MXPA01010618A - Methods and compositions for inhibiting the function of polynucleotide sequences - Google Patents

Abstract

A therapeutic composition for inhibiting the function of a target polynucleotide sequence in a mammalian cell includes an agent that provides to a mammalian cell an at least partially double-stranded RNA molecule comprising a polynucleotide sequence of at least about 200 nucleotides in length, said polynucleotide sequence being substantially homologous to a target polynucleotide sequence. This RNA molecule desirably does not produce a functional protein. The agents useful in the composition can be RNA molecules made by enzymatic synthetic methods or chemical synthetic methods in vitro;or made in recombinant cultures of microorganisms and isolated therefrom, or alternatively, can be capable of generating the desired RNA molecule in vivo after delivery to the mammalian cell. In methods of treatment of prophylaxis of virus infections, other pathogenic infections or certain cancers, these compositions are administered in amounts effective to reduce or inhibit the function of the target polynucleotide sequence, which can be of pathogenic origin or produced in response to a tumor or other cancer, among other sources.

Description

METHODS AND COMPOSITIONS TO INHIBIT THE FUNCTION OF POLYUCLEOTHYDIC SEQUENCES FIELD OF THE INVENTION The present invention relates to polynucleotide compositions having an inhibitory or regulatory effect of another type, on the function of certain target polynucleotide sequences present in a mammalian cell, and for methods of using the compositions in therapeutic, prophylactic, diagnosis and research.

BACKGROUND OF THE INVENTION The polynucleotide compositions have been described for pharmaceutical uses, mainly for the treatment or prophylaxis of diseases in mammals, as well as research in such fields. Specifically, a high interest of activity currently surrounds the use of polynucleotide compositions in the treatment of pathogenic extracellular and intracellular infections, such as viral, bacterial, fungal infections, and the like. As an example, DNA vaccines are described for distributing or administering to a REF cell: 133502 mammal in vivo an agent that combats a pathogen, by boosting the mammalian immune system. Thus, such vaccines are designed to express, for example, a viral protein or polypeptide, and promote a humoral or cellular immune response after challenge with the infectious agent. Genetic therapeutic vectors, on the other hand, are polynucleotide compositions generally designed to deliver to a mammalian cell, a protein that is either not expressed, inappropriately expressed or underexpressed in a mammal. Such vectors must frequently be directed to the species-specific immune responses, to those polynucleotide sequences that are recognized as antigenic, or which evoke an undesired, cellular immune response. Other additional therapeutic uses of the polynucleotide compositions are for the administration of missing or underexpressed proteins to a diseased mammalian patient. In addition, the polynucleotides are useful in themselves as in vivo reagents, in diagnostic / imaging protocols, as reagents in gene therapy, in antisense protocols and in vaccine applications, or otherwise as pharmaceuticals used to treat or prevent a variety of conditions such as genetic defects, infectious diseases, cancer, and auto-immune diseases. The polynucleotides are also useful as in vitro reagents in assays such as biological screening assays, diagnostic and selection medical assays, and contamination detection assays. A host of problems well known in the art has prevented numerous polynucleotide compositions from becoming widely accepted as useful pharmaceuticals. Thus, there are few such DNA vaccines or therapeutic products that have still been accepted by the medical community for the treatment of diseases in mammals. Phenomena have been observed in plants and in nematodes that are mediated by polynucleotide compositions, and are termed as post-translational gene silencing and transcriptional silencing. This phenomenon demonstrates that the transfection or infection of a plant, nematode or Drosophila with a virus, viroid, plasmid or RNA expressing a polynucleotide sequence having some homology with a regulatory element, such as a promoter or a native gene or a portion of the same already expressed in that cell, can result in the permanent inhibition of the expression of the endogenous regulatory element or of the gene and of the exogenous sequence. The effect of silencing was shown to be gene-specific. See for example, L Timmons and A. Fire, Nature, 395: 354 (October 29, 1998); A. Fire et al., Nature, 391: 806-810 (February 19, 1998); and R. Jorgensen et al., Science, 279: 1486-1487 (March 6, 1998). A DNA plasmid encoding a full-length pro-alpha 1 collagen gene was transiently transfected into a rodent fibroblast tissue cell line, and a "silencing" effect was observed on the native collagen gene and the transiently expressed gene [Bahramina and Zarbl, Mol. Cell. Biol., 19 (1): 274-283 (January 1999)]. See also, International Patent Application No. O98 / 05770, published February 12, 1998, which refers to the inhibition of the gene by the use of an antisense RNA with secondary structures and / or in combination with the RNAse of double strand International Patent Application No. O99 / 53050, published on October 21, 1999, also refers to the reduction of the phenotypic expression of a nucleic acid, particularly in plant cells, by the introduction of chimeric genes that code for the molecules of RNA in sense and antisense. There is a need in the art for polynucleotide compositions and methods of using same, to inhibit the function of the polynucleotide sequences that are causing disease in mammals, such as the polynucleotide sequences essential for the replication of viruses and other intracellular pathogens in human cells. mammal, or sequences of extracellular mammalian pathogens, or sequences of tumor antigens that mediate the spread of cancer in a mammal, and the like, without adversely affecting the essential gene sequences in the mammal.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the invention provides a composition for inhibiting the function of an objective polynucleotide sequence in a mammalian cell. The composition comprises an agent that provides a mammalian cell with at least one partially double-stranded RNA molecule, comprising a polynucleotide sequence of at least about 200 nucleotides in length. The polynucleotide sequence is substantially homologous to the target polynucleotide sequence, which may be a polynucleotide sequence, for example, of a virus or other intracellular pathogen, a polynucleotide sequence of a cancerous antigen or of an essential tumorigenic regulatory sequence, a polynucleotide sequence of an extracellular pathogen present in a mammal, or any other polynucleotide sequence that is desired to "turn off" in a cell. This RNA molecule preferably does not produce a functional protein. This RNA molecule is preferably substantially non-homologous to the mammalian, essential polynucleotide sequences of natural origin. In one embodiment, the agent of this composition is an RNA molecule made by synthetic enzymatic methods or synthetic chemical methods in vi tro. In yet another embodiment, the RNA molecule can be generated in a recombinant culture, for example, bacterial cells, isolated therefrom, and used in the methods discussed below. In yet another embodiment, the agent of this composition generates the RNA molecule in vivo after administration to the mammalian cell. In yet another aspect, the invention provides a pharmaceutical composition comprising one or more of the compositions described immediately above and specifically hereinafter, and a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier . Such compositions are useful for the treatment of intracellular pathogenic infections, such as viruses. Other such compositions are useful for the treatment of certain cancers. Other such compositions are useful for the treatment of certain extracellular pathogens. Other such compositions are useful for the treatment of any disease or disorder where inhibition of the function of a polynucleotide sequence in a mammal is desirable, for use in therapy or in vaccine. In yet another aspect, the invention provides a method for treating viral information in a mammal by administering to the mammal of one or more of the compositions described above, wherein the target polynucleotide is a viral polynucleotide sequence necessary for replication and / or pathogenesis of the virus in an infected mammalian cell, together with a second optional agent that facilitates the uptake of the polynucleotide into a cell, in a pharmaceutically acceptable carrier. This composition is administered in an amount effective to reduce or inhibit the function of the viral sequence in the cells of the mammal. In a further aspect, the invention provides a method for preventing a viral infection in a mammal, by administering to the mammal of one or more of the compositions described above, wherein the target polynucleotide is a viral polynucleotide sequence necessary for replication and / or pathogenesis of the virus in an infected mammalian cell, with a second optional agent that facilitates the uptake of polynucleotide into a cell, in a pharmaceutically acceptable carrier. This composition is administered in an amount effective to reduce or inhibit the function of the viral sequence after the subsequent introduction of the virus into the cells of the mammal. In another aspect, the invention provides a method for the treatment or prophylaxis of a virally induced cancer in a mammal, by administration to the mammal, of one or more of the compositions described above in which the target polynucleotide is a sequence encoding a tumor antigen or a functional fragment thereof or a regulatory sequence, whose function of sequence is required for the maintenance of the tumor in the mammal. The compositions may contain a second optional agent that facilitates uptake of the polynucleotide into a cell, and a pharmaceutically acceptable carrier. The composition is administered in an amount effective to reduce or inhibit the function of the sequence that maintains the tumor in the mammal. In still another aspect, the invention provides a method for the treatment or prophylaxis of infection of a mammal by an intracellular pathogen. The mammal is administered with one or more of the compositions described herein, wherein the target polynucleotide is a polynucleotide sequence of the intracellular pathogen, necessary for the replication and / or pathogenesis of the pathogen in an infected mammalian cell. The composition is administered with a second optional agent that facilitates the uptake of the polynucleotide into a cell, in a pharmaceutically acceptable carrier, in an amount effective to reduce or inhibit the function of the sequence in the mammal. . In still another aspect, the invention provides a method for the treatment or prophylaxis of infection of a mammal by an extracellular mammalian pathogen. The mammal is administered one or more of the compositions described herein, wherein the target polynucleotide is a polynucleotide sequence of the extracellular pathogen, necessary for the replication and / or pathogenesis of the pathogen in an infected mammal. The composition is administered in a pharmaceutically acceptable carrier, in an amount effective to reduce or inhibit the function of the sequence in a mammal. This can be administered with a second optional agent that facilitates the uptake of the polynucleotide by the pathogenic cell. In still another aspect, the invention provides a method of treatment or prophylaxis of cancer in a mammal. The mammal is administered one or more of the compositions described above, wherein the target polynucleotide is a polynucleotide sequence of an abnormal gene that causes cancer or a regulatory sequence not expressed, in a mammal, which also possesses a normal copy of the gene or of the regulatory sequence. According to this aspect, the differences between the abnormal sequence and the normal sequence are differences in the polynucleotides. The composition is administered with a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier, and in an amount effective to reduce or inhibit the function of the abnormal sequence in the mammal. In a further aspect, the invention involves a method for treating a disease or disorder in a mammal, comprising administering the mammal having a disease or disorder, characterized by the expression of the polynucleotide product not found in a healthy mammal, one or more of the compositions as described above, in which the target polynucleotide sequence is the polynucleotide sequence expressing that polynucleotide product or a non-expressed regulatory sequence essential for the expression of that product. The composition is administered with or without a second agent that facilitates the uptake of the polynucleotide into a cell, and into a pharmaceutically acceptable carrier, in an amount effective to reduce or inhibit the function of the target polynucleotide product or the regulatory sequence in the mammalian cells. . Yet another aspect of the present invention provides such compositions for use in research methods, such as a reagent for reducing or inhibiting undesired expression of the gene in mammalian cells or tissues, in vi tro for use in diagnostic assays or other research trials, or ex vivo for the return to the mammal for therapy or other medical uses. Other aspects of the invention are further described in the following detailed description of the preferred embodiment thereof.

BRIEF DESCRIPTION OF THE FIGURES Figure IA is an illustration of a PCR product generated using the forward gag primer of the bacteriophage T7 RNA polymerase promoter (T7F) and the reverse gag primer (R). Transcription from this PCR template, using T7 RNA polymerase generates a strand in the gag sense, of RNA sequence. Figure IB is an illustration of a PCR product generated using a forward gag primer (F) and the reverse gag primer of the T7 promoter (T7R). Transcription of this template using a T7 RNA polymerase generates a gag antisense strand of the RNA sequence. The use of the template of FIG. IA and the template of FIG. IB produces the double-stranded gag RNA sequence.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the novel polynucleotide compositions and methods for therapy, prophylaxis, research and diagnosis in diseases and disorders affecting mammalian species, in which the goal is to reduce or inhibit the function of a selected target polynucleotide sequence. These compositions and methods have utility both in vi tro and in vivo. These compositions and methods also make it possible to take advantage of the molecular mechanisms of the cell to achieve the therapeutic goals without requiring any stimulation of the immune system of the mammal involved. As used herein, the phrases "objective" or "target polynucleotide sequence" refer to any sequence present in a mammalian cell or mammalian organism, either naturally occurring, and possibly a mammalian defective polynucleotide sequence. or a heterologous sequence, present, due to an intracellular or extracellular pathogenic infection or a disease, whose polynucleotide sequence has a function that is desired to be reduced or inhibited. This target sequence can be a coding sequence, ie, it is translated to express a protein or a functional fragment thereof. Alternatively, the target sequence may be non-coding, but may have a regulatory function. An objective polynucleotide sequence is a viral polynucleotide sequence necessary for the replication and / or pathogenesis of the virus in an infected mammalian cell. Another embodiment of a target polynucleotide sequence is a tumor antigen or a functional fragment thereof, or a non-expressed regulatory sequence of a cancer induced by the virus, whose sequence is required for the maintenance of the tumor in the mammal. Yet another embodiment of an objective polynucleotide sequence is a polynucleotide sequence of an intracellular or extracellular pathogen necessary for the replication and / or pathogenesis of that pathogen in an infected mammal. Yet another embodiment of an objective polynucleotide sequence is a polynucleotide sequence of an abnormal gene that causes cancer (or a regulatory sequence not expressed) in a mammal, which also has a normal copy of the gene or sequence. The differences between the abnormal sequence and the normal sequence are differences at the level of the polynucleotide sequence. Such a normal sequence may be, for example, a fusion of two normal genes, and the target sequence may be the sequence encompassing that fusion, for example, the sequence of the BCR-abl gene characteristic of certain leukemias. The term "gene" is intended to include any target sequence intended to be "silenced", whether transcribed and / or translated or not, including regulatory sequences, such as promoters. The term "mammal" or "mammals" is intended to encompass its normal meaning. While the invention is most desirably aimed for efficacy in humans, it can also be employed in domestic mammals such as canines, felines, and equines, as well as mammals of particular interest, eg, zoo animals, farm animals and the like. .

A. The compositions of the invention A composition for inhibiting the function of an objective polynucleotide sequence in a mammalian cell, according to this invention, comprises an agent that provides a mammalian cell with at least one partially double-stranded RNA molecule. In general, the term "RNA" may also include RNA-DNA hybrids, except where otherwise specified, for example, where a 2'-OH group of ribose is required for a particular bond. The RNA molecule comprises a polynucleotide sequence of at least about 200 nucleotides in length. Importantly, this polynucleotide sequence of the RNA molecule is substantially homologous to the target polynucleotide sequence. This polynucleotide sequence also preferably contains exon sequences or portions thereof. Desirably, the polynucleotide sequence does not contain intron sequences. Preferably, the RNA molecule does not produce a functional protein, and more preferably, it is not translated. The polynucleotide sequence of the RNA molecule is preferably substantially non-homologous to any essential, normally functional, mammalian polynucleotide sequence of natural origin. The polynucleotide sequences described herein may employ a multi-target or polyepitope method, for example, coding sequences for more than one gene of a single target pathogen or against more than one target pathogen, or another desired target category that goes be silenced The "at least partially double-stranded RNA molecule" includes a polynucleotide sequence of RNA of between about 100 to 10,000 polynucleotides in length. To date, the sequence is most desirably at least 200 polynucleotides in length, but can range from 200 to 8,000 polynucleotides in length. In yet another embodiment, the RNA molecule may be less than 7,500 polynucleotides in length. In yet another embodiment, the RNA molecule may have a sequence length of less than about 5,000 polynucleotides. In yet another embodiment, the RNA molecule may have a sequence length of less than about 2,000 polynucleotides. In yet another embodiment, the RNA molecule may have a sequence length of less than about 1,000 polynucleotides. In yet another embodiment, the RNA molecule may have a sequence length of less than about 750 polynucleotides. Minimally, to keep the RNA molecule stable, it has a minimum of 11 to 30 nucleotides involved in a double-stranded sequence, depending on the composition of the polynucleotide sequence and a? G of approximately -9.2 kcal / mol. As is known in the art,? G defines the state of minimum external energy required to maintain a stable molecular configuration [see for example, Jaeger et al., Proc. Nati Acad.

Sci., USA, 20: 7706-7710 (1989); and Soler and Jankowski, Math.

Biosci., 2: 167-190 (1991)]. Based on this minimum, preferably at least 10% of this sequence of the double-stranded RNA molecule is partially double-stranded. Alternatively, the double-stranded portion of these RNA molecules can be at least 30% of the sequence. In yet another embodiment, the double-stranded portion of these molecules can be at least 50% of the sequence. In yet another embodiment, the double-stranded portion of these molecules can be at least 70% of the sequence. In yet another embodiment, the double-stranded portion of these molecules can be at least 90% of the sequence. In another modality, the complete sequence can be double-stranded. Alternatively, the double-stranded portion of these molecules can appear at one or both ends, or at an intermediate portion of the sequence, if the molecule is linear. Similarly, the double-stranded portion can be anywhere if the molecule is circular. In certain embodiments of the present invention, the double-stranded portion of the RNA molecule becomes double-stranded only when the molecule is in the mammalian cell. In yet another embodiment of this invention, the partially double-stranded molecule is an RNA / DNA hybrid, for example, a strand containing RNA and DNA, prepared in vi tro or in vivo.; or a duplex of two such simple chains or portions thereof. In yet another embodiment, the RNA molecule, made in vivo or in vi tro, is a duplex comprised of a single strand of RNA and a single strand of DNA.

The polynucleotide sequence of the partially double-stranded RNA molecule must be substantially homologous to the target polynucleotide sequence in order to effectively reduce or inhibit the function thereof. The necessary homology can be adequately defined by the use of a computer algorithm. As is known in the art, "homology" or "identity" means the degree of sequential relativity between two polypeptide sequences or two polynucleotides as determined by the identity of the agreement between two lengths of such sequences. Identity and homology can be easily calculated by the methods described in the prior art [See, for example, COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York (1988); BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D., ed. , Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M. , and Griffin, H.G., eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, (1991)]. While there are a number of methods for measuring the identity and homology between two polynucleotide sequences, the terms "identity", "similarity", and homology are well known to those skilled in the art [H. Carillo and D. Lipton, SIAM J. Applied Math., 49: 1073 (1988)]. Methods commonly employed to determine the identity of homology between two sequences include, but are not limited to, those described in Guide to Huge Computers, Martin J. Bishop, ed. , Academic Press, San Diego, 1994, and H. Carillo and D. Lipton, SIAM J. Applied Math., 48: 1073 (1988). Preferred methods for determining identity or homology are designed to give the greatest agreement between the two sequences tested. Methods for determining identity and similarity are codified in computer programs. The preferred computer program for determining the identity and homology between two sequences includes, but is not limited to, the BESTFIT algorithm from the GCG program package [J.

Devereux and collaborators, Nucí. Acids Res., 12 (1): 387 (1984)], the related MACVECTOR program (Oxford), and the FASTA (Pearson) programs. For example, searches for sequential similarities in databases between mammalian polynucleotide sequences, naturally occurring, significant and target polynucleotide sequences, make it possible to design suitable RNA molecules, desired for use in the invention. The algorithm and / or degree of homology necessary for any particular RNA molecule can be selected by a person skilled in the art, depending on the identity of the target and / or the closeness of homology of the target sequence to any sequence of mammal of natural origin, which is desired to be left functioning normally after the use of the methods of this invention. In a preferred embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 10% of the target sequence and contains at least one segment (window) of 30 contiguous nucleotides with a homology in that window of at least 50% to a similar region of 30 nucleotides of the target sequence, using the MACVECTOR program with a default firing temperature of 37 ° C. In yet another embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 30% of the sequence target and contains at least one window of 30 contiguous nucleotides with a homology in that window of at least 50% to a similar region of 30 nucleotides of the target sequence. In another preferred embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 50% to the target sequence and contains at least one window of 30 contiguous nucleotides with a homology in that window of at least 50% to a region similar to 30 nucleotides of the target sequence. In another modality more, the polynucleotide sequence of RNA desirably has a total homology of at least 70% to the target sequence and contains at least one window of 30 contiguous nucleotides with a homology in that window of at least 50% to a similar region of 30 nucleotides of the objective sequence. In yet another embodiment, the polynucleotide sequence of RNA desirably has a total homology of at least 90% to the target sequence and contains at least one window of 30 contiguous nucleotides with a homology in that window of at least 50% to a region similar to 30 nucleotides of the target sequence. In yet another embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 10% to the target sequence and contains at least one window of 30 contiguous nucleotides with a homology in that window of at least 70% to a region similar to 30 nucleotides of the target sequence. In another embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 10% to the target sequence, and contains at least one segment (window) of 30 contiguous nucleotides with a homology in that window of at least 90% to a similar region of 30 nucleotides of the target sequence. In yet another embodiment, the polynucleotide sequence of RNA desirably has a total homology of at least 10% to the target sequence, and contains at least two windows of 30 contiguous nucleotides with a homology in the windows of at least 50% to regions similar to 30 nucleotides of the target sequence. Other embodiments of this formula may be developed by a person skilled in the art. In a second preferred embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 10% to the target sequence, and contains at least one segment (window) of 5 contiguous nucleotides with absolute homology in that window, at a region of 5 nucleotides of the target sequence, using the MACVECTOR program with an annealing temperature by default of 37 ° C. In yet another variant of this embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 30% to the target sequence, and contains at least one window of 5 contiguous nucleotides with absolute homology to a region of 5 nucleotides of the objective sequence. In yet another embodiment, the RNA polynucleotide sequence desirably has a total homology of at least 50% to the target sequence, and contains the absolutely homologous window of 5 nucleotides, described above. Other variants of this modality can be developed by a person skilled in the art.

The presence of the windows referred to in the above formulas allows the total homology of the rest of the sequence to be low, however, it is anticipated that a low total complete homology is likely to affect the dosage of the therapeutic compositions described below, so adverse An increase in the number of such windows in the polynucleotide sequence of RNA is likely to allow the total homology of the rest of the sequences to be low, but does not affect the dosage. It should be understood that the selection of the necessary homology, the selection of the omissions for the program and the selection of the program used to calculate homology is within the experience of the technique, given the teachings of this specification and the knowledge that exists in the scientific literature The polynucleotide sequence of the RNA molecule is also desirably substantially non-homologous to any normal mammalian, naturally occurring, essential polynucleotide sequence, so that the polynucleotide sequence of the RNA molecule does not adversely affect function of any mammalian polynucleotide sequence, of natural, essential origin, when the methods of this invention are used. Such functional, mammalian, mammalian polynucleotide sequences include mammalian sequences that encode desired proteins, as well as mammalian sequences that are non-coding but provide the essential regulatory sequences in a healthy mammal. Essentially, the RNA molecule useful in this invention must be sufficiently distinct in sequence from any mammalian polynucleotide sequence for which the function is intended to be undisturbed after any of the methods of the invention are performed. As described for the determination of homology to the above target sequence, a person skilled in the art may have resorted to previously identified computer algorithms to define the essential lack of homology between the polynucleotide sequence of the RNA molecule and the sequences of normal mammals. Thus, in an exemplary embodiment, the homology between the RNA polynucleotide and the normal sequence selected is less than the homologies of the formulas described above. More preferably, there is almost no homology at all between the RNA polynucleotide and any normal mammalian sequence. It must be understood that the selection of the necessary homology is within the experience in the technique, given the teachings of this specification and the knowledge that exists in the scientific literature.

Finally, another desirable attribute of the RNA molecule of the composition of the present invention is that it does not produce a functional protein, or alternatively, it is not translated. The RNA molecule or the delivery agent can be engineered in a variety of known ways, so as not to optionally express a functional protein or to not optionally interact with cellular factors involved in the translation. Thus, for example, the agent whether it is a synthesized RNA molecule or an agent that becomes an RNA molecule in vivo, lacks a polyadenylation sequence. Similarly, the agent may lack a Kozak region necessary for the translation of the protein. In yet another embodiment, the RNA molecule may also lack the native, start methionine codon. In yet another embodiment, the polynucleotide sequence of the RNA molecule lacks a cap structure. In another additional embodiment, the RNA molecule has no signals for the synthesis of the protein. In yet another embodiment, the RNA molecule does not contain a coding sequence or a functionally non-operative coding sequence. In yet another embodiment, the RNA sequence can be scored with intronic sequences. In another additional modality, a hhorquilla type sequence can be placed before the native initiation codon, if present. In still another embodiment, the RNA molecule can be an RNA / DNA hybrid as described above. All such modalities can be designed to resort to the teachings known, for example, from such texts as cited below. The following are various specific embodiments that can be used to achieve polynucleotide inhibition as described herein. It should be recognized that the various RNA structures (and RNA / DNA hybrids) described below, can be used alone or in any combination of two or more, for example, a loop molecule (sense or antisense) and / or a circular and / or linear complementary molecule. The loop or antisense circle structures can also be used alone. In addition, these structures may include regions of self-complementarity (eg, sense and antisense sequences, in tandem) as well as additional antisense sequences relative to a desired objective. Throughout this document, the term "antisense" is used to mean complementary to and capable of hybridizing with any mRNA. In one embodiment, the polynucleotides in the form of "loops" can be used. The loops contain a 2'-5 'phosphodiester linkage as opposed to the usual 3'-5'-linkage. Such structures are formed in splicing reactions characterized by spliceosomes and self-excision ribozymes. These structures are either intermediates or by-products of the splicing reactions. These can be prepared in vivo through expression (transcription) in a cell or prepared in vi tro. Loops are formed when a free 5 'phosphoryl group of either ribose or deoxyribose becomes bound to the 2' -0H of a ribose in a loop fashion. The loops may contain 10 or more nucleotides in the loop or they may be a complete circle, with the loop link in each case being 2 '-5'. A loop that links the terminal nucleotides produces a circle-like structure. The curls and / or the stem can contain either the sense and antisense sequences in tandem in a single molecule, or each single loop contains either a sense sequence or an antisense. The loops that contain the sense and the antisense in separate molecules can be administered together as a double-stranded form or the antisense loop can be used only to form a double strand with the mRNA in the cell. The loops can be RNA or a hybrid of DNA, with the 2'-5'-linkage effected through 2'-0H of the RNA portion of the hybrid [Rees C and Song Q. Nucí. Acd. Res., 27, 2672-2681 (1999); Dame E et al., Biochemistry, 38, 3157-3167, 1999; Clement J. Q. et al., RNA, 5, 206-220, 1999; Block T and Hill J. J. Neurovirol, 3, 313-321, 1997; Schindewolf CA and Domdey H., Nucí. Acid Res., 23, 1133-1139 (1995)]. In yet another embodiment, a circular RNA (or circular RNA-DNA hybrid) can be generated through a 2? -5 'linkage or a 3' -5 'linkage of the ends. These can be generated enzymatically through RNA-ligase reactions using a splinter to put the ends in close proximity in vi tro, or through the use of autoempalme ribozymes (in vivo and in vi tro). The desired inhibition can be achieved by the provision of one or more RNA circles, elaborated in vi tro or expressed in vivo, including simple circles with or without self-complementarity, as well as circular double-stranded RNA (strands in sense and antisense in relation to to the target polynucleotide), or two circles of single-stranded RNA having regions of complementarity to each other, as well as having complementarity to a target. Yet another embodiment uses the antisense circles of single RNA (or RNA-DNA hybrid) (circular RNA without self-complementarity that is complementary to the target mRNA). Yet another embodiment uses the RNA-DNA circles or a circular DNA molecule complementary to a target mRNA molecule. Simple circles with tandem sense and antisense sequences (in any order) that have complementarity to an objective message can be used as the composition that inhibits the function of the target sequence. It may be preferred to use circular molecules having such self-complementary sequences which can form rod-shaped sections, as well as additional antisense sequences to the target [Schindewolf CA and Domdey H. Nucí. Acid Res., 23, 1133-1139 (1995); Rees C and Song Q., Nucí. Acid Res., 27, 2672-2681 (1999); Block T and Hill J., Neurovirol., 3, 313-321 (1997)]. In a further embodiment, the composition that inhibits the target sequence is a linear RNA cased. Whether the dsRNA is formed in vi tro or in vivo, one or both strands can be cascaded. In circumstances where cytoplasmic expression could ordinarily not result in stapling of the RNA molecule, pouching can be accomplished by a variety of means including the use of a stacking enzyme, such as a vaccinia stacking enzyme or a stacking enzyme. of alphavirus. A nested antisense molecule can be used to achieve the desired post-transcriptional silencing of the target gene. Cased RNA can be prepared in vi tro or in vivo. The RNA elaborated in the nucleus by the RNA polll is ordinarily encasquetado. The cytoplasmically expressed RNA may or may not be encased. The packing can be achieved by the expression of the enzymes of encapsulation of the cytoplasmic viruses. In these compositions RNA or hybrid RNA-DNA sequences can be used, both encased or one cased, and one non-cased or both uncaged. The antisense molecule encapsulated or uncaged can be used, alone or in any combination with the polynucleotide structures described herein. The RNA molecule according to this invention can be distributed to the mammal, or the extracellular pathogen present in the mammalian cell in the composition, as an RNA molecule or a partially double-stranded RNA sequence, or an RNA / hybrid. DNA, which was elaborated in vi tro by means of synthetic, conventional enzymatic methods using, for example, the bacteriophage T7, the T3 or SP6 RNA polymerases according to the conventional methods described by texts such as Promega Protocols and Applications Guide, (3a. ed. 1996), eds. Doyle, ISBN No. 1-882274-57-1. Alternatively, these molecules can be elaborated by chemical synthetic methods in vi tro [see, for example, Q. Xu et al., Nucí. Acids Res., 24 (18): 3643-4 (September 1996); N. Naryshkin et al., Bioorg, Khim. , 22 (9): 691-8 (September 1996); J. A. Grasby et al., Nucí. Acids Res., 21 (19): 4444-50 (September 1993); C. Chaix et al., Nucí. Acids Res., 17 (18): 7381-93 (1989); S.H. Chou et al., Biochem. , 28 (6): 2422-35 (March 1989); 0. Odai and collaborators, Nucí. Acids Symp. Ser., 21: 105-6 (1989); N.A. Naryshkin et al., Bioorg. Khim, 22 (9): 691-8 (September 1996); S. Sun et al., RNA, 3 (11): 1352-1363 (November 1997); X. Zhang et al., Nucí. Acids Res., 25 (20); 3980-3 (October 1997); YE.

Grvaznov et al., Nucí. Acids Res., 26 (18): 4160-7 (September 1998); M. Kadokura et al., Nucí. Acids Symp. Ser., 37: 77-8 (1997); A. Davison et al, Biomed. Pept. Proteins. Nucí Acids, 2 (1): 1-6 (1996); and A. V. Mudrakovskaia et al., Bioorg. Khim., 17 (6): 819-22 (June 1991)]. Alternatively, the RNA molecule of this invention can be made in a recombinant microorganism, for example, bacteria and yeast or in a recombinant host cell, for example, mammalian cells, and isolated from the cultures thereof by conventional techniques. See, for example, the techniques described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2a. Ed .; Col Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, which is exemplary of the laboratory manuals detailing these techniques, and the techniques described in U.S. Patent Nos. 5,824,538; 5,877,159 and 65,643,771, incorporated by reference herein. Such RNA molecules prepared or synthesized in vi tro can be directly administered to the mammalian or mammalian cells, since these are elaborated in vi tro. The above references provide a person skilled in the art with the techniques necessary to produce any of the following specific embodiments, given the teachings provided herein. Therefore, in one embodiment, the "agent" of the composition is a duplex (for example, it is made up of up to two strands), either fully or partially double-stranded RNA. In yet another embodiment, the agent is a strand in the sense of single-stranded RNA. In yet another embodiment, the agent of the composition is an antisense strand of single-stranded RNA. Preferably, the sense or antisense strand of single-stranded RNA forms a hairpin at one or both ends. Desirably, the sense or antisense strand of single-stranded RNA forms a hairpin at some intermediate portion between the ends. Such a strand in sense or antisense of single-stranded RNA can also be designed to fold on itself to become partially double-stranded in vi tro or in vivo.

Yet another embodiment of an existing RNA molecule as the effective agent used in the compositions is a single-stranded RNA sequence comprising a polynucleotide sequence in sense and an antisense polynucleotide sequence, optionally separated by a polynucleotide sequence of unpaired bases. . Preferably, this single-stranded RNA sequence has the ability to become double-stranded once it is in the cell, or in vi tro during its synthesis. Yet another embodiment of this invention is a hybrid of RNA / DNA as described above. Yet another embodiment of the synthetic RNA molecule is a circular RNA molecule that optionally forms a rod structure [see for example, K.S. Wang et al., Nature, 323: 508-514 (1986)] or is partially double-stranded, and can be prepared according to the techniques described in S. Wang et al., Nucí. Acids Res., 22 (12): 2326-33 (June 1994); Y. Matsumoto et al., Proc. Nati Acad. Sci., USA, 87 (19) 7628-32 (October 1990); Proc. Nati Acad. Sci., USA, 91 (8) .3117-21 (April 1994); M. Tsagris and collaborators, Nucí. Acids Res., 19 (7): 1605.12 (April 1991); S. Braun et al., Nucí. Acids Res., 24 (21): 4172-7 (November 1996); Z. Pasman et al., RNA, 2 (6): 603-10 (June 1996); P.G. Zaphiropoulos, Proc. Nati Acad. Sci., USA, 93 (13): 6536-41 (June 1996); D. Beaudry et al., Nucí. Acids Res., 23 (15): 3064-6 (August, 1995), all incorporated by reference herein. Another agent is a double-stranded molecule comprised of RNA and DNA present on separate strands, or interspersed on the same strand. Alternatively, the RNA molecule can be formed in vivo and thus administered by a "delivery agent" that generates such a partially double-stranded RNA molecule in vivo., after the distribution of the agent to the mammalian cell or to the mammal. Thus, the agent forming the composition of this invention is, in one embodiment, a double-stranded DNA molecule "encoding" one of the RNA molecules described above. The DNA agent that provides the nucleotide sequence that is transcribed within the cell to become a double-stranded DNA. In yet another embodiment, the DNA sequence provides a deoxyribonucleotide sequence which within the cell is transcribed into the sense or antisense strand of single-stranded RNA, described above, which optionally forms a hairpin at one or both ends or it folds over itself to become partially double-stranded. The DNA molecule which is the agent for distributing the composition, can provide a single-stranded RNA sequence, comprising a polynucleotide sequence in sense and an antisense polynucleotide sequence, optionally separated by a polynucleotide sequence of unpaired bases, and where the single-stranded RNA sequence has the ability to become double-stranded. Alternatively, the DNA molecule which is the distribution agent provides the transcription of the above-described circular RNA molecule which optionally forms a rod-shaped structure or a partial double strand in vivo. The DNA molecule can also provide the in vivo production of an RNA / DNA hybrid as described above, or a duplex containing an RNA strand and a strand of DNA. These various DNA molecules can be designed by resorting to conventional techniques such as those described in Sambrook, cited above or in the Promega reference, cited above. A further distribution agent of the present invention, which enables the formation in mammalian cells of any RNA molecules described above, can be a single-stranded or double-stranded DNA or plasmid vector. Expression vectors designed to produce RNAs as described herein or in vivo may contain sequences under the control of any RNA polymerase, including mitochondrial RNA polymerase, poly RNA, polyI RNA, and RNA polIII. These vectors can be used to transcribe the desired RNA molecule in the cell according to this invention. Vectors can be desirably designed to utilize an endogenous mitochondrial RNA polymerase (e.g., human mitochondrial RNA polymerase, in which case such vectors can utilize the corresponding human mitochondrial promoter). Mitochondrial polymerases can be used to generate messages cascaded (through the expression of a packaging enzyme) or uncaged, in vivo. The transcripts of poly RNA, RNA pol11 and RNA polIII can also be generated in vivo. Such RNAs can be cascaded or not, and if desired, the cytoplasmic clumping can be carried out by various means including the use of a sheathing enzyme such as a vaccinia sheath enzyme or an alphavirus sheath enZyme. The DNA vector is designed to contain one of the multiple promoters or promoters in combination (mitochondrial, poly RNA, polll, or polIII, or viral, bacterial or bacteriophage promoters together with the cognate polymerases). Preferably, where the promoter is RNA poly, the sequence encoding the RNA molecule has an open reading structure greater than about 300 nucleotides to prevent degradation in the nucleus. Such plasmids or vectors may include plasmid sequences from bacteria, viruses or phages. Such vectors include chromosomal, episomal and virus derivatives, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes., yeast chromosomal elements, and viruses, vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage, cosmid and phagemid genetic elements. Thus, an exemplary vector is a single-stranded or double-stranded phage vector. Another exemplary vector is a single-strand or double-stranded DNA or RNA viral vector. Such vectors can be introduced into cells as polynucleotides, preferably DNA, by well-known techniques for introducing DNA and RNA into cells. The vectors in the case of phage and viral vectors can also be and preferably are introduced into cells as packaged or encapsidated viruses by well known techniques, for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally occurs only in complementing host cells. In yet another embodiment, the distribution agent comprises more than one plasmid or vector of DNA or simple RNA. As an example, a first DNA plasmid can provide a single-stranded RNA sense polynucleotide sequence, as described above, and a second DNA plasmid can provide a single-stranded RNA antisense polynucleotide sequence, such as described above, wherein the sense and antisense RNA sequences have the ability to pair the bases and become double-stranded. Such or such plasmids may comprise other conventional plasmid sequences, for example bacterial sequences such as the well-known sequences used to construct plasmids and vectors for the recombinant expression of a protein. However, it is desirable that the sequences that make possible the expression of the protein, for example, the Kozak regions, etc., are not included in these plasmid structures. Vectors designed to produce dsRNAs of the invention can be desirably designed to generate two or more, including a number of different dsRNAs, homologous and complementary to a target sequence. This procedure is desirable, since a single vector can produce many independently operative dsRNAs instead of a single dsRNA molecule from a single transcription unit and by producing a plurality of different dsRNAs, it is possible to self-select the optimal effectiveness. Various means can be employed to accomplish this, including autocatalytic sequences as well as sequences for cleavage, to create random and / or predetermined splice sites. Other distribution agents to provide the information necessary for the formation of the desired RNA molecules, described above in the mammalian cell include live, attenuated or killed, inactivated recombinant bacteria, which are designed to contain the necessary sequences for the molecules of RNA required of this invention. Such recombinant bacterial cells, fungal cells, and the like can be prepared by the use of conventional techniques such as described in U.S. Patent Nos. 5,824,538; 5,877,159 and 65,643,771, incorporated by reference herein. Microorganisms useful in the preparation of these distribution agents include those listed in the aforementioned reference, including, without limitation, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species of Pseudomonas, Streptomyces and Staphylococcus. Other additional distribution agents to provide the information necessary for the formation of the above-described, desired RNA molecules in the mammalian cell include live, attenuated or killed, inactivated viruses, and particularly recombinant viruses that possess the RNA polynucleotide sequence discussed previously. Such viruses can be designed similarly to the recombinant viruses currently used to distribute the genes to the cells for gene therapy and the like, but preferably do not have the ability to express a protein or functional fragment of a protein. Among the useful viruses or useful viral sequences that can be manipulated to provide the molecule required for the mammalian cell in vivo are, without limitation, alphaviruses, adenoviruses, adeno-associated viruses, baculoviruses, delta viruses, smallpox viruses ( pox virus), hepatitis virus, herpes virus, papovavirus, (such as SV40), poliovirus, pseudorabies virus, retrovirus, vaccinia virus, positive and negative strand RNA virus, viroids, and virusoids, or portions thereof. These various viral delivery agents can be designed by the application of conventional techniques such as described in M. Di Nicola et al., Cancer Gene Ther. , 5 (6): 350-6 (1998), among others, with the teachings of the present invention. Another distribution agent to provide the information necessary for the formation of the RNA molecules described above, desired in the mammalian cell include inactivated, live, attenuated or killed donor cells, which have been transfected or infected in vi tro with a molecule of synthetic RNA or a DNA distribution molecule or a recombinant virus distribution, as described above. These donor cells can be administered to a mammal, as described in detail below, to stimulate the mechanism in the mammal that mediates this inhibitory effect. These donor cells are desirably mammalian cells, such as the C127, 3T3, CHO, HeLa, 293 human kidney, BHK, and COS-7 cell lines, and are preferably of the same mammalian species as the mammalian recipient. Such donor cells can be made using techniques similar to those described for example in, Emerich et al., J. Neurosci., 16: 5168-81 (1996). More preferably, donor cells can be harvested from a specific mammal to be treated and processed in donor cells, by ex vivo manipulation, akin to adoptive transfer techniques, such as those described in D.B. Kohn et al., Nature Med., 4 (7): 775-80 (1998). The donor cells can also be from non-mammalian species, if desired. Finally, the composition of this invention may also include one or more of the selected agents described above. The composition may contain a mixture of synthetic RNA molecules described above, synthetic DNA distribution molecules, described above, and any of the other distribution agents described above, such as recombinant bacteria, cells, and viruses. The identity of the composition mixture can be easily selected by a person skilled in the art.

B. Pharmaceutical Compositions (Therapeutic or Prophylactic) of the Invention The compositions of this invention for pharmaceutical use desirably contain the synthetic RNA molecule as described above or the agent that provides the RNA molecule to the mammalian cell in vivo, in a pharmaceutically acceptable carrier, with additional optional components for the pharmaceutical distribution. The specific formulation of the pharmaceutical composition depends on the form of the agent that distributes the RNA molecule. Suitable pharmaceutically acceptable carriers facilitate administration of the polynucleotide compositions of this invention, but are physiologically inert and / or harmful. The carriers can be selected by a person of skill in the art. Such carriers include but are not limited to, sterile saline, phosphate, buffered saline, dextrose, sterile water, glycerol, ethanol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, oil. olive, sesame oil, and water, and combinations thereof. Additionally, the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone, or with a wax. In addition, slow-release polymer formulations can be used. The formulation must adapt not only to the form of the distribution agent, but also to the mode of administration. The selection of an appropriate carrier according to the mode of administration is routinely carried out by those skilled in the art. Where the composition contains the synthetic RNA molecule or where the agent is another polynucleotide, such as a DNA molecule, plasmid, viral vector, or recombinant virus, or multiple copies of the polynucleotide or different polynucleotides, etc., as described above, the composition can be desirably formulated as a "naked" polynucleotide, with only one carrier. Alternatively, such compositions desirably contain optional polynucleotide facilitating agents or "co-agents", such as a local anesthetic, a peptide, a lipid including cationic lipids, a liposome or lipid particle, a polycation such as polylysine, a three-dimensional polycation branched such as a dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or another compound that facilitates the transfer of the polynucleotide to the cells. Non-exclusive examples of such facilitators or co-agents useful in this invention are described in U.S. Patent Nos. 5,593,972; 5,703,055; 5,739,118; 5,837,533 and International Patent Application No. WO96 / 10038, published April 4, 1996; and International Patent Application No. W094 / 16737, published August 8, 1994, which are incorporated by reference herein. When the facilitating agent used is a local anesthetic, bupivacaine preferably, an amount of about 0.1 weight percent to about 1.0 weight percent is preferred based on the total weight of the polynucleotide composition. See, also International Patent Application No. PCT / U98 / 22841, which teaches the incorporation of benzylammonium surfactants as co-agents, administered in an amount of between about 0.001-0.03% by weight, the teaching of which it is incorporated by reference herein. Where the agent for distributing the composition is different from a polynucleotide composition, for example, it is a transfected donor cell or a bacterium as described above, the composition may also contain other additional agents, such as those discussed in the Patents of the United States. United Nos. 5,824,538; 5,643,771; 5,877,159, incorporated by reference herein. Other additional components that may be present in any of the compositions are, adjuvants, preservatives, chemical stabilizers, or other antigenic proteins. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficiency in the human or animal target. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable stabilizing ingredients that can be used include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z-amine, monopotassium diphosphate, lactose, lactalbumin hydrolyzate, and milk powder. A conventional adjuvant is used to attract leukocytes or improve an immune response. Such adjuvants include, inter alia, Ribi, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121 / squalene, D-lactide-piolulactide / glucoside, Pluronic polyols, muramyl dipeptide, Bordetella dead, and saponins, such as Quil A. In addition, other agents that can function as transfection agents and / or replication agents and / or inflammatory agents and that can be co-administered with the composition of this invention, include growth factors, cytokines and lymphokines such as alpha- interferon, gamma-interferon, platelet-derived growth factor (PDGF), colony-stimulating factors, such as G-CSF, GM-CSF, tumor necrosis factor (TNF), epidermal growth factor (EGF), and interleukins , such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12. In addition, fibroblast growth factor, surface active agents such as immune stimulatory complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog including monophosphoryl-Lipid A (MPL), muramyl-peptides, analogues of quinone, and vesicular complexes such as squalene, and hyaluronic acid, can be used administered in conjunction with the compositions of the invention. The pharmaceutical compositions may also contain other additives suitable for the mode of administration selected from the composition. Thus, these compositions may contain additives suitable for administration via any conventional route of administration, including without limitation, parenteral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal, intra-pulmonary administration, rectal administration, vaginal administration, and the like. All such routes are suitable for the administration of these compositions, and can be selected depending on the agent used, the patient and the condition treated, and similar factors by a attending physician. The composition of the invention can also involve lyophilized polynucleotides, which can be used with other pharmaceutically acceptable excipients, for the development of powder, liquid or suspension dosage forms, including those for intranasal or pulmonary applications. See, for example, Remington: The Science and Practice of Pharmacy, Vol. 2, 19a. edition (1995), for example, Chapter 95 Aerosols; and International Patent Application No. PCT / US99 / 05547, the teachings of which are incorporated by reference herein. The administration routes for these compositions can be combined, if desired, or adjusted. In some preferred embodiments, the pharmaceutical compositions of the invention are prepared for administration to mammalian subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric coated tablets or capsules, or suppositories. The compositions of the present invention, when used as pharmaceutical compositions, may comprise about 1 ng to about 20 mgs of polynucleotide molecules as the agent for distributing the compositions, for example, synthetic RNA molecules or distribution agents that can be DNA molecules, plasmids, viral vectors, recombinant viruses, and mixtures thereof. In some preferred embodiments, the compositions contain about 10 ng to about 10 mg of polynucleotide sequences. In other embodiments, the pharmaceutical compositions contain about 0.1 to about 500 μg of polynucleotide sequences. In some preferred embodiments, the compositions comprise about 1 to about 350 μg of polynucleotide sequences. In other preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 μg of the polynucleotide sequences. In some preferred embodiments, vaccines and therapeutic products contain approximately 100 μg of the polynucleotide sequences. The compositions of the present invention in which the distribution agents are donor cells or bacteria, can be administered in doses of between about 1 cell to about 10 7 cells / dose. Similarly, where the distribution agent is a living recombinant virus, a composition based on the appropriate vector contains between 1 x 102 pfu up to 1 x 1012 pfu per dose. Given the teachings of this invention, and the observed ability of the inhibitory effect of the methods and compositions of this invention to be propagated to more cells than cells transfected or infected with the composition of this invention, it is likely that adjustments of suitable dosage down from the doses noted above. Thus, the dose ranges are guidelines only. In general, the pharmaceutical compositions are administered in an amount effective to inhibit or reduce the function of the target polynucleotide sequence, for the treatment or prophylaxis of diseases, disorders or infections for which such objective functions are necessary for the further propagation of the disease or the causative agent of the disease. The amount of the pharmaceutical composition in a dosage unit employed is determined empirically, based on the responses of the cells in vi tro and the response of the experimental animals to the compositions of this invention. The optimal dose is determined by standard methods for each modality and indication of treatment. In this way the dose, the time, the route of administration, and the need for the readministration of these compositions, can be determined by a person of experience in the art, taking into account the condition in question, its severity, the conditions of complication, and factors such as age, and the physical condition of the mammalian subject, the use of other active compounds, and the like.

C. Therapeutic and Prophylactic Methods of the Invention The methods of this invention can employ the compositions described in detail above, and possibly other polynucleotide sequences currently used in the art (eg, polynucleotide molecules which do code for proteins, whether functional or non-functional, or RNA catalytic sequences). known, such as ribozymes) which can provide partially double-stranded RNA molecules to a mammalian cell. It is anticipated, however, that the efficiency of these methods is increased by the use of RNA molecules that do not produce protein. These methods reduce or inhibit the function of one or more target polynucleotide sequences in a mammal or in the cell of a mammal. The compositions, pharmaceutical compositions, doses and modes of administration described above are particularly desirable for the treatment of a variety of disorders that plague mammals, including infections by heterologous pathogenic organisms, whether intracellular or extracellular pathogens. In addition, the compositions of this invention are useful in the prevention of the infection of a mammal with a pathogen, or the prevention of the occurrence of disorders caused by the reactivation of a latent pathogen. These compositions are also useful for the treatment of pathogenically induced cancers. One embodiment of a method of this invention is a method for treating a viral infection in a mammal. DNA viruses or viruses having an intermediate DNA are particularly suitable for such a treatment area. Such viruses include, without limitation, viruses of the Retrovirus, Herpesvirus, Hepadanovirus, Poxvirus, Parvovirus, Papilomavirus, and Papovavirus species. Specifically, some of the most desirable viruses to treat with this method include, without limitation, HIV, HBV, HSV, CMV, HPV, HTLV and EBV. The agent used in this method provides the mammalian cell with at least one partially double-stranded RNA molecule as described above, which is substantially homologous to an objective polynucleotide which is a viral polynucleotide sequence necessary for replication and / or pathogenesis of the virus in an infected mammalian cell. Among such target polynucleotide sequences are the sequences coding for the proteins necessary for the propagation of the virus, for example, the gag, env and pol genes of HIV, the Ll and E2 genes of HPV6, the Ll and E2 genes of HPV11, the E6 and E7 genes of HPV16, E6 and E7 genes of HPV18, HBV surface antigens, HBV nuclear antigen, HBV reverse transcriptase, HSV gD gene, HSVvp gene 16, gC genes, gH, gL and gB of HSV, the genes ICPO, ICP4 and ICP6 of HSV, genes gB, gC and GH of Varicella zoster, and the chromosomal sequences of BCR-abl, and the non-coding viral polynucleotide sequences which provide the regulatory functions necessary for the transfer of infection from cell to cell, for example, HIV LTR, and other viral promoter sequences, such as the HSV vpl6 promoter, the HSV ICPO promoter, the ICP4, ICP6 and gD promoters. HSV, the surface antigen promoter of HBV, the pre-genomic promoter of HBV, among others. As described above, the composition is administered with a polynucleotide uptake enhancer or facilitator, and an optional pharmaceutically acceptable carrier. The amount or dose that is administered to the mammal is effective to reduce or inhibit the function of the viral sequence in the cells of the mammal. While not wishing to be committed to any theory, once the RNA molecule is distributed to or produced in a virus-infected cell, the exogenous RNA molecule reduces or inhibits (eg turns off) the homologous viral sequence and is itself same inhibited, so that the function of the viral sequence is reduced or inhibited. As demonstrated in the examples below, the inhibition of the function effect is transferred from the mammalian cell receiving the exogenous RNA molecule to other mammalian cells on the assumption that these have not been directly provided with the RNA molecule. exogenous The current theory is that these results occur at the level of RNA degradation. Thus, this method can be used to treat mammalian subjects already infected with a virus, such as HIV, in order to decrease or inhibit a viral gene function, essential for the replication and / or pathogenesis of the virus, such as HIV gag. Alternatively, this method can be used to inhibit the functions of viruses that exist in mammals such as latent viruses, for example, Varicella zoster virus, and are the causative agents of the disease known as herpes. Similarly, diseases such as atherosclerosis, ulcers, chronic fatigue syndrome, and autoimmune disorders, recurrences of HSV-1 and HSV-2, persistent HPV infection, for example, genital warts, and chronic HBV infection among others. , which have been shown to be caused, at least in part, by viruses, bacteria or other pathogens, can be treated according to this method by targeting certain essential viral polynucleotide sequences towards viral replication and / or pathogenesis in the mammalian subject. In yet another embodiment of this invention, the compositions described above can be employed in a method for preventing viral infection in a mammal. When the method described above, for example, administration of a composition described above in an amount effective to reduce or inhibit the function of the target viral polynucleotide sequence, essential to a mammal, is administered prior to mammalian exposure to the virus, it is it expects the exogenous RNA molecule to remain in the mammal and function to inhibit any homologous viral sequence that presents itself to the mammal thereafter. Thus, the compositions of the present invention can be used to inhibit or reduce the function of a viral polynucleotide sequence for use in a vaccine. An analogous embodiment of the aforementioned "antiviral" methods of the invention includes a method for the treatment or prophylaxis of a virally induced cancer in a mammal. Such cancers include cervical carcinoma induced by HPV E6 / E7 virus, cancer induced by HTLV, and EBV-induced cancers, such as Burkitts lymphoma, among others. This method is carried out by administering to the mammal a composition as described above, in which the target polynucleotide is a sequence encoding a tumor antigen or a functional fragment thereof, or a non-expressed regulatory sequence, whose function of antigen or sequence is required for the maintenance of the tumor in the mammal. Such sequences include, without limitation, the E6 and E7 sequences of HPV16 and the E6 and E7 sequences of HPV18. Others can be easily selected by a person skilled in the art. The composition is administered in an amount effective to reduce or inhibit the function of the antigen in the mammal, and preferably employ the components of the composition, the doses and the routes of administration as described above. The molecular mechanism underlying this method is the same as that described above. In yet another embodiment of the invention, the compositions of this invention can be employed in a method for the treatment or prophylaxis of infection of a mammal by a non-viral pathogen, either intracellular or extracellular. As used herein, the term "intracellular pathogen" is understood to refer to a virus, bacterium, protozoan or other pathogenic organism that, for at least part of its reproductive or life cycle, exists inside a host cell and in it produces or causes pathogenic proteins to be produced. Intracellular pathogens that infect cells that include a stage in the life cycle where these are intracellular pathogens, include, without limitation, Listeria, Chlamydia, Leishmania, Brucella, Mycobacteria, Shigella, as well as Plasmodia, for example, the causative agent of malaria, P. falciparum. Extracellular pathogens are those that replicate and / or propagate outside the mammalian cell, eg, Gonorrhoeae, and Borrelia, among others. According to this embodiment, such infection by a pathogen can be treated or possibly prevented by administration to a mammalian subject, either already infected or anticipating exposure to the pathogen, with a composition as described above, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, in a pharmaceutically acceptable carrier. In this case, the RNA molecule of the composition has a polynucleotide sequence that is substantially homologous to an objective polynucleotide sequence of the pathogen, which is necessary for the replication and / or pathogenesis of the pathogen in an infected mammal or in an infected mammalian cell. . As mentioned above, the amount of the composition administered is an amount effective to reduce or inhibit the function of the pathogenic sequence in the mammal. Dosages, times, routes of administration and the like are as described above. A person skilled in the art, given this description, can easily select the viral families and genera, or the pathogens including prokaryotic and eukaryotic protozoan pathogens as well as multicellular parasites, for which the therapeutic or prophylactic compositions can be elaborated in accordance to the present invention. See for example the tables of such pathogens in general immunology texts and in U.S. Patent No. 5,593,972 incorporated by reference herein. The compositions of this invention and possibly the molecules coding for the proteins of the prior art can also be employed in another novel method of this invention. Such compositions are also useful in the treatment of certain diseases or non-pathogenic disorders of some mammals, such as certain cancers or inherited disorders. Among the conditions particularly susceptible to treatment or prophylaxis according to this invention, are those conditions which are characterized by the presence of a mammalian aberrant polynucleotide sequence, the function of which is necessary for the initiation or progression of the disorder, but may be inhibited without causing harm or otherwise unduly adverse to the health of the mammal. In other words, a characteristic of an appropriate disorder for this treatment is that the mammal can survive without the function of the gene, or it can survive if the function of the gene was substantially reduced. In such cases, the function of the aberrant or normal polynucleotide sequence can be replaced exogenously by therapy. In yet another case, the disease can be caused by the presence or function of an abnormal polynucleotide sequence or an abnormal gene in a mammal, where the mammal also possesses a normal copy of the gene or polynucleotide sequence, and where the differences between the gene Abnormal and normal gel are differences in the nucleotide sequence. In such cases, inhibition of the function of the abnormal polynucleotide sequence by the method of this invention is likely to allow the normal polynucleotide sequence to function, without exogenous treatment. Thus, in one embodiment, a method of treating or prophylaxis of a cancer in a mammal involves administering to the mammal a composition of this invention in which the target polynucleotide sequence is an abnormal polynucleotide sequence that causes cancer., or an abnormal gene in a mammal. The composition is this invention is administered in an amount effective to reduce or inhibit the abnormal sequence function in the mammal. As described above, the composition may contain a second optional agent that facilitates uptake of the polynucleotide into a cell, and a pharmaceutically acceptable carrier, and be administered in doses, regimens and via routes as described above. Mammalian cancers, which are characterized by the presence of normal and abnormal polynucleotide sequences include chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL), where the abnormal sequence is a fusion of the two normal genes, for example , ibcr-ai)! See, for example, the description of these cancers in International Patent Publication No. W094 / 13793, published June 23, 1994, and incorporated by reference herein, for a description of these diseases. In such cancers or diseases, such as CML, the affected mammal also possesses a normal copy of the gene or polynucleotide sequence, and the differences between genes and normal and abnormal sequences are differences in the nucleotide sequence. For example, for CML, the abnormal sequence is the bcr-abl fusion, while the normal sequence is bcr and abl. In this way, the above method can be employed with the target polynucleotide sequence which is the sequence encompassing the fusion. A method of treating or prophylaxis of such a cancer in a mammal comprises administering to the mammal a composition of this invention wherein the target polynucleotide is a polynucleotide sequence of an abnormal gene that causes cancer in a mammal, which also possesses a normal copy of the gene, and where the differences between the normal gene and the abnormal gene are differences in the polynucleotide sequence. The composition is administered as described above, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, and into a pharmaceutically acceptable carrier and in an amount effective to reduce or inhibit the abnormal sequence function in the mammal. The present invention thus encompasses methods for evoking the molecular mechanism described above for the treatment of any disease or disorder in a mammal, characterized by the expression of an undesirable function mediated by a polynucleotide or polynucleotide product not found in a healthy mammal, by using a composition that can distribute to the cells of a mammal the partially double-stranded RNA molecule, substantially homologous to the target polynucleotide sequence that expresses or mediates the undesired product or function, in an amount effective to reduce or inhibit the function of that polynucleotide in the cells of the mammal. Provided that the RNA molecule is sufficiently non-homologous to the essential polynucleotide sequences of the mammal, so that it does not inhibit the function of those essential sequences, this method can be clearly observed as having many therapeutic and prophylactic uses. A person skilled in the art can easily select the disorders described above, and can also easily select the target polynucleotide sequences against which the compositions of the present invention are directed.

D. Other Methods of the Present Invention The compositions described above, and the general methods of using these compositions to inhibit or reduce the function of an objective polynucleotide sequence, can also be applied by a variety of research applications, and in vi tro. For example, the method of this invention can be applied to the investigation to determine the function of a selected polynucleotide sequence in a cell line, or a mammalian laboratory animal, by administration to that cell in tissue culture or to that animal, in vivo, of a composition of the invention wherein the polynucleotide sequence of the RNA molecule is substantially homologous to the selected sequence, and preferably substantially not homologous to other polynucleotide sequences in the animal. The inhibition of the function of that target sequence allows the study of its influence on the biology and physiology of the animal. Similarly, the application of this method can be used to elaborate mammalian, bacterial, yeast, fungal, insect and other origins lines, defective in the selected pathways by "silencing" a selected functional sequence, such as an enzymatic sequence, a sequence expressing protein, or regulatory sequences necessary for the expression thereof. Such manipulated cells can be used in conventional assays or in drug selection assays, etc. In an analogous method, a laboratory animal "suppressed in some gene" can be prepared by altering the dosage of administration, sufficient to permanently quench the function of a selected gene. Thus, the method of the present invention, in the distribution of an RNA molecule with a polynucleotide sequence sufficiently homologous to the selected sequence that is to be "deleted in some gene" in the laboratory animal as described above, provides a simpler technique to develop "suppressed in some gene" mice and other laboratory animals useful for pharmaceutical and genetic research. Other methods of investigation for the use of the compositions and methods of this invention include the preparation of the mutants of the microorganisms, eukaryotic and prokaryotic, for use as research agents or as industrially produced strains for the microbial production of desired proteins. Other additional uses are expected to be obvious to the person skilled in the art given the teachings herein. The following examples illustrate the methods for preparing the compositions and using the compositions of this invention to reduce or inhibit the target polynucleotide sequences. These examples which employ as the agent of the composition, the double-stranded RNA molecules made by in vi tro synthesis and the target polynucleotide sequences of HIV gag or HSV gD2 merely illustrate the embodiments of this invention. It is understood by those of skill in the art that other selections for the various agents of the compositions, and the identity of the target polynucleotide sequences can be easily selected as taught by this specification. These examples are illustrative only and do not limit the scope of the invention.

EXAMPLE 1: REDUCTION OR INHIBITION OF THE FUNCTION OF HIV p24 IN VIRALLY INFECTED CELLS During the course of HIV infection, the viral genome is reverse transcribed into a template DNA that is integrated into the host chromosome of the infected cells in division. The integrated copy is now a negative copy of which more HIV particles are made. According to this invention, if the function of a polynucleotide sequence essential for the replication and / or pathogenesis of HIV is reduced or inhibited, the viral infection can be treated. This example demonstrates the operation of one embodiment of the method of this invention. The plasmid, HlVgpt (Catalog of AIDS Reference Research Reagents Program) was used to generate stable integrated Rhabdomyosarcoma (RD) of C0S7 cell lines containing the integrated copies of the defective HIV genome, HlVgpt. The genome of HlVgpt codes for a mycophenolic acid (MPA) resistance gene instead of the envelope gene and thereby confers resistance to MPA. The cell lines are made by transfecting the cells with the plasmid followed by the selection of the cells in MPA. The MPA-resistant cells were clonally amplified. The media from the clonally expanded cultured cells that were then evaluated for the presence of p24 (an HIV gag polypeptide that is secreted extracellularly) using the. ELISA p24 (Coulter Corporation). All cells were positive for p24.

Two RD cell lines and two cell lines C0S7 are used to demonstrate an embodiment of the method of the present invention, for example, the reduction or inhibition of the function of the HIV p24 target polynucleotide, which controls the synthesis of p24 in these cells. To generate a reagent of the present invention, an RNA in the sense of 600 polynucleotides (nt), an antisense RNA of 600 nt, and a double-stranded RNA of 600 bp (dsRNA) are used to map the cells. the same coordinates of the HIV gag gene strain HXB2 lacking a cap, a polyadenylation sequence, and a native initiation codon are used to transfect the cells. RNA molecules are generated through the transcription of PCR products that possess a T7 polymerase promoter from the bacteriophage at one end (see Figures IA and IB). The coordinates of the primers were derived from the complete HIV genome map (HXB2), Genbank Accession number K03455 [see also, L. Ratner et al., AIDS Res. Hum. Retroviruses, 31 (1): 57-69 (1987)]. The forward gag primer maps the map to coordinates 901-904 and this sequence follows the T7 promoter in the forward gag primer T7. The reverse gag primer maps the map to coordinates 1476-1500 and follows the T7 promoter in the reverse gag primer T7.

To generate a composition of this invention where the agent is the single-strand sense RNA, a T7 promoter is located at the 5 'end of the forward PCR primer. The PCR primers used to generate the DNA template encoding the RNA in the sense of a single strand, written 5 'to 3' with the upper strand of the T7 promoter underlined, are the forward gag primer T7 [SEQ ID NO. 1]: 5 'GTAATACGACTCACTATAGGGCGGCAGGGAGCTAGAACGATTCGCAG 3' and the reverse gag primer [SEQ ID NO. 2]: 5 'CTGCTATGTCACTTCCCCTTGGTTC 3' To generate a composition where the agent is a single-stranded antisense RNA molecule, the T7 promoter is located at the 5 'end of the reverse PCR primer. These primers are the reverse gag primer T7 [SEQ ID NO. 3]: 5 'GTAATACGACTCACTATAGGGCGCTGCTATGTCACTTCCCCTTGGTTC 3' and the forward gag primer [SEQ ID NO. 4]: 5 'GCAGGGAGCTAGAACGATTCGCAG 3' Both types of PCR products described above are included in the transcription reaction of T7 to generate a composition where the agent is the double-stranded RNA molecule. Alternatively, an agent of the composition according to this invention is prepared by co-mixing the sense and antisense RNA, after transcription. As a control, RNA molecules in sense, antisense RNA, and dsRNA of similar size, are derived from the gD gene of a Herpes Simplex virus, genome type 2, are generated by the same transcription techniques of PCR and T7 . The coordinates of the PCR primers for gD of HSV are derived from the GenBank accession number map K01408, the gD2 gene of HSV. The forward gD primer maps the map to coordinates 313-336; this sequence follows the T7 promoter in the forward gD primer of T7. The inverse gD primer maps the map to coordinates 849-872, and follows the T7 promoter in the inverse gD primer of T7. The groups of primers used to generate these control molecules were: forward gd primer of T7 [SEQ ID NO. 5]: 5 'GTAATACGACTCACTATAGGGCGGTCGCGGTGGGACTCCGCGTCGTC 3' and forward gD primer [SEQ ID NO. 6]: 5 'GTCGCGGTGGGACTCCGCGTCGTC 3' and inverse gD primer [SEQ ID NO. 7]: 5 'GTAATACGACTCACTATAGGGCGGTGATCTCCGTCCAGTCGTTTATC 3' and reverse gD primer [SEQ ID NO. 8]: 5 'GTGATCTCCGTCCAGTCGTTTATC 3' These RNA molecules of the invention and the control molecules described above are evaluated with the cell lines RD and C0S7 as follows: between 5-6 x 10 5 cells / well in six-well plates are cultured to a confluence of approximately 80-90%, and are transfected with 2-3 μg of a selected RNA molecule or a control molecule, using 10 μl of lipofectamine (Gibco-BRL) as a transfection agent. The transfected cells were incubated by times in the range between 1 to 17 hours. Another cell culture was transfected with doses of RNA in the range between 1 μg to 500 μg, distributed without the known transfection agent and incubated on the cells from 0.5 minutes to approximately two days. For example, one group of cells is transfected with gag RNA in sense, another with antisense gag RNA, and another with gag RNA from ds, another with gD RNA in sense, as a control, another with gD antisense RNA as control, and another with the gD RNA of ds as control. Also the additional negative controls are cells that do not receive RNA molecules. The cells are cultured at 37 ° C and checked periodically for the synthesis of p24 over the course of several weeks. The cells are evaluated three times a week after two days post-administration of the RNA, by measuring p24 in the cell media using the ELISA p24 assay kit (Coulter Corp) and by immunostaining the fixed cells for p24 using a polyclonal rabbit anti-p24 serum (Intracell Corp.), and anti-rabbit IgG that is conjugated to FITC (Sigma). According to the present invention, none of the gD RNA molecules demonstrate the ability to retard or inhibit the synthesis of p24. However, according to the invention the ds gag RNA inhibits or sub-regulates the synthesis of p24. Sense and antisense RNA molecules are expected to cause only a modest, if not inhibitory, effect on p24 synthesis, unless these RNAs are capable of forming some double-stranded type of degree.

EXAMPLE 2: DETERMINATION OF THE DEGREE OF REDUCTION OF THE SYNTHESIS OF P24 FROM ONE CELLULAR CROP TO ANOTHER To demonstrate that the sub-regulated signal can be transmitted to cells that have not been sub-regulated, this example demonstrates that the reduction / inhibition effect (for example, the inhibition or reduction of p24 synthesis) is transmitted to cells in culture that are not transfected by the agent.

A. Co-Culture of C0S7 and RD cells The cells from the cultures of Example 1 demonstrating the reduction of p24 synthesis are co-cultured with the control cells of cells that have not been previously incubated with any RNA molecule and are, in fact, synthesizing p24 at levels of wild type. According to the present invention, the previously transfected cells can transfer the inhibition of the target polynucleotide function to the non-transfected cells, and the control cells in the co-culture are characterized by a reduction in the synthesis of p24. In order to distinguish control cells from cells previously transfected in the culture, a first protocol is followed: the COS7 cells of Example 1 demonstrating the inhibition of p24 synthesis are cultured with untransfected RD cells, which express p24 at wild-type levels at various ratios of cell types, for example, the ratios are in the range of 1/1000 to 1/10 (COS 7 / RD) up to a total of 6-7 x 10 5 cells in plates of 6 wells After 2 days of culture under the conditions specified in Example 1, the RD cells in the cultures are examined for the synthesis of p24. The cells are examined approximately 3 times per week, for 3 weeks.

The synthesis of p24 is evaluated by two methods. In the first method, media from co-cultured cells are evaluated for p24 using the ELISA assay for p24 (Coulter). In the second method, the cells are immunostained for p24 using rabbit polyclonal sera (Intracell Corp.) against p24 and anti-rabbit IgG conjugated to FITC. Because the C0S7 and RD cells are distinguishable by morphology, a loss of staining in RD cells can be easily distinguished from C0S7 cells. Because the C0S7 cells express the T antigen while the RD cells do not, the co-cultured cells are also stained for the T antigen using monoclonal sera against the SV40 T antigen (Pharmagen Corp.) and anti-mouse IgG conjugated to r-phycoerythrin (PE). Only C0S7 cells are stained under these conditions. This cell staining is determined by fluorescence microscopy or FLOW cytometry. Inhibition of p24 function in RD cells in co-culture is demonstrated by comparison to a control culture containing only RD cells by a loss of FITC staining in co-cultured RD cells. RD cells in the co-culture that are not stained with FITC or PE are evidence of the reduction or inhibition of the function of p24 synthesis of the target polynucleotide p24 by the RNA molecules (particularly the ds RNA molecules) of the Example 1.

B. Cultures of RD cells transfected with non-transfected RD cells In a second protocol, the transfected RD cells of Example 1, which demonstrate reduced production of p24 are co-cultured with untransfected RD cells which are engineered to contain an integrated hygromycin resistance gene, and express normal levels of p24 using different cell ratios, with ratios in the range of 1/1000 to 1/10 (RD / RD control) up to a total cell number of 6-7 x 105 in a 6-well plate . The hygromycin-resistant RD cells are elaborated as follows: the RD cells (5-6 x 10 5 cells) are cultured at 80-90% confluence in a six-well plate and are transfected with 2.5 μg of the Nru I-fragment. Sal 1 from pCEP4 (Invitrogen Corp.) containing the hygromycin resistance gene under the control of a thymidine kinase (TK) promoter. Transfections are performed using the transfection agent, lipofectamine (Gibco BRL). Two days after transfection, the cells are incubated in the presence of 400 μg / ml hygromycin. The resistant cells are clonally expanded. One or more of the clonally expanded cell lines are used as the control in the experiment. From day 1 to several days after co-cultivation under the conditions specified in Example 1, duplicate co-cultures are incubated with 400 μg / ml hygromycin. This concentration of hygromycin kills RD cells that are not resistant to hygromycin, leaving only RD cells resistant to hygromycin, control. The remaining resistant cells are derived from the control cells. The levels of P24 are measured directly from the control cells, for example, using the ELISA of Example 1, as well as by immunostaining as described above. According to the present invention, the inhibition of p24 production is revealed in at least a subset of control cells.

EXAMPLE 3: JN LIVE INHIBITION OF ENDOGENOUS INTERLEUCINE 12 PRODUCTION BY THE METHOD OF THIS INVENTION A. Design of the RNA molecules as compositions of the invention All RNA molecules in this experiment are about 600 nts in length, and all RNA molecules are designed to be capable of producing the p40 chain of IL-12. The molecules do not have a cap and do not have poly-A sequence; the native start codon is not present, and the RNA does not code for the full length product. The following RNA molecules are designed: (1) a polynucleotide sequence of RNA in the sense (single strand) (ss) homologous to the murine messenger RNA of p40 of IL-12 (mRNA); (2) a polynucleotide sequence of antisense (ss) RNA complementary to the murine p40 RNA of IL-12, (3) a double-stranded RNA (ds) molecule comprised of the polynucleotide sequences of murine mRNA of IL-12, p40, in sense and antisense, (4) a polynucleotide sequence of RNA in the ss sense homologous to the murine heterogeneous RNA of p40 of IL-12 (ARNhn) (5) a polymucleotide sequence of antisense RNA ss complementary to A .-- murmo of p40 of IL-12, (6) a ds RNA molecule comprised of the polynucleotide sequences of mnn mRNA of p40 of IL-12, in sense and antisense, (7) a polymucleotide sequence of murine RNA ss homologous to the upper strand of the p40 promoter of IL-12 , (8) a polymucleotide sequence of murine RNA ss homologous to the lower strand of the p40 promoter of IL-12, and (9) a ds RNA molecule comprised of the polynucleotide sequences of munic RNA homologous to the upper and lower strands of the promoter. p40 of IL-12. As a negative control the sense, antisense and ds RNAs derived from the HSV2 gd gene described in Example 1 are also used. Another control group is composed of mice that did not receive RNA. As described in Example 1, the various RNA molecules of sections (l) - (9) above are generated through transcription by T7 RNA polymerase of PCR products possessing a T7 promoter at one end. In the case where a sense RNA is desired, a T7 promoter is located at the 5 'end of the forward PCR primer. In the case where an antisense RNA is desired, the T7 promoter is located at the 5 'end of the reverse PCR primer. When the dsRNA is desired, both types of PCR products are included in the transcription reaction of T7. Alternatively, sense and antisense RNAs are mixed together after transcription. The PCR primers used in the construction of the RNA molecules of this Example are 5 'to 3', with the upper strand of the T7 promoter underlined. (ARNhn) genomic of forward IL-12 (SEQ ID NO: 9): 5 'TCAGCAAGCACTTGCCAAACTCCTG 3' and genomic (ARNhn) of reverse IL-12 [SEQ ID NO: 10]: 5 'GAGACAAGGTCTCTGGATGTTATTG 3', (ARNhn) genomic of forward IL-12 of T7 (SEQ ID NO: 11): 5 'GTAATACGACTCACTATAGGGTCAGCAAGCACTTGCCAAACTCCTG 3' (ARNhn) T7 reverse IL-12 genomic (SEQ ID NO.12): 5 'GTAATACGACTCACTATAGGGGAGACAAGGTCTCTGGATGTTATTG 3' forward IL-12 promoter T7 (SEQ ID NO: 13): 5 'GTAATACGACTCACTATAGGGCCTATAAGCATAAGAGACGCCCTC 3' and forward IL-12 promoter (SEQ ID NO.14): 5 'CCTATAAGCATAAGAGACGCCCTC 3' inverse IL-12 promoter (SEQ ID NO. 15]: 5 'GGCTGCTCCTGGTGCTTATATAC 3 'and reverse promoter IL-12 of T7 (SEQ ID NO: 16): 5' GTAATACGACTCACTATAGGGGGCTGCTCCTGGTGCTTATATAC 3 'cDNA of T7 forward IL-12 (mRNA) (SEQ ID NO: 17): 5' GTAATACGACTCACTATAGGGTGTGTCCTCAGAAGCTAACCATC 3 'and IL cDNA -12 forward (mRNA) (SEQ ID NO: 18): 5 'TGTGTCCTCAGAAGCTAACCATC 3', reverse IL-12 cDNA (mRNA) (SEQ ID NO. 19): 5 'GCAGGTGA CATCCTCCTGGCAGGA 3 'and T7 reverse IL-12 cDNA (mRNA) (SEQ ID NO. 20]: 5 'GTAATACGACTCACTATAGGGGCAGGTGACATCCTCCTGGCAGGA 3' The coordinates of the genomic and PCR primers are based on the map provided in the following quote: Tone et al., Eur. J. Immunol, 26: 1222-1227 (1996). The front IL-12 genomic primer maps the map to coordinates 8301-8325. The reverse IL-12 genomic primer maps the map to coordinates 8889-8913. The forward IL-12 promoter primer maps the map to coordinates 83-106. The reverse IL-12 promoter primer maps the map to coordinates 659-682. The coordinates for the cDNA PCR primers are based on GenBank Accession No. M86671. The forward IL-12 cDNA primer maps the map to coordinates 36-58. The reverse IL-12 cDNA primer maps the map to coordinates 659-682.

B. Test Balb / c mice (5 mice / group) are injected intramuscularly or intraperitoneally with the specific RNAs of the p40 chain of murine IL-12, described above or with the controls identified above, at doses in the range between 10 μg and 500 μg. The sera are collected from the mice every four days for a period of three weeks and evaluated for the levels of the p40 chain of IL-12 using the ELISA assay for p40 Quantikine M-IL-12 (Genzyme). According to the present invention, the mice receiving the ds RNA molecules derived from the IL-12 mRNA, the IL-12 RNAhn and the ds RNA derived from the IL-12 promoter, demonstrate a reduction or inhibition in the production of IL-12. A modest, if not inhibitory, effect is observed in sera from mice that receive RNA molecules derived from single-stranded IL-12, unless the RNA molecules have the ability to form some level of type of RNA. double strand None of the HSD gD-derived RNAs are expected to reduce or inhibit IL-12 in vivo in a specific manner.

EXAMPLE 4: METHOD OF THE INVENTION IN THE PROPHYLAXIS OF THE DISEASE A. Essay in vi tro Vero and / or BHK cells, seeded at a density of -30% confluence, are grown in six-well plates at 37 ° C in DMEM with 10% FBS. When the cells are 80-90% confluent, they are transfected with 2-3 μg of the HIV-gag and gD-specific RNA molecules of HSV, described in Example 1 using lipofectamine (Gibco-BRL) as an agent of transfection The RNA molecules are also distributed in the absence of any known transfection agent in amounts ranging from 5 to 100 μg. Another group of cells do not receive RNA. Other groups of vero and / or BHK cells are similarly transfected with 2-3 μg of a double-stranded DNA plasmid, plasmid 24, which is described in U.S. Patent No. 5,851,804, incorporated by reference in the present, which contains a sequence encoding the gD protein of HSV2, under the control of the HCMV promoter and a polyA SV40 sequence. The transfected cells are cultured at 37 ° C in DMEM with 10% FBS. On days 1, 2, 4 and 7 after transfection, the cells are infected with HSV2 at a multiplicity of infection (MOI) of 0.1 in an inoculum of 250 μl of DMEM. The inoculum is allowed to adsorb for 1 hour after which 2 ml of DMEM (10% FBS) is added per well. For those cells infected on days 4 and 7 after transfection, the cells are passed to a new six well plate such that they are confluent at the time of infection. If the cells do not pass, they become overpopulated.

At 36-48 hours after infection, cell lysates are evaluated for viral titer by conventional plaque assay on vero cells.

[Clinical Virology Manual, 2a. edic, eds. S. Specter and G. Lancz, pp. 473-94 (1992)]. According to this invention, cells transfected with the DNA plasmid ds, APL-400-024, and with the ds RNA molecule containing a polynucleotide sequence of the gD2 antigen, can not be productively infected with HSV2. It is anticipated that all other cells will become productively infected with HSV-2.

B. In vivo assay Using the gD-specific RNA molecules of HSV described in Example 1, which do not have the ability to make HSV gD protein and HIV gag-specific RNA molecules as controls, mice are evaluated for protection against starting from the challenge with HSV through the use of the RNA molecules specific to the gD gene of the invention. Balb / c mice (5 mice / group) are immunized intramuscularly or intraperitoneally with the .RNA molecules described at doses in the range between 10 and 500 μg of RNA. On days 1, 2, 4 and 7 after RNA injection, mice are challenged with HSV-2 (105 pfu in 30 μl) by intravaginal inoculation. Each day after inoculation of HSV-2, mice are observed for signs of infection and scored on a scale of 0-4. Zero is no sign of infection; 1 denotes redness; 2 denotes vesicles and redness; 3 denotes vesicles, redness and incontinence; and 4 denotes paralysis. According to the present invention, because the mice receiving the dsRNA molecules of the present invention which contain the gD sequences of HSV, are shown to be protected against challenge. The mice that receive the control RNA molecules from the HIV gag are not protected. Mice receiving the ss RNA molecules containing the gD sequence of HSV are expected to be minimally protected if they are, unless these molecules have the ability to become at least partially double stranded in vivo. According to this invention, because the dsRNA molecules of the invention have no ability to make the HSV gD protein, the protection provided by the distribution of RNA molecules to the animal is due to a mechanism not mediated by the system. immune that is specific to the gene. All references published previously, noted, are incorporated herein by reference. Numerous modifications and variations of the present invention are included in the previously identified specification, and are expected to be obvious to a person skilled in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed within the scope of the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > American Home Products Corporation Pachuk, Catherine Satishchandran, C. < 120 > Methods and Compositions for Inhibiting the Function of Polynucleotide Sequences < 130 > AHP28APCT < 140 > < 141 > < 150 > 60 / 130,377 < 151 > 1999-04-21 < 160 > 20 < 170 > PatentIn Ver. 2.1 < 210 > 1 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: T7 front gag starter < 400 > 1 gtaatacgac tcactatagg gcggcaggga gctagaacga ttcgcag 47 < 210 > 2 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: reverse gag primer < 400 > 2 ctgctatgtc acttcccctt ggttc 25 < 210 > 3 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: T7 inverse gag primer < 400 > 3 gtaatacgac tcactatagg gcgctgctat gtcacttccc cttggttc 48 < 210 > 4 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: forward gag primer < 400 > 4 gcagggagct agaacgattc gcag 24 < 210 > 5 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: delever gD primer of T7 < 400 > 5 gtaatacgac tcactatagg gcggtcgcgg tgggactccg cgtcgtc 47 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: forward gD primer < 400 > 6 gtcgcggtgg gactccgcgt cgtc 24 < 210 > 7 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse gD primer of T7 < 400 > 7 gtaatacgac tcactatagg gcggtgatct ccgtccagtc gtttatc 47 < 210 > 8 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: inverse gD primer < 400 > 8 gtgatctccg tccagtcgtt tatc 24 < 210 > 9 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: genomic forward IL-12 < 400 > 9 tcagcaagca cttgccaaac tcctg 2 < 210 > 10 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse genomic IL-12 < 400 > 10 gagacaaggt ctctggatgt tattg 2 < 210 > 11 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: T7 front genomic IL-12 < 400 > eleven gtaatacgac tcactatagg gtcagcaagc acttgccaaa ctcctg 4 < 210 > 12 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse genomic IL-12 of T7 < 400 > 12 gtaatacgac tcactatagg ggagacaagg tctctggatg ttattg 4 < 210 > 13 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: T7 front IL-12 primer < 400 > 13 gtaatacgac tcactatagg gcctataagc ataagagacg ccctc 45 < 210 > 14 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: forward IL-12 promoter < 400 > 14 cctataagca taagagacgc cctc 2 < 210 > 15 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: inverse IL-12 promoter < 400 > 15 ggctgctcct ggtgcttata tac 23 < 210 > 16 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse IL-12 Promoter of T7 < 400 > 16 gtaatacgac tcactatagg gggctgctcc tggtgcttat atac 44 < 210 > 17 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: T7 IL-12 forward cDNA < 400 > 17 gtaatacgac tcactatagg gtgtgtcctc agaagctaac cate 44 < 210 > 18 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Front IL-12 cDNA < 400 > 18 tgtgtcctca gaagctaacc ate 23 < 210 > 19 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse IL-12 cDNA < 400 > 19 gcaggtgaca tcctcctggc agga 24 < 210 > 20 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Inverse IL-12 cDNA of T7 < 400 > twenty gtaatacgac tcactatagg ggcaggtgac atcctcctgg cagga 45

Claims (67)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A composition for inhibiting the function of an objective polynucleotide sequence in a mammalian cell, characterized in that the composition comprises an agent that provides a mammalian cell with at least one partially double-stranded RNA molecule that does not produce a functional protein, and it comprises a polynucleotide sequence of at least 200 nucleotides in length, said polynucleotide sequence being substantially homologous to the target polynucleotide sequence, and substantially not homologous to an essential mammalian polynucleotide sequence, of natural origin, selected.

2. The composition according to claim 1, characterized in that at least 11 contiguous nucleotides of the polynucleotide sequence of the RNA molecule are present in a double-stranded sequence, depending on the composition of the polynucleotide sequence and a? G of about -9.2 kcal / mol.

3. The composition according to claim 2, characterized in that substantially the complete polynucleotide sequence of the RNA molecule is double-stranded.

The composition according to claim 1, characterized in that the polynucleotide sequence of the RNA molecule has a sequence of at least about 12 to about 16 contiguous nucleotides in exact homology to the target polynucleotide sequence, and wherein the complete homology of the polynucleotide sequence of the RNA molecule to the target sequence is greater than about 10%.

5. The composition according to claim 4, characterized in that the homology is greater than about 50%.

6. The composition according to claim 1, characterized in that the agent is an RNA molecule made by the synthetic enzymatic methods or the synthetic chemical methods in vi tro. 1 .

The composition according to claim 1, characterized in that the agent is an RNA molecule made in vi tro through the isolation of a recombinant microorganism or the culture media in which the microorganism develops.

The composition according to claim 1, characterized in that the agent generates the RNA molecule in vivo after administration to the mammalian cell.

9. The composition according to claim 1, characterized in that the agent is a double-stranded RNA.

10. The composition according to claim 1, characterized in that the agent is a strand in the direction of the single-stranded RNA.

11. The composition according to claim 10, characterized in that the strand in the direction of the single-stranded RNA forms a fork at one or both ends or intermediate between the ends.

12. The composition according to claim 10, characterized in that the strand in the direction of the single-stranded RNA is folded on itself to become partially double-stranded.

The composition according to claim 1, characterized in that the agent is an antisense strand of single-stranded RNA.

The composition according to claim 13, characterized in that the antisense strand of single-stranded RNA forms a hairpin at one or both ends or intermediate between said ends.

15. The composition according to claim 13, characterized in that antisense latebra of single-stranded RNA is folded on itself to become partially double-stranded.

16. The composition according to claim 1, characterized in that the agent is a single-stranded RNA sequence comprising a polynucleotide sequence in sense and an antisense polynucleotide sequence, optionally separated by a polynucleotide sequence of unpaired bases, and the single-stranded RNA sequence has the ability to become double-stranded.

17. The composition according to claim 1, characterized in that the agent is a circular RNA molecule that forms a rod structure.

18. The composition according to claim 8, characterized in that the agent is a double-stranded DNA molecule that codes for the RNA molecule.

19. The composition according to claim 18, characterized in that the DNA encodes a double-stranded RNA.

20. The composition according to claim 18, characterized in that the DNA codes for a strand in the direction of the single-stranded RNA.

21. The composition according to claim 20, characterized in that the DNA encodes a strand in the RNA direction, of a single strand, forming a hairpin at one or both ends, or intermediate therebetween.

22. The composition according to claim 20, characterized in that the DNA codes for a strand in the RNA sense, of a single strand, which folds on itself to become partially double-stranded.

23. The composition according to claim 18, characterized in that the DNA encodes an antisense strand of single-stranded RNA.

24. The composition according to claim 23, characterized in that the DNA encodes an antisense strand of the single-stranded RNA that forms a hairpin at one or both ends or intermediate therebetween.

25. The composition according to claim 23, characterized in that the DNA codes for an antisense strand of the single-stranded RNA that folds on itself to become partially double-stranded.

26. The composition according to claim 18, characterized in that the DNA encodes a single-stranded RNA sequence, comprising a polynucleotide sequence in sense and an antisense polynucleotide sequence, optionally separated by a polynucleotide sequence of unpaired bases, the single-stranded RNA sequence has the ability to become double-stranded.

27. The composition according to claim 18, characterized in that the DNA encodes a circular RNA molecule that forms a rod structure.

28. The composition according to claim 1, characterized in that the agent is a plasmid.

29. The composition according to claim 1, characterized in that the agent comprises a first DNA plasmid encoding a polynucleotide sequence in the RNA sense of one. single strand, and a second DNA plasmid encoding a single stranded RNA antisense polynucleotide sequence, wherein the sense and antisense RNA sequences have the ability to base pairing and become double stranded.

30. The composition according to claim 28, characterized in that the plasmid comprises bacterial sequences.

31. The composition according to claim 1, characterized in that the agent is a recombinant bacterium.

32. The composition according to claim 1, characterized in that the agent is a recombinant virus.

33. The composition according to claim 1, characterized in that the agent is a donor cell transfected in vi tro with the molecule described according to any of claims 2 to 32.

34. The composition according to any of the claims 30-32, characterized in that the agent is selected from the group consisting of a recombinant virus or live bacterium or cell, a dead virus or bacterium or cell, or an inactivated virus or bacterium or cell.

35. The composition according to claim 1, characterized in that the agent lacks a polyadenylation sequence.

36. The composition according to claim 1, characterized in that the RNA molecule is not translated.

37. The composition according to claim 1, characterized in that the agent lacks a Kozak region.

38. The composition according to claim 1, characterized in that the agent lacks a start methionine codon.

39. The composition according to claim 1, characterized in that the RNA molecule lacks a cap structure.

40. The composition according to claim 1, characterized in that the agent lacks the signals for the synthesis of proteins.

41. The composition according to claim 1, characterized in that it comprises a mixture of different agents.

42. The composition according to claim 1, characterized in that the target polynucleotide sequence is a viral polynucleotide sequence necessary for the replication and / or pathogenesis of the virus of an infected mammalian cell.

43. The composition according to claim 42, characterized in that the virus is selected from the group consisting of a DNA virus and a virus having an intermediate DNA stage.

44. The composition according to claim 43, characterized in that the virus is selected from the group consisting of Retroviruses, Herpesviruses, Hepadenovirus, Poxviruses, Parvoviruses, Papilomaviruses and Papovaviruses.

45. The composition according to claim 44, characterized in that the virus is selected from the group consisting of HIV, HBV, HSV, CMV, HPV, HTLV and EBV.

46. The composition according to claim 1, characterized in that the target polynucleotide sequence is a tumor antigen or functional fragment thereof, or a regulatory sequence of a cancer induced by virus, whose antigen or sequence is required for the maintenance of the tumor in the mammal

47. The composition according to claim 46, characterized in that the cancer is selected from the group consisting of cervical carcinoma induced by HPV E6 / E7 virus, HTLV-induced cancer and EBV-induced cancer.

48. The composition according to claim 1, characterized in that the target polynucleotide sequence is a polynucleotide sequence of an intracellular or extracellular pathogen necessary for the replication and / or pathogenesis of said pathogen in an infected mammalian cell.

49. The composition according to claim 1, characterized in that the target polynucleotide sequence is a polynucleotide sequence of an abnormal sequence that causes cancer, in a mammal, which also possesses a normal copy of said sequence, and wherein the differences between the abnormal and normal sequences are differences in the polynucleotides.

50. The composition according to claim 49, characterized in that the abnormal sequence is a fusion of two normal genes.

51. The composition according to claim 50, characterized in that the target polynucleotide is the polynucleotide sequence spanning said fusion.

52. A pharmaceutical composition, characterized in that it comprises a composition according to any of claims 1-51, and a second optional agent that facilitates the uptake of the polynucleotide into a cell, in a pharmaceutically acceptable carrier.

53. The composition according to claim 52, characterized in that the second agent is selected from the group consisting of a local anesthetic, a peptide, a lipid including cationic lipids, a liposome or lipid particle, a polycation, a branched polycation, three-dimensional, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or another compound that facilitates the transfer of polynucleotides to cells.

54. The composition according to claim 53, characterized in that the second agent is bupivacaine.

55. A method for treating a viral infection in a mammal, characterized in that it comprises: administering to said mammal a composition according to claim 1, with a second optional agent that facilitates uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier , wherein the target polynucleotide is a viral polynucleotide sequence necessary for the replication and / or pathogenesis of said virus in an infected mammalian cell, in an amount effective to reduce or inhibit the function of the viral sequence in the cells of the mammal.

56. A method for preventing a viral infection in a mammal, characterized in that it comprises: administering to said mammal a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier , wherein the target polynucleotide is a viral polynucleotide sequence necessary for the replication and / or pathogenesis of said virus in an infected mammalian cell, in an amount effective to reduce or inhibit the function of the viral sequence after the subsequent introduction of the virus inside the cells of the mammal.

57. A method for the treatment or prophylaxis of a virally induced cancer in a mammal, characterized in that the method comprises: administering to said mammal a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a sequence that encodes a tumor antigen, a regulatory sequence, or a functional fragment thereof, whose antigen or sequence function is required for the maintenance of the tumor in the mammal, in an amount effective to reduce or inhibit the function of the antigen in the mammal ,

58. A method for the treatment or prophylaxis of infection of a mammal by an intracellular or extracellular pathogen, characterized in that the method comprises administering to the mammal a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a pathogenic or mammalian cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a polynucleotide sequence of said pathogen, necessary for the replication and / or pathogenesis of the pathogen in an infected mammalian mammal or mammalian cell, in an amount effective to reduce or inhibit the function of the sequence in said mammal.

59. A method for the treatment or prophylaxis of cancer in a mammal, characterized in that it comprises administration to the mammal of a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide in a cell, in a carrier pharmaceutically acceptable, wherein the target polynucleotide is a polynucleotide sequence of an abnormal sequence that causes cancer, in a mammal, which also possesses a normal copy of said sequence, and wherein the differences between the abnormal sequence and the normal sequence are differences in the polynucleotides, in an amount effective to reduce or inhibit the function of the abnormal sequence in the mammal.

60. A method for the treatment of a disease or disorder in a mammal, comprising: administering to the mammal having a disease or disorder characterized by the expression of the polynucleotide product not found in a healthy mammal, a composition according to claim 1 , wherein the target polynucleotide sequence is a polynucleotide sequence that expresses the polynucleotide product or regulatory sequence necessary for the expression of said product, in an amount effective to reduce or inhibit the function of the target polynucleotide product in the cells of the mammal.

61. The use of a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide from a cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a viral polynucleotide sequence necessary for replication and / or pathogenesis of the virus in an infected mammalian cell, in the preparation of a medicament for treating a viral infection in a mammal.

62. The use according to claim 61, wherein the composition is in an amount effective to reduce or inhibit the function of the viral sequence in the cells of the mammal.

63. The use according to claim 61, wherein the composition is in an amount effective to reduce or inhibit the function of the viral sequence after subsequent introduction into the virus within the cells of the mammal.

64. The use of a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a sequence encoding a tumor antigen, a sequence regulatory, or a functional fragment thereof, whose function of the sequence antigen is required for the maintenance of the tumor in the mammal, in an amount effective to reduce or inhibit the function of the antigen in the mammal, in the preparation of a drug for the treatment or prophylaxis of a virally induced cancer, in a mammal.

65. The use of a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a pathogenic or mammalian cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a polynucleotide sequence of the pathogen , necessary for the replication and / or pathogenesis of the pathogen in an infected mammal or mammalian cell, in an amount effective to reduce or inhibit the function of the sequence in the mammal, in the preparation of a medicament for the treatment or prophylaxis of the infection of a mammal by an intracellular or extracellular pathogen.

66. The use of a composition according to claim 1, with a second optional agent that facilitates the uptake of the polynucleotide into a cell, into a pharmaceutically acceptable carrier, wherein the target polynucleotide is a polynucleotide sequence of an abnormal sequence that causes cancer, in a mammal, which also possesses a normal copy of said sequence, and wherein the differences between the abnormal sequence and the normal sequence are differences in the polynucleotides, in an amount effective to reduce or inhibit the abnormal sequence function in the mammal , in the preparation of a medicament for the treatment or prophylaxis of cancer in a mammal.

67. The use of a composition according to claim 1, wherein the target polynucleotide sequence is a polynucleotide sequence expressing the polynucleotide product or the regulatory sequence necessary for the expression of a polynucleotide product not found in a healthy mammal, in a an effective amount to reduce or inhibit the function of the target polynucleotide product in mammalian cells, in the preparation of a medicament for treating a disease or disorder in a mammal, characterized by the expression of said product.