MXPA01003643A - PRODUCTION OF ssDNA IN VIVO. - Google Patents

Abstract

Methods and compositions for producing single-strand cDNA (cDNA-ss) in eukaryotic cells, specifically a DNA cartridge that produces cDNA-ss in vivo. The cartridge contains the gene encoding reverse transcriptase / RNase H of murine Moloney leukemia virus, a bacterial restriction endonuclease gene, and a sequence of interest that produces an RNA template from which reverse transcriptase synthesizes cDNA -ss of a specified sequence. The cDNA-ss is then modified to remove all flank vector sequences by taking advantage of the "stem-loop" structure of the ss-cDNA, which is formed as a result of the inclusion of an inverted tandem repeat that allows the cDNA ss is doubled on itself, forming a DNA stem of double helix, in the sequence of interest. The double helical stem contains one or more functional genetic elements such as a restriction endonuclease recognition site and the loop, which remains as ss-DNA, constituted by any desired nucleotide sequence. This design allows the double helix stem of the intermediate stem loop to be broken to the corresponding restriction endonuclease (s) specific to the site in the stem and the loop portion, or sequence of interest, it is then released as a piece of linearized DNA, of a single filament. This piece of ss-DNA released (or broken) contains minimal sequence information, if at all, either upstream of 5'o downstream of 3'of the detailed double helix portion containing the endonuclease cleavage site of restriction. In vivo transfections using the DNA vector system described herein demonstrate the use of this system to produce ss-DNA in host cells

Description

IN-LIVE sDNA PRODUCTION Description The present invention relates to a stable DNA construct, which is conveniently referred to as a cartridge, within which a nucleic acid sequence is incorporated to be used as a template for the subsequent production of that sequence in a host cell prokaryotic or eukaryotic, and vector systems for the expression of that sequence inside the eukaryotic host cells without (or with minimal) flanking sequences. The cartridge includes inverted a tandem repeat that forms the stem of a stem-spire intermediate that functions in vivo to cause the expression of the sequence as a single-stranded DNA sequence (ssDNA), referred to as the sequence of interest. The vector system of the present invention removes the contiguous plasmid (or other vector sequences) from the ssDNA sequence of interest, either by stalk-turn formation with the subsequent termination of a reverse transcription reaction via the stem , or by dissociation of the stem-spire intermediary. The ssDNA that is produced by this method can be designed in such a way that it is complementary to any target endogenous nucleic acid sequence.

As far as is known, there is no method available to produce single chain deoxyribonucleic acid (ssDNA) species in eukaryotic cells, which do not contain intervening and / or flanking vector sequences. The scientific and patent literature includes the description of cDNA-producing vectors (see A. Ohshima, et al., 89 Proc. Nati, Acad. Sci. USA 1016-1020 (1992), S. Inouye, et al., 3 Current Opin. Genet, Develop, 713-718 (1993), O. Mirochnitchenko, et al., 269 J. 3iol, Chem. 2380-2383 (1994), JR Mao, et al., 270 J. Biol. Chem. 19684-19687 ( 1995) and U.S. Patent Nos. 5,436,141 and 5,714,323), but the systems described in these references do not appear to have demonstrated the ability to produce ssDNA in eukaryotic cells without intervening vector sequences that may interfere with the intended function of the ssDNA product. It is therefore an object of the present invention to provide a method for producing single-stranded nucleic acid in yeast, prokaryotic and / or eukaryotic cells, which overcomes this limitation by providing a method, and a DNA construct that directs the ssDNA synthesis of any desired nucleotide sequence in vivo, without undesirable intervening nucleotide bases or flanking. The ssDNA can be (but is not limited to) an inhibitory nucleic acid such as an anti-sense sequence for binding to the mRNA in an anti-sense fashion to down-regulate a genetic product or a viral genetic product of interest, or for binding to the duplex (native DNA) to form triple structures that may interfere with the transcription and regulation of normal genes. The ssDNA produced in this way can also function to interrupt one or more of many highly regulated cellular functions. For example, the ssDNA tails of telomeric repeats can be altered, through the production of a ssDNA having a nucleotide base composition identical or complementary to the native DNA sequence in the telomer repeats or other regulatory sequence. This objective, and the many others that will be apparent to those skilled in the art, by the following description of many embodiments of the invention, are achieved by providing a cartridge, or nucleic acid construct, comprising a nucleic acid sequence , comprising a sequence of interest flanked by inverted tandem repeats, a gene encoding an RNA-dependent DNA polymerase, and a gene encoding a restriction endonuclease. The cartridge also preferably includes a gene encoding an NRase H and eukaryotic promoter (s) / promoter (s) either constitutive or inducible for the RNA-dependent DNA polymerase and the restriction endonuclease genes. The invention also contemplates that the cartridge is incorporated within a plasmid, and that the plasmid is incorporated into a suitable host cell. In another aspect, the present invention comprises a method for producing single-stranded DNA in vivo, comprising the steps of transcription and translation of a cartridge comprising an RNA-dependent DNA polymerase gene, and a sequence of interest, in a eukaryotic cell, and the conversion of the mRNA transcript of the sequence of interest to cDNA, with the polymerase produced by the RNA-dependent DNA polymerase gene, with the simultaneous digestion of the template of the mRNA component with an expressed enzyme of NRase H. The sequence of interest also includes an inverted tandem repeat. The cartridge may also include a restriction endonuclease gene that, when transcribed or translated, produces a restriction endonuclease that linearizes the transcript of the sequence of interest, by cutting the DNA-ss at the site of the restriction endonuclease formed when the inverted tandem repeat causes the transcript to form a stem-spire intermediate. In another aspect, the present invention comprises a method for producing a single-stranded oligonucleotide in a target cell. In one embodiment, this method is intended to send an anti-sense sequence. In other embodiments, the method is used to send triple-forming sequences, or sequences that are recognized and fixed by specific DNA binding proteins, or other nucleic acids and / or proteins that function in metabolism and / or cell replication . The method comprises encoding the oligonucleotide into a complementary sequence of interest, into a cartridge that includes a gene encoding an RNA-dependent DNA polymerase, preferably including a NRase H gene, and a eukaryotic promoter / enhancer that can be inducible or constitutive, appropriate for that polymerase / NRase H gene. The cartridge includes a gene encoding a restriction endonuclease (RE) and, in the preferred embodiment, a promoter / enhancer appropriate for that RE gene. The cartridge further comprises an inverted tandem repeat and, when assimilated into the target cell, the cell transcribes the cartridge (including the sequence of interest and inverted tandem repeats), under the control of the promoter (s) / enhancer (is) . The normal function of the target cell causes the transcript of mRNA resulting from the polymerase and the RE genes to be transferred, providing whatever is necessary for the production of ss-DNA from the mRNA transcript of the sequence of interest. Specifically, the RNA-dependent DNA polymerase produced from the cartridge converts the mRNA transcript of the sequence of interest, and the inverted tandem repeats to cDNA-ss, the ss-cDNA forms a stem-loop intermediate as the bases of the nucleotide comprising the inverted tandem repeats in pairs, and the restriction endonuclease produced from the RE gene in the cartridge digests the double-stranded portion of the stem-loop intermediate, to release the DNA oligonucleotide from a single strand of the cycle portion of the stem-turn intermediary. Multiple systems can be used to send the cartridge to the target cell, to direct ssDNA synthesis inside the cell, including plasmid-based or plasmid-based vector systems, or virus-based vector systems, and these systems are adapted to that purpose in accordance with a practicing technician. These systems include, but are not limited to, virus-based systems such as adenoviruses, adeno-associated viruses, retroviral vectors, and conjugated vectors using transfection systems based on double-stranded plasma DNA. Once inside the cell, the cartridge is transcribed in the normal course of cellular metabolism, producing a mRNA transcript of the sequence of interest, which is then converted to cDNA by reverse transcriptase, which is also produced by the cell from the reverse transcriptase / NRase H gene, included in the cartridge, under the control of the promoter. In accordance with the present invention, there is provided a nucleic acid construct comprising an inverted tandem repeat, a primer binding site for a reverse transcriptase located at a 3-position with respect to the inverted tandem repeat, and a localized sequence of interest. either between the inverted tandem repeat, or between the inverted tandem repeat and the 3 'primer binding site.

The nucleic acid construct of the present invention allows a single-stranded nucleic acid sequence to be produced reliably and stably in a target cell. In particular, the inverted tandem repeat can be used to create a stem-loop intermediate of nucleic acid, to produce and / or control the production of ssDNA in vivo, with contiguous nucleotide sequences reduced or eliminated. Due to the configuration of the nucleic acid construct, with the primer binding site in a position that is 3 'to the sequence of interest, there is no limit to the size or type of sequence of interest that can be produced using the nucleic acid construct of the present invention, and the construct can be easily incorporated into a vector for delivery by any desired route to a target cell. Where the sequence of interest is located between the inverted tandem repetition, the inverted tandem repetition is preferably able to form a stem-loop intermediate, with the sequence of interest in the loop, and the inverted tandem repetition forming the stem . An advantage of this stem-loop structure is that the sequence of interest can be released from the loop as a single-stranded cDNA, with reduced or deleted flanking and / or intervening vector sequences, by cutting the loop where it joins the stem. The ssDNA thus obtained is free or substantially free of any intervening and / or flanking nucleotide sequences, for example, vector nucleotide sequences, which could interfere with the intended function of the ssDNA product. Where a sequence of interest is located between the inverted tandem repeat, a second sequence of interest can be additionally provided between the inverted tandem repeat and the 3 'primer binding site. This embodiment of the invention has the advantage that the inverted tandem repeat can be selected to produce stems of different stabilities, by means of which variable amounts of reading can be achieved through the mRNA transcript, or early termination of the transcription, providing by the same a method for regulating the production of two or more sequences of interest by secondary bending of the intermediate mRNA transcript. Preferably, the inverted tandem repeat comprises one or more specific enzyme recognition sequence (s). More preferably, the specific enzyme recognition sequence comprises a restriction endonuclease site. The restriction endonuclease site can either be recognized by an endogenous restriction endonuclease, or the nucleic acid construct of the present invention can additionally comprise a gene encoding a restriction endonuclease for that restriction endonuclease site. In the case that the nucleic acid construct further comprises a restriction endonuclease gene, the restriction endonuclease gene is preferably located at a 5 'position with respect to the inverted tandem repeat. The specific enzyme recognition sequence may include, for example, the restriction sites either Hind III and Not I, Hind III, or Not I. In fact, the specific enzyme recognition sequence may be any of a restriction endonuclease. Type I, endonuclease type II, endonuclease type III, recognition of eukaryotic receptor, recognition of prokaryotic receptor, promoter, promoter / enhancer, and cantilever PCR site in T, or combinations thereof. Where the sequence of interest is located between the inverted tandem repeat and the 3 'primer binding site, or where a first sequence of interest is located between the inverted tandem repeat, and a second sequence of interest is located between the inverted tandem repeat and the primer binding site 3 ', the inverted tandem repetition is, preferably, capable of forming a stable stalk-spire, an unstable stem-spire or a stalk-spire of intermediate stability. The inverted tandem repeats are selected to form a stable stem-turn in the mRNA transcript as a means to cause the premature termination of the transcript, such that if a sequence of interest is located in the mRNA transcript between the start site for reverse transcription and the stem-spire structure, ssDNA can be produced again substantially free of contiguous vector sequences. The inverted double repeat (s) of the present invention allow the use of a stem-loop structure as a vehicle to remove unwanted contiguous nucleotide sequences, for example, either by of causing the premature termination of the reverse transcription of the mRNA transcripts, or by cutting the unwanted sequences of the cDNA by means of restriction endonuclease sites in the inverted tandem repeats. According to another preferred embodiment of the present invention, the inverted tandem repeat may comprise one or more sites of DNA binding of eukaryotic, prokaryotic, and / or viral protein. The stem-coil structure of the present invention, therefore, has the further advantage that it can also serve as a functional entity in itself, for example, to protect the ssDNA in the turn of degradation by intracellular nucleases, or to perform certain applications by means of functional elements (for example, protein binding sites) in inverted tandem repeats. Preferably, the inverted tandem repeat acts in the cis-oriented manner. The primer binding site may be specific for an endogenous reverse transcriptase (e.g., in the case of a cell infected with the human immunodeficiency virus or the simian immunodeficiency virus). Alternatively, the nucleic acid construct may additionally comprise a gene encoding a reverse transcriptase. The reverse transcriptase gene is preferably located at a 5 'position, with respect to the inverted tandem repeat. Alternatively, the nucleic acid construct may additionally comprise a gene encoding a reverse transcriptase / RNase polyprotein. The reverse transcriptase polyprotein / RNase H gene is preferably located at a 5 position, relative to the tandem repeat. inverted The reverse transcriptase polyprotein / RNase H gene can be derived from the Moloney murine leukemia virus, the human immunodeficiency virus, or the simian immunodeficiency virus. In the case that the nucleic acid construct of the present invention is provided with a reverse transcriptase or reverse transcriptase polyprotein / RNase H gene, the primer binding site is preferably specific for reverse transcriptase or for the reverse transcriptase polyprotein / RNase H encoded by their respective genes. Preferably, the nucleic acid of the present invention further comprises a promoter and, optionally, an enhancer for each of the first or second sequence of interest, that restriction endonuclease, the reverse transcriptase and / or the reverse transcriptase gene. RNAase H.

More preferably, the promoter and / or enhancer is a eukaryotic promoter / enhancer. The promoter can be a constitutive promoter, which can be induced broadly, or specific to the tissue. Preferably, the nucleic acid construct of the present invention additionally comprises a polyadenylation tail sequence, located at a 3 'position with respect to the 3' primer binding site. The polyA tail allows the stabilization of the mRNA transcript. Preferably, the first or second sequence of interest includes a sequence encoding a single-stranded DNA having enzymatic activity. An example of such single-stranded DNA having enzymatic activity comprises the 5'-GGCTAGCTACAACGA-3 'sequence, which has RNAse activity. This sequence is flanked in both 5 'and 3' directions by one or more sequence (s) that are complementary to a target mRNA species, eg, h-ras kinase, c-raf, anti-sense sequence to the growth factor angiogenic pleiotrophin, or the tat region of the simian immunodeficiency virus. Preferably, the primer binding site is complementary to a transfer RNA (tRNA). Preferably, the nucleic acid construct is DNA. The nucleic acid construct of the present invention thus provides an efficient system for directing the synthesis of a stable, single-stranded nucleic acid sequence, both in vivo and in vitro. The nucleic acid sequence can be used to provide a desired effect in a cell, tissue or organism. Because the production of the nucleic acid sequence of a single chain of interest takes place within the cell, the problems of the prior art arising from the delivery of the nucleic acid sequence of the cells are overcome, or at least mitigated. a single chain to the cell. A mRNA transcript of the nucleic acid construct of the present invention is also provided. In accordance with another aspect of the present invention, there is provided an mRNA transcript comprising a sequence of interest flanked by an inverted tandem repeat, and further comprising a primer binding site located 3 'to the inverted tandem repeat. In accordance with this aspect of the present invention, there is also provided an mRNA transcript comprising an inverted tandem repeat and a primer binding site located at 31 to the inverted tandem repeat, and a sequence of interest located between the inverted tandem repeat and the 3 'primer binding site. In accordance with this aspect of the present invention, there is further provided an mRNA transcript comprising a first sequence of interest flanked by an inverted tandem repeat, a primer binding site located 3 'to the inverted tandem repeat, and a second sequence of interest between the inverted tandem repeat and the 3 'primer binding site. In addition, a single-stranded DNA transcript of the mRNA of the present invention is provided. A vector comprising a nucleic acid construct according to the present invention is also provided. The nucleic acid constructs of the present invention are such that these can be incorporated into commercially available delivery vectors for mammalian and human therapeutic purposes, and can be administered by any feasible route, depending on the target cell. In accordance with the present invention, there is also provided a vector comprising an inverted tandem repeat, a primer binding site for a reverse transcriptase located at a 3 'position with respect to the inverted tandem repeat, and an insertion site for a sequence of interest between the inverted tandem repeat, or between the inverted tandem repeat and the primer binding site. Preferably, the vector comprises a first insertion site between the inverted tandem repeat, and a second insertion site between the inverted tandem repeat and the 3 'primer binding site. This allows the user to insert any desired sequence of interest into either or both of the insertion sites. Due to the configuration of the nucleic acid construct, with the primer binding site in a position that is 3 'to the inverted tandem repeat, there is no limit on the size or type of the sequence of interest that can be Insert inside the insertion site. Preferably, the vector further comprises a gene encoding a reverse transcriptase, or a gene encoding a reverse transcriptase / H-RNA polyprotein. Preferably, the reverse transcriptase or reverse transcriptase polyprotein / H-RNA polyprotein gene is located in a position 5 'with respect to the inverted tandem repetition. In accordance with the present invention, there is further provided a vector system, comprising a first vector in accordance with the present invention, (i.e., a vector comprising an inverted tandem repeat, a primer binding site for a reverse transcriptase located at a 3 'position with respect to the inverted tandem repeat, and an insertion site for a sequence of interest between the inverted tandem repeat, or between the inverted tandem repeat and the primer binding site), and a second vector that it comprises a gene encoding a reverse transcriptase, a reverse transcriptase polyprotein / RNase H, or a reverse transcriptase / RNase polyprotein linked by means of a proline-rich linker to a restriction endonuclease. The vector or vector system of the present invention can also be designed to allow the primer binding site to be removed and interchanged, so that different primer binding sites can be used, depending on the user's requirements and the specificity of the reverse transcriptase that is being used. Preferably, the reverse transcriptase gene, reverse transcriptase polyprotein / RNase H, or reverse transcriptase / RNase H / restriction endonuclease, is operably linked to an expression control sequence. The vector or system of vectors of the present invention can be conveniently employed to send an antisense, sense, triplex, or any other sequence of nucleotides of a single chain of interest, within a cell, using known digestion and ligation techniques for splice the sequence of interest within the vector. The vector or vector system described herein provides all the signaling instructions and enzymatic functions necessary to allow a host cell to produce a single-stranded nucleic acid molecule having a desired sequence. A host cell stably transformed or transfected with a vector or system of vectors according to the present invention is also provided. In particular, the host cell can be a eukaryotic or prokaryotic cell. Eukaryotic cells include yeast, plant and mammalian cells. Prokaryotic cells include bacterial cells. In accordance with the present invention, a kit for producing a single-stranded nucleic acid sequence, kit comprising a vector or system of vectors according to the present invention, and a restriction endonuclease for each insertion site is further provided. . In accordance with another aspect of the present invention, there is provided a kit for producing a single-stranded nucleic acid sequence, kit comprising a vector or system of vectors according to the present invention, a container for the vector / system of vectors, and instructions for the use of the vector / vector system. In accordance with the present invention, an in vivo or in vitro method is also provided for producing a single chain of interest nucleic acid sequence, which method comprises the steps of introducing a nucleic acid construct according to the present invention, into a target cell, transcribe the nucleic acid construct within the mRNA, and reverse transcribe the mRNA transcript within the cDNA. Preferably, the method also comprises the step of removing the mRNA transcript from the mRNA / cDNA heteroduplex, formed by the reverse transcription of the mRNA. Reverse transcription can be performed either by a reverse transcriptase that is endogenous to the target cell, or the method of the present invention can further comprise the step of introducing a gene encoding a reverse transcriptase, or a gene encoding a polyprotein of reverse transcriptase / RNase H, within the target cell. Preferably, the method of the present invention further comprises the step of making the cDNA transcript of the sequence of interest linear, by cutting the stem-spire structure of the cDNA, formed by the inverted tandem repeat wherein the structure of the Spire joins the stem. In accordance with this latter embodiment of the present invention, the method further comprises the step of introducing a gene encoding a restriction endonuclease within the target cell. By providing one or more restriction endonuclease sites in the inverted tandem repeat, the restriction endonuclease expressed by the restriction endonuclease gene can then be used to cut the stem-spire structure of the cDNA. Alternatively, the cutting of the stem-spire structure of the cDNA may depend on the restriction endonucleases that are endogenous to the target cell. Restriction endonuclease can also be provided by the step of introducing a gene encoding a reverse transcriptase / RNase polyprotein, linked via a proline-rich linker, to a restriction endonuclease within the target cell. Where the single-stranded nucleic acid sequence is prepared by an in vivo method of the present invention, the method may further comprise the step of isolating the mRNA transcript, the mRNA / cDNA heteroduplex and / or the single-stranded cDNA. target cell chain. Also provided is a single-stranded cDNA transcript, an inhibitory nucleic acid molecule (e.g., anti-sense or aptamer sequence), a single-stranded DNA having enzymatic activity (e.g., RNase activity), a mRNA transcript and / or a heteroduplex molecule produced by the methods of the present invention. An inhibitory nucleic acid molecule can be single stranded DNA synthesized from the mRNA transcript, or the mRNA transcript itself, which can be specifically bound to a complementary nucleic acid sequence. These inhibitory nucleic acid molecules can be "sense" or "antisense" sequences, and are particularly useful for regulating gene function. An inhibitory nucleic acid molecule can also be an oligonucleotide that binds specifically to a biomolecule, for example, thrombin, bradykinin or PGF2a, which is not normally bound to RNA or DNA. "In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid construct, the vector or vector system, or the host cell according to the present invention, together with an auxiliary, diluent or carrier. pharmacologically acceptable.

In accordance with the present invention, there is provided a nucleic acid construct, vector or vector system, or host cell according to the present invention, for use in therapy, especially for use in sending an inhibitory nucleic acid molecule to a host cell The nucleic acid construct, the vector or vector system and the host cell of the present invention are particularly useful for alleviating a pathological condition by regulating gene expression. In accordance with another aspect of the present invention, there is further provided the use of a nucleic acid construct, vector or vector system, or host cell according to the present invention, for the manufacture of a medicament for alleviating a pathological condition by regulation of gene expression, especially to alleviate a pathological condition by sending an inhibitory nucleic acid molecule to a target cell. Other uses are also described. The sets of genetic elements, vectors, vector systems and host cells of the present invention can be used for the prophylactic or therapeutic treatment of a wide range of conditions or diseases, particularly conditions or diseases that are caused by the abnormal or altered expression of genes, or conditions or diseases that can be alleviated by regulating gene expression.

The sets of genetic elements, vectors, host cells, kits and methods of the present invention can be used to produce single-stranded nucleic acid molecules, or virtually any previously defined or desired nucleotide base composition in a host cell, and They can adapt and apply to any system in vivo or in vitro. According to a preferred embodiment, the nucleic acid construct of the present invention is an artificially synthesized, recombinant, chimeric, and / or heterologous product, and the sequence of interest may be foreign to the cell in which it is produced. . The many embodiments of the invention are illustrated in the figures in which Figure 1 is a schematic illustration of the production of cDNA-ss in a host cell, according to the present invention. Figure 2 is a schematic illustration of the stem-spire intermediate formed by the method illustrated in Figure 1. Figure 3A is a schematic representation of the attachment of an anti-sense sequence, produced in accordance with the present invention, that incorporates the enzyme DNA 10-23 that binds to a target mRNA, and the subsequent dissociation of the target mRNA. Figure 3B is an enlarged representation of the binding of the anti-sense sequence of Figure 3A to the target mRNA, showing the interaction of the enzyme DNA 10-23 (included in a generalized inhibitory nucleic acid sequence) with the cleavage site of the target mRNA. Figure 4 is a schematic map of the plasmid ADNpss-Express A constructed in accordance with the present invention. Figures 5A, 5B, and 5C depict a schematic map of the plasmid DNApss-Express B constructed in accordance with the present invention, an enlarged portion of the plasmid DNApss-Express B, and the sequence of the insertion region of the plasmid DNApss-Express B , respectively. Figures 6A and 6B depict the sequence of the insertion regions between the Apa I and Nhe I sites of the pTest and pTelo plasmids constructed from the plasmid DNApss-Express B, according to the present invention. Figure 7A is a schematic map of the starting plasmid ADNpc3.1Zeo + (Invitrogen, Inc.) for the plasmid DNApc3. lZeo + / NM-link2-gag / ADNpss-Express-4B constructed in accordance with the present invention. Figure 7B is a schematic map of cDNA3. lZeo + / NM-link2-gag (also designated pssADN-Express-4B). Figure 7C is a schematic representation of the cloning sites, the inverted repeats, and the PBS region (arrow) for ADNpc3. lZeo + / NM-link2-gag / ADNpss-Express-4B. Figure 7D is a sequence map of the cloning sites, the inverted repeats, and the PBS region (arrow) for cDNA3. lZeo + / NM-link2-gag / ADNpss-Express-4B. Figure 8A is a schematic map of the plasmid ADNpss-Express A constructed in accordance with the present invention. Figure 8B is a schematic map of the plasmid ADNpss-Express A after deletion of the Mbo II gene by digestion with Xma I and Sac II. Figure 9A is a schematic map of the plasmid ADNpss-Express-C constructed in accordance with the present invention. Figure 9B is a schematic representation of the cloning sites, the inverted repeats, and the PBS region (arrow) for the plasmid DNApss-Express-C. Figure 9C is a sequence map of the cloning sites, the inverted repeats, and the PBS region (arrow) for the plasmid DNApss-Express-C. Figure 10A shows the partial sequence of the 23rd codon of the h-ras anti-sense binding sequence, with the sequence of the DNA enzyme 10-23 inserted between the complementary sequences 5 'and 3'. Figure 10B shows the partial sequence of the c-raf kinase anti-sense binding sequence, with the sequence of the DNA enzyme 10-23 inserted between the complementary 5 'and 3' sequences. Figure 10C shows the partial sequence of the pleiotropin anti-sense binding sequence, with the sequence of the DNA enzyme 10-23 inserted between the complementary sequences 5 'and 3'. Figure 10D shows the partial sequence of the anti-sense binding region tat of the SIV sequence, with the sequence of the DNA enzyme 10-23 inserted between the complementary sequences 5 'and 3'. Figures 11A and 11B are gels showing the results of PCR reverse transcriptase assays (J. Silver, et al., 21 Nucleic Acids Res. 3593-3594 (1993)) in RNA / ssDNA extracts from HeLa cell lines, transformed with the pTest plasmid constructed in accordance with the present invention. Lanes, Figure 11A: (1) size markers, (2) untransformed HeLa cells, (3) HeLa cells transformed with the vector ADNpc3. lZeo +, (4) HeLa cells transformed with pssXa, (5) positive control containing 2 milliunits of MoMuLV reverse transcriptase, and (6) negative control that does not contain any protein extract. The arrow indicates the amplification product of 150 base pairs. Lanes, Figure 11B: (1) clone A12, (2) B8, (3) B12, (4) D7, (5) positive control containing 2 milliunits of MoMuLV reverse transcriptase, and (6) negative control containing no protein extract. Figure 12 are gels showing the PCR amplification of the DNA of a single strand of extracts of HeLa cell lines transformed with the pTest plasmid constructed in accordance with the present invention. RNA / ssDNA preparations were used as templates in PCR reactions, using primers specific to the expected ssDNA product. Left panel, lanes 1-3: total RNA / ssDNA PCR template isolated from the stably transformed Al2 colony (stable HeLa cell line transfected with the plasmid ADNpss-Express-A) (1) used without any previous treatment, (2) treated with nuclease SI, (3) treated with RNase A. Lane (4) total RNA / ssDNA from a colony stably transformed with the vector pcpc3. lZeo + solo. Right panel, lanes 1-3: Total RNA / ssDNA PCR template isolated from the stably transformed B12 colony (HeLa cell line stably transfected with the plasmid ADNpss-Express-A) (1) used without any previous treatment, (2) treated with nuclease SI, (3) treated with RNase A. Lane (4) total RNA / ssDNA from a colony stably transformed with the vector ADNpc3.1Zeo + alone. Lane (5), positive control template, pTest plasmid. Figure 13 is a gel showing PCR amplification of DNA from a single strand of extracts from cells transformed with the pTelo plasmid constructed in accordance with the present invention. RNA / ssDNA was harvested from cultures of transfected cells, at 36 hours after the transference, and were used as templates for the PCR reactions, using primers specific to the expected ssDNA product. The rail marker of 25 base pairs was used. Lanes 1-5 with isolation of the HeLa A12 cell line, (1) total RNA / ssDNA fraction, (2) total RNA treated with nuclease SI, (3) total RNA treated with RNase A, (4) negative control, (5) positive control telomere DNA plasmid. Lanes 6-9, RNA isolated from B12 cell line, (6) total RNA / ssDNA fraction, (7) total RNA treated with nuclease SI, (8) total RNA treated with RNase A, (9) negative control, (10) positive control telomere DNA plasmid. Lanes 11-12, negative repetition controls. Acrylamide gel at 8 percent at 45 V for 20 minutes.

Figure 14 is a gel showing the truncation of ss-cDNA transcription and reading products by in vitro reverse transcriptase reaction. Lanes 1-5 represent plasmid constructions with stem-spire structures constructed in accordance with the present invention, and different sequences of interest cloned within the loop region. The RNA template was produced by in vitro transcription with the T7 RNA polymerase under standard conditions, then treated with DNase to remove the template from the plasmid. Then RNA groups extracted by phenol / chloroform were transcribed in reverse, with mouse Moloney reverse transcriptase, treated with RNAse A for 15 minutes, and resolved on a 6 percent acrylamide gel at 45 V for 30 minutes. . Marker of 25 base pairs on the edges. (1) RT reaction of 10 μ? of the original plasmid ADNpss-Express-B constructed in accordance with the present invention, (2) RT reaction of 10 μ? of the original pTest plasmid constructed in accordance with the present invention, (3) RT reaction of 10 μ? of the original plasmid constructed in accordance with the present invention, (4) negative control plasmid pSDc-Zeo (Invitrogen), (5) repeat of 3 μ? ADNpss-Express-B. Figure 15 shows a Northern blot of an anti-sense producing vector, constructed in accordance with the present invention, which produces an anti-sense sequence against the c-raf kinase (Figure 10B), and which includes the "DNA enzyme". -23"after transfection of the plasmid DNApss-Express-C (Figure 9) that includes that sequence within a cell line of lung cancer in vi tro. In this description of the preferred embodiments of the present invention, the nucleic acid methods and constructs are described for use to produce single chain (cDNA-ss) deoxyribonucleic acid oligonucleotides of virtually any previously defined nucleotide base composition or desired in vitro, in yeast, prokaryotic cells, and / or eukaryotic cells, with or without flanking nucleotide sequences. Methods and constructs using biological synthesis are described rather than in vitro, or artificial synthesis of the ss-DNA of the desired nucleotide base composition. Because biological reactions are used, that is, enzymatic in these methods, they can be applied to any system in vivo. A vector system was designed (as used herein, the term "vector" refers to a plasmid or viral construct modified to send and manipulate DNA segments of interest) to produce any DNA sequence such as a DNA-cDNA molecule. ss, free of most contiguous vector sequences, inside yeast, prokaryotic, and / or eukaryotic cells. The vector system contains all the enzymatic functions and signaling instructions necessary to enable the host cell to which the vector is sent, to produce the cDNA-ss. The cell to which the vector of the present invention is sent produces an RNA transcript (Figure 1), driven by a eukaryotic promoter, just as the eukaryotic promoters drive the enzymes described above, which is used as a template to direct the synthesis of any desired single-stranded DNA sequence (a "sequence of interest"). In more detail, a first system is described in which the vector comprises two plasmids that are co-transfected into a suitable host cell, which can be yeast or any prokaryotic or eukaryotic cell, to produce a ssDNA that includes the sequence of interest in the cell. A second system is described in which the vector system comprises a single plasmid containing the sequence of interest that is transfected into a host cell suitable for the production of an inhibitory nucleic acid, such as the anti-sense sequence, of the sequence of interest. The inhibitory nucleic acids can be synthesized to ssDNA from the mRNA template or the mRNA template itself, which can be specifically bound to a complementary nucleic acid sequence. By binding to the appropriate target sequence, a double or triple of RNA-RNA, AD-DNA, or RNA-DNA is formed. These nucleic acids are often referred to as "anti-sense" because they are usually complementary to the sense or coding strand of the gene, but the "sense" sequence is also used in the cell for therapeutic purposes. For example, the identification of oligonucleotides that bind specifically to biomolecules that normally do not bind to RNA or DNA has now been demonstrated for many biomolecules that vary widely in size, structure and composition. Examples of those molecules include (but are not limited to): (1) thrombin, a multi-function regulatory protein that converts fibrinogen to fibrin in the process of clot formation; (2) bradykinin, a nonapeptide quinine involved in the regulation of blood pressure, and involved in hypertension; (3) PGF2 alpha, a prostglandin or fatty acid derivative that exhibits hormonal activity. Additionally, the interaction of oligonucleotides with biomolecules whose natural biological function is mainly extracellular has now been demonstrated (see, for example, U.S. Patent No. 5,840,867). The term "inhibitory nucleic acids", as used herein, therefore refers to both "sense" and "antisense" nucleic acids. By binding to the target nucleic acid, an inhibitory nucleic acid inhibits the function of the target nucleic acid. This inhibitory effect is the result of, for example, blockage of DNA transcription, processing or addition of poly (A) to mRNA, inhibitory mechanisms of replication, translation or promotion of cell DNA (such as promotion of RNA degradation). The inhibitory nucleic acid methods, therefore, encompass many different approaches to alter gene expression. These different types of inhibitory nucleic acids are described in Helene, C. and J. Toulme, 1049 Biochim. Biophys. Acta. 99-125 (1990). Briefly, the approaches of inhibitory nucleic acid therapy can be classified into (1) those that target DNA sequences, (2) those that target RNA sequences (including pre-mRNA and mRNA), (3) those that they target proteins (sense chain approaches), and (4) those that cause the dissociation or chemical modification of target nucleic acids such as ssDNA enzymes, including the so-called "enzyme 10-23", as described at the moment. The first approach contemplates many categories. The nucleic acids are designed to attach to the major groove of the double DNA to form a triple helical structure or "triplex". Alternatively, the inhibitory nucleic acids are designed to bind to single stranded DNA regions that are the result of double DNA opening during replication or transcription. Most commonly, inhibitory nucleic acids are designed to bind to mRNA or mRNA precursors. The inhibitory nucleic acids are used to prevent maturation of the previous mRNA. The inhibitory nucleic acids may be designed to interfere with the processing, splicing, or translation of the AR. In the second approach, the inhibitory nucleic acids are directed to the mRNA. In this approach, the inhibitory nucleic acids are designed to specifically block the translation of the encoded protein. Using this second approach, the inhibitory nucleic acid is used to selectively suppress certain cellular functions by inhibiting the translation of the mRNA encoding critical proteins. It has been shown that the inhibitory nucleic acids that target the mRNA work through many different mechanisms to inhibit the translation of the encoded protein (s). An example of such an inhibitory nucleic acid is the sequence that is complementary to regions of the c-myc mRNA, which inhibits the expression of the c-myc protein in a human promyelocytic leukemia cell line, HL60, which over-expresses the proto- oncogene c-myc. icktrom E. L., et al., 85 Proc. Nati Acad. Sci. USA 1028-1032 (1988), Harel-Bellan, A., et al., 168 Exp. Med.2309-2318 (1988). The nucleic acid inhibitors produced in the cell can also use the third approach to design the "sense" chain of the gene or mRNA to trap or compete for the enzymes or binding proteins involved in the translation of mRNA. Finally, nucleic acid inhibitors are used to induce chemical inactivation or dissociation of genes or mRNA. Objective. Chemical inactivation occurs, for example, by inducing cross-links between the inhibitory nucleic acid and the target nucleic acid inside the cell, and by the method contemplated herein, namely, the dissociation of the target mRNA by the sequence having enzymatic activity against the mRNA that is incorporated within the cartridge of the present invention. Briefly, in a first aspect, the present invention comprises a set of genetic elements, adapted to be sent within a cell, to produce ss-DNA in vitro or in vivo. The set of genetic elements is incorporated into at least one vector for delivery within the cell and includes an RNA-dependent DNA polymerase gene (trans-reverse cryptase), and a cartridge that includes (1) an inverted tandem repeat ( IR), (2) a sequence of interest located either between the inverted repeat (IR) or at 3 'to the inverted repeat, and (3) a primer binding site (PBS) for the reverse transcriptase which is located at 31 to the inverted repetition. The vector system also preferably includes the signaling functions and instructions for the transcription of these elements in vivo, and the functions and signaling instructions for the translation of the reverse transcriptase (RT) gene. Additional elements that may be included in the set of genetic elements of the present invention include a restriction endonuclease (RE) gene, which may be included for a purpose that is described below, and a sequence that has enzymatic activity when the DNA -ss linearized is doubled to the secondary configuration that confers enzymatic activity on the sequence. The enzymatic sequence is preferably located within a sequence of interest, regardless of whether the sequence of interest is located between the inverted tandem repeats, or between the 3 'aspect of the inverted repeat and the PBS. In a first embodiment of the vector system described herein, the vector comprises a system of two plasmids, and the first plasmid includes an RNA-dependent DNA polymerase gene (reverse transcriptase) and an RNAse H gene, linked to a histidine-proline linker to a restriction endonuclease gene. The plasmid within which these genes were inserted contains the transcriptional and translational control elements necessary for these genes, together with polyadenylation tail sequences. Herein, this plasmid is referred to as the "A" plasmid (ADNpss-Express-A as shown in Figure 4 in one of the preferred embodiments described herein). A second "B" plasmid was constructed which, in the embodiment described herein, includes the cartridge comprising three of the elements listed above, namely, a primer binding sequence (PBS) coupled to the reverse transcriptase, a sequence of interest, and an inverted tandem repeat. In two embodiments described herein (pMN-link-n evo and pssDNA-Express-4B, the latter is shown in Figure 7B), the sequence of interest is placed either between the inverted tandem repeats, or at a 5 'position (relative to the mRNA transcript) to the primer binding sequence (PBS), the PBS being located in the most 3' aspect of the mRNA transcript. In other words, the sequence of interest is located (1) in the middle of the inverted repeats, (2) between the inverted repeats and the PBS, and / or (3) both between the inverted repeats and in the middle of the inverted repeats and the PBS. Like plasmid A, plasmid B also includes a combination of transcription control elements. However, in at least one preferred embodiment described herein, plasmid B does not include (or require) the translation control elements, since no protein product is produced from this construction. In another embodiment as described herein, the vector system of the present invention comprises a single plasmid, designated as plasmid C, in which all the elements of the set of genetic elements listed above are incorporated, with the exception of the RE gene, which was not included for reasons that will become clear in the following discussion. Additionally, the components of the cartridge included in the plasmid B of the two plasmid system described above, for example, the primer binding sequence (PBS), the sequence of interest, and the inverted tandem repeat, reside in the 3 'portion not translated from the reverse transcriptase polyprotein in plasmid C. In other words, when the RT-RNase H component of the plasmid C is transcribed under the control of an appropriate promoter (in the preferred embodiments described herein, the RSV promoter was used). ), the resulting mRNA transcript contains the coding region for the reverse transcriptase polyprotein-RNase H and, at the end of the translation in the stop signals, the additional mRNA transcript contains (3 'to this translated protein) the elements of plasmid B with downstream signaling events another 3 'for the polyadenylation signals, which remain intact from the RT-RNase H component. The single plasmid vector system described herein (ADNpss-Express-C as shown in Figure 9) does not contain the restriction endonuclease gene, and therefore, does not include the ability to digest the stem of the Stem-spiral intermediary formed by inverted repeats. Accordingly, the sequence of interest (including the DNA enzyme) is inserted into plasmid C only at a 3 'position to the inverted repeats, and the sequences of unwanted vectors are removed by premature truncation of the cDNA-ss product, as the transcript finds the stem relatively stable, and is unable to continue transcribing the cDNA-ss from the mRNA transcript. More specifically, as will be apparent from the following description, each of the sequences of interest was inserted only within the Bam H 1-Pac 1 restriction sites of the plasmid DNApss-Express-C. As will also be apparent from the following description of plasmids B and C, plasmids include cloning sites for insertion of the sequence of interest. In the preferred embodiment of plasmid B described herein both Not 1 sites (located between the inverted repeats) and the Pac I / BamH sites are provided (3 'to the inverted repeats, eg, between the inverted repeats and the PBS) . The preferred plasmid C described herein includes only the Pac I / BamH sites for this purpose. However, those skilled in the art who have the benefit of this disclosure will recognize that these particular cloning sites were selected for the particular systems described herein, and that other cloning sites may be equally useful for this same purpose. It was not intended that the particular plasmid A comprising the two plasmid vector system described herein would include the sequence of interest, but those skilled in the art having the benefit of this description will recognize that, if a system is to be used. of two plasmid vectors, the set of genetic elements of the present invention, and particularly the sequence of interest, can be inserted into any plasmid as convenient. The nucleic acid sequence referred to herein as a cartridge provides the template for the synthesis of cDNA-ss in the target cells. It is this element that includes the sequence of interest, the inverted tandem repeat, and the primer binding site. As is the case for the other elements of the set of genetic elements of the present invention, this genetic element is preferably regulated by means of a broad spectrum or tissue-specific promoter / enhancer., such as the CMV promoter, or the combination of promoters / enhancers, located upstream of the genetic element. Also as is the case for the other genetic elements, the promoter / enhancer can be a promoter either constitutive or inducible. Those skilled in the art who have the benefit of this disclosure will recognize that many other eukaryotic promoters can be used to conveniently control the expression of the sequence of interest, including SV-40, RSV (specific to non-cellular type) or glial fibular acidic protein. (GFPA) specific. As shown in Figure 1, for expression in eukaryotic cells, the cartridge also includes a downstream polyadenylation signal sequence, such that the mRNA produced by means of the sequence of interest has a poly (A) tail. The primer binding site (PBS) for initiation of priming for cDNA synthesis is located between the inverted tandem repeat 31 and the polyadenylation signal. PBS is a sequence that is complementary to a transfer RNA (tRNA) that is resident within the target eukaryotic cell. In the case of the Maloney mouse reverse transcriptase described herein, as has been used in conjunction with the present invention, PBS takes advantage of the proline tRNA. The PBS that is used in connection with the presently preferred embodiment of the invention, which is described herein, was taken from the current 18 nucleotide sequence region of the Moloney mouse virus. See 293 Nature 81. In the case of the human immunodeficiency virus reverse transcriptase gene that was also tested as noted above, the PBS that was used was taken from the HIV nucleotide sequence. Y. Li, et al., 66 J. Virology 6587-6600 (1992). Briefly, any PBS that is coupled to the reverse transcriptase, which is used in connection with the method of the present invention, is used for this purpose. The cartridge of the present invention also comprises a pair of inverted tandem repeats. After digestion of the mRNA from the heteroduplex of mRNA-cDNA, by means of RNase H and the release of the cDNA-ss, the inverted repeats cause the cDNA-ss to bend back on themselves, to form the stem of a stem-spire structure, the stem structure comprising double-stranded anti-parallel DNA. The produced ss cDNA is transcribed with the 3 'and 5' encoded regions flanking the stem (consisting of the inverted repeat) and a loop containing the sequence of interest. One or more functional genetic elements can be designed within the double-stranded portion, ie, the inverted repeat, which forms the stalk of the stem-spire intermediate. In the examples described herein, the functional genetic element is one or more restriction endonuclease site (s) that is cut out by means of the restriction endonuclease produced from the restriction endonuclease gene described above (in FIG. the case of those plasmids that include a RE gene), but those skilled in the art will recognize that the inverted repeat can be designed to include other functional genetic elements, such as a specific enzyme recognition sequence (apart from an endonuclease site restriction sites), eukaryotic and / or prokaryotic receptor recognition sites, promoter or promoter / enhancer sites to drive expression of an objective sequence (which may or may not be equally designed within the stem) in an isolated cis-oriented manner. In the event that the functional genetic element comprises a specific enzyme recognition sequence such as a restriction endonuclease site, then the stem is cut (also called digested or dissociated) by means of any of the many restriction endonuclease enzymes. that recognize the designated cut site within the stem (note that the endonuclease recognition site can be designed within the stem, although the ER gene is not included in the vector system of the present invention), to release the ss-cDNA loop. The loop portion of the cDNA-ss, which does not form any apparent double DNA, is immune to the activity of the restriction endonuclease, since the restriction endonucleases recognize only the double-stranded DNA as a target substrate. Those skilled in the art will recognize that, if the second aspect of the present invention is being used, the restriction endonuclease site (s) need not be designed within the inverted repeats that form the stalk of the stem intermediate. turn. In other words, if it is desired to produce ssDNA from a second sequence of interest located between the primer binding site and the inverted repeats, with the transcription of the cartridge to end up in the stem formed by the inverted repeats, there is no need to a restriction endonuclease site in the stem. Another option is to design the inverted repeats to contain sites of eukaryotic, prokaryotic or viral protein binding sites, which act to competitively titrate selected cellular proteins. The combinations of the restriction sites or other specific elements of sequence can be included in the inverted tandem repeats, depending on the composition of base pairs selected for the construction of inverted repetitions, in such a way that intermediate linear or stem forms are produced -spray cut accurately from the DNA-ss. It is generally preferred to use functional genetic elements constructed in a synthetic manner in the inverted tandem repeat since (if the functional genetic element is a restriction endonuclease site) it is unlikely that a naturally occurring inverted repeat would have the restriction sites properly aligned. As noted above, the cartridge comprising one of the elements of the set of genetic elements of the present invention, further optionally includes a DNA sequence with catalytic activity. Due to the inclusion of the so-called "DNA enzyme" in the cartridge, and specifically, to the fact that the DNA enzyme is contemplated to be located within the sequence of interest, the present invention is used for particular benefit when the sequence of interest it serves as the template for the synthesis of an inhibitory nucleic acid, which is an anti-sense sequence. For that reason, the examples set forth herein describe the production of four anti-sense interest sequences, as set forth in Figure 6, each including a sequence having enzymatic activity against the mRNA, including the C-raf kinase, the anti-sense lira sequence, the anti-sense sequence to the angiogenic growth factor pleiotrophin, and the tat region of the simian immunodeficiency virus (SIV). Those skilled in the art will recognize, however, that the present invention is not limited to anti-sense sequences only, and that the inhibitory nucleic acid sequence can be any of the other types of inhibitory nucleic acid sequences described above. The nucleic acid sequence having enzymatic activity, which comprises the cartridge of the present invention, can be any of those enzymatic sequences. The sequence having the desired catalytic activity is inserted into the cartridge in either or both of the two locations, for example, (a) between the inverted repeats and within the sequence of interest, or (b) within the second sequence of interest that is located 3 'to the inverted repetitions and 5' to the PBS. Either way, the resulting aptamer is specific to the target of the sequence of interest, and is therefore used to direct other DNA sequences, mRNA sequences, and any other suitable substrate, to inhibit or change the splicing mechanisms of DNA or mRNA, or even to directly alter the cellular genome in a specific manner. Nucleic acid sequences with enzymatic activity, which are suitable for use in connection with the present invention include, but are not limited to, sequences that have RNAse activity such as the so-called "enzymes 10-23" and "8-17" (Santoro, SW, and GF Joyce, supra (1997)) and other metal-dependent RNAases (Breaker, RR, and GF Joyce, 1 Biol. Chem. 223-229 (1994), Breaker, RR and GF Joyce, 2 Biol. Chem. 655-660 (1995)) and histidine-dependent RNase (Roth, A., and RR Breaker, 95 Proc. Nati, Acad. Sci. USA 6027-6031 (1998)); sequences having DNase activity such as copper-dependent DNase (Carmi, N., et al., 3 Chem. Biol. 1039-1046 (1996), Carmi, N., et al., 95 Proc. Nati. Acad. Sci USA 2233-2237 (1998), Sen, D. and CR Geyer, 2 Curr. Opin. Chem. Biol. 680-687 (1998)) and DNAases that require divalent metal ions as co-factors, or hydrolyze the substrate independently of the divalent metal ions as reported by Faulhammer, D. and. Famulok (269 J. Molec. Bio. 18-203 (1997)); sequences with DNA ligase activity, 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 DNA kinase activity such as the calcium-dependent DNA kinase (Li, Y. and R. R. Breaker, 96 Proc. Nati, Acad. Sci. USA 2746-2751 (1999)); and sequences with RNA kinase activity such as the calcium-dependent DNA kinase (Li, Y., supra (1999)). The particular nucleic acid sequence having enzymatic activity, which is used in the examples described herein, is "enzyme 10-23" (Santoro, SW and GF Joyce, supra (1997)), and it is this enzymatic sequence that it is represented schematically in Figures 3A and 3B. However, those skilled in the art will recognize, by this disclosure, that any of the sequences reported in the literature listed above will function for the intended purpose, when inserted into the cartridge of the present invention. Regarding the RNA-dependent DNA polymerase, or the reverse transcriptase (RT) gene which also comprises an element of the set of genetic elements of the present invention, as noted above, the reverse transcriptase / RNase H gene was used. oloney murine leukemia virus, for benefit in the examples described herein. The human immunodeficiency virus (HIV) reverse transcriptase / RNase H gene has also been tested. Many other retroviral reverse transcriptase / RNase H genes can be used for benefit in connection with the present invention, including those of retroviruses such as SIV, hepatitis B strains, hepatitis C, bacterial retrone elements, and retrons isolated from different species of yeast and bacterial, it being preferable that the reverse transcriptase / RNase H gene is a reverse transcriptase / NRase H gene, which is regulated by means of an appropriate upstream promoter / enhancer such as the cytomegalovirus (CMV) promoter or Rouse sarcoma virus (RSV) for expression in eukaryotic cells. As found in nature, these RNA-dependent DNA polymerases usually have an associated NRase component enzyme within the same coding transcript. However, the present invention does not require the naturally occurring NRase H gene for a particular reverse transcriptase. In other words, those skilled in the art will recognize, by this disclosure, that different combinations of reverse transcriptase and Rasa H genes can be spliced together, to be used in connection with the present invention, to accomplish this function, and that they are available , and / or that those skilled in the art know, modify and / or hybrid versions of these two enzyme systems, which will function in the intended manner. Those skilled in the art will also recognize that the target cell can itself have sufficient endogenous NRase H to accomplish this function. Similarly, those skilled in the art will recognize that the target cell can itself have sufficient endogenous reverse transcriptase activity of, for example, retroviral infection prior, to fulfill this function. The reverse transcriptase / NRase H gene also preferably includes a downstream polyadenylation signal sequence, such that the mRNA produced from the reverse transcriptase / NRase H gene includes a poly (A) 3 'tail for stability of the MRNA. As those skilled in the art know, multiple poly (A) tails are available, and are routinely used for the production of expressed eukaryotic genes.

The reverse transcriptase produced in the cell synthesizes a complementary DNA (cDNA), using as the template the genetic element that includes the sequence of interest described later. The RNAse H activity of the reverse transcriptase, together with the activity of the endogenous RNase within the cell, degrades the mRNA template component of the RNA / cDNA hybrid, to produce ss-DNA in vivo. The gene encoding the restriction endonuclease (which is used only in the two plasmid system, and is not even a required component of that system) can be any of many genes that code for restriction endonucleases, and preferably those that are controlled by one or more constitutive or inducible, broad spectrum and / or tissue-specific promoters / speakers as described below. The particular restriction endonucleases that were used herein were Mbo II and Fok I, but those skilled in the art having the benefit of this disclosure will recognize that any restriction endonuclease site (type I, II, IIS, or III) in the inverted tandem repetition. These enzymes "embrace", or digest, the stalk of the stem-spire intermediate to make the sequence of interest linear as single-stranded DNA. Although the expression of the ER gene can be regulated by means of an appropriate constitutive or inducible promoter / enhancer, located upstream of the restriction endonuclease gene such as the CMV or RSV promoter for expression in human cells, in the plasmid DNApss-Express-A described herein, the RE (Mbo II) gene is linked to the RT-RNase H polypeptide. The RE gene also preferably includes a downstream polyadenylation signal sequence, such that the mRNA transcript of the restriction endonuclease gene will have a poly (A) 3 'tail. Those skilled in the art who have the benefit of this disclosure will also recognize that many tissue-specific or broad-spectrum promoters / enhancers, or combinations of promoters / enhancers other than those listed above, may also be used for the purpose of regulating the gene for reverse transcriptase / NRase H, the RE gene (if used), and the sequence of interest. Although a list of all promoters / enhancers available to exemplify the invention is not necessary, as noted above, the promoters / enhancers may be constitutive or inducible, and may include CMV or RSV promoters / enhancers (not specific to type of promoter / enhancer). cell) or GFAP (tissue specific), and many other viral or mammalian promoters. Representative promoters / enhancers that are suitable for use in connection with the elements of the present invention can include, but are not limited to, 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. Nati, Acad. Sci. USA 8283 (1986)), MMTV (A.L. Huang, et al., 27 Cell 245 (1981)), adenovirus 5 E2 (M.J. 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 Nucí Acids Res. 1121 (1989)), MHC class gene I H-2kb (MA Blanar, et al., 8 EMBO J. 1139 (1989)), and thyroid stimulating hormone (V. K. Chatterjee, et al., 86 Proc. Nati, Acad. Sci. USA 9114 (1989)). When the elements comprising the set of genetic elements of the present invention are incorporated within a vector, it is preferred that the vector contains other specialized genetic elements, to facilitate the identification of cells carrying the vector and / or to increase the level of expression of the cartridge. Specialized genetic elements include marker genes that can be selected, in such a way that the vector can be transformed and amplified in a prokaryotic system. For example, the most commonly used selectable markers are genes that confer bacteria (eg, E. coli) resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin (neomycin), or tetracycline. It is also preferred that the vector contain specialized genetic elements for transiation, identification and subsequent expression in a eukaryotic system. For expression in eukaryotic cells, multiple selection strategies (eg, Chinese OHS Hamster Ovary), which confer resistance to an antibiotic or other drug to the cell, or which alter the cell phenotype such as morphological changes may be used. , loss of contact inhibition, or increased growth rate. Selectable markers that can be used in eukaryotic systems include, but are not limited to, resistance markers for Zeocin, resistance to G418, resistance to aminoglycoside antibiotics, or phenotypic selection markers such as β-gal or protein. of green fluorescence. The incorporation of these components into an appropriate vector allows two convenient methods to remove sequences of previously determined vectors, after the production of ssDNA. In the first method, the turn-off portion of the stem-turn intermediary of the ssDNA that is produced comprises the nucleotide sequence of interest and, after digestion with the restriction endonuclease, the loop is released as single-stranded cDNA, linearized, without flanking sequences. In the second method in which a second sequence of interest is included in the 3 'cartridge to the inverted tandem repeat, the reverse transcription is terminated in the stem of the stem-coil structure, such that the resulting ssDNA is produced without flanking sequences. If it is desired to produce ssDNA using the second method, the cartridge is designed with an inverted repeat, which forms a stem that is more stable than the stalk of the stem-spire intermediate from which the ssDNA is produced, by digestion of the stem , in accordance with the first aspect of the present invention. By designing the cartridge with an inverted repeat that forms a stem that is easily denatured, according to the first aspect of the invention, the reverse transcription proceeds through the second sequence of interest (if it is designed inside the cartridge). ) to the sequence of interest located between the inverted repetition. A stem that is of intermediate stability allows the production of both the first and the second sequences of interest. This premature termination of the reverse transcriptase cDNA transcript was discovered in the 3 'aspect of the stem structure, when a vector with a stem including 29 base pairs was used in in vitro experiments, the additional base pairs included in the Stem providing the additional stability that causes the production of both the first and second sequences of interest. The design of a stem-spire intermediate having a stem of the desired stability to obtain either the production of the first (between the inverted repeat) or the second (between the 3 'aspect of the inverted repeat and the PBS), It is within the experience of those technicians in the field who have the benefit of this description. Briefly, factors such as the length of the inverted repeat (e.g., the number of nucleotides comprising the repeat) and the identity of the bases comprising the invention can be manipulated (using, for example, the in vitro system described herein). repetition, to obtain a stem that has as little or as much stability as may be desired, to obtain the production of the first or second sequence of interest, or a mixture of both sequences. From this description it will also be apparent to those skilled in the art that the intact stem-spire cDNA-ss structure can function similarly in many applications such as the linearized ss-cDNA form. In consecuense, the cartridge is also used for benefit without the restriction endonuclease gene and associated regulatory elements and / or with a sequence of interest lacking the corresponding restriction endonuclease site. It will also be apparent to those skilled in the art, by this description of the preferred embodiments of the present invention, that a cartridge encoding a cDNA-ss having an "arranged" stem-spire structure can be made. The restriction endonuclease sites encoded in the inverted repeats flanking the sequence of interest are designed in such a way that the stem portion (after the formation of the duplex) is digested with the corresponding restriction endonuclease, in order to cut the DsDNA comprising the stem, in a manner that removes a portion of the stem, and the associated flanking sequences still leave enough DNA duplex, such that the transcript retains the stem-spire structure described above. That cDNA-ss structure may be more resistant to intracellular nucleases by retaining the "ends" of a ssDNA in the form of a double strand. It will also be apparent to those skilled in the art that the stem (duplex DNA) can be designed to contain a previously determined sequence (or sequences), i.e., aptamers, which are recognized and fixed by means of the DNA binding proteins. specific. Among other uses, that stem structure is used in the cell as a competitor to title a selected protein (s) that regulate (n) the specific function of the gene. For example, a cDNA-ss stem-splice according to the present invention is produced in a cell that contains a binding site for a selected positive transcription factor such as the adenovirus Ela. The adenovirus Ela, like other oncogenes, modulates the expression of many adenoviral and cellular genes, by affecting the activity of the transcription factors encoded by the cells, resulting in the change of normal cells to transformed cells. Jones, et al., 2 Genes Dev. 267-281 (1988). The duplex stem of the stem-spire intermediate produced in accordance with the present invention, therefore, is designed to function to "bind" this transcription factor, preventing the protein from binding a promoter, and thus, inhibiting expression of the particular harmful gene, for those skilled in the art, it will be clear that the duplex stem structure may optionally contain multiple binding sites, eg, sites that are recognized by means of different transcription factors that actively regulate the expression of the particular gene . For example, adenovirus Ela has been found to express transcription of the collagenase gene by means of the element responsive to phorbol ester, a promoter element responsible for the transcription induction by 12-0-tetradecanoliforbol 13-acetate (TPA). , by means of many other nitrogens, and by means of oncogenes ras, mos, src, and trk. The mechanism involves the inhibition of the family function of the transcription factor AP-1. Offringa, et al., 62 Cell 527-538 (1990). Any desired nucleotide sequence can be inserted into the genetic element encoding the "turn" portion of the stem-spire intermediate, to ultimately perform a desired inhibitory function, eg, anti-sense binding, down-regulation of a gene, etc. , as described herein. In another aspect that will be recognized by those skilled in the art, the present invention is used to construct complex secondary ssDNA structures that confer biological reactions on the cDNA transcript, based on the folding of the secondary conformational structure. That secondary structure can be designed to satisfy any of many functions. For example, the sequence of interest may include (but is not limited to) a sequence that is incorporated within the loop portion of the single-stranded cDNA transcript, which forms so-called "cloverleaf" or "crucible" structures. , such as those found in the long terminal repeats of viruses associated with adeno or retro-transposons. Under the right circumstances, that structure integrates in a specific way into the site within the host genome. Because the set of genetic elements of the present invention can be adapted for incorporation into multiple commercially available delivery vectors, for therapeutic purposes of mammals and humans, multiple shipping routes are feasible, depending on the vector selected for a particular target cell . For example, currently viral vectors are the most frequently used means to transform the patient's cells and introduce the DNA into the genome. In an indirect method, viral vectors are used that carry new genetic information, to infect target cells removed from the body, and then the infected cells are re-implanted (i.e., ex vivo). Direct gene transfer has been reported in vivo, within postnatal animals, for DNA formulations encapsulated in liposomes, and DNA trapped in proteo-liposomes containing viral envelope receptor proteins. Nicolau, et al., 80 Proc. Nati Acad. Sci. USA 1068-1072 (1983); Kaneda, et al., 243 Science 375-378 (1989), Mannino, et al., 6 Biotech-niques 682-690 (1988). Positive results have also been described with DNA co-precipitated with calcium phosphate. Benve-nisty and Reshef, 83 Proc. Nati Acad. Sci. USA 9551-9555 (1986). Other systems that are used for benefit include intravenous, intramuscular, and subcutaneous injection, as well as intra-tumoral and direct intra-cavity injection. The set of genetic elements, when incorporated within the vector of choice, is also conveniently administered through local, trans-mucosal, rectal, oral, or inhalation-type application methods. The vector that includes the set of genetic elements of the present invention is conveniently employed to send a sequence of interest of anti-sense, triple nucleotides, or any other nucleic acid sequence of inhibitory nucleic acids or of a single chain, using digestion techniques and known ligation, for splicing the particular sequence of interest within the center cartridge to the inverted tandem repeat or between the PBS and the inverted tandem repeat). Those skilled in the art who have the benefit of this disclosure will also recognize that the signals described above that are used for expression within eukaryotic cells can be modified in manners known in the art, depending on the particular sequence of interest, for example. example, a probable change is to change the promoter, in order to confer convenient expression characteristics in the cartridge, in the system in which it is desired to express the sequence of interest. There are also too many possible promoters and other signals, and these are so dependent on the particular target cell for which the sequence of interest has been selected, that it is impossible to list all potential enhancers, inducible and constitutive promoter systems, and / or delivery systems. poly (A) tail that could be preferred for a particular target cell and sequence of interest. In another embodiment, the present invention takes the form of a kit comprising a plasmid having the RNA-dependent DNA polymerase described above, and restriction endonuclease genes cloned therein, as well as a multiple cloning site (MCS) inside which the user of the case inserts a sequence (s) of particular interest (s). The cloning site into which the sequence (s) of interest is inserted is located between the inverted tandem repeats described above, eg, upstream from the genetic element encoding the primer binding site. The resulting plasmid is then purified from the cell culture in which it is maintained, lyophilized or otherwise conserved for packaging and shipping, and sent to the user.

The kit also preferably includes the restriction endonuclease for the cloning site within which the sequence of interest, ligases and other enzymes, together with appropriate pH regulators, are to be cloned to ligate the sequence of interest within the plasmid, and a map of the plasmid and / or other instructions for the user. In the embodiment described herein, the sequences of interest are sent to a host cell, either by co-transfecting the cells with two plasmids, designated A and B, each plasmid being designed and constructed to include one or more of the genetic elements listed above, or a single "C" plasmid. To summarize, in the two-plasmid system, plasmid B encodes the cartridge that includes the sequence of interest, nested within flanking sequences that include the inverted repeat, and the primary binding site that provides the processing signals after transcription , which mediate the conversion of mRNA to single-stranded DNA. Plasmid B also includes the second sequence of interest when this second aspect of the invention is used (as stated above, when the RE gene of the construction of the present invention is omitted, for example in the individual plasmid "C" described in the present, is this second sequence of interest encoding the particular inhibitory nucleic acid having the desired activity). The activities required to process the primary genetic product from plasmid B to single-stranded DNA, with the removal of vector sequences and processing signals, specifically reverse transcriptase / RNAse H, and restriction endonuclease, are expressed at from plasmid A. The single-stranded DNA sequence that is released by the interaction of the transcription products of these component in vivo is free to fix intracellular targets such as mRNA species and DNA promoters in anti-sense strategies and triples As noted above, as described herein, plasmid B includes cloning sites (Not I sites were used in a mode of plasmid B described herein) among which any DNA sequence of interest is placed (as noted above, in the examples described herein, the sequences include a "filler", or test, sequence, telomer repeats, h-ras, c-raf kinase, a region encoding the angiogenic growth factor pleiotrophin, and the region that codifies SIV tat). Flanking the cloning sites are the signals that direct the processing of the primary mRNA transcript, produced from a promoter (a CMV promoter was used in the preferred B plasmid described herein), inside the single chain inhibitory nucleic acid wanted. After the cloning of the desired sequence of interest into plasmid B, plasmids A and B are co-transfected into a cell line of choice, for the constitutive expression of the ssDNA. Similarly, in the single plasmid ("C") system described herein, the sequence of interest within that plasmid is cloned, and transfected into the cell line for further processing. Regardless of the distribution of the elements of the previously described set of genetic elements between two (or even more) plasmids, or if all the elements are contained in a single plasmid, this processing precedes in three steps after the transcription of the DNA region of a single strand (i.e., the sequence of interest, the inverted repeats and the PBS): (1) reverse transcription of the RNA transcript of plasmid B or C, by means of a reverse transcriptase, which in the preferred embodiment described in the present is the reverse transcriptase expressed by plasmid A or B (in the preferred embodiment described herein, the reverse transcriptase is reverse transcriptase of the Moloney mouse leukemia virus (MoMuLV)), proceeding from the primer binding site which is 3 'to the sequence of interest (including the sequence with enzymatic activity), the inverted repeats, and the primer binding site, as shown in Figure 1; (2) digestion of RNAse H from the resulting heteroduplex, either by means of the activity of RNase H of the reverse transcriptase polyprotein, or by means of the activity of endogenous RNase H, to release the DNA precursor from a single chain of its RNA complement; and (3) removal of the flanking sequences by digestion of any stem restriction endonuclease from a stem-spire intermediate formed after base-atson-Crick base formation comprising the inverted repeat, or by means of the premature termination of the cDNA transcript, by forming the secondary stem-spire structure by means of the self-complementary inverted tandem repeats. Those skilled in the art will recognize, by this disclosure, that particular cloning sites flanking the sequence of interest, the particular reverse transcriptase, the restriction endonuclease (if used), the promoter, the primer binding site, and all other elements of the set of genetic elements of the present invention are selected depending on the sequence of interest and / or particular system in which the ssDNA is going to be expressed. Except where otherwise indicated, standard techniques are used in the examples described below, as described by Seabrook, et al. (1989) (J. Seabrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press (1989), referred to hereafter as "Maniatis, et al. (1989)") and Ausubel, et al. (1987) (FM Ausubel, et al., Current Protocols in Molecular Biology, New York : John Wiley & Sons (1987)), both of which are incorporated in their entirety by this specific reference to them. It should be understood that other methods of ssDNA production can also be used, both by natural processes and by artificially designed methods, which use different products of enzymes or systems, in connection with the method of the present invention, and that the examples set out in the present are set forth for purposes of exemplification, and are not intended to limit the scope of this description or of the invention described herein. The plasmid DNApc3.1 / Zeo (+) was purchased with Invitrogen Corp. (Carlsbad, California, United States) and the plasmid pBK-RSV with Statagene (La Jolla, California, United States). The oligodeoxynucleotides (ODN) were synthesized by Midland Certified Reagent Co. (Midland, Texas, United States). Polymerase chain reactions (PCR) were performed using the Taq DNA polymerase, which was purchased from Boehringer Mannheim Corp. (Indianapolis, Indiana, United States) on a ROBO gradient thermal cycler (Stratagene (La Jolla, California, United States). United) ) . Restriction endonucleases and T4 DNA ligase were obtained with Boehringer Mannheim Corp. (Indianapolis, Indiana, United States). The ODNs that were used are listed in the attached Sequence Listing. EXAMPLE 1. In Vitro-ss DNA synthesis Four single-stranded ODNs (129, 121, 111, and 103 bases in length, Sequence ID Nos: 4, 5, 15, and 16, respectively) were designed (see Sequence Listing in Table 1) so that they contained an inverted tandem repeat structure with a region called "padding" of random nucleotide sequence composition, inserted as a sequence of interest between the inverted tandem repeat. The inverted tandem repeat was designed in such a manner (Figure 2) as to have a pair of restriction endonuclease recognition cleavage sites within the repeats. The designated cutting sites for ?? I and Fok I (restriction endonuclease type II and type IIS recognition slices, respectively) were the oligonucleotides of base length 111 (Sequence ID 15) and 102 (Sequence ID 16), and the endonuclease cleavage sites Oligonucleotides of length 129 (Sequence ID 4) and 121 (Sequence ID 5) bases were designated restriction for Not I and Mbo II. In addition, a primer binding sequence (PBS) of tRNA was designed for recognition by the primer for reverse transcriptase, inside the oligonucleotides. The PBS was located 3 'downstream from the inverted tandem repeat. For each of the synthetic oligonucleotides, 1 μ? (5 μg μl in water) from aliquots to four separate tubes, and heated at 70 ° C for 5 minutes, and allowed to auto-temper for 15 minutes at room temperature. This process allows optimal hybridization for the formation of a stem-spire structure; the stem portion, which has no sections of complementary sequence, must not experience significant self-tempering, and remains predominantly of a single chain. Then the standard restriction endonuclease digestions were performed with Not I, Fok I, bo II (appropriate for each oligonucleotide for which a restriction endonuclease cleavage site was present) and EcoR I (as a negative control) with 10 enzyme units, in a total reaction volume of 15 μ? and appropriate reaction pH regulators. Digestions were confirmed by electrophoretic gel analysis. The results of this experiment showed that a synthetic cDNA-ss with inverted repeats formed double DNA. The double DNA, presumably the stem of a stem-spire structure, formed specific and recognizable restriction endonuclease sites, from the sequences in the inverted tandem repeats, which were designed for the purpose of forming the restriction endonuclease sites Not I and Fok I by forming Watson-Crick base pairs of the bases comprising the inverted tandem repeats. When the cDNA-ss was incubated with Not I and Fok I, the DNA was digested. When incubated with EcoR I (negative control), the same DNA was not digested. EXAMPLE 2. In vltro Formation of DNA-ss from Cartridge Transcripts To conduct this experiment, two test plasmids were constructed. ADNpc3.1 / Zeo (+) was digested with Nhe I and Apa I under standard conditions. Two sets (A and B) of 5 'phosphorylated oligonucleotides, which were designed to be complementary to one another, were allowed to hybridize (see listings of attached sequences). Hybridization was performed by heating the complementary oligonucleotides at 95 ° C, and then allowed to cool to room temperature for 15 minutes. The resulting duplex oligonucleotide linkers with appropriate cohesion ends were ligated under standard conditions to the previously prepared DNApc3.1 / Zeo (+) vector. The selection of positive clones in ampicillin plates was carried out, after transformation to XLl-Blue MRF cells (Stratagene), as described by Maniatis, et al. (1989) and the accompanying instruction. After the positive clones were captured, the plasmid DNA was isolated using a commercially available plasmid isolation kit (Quiagen, Inc., Sant Caita, California, United States). Confirmation of DNA ligation was performed by DNA sequencing. Positive circular test plasmids were linearized by digestion with Pme I, and standard reverse transcription reactions were performed as follows. To each tube was added 0.1 μg of linearized plasmid DNA, 25 units of T7 polymerase (ie, a DNA-dependent RNA polymerase), 2.5 rtiM of rNTPs (ribonucleotide triphosphates rUTP, rCTP, rGTP, rATP) and a pH regulator appropriate. The reaction tube was incubated at 37 ° C for 90 minutes. After incubation, 10 units of DNase were added and incubated for 15 minutes at 37 ° C. the reaction was terminated by incubation at 70 ° C for 10 minutes. Standard cDNA synthesis reactions were performed, using the above reactions. To a fresh tube 5 μ? of the previous reverse transcriptase reaction, 0.2 μg of primer (see sequence listing) complementary to the PBS region (downstream from the inverted tandem repeats). To this mixture were added 25 units of Moloney murine leukemia virus reverse transcriptase, 2.5 mM dNTPs (deoxyribonucleotide triphosphates dTTP, dCTP, dGTP, dATP), and the appropriate reaction pH regulator. The reaction mixtures were incubated at 37 ° C for 1.5 hours. After the incubation period, 100 units of NRase H were added and the tubes were incubated at 37 ° C for 15 minutes. The reaction tubes were incubated at 70 ° C and allowed to warm by cooling to room temperature for 15 minutes. The mixture was divided equally into four tubes, in which either 10 units of Not I, Fok I, Mbo II, or EcoR I restriction enzyme were added, together with the appropriate reaction pH regulator. The tubes were incubated at 37 ° C for 1 hour. Reaction reactions were treated with nuclease SI (specific for ssDNA). The DNA of the above reactions was resolved by gel electrophoresis. This experiment demonstrated the in vitro production of ssDNA by the enzymatic sequential activities of the T7 RNA polymerase, reverse transcriptase, and NRase H. The linker contained inverted tandem repeats with the restriction sites Not I, Fok I, and Mbo II, a "fill" region (which included the turn of the stem-turn intermediary), and a PBS of AR t. The plasmid contained the T7 promoter immediately upstream of the linker region. The agarose gel electrophoresis analysis showed the stepwise production of (1) an intermediate of mRNA of predicted length, (s) a cDNA of predicted length, and (3) the subsequent digestion of the ssDNA by the corresponding restriction endonucleases Not I, Fok I, and Mbo II. A negative control in which EcoRI was used did not digest the cDNA-ss. EXAMPLE 3. In vivo synthesis of cDNA-ss in Eukaryotic Cells The following in vivo experiments were designed to test the plasmids made in Example 2, which contained different "filler" regions that were inserted between the inverted tandem repeats. Tissue culture cells that were used in these experiments encoded and endogenously expressed RNA polymerase II dependent on eukaryotic DNA, which uses the RSV promoter located upstream of the cloned genetic element which is the linker / filler segment described in Example 1, previous. Those technicians in the field who have the benefit of this description, will recognize that any eukaryotic promoter can be used for this purpose. Furthermore, these people recognize that a DNA-dependent RNA polymerase encoded by vector will also function in this capacity. Plasmid constructions. The ODNs were allowed to hybridize in 1 μ? (5 μ? / Μ? In water) in four separate tubes that were incubated at 70 ° C for 5 minutes, and allowed to hybridize for 15 minutes at room temperature. Standard restriction endonuclease digestions (EcoR I used as a negative control) were performed with 10 units of enzyme in a total reaction volume of 15 μ? and appropriate reaction pH regulators. DNA fragments were resolved, and isolated from agarose gels. The selection of positive clones in ampicillin plates was performed after transformation to competent XLl-Blue MRF cells (Stratagene) as described by Maniatis, et al. (1989). After the positive clones were selected, the plasmid DNA was isolated using the Quiagen plasmid isolation kit described above. The construction of three expression plasmids is described. The first plasmid B was derived from pDNA3.1 / Zeo (+) (Figure 7A) by digestion with the restriction endonucleases Nhe I and Apa I, which are located at the multiple cloning site (MCS). The double-stranded oligodeoxynucleotide having the compatible ends Nhe I and Apa I was ligated, which was formed by annealing the synthetic, single-strand oligodeoxynucleotides with Sequence ID 4 / ODN-PMMV (+) and Sequence ID 5 / ODN-PMMV (-), within the ADNpc3.1 / Zeo (+) digested, to give pMMV. This insert contains the promoter / primer binding site (PBS) region of the Moloney Mouse leukemia virus reverse transcriptase (MoMuLV-RT). It also contains two Not I sites, unique to pMMV, and one Mbo II site. In this plasmid, and the plasmids derived from this construction, the designated chains (+) are placed so that they are transcribed within the RNA of the cytomegalovirus promoter of cp133.1 / Zeo (+). Plasmid B, ADNpss-Express-B, containing sites for convenient insertion of a sequence of interest to be expressed in vivo as ssDNA, was obtained by ligation of the double-stranded sequence formed by means of an ODN-annealing XB (+) and ODN-XB (-), which had Not I compatible cantilevers, between the Not I sites of pMMV. The structure of the ADNpss-Express-B is shown in Figure 5. The mammalian cells containing the plasmid can be selected with the antibiotic zeocin. The position and general configuration of the key regions of the insert are shown in Figure 5A and the specific configuration of the insert sequences are shown in Figure 5B. Transcription of the insert region is activated by the cytomegalo-virus promoter and terminated in the polyA region of BGH. The RNA transcript contains the core promoter of MoMuL V together with some flanking regions of this promoter, whose positions are indicated in Figure 5B. The reverse transcriptase synthesizes a copy of the (+) (top) chain, using the pro tRNA as a primer, starting at the position of the core promoter. Digestion of the RNA strand by RNase H releases a single-strand DNA sequence containing the complementary inverted repeats IR-L and IR-R (Figure 1). The double formation by these repetitions in Figure 2, creates a stem-loop with the sequence of interest in the cycle. The stem contains a recognition site for Mbo II, GAAGA, which is placed in such a way that the enzyme, which dissociates 8/7 bases 3 'for the GAAGA recognition site, releases the sequence of interest from the sequences of the flanking vector. To obtain the pTest of plasmid B, from which a sequence of interest that served as a control sequence is expressed, the double-stranded oligonucleotide that is formed by the softened synthetic, Seq. ID 11 / ODN-Test (+) and Seq. ID 12 / ODN-Test (-) of the single-stranded oligonucleotides, which have compatible Not 1 ends, was inserted between the Not 1 sites of the pMMV. Plasmid B expressing the telomeric repeat sequence as the sequence of interest was obtained in a similar manner, by inserting the Seq. ID 13 / 0DN-Telo (+) and Seq. ID 14 / ODN-Telo (-) of the Not 1 compatible oligonucleotides, softened between the Not 1 sites of pMMV (Figure 6A). The last linker sequence contains nine repeats of the telomere sequence of the vertebrate 5 '-AGGGTT-3' (Figure 6B). Blackburn, E.H., 350 Nature 569-575 (1991). A third plasmid B, which is called pMN-new-link, was constructed in which the stem comprised 29 base pairs (rather than the stem site of 27 base pairs of p NV) because the stem of 29 pairs of base gives a more stable stem-spire structure in the stem-spire intermediate, than the stem site of 27 base pairs of pMNV. The pNM-new-bond was formed by ligating two self-complementary ODN's, the new-link-NMD-ODN (+) and the new-link-NMD-ODN, between the two Not 1 sites within the pMNV. Plasmid A, ADNpss-Express-A (Figure 4), was prepared from pBK-RSV (Stratagene) using Blue RF XL-1 as the host cell. A mouse cell line expressing Moloney murine leukemia virus was obtained from the American Culture Type Collection (# CRL-1858). Virus RNA was isolated and prepared for the reverse transcriptase-Polymerase Chain Reaction (RT-PCR). A 2.4 kb fragment containing the coding sequence of MoMuL V-RT was amplified by polymerase chain reaction, using primers as start in Seq. ID l / ODN-RT (-) (position of the primer in nucleotide # 2545) and Seq. ID 2 / ODN-RT (+) (position of the primer in nucleotide # 4908), to produce a DNA fragment with a 5 '-Sac and a compatible end-hind III. The 2.4 kb product that was obtained included the sequence of the MoMuLV genome between positions 2546 and 4908. The reverse transcriptase of the mature virus was coded by the sequence between positions 2337 and 4349 (Petropoulos, CJ, retroviral taxonomy, structure of the protein , sequences and genetic maps, in JM Coffin (ed.), Retrovirus, 757, Appendix 2, New York; Cold Spring Harbor Press (1997)), but peptides that were truncated at the amino terminus retain complete activity (N. Tañese et al., 85 Proc. Nati, Acad. Sci. USA 1777-1781 (1998)). The peptide that was coded by this construct includes a part of the integrase gene, which follows the reverse transcriptase in the MoMuLV polyprotein, but is not relevant here so that the length of the construct was selected due to the capacity of a site of convenient restriction for cloning. The Moraxella bovis bacterium, which encodes the restriction endonuclease MboII (Bocklage, H. et al., 19 Nucleic Acids Res. 1007-1013 (1991)), was obtained from the American Type Culture Collection (ATCC # 10900). Genomic DNA was isolated from M. bovis and used as the template DNA in the polymerase chain reaction. A 1.2 kb fragment containing the Mbo II gene was amplified by the polymerase chain reaction, using Seq.

ID 3 / 0DN-Mbo (+) (position of the primer in nucleotide # 887) and Seq. ID 8 / 0DN-Mbo (-) (position of the primer in nucleotide # 2206). These primers contain inequalities that are designed to introduce a Hind III site into the 5 'primer and an Xba site into the 3' downstream primer. The 1.2 kb DNA amplification product, which copies the genome of M. bovis between positions 888 and 2206, thus contains the coding region for the Mbo II protein. The amplification product was digested with Hind III and Xba I. pBK-RSV was digested with Xba and Nhe I to remove the promoter region. The Nhe I terminus became an Sac I terminus, using the linker that was formed by Seq. ID 6 / ODN-N > S (+) and Seq. ID 7 / ODN-N > S (-) of the softened oligonucleotides. The reverse transcriptase and the amplifiers of the Mbo II were ligated through the Hind III sites and this construction was subsequently ligated between the Sac I and Xba I sites of the p-BK-RSV, to produce the pBK-RSV-RT / Mbo. To insert a flexible linker between the reverse transcriptase and the Mbo II domains of the polyprotein and to provide a useful label for the purification of the protein, the double chain sequence that was formed by means of smoothing the Seq was inserted. ID 9 / ODN-HisPro (+) and Seq. ID 10 / ODN-HisPro (-) of the oligonucleotides, which encode the alternative histidine and proline amino acids, within pBK-RSV-RT / Mbo by means of digesting it with Hind III. The his-pro linker, with compatible Hind III ends, was inserted into the Hind III site to produce the pBK-RSV-RT / Mbo-L plasmid and orientation was confirmed by sequencing. However, pBK-RSV-RT / Mbo-L sequencing revealed a mutation of structure change at the 5 'end of the Mbo II domain. This mutation was corrected and the foreign part was removed simultaneously, by removing the plasmid fragment lying between the Ase I and Bgl II sites, which encode the 5 'end of the Mbo II gene, the his-pro linker region. and the gene fragment 51 -Mbo II. The modified his-pro linker increased the number of histidines by one, to six, and included in the 5 'end a number of unique restriction sites. The 5 'end of the Mbo II gene was modified to replace the leucine in the N term that was introduced, by the inequality in the polymerase chain reaction primer, to the original methionine and to optimize the use of the codon for the expression of this segment of the gene in mammalian cells. The repair construct was obtained by mutually primed DNA synthesis from two templates, ODN-Rep (+) and ODN-Rep (-), which have complementary sequences of 16 bases at the 3 'ends. These oligonucleotides were smoothed and extended with the modified SEQUENASE® DNA polymerase enzyme (United States Biochemical Corp.). The double-stranded product was digested with Ase I and Bgl II and inserted into the plasmid to give the DNApss-Express-A (plasmid A).

The structure of the ADNpss-Express-A is shown in Figure 8A. As stated above, to construct this plasmid, the sequences encoding an active fragment of the reverse transcriptase of MoMuLV and the restriction enzyme Mbo II of M. bovis were cloned between the Nhe I and Xma I sites of the eukaryotic expression vector pBK-RSV. The transcription of the cloned region is activated by the RSV promoter and the selection for the transformed cells is carried out in the presence of the antibiotic G418 (neomycin). Reverse transcriptase and Mbo II are expressed as a single chain of proteins, difunctional with the two functional domains separated by a linker rich in histidine and proline, short. Tissue Culture Studies. Stable and transient transfections were performed by the use of lipofectant (Boehringer Mannhiem Corp.), using the manufacturer's accompanying instructions. All constructs of the plasmids were transfected into the HeLa cell lines. The assays for the ssDNA were carried out by means of the polymerase chain reaction and by spot-spot analysis from 24 to 48 hours after transfection. The reverse transcriptase activity was assayed using the RT-polymerase chain reaction assay that developed Silver, J. and collaborators (21 Nucleic Acids Res. 3593-3594 (1993)). After transfection with the plasmid DNApss-Express-A (Figure 11, panel A). Individual colony isolates of the stably substituted HeLa cell lines (A12 and B12) were further tested for RT activity (Figure 11B). The ssDNA-c was isolated from the cells that were transfected 48 to 72 hours before. As described above, the cDNA-c, which is localized together with the ARB, was made using trizol reagent (Gibco Life Technologies, Gaithersburg, Maryland, United States). The assays for the specific ssDNA-c species were performed both by means of assays based on the polymerase chain reaction (Figure 12 for pTest and Figure 13 for pTelO) for the internal fragment, and by means of gel electrophoresis single denatured chain with spotting and subsequent nylon sounding, with a probe labeled with internal biotin. This experiment showed that human tissue culture cells (the HeLa and Cos-7 cell lines), which were co-transfected with plasmids A and B, produced the ss-cDNA of the predicted size. Those skilled in the art, however, will recognize that a single plasmid can also be used that includes the RNA template for the stem-spire intermediary and the genes for reverse transcriptase and restriction endonucleases for this purpose, as it is described in other examples that are described herein. The pNM-Link-New vector (which after the single-chain conversion contains the most stable base pair structure of 29 base pairs, was used for the in vitro experiments to demonstrate the premature termination of the transcriptase cDNA transcript Inverse in aspect 3 of the stem structure As shown in Figure 14, there was also some "through" reading of this transcript through the stem structure as well (see the larger bands in Figure 14). sequence of interest that occurred from this premature termination, is the second sequence of interest referred to in the present example 4. In vivo synthesis of cDNA-c That includes the DNA Enzyme in the eukaryotic cells. The following in vivo experiments were designed to test whether the vector system of the present invention can be used to produce the ssDNA including a DNA enzyme sequence in the cells of the culture of eukaryotic tissue. Plasmid constructions. ODNs were prepared and the plasmids were constructed in the same manner as described in Example 3, above. Construction of Plasmid B. A second embodiment of the first of the two plasmids comprising a two-plasmid mode of the vector system of the present invention, is plasmid "4B". Plasmid 4B, like the plasmid DNApss-Express-B that is described in Example 3, was derived from ADNpcA3.1 / Zeo (+) (Invitrogen Corp.), which is shown in Figure 7. The ADNpss-Express-4B was constructed by being digested with the restriction endonucleases Hind III and Not I at positions 911 and 978, respectively. The double-stranded linker region having the Hind III and Not I ends that are formed by means of softening the synthetic oligonucleotides, of a single chain ODN-5 '-N / M (bond) 2-H / N and ODN- 3'-N / M (link) 2-H / N from a single chain, was ligated under standard conditions into the digested ADNpcA3.1 / Zeo (+) which was transformed into Sure II cells (Stratagene, Inc.). The ODNs were allowed to hybridize in 1 μ? (5 μg / μl in water) in Ependorf tubes that were incubated at 70 ° C for 5 minutes and allowed to hybridize for 15 minutes at room temperature. Appropriate clones were selected and sequenced to ensure proper insertion of the linker region. The resulting plasmid was called ADNpcA3.1 / Zeo (+) / NM-bond2-gag and it was renamed ADNpss-Express-4B. The DNApss-Express-4B is shown in Figure 7B and is the plasmid within which the sequence of interest is cloned. To clone the sequences of interest between inverted tandem repeats, the two Not I sites were used at positions 935 and 978, respectively (see Figure 7B). These two sites are contained within the inverted tandem repeats. To insert the sequences of interest between the inverted tandem repeats and the primer binding site, two convenient restriction endonuclease sites, Pac I and Bam H I, were used at positions 1004 and 1021, respectively. Construction of Plasmid A. The second plasmid of the two plasmid system comprising this second embodiment of the vector of the present invention, is plasmid A, ADNpss-Express-A, which is shown in Figure 8A, which was prepared as described in Example 3. Construction of Plasmid C. As noted above, the vector system of the present invention can also take the form of a single plasmid. To produce a vector system of a single plasmid, or "C" plasmid, the plasmid DNApss-Express-A was digested with Sac I and Xma I to remove the Mbo II gene (Figure 8B). A region of the linker comprising the oligonucleotides 5 '- (link) 2-Hind / Xba and 31- (link) 2-Hind / Xba (Table 1), which were allowed to soften at 70 ° C for 15 minutes and which were cooled slowly to room temperature, bound within the plasmid after digestion under standard conditions. The positive clones were cultured and sequenced to verify the placement of the linker and then this plasmid was digested with Xba and Hind III. The plasmid DNApss-Express-B was then digested with the Hind III and the Xba and the corresponding DNA fragments of 300 base pairs containing the inverted tandem repeats previously described, the multiple cloning site, and the PBS, they were cloned into the digested plasmid to give the ADNpss-Express-C (Figure 9A). Standard ligation reactions were performed and transformed into Sure II cells (Stratagene, Inc.). The transformed positive colonies were cultured and positive clones were identified by restriction analysis.

The sequences of interest within the multiple cloning site of the ADNpss-Express-C were cloned by using the Bam H I and Pac I sites at the multiple cloning site (Figure 9B). The four different sequences of interest as listed in Table 1, each including the "DNA enzyme 10-23" inserted between the 5 'and 3' aspects of the anti-sense sequence (Figure 9C) and shown in the Figures 10A-10D were synthesized for these constructs, and similar procedures were used to insert each of the four sequences of interest. Each construct was prepared by allowing the paired oligonucleotides to soften at 70 ° C for 15 minutes and to cool to room temperature, followed by ligation into the plasmid under standard conditions. After transformation into Sure II cells, the appropriate colonies were selected by sequencing verification for the individual inserts. The antisense capacity of each of these plasmids was tested by transfecting each plasmid with the appropriate controls, which contained a random sequence of equal length and did not contain anti-sense inserts or the "DNA enzyme 10-23", within HeLa cells, using the lipofectant reagent (Boehringer Mannheim) under standard conditions. The Trizol reagent was used to grow the cells and the RNA fraction, and a subsequent Northern spotting analysis was performed to demonstrate the anti-sense specific expression.

Tissue culture studies. Stable and transient transfections were performed using the lipofectant (Boehringer Mannheim Corp.), using the manufacturer's accompanying instructions. All constructs of the plasmids were transfected in HeLa cell lines. The ssDNA assays were performed by the polymerase chain reaction and by spot-spot analysis from 24 to 48 hours after transfection. The activity of the reverse transcriptase was assayed, using the RT-PCR assay developed by Silver, J. et al., Supra, after transfection with the plasmid of the ADNpss-Express-A. Isolates from the individual colony of the HeLa cell lines that were stably replaced (A12 and B12) were further tested for RT activity. The ss-cDNA was isolated from the cells that were transfected 48 to 72 hours before, using the Trizol reagent. The assays for the ssDNA-c species were performed by the two tests based on the polymerase chain reaction for the internal fragment and by denatured single-chain gel electrophoresis with nylon staining and probing with a probe labeled with internal biotin . This experiment showed that human tissue culture cells (HeLa cell line) that were transfected with the plasmids that were designed to synthesize a processed ss-c DNA produced the ssDNA of the predicted size. As described in Example 3, the ssDNA sequence of interest that was produced in accordance with the method of the present invention, is produced from either the position between the inverted repeats after digestion of the stalk intermediate of the stem intermediate -spray, or from the position between the inverted repeats and the primer binding site, by premature termination of the transcript of the reverse transcriptase cDNA in the 3 'aspect of the stem structure. The sequence of interest that occurred from this premature termination is the second sequence of interest to which reference is made herein. Figure 15 shows that the cells that are transfected with the plasmid DNApss-Express-4B which has the enzyme 10-23 included in the anti-sense sequence against the c-raf kinase that was used as the sequence of interest, produced a sequence anti-sense against the c-raf kinase that included the DNA enzyme 10-23 from the position between the inverted tandem repeats and the primer binding site. * * * * * * * * * * * *ents described above demonstrate a method of producing ssDNA in vitro and in vivo by multiple reactions per step, using eukaryotic reverse transcriptase reactions and different cDNA priming reactions. This reaction was followed by the formation of a "stem-loop" intermediate, which can be used to eliminate any unwanted sequences either upstream 5 'or downstream 3' from a designed "stem" (and formed ) after being dissociated subsequently by a restriction endonuclease. Any nucleotide sequence of interest could be produced by this method in a eukaryotic cell. This sequence of interest is cloned (or synthesized) between the inverted tandem repeats designed and represents the sequence in the "turn", after the production of the ssDNA and the subsequent formation of the stem-spiral. The sequence of interest to be produced can be of any base composition (ie,?, T, G, C), provided that the sequence does not interfere with the stalk formation of the stable stem-spire intermediate, which can optionally including the functional genetic element such as a specific enzyme recognition sequence, e.g., a site that serves as a substrate for a particular restriction endonuclease. Again, any restriction endonuclease can also be used to digest (or dissociate) the stem portion of the stem-spire intermediate, provided that the recognition site for that particular restriction endonuclease has been designed within the inverted tandem repeat. Although described with reference to the specific figures and examples set forth herein, those skilled in the art will recognize that certain changes can be made to the specific elements described herein, without changing the manner in which they are described. elements work to achieve their respective proposed results. For example, the cartridge described herein is comprised of three genetic elements, a sequence of interest, an inverted tandem repeat, and a primer binding site. Other genetic elements include an optional restriction endonuclease gene and a reverse transcriptase gene, each of these genes being provided with the appropriate promoters, as described herein. Those skilled in the art will recognize that, for example, the reverse transcriptase gene of the Moloney leukemia virus that was described for use as the reverse transcriptase gene of the cartridge, can be replaced with other reverse transcriptase genes (the Reverse transcriptase gene from human immunodeficiency virus was one of these genes, which was noted above) and promoters other than the CMV promoter can be conveniently used. In addition, different restriction endonuclease genes were previously enlisted, but those skilled in the art will recognize from this description that the list set forth above is not exhaustive, and that many other restriction endonuclease genes will function conveniently in connection with the present invention. Similarly, the RSV promoter that is described being used in connection with the restriction endonuclease genes described herein, is not the only promoter that can be used conveniently. It is intended that all such changes and modifications not departing from the spirit of the present invention fall within the scope of the following non-limiting claims.

Table 1 ODN- PMMV (+) 5'-CTAGGTCGGCGGCCGCGAAGATTGGTGCGCACACACACAACGCGCA 129 bases (# 23) CCAATCTTCGCGGCCGCCGACCCGTCAGCGGGGGTCTTTCATTTGGGGG CTCGTCCGGGATCGGGAGACCCCTGCCCAGGGCC-3 'ODN-PMMV (-) 5'-CTGGGCAGGGGTCTCCCGATCCCGGACGAGCCCCCAAATGAAAGAC 121 bases (# 24) CCCCGCTGACGGGTCGGCGGCCGCGAAGATTGGTGCGCGTTGTGTGTGT GCGCACCAATCTTCGCGGCCGCCGAC-3' ODN-Test (+) 5 ' -GGCCGGAAGATTGGGGCGCCAAAGAGTAACTCTCAAAGGCACGCGC 5 7 bases (# 38) CCCAATCTTCC-3 'ODN-Test (-) 5' -GGCCGGAAGATTGGGGCGCGTGCCTTTGAGAGTTACTCTTTGGCGC 57 bases (# 39) CCCAATCTTCC-3 'ODN-Telo (+) 5'-GGCCGGAAGATTGGGGCGTTAGGGTTAGGGTTAGGGTTAGGGTTAG_92_bases (# 40) GGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGCGCCCCAATCTTCC -3' ODN-Telo (-) 5 '-GGCCGGAAGATTGGGGCGCCCTAACCCTAACCCTAACCCTAACCCT 92 bases (# 41) AACCCTAACCCTAACCCTAACCCTAACCCTAACGCCCCAATCTTCC-3' ODN-XB (+) 5'-GGCCTTGAAGAGCGGCCGCACTAACACCACCACAGTGCGGCCGCTC 51 bases TTCAA-3 'ODN-XB (-) 5'-GGCCTTGAAGAGCGGCCGCACTGTGGTGGTGTTAGTGCGGCCGCTC 51 bases TTCAA-3' ODN-RT (+) 5 '-GGGATCAGGAGCTCAGATCATGGGACACATGG-3' 32 bases (# 13 ) ODN-RT (-) 5 '-CTTGTGCACAAGCTTTGCAGGTCT-3' 24 bases (# 12) ODN-N > S (+) 5 '-CTAGCGGCAAGCGTAGCT-3' 18 bases (# 25) ODN-N > S (-) 5'-ACGCTTGCCG-3 '10 bases (# 26) ODN-Mbo (+) 5' -CAATTAAGGAAAGCTTTGAAAAATTATGTC-3 '30 bases (# 16) ODN -Mbo (-) 5' -TAATGGCCCGGGCATAGTCGGGTAGGG-3 '27 bases (# 33) ODN-HisPro (+) 5 '-AGCTGGATCCCCCGCTCCCCACCACCACCACCACCCTGCCCCT-3' 43 bases (# 36) ODN-HisPro (-) 5 '-AGCAGGGGCAGGGTGGTGGTGGTGGTGGGGAGCGGGGGATCC-3' 42 bases (# 37) ODN-Rep (+) 5 '- ATATCTATTAATTTTGGCAAATCATAGCGGTTATGCTGACTCAGGT 121 base GAATGCCGCGATAATTTTCAGATTGCAATCTTTCATCAATGAATTTCAG TGATGAATTGCCAAG ATTGATGTTGC-3' ODN-Rep (-) 111 base 5'-GACGAGATCTCCTCCAGGAATTCTCGAGAATTCGGATCCCCCGCTC AACATCAATCTTGGC CCCACCACCACCACCACCACCCTGCCCCGCGGATGAAAAATTATGTGAG C-3 '. Name: 3 '- T Mol-Hind III (24-mer) Sequence: 5'-CTT GTG CAC AAG CTT TGC AGG TCT-3' 2. Name: 5'-RT / Mol-Sac I (32-mer) Sequence : 5'-GGG ATC AGG AGC TCA GAT CAT GGG ACC AAT GG-3 '3. Nombe: 5' -Mbo II-Hind III (30-mer) Sequence: 5 '-CAA TTA AGG AAA GCT TTG AAA AAT TAT GTC -3 '4. Name: 5'-RT-Not-Mbo-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: 5'-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 TGT TGT GTG TGT GCG CAC CAA TCT TCG CGG CCG CCG AC-3 '6. Name: 5'-Nhe-Sac -Link (18-mer) Sequence: 5'-CTA GCG GCA AGC GTA GCT-3 '7. Name: 3' -Nhe-Sac-Link (10-mer) Sequence: 5'-ACG CTT GCC G-3 ' 8. Name: 3 '-Mbo II-Xba l (27-mer) Sequence: 5'-TAA TGG CCC GGG CAT AGT CGG GTA GGG -3' 9. Name: 5'-Hi nd-link-Histag (43-mer) Sequence: 5'-A GCT GGA TCC CCC GCT CCC CAC 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 '1 1. Name: 5'-Not-link-testl (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-testl (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 ITA 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-Fokl -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-Fokl -RT (103-mer) Sequence: 5'-CTG GGC AGG GGT CTC COG 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 COG A-3 '

Claims (98)

1. CLAIMS 1. A set of genetic elements adapted for incorporation into a vector for delivery to a cell, comprising: a cartridge comprising (a) a sequence coding for a sequence of interest flanked by complementary sequences 31 and 5 'comprising a inverted tandem repeat and (b) a sequence encoding a first binding site at a 3 'position relative to the inverted tandem repeat; and a gene that encodes a reverse transcriptase.
2. 2. The set of genetic elements of claim 1, wherein said reverse transcriptase gene is selected from the group consisting of the reverse transcriptase genes of the Moloney murine leukemia virus or the human immunodeficiency virus.
3. 3. The set of genetic elements of claim 1, further comprising a eukaryotic promoter for said reverse transcriptase gene.
4. 4. The set of genetic elements of claim 1, further comprising a eukaryotic promoter for said sequence of interest.
5. 5. The set of genetic elements according to either claim 3 or 4, wherein the promoter is selected from the group of promoters comprising constitutive, inducible, broad-spectrum, or tissue-specific promoters.
6. 6. The set of genetic elements according to any of claims 1-5, further comprising a second sequence coding for a sequence of interest between the sequence encoding PBS and the inverted tandem repeat of said cartridge.
7. 7. A set of genetic elements according to any of the preceding claims, wherein the sequence of interest is a single-stranded nucleic acid molecule.
8. 8. A set of genetic elements according to claim 7, wherein the single-stranded nucleic acid molecule is cDNA.
9. 9. A set of genetic elements according to claim 7, wherein the nucleic acid molecule of a single strand in mRNA.
10. 10. A set of genetic elements according to any of claims 7 to 9, wherein the single-stranded nucleic acid molecule is an inhibitory nucleic acid molecule.
11. 11. A set of genetic elements according to claim 10, wherein the inhibitory nucleic acid molecule is an anti-sense or aptamer sequence.
12. 12. A transcription of mRNA from the set of genetic elements according to any of the preceding claims.
13. 13. A method of producing single-stranded DNA having a sequence of interest, comprising the steps of transcribing a reverse transcriptase gene and a cartridge comprising (a) a sequence coding for a sequence of interest flanked by 3 'complementary sequences. and 5 'comprising an inverted tandem repeat and (b) a sequence encoding a first binding site at a 3' position with respect to the inverted tandem repeat in the nucleus of a cell, and reverse transcribing the mRNA transcription of the cartridge with the reverse transcriptase produced by the reverse transcriptase gene.
14. A method according to claim 13, further comprising the step of removing mRNA from the heteroduplex mRNA / cDNA formed by reverse transcription of the mRNA.
15. 15. The method of claim 13 or 14, further comprising linearizing the resulting cDNA.
16. 16. The method of claim 15, wherein the cDNA is linearized including a restriction endonuclease site in the inverted tandem repeat, the cDNA forming an intermediate spike through the Watson-Crick base pair formation of the inverted tandem repeat, and cutting the stem of the intermediate stalk-turn with a restriction endonuclease.
17. The method of claim 13, further comprising inducibly promoting the transcription of the reverse transcriptase gene.
18. 18. The method of claim 17, wherein the transcription of the reverse transcriptase gene is promoted with a eukaryotic promoter.
19. 19. A nucleic acid construct for delivery to a cell, comprising: complementary sequences 3 'and 5' comprising an inverted tandem repeat, a sequence encoding a first binding site for a reverse transcriptase located at a 3 'position with with respect to inverted tandem repetition, and sequence coding for a sequence of interest located either between the complementary sequences 3 'and 5' of the inverted tandem repeat, or between the inverted tandem repeat and the sequence encoding the binding site of primer 31.
20. 20. A nucleic acid construct according to claim 19, wherein the sequence encoding the sequence of interest is located between the complementary sequences 3 'and 5' of the inverted tandem repeat.
21. 21. A nucleic acid construct according to claim 20, wherein the inverted tandem repeat is capable of forming an intermediate stem-spindle in a single-strand nucleic acid product encoded by said nucleic acid construct, said sequence of interest or sequence encoding said sequence of interest in the loop and said inverted tandem repetition forming the stem.
22. 22. A nucleic acid construct according to claim 20 or 21, which comprises a second sequence coding for a sequence of interest located between the inverted tandem repeat and the sequence encoding the 3 'primer ligation site.
23. 23. A nucleic acid construct according to any of claims 20 to 22, wherein the inverted tandem repeat comprises a sequence encoding one or more specific enzyme recognition sequences.
24. 24. A nucleic acid construct according to claim 23, wherein the specific enzyme recognition sequence comprises a restriction endonuclease site.
25. 25. A nucleic acid construct according to claim 24, further comprising a gene encoding a restriction endonuclease.
26. 26. A nucleic acid construct according to claim 25, wherein the restriction endonuclease gene is located 5 'to the inverted tandem repeat.
27. 27. A nucleic acid construct according to any of claims 23 to 26, wherein the specific enzyme recognition sequence includes restriction sites either Hind III and Not I, Hind III, or Not I.
28. 28. A nucleic acid construct according to any of claims 23 to 26, wherein the specific enzyme recognition sequence is selected from the group consisting of a type I restriction endonuclease, type II endonuclease, endonuclease type III, eukaryotic receptor recognition , recognition of prokaryotic receptor, promoter, promoter / enhancer and PCR site with hanging T, and their combinations.
29. 29. A nucleic acid construct according to claim 19, wherein the sequence encoding the sequence of interest is located between the inverted tandem repeat and the sequence encoding the 3 'primer ligation site.
30. 30. A nucleic acid construct according to claim 19 or 29, wherein the inverted tandem repeat is capable of forming a stable stem-spindle, an unstable stem-spire, or a stalk-coil of intermediate stability in an acid product. single-stranded nucleic nucleus encoded by said nucleic acid construct.
31. 31. A nucleic acid construct according to any of claims 19 to 30, wherein the inverted tandem repeat comprises a sequence encoding one or more eukaryotic, prokaryotic and / or viral protein binding sites.
32. 32. A nucleic acid construct according to any of claims 19 to 31, wherein the inverted tandem repeat acts in cis-oriented form.
33. 33. A nucleic acid construct according to any of claims 19 to 32, wherein the primer ligation site is specific for an endogenous reverse transcriptase.
34. 34. A nucleic acid construct according to any of claims 19 to 32, further comprising a gene encoding a reverse transcriptase.
35. 35. A nucleic acid construct according to claim 34, wherein the reverse transcriptase gene is located 5 'to the inverted tandem repeat.
36. 36. A nucleic acid construct according to any of claims 19 to 32, further comprising a gene encoding a reverse transcriptase / RNase polyprotein.
37. 37. A nucleic acid construct according to claim 36, wherein the gene which encodes the reverse transcriptase polyprotein / RNase H is located 5 'to the inverted tandem repeat.
38. 38. A nucleic acid construct according to claim 36 or 37, wherein the gene encoding the reverse transcriptase polyprotein / RNase H is from Moloney murine leukemia virus, human immunodeficiency virus, or simian immunodeficiency virus.
39. 39. A nucleic acid construct according to any of claims 34 to 38, wherein the primer binding site is specific for a reverse transcriptase encoded by the reverse transcriptase gel or reverse transcriptase polyprotein / RNase
40. 40. A nucleic acid construct according to any of the preceding claims, further comprising a promoter and, optionally, a enhancer for each of said first or second sequences encoding a sequence of interest, said restriction endonuclease, said reverse transcriptase and / or said reverse transcriptase / RNase gene.
41. 41. A nucleic acid construct according to claim 40, wherein the promoter and / or enhancer is a eukaryotic promoter / enhancer.
42. 42. A nucleic acid construct according to claim 40 or 41, wherein the promoter is a constitutive, inducible, broad-spectrum or tissue-specific promoter.
43. 43. A nucleic acid construct according to any of the preceding claims, further comprising a sequence encoding a polyadenylation tail sequence located 3 'to the 3' primer ligation site.
44. 44. A nucleic acid construct according to any of the preceding claims, wherein the first or second sequence encoding a sequence of interest includes a sequence encoding a ssDNA having enzymatic activity.
45. 45. A nucleic acid construct according to claim 44, wherein the first or second sequence encoding a sequence of interest includes the sequence 5'-GGCTAGCTACAACGA-3 ', flanked in both the 3' and 5 'directions by sequences encoding one or more sequences complementary to the target mRNA species.
46. 46. A nucleic acid construct according to claim 45, wherein the target mRNA species is for: (i) h-ras, (ii) c-raf kinase, (iii) anti-sense sequence to angiogenic growth factor of pleiotrophin, or (iv) the tat region of the simian immunodeficiency virus (SIV).
47. 47. A nucleic acid construct according to any of the preceding claims, wherein the 3 'primer ligation site is complementary to a transfer RNA (AR t).
48. 48. A nucleic acid construct according to any of claims 19 to 47, wherein the sequence of interest is a single-stranded nucleic acid molecule.
49. 49. A nucleic acid construct according to claim 48, wherein the single-stranded nucleic acid molecule is cDNA.
50. 50. A nucleic acid construct according to claim 48, wherein the nucleic acid molecule of a single strand in mRNA.
51. 51. A nucleic acid construct according to any of claims 48 to 50, wherein the single-stranded nucleic acid molecule is an inhibitory nucleic acid molecule.
52. 52. A nucleic acid construct according to claim 51, wherein the inhibitory nucleic acid molecule is an anti-sense or aptamer sequence.
53. 53. A nucleic acid construct according to any of claims 19 to 52, wherein the nucleic acid is DNA.
54. 54. A transcription of DNA mRNA of claim 52.
55. 55. A transcription of mRNA, comprising (a) a sequence coding for a sequence of interest flanked by complementary sequences 3 'and 5' comprising an inverted tandem repeat, and further comprising a primer ligation site located 3 'to the inverted tandem repeat.
56. 56. A mRNA transcript, comprising (a) complementary sequences 3 'and 5' comprising an inverted tandem repeat and (b) a sequence coding for a sequence of interest located between the inverted tandem repeat and the binding site of 3 'primer
57. 57. A mRNA transcript, comprising (a) a first sequence coding for a sequence of interest flanked by complementary 3 'and 5' sequences comprising an inverted tandem repeat, (b) a 3 'located primer ligation site. regarding tandem repeat inverted, and (c) a second coding sequence for a sequence of interest between the inverted tandem repeat and the primer binding site 3 '.
58. 58. A mRNA transcription according to any of claims 54 to 57, wherein the inverted tandem repeat is capable of forming a stable stem-spindle, an unstable stem-spire or a stalk-coil of intermediate stability.
59. 59. The mRNA transcription of any of claims 55 to 58, wherein the sequence encoding the sequence of interest includes a sequence encoding a ssDNA having enzymatic activity.
60. 60. A ssDNA transcript of the mRNA of any of claims 55 to 59.
61. 61. A vector, comprising the nucleic acid construct of any of claims 19 to 53.
62. 62. A vector, comprising: 3 'complementary sequences. and 5 'comprising an inverted tandem repeat, a sequence encoding a primer binding site for a reverse transcriptase located at a 3' position relative to the inverted tandem repeat, and an insertion site for a sequence coding for a sequence of interest between the complementary sequences 3 'and 5' of the inverted tandem repeat, or between the inverted tandem repeat and the sequence encoding the primer binding site 31.
63. 63. A vector according to claim 62, which comprises a first insertion site between the complementary 3 'and 5' sequences of the inverted tandem repeat and a second insertion site between the repeat inverted tandem and the sequence encoding the 3 'primer ligation site.
64. 64. A vector according to claim 62 or 63, further comprising a gene encoding a reverse transcriptase.
65. 65. A vector according to claim 62 or 63, further comprising a gene encoding a reverse transcriptase / RNase polyprotein.
66. 66. A vector according to claim 64 or 65, wherein the reverse transcriptase or the gene of the Reverse transcriptase polyprotein / RNase H is located 5 'to the inverted tandem repeat.
67. 67. A vector system, comprising a first vector according to claim 62 or 63, and a second vector comprising a gene encoding a reverse transcriptase.
68. 68. A vector or vector system according to any of claims 62 to 67, wherein the vector is a plasmid or a modified viral construct.
69. 69. A vector or vector system according to any of claims 62 to 68, wherein the gene is operably linked to an expression control sequence.
70. 70. A host cell stably transformed or transfected with a vector or vector system according to any of claims 62 to 69.
71. 71. A host cell according to claim 70, which is a eukaryotic cell.
72. 72. A host cell according to claim 70, which is a bacterial cell.
73. 73. A kit for producing a single-stranded nucleic acid molecule, which kit comprises a vector or a vector system according to any of claims 62 to 69, and a restriction endonuclease for each insertion site.
74. 74. A kit for producing a single-stranded nucleic acid molecule, which kit comprises a vector or vector system according to any of claims 62 to 69, a container for the vector / vector system and instructions for use of the vector / vector system.
75. 75. An in vivo or in vitro method of producing a single-stranded nucleic acid molecule having a sequence of interest, which method comprises the steps of introducing a nucleic acid construct according to any of claims 19 to 53 in a target cell, transcribing the construction of nucleic acid into mRNA, and reverse transcribing the transcription of mRNA into cDNA.
76. 76. A method according to claim 75, further comprising the step of removing the mRNA transcript from the mRNA / cDNA heteroduplex formed by reverse transcription of the mRNA.
77. 77. A method according to claim 75 or 76, wherein the reverse transcription is carried out by a reverse transcrip-rate that is endogenous to the target cell.
78. 78. A method according to claim 75 or 76, further comprising the step of introducing a gene encoding a reverse transcriptase into a target cell.
79. 79. A method according to claim 75 or 76, further comprising the step of introducing a gene encoding a reverse transcriptase / RNase polyprotein in the target cell.
80. 80. A method according to any of claims 75 to 79, further comprising the step of linearizing cDNA transcription by cutting the stem-spire structure of cDNA formed by the inverted tandem repeat where the loop structure is attached to the stem.
81. 81. A method according to claim 80, further comprising the step of introducing a gene encoding a restriction endonuclease in the target cell.
82. 82. A method according to claim 80, further comprising the step of introducing a gene encoding a restriction endonuclease into the target cell.
83. 83. A method according to any of claims 75 to 82, further comprising the step of isolating the mRNA transcript, the mRNA / ADC heteroduplex and / or the single-stranded cDNA from the target cell.
84. 84. A transcription of single-stranded cDNA, produced by the method of any of claims 75 to 83.
85. 85. An inhibitory nucleic acid molecule, produced by the method of any of claims 75 to 83.
86. 86. A molecule of inhibitory nucleic acid according to claim 85, which is an antisense or aptamer sequence.
87. 87. A single-strand cDNA, having enzymatic activity, produced according to the method of any of claims 75 to 83.
88. 88. A mRNA transcript produced according to the method of any of claims 75 to 83.
89. 89. A heteroduplex molecule produced by the method of any of claims 75 to 83.
90. 90. A pharmaceutical composition, comprising a nucleic acid construct according to any of claims 19 to 53, together with an adjuvant, diluent or carrier. pharmacologically acceptable.
91. 91. A pharmaceutical composition, comprising a vector or vector system according to any of claims 62 to 69, together with a pharmacologically acceptable adjuvant, diluent or carrier.
92. 92. A pharmaceutical composition, comprising a host cell according to any of claims 70 to 72, together with a pharmacologically acceptable adjuvant, diluent or carrier.
93. 93. A nucleic acid construct according to any of claims 19 to 53, for use in therapy, especially for use in delivery of an inhibitory nucleic acid molecule to a target cell.
94. 94. A vector or vector system according to any of claims 62 to 69, for use in therapy, especially for use in delivery of an inhibitory nucleic acid molecule to a target cell.
95. 95. A host cell according to any of claims 70 to 72 for use in therapy, especially for use in delivery of an inhibitory nucleic acid molecule to a target cell.
96. 96. The use of a nucleic acid construct according to any of claims 19 to 53 for the manufacture of a medicament for alleviating a pathological condition by regulating the expression of genes, especially for alleviating a pathological condition by delivering a molecule of inhibitory nucleic acid to a target cell.
97. 97. The use of a vector or vector system according to any of claims 62 to 69 for the manufacture of a medicament for alleviating a pathological condition by regulating the expression of genes, especially for alleviating a pathological condition by delivering a molecule of inhibitory nucleic acid to a target cell.
98. 98. The use of a target cell according to any of claims 70 to 72 for the manufacture of a medicament for alleviating a pathological condition by regulating gene expression, especially for alleviating a pathological condition by delivering a nucleic acid molecule inhibitory to a target cell. Summary Methods and compositions for producing single-strand cDNA (cDNA-ss) in eukaryotic cells, specifically a DNA cassette that produces cDNA-ss in vivo. The cartridge contains the gene encoding reverse transcriptase / RNase H of murine Moloney leukemia virus, a bacterial restriction endonuclease gene, and a sequence of interest that produces an RNA template from which reverse transcriptase synthesizes cDNA -ss of a specified sequence. The cDNA-ss is then modified to remove all flank vector sequences by taking advantage of the "stem-loop" structure of the ss-cDNA, which is formed as a result of the inclusion of an inverted tandem repeat that allows the cDNA ss is doubled over itself, forming a DNA stem of double helix, in the sequence of interest. The double helical stem contains one or more functional genetic elements such as a restriction endonuclease recognition site and the loop, which remains as ss-DNA, constituted by any desired nucleotide sequence. This design allows the double helix stem of the intermediate stem loop to be broken to the corresponding restriction endonuclease (s) specific to the site in the stem and the loop portion, or sequence of interest, it is then released as a piece of linearized DNA, of a single filament. This piece of ss-DNA released (or broken) contains minimal sequence information, if at all, either upstream of 51 or downstream of the 3 'portion of the previous double helix stem containing the endonuclease cleavage site of restriction. In vivo transfections using the DNA vector system described herein demonstrate the use of this system to produce ss-DNA in host cells.