KR20020059608A - Altering Gene Expression with ssDNA Produced in vivo - Google Patents

Abstract

The present invention relates to a method of changing the expression of a target nucleic acid sequence in a target cell by generating a single-stranded cDNA (ss-cDNA) in vivo of the target cell. The target cell is transfected with a cassette comprising a target sequence, an inverted tandem repeat sequence, and a primer binding site located 3 ′ of the inverted tandem repeat sequence. Transcription of the cassette by the target cell produces an RNA template, which is reverse transcribed to produce ss-cDNA of a particular sequence. Reverse transcriptase / RNase H coding genes can also be transfected into target cells. The ss-cDNA is modified to remove all vector contiguous sequences by using the "stem-loop" structure of the ss-cDNA, where the stem-loop structure is folded into a double-folded ss-cDNA by itself due to an inverted tandem repeat sequence. Stranded DNA stems formed. The double-stranded stem contains one or more restriction enzyme recognition sites and the loop remaining with ssDNA contains the desired sequence, which can be any desired nucleotide. Due to this design, the double-stranded stem of the stem-loop intermediate is cleaved with the desired corresponding restriction enzyme (s) and the loop portion (ie, the desired sequence) is then released as a linearized single-stranded DNA fragment. This released (or truncated) ssDNA fragment, if present, contains minimal sequence information upstream 5 'or downstream 3' from the double stranded stem. The resulting ssDNA binds to an endogenous target nucleic acid sequence, thereby causing duplication or triplet nucleic acid binding, site-directed mutagenesis, impairment of cellular function by binding to specific cellular proteins, and RNA splicing functions for therapeutic purposes. Damage or the like is used to inactivate the gene to change the expression of the sequence.

Description

Altering Gene Expression with ssDNA Produced in vivo

The present invention relates to alterations in gene expression using stable DNA constructs (called for convenience cassettes) containing nucleic acid sequences incorporated for use as templates for subsequent nucleic acid sequence generation in prokaryotic or eukaryotic host cells, and eukaryotic host cells. To a system that expresses the sequence so that there is no contiguous sequence within (or to include the least contiguous sequence). The construct, or cassette, is a stem-loop that functions to express a sequence (also called a target sequence) in vivo as a single stranded DNA (ssDNA) sequence that changes the expression of the target gene by binding to or interacting with the target gene. Inverted tandem repeats forming the stem structure of the intermediate. The expression system of the present invention is capable of removing most or all of the adjacent plasmid (or other vector) sequences from ssDNA by formation of stem-loops and termination of reverse transcription reactions by stems thereafter, or by cleavage of stem-loop intermediates. Remove The ssDNA generated by this method is designed to be complementary to any endogenous nucleic acid sequence target and / or to bind to the sequence target, thereby targeting any desired gene.

Antisense gene therapies have been successfully used in various fields for down regulation of gene function (Jain, KK, Handbook of Gene Therapy, New York: Hofgrefe & Huber Publishing (1998)). However, to date, such therapies include a number of disadvantages and limiting factors that reduce the usefulness of these types of therapies, including the short half-life of the antisense molecule, the nonspecificity of the effects, the uncertainty of the antisense sequence of action, and the potential toxic effects in animal studies. Is serious). For example, antisense oligonucleotides (ODNs) and analogs thereof should be administered intravenously, which is a problem in cellular uptake and distribution (Cossum, PA, et al., Disposition of the ¹⁴ C-labeled phosphorothioate oligonucleotide ISIS 2105 after intravenous administration to rats, 267 J. Pharmacol.Exp. Ther. 1181-1190 (1993), Sands, H., et al., Biodistribution and metabolism of internally ³ H-labeled oligonucleotides.II. 3 ', 5'-blocked oligonucleotides, 47 Mol. Pharmacol. 636-646 (1995), as well as problems with toxicity due to high blood concentrations (Henry, SP, et al., Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4- week study in CD-1 mice, 7 Antisense Nucleic Acid Drug Dev. 473-481 (1997), Henry, SP, et al., Comparison of the toxicity profiles of ISIS 1082 and ISIS 2105, phosphorothioate oligonucleotides, following subacute intradermal administration in Sprague-Dawley rates, 116 Toxicology 77-88 (1997) The.

The antisense ODN analogs used to date for most antisense therapies are phosphorothioates or methylphosphonates. However, phosphorothioate ODN tends to nonspecifically bind to serum and intracellular proteins (Crooke, ST, et al., Pharmocokinetic properties of several novel oligonucleotide analogs in mice, 227 J. Pharmacol. Exp. Ther. 923 -937 (1996), Gao, WY, et al., Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology, 41 Mol.Pharmacol. 223-229 (1992)), RNase H activity at high concentrations (Crooke, ST, et al., Kinetic characteristic of Escherichia coli Rnase H: Cleavage of various antisense oligonucleotide-RNA duplexes, 312 Biochem. J. 599-608 (1995)). Phosphorothioate ODN has a lower Tm for RNA (average 0.5 ° C per base pair) than for DNA (Crooke, S.T. and B. LeBleu, Antisense research and application, Boca Raton: CRC Press (1993)). Due to this low Tm, phosphorothioate ODN should typically be longer than phosphodiester DNA for effective binding. However, increasing ODN length can cause loss of hybridization specificity (Toulme, JJ, et al., Antisense technology: A practical approach, in C. Lichtenstein and W. Nellen (Eds.), New York: IRL Press, pp 39-74 (1997). In addition, methylphosphonate ODN does not activate RNase H enzyme activity (Maher, LJ, et al., Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation, 245 Science 725-730 (1989), Miller, PS Oligodeoxynucleotides: Antisense inhibitors of gene expression, in JS Cohen (Ed.), Boca Raton: CRC Press, p. 79 (1989)), are rapidly removed (Chen, TL, etal., Disposition and metabolism of oligodeoxynucleoside methylphosphonate following a single iv injection in mice., 18 Drug Metab. Dispos. 815-818 (1990)).

Another approach to gene therapy is to administer molecules with catalytic activity that inhibit the gene and / or its transcription products. Ribozymes contain only RNA molecules and catalyze the cleavage of specific mRNA sequences, and are probably more effective than antisense ODN due to their catalytic capacity (Woolf, TM, To cleave or not to cleave: Ribozymes and antisense). , 5 Antisense Res. Dev. 227-232 (1995)). Ribozymes have been used as inhibitors of gene expression and viral replication (Jain, supra) (1998). Unlike antisense ODN, ribozymes can be delivered internally (eg, using viral vectors) or externally. However, ribozymes are limited in their stability because they are degraded by RNase in vivo (Jain, supra) (1998).

Several small-stranded single-stranded DNA catalyzes the cleavage of RNA, and has recently been demonstrated using in vitro screening (Breaker, RR, Catalytic DNA: In training and seeking employment, 17 Nature Biotechnology 422-423 (1999)). This has led to the prospect of providing targeted activity that inhibits certain genes. In the patent and scientific literature, many of these short have been found to have catalytic activity, including so-called "10-23 DNA enzymes" and other ssDNA sequences that act as, for example, copper-dependent DNA ligase and calcium-dependent DNA kinases. Deoxynucleic acid sequences are described (Breaker, RR and GF Joyce, 1 Chem. Biol. 223-229 (1994); Cuenoud, B. and JW Szostak, 375 Nature 611-613 (1995), Santoro, SW and GF Joyce, 94 Proc. Natl. Acad. Sci. USA 4262-4266 (1997); Faulhammer and M. Famulok, 269 J. Molec. Bio. 188-203 (1997); Carmi, N, et al., 95 Proc Natl.Acad. Sci. USA (1998); Li, Y. and RR Breaker, 96 Proc. Natl. Acad. Sci. USA 2746-2751 (1999); and US Pat. Nos. 5,807,718 and 5,910,408). The catalytic efficiency of such sequences has been demonstrated to cleave the mRNA target to 10 ⁹ m ⁻¹ / min ⁻¹ in the presence of divalent magnesium, thus providing an opportunity to target and destroy substrate molecules (eg See, for example, RR Breaker, supra (1999). Although it appears in the art to recognize the potential of using this enzyme activity for therapeutic purposes, so far known systems are available that can be used to generate target-specific enzymatic nucleic acid sequences that exhibit therapeutic effects in vivo. none.

Thus, there is no system that can utilize the efficient catalytic activity of these enzymatic nucleic acid sequences for changing gene expression. It is therefore an object of the present invention to provide a DNA construct capable of synthesizing an ssDNA containing a sequence that specifically cleaves an mRNA target in vivo and changes the expression of the gene producing the target mRNA.

Since secondary structure folding may be essential for the catalytic function of the enzymatic sequence of ssDNA, another object of the present invention is to use a target nucleic acid for use in altering the expression of a gene comprising the target nucleic acid. Provided are methods and DNA constructs for generating ssDNA comprising DNA enzyme sequences of any desired nucleotide sequence without unwanted intervening or contiguous nucleotide bases in eukaryotic cells to preserve the enzymatic function of the inhibiting ssDNA. It is.

Another object of the present invention is a method for the production of ssDNA containing DNA enzyme sequences in eukaryotic cells to overcome the serious problems encountered with the use of standard oligonucleotide delivery methods for therapeutic purposes, and DNA constructs that can be used in such methods. To provide.

Another object of the present invention is to inhibit, for example, binding to mRNA in an antisense manner to downregulate the desired gene product or viral gene product, or inhibit specific cellular function through binding to a protein that recognizes a nucleic acid sequence. Provided are methods and DNA constructs for generating ssDNA for any in vivo nucleotide sequence that functions as, but is not limited to, a sexual nucleic acid.

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

Another object of the present invention is to produce ssDNA in eukaryotic cells for the purpose of interfering with one or more cellular functions.

It is another object of the present invention that the ssDNA oligonucleotide binds to and / or binds a nucleic acid that exhibits the function of various cells depending on the catalysis of the protein or on nucleic acid protein interactions such as transcription, translation and DNA replication. Or to provide a method and DNA construct for generating ssDNA whose secondary structure is designed to inhibit or activate such function.

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

Another object of the present invention is to provide methods and DNA constructs for producing ssDNA of precisely defined composition which induce site-specific insertion into the genome for therapeutic purposes.

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

Another object of the present invention is to provide an mRNA target for transfection into eukaryotic cells to overcome obstacles to direct administration of ssDNA by lipofection, direct inhalation of cells and / or microinjection. It is to provide a method for producing ssDNA in vivo comprising a sequence exhibiting catalytic activity for, and the DNA expression used in such a method.

It is another object of the present invention to provide all the enzymatic functions necessary for in vivo production of ssDNA containing sequences having enzymatic activity for selected target mRNAs in single or dual plasmid expression systems.

Another object of the present invention is to provide a method for delivering an inhibitory nucleic acid sequence comprising a sequence having enzymatic activity to a target cell in a manner that produces a therapeutic effect, and a pharmaceutically acceptable composition.

The above list listing the objects of the present invention is not a list of all the objects of the present invention. There are many other cellular functions that are mediated by the genome of the cell that are not mentioned herein for brevity and practicality but that can be regulated by in vivo ssDNA production. Exonucleases, for example, degrade ssDNA much better than double-stranded DNA (dsDNA). Accordingly, another object of the present invention is to provide ssDNA constructs that are not degraded by natural exonucleases such as dsDNA in cells, and methods of producing such constructs in vivo. From this fact, it can be seen that the above list listing some of the objects of the present invention is provided for illustration, and is not intended to limit the scope of the present invention.

These objectives include introducing a cassette comprising a target sequence, an inverted tandem repeat sequence located at both ends thereof, and a 3 ′ primer binding site (PBS) into a target cell, and reverse transcription of the mRNA transcript of the cassette from PBS to mono- Achieved by a method of altering the expression of an endogenous nucleic acid target sequence in a target cell, comprising releasing the strand cDNA transcript. The sequence of interest includes nucleic acid sequences that, when reverse transcribed to alter the expression of a target sequence, produce a nucleic acid sequence that binds to an endogenous target nucleic acid sequence.

Some embodiments of the invention are described in the following figures.

1 is a schematic diagram of ss-cDNA production 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.

3 is a schematic of a pssXA plasmid comprising the first component of the first embodiment in an expression system of the present invention. Reverse transcriptase (RT) and MboII genes were subcloned into mammalian expression vector pBK-RSV (Stratagene) to make pssXA and expressed as a single polypeptide. RT and MboII domains are separated by histidine-rich linkers.

4A and 4B are schematic representations (p. 4A) of a pssXB plasmid comprising the second component of the first embodiment in the expression system of the present invention comprising the sequence of interest, (1) MoMuLV reverse transcriptase promoter region, (2) Insert region regions of two NotI sites, one PacI site and one BamHI site for subcloning the DNA sequence of interest, and (3) the IR-L and IR-R, and pssXB plasmids, which are tandem inversion repeats (FIG. 4B).

FIG. 5A is a schematic of the pssXC plasmid comprising the second embodiment in the expression system of the present invention, which includes the 10-23 DNA enzyme sequence schematically depicted in FIG. 5B.

6A and 6B are schematic diagrams of a pssXD plasmid comprising a third embodiment (FIG. 6A) and an enlarged portion of the pssXD plasmid (FIG. 6B) in the expression system of the present invention.

Figure 7 shows the results of PCR analysis of RT activity in cell lysates transfected with pssXA. Lanes 1 and 2: A549 cells transiently transfected with a pBK-RSV vector; Lanes 3 and 4: A549 cells transiently infected with pssXA; Lanes 5 and 6: A549 cells stably transfected with pssXA (E10). Prior to 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) and 30 minutes, respectively.

8 shows the results of ssDNA detection assay by PCR analysis. Total RNA was isolated from E10 cells transiently transfected with pssXB vector, pssXB-I or pssXB-II. Prior to PCR amplification, total RNA was pretreated with 37 nucleases (lanes 1 and 3) or RNases (lanes 2,4 and 5) at 37 ° C. for 30 minutes. Lanes 1 and 2: pssXB-I, lanes 3 and 4: pssSB-II; Lane 5: pssXB vector.

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 cell.

FIG. 10 shows a bar graph quantifying the northern blot of an ssDNA-producing vector that produces an antisense sequence for c-raf kinase, constructed according to the present invention. Lanes 1-3: cells harvested 24 hours after transfection; Lanes 4-6: cells harvested 48 hours after transfection. Lane 1: E10 cells transfected with a pssXB vector; Lanes 2 and 5: E10 cells transfected with pssXB-I; Lanes 3 and 6: E10 cells transfected with pssXB-II.

11 shows the results of dot blot analysis for the detection of ssDNA in A549 cells transfected with pssXD-II containing control pssXD-I or c-raf DNA enzyme sequences. In the presence of S1 nuclease, no detectable signal was produced because the ssDNA enzyme was specifically degraded by this S1 nuclease.

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

FIG. 13 shows the results of western blot for 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.

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

FIG. 15 shows the results of Western blot for PARP cleavage inducing cellular apoptosis by inhibition of c-raf gene expression. Lane 1: pssXD-II; Lane 2: control pssXD-I; Lane 3: untransfected cells.

The methods and nucleic acid constructs in this description of the present invention may or may not include contiguous nucleotide sequences for use in altering the expression of a target gene in vivo, such as yeast, prokaryotic and / or eukaryotic cells, It is described to generate single-stranded deoxyribonucleic acid (ss-DNA) oligonucleotides of virtually any predetermined or desired nucleotide base composition. The methods and compositions of the present invention describe the use of biological synthesis rather than in vitro or artificially synthesizing ss-cDNA of the desired nucleotide base composition. Since biological reactions, ie enzymatic reactions, are used in the methods of the invention, they can be applied to any in vivo system.

In one embodiment, an expression system of the present invention is a vector designed to generate any desired sequence as an ss-cDNA molecule (the term “vector” as used herein is intended to synthesize and / or transfer naturally occurring nucleic acid sequences and Plasmid used in the manipulation or modified viral or non-viral recombinant biological constructs), preferably with few contiguous vector sequences. This vector system has all the enzymatic functions and signaling commands required to produce ss-cDNA in host cells. The host cell to which the vector of the present invention is delivered produces an RNA transcript derived by the eukaryotic cell promoter (FIG. 1), which is used as a template for synthesizing any desired single-stranded DNA sequence (“target sequence”). do.

More specifically, by including two plasmids co-transfected into a suitable host cell (which may be yeast or any prokaryotic or eukaryotic cell), it is possible to generate the desired ssDNA sequence in a cell for changing gene expression. Described herein is a first expression system comprising a vector. The second expression system is also described as comprising a single plasmid transfected into a host cell to generate the desired ssDNA sequence in a cell for altering gene expression.

The ssDNA generated in vivo using the expression system described herein can be an inhibitory nucleic acid. The inhibitory nucleic acid may be ssDNA synthesized from an mRNA template or the mRNA template itself, which may specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target nucleic acid sequence, RNA--RNA, DNA--DNA or RNA--DNA duplexes or triplets are formed. More generally, these nucleic acids are generally termed "antisense" because they are complementary to the sense or coding strand of a gene, although "sense" sequences can also be used in cells for therapeutic purposes. Thus, the term "inhibitory nucleic acid" as used herein refers to both "sense" and "antisense" nucleic acids.

By binding to the target nucleic acid, the inhibitory nucleic acid changes the function of the target nucleic acid. This change (usually an inhibitory effect) may be, for example, blocking DNA transcription, processing or addition of poly (A) to mRNA, DNA replication, translation, or promotion of the inhibitory mechanism of the cell (eg, RNA degradation). Due to the promotion of). Thus, inhibitory nucleic acid methods include a number of different approaches for altering gene expression. These different types of inhibitory nucleic acid techniques are described in Helene, C. and J. Toulme, 1049 Biochim. Biophys. Acta. 99-125 (1990), hereinafter referred to as "Helene and Toulme," which is hereby incorporated by reference in its entirety.

In sum, inhibitory nucleic acid therapies include (1) targeting a DNA sequence, (2) targeting an RNA sequence (including pre-mRNA and mRNA), and (3) targeting a protein (a sense strand approach). ), And (4) result in cleavage or chemical alteration of the target nucleic acid, such as the ssDNA enzyme, including so-called "10-23 enzymes" herein. The first approach considers several categories. Nucleic acids are designed to bind to the major grooves of the duplex DNA to form triple helix or "triple" structures. Alternatively, the inhibitory nucleic acid is designed to bind to regions of single stranded DNA resulting from the spreading of the duplex DNA upon replication or transcription. More typically, inhibitory nucleic acids are designed to bind mRNA or mRNA precursors. Inhibitory nucleic acids are also used to prevent the maturation of pre-mRNAs. Inhibitory nucleic acids may be designed to interfere with RNA processing, splicing or translation. In a second approach, inhibitory nucleic acids target mRNA. In this approach, inhibitory nucleic acids are designed to specifically interfere with the translation of the encoded protein. Using this second approach, inhibitory nucleic acids are used to selectively inhibit specific cellular functions by inhibiting translation of mRNA encoding important proteins. An example of such inhibitory nucleic acid is a sequence complementary to the c-myc mRNA region, which is a c-myc primitive-oncogene (Wickstrom EL, et al., 85 Proc. Natl. Acad. Sci. USA 1028-1032 (1988). and Harel-Bellan, A., et al., 168 Exp. Med. 2309-2318 (1988)) inhibit the expression of c-myc protein in HL60, a human promyelocytic leukemia cell line. As described in Helen and Toulme's literature, inhibitory nucleic acids targeting mRNA have been found to act by several different mechanisms to inhibit translation of encoded protein (s).

Inhibitory nucleic acids can also capture or compete with enzymes or binding proteins involved in mRNA translation described in Helen and Toulme's literature using a third approach to designing the "sense" strand of a gene or mRNA. Finally, inhibitory nucleic acids are used to induce chemical inactivation or cleavage of a target gene or mRNA. Chemical inactivation occurs, for example, by inducing crosslinking between an inhibitory nucleic acid and a target nucleic acid in a cell, and the target nucleic acid is introduced by a method contemplated herein, ie, by a sequence incorporated in the cassette of the invention and having enzymatic activity. Occurs by cutting.

In short, in a first aspect, the present invention provides an expression system comprising a set of gene components, said set of gene components adapted for delivery into cells to produce ssDNA in vitro or in vivo for alteration of gene expression. And one or more cell (s) stably transfected comprising the set of gene components. The set of gene components is incorporated into an expression system for delivery into cells,

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

(B) (1) inversion tandem repetition sequence (IR), (2) (a) between inversion repetition sequence (IR), (b) at 3 'of IR, or (c) between IR and 3' of IR A cassette comprising at least one target sequence located at and (3) a primer binding site (PBS) for reverse transcriptase located at 3 ′ of the IR as shown in FIG. 2.

It includes.

Although not essential, the expression system preferably includes functions and signaling instructions for in vivo transcription of these components, and functions and signaling instructions for translation of reverse transcriptase (RT) genes. Additional components that may optionally be included in the set of gene components of the invention include, generally, one or more RNase genes, restriction enzyme (RE) genes (for the purpose described below), expression in eukaryotic cells involved in the RT gene. For the downstream polyadenylation signal sequence, which causes the mRNA to be produced such that the target sequence comprises a poly (A) tail (see FIG. 1), and a DNA sequence with enzymatic activity if the ssDNA is folded into a suitable secondary structure. May be included. Although the present invention is not so limited, in one embodiment for the gene components, the DNA enzyme sequence may be used regardless of whether the target sequence is located between inverted repeats (IR) or between the 3 'side of the IR and PBS. Located in the sequence.

In a first embodiment of the expression system described herein, a vector system comprising two plasmids is provided wherein the set of gene components described above adapted for delivery into cells to produce ssDNA in vivo is an RNA-dependent DNA polymerization Enzyme (reverse transcriptase), which additionally contains an RNase H gene linked to a restriction enzyme gene with a histidine-proline linker. These genes are constructed and then inserted into plasmid vectors containing the necessary transcriptional and translational regulatory components along with the polyadenylation tail sequence. This plasmid is referred to herein as the "A" plasmid, pssXA, as shown in FIG. The second “B” plasmid, in the embodiments described herein, comprises a primer binding site (PBS), a target sequence (SOI) and an inversion repeat (IR) that match the three components of the cassette, namely reverse transcriptase (RT). It is designed to include). In the second plasmid shown by plasmid pssXB of Figure 4, the SOI is located between inverted tandem repeats, or at the 5 'position (for mRNA transcript) with respect to PBS, where PBS is before mRNA Located at the 3 'end of the body. That is, the SOI is located between (1) IR, (2) between IR and PBS, and / or (3) between IR and between IR and PBS, as described below. Plasmids are described, one of which (pssXB-I) has an SOI between IR (eg, NotI site) and the other (pssXB-II) is located between IR and PBS (eg, PacI / BamHI site) Cloning). Similar to plasmid A, plasmid B also contains a combination of transcriptional regulatory components. However, in another preferred embodiment herein, plasmid B does not contain (or need) translational control components because no protein product is produced from this construct.

In other embodiments described herein, the expression system of the present invention, shown in FIGS. 5 and 6, called plasmids pssXC and pssXD, respectively, comprises a single plasmid vector comprising a set of described gene components. The components of the B plasmid described above, such as PBS, SOI and IR, are present in the untranslated 3 '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 a suitable promoter (in the embodiments described herein, the RSV promoter was used), the resulting mRNA transcripts were directed to the coding region for RT-RNase H polyprotein. And additional mRNA transcripts at the stop signal at the end of the translation are further 3 'downstream for the polyadenylation signal, which remains intact from the RT-RNase H component (to 3' for the translated protein). It contains components from the B plasmid that deliver the signal to the.

Certain single plasmid expression systems described herein do not contain restriction enzyme (RE) genes and therefore do not degrade the stem of stem-loop intermediates formed by inversion repeats. Thus, the SOI (including DNA enzymes) is inserted only at the 3 'position for the IR in the C or D plasmid, and unwanted vector sequences mature the ss-cDNA product when the transcript is in contact with the relatively stable stem of the stem-loop intermediate. It is not possible to maintain transcription of ss-cDNA from mRNA transcripts by elimination by whole terminal truncation. More specifically, as will be apparent from the description below, each SOI was inserted only at the PacI / BamHI restriction sites of the pssXC and pssXD plasmids.

As will be apparent from the description below for B, C and D plasmids, these plasmids comprise a cloning site for insertion of SOI. Both the NotI site (located between IRs) and the PacI / BamHI site (3 'relative to the IR, eg, located between IR and PBS) are provided in preferred embodiments of the B plasmid described herein. The C and D plasmids described herein contain only PacI / BamHI sites for this purpose. However, one of ordinary skill in the art using the present disclosure will appreciate that these particular cloning sites have been selected for the particular systems described herein, and that other cloning sites may equally be used for the same purpose. Although A plasmid comprising the two plasmid vector systems described herein does not comprise SOI, those skilled in the art will appreciate that if two plasmid vector systems are used, the set of genetic components of the present invention, and in particular SOI, may be It will be appreciated that it may be inserted.

Nucleic acid sequences, referred to herein as cassettes, provide templates for the synthesis of ss-cDNA in target cells. This includes SOI, IR and PBS as components. As with other components of the set of gene components of the invention, this gene component is preferably located upstream of the gene component, or a wide range or tissue-specific suitable promoter / enhancer (e.g., a CMV promoter). Controlled by a combination of promoter / enhancer. In addition, as is the case with other genetic components, the promoter / enhancer may be a constitutive promoter or an inducible promoter. Those of ordinary skill in the art using the present disclosure, a number of eukaryotic promoters, including SV-40, RSV (cell-free specific) or tissue-specific glial fibulary acidic protein (GFAP), regulate the expression of SOI. It will be appreciated that it can be used to advantage.

A primer binding site (PBS) for initiating priming to synthesize cDNA is located between the 3 ′ IR and the polyadenylation signal. PBS is a sequence complementary to transfer RNAs (tRNAs) present in eukaryotic target cells. For the mouse Moloney reverse transcriptase (MoMULV RT) described herein as used in the present invention, PBS uses proline tRNA. The PBS used according to the presently preferred embodiments of the invention described herein was obtained from the actual 18 nucleotides of the mouse moronivirus region (Shinnick, TM, et al., Nucleotide sequence of Moloney murine leukemia virus, 293 Nature 543-548) (1981). For the RT gene from human immunodeficiency virus tested as mentioned below, the PBS used was from HIV (Y. Li, et al., 66 J. Virology 6587-6600 (1992)). In short, any PBS that matches a particular RT can be used for this purpose. PBS is exclusively recognized by primer tRNAs that are endogenous to target cells. Each tRNA has the ability to recognize a unique sequence (ie codon) on an mRNA transcript that encodes an amino acid, and has the ability to covalently bind to a specific amino acid (ie, when bound to a specific amino acid, the tRNA is “charged”). "Status). However, when the primer tRNA binds to the mRNA transcript PBS and is not covalently bound to an amino acid (ie, in an "uncharged" state), the primer tRNA can be used to initiate ssDNA synthesis by RT. For example, MoMULV RT used in the Examples herein recognizes and utilizes unfilled lysine tRNAs. In other words, PBS recognizes and binds unique sequences. Thus, each PBS incorporated 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.

RT / RNase H genes of other retroviruses may also be used in accordance with the present invention, in which case the RT / RNase H genes may be used by appropriate upstream eukaryotic cell promoters / enhancers such as CMV or RSV promoters for expression in human cells. It is preferred to be adjusted. RNA-dependent DNA polymerase / RT genes suitable for the present invention include retroviruses, hepatitis B virus, hepatitis C virus, bacterial retron components, and genes from retrons isolated from various yeast and bacterial species. Included. As it appears in nature, these RNA-dependent DNA polymerases generally contain complex RNase H component enzymes within the same coding transcript. However, the present invention does not require a naturally occurring RNase H gene for a particular RT. That is, those skilled in the art will appreciate from the present disclosure that combinations of various RT and RNaseH genes can be made to be used in accordance with the present invention to perform their functions, and modified and / or hybrid versions of these two systems are available. And / or may be known to those skilled in the art and function in the intended manner. Those skilled in the art will also recognize that the target cell itself has sufficient endogenous RNase H activity to perform the functions of the present invention. Similarly, those skilled in the art will appreciate that the target cells themselves may have sufficient endogenous RT activity, for example prior to retroviral infection, to perform the functions of the present invention.

In addition, the RT / RNase H gene preferably comprises a downstream polyadenylation signal sequence, such that mRNA generated from this RT / RNase H gene includes the 3 ′ poly (A) tail to ensure stability of the mRNA. As is well known in the art, multiple poly (A) tails are available, which are commonly used for the production of expressed eukaryotic cell genes.

In addition, one of ordinary skill in the art will appreciate that multiple tissue-specific or wide ranges of promoters / enhancers, or combinations of promoters / enhancers not described above, may be used to modulate the RT / RNase H gene, RE gene (if used) and the desired sequence. You will know. The list of all available promoters / enhancers need not be exemplified in the present invention, but as mentioned above the promoters / enhancers may be constitutive or inducible and may include CMV or RSV (cell-free specific) described herein. ) Or GFAP (tissue specific) promoters / enhancers and myriad other viral or mammalian promoters. Representative promoters / enhancers suitable for use in the cassettes of the invention include HSVtk (SL McKnight, et al., 217 Science 316 (1982)), human β-globulin promoter (R. Breathnach, et al., 50 Ann. Rev. of Biochem. 349 (1981)), β-actin (T. Kawamoto, et al., 8 Mol. Cell Biol. 267 (1988)), rat growth hormone (PR Larsen, et al., 83 Proc. Natl. Acad Sci. USA 8283 (1986)), MMTV (AL Huang, etal., 27 Cell 245 (1981)), adenovirus 5 E2 (MJ Imperiale, et al., 4 Mol. Cell. Biol. 875 (1984)) , SV40 (P. Angel, et al., 49 Cell 729 (1987)), α-2-macroglobulin (D. Kunz, et al., 17 Nucl. Acids Res. 1121 (1989)), MHC class I Gene H-2kb (MA Blanar. Et al., 8 EMBO J. 1139 (1989)) and thyroid stimulating hormone (VK Chatterjee, et al., 86 Proc. Natl. Acad. Sci. USA 9114 (1989)) But not limited to.

The RT generated in the cells synthesizes complementary DNA (cDNA) using the genetic component comprising SOI described below as a template. The RNase H activity of RT degrades the mRNA template component of the RNA / cDNA hybrid to produce ss-cDNA in vivo.

The gene encoding the RE (used in two plasmid expression systems, but not an essential component of the system) may be any of several genes encoding REs, and may include one or more of the constitutive or inducible broad ranges described above and ( Or) controlled by a tissue-specific promoter / enhancer. While the specific REs tested were MboII and FokI, those skilled in the art using the present disclosure will appreciate that any RE (type I, II, IIs or III) site may be included in the IR. These enzymes “clip” or digest the stem of the stem-loop intermediate described below to linearize SOI into single-stranded DNA.

Although expression of the RE gene is regulated by a suitable constitutive or inducible promoter / enhancer (e.g., CMV or RSV promoter for expression in human cells) upstream of the restriction enzyme gene, for the plasmid pssXA The gene (MboII) is linked to the RT-RNase H polyprotein. In addition, the RE gene preferably comprises a downstream polyadenylation signal sequence such that the mRNA transcript from the RE gene has a 3 'poly (A) tail.

The cassettes of the invention also include inverted tandem repeats (IR). After digesting mRNA by RNase H from mRNA-cDNA heteroduplex, the IR is ss-cDNA itself folded in the manner described in US Pat. No. 6,054,299 and shown in FIG. Wherein the stem structure comprises double stranded non-parallel DNA, the cassette being transcribed in the cell and the RT / RNase H generated by transcription of the gene, followed by the desired ss-cDNA sequence. It is produced from mRNA transcripts in these cells. The one or more RE site (s) cleaved by the RE generated from the RE gene (in the case of a plasmid comprising the RE gene) may be designed to be a double stranded portion, ie, an IR, forming the stem of the stem-loop intermediate. The resulting ss-cDNA is transcribed with a loop containing the SOI and coded 5 'and 3' regions adjacent to the stem (IR). Subsequently, the stem is designed by a number of RE enzymes that recognize the cleavage site designed inside the stem (note that the endonuclease recognition site may be designed inside the stem, even if the RE gene is not included in the vector system of the present invention). Is cut (also called digestion or cleavage) to release the ss-cDNA loop (see FIG. 1). The loop portion of ss-cDNA that does not form any apparent duplex DNA is not affected by RE activity since RE recognizes only double stranded DNA as the target substrate.

As mentioned above, one of skill in the art would appreciate that if the RE site (s) form the stem of the stem-loop intermediate, the transcription of the cassette in the stem formed by the IR may be desired even if one wishes to generate ssDNA from the SOI located between the PBS and the IR. Will not need to be designed inside). Another option is to design the IR at the DNA binding site of a particular eukaryotic, prokaryotic or viral protein, which competes with the protein of the selected cell to interfere with the binding of that protein. Combinations of restriction sites or other sequence specific components may be included in the IR depending on the base pair composition selected for the IR such that a stem-loop intermediate form of linear or correctly truncated ssDNA is produced. Generally, since naturally occurring inversion repeats are not properly aligned with restriction sites, it is preferable to use synthetically produced sequence specific components for IR.

As mentioned above, a cassette comprising one component of the set of gene components of the invention may also comprise a DNA sequence with catalytic activity. Since the cassette contains a so-called "DNA enzyme" (in the embodiments described herein, the DNA enzyme is located within the target sequence), the present invention acts as a template for the synthesis of the inhibitory nucleic acid (antisense sequence). It is particularly advantageous in this case. For this reason, the examples described herein include c-targeted by antisense ISIS 5132 (Monia, BP, et al., 2 Nature Medicine 668-675 (1996); incorporated herein by reference in its entirety). The generation of antisense SOI of FIG. 5B comprising a sequence with enzymatic activity for mRNA comprising c-raf cleavage enzyme designed to specifically bind to the 3 'untranslated region of raf mRNA is described. Two 9 bp target specific binding arms are adjacent to a 15 bp catalytic domain (Santoro, SW and GF Joyce, Mechanism and utility of an RNAcleaving DNA enzyme, 37 Biochemistry 13330-13342 (1998); The entirety of which is incorporated herein by reference). A compatibility restriction site was added to the DNA enzyme oligonucleotide to allow insertion into the NotI site or the PacI and BamHI sites, and the resulting plasmids were named pssXB-I and pssXB-II, respectively.

Those skilled in the art will appreciate that the present invention is not limited to antisense sequences, and that the antisense sequences do not necessarily contain nucleic acid sequences with catalytic activity, and that the inhibitory nucleic acid sequences may be any other type of inhibitory nucleic acid sequences described above. . Since c-raf kinase in the A549 lung cancer cell system is well characterized, the SOI was chosen to demonstrate the present invention (Monia, et al., Supra (1996)). Raf protein is a serine / threonine protein kinase that acts as a direct downstream effector for Ras protein in the MAP kinase signaling pathway, including downstream MEK1 / MEK2 activation and subsequent ERK1 and ERK2 activation (Daum, G., et al., The ins and outs of raf kinases, 19 Trends Biol. Sci. 474-480 (1994)). Many loyal tumors and leukemias have been shown to be mutagenic or upregulated in the MAP kinase signaling pathway. Signaling pathways such as c-raf associated with tumors have been excellent targets in oncological therapies, and the aforementioned phosphorothioate ODN ISIS 5132 has proven to be a potential antisense inhibitor (Monia, et al., Supra (1996) )). In addition, ISIS 5132 has been shown to induce apoptosis (Lau, QC, et al., 16 Oncogene 1899-1902 (1998); incorporated herein by reference in its entirety) and also for such tumors. It seems to indicate a potential therapeutic effect. This antisense ODN has recently entered Phase I clinical trials (O'Dwyer, PJ, et al., C-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligonucleotide ISIS 5132 (CGP 69846A) , 5 Clinical Cancer Res. 3977-3982 (1999)), and will also prove useful in the treatment of c-raf-associated tumors. Another SOI cloned into the plasmid for expression using the expression system of the present invention is a h- with a 10-23 DNA enzyme sequence (Santoro and Joyce, supra (1997)) inserted between the 5 'and 3' complementary sequences. ras antisense binding sequence, 23rd codon subsequence, 10-23 DNA enzyme sequence, partial sequence of pleiotropin antisense binding sequence inserted between 5 'and 3' complementary sequences, and 10-23 DNA enzyme The sequence comprises a tat antisense binding region subsequence of the SIV sequence inserted between the 5 'and 3' complementary sequences. Although each of these sequences is included in the DNA enzyme sequence, those skilled in the art will appreciate from the present disclosure that the DNA enzyme sequences need not necessarily be included in these SOIs or any other SOIs.

Nucleic acid sequences with enzymatic activity used in the methods of changing gene expression described herein are 10-23 DNA enzymes (Santoro and Joyce, supra) (1997). This enzyme sequence can either be (a) inside the SOI (NotI site) between IRs or (b) inside the second SOI (PacI / BamHI site) located 3 'for IR and 5' for PBS, or It is inserted in both. In any case, because the resulting aptamer is specific for the target of the SOI, it can be used to target other DNA sequences, mRNA sequences, and any other suitable substrate to inhibit or change the DNA or mRNA splicing mechanism or even Can directly alter the genome of a cell in a specific way.

Those skilled in the art will appreciate from the present disclosure that any DNA sequence having enzymatic activity may function for the intended purpose when inserted into the cassette of the present invention. Numerous nuclear line sequences with enzymatic activity have been reported in the literature.

Sequences with RNase activity (Santoro, SW and GF Joyce, supra (1997)) and other metal-dependent RNase activities such as so-called "10-23 enzyme" and "8-17 enzyme" (Breaker, RR and GF Joyce, 1 Biol. Chem. 223-229 (1994) and Breaker, RR and GF Joyce, 2 Biol. Chem. 655-660 (1995)) and sequences with histidine-dependent RNase activity (Roth, A. and RR Breaker, 95 Proc. Natl Acad. Sci. USA 6027-6031 (1998);

Sequences with DNase activity such as copper-dependent DNase (Carmi, N., et al., 3 Chem. Biol. 1039-1046 (1996), Carmi, et al., Supra, (1997); Sen, D. and CR Geyer, 2 Curr. Opin. Chem. Biol. 680-687 (1998)) and sequences with DNase activity that require divalent sonic ions as cofactors or hydrolyze divalent metal ions independently of the substrate ( Faulhammer, D. and M. Famulok 269 J. Molec. Bio. 18-203 (1997));

DNA ligase such as copper-dependent DNase (Breaker, RR, 97 Chem. Rev. 371-390 (1997)) and zinc-dependent E47 ligase (Cuenoud, B. and JW Szostak, 375 Nature 611-613 (1995)) Sequences with an aze activity;

Sequences with DNA kinase activity such as calcium-dependent DNA kinases (Li, Y. and R. R. Breaker, 96 Proc. Natl. Acad. Sci. USA 2746-2751 (1999)); And

Included are sequences with RNA kinase activity such as calcium-dependent DNA kinases (Li, Y., supra (1999)).

In general, these DNA sequences have enzymatic activity derived from physiological conditions and are preferably used in the cassette of the present invention.

When components comprising a set of gene components of the invention are incorporated into an expression vector in a target cell, the vector facilitates identification of and / or the expression of the set of gene components comprising the cassette. It is desirable to contain other special genetic components that increase the amount. Particular genetic components include selectable marker genes that allow vectors to be transformed and amplified in prokaryotic system. For example, most of the commonly used selectable markers are resistant to bacteria (eg, E. coli ) to antibiotics such as ampicillin, chloramphenicol, kanamicin (neomycin) or tetracycline. It is a gene to be given. The vector also preferably contains special genetic components for subsequent transfection, identification and expression in eukaryotic cell systems. For expression in eukaryotic cells, multiple selection strategies (e.g., Chinese hamster ovary cells: CHO) are used to confer the cells resistance to antibiotics or other drugs, or morphological changes, loss of contact inhibition or growth rate It can lead to phenotypic changes such as increases. Selectable markers used in eukaryotic cell systems include resistance markers for zeosin, resistance markers for G418, resistance markers for aminoglycoside antibiotics, or phenotypic selection markers such as β-gal or green fluorescent protein, It is not limited to this.

Incorporation of these components into plasmids comprising the expression system of the present invention provides two convenient methods for removing a predetermined vector sequence after generation of ssDNA. In the first method, the cassette is reverse transcribed from PBS, and the SOI between the IRs is an ssDNA stem-loop intermediate (stem contains an RE site) generated when the nucleotides comprising the IR pair to form a stem of the stem-loop vector. ), And after digestion with a suitable RE, the loop releases the linearized single-stranded cDNA without contiguous sequences (and / or with minimal contiguous sequences). In the second method, the cassette is reverse transcribed from PBS and the SOI contained 3 ′ to IR is similarly transcribed, but reverse transcription is terminated in the stem of the stem-loop structure formed by pairing the nucleotides of the IR. In any case, the resulting ssDNA is produced without contiguous sequences (and / or with minimal contiguous sequences). If it is desired to generate ssDNA using the second method, the cassette is in the case of ssDNA (eg, designed to not include an RE site) produced by digesting the stem according to the first aspect of the invention. It is designed to include an IR that forms a more stable stem than the stem needed. By designing the cassette to include an IR forming a stem that is readily denatured according to the first aspect of the present invention, reverse transcription proceeds through the second SOI (if designed into the cassette) to the SOI located between the IRs. Thus, "premature termination" of the reverse transcriptase cDNA transcript, appearing on the 3 'side of the stem structure, provides a second method of limiting intervening vector sequences contained in in vivo-produced ss-cDNA. It is possible to generate both the first SOI and the second SOI.

It will also be apparent to those skilled in the art from this description that the intact stem-loop ss-cDNA structure can function similarly in many fields, such as in the form of a linear ss-cDNA. Thus, cassettes can also be used to include a target sequence that does not contain restriction enzyme genes and related regulatory components and / or lacks a corresponding restriction enzyme site.

It will also be apparent to those skilled in the art from the description of the preferred embodiment of the present invention that the cassette can be made to encode ss-cDNA having a “trimmed” stem-loop structure. The RE region encoded in the IR adjacent to the SOI is such that the dsDNA comprising the stem is cleaved in such a way as to remove the stem portion and related contiguous sequences, leaving the transcript leaving sufficient duplex DNA to retain the stem-loop structure. The stem portion (after duplex formation) is designed to decompose into the corresponding RE. Such ss-cDNA structures may be more resistant to intracellular nucleases by retaining the double stranded form of ssDNA "end".

In addition, those skilled in the art from the description of the preferred embodiment of the present invention can be designed such that the stem (duplex DNA) contains a predetermined sequence (or sequences), ie, aptamers that are recognized and bound by a particular DNA-binding protein. It is obvious. Among other uses, such stem structures are used as competitors in cells, interfering with the binding of selected protein (s) to regulate specific gene functions. For example, ss-cDNA stem-loops are produced according to the invention in cells containing selected positive transcription factors, such as adenovirus El. Similar to other oncogenes, adenovirus E1a also regulates the expression of various adenovirus genes and cellular genes by affecting the activity of cell-coding transcription factors, transforming normal cells into transformed cells (Jones, et. al, 2 Genes Dev. 267-281 (1988)). Thus, the duplex stems of stem-loop intermediates produced according to the present invention are designed to "bind up" this transcription factor, preventing the protein from binding to the promoter, thereby preventing the expression of certain harmful genes. Suppress It will be apparent to those skilled in the art that the duplex stem structure may optionally contain multiple binding sites, eg, sites recognized by various transcription factors that actively regulate the expression of a particular gene. For example, adenovirus E1a is transcribed by 12-O-tetradecanoylphorball 13-acetate (TPA), by a number of other mitogens, and by ras, mos, src and trk oncogenes. It has been shown to inhibit transcription of the collagenase gene through the phorbol ester-reactive component, a promoter component responsible for the induction of. This mechanism involves the inhibition of function of the transcription factor family AP-1 (Offringa, et al., 62 Cell 527-538 (1990)). Any preferred nucleotide sequence can be inserted into the genetic component encoding the "loop" portion of the stem-loop intermediate to perform the desired inhibitory functions such as the antisense binding described herein and down regulation of the gene.

In another aspect that will be appreciated by those skilled in the art, the present invention is used to construct complex ssDNA structures that impart biological responses on cDNA transcripts based on the folding of secondary structures. Such secondary structures can be engineered to perform any of a number of functions. For example, the desired sequence includes, but is not limited to, sequences incorporated into the loop portion of a single-stranded cDNA transcript. It may form a so-called "clover leaf" or "crucible-like" structure found in long terminal repeats of related viruses or retrotransposons. Under the right conditions, such structures are integrated into the host genome in a site-specific manner.

Since the cassettes of the invention can be applied to incorporation into a number of commercially available delivery vectors for the treatment of mammals and humans, multiple delivery pathways are possible depending on the vector chosen for a particular target cell. For example, viral vectors are generally used to transform cells of a patient to introduce DNA into the genome. In an indirect manner, viral vectors with new genetic information are used to infect target cells isolated from the body, and then to re-infect (ie ex vivo) the infected cells. Direct delivery of genes in vivo to animals after birth has been reported for formulation of DNA encapsulated in liposomes and formulation of DNA captured in proteoliposomes containing viral envelope receptor proteins (Nicolau, et al., 80 Proc. Natl. Acad Sci USA 1068-1072 (1983); Kaneda, et al., 243 Science 375-378 (1989); Mannino, et al., 6 Biotechniques 682-690 (1988)). In addition, the use of DNA precipitated with calcium phosphate has been shown to yield positive results (Benvenisty and Reshef, 83 Proc. Natl. Acad. Sci. USA 9551-9555 (1986)). Other systems used to administer expression systems comprising the set of genetic components of the invention include intravenous and intracavitary injections, as well as intravenous, intramuscular and subcutaneous injections. When inserted into the chosen expression system, the cassette is preferably administered via topical, mucosal, rectal, oral or inhalational delivery methods.

Cassettes of the invention are advantageous for delivering antisense, triplex, or any inhibitory nucleic acid or single-stranded target nucleotides using known digestion and ligation techniques to splice specific target sequences into the cassette. Can be used. In addition, those skilled in the art using the present disclosure will appreciate that the signals used for expression in eukaryotic cells may be modified using methods known in the art, depending on the particular sequence of interest. For example, a possible modification is to alter the promoter to confer favorable expression properties on the cassette in the system that is desirable to express the desired sequence. In addition, there are a number of modifiable promoters and other signals, which are dependent on the particular target cell selected for the target sequence, and therefore all potential enhancer, inducible and constitutive promoter systems that may be desirable for the particular target cell and target sequence. It is not possible to list both and / or poly (A) tail systems.

In one particularly preferred embodiment, the invention takes the form of a kit comprising said RNA-dependent DNA polymerase and the RE gene cloned therein, and multiple cloning sites (MCS) into which the user of the kit has inserted a particular SOI. . The cloning site into which the SOI is inserted is located between the IRs. The resulting plasmid can then be purified, lyophilized, or packaged from cell cultures holding the cells and stored for delivery to the user. Also preferably, the kit includes RE (s), ligase and other enzymes (with suitable buffer for ligating the SOI into the plasmid), and a map of the plasmid for the MCS to which the SOI is cloned.

In certain embodiments described herein, the SOI (s) are delivered to the host cell by co-transfecting the cells with two plasmids (designed and constructed to include the components listed above, respectively called A and B). Or is delivered to host cells by a single C or D plasmid. In two plasmid systems, the B plasmid contains a cassette contained within contiguous sequences comprising the IR, or comprising an SOI present between the IR and PBS, and providing a post-transcriptional processing signal that mediates the conversion of mRNA to ssDNA. Coding The activities required to remove the vector sequence and processing signals to process the primary gene product of the B plasmid into ssDNA, specifically RT / RNase H and RE (if used), are expressed from the A plasmid. Single-stranded DNA sequences released by the interaction of transcription products to these components in vivo are free to bind intracellular targets such as mRNA species and DNA promoters in antisense and triplex strategies.

As mentioned above herein, the B plasmid comprises a cloning site (where the NotI site is used in the B plasmid herein) where any DNA SOI is located (as mentioned above, in the examples described herein, the SOI is An antisense sequence for c-raf kinase comprising a 10-23 enzyme sequence, but other sequences as described above generated in vivo using the expression system described herein may be a "stuffer" or test sequence, Telomer repeat, h-ras, angiogenic growth factor iotropin, and regions encoding tat (from SIV). The sequence adjacent to the cloning site is a signal that induces the processing of the primary mRNA transcript generated from the promoter (using the CMV promoter in the B plasmid herein) into the desired single-strand inhibitory nucleic acid. After cloning the desired SOI into the B plasmid, A and B plasmids were co-transfected into selected cell lines to constitutively express ssDNA. Similarly, in the single plasmid expression system described herein, SOI is cloned into the plasmid and transfected into cell lines for further processing. Regardless of the distribution of the components in the above-mentioned set of genetic components between two (or more) plasmids, or if they are all contained in a single plasmid, this processing is performed in a single-stranded DNA region (ie, SOI, IR and PBS) following transcription in three steps:

(1) Reverse transcription step of plasmid RNA transcript by RT [in embodiments herein RT is expressed by A, C or D plasmids (in embodiments herein RT is MoMuLV RT) and the SOI shown in FIG. 1 (SOI may optionally include sequences with enzymatic activity), proceeding from primer binding sites located 3 ′ to IR and PBS;

(2) the resulting heterologous duplexes are resolved by RT polyproteins by RNase H activity or endogenous RNase H activity to release single-stranded DNA precursors from RNA complementary sequences; And

(3) prior to maturation by degrading the stem of the stem-loop intermediate formed based on Watson-Crick base pairs of bases comprising IR, or by forming a stem-loop secondary structure by self-complementary IR Removing contiguous sequences by terminating.

Those skilled in the art will appreciate from the present disclosure that a particular cloning site adjacent to an SOI, a specific RT, RE (if used), a promoter, PBS, and all other components of the set of gene components of the invention expresses a particular SOI and / or ssDNA. It will be appreciated that the choice depends on the system being created.

Unless stated otherwise, in the Examples set forth below, this disclosure is incorporated by reference in its entirety. Seabrook, et al, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press (1989), hereinafter referred to as "Maniatis, et al. (1989)" and [F.M. Ausubel, et al., Current Protocols in Molecular Biology, New York: John Wiley & Sons (1987). It is to be understood that other methods of producing ssDNA by natural processes and by designing artificial methods using different enzyme products or systems may also be used in accordance with the methods of the present invention, and the examples described herein may also be used in the present invention. It is to be understood that the purpose of the description is not to limit the scope of the disclosure or the invention as described herein.

Plasmid pcDNA3.1Zeo + was purchased from Invitrogen Corp., Carlsbad, Calif., And plasmid pBK-RSV was purchased from Stratagene, La Jolla, Calif. Oligodeoxynucleotides (ODNs) were synthesized in Midland Certified Reagent Co., Midland, TX. Polymerase chain reaction (PCR) was performed using a Taq DNA polymerase purchased from Boehringer Mannheim Corp., Indianapolis, Ind. (Robo-gradient thermal cycler; Stratagene, La). Jolla, CA). Restriction enzymes and T4 DNA ligase were obtained from Boehringer Mannheim Corp., Indianapolis, IN. The ODN used is listed in the attached sequence listing.

All ODNs were hybridized to 1 μl (5 μg / μl in water) doses in separate tubes, incubated at 70 ° C. for 5 minutes and hybridized at room temperature for 15 minutes. Standard restriction enzyme cleavage was performed with 10 units of enzyme in a total reaction volume of 15 μL and appropriate reaction buffer (EcoRl was used as negative control). Selection of positive clones on ampicillin plates was performed after transformation with water-soluble XL1-Blue MRF cells (Stratagene) as described in Maniatis, et al (1989). After selecting positive clones, plasmid DNA was isolated using the Qiagen plasmid isolation kit.

<Production of plasmid>

The production of four expression plasmids is described. The first plasmid, pssXB (FIG. 3), is derived from pcDNA3.1Zeo (+) (Invitrogen Corp.) and contains the genetic component encoding the target ss-cDNA sequence used herein. pcDNA3.1Zeo (+) was digested with restriction enzymes HindIII and NotI at positions 911 and 978, respectively. Formed by synthesized single stranded oligodeoxynucleotides ODN-5'-N / M (link) 2-H / N and ODN-3'-N / M (link) 2-H / N Double-stranded linker regions with Notl ends were ligated under standard conditions into HindIII / Notl double-lyzed pcDNA3.1Zeo (+) transformed with SureII cells (Stratagene, Inc.). ODN was hybridized to 1 μl (5 μg / μl in water) dose in an Eppendorf tube, incubated at 70 ° C. for 5 minutes and hybridized at room temperature for 15 minutes. Appropriate clones were selected and sequenced to confirm proper insertion of linker regions. The resulting plasmid was named pssXB. pssXB is shown in FIG. 4A and is a plasmid in which the target sequence (FIG. 4B) has been cloned. To clone the desired sequence between inverted tandem repeats, two NotI sites (FIG. 4A) located at 935 and 978, respectively, were used. These two sites are contained between inverted tandem repeats. To insert the desired sequence between the inverted tandem repeat sequence and the primer binding site, two convenient restriction sites, PacI and BamHI sites, located at 1004 and 1021, respectively, were used.

The second plasmid is also pssXA (FIG. 3), which is a component of the two plasmid vector systems described herein. This "A" plasmid contains Mo-MuLV-RT (Shinnick, TM, et al., 293 Nature 543-548 (1981)) and restriction enzyme genes and is derived from pBK-RSV (Stratagene) and also XL- 1 Blue MRF 'is used as host cell. Mouse cell lines expressing Moroni rat leukemia virus were obtained from the American Type Culture Collection (# CRL-1858). Viral RNA is described in Chomczymski, P. and N. Sacchi, 162 Anal. Biochem. 156-159 (1987)] was isolated using a Trizol reagent (GibcoBRL), and reverse transcription using primer 3'-RT-HindIII (5'-CTTGTGCACAAGCTTTGCAGGTCT-3 '). Subsequently, the transcript was amplified by PCR using a TaqPlus long chain polymerase system (Stratagene) and repeating the cycle of 94 ° C 1 minute, 67 ° C 1 minute, and 72 ° C 2.5 minutes. Primers used for the PCR reaction were 5'-RT-SacI (5'-GGGATCAGGAGCTCAGATCATGGGAC-CAATGG-3 ') and 3'-RT-HindIII (the same as used for reverse transcription). These primers contain compatible Sac I and HindIII sites, respectively. The resulting 2.4 kb product contained a sequence of Mo-MuLV between 2546 and 4097 positions. Mature viral RT peptides are encoded by sequences between positions 2337 and 4349 (Petropoulos, CJ, Retroviral taxonomy, protein structure, sequences and genetic maps, in JM Coffin, et al. (Eds.), Retroviruses, New York: Cold Spring Harbor Laboratory Press, pp. 757-805 (1997)), but peptides truncated at the amino terminus retained full activity (Tanese, N. and SP Golf, 85 Proc. Natl. Acad. Sci. USA 1777- 1781 (1988)). The peptide encoded by this construct includes a portion of the integrase gene, which is followed by RT in the MoMuLV polyprotein (Petropoulos, supra (1997)).

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

pBK-RSV vectors were digested with XmaI and NheI. NheI wheat tips using a linker formed by annealing two oligonucleotides of 5'-Nhe-Sac-link (5'-CTAGCGGCAAGCGTAGCT-3 ') and 3'-Nhe-Sac-link (5'-ACGCTTGCCG-3') Was converted to the Sac I terminal. RT and MboII amplifications were ligated through the HindIII site and the construct was then ligated between the SacI and XmaI sites of pBK-RSV to obtain pBK-RSV-RT / MboII.

To insert a flexible linker between the RT and MboII domains of the polyprotein, a fragment of the pBK-RSV-RT / MboII plasmid between the AseI and BglII sites (encoding the 5 'end of the MboII gene and part of the integrase) ) Was cut out and replaced with inserts containing 6-His-linker and 5'-MboII DNA fragment (deleted by double digestion). The insert is 3 '17 bases at the terminal complementary to the two mold Rep (+) (5'-ATACTATTAATTTTGGCAAATCATAGCGGTTATGCTGACTCAGGTGAATGCCGCGATAATTTTCAGATTGCAATCTTTCATCAATGAATTTCAGTGATGAATTGCCAAGATTGATGTTGC-3') and Rep - mutual from (5'-GACGAGATCTCCTCCAGGAATTCTCGAGAATTCGGATCCCCCGCTCCCCACCACCACCACCACCACCCTGCCCCGCGGATGAAAAATTATGTGAGCAACATCAATCTTGGC-3 ') () - priming Obtained by DNA synthesis. These two oligonucleotides were annealed, stretched with modified T7 DNA polymerase (USB), and then double-stranded oligonucleotides were cleaved with AseI and BglII and inserted into the pBK-RSV vector to obtain pssXA (FIG. 3). .

In a first embodiment of a single plasmid expression vector system constructed in accordance with the present invention, the "B" plasmid derived from pcDNA3.1Zeo (+) and the "A" plasmid derived from pBK-RSV are fused to obtain the resulting plasmid. All components of the set of gene components of the invention were encoded (ss-cDNA-coding sequence, tandem inversion repeat sequence, Mo-MuLV-RT gene, and restriction enzyme (MboII) gene). To generate a C plasmid, plasmid pssDNA-Express-A was digested with Sac I and Xma I to isolate the MboII gene. Linker region containing nucleotides 5 '-(link) 2-Hind / Xba (5'-CCGGATCTAGACCGCAAG-CTTCACCGC-3') and 3 '-(link) 2-Hind / Xba (5'-GGTGAAGCTTGCGGTCTAGAT-3') Annealed at 75 ° C. for 15 minutes and slowly cooled to room temperature) was digested under standard conditions and then ligated into the plasmid. Positive clones were harvested and sequenced to confirm the location of the linker, and then the plasmids were digested with Xba and HindIII. The plasmid pssDNA-Express-B was then digested with HindIII and Xba, and the corresponding 300 base pair DNA fragment comprising the inverted tandem repeat sequence, multiple cloning sites and PBS described above was cloned into the digested plasmid to pssXC (FIG. 5A). Got. Standard ligation reactions were performed to transform Sure II cells (Stratagene, Inc.). Transformed positive colonies were harvested and positive clones confirmed by restriction enzyme analysis.

The desired sequence was cloned into multiple cloning sites of pssXC using BamHI and PacI in multiple cloning sites (FIG. 5B). Four different target sequences were synthesized for each construct as above, and similar procedures were used to insert each of the four target sequences. Each construct was prepared by annealing the paired oligonucleotides at 70 ° C. for 15 minutes and then cooling to room temperature, which was ligated into the plasmid under standard conditions. After transformation into SureII cells, appropriate colonies were selected by sequencing of individual inserts.

The second plasmid was constructed by combining two plasmids pssXA and pssXB in the following manner using a single plasmid expression system pssXD. PssXA containing Mo-MuLV reverse transcriptase (RT) was digested with XmaI and BglII, and the resulting GapI-BglII fragment was digested with two oligos [XmaI-BglII-Stop 1 (5'-CCGGATCTAGACCGCAAGCTTCATTTAAA-3 ') and XmaI- BglII-Stop 2 (GATCTTTAAATGAAGCTTGCGGTCTCGAT-3 ')] was replaced with a double-stranded DNA adapter formed by annealing. The adapter contains a protein translational stop codon and the XbaI and HindIII sites, which are subcloning sites. The resulting plasmid was named pssXD (FIG. 6A). XbaI-HindIII fragments were cleaved from both pssXB and pssXB-II and cloned between the XbaI and HindIII sites of pssXD. These DNA fragments contain 1) RT primer binding site (PBS), 2) stem-loop structure, and 3) random control sequence (pssXB) or c-raf DNA enzyme sequence (pssXB-II). The resulting plasmids were named pssXD-I and pssXD-II, respectively. The RSV promoter regulates the gene expression of all components necessary for single-stranded DNA expression, all components transcribed into a single mRNA molecule. Endogenous tRNA ^pro binds to the 3 'end of the transcript and is used as a primer for single-stranded DNA synthesis (Marquet, et al., 77 Biochimie 113-124 (1995)). After reverse transcription by RT of single-stranded DNA, ssDNA is released when template mRNA is degraded by endogenous RNase H or RNase H activity of RT (Tanase and Goff, 85 Proc. Natl. Acad. Sci. USA 1777 -1781 (1988)).

Tissue Culture Research

Stable transfection and transient transfection were performed according to the manufacturer's instructions using DOTAP liposome transfection reagent (Boehringer Mannhiem Corp., Indianapolis, IN). All plasmid constructs were transfected into A549 lung cancer cell line (ATCC CCL-185) and HeLa cell line conserved in Dulbelco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS; GibcoBRL, Gaithersburg, MD). . Assays for ssDNA were performed by PCR and dot-blot analysis 24 to 48 hours after transfection. Ss-cDNA (Mitrochnitchenko, O., et al., Production of single-stranded DNA in mammalian cells by use of a bacterial retron, 269 J. Biol. Chem. 2380-2383 (1994)) Made using Trizol Reagent (Gibco Life Technologies, Gaithersburg, MD). Assays for specific ss-cDNA species were performed by PCR-based analysis of internal fragments and both denatured single stranded gel electrophoresis and subsequent nylon blotting and probing with internal biotin-labeled probes.

In more detail, reverse transcription activity is described by Silver, J .. et al., An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions, 21 Nucleic Acids Res. 3593-3594 (1993), using the RT-PCR assay described above, modified as follows. Cells transfected with pssXA 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 18,000 g for 30 minutes, the supernatant was collected and frozen to -80 ° C until use. Brome mosaic virus (BMV) RNA used as a template was incubated for 10 or 30 minutes at 37 ° C with a lysate that would have RT activity to perform reverse transcription. Using primers of 5'-CGTGGTTGACACGCAGACCTCTTAC-3 'and 5'-TCAACACTGTACGGCACCCGCATTC-3', the products of reverse transcription were amplified by PCR (40 cycles of 94 ° C 20 seconds, 56 ° C 20 seconds and 72 ° C 20 seconds). RT-PCR supernatants were analyzed on a 1.5% agarose gel as shown in FIG. 6.

This RT-PCR assay is dependent on the RT activity present in the cell lysates of the transfected cells to produce cDNA transcripts of the BMV RNA substrate. Since the DNA intermediate does not appear in the virus's replication cycle, there is no chance of amplification products unless reverse transcription occurs first. RT activity was measured in lysates of A549 cells transfected with pssXA plasmids (lanes 3 and 4) and E10 clones and showed relatively high expression (lanes 5 and 6). RT activity was also measured from A549 cells transiently transfected with the control pBK-RSV plasmids (lanes 1 and 2). For transient transfection, lysates were prepared 48 hours after transfection. As a result, 150 bp bands (lanes 3 to 6) of expected size were produced in both lysates from transiently and stably transfected (E10) cells, while not in control lysates (lane 1 And 2).

To detect ssDNA expressed in mammalian cells by pssXB-I and pssXB-II plasmids when co-transfecting A549 cells (E10) with pssXA, a T7 primer and a c-raf DNA enzyme (primer 5'- PCR reactions were performed using CTAGCTACAACGAGACATGC-3 'specific. The entire RNA fraction was used as a template and left with or without pre-treatment with S1 nuclease or RNase A for 30 minutes at 37 ° C. The pre-treated RNA samples were then PCR amplified (30 cycles of 94 ° C. 45 seconds, 55 ° C. 45 seconds and 72 ° C. 30 seconds). PCR products were analyzed on 8% acrylamide gels as shown in Figure 7 (lanes 1 and 3: S1 nucleases; lanes 2, 4 and 5: RNase). Bands of expected size were generated in both the total RNA preparations treated (lanes 2 and 4) and the untreated preparations (data not shown). The product was not amplified in the control formulation treated with the highly specific ssDNA endonuclease S1 nuclease (lanes 1 and 3).

The presence of c-raf DNA enzyme was further confirmed by performing dot-blot detection of ssDNA using North2South chemiluminescent nucleic acid hybridization and detection kit (Pierce) according to the manufacturer's instructions. 2 μg of total RNA isolated from cells transfected with pssXA / pssXB-I or pssXA / pssXB-II, or isolated from pssXA or untransfected cells were used. The biotin-labeled probe specific for c-raf was 5'-GGCCGCACTAATGCATGTCTCGTTGTAGCTA-GCCCAGGCGGGAAGTGC-3 '. As shown in FIG. 8, biotin-labeled c-raf specific oligo probes could only be detected in RNA preparations isolated from E10 cells transfected with pssXB-I or pssXB-II and not transfected E10 cells. Or not detected in A549 cells.

Northern blot analysis was performed to determine whether c-raf mRNA expression was altered in single-stranded c-raf DNA enzymes expressed with the pssXA / pssXB vector system of the present invention in mammalian cells. E10 cell lines were transiently transfected with pssXB-I or pssXB-II. After 24 and 48 hours, cells were harvested to prepare total RNA. 15 μg of total RNA was isolated on denatured agarose gel for Northern blot analysis. After delivering RNA overnight, the membranes were fixed and probed with ³² P-labeled c-raf DNA fragments and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Using a random-priming marker kit (Boehringer Mannheim), a c-raf probe was prepared from an IMAGE ™ cDNA clone (ID 645539, Research Genetics) comprising a coding region at positions 571-2028 of the c-raf kinase gene. G3PDH was also labeled with ³² P and used for standardization of RNA blots. Membranes were washed with 2 × SSC, 0.1% SDS for 15 minutes and with 0.1 × SSC for 5 minutes. The blots were then exposed to X-ray films or quantified by a molecular kinetic phosphomizer (trade name, Melecular Dynamics PhosphoImager ™). The results of representative experiments with phosphorus images are shown in visual form in FIG. 9. Compared to the control group transfected with pssXB containing an unrelated sequence, pssXB-II decreased the amount of c-raf mRNA to 81% at 24 hours and 66% at 48 hours. pssXB-I showed a similar effect, and the amount of c-raf mRNA was reduced to 35% after 48 hours. In addition, significantly increased cell death (about 1/3 increase) was observed in cells transfected with pssXA / pssXB vectors expressing c-raf DNA enzyme compared to the control. Only the remaining adherent cells were harvested except those that started to "float", and the extent of mRNA reduction exceeded 34-36%.

PssXC, a single plasmid vector system, was transfected into the HeLa cell line. Assays for ssDNA were performed by PCR and dot-blot analysis 24 to 48 hours after transfection as described above. Reverse transcriptase activity was performed as described above using the RT-PCR assay developed by Silver et al. (1993). RT activity was further analyzed for individual colony isolates of stably substituted HeLa cell lines (A12 and B12). ss-cDNA was isolated from the transfected cells at 48-72 hours. Ss-cDNA co-located with RNA was made using Trizol reagent (Gibco Life Technologies, Gaithersburg, MD). Assays for specific ss-cDNA species were performed by PCR-based analysis of internal fragments and both denatured single stranded gel electrophoresis and subsequent nylon blotting and probing with internal biotin-labeled probes.

This experiment demonstrated that human tissue culture cells (HeLa cell line) transfected with plasmids designed to synthesize processed ss-cDNA produced ss-cDNA of expected size. As described in Application No. 09 / 397,782, incorporated herein by this nomenclature, the desired ssDNA sequence generated in vivo from pssXC is generated from positions between inverted repeats after digesting the stem of the stem-loop intermediate. Or from the position between the inversion repeat sequence and the primer binding site by terminating the reverse transcriptase at the 3 'side of the stem structure prior to maturation of the cDNA transcript.

Using the total RNA fraction, expression of intracellular single-stranded c-raf DNA enzyme was measured by single dot-blot analysis. Biotin-labeled c-raf specific oligonucleotide probes were synthesized at Integrated DNA Technologies, Coralville, IA and transfected with control pssXD-I or pssXD-II containing c-raf DNA enzyme sequences. It was used to detect signals in RNA samples isolated from A549 cells. 2 μg of total RNA was pretreated with RNase A for 30 minutes at 37 ° C. in the presence or absence of S1 nuclease to completely exclude nonspecific hybridization to RNA. The samples were then loaded onto a Hybond-N + membrane (Amersham Pharmacia Biotec, Piscataway, NJ) and then fixed by exposure to UV for 3 minutes. Hybridization and signal detection were performed using the North2South chemiluminescent nucleic acid hybridization and detection kit (Pierce, Rockford, IL). 11 shows that only cells transfected with pssXD-II showed a positive signal and no detectable signal was observed due to the specific degradation of ssDNA by S1 nuclease in the presence of S1 nuclease.

Quantitative RT-PCR was performed to determine whether single-stranded DNA enzyme expressed in A549 cells changed the amount of c-raf mRNA. c-raf mRNA was quantified by RT-PCR with a slight modification of the method described in Li, et al., 7 Gene Therapy 321-328 (2000). In sum, 1 μg of total RNA was reverse transcribed into the Reverse Transcription System (Promega Corp., Madison, Wis.). Fractions containing the resulting cDNA were PCR amplified using the template. 40 cycles of PCR (95 ° C. 30 sec, 50 ° C. 30 sec and 72 ° C. 60 sec) were performed using specific primers. Specific primer sequences are as follows: 1) c-raf primers: 5'TCAGAGAAGCTCTGCTAAG-3 'and 5'-CAATGCACTGGACACCTTA-3'; 2) Actin primers: 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 c-raf DNA enzyme 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 the amount of loading between different samples. As shown in FIG. 12, a significant decrease in c-raf mRNA amount (about 70-80%) was detected in cells transfected with pssXD-II (lane 2) compared to the control (lane 1).

The amount of c-raf protein in A549 cells transfected with pssXD-I or pssXD-II was assessed by Western blot analysis. 30 μg of cell extracts were electrophoresed (SDS-PAGE) on 12% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred to high-bond ECL membranes (Amersham Pharmacia Biotec, Piscataway, NJ) using a Mini Trans-Blot Electrophoretic Transfer Cell as directed by the manufacturer (BioRad Laboratories, Hercules, CA). Delivered to. The membranes were then blocked in a buffer containing 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween-20 and 5% skim milk, followed by 45 minutes each with the primary and HRP-conjugated secondary antibodies. During incubation. Polyclonal antibodies against c-raf (anti-raf) and monoclonal antibodies against actin (Ab-1) were purchased from Calbiochem-Nova Biochem Corp., San Diego, CA It was. Monoclonal antibodies against poly-ADP ribose polymerase (PARP) (IgG1, C-2-10) are available from Clontech Laboratories, Inc. (Clontech Laboratories, Inc., Palo Alto, Calif.). Proteins were visualized using the SuperSignal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL). As shown in FIG. 13, the amount of c-raf protein in cells transfected with control pssXD-I (lane 2) was similar to the amount in untransfected cells (lane 3). However, the amount of c-raf protein in cells transfected with pssXD-II expressing c-raf DNA enzyme (lane 1) was less than that in the control group (about 20-30%).

To determine whether expression of the c-raf DNA enzyme induces apoptosis in A549 cells, assays for two standard apoptosis assays, genomic DNA cleavage and pARP cleavage, were performed. Genomic DNA cleavage was measured according to the manufacturer's instructions using the LM-PCR Ladder Assay Kit (Clontech Laboratories, Inc., Palo Alto, Calif.). In short, 0.5 μg of genomic DNA was ligated overnight at 4 ° C. using T4 DNA ligase to adapters supplied by Clontech Laboratories, Inc. Fractions of adapter ligated DNA were used as templates in LM-PCR. 25 cycles of PCR (95 ° C. 1 minute and 72 ° C. 3 minutes) were performed at 72 ° C. with extension for 15 minutes. Genomic DNA isolated from cells transiently transfected with pssXD-I (control) or pssXD-II (DNA enzyme) was ligated to specific adapters. LM-PCR was then performed using c-raf primers and specific primers. As shown in FIG. 14, genomic DNA fragmented in cells transfected with pssXD-II (lane 1) was either transfected with the control plasmid pssXD-I (lane 2) or untransfected cells (lane 3). Significantly increased compared to These results suggest that the increase in fragmented genomic DNA is the result of DNA cleavage induced by inhibition of c-raf gene expression altered by the presence of c-raf DNA enzymes.

Another apoptosis assay, PARP cleavage analysis, was performed using Western blot analysis. The amount of full-length PARP in cells transfected with pssXD-II was reduced (FIG. 15) compared to the controls (lanes 2 and 3), which also means induction of apoptosis by inhibition of c-raf gene expression. As measured by the presence of actin, a similar amount of protein is loaded in each lane (lanes 1-3).

* * * * *

The experiments demonstrate that the method of producing ssDNA in vitro and in vivo by multistep reaction using eukaryotic cell RT response and various cDNA priming reactions successfully reduced the expression of c-raf kinase in vivo. Those skilled in the art will appreciate that they are not limited to this particular embodiment of the invention. For example, it will be appreciated that many nucleic acid sequences may be used depending on the particular target and / or mode of inhibitory action of the SOI. Similarly, the SOI may be located at either or both of the two positions, for example between the IR and / or between the PBS and the 3 'side of the IR. In addition, the SOI may or may not include DNA enzyme sequences depending on the particular target and / or mode of action of the SOI and / or mode of action of the DNA enzyme sequence. Those skilled in the art using the present disclosure will appreciate that any desired therapeutic effect can be produced by transfecting eukaryotic cells with the appropriate SOI using the vector system of the present term. For example, the following inhibitory nucleic acids are known in the art and can be used as SOI to alter gene expression in accordance with the present invention:

(1) A sequence that acts as an antisense oligonucleotide for one or more RNA molecules encoding any of a variety of dopamine receptors for the treatment of Parkinson's disease. This antisense oligonucleotide binds specifically to the expression-regulating sequence of the RNA molecule, thereby selectively regulating the expression of one or more dopamine receptor subtypes and alleviating the pathological conditions associated with their expression;

(2) Expression of KSHV virion protein 26, comprising a sequence that acts as an antisense and / or triplet oligonucleotide for the treatment of Karposi's syndrome, as described in US Pat. No. 5,856,903. Sequences that inhibit;

(3) oligonucleotides that regulate the expression of IL-8 and / or IL-8 receptors that control tumor proliferation, metastasis and / or angiogenesis, as described in US Pat. No. 5,856,903;

(4) Nucleotide bases that specifically hybridize to selected sequences of cytomegalovirus DNA or RNA, specifically to sequences targeting cytomegalovirus DNA or RNA encoding IE1, IE2 or DNA polymerase proteins. Oligonucleotides with Sequences. As described in US Pat. No. 5,442,049, such oligonucleotides preferably have about 5 to about 50 nucleic acid base units;

(5) Herpes simplex virus type 1 UL5, UL8, UL9, UL20, UL27, UL29, UL30, UL42, comprising nucleotide units with sufficient identity and number to allow for specific hybridization; An oligonucleotide that specifically hybridizes to RNA or DNA derived from a gene corresponding to either of UL52 and IE175 open reading frames. This oligonucleotide is preferably hybridized specifically to the translation initiation site, coding region or 5 'untranslated region. The oligonucleotides include Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus, human herpes virus type 6, Epstein Barr virus; EBV) or varicella zostr virus (VZV) is designed to specifically hybridize to DNA, or preferably RNA, derived from either species. Such oligonucleotides are conveniently and preferably formulated as pharmaceutical compositions in pharmaceutically acceptable carriers, as described in US Pat. No. 5,514,577. Those skilled in the art will appreciate that the particular open reading frame described for Herpes simplex virus type 1 may find corresponding sequences in other viruses. Thus, each of the Herpes Simplex Virus Type 2, Cytomegalovirus, Human Herpes Virus Type 6, Epstein Barr Virus or Varicella Zoster Virus will each have a number of similar open reading frames that encode proteins with similar functions. It is believed. Accordingly, the present invention relates to antisense oligonucleotide therapies wherein the oligonucleotides are derived for any of the above viruses, or any similar virus known below (with one or more similar open reading frames). . In the present invention, all such viruses are referred to as herpes viruses for convenience;

(6) antisense oligonucleotides for the proto-oncogene, and especially for the c-myb gene, for use as anti-tumor and immunosuppressive agents, as described in US Pat. No. 5,098,890;

(7) interleukin-1 beta, for use as an anti-inflammatory agent with activity against various inflammatory diseases or diseases with inflammatory components such as asthma, rheumatoid arthritis, taga transplant rejection, inflammatory bowel disease, various dermatological symptoms and psoriasis Antisense oligonucleotides that interfere with ICAM-1 gene expression in cells stimulated with. In addition, as described in US Pat. No. 5,843,738, inhibitors of ICAM-1, VCAM-1 and ELAM-1 also treat rhinovirus infection, AIDS, Kaposi's sarcoma, and treatment of some tumors and colds due to their metastases. Can be effective. Similarly, International Application No. PCT / US90 / 02357 discloses DNA sequences encoding endothelial adhesion molecules (ELAM), including ELAM-1, VCAM-1 and VCAM-1b. It is contemplated that oligonucleotides designated ISIS 1570 and ISIS 2302 may be used as target sequences in the methods of the present invention for reducing the metastatic capacity of target cells; And

(8) Protein-binding oligonucleotides (aptamers) that specifically bind to target molecules (especially thrombin), such as proteins, in host cells, as described in US Pat. No. 5,840,867. These non-oligonucleotide target molecules bind to nucleic acids that specifically regulate the biological activity of the protein (Blackwell, TK, et al., 250 Science 1104-1110 (1990); Blackwell, TK, et al., 250 Science 1149-1152 (1990); Turek, C. and L. Gold, 249 Science 505-510 (1990); Joyce, GF, 82 Gene 83-87 (1989)).

Although the present invention has been described with reference to the drawings and specific examples described herein, one of ordinary skill in the art appreciates that certain components described herein can be changed in a manner that does not alter the function of the components achieving each intended result. will be. For example, the cassettes described herein have been described as comprising three gene components (target sequence, primer binding sequence and tandem inversion repeat sequence), and are directed to a target cell with a reverse transcriptase gene under the control of a suitable promoter. Upon infection, the inhibitory nucleic acids described herein are produced. However, those skilled in the art will appreciate that the mouse molony leukemia virus reverse transcriptase gene described for use as a reverse transcriptase gene in a cassette, for example, is a reverse transcriptase gene (the reverse transcriptase gene from human immunodeficiency virus is one of the genes mentioned above). It will be appreciated that it may be substituted, and it will also be appreciated that promoters other than the CMV promoter described herein may be advantageously used. As noted above, the formed stem-loop intermediate may or may not include a restriction enzyme site and may advantageously manipulate its susceptibility to denaturation depending on the particular desired sequence intended to be produced from the intermediate. have. All such changes and other modifications of the present specification that will become apparent to those skilled in the art without departing from the scope of the invention are intended to be included within the scope of the following claims.

1.Name: 3'-RT / Mol-HindIII (24-mer)

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

2. Name: 5'-RT / Mol-SacⅠ (32-mer)

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

3. Name: 5'-MboⅡ-HindIII (30-mer)

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

4. Name: 5'-RT-Not-Mbo-Link (129-Mer)

Sequence: 5'-CTA GGT CGG CGG CCG CGA AGA TTG GTG CGC ACA CAC ACA ACG CGC ACC AAT CTT CGC GGC CGC CGA CCC GTC AGC GGG GGT CTT TCA TTT GGG GGC TCG TCC GGG ATC GGG AGA CCC CTG CCC AGG GCC

5. Name: 3'-RT-Not-Mbo-Link (121-Mer)

Sequence: 5'-CT GGG CAG GGG TCT CCC GAT CCC GGA CGA GCC CCC AAA TGA AAG ACC CCC GCT GAC GGG TCG GCG GCC GCG AAG ATT GGT GCG CGT TGT GTG TGT GCG CAC CAA TCT TCG CGG CCG CCG AC-3 '

6. Name: 5'-Nhe-Sac-Link (18-mer)

SEQ ID NO: 5'-CTA GCG GCA AGC GTA GCT-3 '

7. Name: 3'-Nhe-Sac-Link (10-mer)

SEQ ID NO: 5'-ACG CTT GCC G-3 '

8. Name: 3'-MboⅡ-XbaⅠ (27-mer)

SEQ ID NO: 5'-TAA TGG CCC GGG CAT AGT CGG GTA GGG-3 '

9. Name: 5'-Hind-link-Histag (43-Mer)

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

10. Name: 3'-Hind-link-Histag (42-mer)

Sequence: 5'-AGC AGG GGC AGG GTG GTG GTG GTG GTG GGG AGC GGG GGA TCC-3 '

11.Name: 5'-Not-link-test1 (57-mer)

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

12.Name: 3'-Not-link-test1 (57-mer)

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

13. Name: 5'-Not-Mbo-link-telo (92-mer)

Sequence: 5'-GGC CGG AAG ATT GGG GCG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG CGC CCC AAT CTT CC-3 '

14. Name: 3'-Not-Mbo-link-telo (92-mer)

Sequence: 5'-GGC CGG AAG ATT GGG GCG CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CCC TAA CGC 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 AAC ACA GCG GCC GCA TCC GAT CAG CGG GGG TCT TTC ATT TGG GGG CTC 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 CAA ATG AAA GAC CCC CGC TGA TCG GAT GCG GCC GCT GTG TTG TTT GTT GTT GTG CAG CGG CCG CAT CCG A-3 '

17. Name: XmaⅠ-BglⅡ-Stop 1

SEQ ID NO: 5'-CCGGATCTAGACCGCAAGCTTCATTTAAA-3 '

18. Name: XmaⅠ-BrlⅡ-Stop 2

SEQ ID NO: 5'-GATCTTTAAATGAAGCTTGCGGTCTCGAT-3 '

<Sequence list>

Claims (8)

A cassette comprising a target sequence comprising a nucleic acid sequence designed to produce a nucleic acid sequence that binds to an endogenous nucleic acid sequence when reverse transcribed, an inverted tandem repeat located at both ends thereof and a 3 ′ primer binding site (PBS) Introducing into the target cell; Reverse transcripting the mRNA transcript of the cassette from PBS to release the single-stranded cDNA transcript into the cell; And binding the cDNA transcript to an endogenous nucleic acid target sequence to change the expression of that target sequence A method for altering the expression of an endogenous nucleic acid target sequence in a target cell, comprising. The method of claim 1, further comprising transfecting the reverse transcriptase / RNase gene into the target cell. The method of claim 1, further comprising linearizing the transcript of the desired sequence. 4. The cDNA transcript of claim 3, wherein the cDNA transcript of the desired sequence forms a stem-loop intermediate by the Watson-Crick base pair formation of an inverted tandem repeat sequence, thereby including a restriction site in the inverted tandem repeat sequence To be linearized. The method of claim 2, further comprising inducibly promoting transcription of a reverse transcriptase gene. The method of claim 5, wherein the transcription of the reverse transcriptase gene is promoted by a promoter of eukaryotic cells. CDNA transcript produced by the method of claim 1. A target cell in which the expression of a nucleic acid target sequence is changed according to the method of claim 1.