17 enzymes" (Santoro, S.W. and G.F.Joyce, supra (1997)) and other metal-dependent RNAses (Breaker, R.R. and G.F.Joyce, Biochemistry, 1, 223-229 (1994) and Breaker, R.R. and G.F.Joyce, Biochemistry, 2, 655-660 (1995)) and histidine-dependent RNAse (Roth, A. and R.R.Breaker, USA Proceedings of the National Academy of Sciences, 95, 6027-6031(1998));

Sequences having DNAse activity, such as copper-dependent DNAse (Carmi, N., etc., Chemical Biology, 3, 1039-1046 (1996), Carmi, etc., cited above (1997); Sen, D. and C.R.Geyer, Current Opinion in Chemical Biology, 2, 680-687 (1998)) and reported in Faulhammer, D. and M. Famulok (J. Molecular Biology, 269, 18-203 (1997)) require divalent metal ions as auxiliary Factors or DNAses that do not rely on divalent metal ions to hydrolyze substrates;

Sequences having DNA ligase activity, such as copper-dependent DNAse (Breaker, R.R., Chemical Reviews, 97, 371-390 (1997)) and zinc-dependent E47 ligase (Cuenoud.B. and J.W.Szostak, Nature, 375, 611-613 (1995));

Sequences having DNA kinase activity, such as calcium-dependent DNA kinase (Li, Y. and R.R. Breaker, Proc. National Academy of Sciences USA, 96, 2746-2751 (1999)); and

A sequence having RNA kinase activity, such as a calcium-dependent DNA kinase (Li, Y., supra (1999)).

In general, it is those enzymatically active DNA sequences derived from physiological conditions that are preferred for use in the cassettes of the invention.

When elements containing the set of genetic elements of the invention are inserted into a vector for expression in target cells, it is preferred that the vector contains other specialized genetic elements to aid in the identification of cells carrying the vector and sequence cassette and/or to increase the number of cells containing the set of genetic elements. The expression level of the sequence cassette of the element. Dedicated genetic elements include selectable marker genes so that the vector can be transformed and amplified in prokaryotic systems. For example, the most commonly used selectable markers are genes that confer resistance to bacteria (eg, E. coli) to antibiotics such as ampicillin, chloramphenicol, kanamycin (neomycin), or tetracycline. It is also preferred that the vector contains dedicated genetic elements for subsequent transfection, identification and expression in eukaryotic systems. For expression in eukaryotic cells, various selection strategies that confer resistance to antibiotics or other drugs or alter the phenotype of the cells, such as morphological changes, loss of contact inhibition, or increased growth rate (e.g., Chinese hamster Ovary: CHO). Selectable markers used in eukaryotic systems include, but are not limited to, resistance markers to Zeocin, resistance to G418, resistance to aminoglycoside antibiotics, or phenotypic selectable markers such as β-gal or green fluorescence protein.

Insertion of these components into plasmids containing the expression system of the present invention creates two convenient methods for removing predetermined vector sequences after production of ssDNA. In the first method, the sequence cassette is reverse transcribed from PBS, and the SOI between the IR contains the loop portion of the ssDNA stem-loop intermediate that is created when the nucleotides containing the IR pair to form the stem of the stem-loop vector The intermediate, stem contains one RE site, and upon digestion with the appropriate RE, the circle is released as linearized single-stranded cDNA with no (and/or minimal) flanking sequences. In the second method, the cassette is reverse transcribed from PBS and contains the SOI at the 3' end of the cassette IR as transcribed, but reverse transcription is terminated at the stem of the stem-loop structure formed by the nucleotide pairing of IR. In each method, ssDNA is produced with no (and/or minimal) flanking sequences. If it is desired to use the second method to produce ssDNA, the cassette is designed with an IR that forms a stem that is more stable than that required when digesting the stem to produce ssDNA according to the first aspect of the invention (e.g., the IR can be designed to contain no RE site). Reverse transcription is carried out from the second SOI (if it is designed into the cassette) to the SOI located between the IR through the cassette designed with the IR forming a stem that is susceptible to denaturation according to the first aspect of the invention. This "pre-mature termination" of reverse transcriptase cDNA transcripts at the 3' end of the stem structure thus provides a second means of restricting in vivo produced ss-cDNA to contain intervening vector sequences. Stems as intermediates allow the creation of first and second SOIs in stability.

It will be apparent to those skilled in the art from this specification that the complete stem-loop ss-cDNA structure is functionally similar to the linearized ss-cDNA form in many applications. Therefore, it is also advantageous to use cassettes that do not contain restriction endonuclease genes and associated regulatory elements and/or have sequences of interest that lack corresponding restriction endonuclease sites.

It will be apparent to those skilled in the art from the description of the preferred embodiments of the present invention that a cassette encoding ss-cDNA with a "trimmed" stem-loop structure can be prepared. RE sites encoded in the IR flanking the SOI were designed to digest the stem portion (after duplex formation) with the corresponding RE to cleave the dsDNA-containing stem by removing part of the stem and associated flanking sequences and leaving Sufficient duplex DNA for the transcript to retain the stem-loop structure described above. This ss-cDNA structure is more resistant to intracellular nucleases by retaining the ssDNA "ends" in double-stranded form.

It will also be apparent to those skilled in the art from the description of the preferred embodiments of the present invention that the stem (double stranded DNA) can be designed to contain a predetermined sequence (or sequences), i.e., aptamers, which are activated by specific DNA binding proteins. Identify and combine. Among other uses, the stem structure is used in cells as a competitor to titrate out selected proteins that regulate the function of a particular gene. For example, ss-cDNA stem-loops produced in cells according to the invention contain binding sites for selected positive transcription factors such as adenovirus Ela. Adenovirus Ela, like other oncogenes, regulates the expression of some adenovirus and cellular genes by affecting the activity of cell-encoded transcription factors, resulting in the transformation of normal cells into transformed cells. Jones, et al., Gene Development, 2, 267-281 (1988). Thus, the double-stranded stem of the stem-loop intermediate produced according to the present invention is designed to function "on" the transcription factor, preventing the protein from binding the promoter, and thus inhibiting the expression of a particular deleterious gene. It will be apparent to those skilled in the art that the double-stranded stem structure may optionally contain multiple binding sites, eg, sites recognized by various transcription factors that efficiently regulate the expression of specific genes. For example, the adenovirus Ela has been found to repress the transcription of the collagenase gene through the phorbol ester response element, a promoter element responsible for the conversion of 12-O-tetradecanoylphorbol 13-acetate (TPA), Transcription is induced by many other mitogens, and by the ras, mos, src and trk oncogenes. This mechanism involves repression of the function of the transcription factor family AP-1. Offringa, et al., Cell, 62, 527-538 (1990). Desired nucleotide sequences encoding the "loop" portion of the stem-loop intermediate can be inserted into the genetic element to achieve the desired repressive function, eg, antisense binding, downregulation of a gene, and the like described herein.

In another aspect that will be appreciated by those skilled in the art, the present invention is useful for constructing complex secondary ssDNA structures that provide biological responses to cDNA transcripts based on the folded conformation of the secondary structure. This secondary structure can be adapted for any of several functions. For example, sequences of interest may include, but are not limited to, insertion into the loop portion of a single-stranded cDNA transcript to form so-called "clover leaf" or "crucible" structures such as those found in adeno-associated virus long terminal repeats or retrotransposons. "-like structure sequence. Where appropriate, the construct is integrated into the host genome in a site-specific manner.

Since the cassettes of the present invention are suitable for insertion into a variety of commercially available delivery vectors for therapeutic purposes in mammals and humans, a variety of delivery routes can be used depending on the vector chosen for the particular target cell. For example, viral vectors are often used to transform patient cells and introduce DNA into the genome. In an indirect approach, a viral vector carrying the new genetic information is used to infect target cells removed from the body and the infected cells are then reimplanted in vivo (ie ex vivo). Direct in vivo gene transfer into postnatal animals has been reported for formulation of DNA encapsulated in liposomes and DNA embedded in proteoliposomes containing viral envelope receptor proteins. Nicolau, et al., Proceedings of the National Academy of Sciences USA, 80, 1068-1072 (1983); Kaneda, et al., Science, 243, 375-378 (1989); Mannino, et al., Biotechnology, 6, 682-690 (1988). Positive results obtained with DNA co-precipitated with calcium phosphate have also been described. Benvenisty and Reshef, Proceedings of the National Academy of Sciences of USA, 83, 9551-9555 (1986). Other systems for facilitating the administration of expression systems containing the set of genetic elements of the invention include intravenous, intramuscular, and subcutaneous injections, as well as direct intratumoral and intracavitary injections. When inserted into an expression system of choice, the cassette also facilitates administration by topical, transmucosal, rectal, oral, or inhalation-type delivery methods.

The use of the cassettes of the present invention facilitates the delivery of antisense, triplex, or any other inhibitory nucleic acid or single-stranded nucleotide sequence of interest, and specific sequences of interest can be spliced into the cassette using known digestion and ligation techniques (in between inverted tandem repeats or between PBS and inverted tandem repeats). Those skilled in the art having the benefit of this description will recognize that the above-described signals for expression in eukaryotic cells can be modified according to the particular sequence of interest in a manner known in the art. For example, one possible modification is to alter the promoter in order to provide favorable expression properties to the cassette in the system in which expression of the sequence of interest is desired. There are so many possible promoters and other signals, and they are also dependent on the specific target cell for which the sequence of interest is selected, that it is not possible to list all potential enhancers, inducible and constitutive, that are preferred for a specific target cell and sequence of interest A promoter system, and/or a poly(A) tailing system.

In a particularly preferred embodiment, the present invention takes the form of a kit consisting of a plasmid having the above-described RNA-dependent DNA polymerase and RE gene cloned therein and which facilitates the user of the kit. Insert multiple cloning sites (MCS) for specific SOIs. The cloning site for inserting the SOI is located between the above IRs. The resulting plasmid is then purified from cell culture, where it can be maintained, lyophilized, or stored for packaging and shipment to the user. The kit preferably also includes RE, ligase and other enzymes for cloning the SOI into MCS, as well as suitable buffers and plasmid maps for ligating the SOI into the plasmid.

In the specific embodiment described herein, the cells are co-transfected with two plasmids designated A and B, respectively, designed and constructed to include the above components, or the SOI is delivered into the host cell through a single C or D plasmid. In both plasmid systems, the B plasmid encodes a sequence cassette that includes the SOI nested within flanking sequences containing the IR or located between the IR and the PBS that provides the post-transcriptional processing signals that mediate the conversion of mRNA to ssDNA. The desired activity is expressed from the A plasmid by processing the first gene product of the B plasmid into ssDNA and removing vector sequences and processing signals, especially RT/RNAse H and RE (if used). Single-stranded DNA sequences released from the interaction of in vivo transcripts of these components freely bind intracellular targets such as mRNA species and DNA promoters in antisense and triplex protocols.

As mentioned above, the B plasmids described herein include a cloning site (the NotI site is used for the B plasmids described herein) between which any DNA SOI is placed (as described above, in the examples described herein Here, the SOI is an antisense sequence to c-raf kinase that includes the 10-23 enzyme sequence, but as noted above, other sequences produced in vivo using the expression system described herein include "stuffer sequences", or detection sequences, telomeric repeats sequence, h-ras, coding region of the angiogenic growth factor pleiotrophin, and tat (from SIV)). The cloning site is flanked by signals directing the processing of the primary mRNA transcript produced from the promoter (CMV promoter is used in the B plasmid described here) into the desired single-stranded inhibitory nucleic acid, and the desired SOI is cloned into Following the B plasmid, the A and B plasmids are co-transfected into selected cell lines for constitutive expression of ssDNA. Also, in the single plasmid expression system described herein, SOI is cloned into the plasmid and transfected into cell lines for further processing. Regardless of how elements of the above-mentioned set of genetic elements are distributed between two (or more) plasmids, or whether the elements are all contained in a single plasmid, in single-stranded DNA regions (ie, SOI, IR, and PBS) After transcription, it is processed in three steps:

(1) with RT reverse transcription plasmid RNA transcript, in the embodiment described herein RT is by A, C, or the RT expressed by D plasmid (in the embodiment described herein, RT is MoMuLV RT), from The primer binding site located at the 3' end of SOI (SOI optionally includes a sequence with enzymatic activity) starts reverse transcription, IR, and PBS as shown in Figure 1;

(2) RNAse H digestion of the resulting heteroduplex with the RNAse H activity of the RT polyprotein or with the endogenous RNAse H activity so that the single-stranded DNA precursor is released from its RNA complementary strand; and

(3) After digesting the stem-loop intermediate formed by the base containing IR according to Watson-Crick base pairing or the stem-loop secondary structure formed by self-complementary IR, the cDNA transcript is terminated before maturation to remove the flanking sequence .

Those skilled in the art will recognize from this specification that the specific cloning sites flanking the SOI, the specific RT, RE (if used), promoter, PBS, and all other elements in the set of genetic elements of the invention can be expressed according to Specific SOIs and/or systems for ssDNA are selected.

Example

Unless otherwise stated, in the examples listed below use according to Seabrook, et al. "Maniatis, et al. (1989)") and Ausubel, et al., (1987) (F.M. Ausubel, et al., Current Methods in Molecular Biology, New York: John Wiley & Sons (1987)), both cited in This particular reference is incorporated herein in its entirety. It should be understood that other methods of producing ssDNA both naturally and artificially with different enzyme products or system design can also be used in the methods of the present invention and that the examples provided herein are provided for illustrative purposes and are not intended to limit the present invention. specification or the scope of the invention described herein.

Plasmid pcDNA3.1Zeo+ was purchased from Invitrogen Corporation (Carlsbad, CA) and plasmid pBK-RSV was from Statagene (La Jolla, CA). Oligodeoxynucleotides (ODNs) were synthesized by Midland Certified Reagents (Midland, TX). Polymerase chain reaction (PCR) was performed in a Robo-gradient thermal cycler (Stratagene (La Jolla, CA)) using Taq DNA polymerase purchased from Boehringer Mannheim (Indianapolis, IN). Restriction endonucleases and T4 DNA ligase were obtained from Boehringer Mannheim (Indianapolis, IN). The ODNs used are listed in the attached sequence listing.

All ODNs were hybridized in 1 μl (5 μg/μl in water) in separate tubes that had been incubated at 70° C. for 5 minutes and hybridized for 15 minutes at room temperature. Standard restriction endonuclease digestion was performed with 10 units of enzyme in a total reaction volume of 15 μl and appropriate reaction buffer (EcoRI was used as negative control). DNA fragments are resolved on and separated from agarose gels. Positive clones were selected on ampicillin plates after transformation into competent XL1-Blue MRF cells (Stratagene) as described by Maniatis, et al., (1989). After selecting positive clones, isolate plasmid DNA using the Quiagen plasmid isolation kit described above.

Plasmid construction. The construction of four expression plasmids is described. The first plasmid, pssXB (Fig. 3), was derived from pcDNA3.1Zeo(+) (Invitrogen) and contained the genetic element encoding the ss-cDNA sequence of interest used herein. pcDNA3.1Zeo(+) was digested with restriction endonucleases HindIII and NotI at positions 911 and 978, respectively. Anneal the synthetic single-stranded oligodeoxynucleotide ODN-5'-N/M (linker) 2-H/N and ODN-3'-N/M (linker) 2-H/N to form a compatible The double-stranded linker region at the end of HindIII and NotI was ligated under standard conditions into HindIII/NotI double-digested pcDNA3.1Zeo(+), which had been transformed into SureII cells (Stratagene). ODNs were hybridized in 1 μl (5 μg/μl in water) in Ependorf tubes that had been incubated at 70° C. for 5 minutes and hybridized for 15 minutes at room temperature. Appropriate clones are selected and sequenced to ensure correct insertion of the linker region. The resulting plasmid was called pssXB. pssXB is shown in Figure 4A and is the plasmid into which the sequence of interest (Figure 4B) was cloned. For cloning the sequence of interest between the inverted tandem repeats, two Notl sites at positions 935 and 978, respectively (see Figure 4A) were used. These two sites are contained within an inverted tandem repeat. To insert the sequence of interest between the inverted tandem repeat and the primer binding site, PacI and BamHI sites at positions 1004 and 1021, respectively, were used.

The second plasmid is also a component of the two-plasmid vector system described herein, pssXA (Figure 3). This "A" plasmid contains Mo-MuLV-RT (Shinnick, T.M., et al., Nature, 293, 543-548 (1981)) and restriction endonuclease genes and is derived from pBK-RSV (Stratagene), also using XL -1 Blue MRF' as host cells. A mouse cell line expressing Moloney murine leukemia virus was obtained from the American Type Culture Collection (#CRL-1858). Viral RNA was isolated from cells using Trizol reagent (Gibcob RL) and primer 3'-RT-HindIII (5 '-CTTGTGCACAAGCTTTGCAGGTCT-3') reverse transcription. Transcripts were then amplified by PCR using the TaqPlus long polymerase system (Stratagene) for 35 cycles: 1 min at 94°C, 1 min at 67°C, and 2.5 min at 72°C. The primers used for the PCR reaction were 5'-RT-Sacl (5'-GGGATCAGGAGCTCAGATCATGGGAC-CAATGG-3') and the same 3'-RT-HindIII as used for reverse transcription. These primers included appropriate SacI and HindIII sites, respectively. The resulting 2.4 kb product included the Mo-MuLV sequence between positions 2546 and 4908. The mature viral RT peptide is encoded by the sequence between positions 2337 and 4349 (Petropoulos, C.J., Classification of Retroviruses, Protein Structure, Sequence and Genetic Map, in J.M. Coffin, et al. (eds.), Retroviruses, New York: Cold Spring Hong Kong Laboratory Publishing, pp. 757-805 (1997)), but the amino-terminal truncated peptide retains full activity (Tanese, N. and S.P. Golf, Proceedings of the National Academy of Sciences, 85, 1777-1781 (1988)). The peptide encoded by this construct includes part of the integrase gene following the RT in the MoMuLV polyprotein (Petropoulos, supra (1997)).

The bacterium Moraxella bovis encoding the restriction endonuclease MboII (Bocklage, H., et al., Nucleic Acids Research, 19, 1007-1013 (1991)) was obtained from the American Type Culture Collection (ATCC #10900 )get. Genomic DNA was isolated from M. bovis using a Stratagene DNA extraction kit according to the manufacturer's instructions and used as template DNA in PCR. The MboII gene was amplified from genomic DNA by PCR for 30 cycles using two primers, 5'-MboII-HindIII (5'-CAATTAAGGAAAGCTTTGAAAAATTATGTC-3') and 3'-MboII-XmaI (5'-TAATGGCCCGGGCATAGTCGGGTAGGG-3'): 94°C, 30 seconds, 58°C, 1 minute, 72°C, 1 minute. These primers were designed to include HindIII and XmaI sites, respectively. The 1.2 kb product obtained by copying the M. bovis genome between positions 888 and 2206 contains the coding region for the MboII enzyme.

The pBK-RSV vector was digested with XmaI and NheI. The NheI ends were linked using a linker formed by two annealed oligonucleotides 5'-Nhe-Sac-linker (5'-CTAGCGGCAAGCGTAGCT-3') and 3'-Nhe-Sac-linker (5'ACGCTTGCCG-3'). Converted to SacI terminus. The RT and MboII amplicons were ligated via a HindIII site and this construct was then ligated into pBK-RSV between the SacI and XmaI sites to generate pBK-RSV-RT/MboII.

To insert an elastic linker between the RT and MboII domains of the polyprotein, the fragment of the pBK-RSV-RT/MboII plasmid encoding the 5′ end of the MboII gene and part of the integrase gene located between the AseI and BglII sites was excised and used Insert substitution containing 6-His-linker and 5′-MboII DNA fragment deleted by double digestion.通过从在3′末端具有17个碱基的互补序列的两个模板，Rep(+)(5′-ATACTATTAATTTTGGCAAATCATAGCGGTTATGCTGACTCAGGTGAATGCCGCGATAATTTTCAGATTGCAATCTTTCATCAATGAATTTCAGTGATGAATTGCCAAGATTGATGTTGC-3′)和Rep(-)(5′-GACGAGATCTCCTCCAGGAATTCTCGAGAATTCGGATCCCCCGCTCCCCACCACCACCACCACCACCCTGCCCCGCGGATGAAAAATTATGTGAGCAACATCAATCTTGGC-3′)以互相引发的DNA synthesis yields the insert sequence. These two oligonucleotides were annealed and extended with modified T7 DNA polymerase (USB), and the double-stranded oligonucleotide was then digested with AseI and BglII and inserted into the pBK-RSV vector to generate pssXA (Figure 3).

In a first embodiment of a single plasmid expression vector system constructed according to the present invention, a pc3.1DNAZeo(+)-derived "B" plasmid and a pBK-RSV-derived "A" plasmid are fused so that the resulting plasmid encodes All elements of this group of genetic elements in the present invention include the target sequence encoding ss-cDNA, tandem inverted repeat sequence, Mo-MuLV-RT gene, and restriction endonuclease (MboII) gene. To generate the C plasmid, plasmid pssDNA-Express-A was digested with SacI XmaI to remove the MboII gene. Make containing oligonucleotides 5'-(linker) 2-Hind/Xba (5'-CCGGATCTAGACCGCAAG-CTTCACCGC-3') and 3'-(linker) 2-Hind/Xba (5'-GGTGAAGCTTGCGGTCTAGAT-3') The linker region was annealed at 70°C for 15 minutes and slowly cooled to room temperature, digested under standard conditions and ligated into the plasmid. Positive clones were harvested and sequenced to confirm the location of the linker, then the plasmid was digested with Xba and HindIII. Plasmid pssDNA-Express-B was digested with HindIII and Xba and the corresponding 300 base pair DNA fragment containing the previously described inverted tandem repeat, multiple cloning site, and PBS was cloned into the digested plasmid to generate pssXC (Fig. 5A). Standard ligation reactions were performed and transformed into Sure II cells (Stratagene). Transformation positive colonies were harvested and positive clones were identified by restriction analysis.

The sequence of interest was cloned into the multiple cloning site of pssXC using the BamHI and PacI sites in the multiple cloning site (Figure 5B). Four different sequences of interest were synthesized for these constructs described above, and a similar procedure was used to insert each of the four sequences of interest. Each construct was prepared by annealing the paired oligonucleotides at 70°C for 15 minutes and cooling to room temperature, followed by ligation into the plasmid under standard conditions. After transformation into SureII cells, appropriate colonies were selected by sequencing each insert.

The second plasmid pssXD for the single plasmid expression system was constructed by combining the two plasmids pssXA and pssXB as follows. pssXA containing Mo-MuLV reverse transcriptase (RT) was digested with XmaI and BglII, and the resulting XmaI-BglII fragment was synthesized by annealing two oligonucleotides XmaI-BglII-terminator 1 (5'-CCGGATCTAGACCGCAAGCTTCATTTAAA-3') and Double stranded DNA adapter substitution formed by XmaI-BglII-terminator 2 (GATCTTTAAATGAAGCTTGCGGTCTCGAT-3'). This adapter contains a protein translation stop codon and subcloning sites Xbal and HindIII. The resulting plasmid was named pssXD (Fig. 6A). The XbaI-HindIII fragment was cleaved from pssXB and pssXB-II and cloned into pssXD between XbaI and HindIII. These DNA fragments contain: 1) RT primer binding sites (PBS); 2) stem-loop structures; and 3) optional control sequences (pssXB) or c-raf DNase sequences (pssXB-II). The resulting plasmids were named pssXD-I and pssXDII, respectively. The RSV promoter regulates gene expression of all elements required for single-stranded DNA expression and transcription of all elements into single-stranded mRNA molecules. Endogenous ^tRNAPro binds to PBS at the 3' end of the transcript and serves as a primer for single-stranded DNA synthesis (Marquet, et al., Biochemistry, 77, 113-124 (1995)). After reverse transcription of single-stranded DNA by RT, ssDNA is released when the template mRNA is degraded by endogenous RNase H or the RNase H activity of RT (Tanase and Goff, Proceedings of the National Academy of Sciences USA, 85, 1777-1781 (1988)).

Tissue culture test. Stable and transient transfections were performed using DOTAP liposome transfection reagent (Boehringer Mannhiem Corp., Indianapolis, IN) according to the manufacturer's attached instructions. All plasmid constructs were transfected into the A549 lung cancer cell line (ATCCCCL-185) and HeLa in the cell line. Measurements of ssDNA were performed by PCR and dot blot analysis 24-48 hours after transfection. ssDNA was isolated from cells transfected 48-72 hours earlier. Co-concentration of sscDNA with total RNA was achieved using Trizol reagent (Gibco Life Technologies, Gaithersburg, MD) (Mitrochnitchenko, O., et al., Production of single-stranded DNA in mammalian cells using bacterial reverse transcripts, J. Biological Chemistry, 269 , 2380-2383 (1994)). Specific ss-cDNA species were determined by PCR detection of internal fragments and by denaturing single-strand gel electrophoresis followed by nylon blotting and probing with biotin-labeled internal probes.

More specifically, using the RT-PCR assay method established by Silver, J., et al. The following modification was used to measure reverse transcriptase activity. pssXA transfected cells were lysed with lysis buffer (1% Triton ^™ , 1 mM MgCl ₂ , 100 mM NaCl, 10 mM TRIS-HCl, pH 8.0 and 2 nM DTT). After centrifugation at 18,000g for 30 minutes, the supernatant was collected and frozen at -80°C until use. Brome mosaic virus (BMV) RNA used as template was reverse transcribed by incubation with cleavage products containing RT activity for 10 or 30 minutes at 37°C. The reverse transcript was then amplified by PCR using primers 5′-CGTGGTTGACACGCAGACCTCTTAC-3′ and 5′-TCAACACTGTA-CGGCACCCGCATTC-3′ for 40 cycles: 94°C for 20 seconds, 56°C for 20 seconds, and 72°C for 20 seconds . The RT-PCR product was analyzed by 1.5% agarose gel, as shown in FIG. 6 .

This RT-PCR assay relies on RT activity in cell lysates of transfected cells to generate cDNA transcripts of BMV RNA substrates. The replication cycle of this virus does not involve DNA intermediates, thus ruling out the possibility of generating amplification products without prior reverse transcription. RT activity was determined in lysates of A549 cells transfected with the pssXA plasmid (lanes 3 and 4) and in E10 clones showing relatively higher expression (lanes 5 and 6). RT activity was also determined from A549 cells transiently transfected with the control pBK-RSV plasmid (lanes 1 and 2). For transient transfections, lysates were prepared 48 hours after transfection. Results showed that cell lysates from both transiently and stably transfected (E10) cells supported a band of the expected size of 150 bp (lanes 3-6), whereas control lysates showed none (lanes 1 and 2).

To detect ssDNA expressed in mammalian cells by pssXB-I and pssXB-II plasmids when co-transfected with pssXA into A549 cells (E10), the T7 primer and the c-raf DNase specific primer 5′-CTAGCTACAACGAGACATGC- 3' PCR reaction. The total RNA fraction was used as template and was pretreated with S1 nuclease or RNAse A at 37°C for 30 minutes or left untreated. The pretreated RNA samples were then PCR amplified for 30 cycles: 94 °C for 45 sec, 55 °C for 45 sec, and 72 °C for 30 sec. PCR products were analyzed with 8% amide gel, as shown in Figure 7 (lanes 1 and 3, S1 nuclease; lanes 2, 4, and 5, RNAse). Bands of the expected size were produced in both treated total RNA preparations (lanes 2 and 4) and untreated preparations (data not shown). Control preparations treated with S1 nuclease, a highly specific ssDNA endonuclease, produced no amplification product (lanes 1 and 3).

The presence of c-raf DNase was further confirmed by dot blot detection of ssDNA using the North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Pierce) according to the manufacturer's instructions. 2 μg of total RNA isolated from pssXA/pssXB-I or pssXA/pssXB-II, or pssXA transfected or untransfected cells was used. The c-raf specific biotinylated probe sequence is 5'-GGCCGCACTAATGCATGTCTCGTTGTAGCTA-GCCCAGGCGGGAAGTGC-3'. As shown in Figure 8, biotin-labeled c-raf-specific oligo probes detected signals only in RNA preparations isolated from pssXB-I or pssXB-II transfected E10 cells but not in untransfected E10 cells or A549 Not in cells.

To determine whether single-stranded c-raf DNase expressed with the pssXA/pssXB vector system of the present invention alters c-raf mRNA expression in mammalian cells, a northern blot analysis was performed. E10 cell line was transiently transfected with pssXB-I or pssXB-II. At 24 and 48 hours, cells were harvested for preparation of total RNA. 15 μg of total RNA was separated on denaturing agarose gel for Northern blot analysis, and after overnight transfer, the membrane was fixed and stained with 32P-labeled c-raf DNA fragments and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probing. A c-raf probe was prepared from an IMAGE ^™ cDNA clone (ID 645539, Research Genetics) encompassing positions 571 to 2028 of the coding region of the c-raf kinase gene using a random priming labeling kit from Boehringer Mannheim. G3PDH was also 32P-labeled and used for Northern blot normalization. Membranes were washed with 2xSSC, 0.1% SDS for 15 minutes and O.lxSSC for 5 minutes. Blots were then exposed to X-ray film or quantified with Molecular Dynamics Phosphor Imager ^™ . Phosphoimaging quantitative results from a representative experiment are shown graphically in Figure 9. pssXB-II reduced c-raf mRNA levels to 81% at 24 hours and to 66% at 48 hours compared to pssXB-transfected controls containing unrelated sequences. pssXB-I had a similar effect, reducing c-raf mRNA levels by 35% after 48 hours of culture. Significantly more cell death (approximately one-third more) was also observed in cells transfected with pssXA/pssXB vectors expressing c-raf DNase compared to controls. Harvesting only cells that remained attached and not those that started to "float" resulted in a greater reduction in mRNA than the 34-36% reduction measured.

The single plasmid vector system pssXC was transfected into the HeLa cell line. Determination of ssDNA was performed by PCR and dot blot analysis at 24-48 hours after transfection as described above. Reverse transcriptase activity was also determined using the RT-PCR assay established by Silver, et al., supra (1993), as described above. RT activity was additionally determined on single colony isolates (A12 and B12) of the stably substituted HeLa cell lines. ss-cDNA was isolated from cells transfected 48-72 hours earlier. Co-concentration of ss-cDNA with RNA was performed using Trizol reagent (Gibco Life Technologies, Gaithersburg, MD). Specific sscDNA species were determined by PCR-based determination of internal fragments and by denaturing single-strand gel electrophoresis followed by nylon blotting and probing with biotin-labeled internal probes.

This experiment shows transfection of human tissue culture (HeLa cell line) which has been engineered to synthesize processed ss-cDNA, resulting in ss-cDNA of the expected size. As described in above-referenced Application Serial No. 09/397,782, the ssDNA sequence of interest generated in vivo from pssXC is generated from a position between the inverted repeats following digestion of the stem of the stem-loop intermediate or from the inverted repeats and The position between the primer binding sites is created by terminating the reverse transcriptase cDNA transcript before the 3' end of the stem structure matures.

Intracellular single-stranded c-Faf DNase expression was determined by simple dot blot analysis using total RNA fractions. The biotin-labeled c-raf-specific oligonucleotide probes used were synthesized by Integrative DNA Technologies (Coralville, IA) and used for transfection with control pssXD-I or pssXD-II containing the c-raf DNase sequence. Signals were detected in RNA samples isolated from transfected A549 cells. 2 μg of total RNA was pretreated with RNase A to exclude any possible non-specific hybridization to RNA, and treated for 30 minutes at 37°C in the presence and absence of S1 nuclease. Subsequently, samples were loaded onto a Hybond-N+ membrane (Amersham Pharmacia Biotec, Piscataway, NJ) and fixed by UV irradiation for 3 minutes. Hybridization and signal detection were performed using the North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Pierce, Rockford, IL). Figure 11 shows that only cells transfected with pssXD-II showed a positive signal and no detectable signal was observed in the presence of S1 nuclease since ssDNase was specifically degraded by S1 nuclease.

To determine whether single-stranded DNase expressed in A549 cells altered c-raf mRNA levels, quantitative RT-PCR was performed. c-raf mRNA was quantified by RT-PCR as described by Li et al. (Gene Therapy, 7, 321-328 (2000)) with some modifications. Briefly, 1 μg of total RNA was reverse transcribed using a reverse transcription system (Promega Corporation, Madison, WI). The resulting cDNA fractions were used as templates for PCR amplification. Forty cycles of PCR (95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 60 seconds) were performed using specific primers. The specific primer sequences used are as follows: 1) c-raf primer: 5′-TCAGAGAAGCTCTGCTAAG-3′ and 5′-CAATGCACTGGACACCTTA-3′; 2) actin primer: 5′-ACCTTCTACAATGAGCTGCG-3′ and 5′-GCTTGCTGATCCACATCTGC -3'. Actin was used as a housekeeping gene control. Total RNA isolated from cells transfected with control pssXD-I or pssXD-II containing the c-raf DNase sequence was reverse transcribed and PCR amplified using a pair of c-raf-specific primers. PCR amplification of actin mRNA was used as a control to normalize loading between different samples. As shown in Figure 12, a significant reduction (approximately 70-80%) of c-raf mRNA was detected in cells transfected with pssXD-II (lane 2) compared to the control (lane 1).

c-raf protein levels in A549 cells transfected with pssXD-I or pssXD-II were determined by Western blot analysis. 30 μg of cell extracts were electrophoresed on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins were electrotransferred onto Hybond ECL membranes (AmershamPharmacia Biotec, Piscataway, NJ) using a miniature transblotting electrophoretic transfer cell according to the manufacturer's instructions (BioRad Laboratories, Hercules, CA). The membrane was subsequently blocked in a buffer containing 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween-20, and 5% skim milk, and then incubated with primary and HRP-conjugated secondary antibodies for 45 minute. Anti-c-raf polyclonal antibody (anti-raf1) and anti-actin monoclonal antibody (Ab-1) were purchased from Calbiochem-NovaBiochem (Sann Diego, CA). Anti-poly-ADP ribose polymerase (PARP) monoclonal antibody (IgG1, C-2-10) was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). Proteins were visualized using the SuperSignal West Pico Chemiluminescence Substrate Kit (Pierce, Rockford, IL). As shown in Figure 13, the level of c-raf protein in pssXD-I transfected control cells (lane 2) was similar to that in untransfected cells (lane 3). However, cells transfected with pssXD-II expressing c-raf DNase (lane 1) had lower c-raf protein levels (approximately 20-30%) compared to controls.

To determine whether expression of c-raf DNase induces apoptosis in A549 cells, two standard apoptosis assays, genomic DNA cleavage and PARP cleavage, were performed. Genomic DNA cleavage was determined using the LM-PCR Ladder Assay Kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer's instructions. Briefly, 0.5 μg of genomic DNA was ligated with adapters provided by Clontech Laboratories, Inc. using T4 DNA ligase overnight at 15°C. Adapter-ligated DNA fractions were used as templates for LM-PCR. 25 cycles of PCR (95°C, 1 min and 72°C, 3 min) with extension at 72°C for 15 min were performed. Genomic DNA isolated from cells transiently transfected with pssXD-I (control) or pssXD-II (DNase) was ligated to specific adapters. Subsequently, LM-PCR was performed using c-raf primers and specific primers. As shown in Figure 14, fragmentation was observed in cells transfected with pssXD-II (lane 1) compared with cells transfected with the control plasmid pssXD-I (lane 2), or untransfected cells (lane 3). Genomic DNA was significantly increased. These results indicate that the increase in fragmented genomic DNA is a consequence of DNA cleavage caused by inhibition of altered c-raf gene expression in the presence of c-raf DNase.

Another apoptosis assay, the PARP cleavage assay, was performed using Western blot analysis. The amount of full-length PARP was reduced in cells transfected with pssXD-II (lane 1 ) compared to controls (lane 2-3) ( FIG. 15 ), again suggesting that inhibition of c-raf gene expression induces apoptosis. Similar amounts of protein were loaded per lane (lanes 1-3) when assayed in the presence of actin.

* * * * * * * * * * * * * * *

The experiments described above demonstrate the success of in vivo reduction of c-raf kinase expression using a multi-step reaction of eukaryotic RT reactions and various cDNA priming reactions to generate ssDNA in vitro and in vivo. Those skilled in the art will recognize that the invention is not limited to this particular embodiment. For example, it is recognized that a number of nucleic acid sequences can be used depending on the particular target and/or mode of SOI inhibitory action. Likewise, the SOI may be located at one or both positions, for example between the IR and/or between the PBS and the 3' end of the IR. Likewise, an SOI may or may not include a DNase sequence depending on the particular target and/or mode of action of the SOI and/or the DNase sequence. Those skilled in the art having the benefit of this description will recognize that any desired therapeutic effect can be produced by transfecting a suitable SOI into eukaryotic cells using the vector system of the present invention. By way of example and not limitation, the following inhibitory nucleic acid sequences are known in the art and can be used as SOIs to alter gene expression according to the invention:

Sequences that serve as antisense oligonucleotides encoding one or more RNA molecules of one of several dopamine receptors for Parkinson's disease treatment. The antisense oligonucleotide specifically binds to the expression control sequence of this type of RNA molecule, thereby selectively regulating the expression of one or more dopamine receptor subtypes, and alleviating pathological symptoms related to its expression;

Sequences that inhibit expression of KSHV virion protein 26 include sequences that function as antisense and/or triplex oligonucleotides for the treatment of Karposi's syndrome as described in US Patent No. 5,856,903.

Oligonucleotides for controlling expression of IL-8 and/or IL-8 receptor to control tumor growth, metastasis and/or angiogenesis in tumors as described in U.S. Patent No. 5,856,903;

Oligonucleotides having a nucleotide base sequence that specifically hybridizes to selected sequences of cytomegalovirus DNA or RNA, specifically, targeting cytomegalovirus DNA encoding IE1, IE2, or DNA polymerase proteins or RNA sequence. Oligonucleotides having from about 5 to about 50 nucleobase units as described in US Pat. No. 5,442,049 are preferred.

Can specifically hybridize with RNA or DNA from a gene corresponding to one of the herpes simplex virus type 1 reading frames UL5, UL8, UL9, UL20, UL27, UL29, UL30, UL42, UL52 and IE175 and contain in identity and quantity An oligonucleotide of nucleotide units sufficient to effect this specific hybridization. Oligonucleotides that specifically hybridize to translation initiation sites, coding regions or 5' untranslated regions are preferred. Oligonucleotides designed to interact with cells from herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus, human herpesvirus type 6, Epstein Barr virus (EBV), or varicella zoster DNA, or preferably RNA, of one of the herpesviruses (VZV) hybridizes specifically. Such oligonucleotides are conveniently and desirably provided as pharmaceutical compositions in a pharmaceutically acceptable carrier, as described in US Patent No. 5,514,577. Those skilled in the art will recognize that specific reading frames described for herpes simplex virus type 1 may find counterparts in other viruses as indicated. It is therefore believed that each of herpes simplex virus type 2 (HSV-2), cytomegalovirus, human herpesvirus 6, Epstein Barr virus, and varicella-zoster virus have many similar reading frames encoding similar functional proteins . Accordingly, the present invention relates to antisense oligonucleotide therapy wherein said oligonucleotides are directed against any of the above viruses, or indeed against any similar virus hereafter known to have one or more such similar reading frames. For the convenience of the present invention, all such viruses are referred to as herpes viruses.

Antisense oligonucleotides of proto-oncogenes, and in particular the c-myb gene, are useful as antineoplastic agents and immunosuppressants, as described in US Pat. No. 5,098,890.

Antisense oligonucleotides against ICAM-1 gene expression in interleukin-1 (stimulated cells are used as anti-inflammatory agents for various inflammatory diseases or diseases with inflammatory components, such as asthma, rheumatoid arthritis, Allograft rejection, inflammatory bowel disease, various dermatological conditions, and psoriasis are active. In addition, inhibitors of ICAM-1, VCAM-1, and ELAM-1 are active in colds caused by rhinovirus infection, AIDS , effective in the treatment of Kaposi's sarcoma and some cancers and their metastases, as described in U.S. Patent No. 5,843,738. Likewise, International Application No. PCT/US90/02357 discloses the The DNA sequence of the endothelial cell adhesion molecule (ELAM) of VCAM-1b. The oligonucleotides designated ISIS 1570 and ISIS 2302 are particularly contemplated as sequences of interest in the methods of the invention for reducing the metastatic potential of target cells.

Protein-binding oligonucleotides (aptamers) that specifically bind target molecules in host cells, such as proteins, and in particular thrombin, as described in US Patent No. 5,840,867. These non-oligonucleotide target molecules can bind nucleic acids (Blackwell, T.K., et al., Science, 250, 1104-1110 (1990); Blackwell, T.K., et al., Science, 250, 1149-1152 (1990); Turek, C. and L. Gold, Science, 249, 505-510 (1990); Joyce, G.F., Genes, 82, 83-87 (1989)), specifically control the biological activity of this protein.

Although described with reference to the figures and specific examples provided herein, those skilled in the art will recognize that certain changes may be made in the specific elements provided herein without altering the ability of these elements to function to achieve their intended respective results. Way. For example, the cassettes described herein are described as containing three genetic elements, a sequence of interest, a primer binding site, and a tandem inverted repeat, that when transfected into a target cell with a reverse transcriptase gene under the control of an appropriate promoter Inhibitory nucleic acid sequences described herein are produced. However, those skilled in the art will recognize that, for example, the mouse Moloney leukemia virus reverse transcriptase gene used as the sequence cassette reverse transcriptase gene may be replaced with other reverse transcriptase genes (reversal from human immunodeficiency virus The transcriptase gene is one such gene described above) and it may also be beneficial to use a promoter other than the CMV promoter described herein. As noted above, the resulting stem-loop intermediate may or may not include restriction endonuclease sites and it may be beneficial to address its susceptibility to denaturation depending on the particular sequence of interest desired to be generated from the intermediate. All such changes and other changes which are obvious to those skilled in the art may be made by modification of this description without departing from the spirit of the invention are intended to be within the scope of the following claims.

Table 1 寡聚脱氧核苷酸(ODN's)名称：5′-N/M(接头)2-H/N5′AGCTTGGTCGGCGGCCTTGAAGAGCGGCCGCACTCACGATAGAGTGGGAGATGGGCGCGAGAAAGTGCGGCCGCTCTTCAAGGCCGCCGACCTTAATTAAGTCAGCGGGGGATCCTTTTTGGGGGCTCGTCCGGGATCGGGAGACCCCT-3′名称：3′-N/M(接头)2-H/N5′GGCCAGGGGTCTCCCGATCCCGGACGAGCCCCCAAAAAGGATCCCCCGCTGACTTAATTAAGGTCGGCGGCCTTGAAGAGCGGCCGCACTTTCTCGCGCCCATCTCCCACTCTATCGTGAGTGCGGCCGCTCTTCAAGGCCGCCGACCA-3 'Name: 5'-polyNM-gag linker-(Pleio)-DNa5e-1023-B/P5'-GATGTAAGTCGTTGTAGCTAGCCTCCCCTG-3'Name: 3'-polyNM-gag linker-(Pleio)-DNAse-1023-B/P5' -GAT CCA GGG GA GGC TAG CTA CAA CGA CTT ACA TCA T-3′ name: 5′-polyNM-gag linker-(hras)-DNAse-1023-B/P5′-GGTGGGCGCCTCGTTGTAGCTAGCCTCGGTGTGGG-3′ name: 3′-polyNM -gag linker-(hras)-DNAse-1023-B/P5'-GATCCCCACACCGAGGCTAGCTACAACGAGGCGCCCACCAT-3' name: 5'-polyNM-gag linker-(rafK)-DNAse-1023-B/P5'-AATGCATGTCTCGTTGTAGCTAGCCCAGGCGGGA-3 name: 3 '-polyNM-gag linker-(rafK)-DNAse-1023-B/P5'-GATCTCCCGCCTGGGCTAGCTACAACGAGACATGCATTAT-3' Name: 5'-polyN/M-gag linker-(tat-SIV)-DNAse-1023-B/P5'-AGATGGAGACTCGTTGTAGCTAGCCCCCTTGAGGGCAGATTGGCGCCCGAACAGGGACTTGAAGGA-3'name:3'-polyN/M-gaglinker-(tat-SIV)-DNAse-1023-B/P5'-GATCTCCTTCAAGTCCCTGTTCGGGCGCCAATCTGCCCTCAAGGGGGCTAGCXTACAACGAGTCTCCATCTAT-3'linker name: 5H-(ind -CCG GAT CTA GAC CGC AAG CTT CAC CGC-3' Name: 3'-(linker)2-Hind/Xb25'-GGT GAA GCT TGC GGT CTA GAT-3' ODN-PMMV(+) 129 bases (#23) 5′-CTAGGTCGGCGGCCGCGAAGATTGGTGCGCACACACACAACGCGCACCAATCTTCGCGGCCGCCGACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGCC-3′ ODN-PMMV(-) 121 bases (#24) 5′-CTGGGCAGGGGTCTCCCGATCCCGGACGAGCCCCCAAATGAAAGACCCCCGCTGACGGGTCGGCGGCCGCGAAGATTGGTGCGCGTTGTGTGTGTGCGCACCAATCTTCGCGGCCGCCGAC-3′ ODN-Test (+) 57 bases (#38) 5′-GGCCGGAAGATTGGGGCGCCAAAGAGTAACTTCTCAAAGGCACGCGCCCCAATCTTCC-3′ ODN-Test(-) 57 bases (#39) 5′-GGCCGGAAGATTGGGGCGCGTGCCTTTGAGAGTTACTCTTTGGCGCCCCAATCTTCC-3′ ODN-Telo (+) 92 bases (#40) 5′-GGCCGGAAGATTGGGGCGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGCGCCCCAATCTTCC-3′ ODN-Telo(-) 92 bases (#41) 5′-GGCCGGAAGATTGGGGCGCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACGCCCCAATCTTCC-3′ ODN-XB(+)51 bases 5′-GGCCTTGAAGAGCGGCCGCACTAACACCACCACAGTGCGGCCGCTCTTCAA-3′ ODN-XB(-)51 bases 5′-GGCCTTGAAGAGCGGCCGCACTGTGGTGGTGTTAGTGCGGCCGCTCTTCAA-3′ ODN-RT(+) 32 bases (#13) 5′-GGGATCAGGAGCTCAGATCATGGGACCAATGG-3′ ODN-RT(-) 24 bases (#12) 5′-CTTGTGCACAAGCTTTGCAGGTCT-3′ ODN-N>S(+)18 bases (#25) 5′-CTAGCGGCAAGCGTAGCT-3′ ODN-N>S(-)10 bases (#26) 5′-ACGCTTGCCG-3′ ODN-Mbo(+) 30 bases (#16) 5′-CAATTAAGGAAAGCTTTGAAAAATTATGTC-3′ ODN-Mbo(-) 27 bases (#33) 5′-TAATGGCCCGGGCATAGTCGGGTAGGG-3′ ODN-HisPro(+) 43 bases (#36) 5′-AGCTGGATCCCCCGCTCCCCACCACCACCACCACCCTGCCCCT-3′ ODN-HisPro(-) 42 bases (#37) 5′-AGCAGGGGCAGGGTGGTGGTGGTGGTGGGGAGCGGGGGATCC-3′ ODN-Rep(+)121 bases 5′-ATATCTATTAATTTTGGCAAATCATAGCGGTTATGCTGACTCAGGTGAATGCCGCGATAATTTTCAGATTGCAATCTTTCATCAATGAATTTCAGTGATGAATTGCCAAGATTGATGTTGC-3′ ODN-Rep(-)111 bases 5′-GACGAGATCTCCTCCAGGAATTCTCGAGAATTCGGATCCCCCGCTCCCCACCACCACCACCACCACCCTGCCCCGCGGATGAAAAATTATGTGAGCAACATCAATCTTGGC-3′

1. Name: 3′-RT/Mol-HindIII (24-mer)

Sequence: 5′-CTT GTG CAC AAG CTT TGC AGG TCT-3′

2Name: 5′-RT/Mol-Sac I (32-mer)

Sequence: 5′-GGG ATC AGG AGC TCA GAT CAT GGG ACC AAT GG-3′

3. Name: 5′-Mbo II-Hind III (30-mer)

Sequence: 5′-CAA TTA AGG AAA GCT TTG AAA AAT TAT GTC-3′

4. Name: 5′-RT-Not-Mbo-linker (129-mer)

Sequence: 5′-CTA GGT CGG CGG CCG CGA AGA TTG GTG CGC ACA CACACA ACG CGC ACC AAT CTT CGC GGC CGC CGA CCC GTC AGC GGGGGT CTT TCA TTT GGG GGC TCG TCC GGG ATC AG GGG AGC CCC G

5. Name: 3′-RT-Not-Mbo-linker (121-mer)

Sequence: 5′-CT GGG CAG GGG TCT CCC GAT CCC GGA CGA GCC CCCAAA TGA AAG ACC CCC GCT GAC GGG TCG GCG GCC GCG AAG ATTGGT GCG CGT TGT GTG TGT GCG CAC CAA TCT TCG CGG CCG -' CCGAC

6. Name: 5′-Nhe-Sac-linker (18-mer)

Sequence: 5′-CTA GCG GCA AGC GTA GCT-3′

7. Name: 3′-Nhe-Sac-linker (10-mer)

Sequence: 5′-ACG CTT GCC G-3′

8. Name: 3′-Mbo II-Xba I (27-mer)

Sequence: 5′-TAA TGG CCC GGG CAT AGT CGG GTA GGG-3′

9. Name: 5′-Hind-linker-Histag (43-mer)

Sequence: 5′-A GCT GGA TCC CCC GCT CCC CAC CAC CAC CAC CACCCT GCC CCT-3′

10. Name: 3′-Hind-linker-Histag (42-mer)

Sequence: 5′-AGC AGG GGC AGG GTG GTG GTG GTG GTG GGG AGCGGG GGA TCC-3’

11. Name: 5′-Not-Linker-Test 1 (57-mer)

Sequence: 5′-G GCC GGA AGA TTG GGG CGC CAA AGA GTA ACT CTCAAA GGC ACG CGC CCC AAT CTT CC-3′

12. Name: 3′-Not-Linker-Test 1 (57-mer)

Sequence: 5′-GGC CGG AAG ATT GGG GCG CGT GCC TTT GAG AGT TACTCT TTG GCG CCC CAA TCT TCC-3′

13. Name: 5′-Not-Mbo-linker-telo (92-mer)

Sequence: 5′-GGC CGG AAG ATT GGG GCG TTA GGG TTA GGG TTA GGGTTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGGCGC CCC AAT CTT CC-3′

14. Name: 3′-Not-Mbo-linker-telo (92-mer)

Sequence: 5′-GGC CGG AAG ATT GGG GCG CCC TAA CCC TAA CCC TAACCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAACGC CCC AAT CTT CC-3′

15.5'-SL-linker-Fok1-RT (111-mer)

Sequence: 5′-CTA GTC GGA TGC GGC CGC TGC ACA ACA ACA CAC AACACA GCG GCC GCA TCC GAT CAG CGG GGG TCT TTC ATT TGG GGGCTC GTC CGG ATC GGG AGA CCC CTG CCC AGC GCC-3′

16.3'-SL-linker-Fok1-RT (103-mer)

Sequence: 5′-CTG GGC AGG GGT CIC CCG ATC CGG ACG AGC CCC CAAATG AAA GAC CCC CGC TGA TCG GAT GCG GCC GCT GTG TTG rrr GTTGTT GTG CAG CGG CCG CAT CCG A-3′

17. Name: XmaI-BglII-Terminator 1

Sequence: 5'-CCGGATCTAGACCGCAAGCTTCATTTAAA-3'

18. Name: XmaI-BrlII-Terminator 2

Sequence: 5'-GATCTTTAAATGAAGCTTGCGGTCTCGAT-3'

Sequence Listing

<110> Citogenex (INGENE, INC.)

<120> Altering gene expression with ssDNA produced in vivo

<130> INGA, 004/CIP/PCT

<140>PCT/US00/27381

<141>2000-10-04

<150>PCT/US99/23936

<151>1999-10-12

<160>34

<170>PatentIn Ver.2.1

<210>1

<211>149

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>1

agcttggtcg gcggccttga agagcggccg cactcacgat agagtggggag atgggcgcga 60

gaaagtgcgg ccgctcttca aggccgccga ccttaattaa gtcagcgggg gatccttttt 120

gggggctcgt ccgggatcgg gagacccct 149

<210>2

<211>149

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>2

ggccagggggt ctcccgatcc cggacgagcc cccaaaaagg atcccccgct gacttaatta 60

aggtcggcgg ccttgaagag cggccgcact ttctcgcgcc catctcccac tctatcgtga 120

gtgcggccgc tcttcaaggc cgccgacca 149

<210>3

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>3

gatgtaagtc gttgtagcta gcctcccctg 30

<210>4

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>4

gatccagggg aggctagcta caacgactta catcat 36

<210>5

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>5

ggtgggcgcc tcgttgtagc tagcctcggt gtggg 35

<210>6

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>6

gatccccaca ccgaggctag ctacaacgag gcgccccacca t 41

<210>7

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>7

aatgcatgtc tcgttgtagc tagcccaggc ggga 34

<210>8

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>8

gatctcccgc ctgggctagc tacaacgaga catgcattat 40

<210>9

<211>66

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>9

agatggagac tcgttgtagc tagccccctt gagggcagat tggcgcccga acagggactt 60

gaagga 66

<210>10

<211>72

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>10

gatctccttc aagtccctgt tcgggcgcca atctgccctc aagggggcta gctacaacga 60

gtctccatct at 72

<210>11

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>11

ccggatctag accgcaagct tcaccgc 27

<210>12

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>12

ggtgaagctt gcggtctaga t 21

<210>13

<211>129

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>13

ctaggtcggc ggccgcgaag attggtgcgc acacacaa cgcgcaccaa tcttcgcggc 60

cgccgacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg gagacccctg 120

cccagggcc 129

<210>14

<211>121

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>14

ctgggcaggg gtctcccgat cccggacgag cccccaaatg aaagacccccc gctgacgggt 60

cggcggccgc gaagattggt gcgcgttgtg tgtgtgcgca ccaatcttcg cggccgccga 120

c 121

<210>15

<211>57

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>15

ggccggaaga ttggggcgcc aaagagtaac tctcaaaggc acgcgcccca atcttcc 57

<210>16

<211>57

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>16

ggccggaaga ttggggcgcg tgcctttgag agttactctt tggcgcccca atcttcc 57

<210>17

<211>92

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>17

ggccggaaga ttggggcgtt agggttaggg ttagggttag ggttagggtt agggttaggg 60

ttagggttag ggttagggcg ccccaatctt cc 92

<210>18

<211>92

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>18

ggccggaaga ttggggcgcc ctaaccctaa ccctaaccct aaccctaacc ctaaccctaa 60

ccctaaccct aaccctaacg ccccaatctt cc 92

<210>19

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>19

ggccttgaag agcggccgca ctaacaccac cacagtgcgg ccgctcttca a 51

<210>20

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>20

ggccttgaag agcggccgca ctgtggtggt gttagtgcgg ccgctcttca a 51

<210>21

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>21

gggatcagga gctcagatca tgggaccaat gg 32

<210>22

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>22

cttgtgcaca agctttgcag gtct 24

<210>23

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>23

ctagcggcaa gcgtagct 18

<210>24

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>24

acgcttgccg 10

<210>25

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>25

caattaagga aagctttgaa aaattatgtc 30

<210>26

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>26

taatggcccg ggcatagtcg ggtaggg 27

<210>27

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>27

agctggatcc cccgctcccc accacccacca ccaccctgcc cct 43

<210>28

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>28

agcaggggca gggtggtggt ggtggtgggg agcgggggat cc 42

<210>29

<211>121

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>29

atatctatta attttggcaa atcatagcgg ttatgctgac tcaggtgaat gccgcgataa 60

ttttcagatt gcaatctttc atcaatgaat ttcagtgatg aattgccaag attgatgttg 120

c 121

<210>30

<211>111

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<400>30

gacgagatct cctccaggaa ttctcgagaa ttcggatccc ccgctcccca ccaccaccac 60

caccaccctg ccccgcggat gaaaaattat gtgagcaaca tcaatcttgg c 111

<210>31

<211>111

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: linkers

<400>31

ctagtcggat caggccgctg cacaacaaca cacaacacag cggccgcatc cgatcagcgg 60

gggtctttca tttgggggct cgtccggatc gggagacccc tgcccagcgc c 111

<210>32

<211>103

<212>DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: linkers

<400>32

ctgggcaggg gtctcccgat ccggacgagc ccccaaatga aagaccccg ctgatcggat 60

gcggccgctg tgttgtttgt tgttgtgcag cggccgcatc cga 103

<210>33

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>33

ccggatctag accgcaagct tcatttaaa 29

<210>34

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>34

gatctttaaa tgaagcttgc ggtctcgat 29

Claims (7)

1. A method for changing the expression of an endogenous nucleic acid target sequence in a target cell, comprising the steps of: A sequence cassette containing the target sequence and its flanking inverted tandem repeat sequence and 3' primer binding site PBS is introduced into the target cell. The target sequence contains the following nucleic acid sequence, which is designed to be reverse-transcribed to produce an endogenous Nucleic acid sequence of sexual nucleic acid sequence; reverse transcribing the mRNA transcript of the cassette starting from PBS to release the single-stranded cDNA transcript in the cell; and The cDNA transcript is bound to an endogenous nucleic acid target sequence to alter expression of the target sequence. 2. The method of claim 1, further comprising transfecting a reverse transcriptase/RNase gene into said target cells. 3. The method of claim 1, further comprising linearizing transcripts of the sequence of interest. 4. The method of claim 3, wherein when the cDNA transcript of the target sequence forms a stem-loop intermediate through the Watson-Crick base pairing of the inverted tandem repeat, the transcript of the target sequence passes through the inverted tandem repeat The introduced restriction endonuclease sites are linearized. 5. The method of claim 2, further comprising inducibly initiating transcription of the reverse transcriptase gene. 6. The method of claim 5, wherein a eukaryotic promoter is used to initiate transcription of the reverse transcriptase gene. 7. A target cell wherein expression of a nucleic acid target sequence has been altered according to the method of claim 1.

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