Treatment and prevention of neurodgenerative diseases with use of gene therapy

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely neurology, and concerns treatment and prevention of neurodegenerative diseases, particularly Alzheimer's disease with the use of gene therapy. That is ensured by the introduction of a composition containing one or more nucleic acids inducing cell immune response. The nucleic acids to be introduced code one or more cytokines specified in a group consisting of IL-4 (interleukine-4), IL-10 (interleukine-10) and TGF-β (transforming growth factor beta).

EFFECT: method provides reduced cerebral accumulations of amyloid formations.

16 cl, 7 ex, 7 dwg

 

The SCOPE of the INVENTION

In this proposed invention compositions and methods of treatment and/or prevention of neurodegenerative diseases such as Alzheimer's disease. In some aspects, the data compositions and methods relate to adoptive cellular therapies and to DNA immunization.

PRIOR art

Neurospine associated with the pathology of Alzheimer's disease (AD) (Chen, K. et al., 281 Peptide J.Biol. Chem. 3651-59 (2006), and Frenkel, D. et al., 115 J.Clin. Inves. 2423-33 (2005)). Neurospine includes the accumulation of a large number of activated microglia and astrocytes, as well as small amounts of T-cells, is attached mainly to the postcapillary venules (Agadjanyan, M.G. et al., 174 J. Immunol. 1580-86 (2005); Dickson, D. et al., 7 Glia 75-83 (1993); and Fillit, H. et al., 129 Neurosci. Lett. 318-20 (1991)). It was shown that microglia and astrocytes generate β-amyloid protein (β), one of the main pathological signs of ad. It was shown that he β acts as an inflammatory agent, causing the activation of many inflammatory components. Related biochemical changes include the appearance or increase regulation of numerous molecules, characteristic of inflammation and free radical attack. Especially important may be the proteins of the complement, the agents of the acute phase and inflammatory cytokines. Patients who are taking nonsteroidal anti-inflammatory Lek is stennie means, have a lower risk of ad, than patients who do not accept them. These results have led to increased interest in conducting anti-inflammatory therapy BA (Fillit, H. 1991).

SUMMARY of the INVENTION

Although neurodegenerative diseases such as Alzheimer's disease, classically not considered to be mediated by inflammation or the immune system, in some cases, the immune system plays an important role in degenerative processes. For the treatment of BA were previously developed immunotherapy approaches designed for the induction of humoral immune response. These studies led to clinical trials in humans, which led to both the beneficial and harmful effects. In animal models it was shown that immunotherapy designed for the induction of cellular immune response may be useful in case of damage of the Central nervous system, although T cells can have either beneficial or harmful effects, depending on the type of induced T-cell response. These studies have given a new direction for research therapy of neurodegenerative diseases on the basis of the immune system.

The adaptive immune system can be broadly classified into two types of responses: cellular and humoral (or antibody-based test) types of responses. Among the cellular responses identified what was aziraphale three main types or class of immune response, which play a crucial role in understanding the mechanisms of regulation of the inflammatory process, for example, Th1-response (involving, for example, IFN-γ (interferon-gamma)) in contrast to Th2 and Th3 responses (involving, for example, IL-4 (interleukin-4), IL-10, IL-13 and TGF-β (transforming growth factor beta)). Different classes of T-cell responses have important implications for the development strategy of vaccination against Alzheimer's disease.

In this proposed invention compositions and methods of treatment and/or prevention of neurodegenerative diseases such as Alzheimer's disease. In some aspects, the data compositions and methods relate to DNA vaccines and adoptive cellular gene therapy to treat or alleviate symptoms of the neurodegenerative disease. In some specific aspects of data compositions and methods relate to DNA vaccines encoding element of the cellular immune response, such as Th2 or Th3 cytokine.

In one aspect, a method of treating or relieving the symptoms of neurodegenerative diseases, which includes the introduction of a composition that induces a cellular immune response. In another aspect, a method of treating or relieving the symptoms of neurodegenerative diseases by introducing a composition that induces a cellular immune response by injection of the composition, on which the element comprises a cellular immune response; preferably the element of the cellular immune response is a protein, peptide, nucleic acid or polynucleotide. In a related aspect, a method of treating or relieving the symptoms of neurodegenerative diseases, which includes the introduction of two or more; three or more, four or more, five or more, or six or more elements of the cellular immune response. In another aspect, a method of manufacturing a composition for the treatment or prevention of neurodegenerative diseases, which may include obtaining polynucleotide or its fragment with promoter/enhancers, transcription-related sequence that encodes a gene element of the cellular immune response or its fragment. In a related aspect, a method for obtaining a composition for expression of polynucleotide element of the cellular immune response or a portion of the subject, which includes obtaining polynucleotide with promoter/enhancers, transcription-related sequence that encodes a gene element of the cellular immune response or its fragment; and combining substances that facilitate transfection, with the specified polynucleotide. Also proposed compositions and methods of administration to a mammal, preferably human. In another aspect, the composition may contain a pharmaceutical is viable media and polynucleotide, containing a sequence encoding the polypeptide of the element of the cellular immune response. In some aspects of the proposed set that may include a container suitable for storing a pharmaceutical preparation for administration to a subject, preferably a human; polynucleotide, including the sequence encoding the polypeptide of item t-cell immune response pharmaceutically acceptable carrier and a label attached to the container, or liner in the package. In other aspects of the proposed ways of introducing polypeptide homologue polypeptide of the element of the cellular immune response or its fragment.

In some embodiments of the proposed composition, which induces an element of the cellular immune response. These methods and compositions can include a protein, nucleic acid or polynucleotide; preferably, polynucleotide and/or nucleic acid is a DNA or RNA; preferably, polynucleotide is a circular DNA; preferably, polynucleotide is a plasmid; preferably, polynucleotide includes the promoter/enhancer, a transcription associated with the sequence that encodes a gene element of the cellular immune response; preferably, polynucleotide includes the site of the beginning of replication (ORI); preferably, polynucleotide VK is uchet a multiple cloning site (MCS); preferably, the promoter is suitable for expression in eukaryotic cells; in some preferred embodiments polynucleotide is a vector, preferably a viral vector; in other preferred embodiments polynucleotide represents RNA; preferably, polynucleotide is a double-stranded RNA; preferably, polynucleotide is a short interfering RNA (kirk); or preferably more than one composition, which reduces inflammation, you can enter at the same time. In some embodiments, one element of the cellular immune response is on the same polynucleotide or plasmids; in other embodiments more than one element of the cellular immune response can be on the same polynucleotide or plasmids. In other embodiments one polynucleotide or plasmid includes one or more than one element of the cellular immune response and further includes one or more polypeptides or fragments, which are known to cure or improve the symptoms of neurodegenerative diseases (such as neurotrophic factor brain (BDNF), nerve growth factor (NGF), β-amyloid, β-amyloid peptide 1-42 (β-amyloid1-42), apolipoprotein E (APOE) or APOE-2.

Other embodiments relate to the introduction of proteins, peptides and/or the floor of the peptides element of the cellular immune response. In other embodiments of the methods of introducing nucleic acids and/or polynucleotides encoding polypeptides of the elements of the cellular immune response. Song data can be obtained and entered in such a way that the subject who is administered a composition, expressed polypeptide element of the cellular immune response. The composition can include expression systems, delivery systems and coding sequences immunoregulatory genes, such as anti-inflammatory cytokines, agonists of cytokines or antibodies against TNF (tumor necrosis factor). Preferably, the element of the cellular immune response of these methods and compositions increases (expression) of the gene, which reduces inflammation; preferably, the increase in gene expression occurs by increasing expression regulation;

preferably, a gene that reduces inflammation, is a Th2-cytokine; preferably, the Th2-cytokine represents IL-4, IL-5, IL-10, IL-13 or TGF-β; in other preferred embodiments, the composition containing an element of the cellular immune response, inhibits or reduces gene, which increases inflammation; preferably, a gene that increases inflammation, is a Th1 cytokine; preferably, the weakening of gene expression is accomplished by down-regulation of the expression is AI; preferably, Th1-cytokine represents IL-2, IL-12 and TNFα, in some embodiments, the composition has an impact on the regulation by stimulating the expression or gene product that reduces inflammation, while in other embodiments the composition has an impact on the regulation by inhibiting gene expression, which increases stimulation, such as Th1 antagonist.

Unexpectedly showed as described in Examples 3 and 4 that the introduction of polynucleotide encoding IFN-γ, can treat or prevent the effects of Alzheimer's disease in animal models. Although IFN-γ is a Th1-cytokine, it has been shown that introducing it as a genetic vaccine minimizes the effects of neurodegenerative diseases. Therefore, compositions and methods that include polynucleotide encoding IFN-γ or polypeptide encoding a homolog of the IFN-γ proposed in this invention as a method of treatment and/or relief of symptoms of neurodegenerative diseases such as Alzheimer's disease, in a subject, preferably a mammal, more preferably human.

In some embodiments the element of the cellular immune response includes a gene or protein encoding autoantigen, a cytokine that reduces autoimmune inflammation, cytokine antagonist, improves autoimmune inflammation, or a gene that Indus is the range of anergy, or fragments thereof; preferably, the element of the cellular immune response is a or homologous to the interleukin (IL)-4, IL-5, IL-10, IL-13, transforming growth factor beta (TFG-β), or interferon-gamma (IFN-γ), or their fragments.

In preferred embodiments additionally introduced the element of the cellular immune response to nucleic acid or protein coding for the polypeptide, which is optionally treats and/or reduces the effects of neurodegenerative disease, or its fragments; preferably, the additional gene or protein that encodes a polypeptide that constitutes or homologous brain neurotrophic factor (BDNF), nerve growth factor (NGF), β-amyloid, β-amyloid peptide 1-42 (β-amyloid1-42), apolipoprotein E (APOE) or APOE-2.

In other preferred embodiments of the compositions and methods of inducing cellular immune response include delivery by adoptive cellular gene therapy. Preferably, the type of cells used for adoptive cell therapy is autologous or neautrogena; preferably, the type of cells used for adoptive cell therapy, is a T-cell, antigen presenting cell, a fibroblast, or a stem cell; preferably, the type of cells used for adoptive cellular gene therapy is dandri the Naya cell; cell NIH3T3, neautrogena stem cells, such as cells from the American type culture collection (ATSS) or autologous stem cell.

In some embodiments described in this invention polynucleotide injected the patient with a pharmaceutically acceptable carrier. In some embodiments polynucleotide includes eukaryotic promoter; preferably, the eukaryotic promoter provides expression in humans. In some embodiments polynucleotide is a plasmid in combination with the promoter/enhancer, a transcription associated with the sequence, the coding element of the cellular immune response. In some embodiments polynucleotide is a viral vector. In some embodiments polynucleotide injected with a reagent of lipofectin. In some embodiments, the methods can include one or more of the techniques proposed in this invention compositions selected from the group consisting of intravenous, intranazalnogo, subcutaneous, by injection, by inhalation or by gene gun.

In some preferred embodiments proposed in this invention means polynucleotide, including the sequence encoding the polypeptide of the element of the cellular immune response, or its fragment, enter mammalian what Osamu; more preferably the mammal is a human; preferably, polynucleotide comprising a sequence encoding a gene element of the cellular immune response or fragment is administered together with a substance to facilitate transfection; preferably, a substance that facilitates transfection, includes a lipid; preferably, polynucleotide administered in pharmaceutically acceptable carrier; in some preferred embodiments polynucleotide injected through viral transduction; preferably, polynucleotide administered by gene gun; preferably, polynucleotide administered by inhalation; or, preferably, polynucleotide injected through an injection, or, preferably, subcutaneous injection, or preferably intramuscular injection.

In some embodiments the compositions contain polynucleotide, including the sequence encoding the polypeptide of the element of the cellular immune response or its fragment; preferably, the composition contains a pharmaceutically acceptable carrier; preferably, the composition contains a substance that facilitates transfection; preferably, a substance that facilitates transfection, includes a lipid; preferably, the composition is administered with an adjuvant; preferably, the composition is suitable for injection to a mammal, preferably the milk is itaudit is man; preferably, the composition is suitable for inhalation to a mammal, preferably a mammal is human; preferably, the composition is enclosed in a pharmaceutically acceptable carrier, preferably a pharmaceutically acceptable carrier has a label indicating its composition, and the statement on the introduction of polynucleotide; preferably, the composition includes an insert in the packaging, preferably an insert in the packaging includes a statement of the composition, more preferably an insert in the packaging includes instructions regarding the dosage.

Used in this invention, the term "antigen" refers in a broad sense to any composition to which the individual can develop an immune response. Used in this invention, the term "antigen" refers in a broad sense to a molecule that contains at least one antigenic determinant, which can be directed immune response. The immune response may be cell-mediated or humoral, or both. As is well known in this field, the antigen can be a protein in nature, the carbohydrate in nature, the lipid in nature, nucleic acid in nature or a combination of these biomolecules. For example, the antigen may include non-natural molecules such as polymers and things under the service. Antigens include autoantigens and foreign antigens, such as antigens produced by other animals, or antigens of the infectious agent. The antigens of the infectious agent can be bacterial, viral, fungal, protozoal, and the like.

Used in this invention, the term "autologous" is used in connection with the release of cells from the subject, possibly changing data of these cells, or preserving cells and re-infusion of these cells back to the subject.

Used in this invention, the term "coding region" or "coding sequence" refers to nucleic acid sequence, its complementary sequence, or part thereof, which encodes a specific gene product or fragment, the expression of which is desired, according to the normal relationships for base pairing and use of codons. Coding sequences include the exons in the genomic DNA or immature primary RNA transcripts, which are connected together by means of biochemical mechanisms in the cell with the formation of Mature mRNA. The antisense chain complementary to such nucleic acid, and the coding sequence can be deduced from it. The coding sequence is placed depending on the elements controlling transcri the tion, and initiation codons and translation termination, which produced the transcript of the correct length that will lead to broadcast in the appropriate reading frame with the formation of the desired functional product.

Used in this invention, the term "complement", "complementary" or "complementarity" refers to polynucleotides (i.e. to the sequence of nucleotides such as an oligonucleotide probe or target nucleic acid) according to standard rules mating Watson/Crick. The sequence complementary to the nucleic acid sequence is in "antiparallel Association", so that the 5'end of one sequence is coupled with 3'-end of the other sequence. For example, the sequence "5'-A-G-T-3'" complementary sequence 3'-T-C-A-5'". As described in this invention nucleic acids can be included some of the nucleotides, usually not found in natural nucleic acids; they include, for example, inosine, 7-deazaguanine, locked nucleic acid (LNA), peptide-nucleic acid (PNA). The complementary sequence may be a sequence of RNA, complementary DNA sequence, or its complementary sequence, and can also be cDNA. Complementarity is not necessarily life is perfect; stable duplexes may contain incorrectly paired base pair degenerate or unpaired bases. Experts in the field of technology of nucleic acids can empirically determine the stability of the duplex, considering a number of variables, including, for example, the length of the oligonucleotide, the composition of the bases and the sequence of the oligonucleotide, ionic strength and the frequency of incorrectly paired base pairs.

Complementarity may be "partial," in which only some nucleotide bases of the two chains of nucleic acids correspond according to the rules of the mating grounds. Complementarity may be "full" or "absolute"when all nucleotide bases of the two chains of nucleic acid conjugate according to the rules of the mating grounds. Complementarity may be absent when any one of the nucleotide bases of the two chains of nucleic acid conjugate according to the rules of the mating grounds. The degree of complementarity between nucleic acid chains has significant effects on the efficiency and strength of hybridization between nucleic acid chains. This is especially important in the reactions of amplification, and detection methods, which depend upon binding between nucleic acids. Any term can also be applied in ividually nucleotides, especially in the context of polynucleotides. For example, a particular nucleotide in the oligonucleotide may be known for their complementarity or her absence to the nucleotide in the other strand of the nucleic acid compared or compared to the complementarity between the rest of the oligonucleotide and the chain nucleic acids.

Used in this invention, the term "essentially complementary" refers to two sequences that hybridize under hard conditions hydridization. The person skilled in the art it will be clear that essentially complementary to the sequence of not necessarily hybridize along their full length. In particular, essentially complementary sequences contain a contiguous sequence of bases that do not hybridize with the target sequence located 3' and 5' with respect to a contiguous sequence of bases, which hybridize with the target sequence in the harsh conditions of hydridization.

Used in this invention, the term "dendritic cell" (DK) refers to an antigen presenting cell (APC), which may originate from hematopoietic stem cells. DK can be obtained from many lymphoid and non-lymphoid tissues, as well as from peripheral blood and bone marrow. People hematopoietic stem the yrs, such as CD34+cells, it is possible to artificially differentiate into DC in vitro. Dendritic cell has a characteristic morphology with thin folds (lamellipodia)extending from the body dendritic cells in multiple directions. Several phenotypic criteria are also typical, but can vary depending on the source of dendritic cells. They include high levels of MHC molecules (major histocompatibility complex) and co-stimulating molecules (e.g. B7-1 and B7-2), the absence of markers that are specific for granulocytes, NK cells, b-cells and T-cells. In mice, some (but not all) dendritic cells Express 33D1 (DC from spleen and of Meyerovich plaques, but not skin or medullary region of the thymus), NLDC145 (DC in skin and T-dependent areas of some lymphoid organs) and CD11C (Cd11c also reacts with macrophage). Dendritic cells are able to initiate primary T-cell responses in vitro and in vivo. These responses are antigen specific. Dendritic cells direct a strong reaction of mixed leukocytes (MLR) compared with peripheral blood leukocytes, splenocytes, B-cells and monocytes.

Used in this invention, the term "expression" refers to the biological production of the product encoded by the coding sequence. In most cases, the DNA sequence including the coding for the total sequence, transcribed with the formation of messenger RNA (mRNA). Messenger RNA is translated with the formation of the polypeptide product, which has biological activity. However, in some cases RNA product may be relevant activity and, thus, could be considered as a gene product. The expression may include additional stages of processing of the product of transcription of RNA, such as splicing to remove introns and/or post-translational processing of the polypeptide product.

The terms related to immunological tolerance, as they are used in this invention relate to the acquisition of tolerance to autoantigens. The ability to distinguish between proteins and non-proteins is essential for maintaining the body. Immunological tolerance is additionally described in Seroogy, C.M., et al., Gene Therapy, vol.7, p.9-13 (2000); Costa, G.L, et al., J. Immunol., vol.164, p.3581-90 (2000); and (Weiner, H.L, etal., NYAcad. Sci., vol.778, p.xiii-xviii (1996).

Used in this invention, the term "element of the cellular immune response" refers to any molecule that induces a cellular immune response. Preferably, the cellular immune response is a response Th2 or Th3-type. Preferably, the molecule is a protein, peptide, polypeptide, nucleic acid, oligonucleotide or polynucleotide. Some elements of the s-cell immune response is well known in this field and include, but not limited to, molecules that can enhance the regulation or to produce polypeptides that reduce autoimmune inflammation, which include, but are not limited to, polypeptides IL-4 (i.e. no access to GenBank M13982; SEQ ID NO: 12) and IL-10 (namely no access GenBank M; SEQ ID NO: 14) and nucleic acids encoding IL-4 and IL-10 (namely, SEQ ID nos: 1 and 3). The elements of the cellular immune response can also lower regulation or inhibition of the polypeptide, which increase autoimmune inflammation, which include, but are not limited to, a polypeptide TGF-β (namely no access GenBank M; SEQ ID NO: 16) and nucleic acid encoding TGF-β (namely, SEQ ID NO: 5). However, it should be understood that other elements of the cellular immune response include elements known in this field, and items not yet identified. Preferably, the polypeptide of the element of the cellular immune response or its fragment has an amino acid sequence that is homologous to the amino acid sequence of the element of the cellular immune response, as proposed in this invention, i.e. SEQ ID NO: 12-22. In some preferred embodiments, the fragment element of the cellular immune response is at least 25 amino acids, more preferably at least 50 amino acids, more prefer is Ino at least 150 amino acids, more preferably at least 200 amino acids, more preferably at least 250 amino acids, more preferably at least 300 amino acids, more preferably at least 400 amino acids, more preferably at least 500 amino acids, more preferably at least 600 amino acids, more preferably at least 700 amino acids, more preferably at least 800 amino acids that are homologous element of the cellular immune response, as proposed in this invention, i.e. SEQ ID NO: 12-22. The term "homologous", when he this invention relates to amino acid sequences, means that the amino acid sequence at least 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98% and most preferably 100% identical to known amino acid sequences (e.g. SEQ ID NO: 12-22). Used in this invention, the term "reagent lipofectin" refers to a substance used to include genetic material into the cell by means of liposomes. Examples lipofection reagents include lipofectin, lipofectamine, cationic lipids and neutral solidity.

Used in this image is hetenyi the term "plasmid" refers to the construction, made of genetic material (i.e. nucleic acids). It includes genetic elements located so that the built-in coding sequence can be transcribed in eukaryotic cells. Although the plasmid may include a sequence of viral nucleic acids, such viral sequence does not cause the activation of the plasmids in the virus particle and therefore the plasmid is a non-viral vector. Preferably, the plasmid is a closed circular nucleic acid. Preferably, the nucleic acid is a DNA or RNA. Preferably, the plasmid can be entered into cells by transformation and can autonomously replicate in the cell.

Used in this invention, the term "pharmaceutically acceptable" refers to a composition suitable for administration to humans. Specialists in this field understand that in order for the composition was suitable for the introduction of man, it must meet certain criteria, for example, the composition preferably corresponds quality laboratory practice (Good Laboratory Practices) (GLP); preferably, this composition corresponds to good manufacturing practices Good Manufacturing Practices ' (GMP); more preferably the composition soo is in accordance with the regulations, such as regulations developed by the United States for control of food and drugs; preferably, this composition corresponds to 21 U.S.. (U.S.C.) §301-392.

Used in this invention, the terms "start replication" or "land of the beginning of replication" refers to a nucleotide sequence, which begins DNA synthesis to replicate the nucleic acid sequence. It is usually called the ORI website. Ring (DNA) of bacteria usually has one site ORI, while each eukaryotic chromosome may be many websites ORI. This term includes replicons, which, when used in this invention, refers to a genetic element, which during DNA replication behaves as an Autonomous unit. In bacteria, chromosome operates as a single replicon, whereas eukaryotic chromosomes contain hundreds of replicons arranged in series.

The term "transcriptional unit" or "expression cassette" refers to a nucleotide sequence that contains at least one coding sequence with sequence elements that control initiation and termination of transcription. However, the transcriptional unit may include additional sequences, which may include sequence, involved in posttranscriptional or posttranslational processes.

Used in this invention, the term "sequence that regulates transcription" refers to the sequence that regulates the rate of transcription of the transcription of the associated coding region. This term may include elements such as promoters, operators, enhancers. Preferably, sequences that regulate transcription will include at least one promoter sequence.

Used in this invention, the term "transcriptional linked" refers to a system suitable for transcription, and transcription is initiated under control of the regulatory sequence and continue until the sequences of transcription associated with this regulatory sequence. Preferably, the resulting transcript is not generated mutations, which would change the resulting product is broadcast. For example, "transcriptional associated" usually means that linked DNA sequences are contiguous and, in the case of a secretory leader sequence are contiguous and in a phase of reading. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites n is exist you can use synthetic oligonucleotide adaptors or linkers according to conventional practice.

Used in this invention, the term "5'-untranslated region or 5'-UTR" refers to a sequence located 3' relative to the promoter region and 5' relative to the downstream coding region. Thus, this sequence at the transcription is above (i.e. 5') of the codon that initiates translation, and, therefore, usually not being the site of the polypeptide product.

Used in this invention, the term "3'-untranslated region/poly(A)signal or the 3'UTR poly(A)signal" represents the sequence below (i.e. 3') of the region encoding the polypeptide. As in the case of 5'-UTR, this area is usually transcribed, but not translated. For expression in eukaryotic cells, it is generally preferable to include a sequence that signals the addition of a poly-A tail. As with other synthetic genetic elements, synthetic 3'-UTR/poly(A)signal has a sequence that differs from naturally occurring UTR elements.

Used in this invention, the term "CMV promoter/enhancer sequences" refers to sequences of cytomegalovirus is, which are functional in eukaryotic cells as a transcriptional promoter and above enhancer sequence. Enhancer sequence allows transcription to occur with a higher frequency associated with the promoter.

Described in this invention, the plasmid may include one or more of the following: a promoter, a 5'-noncoding region (5'-UTR), 3'-UTR/poly(A)signal and introns can be a synthetic sequence. In this context, the term "synthetic" refers to sequences that are not represented directly by the sequence of naturally occurring genetic element of this type, but rather is an artificially created sequence (i.e. created by the individual molecular biological methods). Despite the fact that one or more areas such synthetic sequences can be identical to the plots occurring sequence, complete sequence within a given genetic element differs from naturally occurring genetic element of this type. The use of such synthetic genetic elements allows appropriately designing the functional characteristics of this cell battery (included) is that for the desired function.

Used in this invention "polynucleotide, including the sequence encoding the polypeptide of the element of the cellular immune response or its fragment" refers to polynucleotide with nucleotide sequence that encodes a peptide or protein capable of inducing cellular immune response, as defined in this invention. Assume that there are many different nucleotide sequences that could encode a single polypeptide sequence based on the normal vzaimosvyazei mating grounds, and the use of codons. As such, this term refers to any nucleic acid sequence that encodes an element of the cellular immune response or its fragment. In some preferred embodiments polynucleotide, including the sequence encoding the polypeptide of the element of the cellular immune response or its fragment includes a nucleotide sequence that encodes a protein with homology to IL-4, IL-5, IL-10, IL-13 or TGF-P, or its fragments. Preferably, polynucleotide, including the sequence encoding the polypeptide of the element of the cellular immune response, or its fragment, which includes a continuous period of at least 50 nucleotides; more preferably at least 100 nucleotides; more prepost is positive at least 300 nucleotides; more preferably at least 600 nucleotides; more preferably at least 1000 nucleotides; more preferably at least 1500 nucleotides; more preferably at least 2000 nucleotides that are homologous to sequences encoding the polypeptides of IL-4, IL-5, IL-10, IL-13, TGF-β or IFN-γ (as shown in SEQ ID NO: 1-6). The term "homologous"when he refers in this invention to a nucleotide sequence, means that the nucleotide sequence is at least 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98% or most preferably 100% identical to the known nucleotide sequence (such as sequences coding for IL-4, IL-5, IL-10, IL-13, TGF-β or IFN-γ, as they are represented in SEQ ID NO: 1-6). Assume that polynucleotide, including the sequence encoding the polypeptide of item t-cell immune response may contain additional nucleotides that are different from the nucleotides that comprise a sequence that encodes an element of the cellular immune response.

Used in this invention, the term "sample" or "sample for testing" refers to any liquid or solid material, which, excited, contains the selected nucleic acid. The sample for testing can be obtained from any biological source (i.e. a biological sample), such as cells in culture or a tissue sample, or obtained synthetically, including chemically synthesized matrix.

Used in this invention, the term "a sequence encoding a gene element of the cellular immune response or its fragment" refers to any nucleic acid sequence that encodes a gene element of the cellular immune response or its fragment. The term "gene element of the cellular immune response" refers to polynucleotide that encodes the amino acid sequence corresponding to the polypeptide, which can influence inflammation. Examples of genes of an element of the cellular immune response include, but are not limited to, IL-4, IL-5, IL-10, IL-13, TGF-β or IFN-γ. Preferably, the gene element of the cellular immune response, as described in this invention encodes a peptide with the amino acid sequence corresponding to the amino acid sequence of any polypeptide of the element of the cellular immune response, or their fragments on the basis of normal relationships for base pairing and use of translational termination codons. Preferably, the coding sequence encodes the fine is complete amino acid sequence of natural gene element of the cellular immune response.

Used in this invention, the term "translationa" refers to the cell with the selected nucleic acid, translational in a cage. The cell is stably transductional selected nucleic acid, when the selected nucleic acid is replicated and transferred to daughter cells. The cell is "transformed" selected nucleic acid, when the selected nucleic acid integrates into the genome of the cell.

Used in this invention, the terms "treat", "treatment" or "therapy" refer to curative therapy, prophylactic therapy, and preventative therapy. An example of a "preventive therapy" or "preventive therapy" is the prevention or relief of the targeted pathologic condition or disorder. Individuals in need of treatment include individuals who already have the disorder, as well as individuals who are prone to have the disorder or those who have this disorder should be warned. The introduction can be "long" introduction, which refers to the introduction of the agent(s) in a continuous way, in contrast to an emergency manner in order to maintain the initial therapeutic effect (activity) for an extended period of time. The introduction can also be "periodic" introduction, which is a treatment that is not consecutively without interruption, but rather is cyclic by nature. The introduction may also be in combination with one or more other therapeutic agent (s) includes the concurrent (simultaneous and sequential introduction in any order.

Used in this invention, the term "improving regulation" refers to the expression of the gene, or level of RNAS or equivalent RNAS encoding one or more protein subunits, or activity of one or more protein subunits, such as Th2-cytokines, greater than that observed in the absence disclosed in the present invention compositions. For example, the expression of the protein, such as IL-4, can be increased in order to cure, prevent, alleviate symptoms, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.

Used in this invention, the term "inhibit" or "to make lower regulation" refers to the expression of the gene, or level of RNAS or equivalent RNAS encoding one or more protein subunits, or activity of one or more protein subunits, such as Th1-cytokines, smaller than those observed in the absence of these nucleic acid molecules. In one embodiment, the inhibition or decreasing regulation molecule, the enzymatic nucleic acid preferably below, the em level, observed in the presence of an enzymatically inactive or attenuated molecule that is able to contact the same site on the RNA target, but unable to cleave that RNA. In another embodiment, the inhibition or decreasing regulation antimyeloma the oligonucleotides preferably below the level observed in the presence of, for example, the oligonucleotide with scrambled sequence or with incorrect base pairing. In another embodiment, the inhibition or decreasing regulation of TM-cytokine disclosed in the present invention compositions is greater in the presence of this composition than in its absence.

Used in this invention, the term "approximately" means in quantitative terms, plus or minus 10% from the specified value.

Other distinctive features and advantages of this invention will be apparent from the following description of preferred embodiments and from the claims.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Figa. Histological slice of the brain, produced at the age of nine months from the RDA transgenic mouse model of Alzheimer's disease with a control vector (group without treatment) after two injections (aged six and eight months). The arrows on the histological slice show staining of amyloid plaques after staining, the antibody is Rotel β-amyloid protein (specific immunohistochemical staining).

Figb. Histological slice of the brain, produced at the age of nine months from the RDA transgenic mouse model of Alzheimer's disease after two immunizations (ages six and eight months), the gene vaccine IL-10. Histology does not show any labeling of amyloid plaques after staining with an antibody against β-amyloid protein (specific immunohistochemical staining).

Figv. Histological slice of the brain, produced at the age of nine months from the RDA transgenic mouse model of Alzheimer's disease after two immunizations (ages six and eight months), the gene vaccine IL-4. Histology does not show any labeling of amyloid plaques after staining with an antibody against β-amyloid protein (specific immunohistochemical staining).

High Histological slice of the brain, produced at the age of nine months from the RDA transgenic mouse model of Alzheimer's disease after two immunizations (ages six and eight months), the gene vaccine TGF-β. Histology does not show any labeling of amyloid plaques after staining with an antibody against β-amyloid protein (specific immunohistochemical staining).

Figg. Histological slice of the brain, produced at the age of nine months from the RDA transgenic mouse model of Alzheimer's disease after two immunize the AI (aged six and eight months), the gene vaccine IFN-γ. The arrows on the histological slice show staining of amyloid plaques after staining with an antibody against β-amyloid protein (specific immunohistochemical staining).

Figa and 2B. Histological slice of the brain in the hippocampus, produced at the age of eighteen months from the APP transgenic mouse model of Alzheimer's disease after six immunizations (ages six, eight, ten, twelve, fourteen and sixteen months) only vector (Figa) or a mixture of genetic vaccines IL-4, IL-10, NGP and the APO-E2 (Figb). Histology after the mixed vaccination does not show any labeling of amyloid plaques after staining with an antibody against β-amyloid protein (specific immunohistochemical staining), which is consistent with the normal, contralateral disease mice, whereas the introduction of a vector really led to the labeling of amyloid plaques.

DETAILED description of the INVENTION

In this proposed invention compositions and methods of treatment and/or prevention of neurodegenerative diseases such as Alzheimer's disease. In some aspects, the compositions and methods relate to DNA vaccines and adoptive cellular gene therapy to treat or alleviate symptoms of a neurodegenerative disease in a subject, preferably a mammal, more preferably of man. In some aspects, the compositions and methods relate to DNA vaccines encoding Th2 or Th3-cytokine. Song data you can access and input so that the sequence, the coding element of the cellular immune response, is expressed in the subject, which enter the composition. These compositions can include expression systems, delivery systems, substances that facilitate transfection, and one or more elements of the cellular immune response.

Allergic diseases have an immune response that deviates to the profile of T-helper type 2 (Th2) and the profile of T-helper cells type 1 (Th1). The Th1 profile characterized by elevated levels of molecules that support an inflammatory response, such as IFN-γ and IL-2. The Th2 profile characterized by elevated levels of specific interleukins (IL)such as IL-4, IL-5, IL-10, IL-13, T-cells, CD4+ and production of antigen specific IgE. IL-4 is important in the synthesis of IgE and the development of the Th2 response and IL-5 in the survival of eosinophils. Immunotherapy leads to address this imbalance, with the increase of Th1-cytokines IFN-γ and IL-12, which, in turn, inhibit the Th2 response. At the same time, this genetic vaccination rapidly evolving as work on the low-affinity IgG receptor, representing FCγRIIB, which, being busy, inhibits lgE-mediated response to mast cells and basophils (Daero, et al., J.Clin. Invest. 95(2): 577-85 (1995)).

Unexpectedly, as described above and illustrated in Examples 3 and 4, the introduction of polynucleotide encoding IFN-γ, can treat and prevent the effects of Alzheimer's disease in animal models. Although IFN-γ is a Th1-cytokine, it has been shown that introducing it as a genetic vaccine minimizes the effects of neurodegenerative diseases. Therefore, compositions and methods that include polynucleotide encoding IFN-γ or polypeptide encoding a homolog of the IFN-γ proposed in this invention as a method of treatment and/or relief of symptoms of neurodegenerative diseases such as Alzheimer's disease, in a subject, preferably a mammal, more preferably human.

In some embodiments of the composition, which reduce inflammation, can stimulate the expression or to produce a gene that reduces inflammation, while in other embodiments the composition may influence the regulation by inhibiting gene expression, which increases inflammation, such as the antagonist. For inhibition of expression you can use double-stranded RNA, in particular miRNAs. RNA can be entered in the living cell to inhibit the expression of target gene in the cell. This process can be performed ex vivo or in vivo. Such compositions R Is For and methods of application additionally described, for example, in U.S. patent No. 6506559.

For introducing DNA into cells, one can use different approaches, including the introduction of "naked" DNA, the DNA in the complex with liposomes and various viral vectors. You can use a "naked" polynucleotide substances, methods and delivery systems, such as described in U.S. patent No. 6040295, 5763270 and 5580859. Polynucleotide are "naked" in the sense that they do not contain any media for delivery, which may act by facilitating penetration into the cell, or any substance that stimulates the transfection, such as liposomal preparations, charged lipids, such as lipofectin, or agents precipitation, such as CaPO4.

Vectors for delivery of nucleic acids can be viral, non-viral or physical. See, for example, Rosenberg et al., Science 242: 1575-1578 (1988) and Wolff et al., Proc. Natl. Acad. Sci. USA 86: 9011-9014 (1989). Discussion of methods and compositions for use in gene therapy includes Eck et al., in Goodman & Gilman''s The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., eds., McGray-Hill, New York, (1996), Chapter 5, pp.77-101; Wilson, Clin. Exp. Immunol. 107 (Suppl.1): 31-32 (1997); Wivel et al., Hematology/Oncology Clinics of North America, Gene Therapy, S.L.Eck, ed., 12(3): 483-501 (1998); Romano et al., Stem Cells, 18: 19-39 (2000) and references cited therein. In U.S. patent No. 6080728 also provides a discussion of several methods of gene delivery and compositions. Ways of delivery include, for example, system introduction and introduction is their in situ. Well-known methods of viral delivery include the use of adenoviral, retroviral, lentiviral transfer vector-based, vectors foamy virus, herpes simplex virus and adeno-associated viral vectors.

Viral vectors can also be used for transfection of mammalian cells, and the introduction of polynucleotide in the genome. In the indirect method, viral vectors that carry genetic information used for infection of target cells separated from the body, and these cells are then re-implanted. Reported direct gene transfer in vivo in animals after birth for drugs, DNA encapsulated in liposomes, DNA encapsulated in proteoliposome containing receptor viral envelope proteins (Nicolau et al., Proc. Natl. Acad. Sci USA 80: 1068-1072 (1983); Kaneda et al., Science 243: 375-378 (1989); Mannino et al., Biotechniques 6: 682-690 (1988). Viral vectors can be injected or transducible in cell owners in vitro, which are then adoptive transfer and serve as carriers for delivery, such as T cells (Nakajima, A., et al., J. Clin. Invest., vol. 17(21), p.1293-1310 (2001) and Tuohy, V.K., et al., J. Neuroimmunol., vol. 17(2), p.226-32 (2000), fibroblasts (Rabinovich, G.A., et al., J. Exp. Med., vol. 19, p.385-98 (1999)), dendritic cells (DC) (Kim, S.H., et al., J. Immunol., vol. 166(21), p.3499-3550 (2001) and Morita, Y., et al., J. Clin. Invest, vol. 17(21), p.1275-84 (2001)) and stem cells (ATSC or autologous).

In some embodiments viral century the PRS is preferably a retroviral vector. Retroviral vectors are plasmids for gene transfer, where the heterologous nucleic acid is between two retroviral LTR (long terminal repeat). Retroviral vectors typically contain appropriate signals for packaging, which make possible the packaging of the retroviral vector or RNA transcribed from the use of retroviral vectors as a matrix, viral virion in an appropriate packaging cell lines (see, for example, U.S. patent 4650764).

Suitable retroviral vectors for use in this invention are described, for example, in U.S. patents 5399346 and 5252479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, which describes effective ways of introducing nucleic acids into human cells using retroviral vectors. Other retroviral vectors include, for example, vectors of virus-based tumor mammary gland of mice (e.g Shackleford et al., Proc. Natl. Acad. Sci. U.S.A. 85: 9655-9659 (1998)), lentiviruses, and the like. Typical viral vector is an plentilox-IRES-GFP.

Adoptive cellular gene therapy

Methods of introducing nucleic acids into cells vary in transfairusa nucleic acid in cultured cell in vitro or in cells of the intended host in vivo. Techniques, suitable d is I the transfer of nucleic acid into mammalian cells in vitro, include the use of liposomes, electroporation (Luxembourg A., et al., Expert Opinion Biol. Ther. 7(11); 1647-1664 (2007); Kesaraju P, et al., Mol. Ther. 14(3): 416-422 (2006); Luxembourg, et al., Vaccine (24(21): 4490-4493 (2006)), microinjection, cell fusion, DEAE-dextran, the precipitation of calcium phosphate, etc. Preferred methods of gene transfer in vivo include transfection with viral (typically retroviral) vectors and transfection mediated by liposomes with proteins of the viral envelope (Dzau, et al., Trends in Biotechnology 11(5): 205-10 (1993)). Suitable vectors can be constructed using any of the methods well known in the field. See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1987 and revised edition). Vectors developed for DNA vaccines, such as pVAX1 (Invitrogen Carlsbad, CA), are also suitable for delivery of nucleic acid to the mammal. It was widely reported the use of cationic liposomes, such as liposome CD-Chol/DOPE, as a suitable carrier for the delivery of DNA to a variety of tissues by intravenous injections of complexes of DNA/cationic liposome. Cm. Caplen et al., Nature Med., 1: 39-46 (1995); Zhu et al., Science, 261: 209-211 (1993). Liposomes carry genes into the target cells by fusion with the plasma membrane. Examples of successful application of liposomal complexes include examples from the Lesson-Wood et al., Human Gene Therapy, 6: 395-405 (1995)and Xu et al., Molecular Genetics and Metbolism, 63: 103-109(1998).

Directed delivery of nucleic acids to the individual can be achieved by any of various methods known in this field. For example, the source of the nucleic acid can be combined with an agent who has as target cells in the damaged tissue, such as an antibody specific for a membrane protein found on the cell surface or the target cell, a ligand for a receptor on the target cells, etc. When using liposomes, proteins that are associated with associated with endocytosis of membrane proteins on the cell surface, can be used for targeted delivery and/or to facilitate uptake, e.g. capsid proteins or fragments thereof, genotype to a particular cell type, antibodies for proteins which undergo internalization in passing cycle proteins that have as a target intracellular localization and enhance the half-life within the cell. The technique of receptor-mediated endocytosis is described, for example, Wu, et al., J. Biol. Chem. 262(10): 4429-32 (1987); and Wagner, et al., Proc. Natl, Acad. Sci. USA 87(9): 3410-4 (1990). For an overview of the protocols of gene marking and gene therapy, see Anderson, Science 256(5058): 808-13 (1992).

In the present invention methods and compositions can also be used in adoptive cellular gene therapy using immune cells modified by genetic engineering, such campervan T-cells, dendritic cells, fibroblasts, and stem cells that have the ability to migrate to sites of inflammation in an organ-specific autoimmune disease for the expression and delivery of immunoregulatory products and/or therapeutic gene products after viral transduction ex vivo. Ex vivo transduction of these cells makes it possible to avoid exposure of the host to vector encoding the transgene, and thus increases the safety of this approach. Were used antigen-specific T-cell hybridoma who expressed anti-inflammatory cytokines, such as IL-4 antagonists of cytokines such as receptor antagonist IL-12, IL-12p40, or single-chain variable fragment (scFv) antibodies against TNF. All these molecules inhibited the development of the disease and reduced the severity of the disease. CIA model of adoptive cellular gene therapy are examples of convenient gene shuttles for mediating anti-inflammatory gene therapy. Additional studies have shown that primary T-cells, which are harder to transducible are equally effective for the expression of IL-12p40, which indicates that successful adoptive cellular gene therapy can be applied regardless of the type of cells. Therefore, to migrate to sites of inflammation can in order to use these cells, as dendritic cells (DC)derived from bone marrow.

Cells from the family of dendritic cells are particularly suitable to perform two different functions in two discrete areas. In peripheral tissues, dendritic cells (DC) act as "guards" against "dangerous" antigens. DK migrate and transport antigens to lymphoid organs where they initiate the activation of T-lymphocytes that are specific for a given antigen. During the migration of the DC switch with antigennegative path to the path sensitization of T cells. DK also affect the nature of the differentiation of T cells, i.e. on the balance of Th1/Th2. DK provides antigenic and co-stimulating signals required for optimal activation of T-lymphocytes. DK and applications additionally described, for example, in U.S. patent No. 6734014.

Stem cells can also be used for adoptive cellular gene therapy. For in the present invention compositions and methods you can use human embryonic stem (ES) cells. ES cells represent a line of cultured cells derived from the inner cell mass of a blastocyst, which can indefinitely grow in the undifferentiated state, but they also are able to differentiate into all cells of the adult body is ZMA. Preferably, the stem cells suitable for use in the present invention methods and compositions come from the subject or subjected to genetic engineering as a way to evade the immune response, such as the nucleus transfer or transfer of a somatic cell, which entails the replacement of the DNA of embryonic stem cell DNA of the subject. Embryonic stem cells are the most versatile stem cells because of their ability to differentiate about 200 different cell types found in the adult human body, and are the only type of stem cells, which have been developed routine protocols for genetic engineering. Ways of generating stem cells ex vivo are well known in this field include U.S. patents№№6326198, 6261549, 6093531, 5935565, 5670351, 5670147, 5646043, 5437994.

Vaccination with DNA requires a relatively small number of injections and has a more rapid phase increase. Can also be a reduced risk of harmful reactions to immunotherapy. Found that the expression of plasmid DNA and genes lasts for a long time (Wolff, et al., Hum. Mol. Genet. 1: 363-69 (1992)), and confirmed that immune responses in primates and rodents last for more than one year after DNA vaccination (Donnelly, et al., J. Immunol. Meth. 176: 145-152 (1994); and Ra, et al., Proc. Natl. Acad. Sci. 91: 9519-9523 (1994)). Apparently, the plasmid DNA is not included in the host genome, but is stored in the form of an episome (Tang, et al., Nature. 356: 152-4 (1992)). The discovery that "naked" DNA and RNA are absorbed and temporarily expressed in muscle cells in vivo, increased interest in the use of non-viral carriers for genetic delivery. Cm. Wolff et al., Science, 247, 1465-1468 (1990); Acsadi et al., Nature, 352, 815-818, (1991). Although "naked" DNA and RNA can be absorbed by mammalian cells, transfection efficiency can be greatly increased if the DNA or RNA forms a complex with liposomes (Chen, et al., Gene Therapy 7(19): 1698-705 (2000)).

The introduction of polynucleotide the mammal in vivo so that the cellular immune response or a fragment expressively this mammal, can be implemented using any of many methods known in the field of mammalian gene expression. For example, such methods of administration expressed polynucleotides mammals, including systems for the expression and delivery systems can be found in U.S. patent No. 6875748, 5763270, 5580859, 6040295 and 6034072.

Described in this invention polynucleotide constructs include the nucleotide sequence encoding the element of the cellular immune response or its fragment. This polynucleotide impose such a way that polynucleotide included in the cell which expresses definable number of prophylactically or therapeutically effective amount of the desired element of the cellular immune response or its fragment. Typical elements of the cellular immune response, suitable for use as proposed in this invention include IL-4, IL-5, IL-10, IL-13, TGF-β and IFN-γ.

Expression systems

Non-viral introduction of nucleic acid in vivo was carried out in various ways. They include lipofectin/liposomal fusion: Proc. Natl. Acad. Sci.84, pp.7413-7417 (1993); polylysine condensation with adenovirus amplification or without Human Gene Therapy 3, pp.147-154 (1992); and the delivery of nucleic acids into cells using system transferrin: atransferrinemia receptor: Proc. Natl. Acad. Sci.87, pp.3410-3414 (1990). The specific composition consisting of polyacrylic acid, has been disclosed in WO 94/24983. "Naked" DNA was introduced, as disclosed in WO 90/11092.

Thus, in one aspect of the proposed plasmid for expression of the element of the cellular immune response, or its fragment, which includes the expression cassette, which may also be called a transcription unit. When the plasmid is placed in a medium that is suitable for gene expression, the transcription unit is, therefore, to Express polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment. The transcription unit includes a sequence controlling transcription, which is transcriptionally linked to a sequence coding element of the cellular immune response. The sequence controlling transcription, may include the sequence of the promoter/enhancer, such as the sequence of the promoter/enhancer of cytomegalovirus (CMV). However, experts in this field it is clear that many other famous promoter sequence suitable for expression in eukaryotic cells, and they can be similarly used in disclosed in this invention designs. The level of expression of a gene product will depend on the associated promore and the presence and activation of the associated enhancer element. In some embodiments, the sequence encoding the gene element of the cellular immune response or its fragment can be cloned in expressing plasmid, which contains regulatory elements for transcription, translation, RNA stability and replication (i.e. includes the sequence controlling transcription). Such expressing plasmids are well known in this field, and the usual specialist would be able to construct a suitable expressing design with polynucleotides, including the sequence encoding the element of the cellular immune response, or its fragment, so that this element of the cellular immune response is expressed. There are many examples of suitable exp is assiduousy plasmids such as pCl-neo, pUMVC or pcDNA3, which could clone polynucleotide comprising a sequence encoding a gene element of the cellular immune response or its fragment.

Large quantities of bacterial host containing the plasmid for expression of the element of the cellular immune response, or its fragment, it is possible to ferment, and this plasmid can be cleaned for further use. In ongoing clinical trials on humans using plasmids used this approach. Recombinant DNA Advisory Committee Data Management Report, Human Gene Therapy 6: 535-548 (1994).

The purpose of the plasmids to be used in human gene therapy, is usually efficient delivery of nucleic acid sequences and expression of therapeutic genes (i.e. elements of the cellular immune response) in a cell or tissue of a mammal. In particular, the appointment of plasmids can be the achievement of a large number of copies, preventing potential causes of instability of the plasmids and the provision of methods of selection plasmid. With regard to the expression cassette nucleic acid contains the necessary elements for expression of the nucleic acid in the cassette. The expression includes an efficient transcription of the gene, nucleic acid sequence or cassette nucleic acid in the plasmid. Products ek is pressia can be a protein, polypeptides or RNA. The sequence of the nucleic acid can be contained in the cassette nucleic acid. Expression of nucleic acid may be continuous or variable.

The initial stage in the process of obtaining, ultimately, the expression of the product encoded by the nucleic acid, is the implementation of absorption of a given nucleic acid by the cells. The uptake of nucleic acid by the cells depends on several factors, one of which is the period of time during which the nucleic acid is in close proximity to the cell surface. For example, after intramuscular (I/m) injection of plasmid DNA in the buffer, there is a significant decrease in the expression of genes, if the muscle is subjected to massage, presumably due to leakage DNA from muscle, either directly or through lymphatic vessels (Human Gene Therapy 4: 151-159; 1993). Accordingly, it may be desirable to prepare nucleic acids as drug compounds that would reduce the rate at which the nucleic acid is diffused or removed from the area in which the absorption of a given nucleic acid cell is desirable. In addition, these compounds could be suitable for introduction into an organism through means such as injection, while maintaining or restore is allenii physical characteristics, necessary to enhance the absorption cell nucleic acids.

The pharmaceutical composition

In the present invention compositions can be introduced in the form of a pharmaceutical composition where the compound prepared in the form of a drug with a pharmaceutically acceptable carrier, as is well known in this field. Methods for the preparation and injection of pharmaceutical compositions can be found, for example, in "Remington''s Pharmaceutical Sciences, (18th ed., Mack Publishing Co., Easton, PA, 1990). Accordingly, these compounds can be used in the manufacture of medicines. Pharmaceutical compositions of these compounds can be prepared as solutions or lyophilized powders for parenteral administration. Before using such powders can be dissolved by adding a suitable diluent or another pharmaceutically acceptable carrier. Such powders can also be sprayed in a dry form. The liquid preparation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, a standard 5%solution of dextrose in water or buffered solution of sodium acetate or ammonium. This drug is particularly suitable for parenteral administration, but also can be used for oral administration or soda is to go on in the dosing egulatory or nebulizer injection. May be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydrocellulose, Arabian gum, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Alternatively, the compositions containing polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Can be added pharmaceutically acceptable solid or liquid carriers to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, gypsum, magnesium stearate or stearic acid, talc, pectin, Arabic gum, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline solution and water. For aqueous compositions used in vivo, it is preferable to use sterile, pyrogen-free water. Such preparations will contain an effective amount of polynucleotide together with a suitable amount of an aqueous solution in order to obtain pharmaceutically acceptable compositions suitable for administration to a mammal, preferably human. The carrier may also include a substance for Zam is Lenogo release such as glycerylmonostearate or glycerylmonostearate, one or wax. The amount of solid carrier will vary, but preferably will be from about 20 mg to about 1 g per unit dosage. Data pharmaceutical drugs do, following the traditional techniques of pharmacy involving milling, mixing, granulating and compressing, when necessary, for tablet forms; or milling, mixing and filling of hard gelatin capsule forms. When using a carrier liquid, the drug may be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspensions. For rectal injection of the compounds can be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols, and cast in suppositories.

Included the introduction of pharmaceutically acceptable salts, described in this invention polynucleotides. Such salts can be derived from pharmaceutically acceptable non-toxic bases include organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, basically the amino acids and the like. For a useful discussion of pharmaceutical salts, see S.M.Berge et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977).

Also proposed in this invention is a pharmaceutical composition for use in providing a polypeptide of the element of the cellular immune response of the mammal may include a pharmaceutically effective amount of polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, container, including the media and polynucleotide in sterile form, and means associated with the container to allow transfer of polynucleotide from the container into the interstitial space of the tissue, causing the cells of this tissue can absorb and Express polynucleotide. Means of providing such a transfer may include traditional septum that can be pierced, for example, by a needle. Alternatively, when the container is a syringe, we can assume that these funds include the piston of the syringe or needle attached to the syringe. To obtain one or more than one standard dose containers may contain at least 1, preferably at least 5 or 10, and more preferably at least 50 or 100 micrograms of polynucleotide. For many applications this container will contain at least 500 micrograms or 1 m is ligram, and often will contain at least 50 or 100 milligrams of polynucleotide.

Also proposed pharmaceutical composition, which may include polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, pharmaceutically acceptable input form in the container, and a notice associated with the container in form prescribed by the state Agency that regulates the manufacture, use or sale of pharmaceuticals, and the notice reflects approval by this body shape polynucleotide for the introduction of a human or animal. Such notice, for example, can be a label, approved by the U.S. food and medicines for prescription medicines or approved leaflet with information about the product.

In the present invention compositions can be introduced in the form of a pharmaceutical composition, where the composition is prepared in the form of a drug with a pharmaceutically acceptable carrier, as is well known in this field. Methods of manufacturing the drug and the introduction can be found, for example, in "Remington''s Pharmaceutical Sciences, (18th ed., Mack Publishing Co., Easton, PA, 1990). Accordingly, the composition can be used in the manufacture of medicines. The units is eveda, what pharmaceutically acceptable carrier or pharmaceutical composition, or any substance that is suitable for administration to a mammal, should be prepared and stored according to the standards of local ordinances. For example, many government organizations have guidelines or rules that regulate different aspects of manufacturing and treatment compositions, which are intended for the introduction of mammals and/or humans, such as sanitary control, process control, equipment and unity of documents, and personnel qualifications. Preferably, in the present invention compositions include pharmaceutical composition or pharmaceutically acceptable carrier suitable for administration to humans and comply with local regulations, guidelines and/or regulations GMP (good manufacturing practices), such as regulations set forth for such purpose by the United States to monitor the quality of food and medicines.

Polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment for injection, the preferred route of delivery, you can get in a standard dosage form in ampoules or in mnogochasovykh containers. These polynucleotide may be present in so the x forms as suspensions, solutions or emulsions in oily or preferably water fillers. Alternatively, polynucleotide in the form of a salt may be in a dried form for dissolution, at the time of delivery, a suitable filler, such as sterile pyrogen-free water. Both liquid and lyophilized forms, which are subject to dissolution, will contain the agents, preferably the buffers in the quantities necessary for appropriate adjustment of pH of the injection solution. For parenteral application, particularly if the drug is subject to intravenous total concentration of solutes should be monitored in order to make the drug isotonic, hypotonic or weakly hypertonic. To adjust toychest preferred are non-ionic substances such as sugar, and sucrose is particularly preferred. Any of these forms can optionally contain suitable agents for the manufacture of drugs, such as starch or sugar, glycerin, or saline. Song data, both liquid and solid, may contain a unit dosage from 0.1% to 99% polynucleotide substances.

Single dose ampoule or mnogorazovye containers in which polynucleotide Packed to the application may include a hermetically sealed container enclosing an amount of polynucleotide or solution, containing polynucleotide suitable for its pharmaceutically effective dose or a multiple of the number of effective doses. This polynucleotide Packed in a sterile preparation, and a hermetically sealed container is designed to preserve sterility of the drug to use.

The container that contains polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment may include packaging that is labeled, and the label may bear a notice in the form prescribed by public authority, for example, control over the quality of food and medicines, and this notice reflects approval by the public Agency, under Federal law, the manufacture, use, sale are in it polynucleotide substances for administration to humans.

Federal law requires the use of pharmaceutical compositions in human therapy was approved by the Federal government. In the United States, enforcement is the responsibility of the Department for control of food and medicine, which publishes instructions for providing such approval, detailed in §301-392 U.S.. 21. Instructions for the biological material, including products, the scientists from animal tissues, given under §262 U.S.. 42. Similar approval is required for most foreign countries. Instructions vary from country to country, but separate procedures well known to specialists in this field, and in the present invention compositions and methods accordingly preferably satisfy them.

Dosage, subject to introduction, to a large extent depends on the condition and size of the subject, which is treated, and also the frequency of treatment and route of administration. Scheme long-term therapy, including dose and frequency, can be determined on the initial response and clinical opinion. Is preferred parenteral route of injection into the interstitial space of tissues, although at a certain introduction may be required by other parenteral routes, such as inhalation aerosol drug, as, for example, on the mucous membranes of the nose, throat, bronchial tissues or lungs.

As such, the present invention is proposed pharmaceutical product, which may include polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, in solution in a pharmaceutically acceptable injectable carrier and suitable for interstitial injection into the tissue to cause expression of an element of the cellular immune response or its fragment is in the cells of the tissue, the container containing the solution, and a notice associated with the container in form prescribed by a governmental Agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects approval by the public Agency of manufacture, use, or sale solution polynucleotide for the introduction of man.

Introduction

In any of those disclosed in the invention methods, it is preferable that the composition containing polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, was delivered to the mammal. More preferably, the mammal is man. The introduction of the compositions according to any one of those disclosed in the invention methods can be performed according to any of various methods known in this field. For example, in U.S. patent No. 5676954 disclosed injection of genetic material in a complex with cationic lipid carriers mice. Also in U.S. patent No. 558966, 5693622, 5580859, 5703055 and in international patent PCT application PCT/US 94/06069 (WO 94/29469) proposed methods for delivering compositions containing naked DNA or DNA complexes with cationic lipids, vertebrate.

In some embodiments, the compound containing polynucleotide, including the sequence encoding the cell element is monogo response or its fragment, you can enter parenterally, for example, vnutrisosudisto, intravenously, intraarterially, intramuscularly, subcutaneously or the like. This connection can be entered in the muscle, skin, brain, lung tissue, liver tissue or spleen. The connection can also be entered in the blood. The introduction can also be oral, intranasal, rectal, dermal, or inhalation by aerosol. This composition can also be entered in the form of a bolus or make a slow infusion.

Polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment can be delivered to the interstitial space of tissues of the animal body, including muscle tissue, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of tissues contains intercellular fluid, mukopolisaharidnyh matrix among reticular fibers organ tissues, elastic fibers in the walls of blood vessels or cavities, the collagen fibers of fibrous tissues or the same matrix in connective tissue surrounding the muscle cells, or in the lacunae of bone. Similarly, it is not only the et a space occupied by the plasma in the circulatory system and the lymphatic fluid in the lymph channels. Shipping in the interstitial space of muscle tissue is preferred for reasons that are discussed below. They can be delivered by injection into tissue containing these cells. They are preferably delivered and expressed in a stable non-dividing cells, which are differentiated, although delivery and expression may be done in undifferentiated or less fully differentiated cells, such as, for example, stem cells of the blood or skin fibroblasts.

In vivo muscle cells are especially competent in their ability to absorb and Express polynucleotide. This ability may be due to the special architecture of muscle tissue containing multinucleated cells, sarcoplasmatic reticulum and transverse reticular system. In the present invention polynucleotide can flow into the muscles through a lateral reticular system, which contains extracellular fluid and extends deep into the muscle cell. It is also possible that polynucleotide penetrate into damaged muscle cells, which are then restored.

Muscle is also mainly used as the site for delivery and expression polynuclear the species in many therapeutic applications, because animals have proportionally more muscle mass, which is conveniently accessed by direct injection through the skin; for this reason, a relatively large dose of polynucleotides can be introduced into the muscle through multiple injection and re-injection to continue therapy for extended periods of time are easy to perform and can be carried out safely and without special training or device.

Tissues other than muscle, also can be used as sites for injection to obtain the elements of the cellular immune response. One such condition is the use of polynucleotide to obtain a polypeptide that, in order to be effective, must be present in Association with cells of a particular type; for example, with receptors on the cell surface of liver cells associated with cholesterol homeostasis (Brown and Goldstein, Science 232: 34-47 (1986)). In this application, and in many others, such as applications in which the enzyme or hormone are the product of the gene may not be the need to achieve high levels of expression in order to provoke a valuable therapeutic result.

In some embodiments polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment is introduced into cloth is using one injectable carrier. This carrier is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, for example, provide a sucrose solution. This drug may further preferably contain the source of the cytokine, which is included in liposomes in the form of a polypeptide or polynucleotide.

Compounds containing polynucleotide, including the sequence encoding the element of the cellular immune response or its fragment can be prepared in such a way as to include other useful medically drugs or biological agents. These compounds can also be entered in conjunction with the introduction of other drugs or biological agents useful for the treatment of a disease or condition against which aims described in this invention compounds (see, for example, U.S. patent No. 6413955 in respect of active ingredients that are useful for the treatment of osteoporosis).

Compounds containing polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, you can also type in a tissue or in cells via gene gun". DNA can be applied to microparticles of gold and can be delivered intradermally by setting for bombarding particles, or "genetically the gun", as described in the literature (see, for example, Tang et al. (1992), Nature 356: 152-154), where the bombarding particles of gold cover therapeutic DNA, then bombarded the skin cells.

Compounds containing polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, you can also enter via intranasal or oral administration. Low-dose favor active suppression, whereas higher doses favor clonal anergy/deletion. Oral and/or intranasal antigen induces T-helper 2 (Th2) T cells, such as IL-4 and IL-10, and T-helper 3 (Th3) T cells, such as TGF-β, plus CD4+ CD25+ regulatory cells and T-cells with latent associated peptide (latency-associated peptide). Thus, induction of oral tolerance can be enhanced by the introduction of IL-4, IL-10, anti-IL-12, TGF-β, A subunit of cholera toxoid, Flt-3 ligand and anti-CD40 ligand.

Oral and intranasal introduction of antigen (e.g mucosal tolerance) suppresses animal models of autoimmune diseases, including experimental autoimmune encephalitis, uveitis, thyroiditis, myasthenia gravis, arthritis and diabetes in diabetic mice, non-obese (NOD), plus pautakan diseases such as asthma, atherosclerosis, graft rejection, allergic (Taka is as contact sensitivity to dinitrochlorobenzene (DNCB) and allergic to Nickel), colitis, stroke and models of Alzheimer's disease (McGeer P. et al., 42 Neurology 447-449 (1992); Okuray Y., 103(25) PNAS USA 9619-24 (Epub June 12, 2006); Qu C. et al., 244(1-2) J. Neurol. Sci. 151-158 (2006)). Mucosal tolerance may be useful for treatment of neurodegenerative diseases such as Alzheimer's disease, due to the ease of administration, lack of toxicity and antigen-specific mechanisms of action.

Mucosal tolerance is an attractive approach for the treatment of neurodegenerative diseases due to the lack of toxicity, ease of injection for a period of time and antigen-specific mechanisms of action. The successful use of oral tolerance for the treatment of human diseases will depend on the dose, the development of immune markers to assess the immunological effects route (intranasal versus oral), drug, mucosal adjuvants, combination therapy and early treatment.

The phrase "effective amount" as used in this invention, refers to a dose sufficient to provide a sufficiently high concentrations to provide useful effect on its recipient. The specific level of therapeutically effective dose for any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound, the route of administration, rate of excretion of the compound, the duration of treatment, drugs used in combination or simultaneously with the compound, the age, body weight, sex, diet and General health of the subject, and like factors well known in the data fields of science and medicine. Various General considerations taken into account when determining the "therapeutically effective amount", well-known experts in this field and are described, for example, Oilman et al., eds., Goodman And Gilman''s: The Pharmacological Bases of Therapeutics, 8th ed., Mack Publishing Co., Easton, Pa., 1990.

Adjuvants

To deliver polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, system of a mammal, it is generally preferable to use the delivery system. Such a system can provide many advantages, including providing stabilization to protect the integrity of DNA, as well as facilitating the absorption cell.

In addition, as illustrated by typical delivery systems described in this invention (i.e. transfectional reagents), non-DNA components of this drug may also increase or activation of the immune system. As a result, to enhance or minimize immunostimulating effect, you can select the components of the delivery system in combination with a specific g the authorized product.

Immunostimulatory effects are also described for specific nucleotide sequences. For example, Sato et al., Science 273: 352-354 (1996) describes the effects of vaccination double-stranded DNA having a specific sequence containing CpG, production of interferon-γ, interferon-β and interleukin-12.

Transfection reagents

Compositions comprising polynucleotide, including the sequence encoding the element of the cellular immune response, or its fragment, as proposed in this invention can also include one or more substance to facilitate transfection, which facilitate the delivery of polynucleotides into the cell and/or to the desired location in the cell. Many such substances, to facilitate transfection, are commercially available, for example, Lipofectin, Lipofectamine, Lipofectamine 2000, Optifect, Superfect. Examples of substances that facilitate the transfection include, but are not limited to, lipids, preferably cationic lipids; inorganic substances such as calcium phosphate and metal particles (e.g., gold or tungsten) (for example, the solutions for the delivery of "powder" type); peptides, including cationic peptides, targeting peptides for selective delivery to specific cells or intracellular organelles such as the nucleus or nucleolus, and amphipatic peptides, i.e. spiralgalaxies or a pore-forming peptides; the basis of the major proteins, such as histones; sialoprotein; viral proteins (for example, the envelope protein of Sendai virus); pore-forming proteins; and polymers, including dendrimers, star polymers, "homogeneous" polyaminoamide (for example, polylysin, polyalanine), "heterogeneous" polyaminoamide (for example, a mixture of lysine and glycine), copolymers, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG). In addition, these auxiliary agents that facilitate and enhance the flow of polynucleotide in cells of the vertebrate in vivo, can also be viewed as "substances that facilitate the transfection".

The transfection, the lightweight lipofectin, well known in this field, as described, for example, in U.S. patent No. 6034072, 6040295 and 6710035. Some embodiments in the present invention compositions can include as substances that facilitate transfection, lipid, including cationic lipids (e.g. DOTMA, DMRIE, DOSPA, DC-Chol, GAP-DLRIE), essential lipids (such as stearylamine), neutral lipids (such as cholesterol), anionic lipids (e.g phosphatidylserine) and zwitterionic lipids (e.g DOPE, DOPC). Preferably, the cationic lipid is mixed with one or more than one zolipidem. In order to define the term "soliped" refers to any hydrophobic substance, which can be combined with the cationic lipid component, and includes aviationsafety, such as phospholipids, and neutral lipids, such as cholesterol. Cationic lipids and solidity can be mixed or combined in various ways to obtain many ecovalence related macroscopic structures, including, for example, liposomes, multi-layered vesicles, single-layer vesicles, micelles and simple film.

Delivery can also be accomplished through the application of DNA-transporters. The term "DNA-carriers" refers to molecules that bind DNA vectors and can be absorbed by epidermal cells. DNA transporters contain a molecular complex, capable ecovalence to bind to DNA and efficient transport of DNA through the cell membrane. DNA conveyor system may consist of particles containing several elements that are independently and ecovalence contact with DNA. Each element consists of a ligand that recognizes a specific receptor or other functional groups, such as protein in complex with a cationic group, which binds to DNA. Examples of cations that can be used are spermin, derivatives spermine, histone, cationic peptides and/or polylysine. The first element is able to communicate both with the DNA vector, and a cell surface receptor on the target cell. Examples of such elements are the PR is adicheskie connection which interact with the receptor asialoglycoprotein, receptor folate receptor mannose-6-phosphate or receptor carnitine. The second element is able to communicate both with the DNA vector, and receptors on the nuclear membrane. Nuclear ligand capable of recognizing and transporting conveyor system through the nuclear membrane. An example of such a ligand is a sequence targeting the core of the large T antigen or histone SV40. The third element is able to communicate both with the DNA vector, and the elements that induce epitomy lysis. Examples include inactivated viral particles, such as adenovirus, peptides, related to the hemagglutinin of influenza virus, or a peptide GALA.

EXAMPLE 1

The study of DNA vaccines in animal models.

To study the effects of DNA vaccination and Alzheimer's disease have used transgenic mice (Tg-2576)containing the double mutation K670N/M671L ARR. Mice were kept in a complex space for the animals at the University of Tennessee according to institutional guidelines for such research. These studies were approved by the Institutional Committee of the University of Tennessee for the care and use of animals in accordance with the policy of the Ministry of health for the care and use of laboratory animals. The results of the experimentation is and animal models provide evidence framework adoptive cellular gene therapy in other organisms, specifically in people.

In mice Tg-2576 in age from four to five months appear brain damage. After nine or ten months of the lesion is fully manifest. Vaccination, beginning around the age of about four months demonstrates the preventive effects of treatment against Alzheimer's disease, whereas vaccination, beginning after five months of age (i.e. from six months onwards) demonstrates the treatment of mice that have already developed Alzheimer's disease.

Four mice were treated DNA vaccine encoding IL-4, IL-10, TGF-β, IFN-γ, or introduced them to the control vector. Each mouse was given 100 μg of the vaccine. Mice were twice subjected to repeated immunization with a two-month intervals. Used both male and female mice Tg-2576, and kept them until 60 weeks of age. Mice were then killed and their brains were removed, quickly frozen in liquid nitrogen and kept at -80°C.

EXAMPLE 2

Assessment of memory

Spatial learning and memory was assessed using tasks for navigation in the water maze Morris water maze). Mice were trained in a round pool of opaque water to learn the location of the platform in order to get out of the water. The pool was located in the center of the container, having distinct distally located spatial orientation. All testing is opisywali on videotape camera, above (pool). Water tank (80 cm wide, 80 cm deep) was filled with water with a temperature of 23°C. a Hidden circular platform (15 cm wide) was placed at a depth of 1 cm below the water surface, and she served for the animal as saving platform. Maximum sailing time for each test was 90 seconds for the next 20-second rest on the platform. Each mouse was trained for five days, carrying four trials per day. Four randomized starting point for every day training. Tests on memorizing (test test) was performed in the absence of the platform within 30 minutes after the last test. Each animal was released from the space located in front of the planned quadrant and allowed him to swim for 60 seconds. After retraining on the 7th day, saving the platform is moved in the opposite quadrant, and procedure training for the changing skill held on 8-10th experimental day. Tests on memorizing for training on changing skill also conducted 30 minutes after the last test on learning. For each test were recorded latent period (the length of time taken by the mouse to reach the platform).

EXAMPLE 3

The results of the water maze for disease prevention

Alzhei the EPA

The results of the water maze eight-month-old mice that were vaccinated, since four months of age.

GroupIdentification number of the mouseThe latent period
Normal untreated mouse16 seconds
Mouse AP-2576 with the control vector12 minutes
Mouse AP-2576 IL-10 vector115 seconds
Mouse AP-2576 with TGF-β vector123 seconds

The results of the water maze nine-month-old mice that were vaccinated, since four months of age.

GroupIdentification number of the mouseThe latent period
Normal untreated mouse16 seconds
Mouse AP-2576 with the control vector2 minutes 40 seconds
252 seconds
Mouse AP-2576 IL-10 vector18 seconds
27 seconds
315 seconds
Mouse AP-2576 with TGF-β vector11 minute 20 seconds
21 minute 10 seconds
Mouse AP-2576 with IL-4110 seconds
vector
Mouse AP-2576 with IFN-γ vector125 seconds

These results indicate that the effects of Alzheimer's disease can be prevented and/or significantly reduced by early treatment with the use of DNA vaccines.

EXAMPLE 4

The results of the water maze for the treatment of Alzheimer's disease

The results of the water maze for mice at the age of seven and a half, nine and a half and eleven months, which vaktsinirovat and at the age of six, eight, ten and twelve months.

Experiment 1: Results mice at the age of seven and a half months

GroupIdentification number of the mouseThe latent period
Normal untreated mouse16 seconds
Mouse AP-2576 with the control vector12 minutes
Mouse AP-2576 with TGF-β vector123 seconds
Mouse AP-2576 c IL-10 vector115 seconds

Experiment 2: Results mice at the age of nine and a half months

GroupIdentification number of the mouseThe latent period
Mouse AP-2576 with the control vector12 minutes 24 seconds
252 seconds
Mouse AP-2576 with TGF-is a vector 11 minute 12 seconds
21 minute 6 seconds
Mouse AP-2576 with IFN-g125 seconds
vector
Mouse AP-2576 with IL-4 vector110 seconds
Mouse AP-2576 IL-10 vector18 seconds
27 seconds

Experiment 3: Results mice at the age of eleven months

GroupIdentification number of the mouseThe latent period
Mouse AP-2576 with the control vector12 minutes 24 seconds
Mouse AP-2576 mixed genes*113 seconds
Mouse AP-2576 with TGF-β vector11 minute 18 with the Kund
26 seconds
Mouse AP-2576 with IFN-γ vector14 seconds
Mouse AP-2576 with IL-4 vector110 seconds
Mouse AP-2576 IL-10 vector12 seconds
27 seconds
* Mixed genes include BDNF, IL-10, IL-4, APOE.

Because brain lesions are formed at the age of about four or five months, these mice have already developed Alzheimer's disease, and these results show that Alzheimer's disease can be cured and its effects are reduced or converted DNA vaccines encoding the element of the cellular immune response.

EXAMPLE 5

Histology of the brain

After anesthesia aventinum was performed by perfusion of mice through the ear with a solution of warm 0.1 M phosphate buffer (PBS), then warm buffered with 1.5%solution of paraformaldehyde in two minutes. Then followed the processing of the second latch containing cold 4%paraformaldehyde for 25 minutes. Animals were decapitated, exposing the brain and bones, and a whole head p which was gregali in a solution of neutral 10%formaldehyde and kept over night. After washing, PBS, the brain dehydrational ethanol and embedded in paraffin. Collected group of coronary slice thickness of 6 μm, starting from the frontal lobe to the cerebellum, and mounted in series with the use of the microtome. All sections were mounted and stained with standard hematoxylin and eosin (H&E), and studied the immunohistochemical staining of beta-amyloid for evidence of injury or neurodegeneration. All sections were examined on a light microscope (Carl Zeiss Axioskop 2 plus HAL 100 with the final magnification × 400). Digital images of sections were obtained using a camera (Leica)associated with the computer.

Protocol for staining with hematoxylin and eosin (H&E)

Place slides containing sections in paraffin, holder for slides (glass or metal).

To deparaffinizing and registrational slices, using:

3×3 min xylene (remove filter paper excess xylene before using ethanol) (StatLab #8400, lab-quality, brand Anapath, Lewisville, TX)

3×3 min 100% ethanol

1×3 min 95% ethanol

1×3 min 80% ethanol

1×5 min with deionized water

When the sections are in water, to remove the surface layer of hematoxylin Kimwipe to remove the oxidized particles. Remove the filter paper excess water from the holder slides before transfer to genetox the lean.

Staining with hematoxylin includes:

1×3 min hematoxylin (Poly Scientific #A, by hematoxylin Harris with glacial acetic acid, Bayshore, NY)

Rinse with deionized water

1×5 min tap water (to ensure the development of dyeing)

Dip 8-12 times (quickly) in acidified ethanol (for discoloration)

Wash 2×1 min tap water

Wash 1×2 min with deionized water (at this stage you can leave for the night).

Remove the filter paper excess water from the holder slides before transfer to eosin.

The eosin staining and dehydration:

1×30 seconds eosin (up to 45 seconds when using eosin from an older batch) (Poly Scientific#s176, Eosin Phloxine stain, Bayshore, NY)

3×5 min 95% ethanol

3×5 min 100% ethanol (to remove the filter paper excess ethanol before transfer to xylene)

2×15 min xylene

You can leave the slide in xylene at night to get a good cleaning from any amount of water.

Put a drop of Permount (based on xylene) (Fisher Scientific #SP15-100, histological enclosing environment) on a glass slide using a glass rod, making sure not to leave bubbles.

To set the angle of the cover glass and to give him gently down on a glass slide. To give Permount be distributed under a cover glass covering the Xu fabric.

To dry overnight in a fume hood.

The Protocol for immunohistochemical staining of beta-amyloid

Beta-amyloid is a deposition of extracellular filament protein found in the brain. He is a major protein component of amyloid cores and Narineh plaques and is also found in the form of deposits in neurofibrillary tangles. People have Alzheimer's disease is the most common cause of dementia and is characterized by abnormal deposits filament protein in the brain. Deposits of beta-amyloid are also determined in dementia with calves Levi, down syndrome, amyloidosis (Dutch type) and the complex of dementia with Parkinson's disease (GUAM). Brain tissue from these diseases, in addition to brain tissue from Alzheimer's disease, were used as positive controls.

Stage fixation: formalin fixed, paraffin slices.

Positive control: brain tissue disease Alzheimer, dementia with calves Levi, down syndrome, amyloidosis (Dutch type) and a complex of dementia with Parkinson's disease (GUAM).

Solutions and reagents:

Primary antibody:

Mouse against beta-amyloid (clone: 6F/3D) (Novocastra, Cat# NCL-B-Amyloid). The optimal dilution of 1:100. Species reactivity: human, mouse (for additional the Noi information, refer to the data table on the antibody).

Secondary antibody:

Horse against mouse IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-2000). The optimal dilution of 1:500.

Reagent for the determination of:

NRR(horseradish peroxidase-streptavidin (Vector Laboratories, Cat# SA-5004). The optimal dilution of 1:500.

Technique:

1. Paraffin sections are transferred into distilled water.

2. Demeterova antigen: use the method of damascenone antigen formic acid.

3. Rinse the sections in 2 changes of wash buffer for 2 minutes each time.

4. Blocking serum: incubate the sections with a blocking solution of normal horse serum for 30 minutes to block non-specific binding of immunoglobulin.

5. Primary antibody: incubate sections with mouse antibodies against beta-amyloid (Novocastra, Cat# NCL-B-Amyloid), diluted 1:100 in buffer for dilution of primary antibody for 1 hour at room temperature.

6. Rinse in wash buffer 2 times in 2 minutes

7. The peroxidase blocking: incubate sections in solution, blocking peroxidase for 10 minutes to block endogenous peroxidase activity.

8. Rinse in wash buffer 3 times for 2 minutes

9. Secondary antibody: incubate sections with biotinylating antibodies horses against mouse IgG, diluted in buffer for dilution of secondary antibodies, those which begins 30 minutes at room temperature.

10. Rinse in wash buffer 3 times for 2 minutes

11. Definition: incubate sections with HRP-streptavidin, diluted in buffer for dilution of HRP-streptavidin for 30 minutes at room temperature.

12. Rinse in wash buffer 3 times for 2 minutes

13. The Chromogen/substrate: incubate the sections in a solution of peroxidase substrate DAB (diaminobenzidine) for 5-10 minutes.

14. Quickly rinse in distilled water,

15. Contrast staining with hematoxylin solution of Gil (Gill) or a solution of hematoxylin Mayer (Mowag), if so desired.

16. Rinse in the current tap water for 5 minutes.

17. To degidratiruth by 95% ethanol for 2 minutes, 100% ethanol 2 times in 3 minutes

18. To lighten in xylene 2 times in 3 minutes

19. To cover with a cover glass with a permanent mount environment.

The results:

Painting staining: positive staining can be observed in the cores of senile plaques, on the periphery of the plaques and diffuse plaques. In some cases of Alzheimer's staining can be observed in the walls of blood vessels and extracellular neurofibrillary tangles.

Notes:

1. Related protocols: Tau (Tau-2) (Novocastra)

2. It may be necessary blocking avidin/Biotin to block the activity of endogenous Biotin for certain fabrics, so is x as (tissue) kidney, liver, prostate, colon and intestines, which may contain endogenous Biotin. For frozen tissues quickly freeze fresh tissue in isopentane pre-cooled in liquid nitrogen, pour in connection OST in triforma. Cut into 4-8 μm slices on Cryosat and mounted on slides Superfrost plus. Store slides at -80°C until they are needed. Before painting to dry slides at room temperature for 30 minutes and fixed in ice-cold acetone for 5 minutes. Air dried for another 30 minutes. Then start from step 3 for routine immunostaining.

EXAMPLE 6

The results of the water maze for disease prevention

Alzheimer's

The results of the water maze in mice at the age of sixteen months, which were vaccinated at the age of six, eight, ten, twelve and fourteen months.

GroupIdentification number of the mouseThe latent period
Normal untreated mouse16 seconds
Mouse AP-2576 with the control vector1 1 minute 2 seconds
Mouse AP-2576 IL-10 vector13 seconds
24 seconds
Mouse AP-2576 with TGF-β vector114 seconds
Mouse AP-2576 with IL-4 vector14 seconds
Mouse AP-2576 with IL-10 and IL-4 vector13 seconds
Mouse AP-2576 with IFN-γ vector124 seconds

The results of the water maze in mice at the age of thirteen months, which were vaccinated at the age of six, eight, ten and twelve months.

GroupIdentification number of the mouseThe latent period
Normal untreated mouse16 seconds
Mouse AP-257613 minutes 40 seconds
pin the Aulnay vector 21 minute 40 seconds
Mouse AP-2576 IL-10 vector11 second
21 second
38 seconds
Mouse AP-2576 with TGF-β vector11 minute 20 seconds
230 seconds
Mouse AP-2576 with IL-4 vector110 seconds
Mouse AP-2576 with IL-10 and IL-4 vector11 second
22 seconds
Mouse AP-2576 with IL-10 and TGF-β vector145 seconds
244 seconds
Mouse AP-2576 with β-amyloid vector148 seconds
22 minutes 35 seconds
Mouse AP-2576 β-AMILO denim and ARO-E vector 136 seconds
Mouse AP-2576 with β-amyloid and NGF vector119 seconds
236 seconds
Mouse AP-2576 with IFN-γ vector12 minutes 8 seconds
236 seconds

EXAMPLE 7

Histology of the brain in vaccination mixed genes

The brain tissue of mice in the hippocampus were processed as described above, an eighteen-month transgenic mice with protein-amyloid precursor (APP). amyloid precursor protein), which received only a vector with a DNA vaccine (Figa) or a mixture of DNA vaccines containing the genes for IL-4, IL-10, nerve growth factor, originating from the brain (NGF) and the APO-E2 (Figb). Treated mice were vaccinated intramuscularly at the age of six, eight, ten, twelve, fourteen and sixteen months. The dosage of each treatment mixed DNA vaccine was approximately 100 micrograms per injection for each of the four genes (a total of approximately 400 micrograms). Frozen brain tissue was stained with a fluorescently labeled antibody against the amyloid protein. In nearabout the Mr. mouse brain there are a large number of amyloid tissue (Figa) and in the murine brain, processed mixed DNA vaccine, there is significantly less amyloid proteins (Figb).

If not defined otherwise, all technical and scientific terms used in this invention, have the same meaning, which is usually clear to the ordinary specialist in the field that belongs to this invention.

The invention described illustrative in this invention can accordingly be used in practice in the absence of any element or elements, limitation or limitations, not specifically disclosed in this invention. Thus, for example, the terms "comprising", "including", "containing", etc. should be read in a General sense and without restrictions. Optionally used in this invention the terms and expressions have been used as terms of description and not of limitation, and the use of such terms and expressions, there is no intention to exclude any equivalents shown or described signs or portions thereof, but it is believed that within the scope of the claimed invention, various modifications are possible.

Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and features, modifications, improvements and modifications of the inventions embodied therein, disclosed in this invention which may be modified by experts in this field, and that such modifications, improvements and changes are considered as being within the scope of this invention. In the present invention, the materials, methods and examples are typical of the preferred embodiments, illustrative and are not intended to restrict the scope of the invention.

The invention described here is widely and in General. Each of the more narrow categories and subradular groups that fall within the General description, also form part of this invention. This includes a General description of the present invention with the proviso or negative limitation removing any subject of this kind, regardless of stated whether the invention specifically excluded material or not.

In addition, when the characteristics or aspects of the present invention is described in the context of Markush groups, experts in this field will understand that the invention thus also described in the context of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned in this invention, directly incorporated by reference in their entirety to the same extent as if each had been individually incorporated by reference. In case of conflict, the present description of the invention, including the determining, will be monitored.

Other embodiments set forth in the following claims.

1. Method for the treatment or relief of symptoms of Alzheimer's disease in a mammal, comprising an introduction to the specified mammal a composition containing one or more nucleic acids that induce cellular immune response, where the specified one or more nucleic acids encode one or more cytokines selected from the group consisting of IL-4 (interleukin-4), IL-10 (interleukin-10) and TGF-β (transforming growth factor beta).

2. The method according to claim 1, where the specified composition further comprises one or more nucleic acids selected from the group consisting of nucleic acids encoding APOE-2, nerve growth factor (NGF) and brain-derived neurotrophic factor brain (BDNF).

3. The method according to claim 1, further comprising introducing one or more nucleic acids encoding a polypeptide selected from the group consisting of NGF, BDNF and APOE-2 or their fragments.

4. The method according to claim 1, where the specified mammal is man.

5. The method according to claim 1, where the aforementioned introduction includes one or bluespoon, selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneally injection, subcutaneous injection and electroporation.

6. The method according to claim 1, where the aforementioned introduction includes one or more methods selected from the group consisting of injection, inhalation, and gene gun.

7. The method according to claim 1, where the specified polynucleotide contains circular DNA that contains the plot of the beginning of replication (ORI), a promoter and a multiple cloning site (MCS).

8. The method according to claim 1, where the specified polynucleotide is a plasmid that contains the promoter/enhancer, a transcription associated with the sequence that encodes a gene element of the cellular immune response.

9. The method of claim 8, where the specified promoter suitable for expression in eukaryotic cells.

10. The method according to claim 1, where the specified nucleic acid is administered together with a substance to facilitate transfection.

11. The method according to claim 1, where the specified polynucleotide delivered by adoptive cellular gene therapy.

12. The method according to claim 1, where the specified adoptive cellular gene therapy involves the introduction of cells capable of targeted delivery of nucleic acids to the site of inflammation.

13. The method according to claim 1, where this nucleic acid is a vector.

14. The method according to item 13, where the specified vector includes one or more, etc) the ditch, selected from the group consisting of pVAX1, pUMCV, pGCy and pLentilox-GFP.

15. The method according to claim 1, where the specified composition inhibits or reduces the expression of a gene, which increases inflammation.

16. The method according to claim 1, where the aforementioned introduction leads to reduced accumulation of amyloid plaques in the brain of the specified mammal.



 

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9 cl, 5 dwg, 1 tbl, 10 ex

The invention relates to organic chemistry, in particular to new compounds 2-hydroxy-2-methyl-N-(4-X-3-(trifluoromethyl)phenyl)-3-(perforazione)propionamide, in which X is nitro, cyano or halogen, and perforazione contains 2-3 carbon atoms and 0-1 hydrogen atom

The invention relates to a new composition has a curing effect in alopecia, hirsutism in women's type, or seborrhea, or having a preventive effect against metastases in bones caused by prostate cancer, containing N-[1-methyl-1-(4-methoxyphenyl)ethyl]-3-oxo-4-Aza - 5-androst-EN-17-carboxamide, or its pharmaceutically acceptable salt or pharmaceutically acceptable derivative

The invention relates to substituted phenylimidazoline, method of production thereof and to pharmaceutical compositions based on them

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Claimed invention relates to method and composition for treatment of central nervous system (CNS) disorder by intranasal application of compounds, which induce RNA interference. Composition by the invention contains molecules of short interfering nucleic acids (siPHK). Disorder is selected from dementia, Alzheimer's disease, Huntington's disease and/or Parkinson's disease, as well as congenital diseases associated with mutations in CNS genes. According to the invention method includes introduction to patient in case of necessity of efficient amount of one or more siPHK in accordance with the invention.

EFFECT: invention ensures suppression of expression of target genes, connected with changed CNS states.

20 cl, 4 ex, 8 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to gastroenterology, and can be used for treatment of intestinal diseases. For this purpose applied is intrarectal introduction of compounds which cause RNA interference. In addition, claimed are RNKi compounds with specified nucleotide sequence, causing RNA-interference and intended for intrarectal introduction, whose target is interleukin-12 gene. Claimed compounds can also be used for treatment of intestinal diseases.

EFFECT: inventions ensure efficient disease treatment due to efficient reaching and impact in action targets.

43 cl, 1 tbl, 5 ex, 9 dwg

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and deals with anti-sense oligonucleotides for treatment and/or prevention of, at least, one of the following diseases: asthma, hypereosinophilia. Essence of the invention includes oligonucleotides aimed against sequences of nucleic acids, coding receptor, selected from group, which consists of receptor CCR3 and common subunit of receptors IL-3, IL-5 and GM-CSF with sequences SEQ ID NO:1 and SEQ ID NO:14.

EFFECT: reduction of toxicity in comparison with hormonal medications.

39 cl, 8 ex, 11 tbl, 21 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. Offered is a vaccine composition containing immune-stimulating oligodeoxynucleic acid containing deoxyinosine residues (I - ODN) and antigens.

EFFECT: vaccine compositions elicit a more effective immune response in comparison with CpG sequence ODN and reduce induction of adverse reactions.

13 cl, 9 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to polymer conjugates of formula (I) comprising nucleotide or oligonucleotide residue, which can be applied for treatment of cancer and methods for their obtaining. where R1 and R2 independently represent H or polyalkylenoxyde, not necessarily having capping group selected from OH, NH2, SH, CO2N, C1-6 alkyls, compounds of formula (II) X2-(L2)n-(L1)o- and the formula (III) -(L4)p-(L3)m-X3, and when (o+n)≥2 each of n and o is a positive integer, each p and m are equal to zero, and R2 represents H, and when (p+m)≥2 each p and m is a positive integer, each n and o are equal to zero and R1 represents H; X1, X2, X3 are independently selected from a single-stranded or double-stranded oligonucleotide residue; L1 and L4 independently represent released link fragments; L2 and L3 are independently selected from bifunctional spacer groups.

EFFECT: developing of new nucleotide conjugates with antitumor activity.

21 cl, 25 ex, 7 tbl, 12 dwg

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to immunostimulating agents representing oligodeoxynucleotides of the formula (I): wherein any X represents oxygen (O) or sulfur (S) atom and wherein NMP represents deoxynucleoside monophosphate or monothiophosphate chosen from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxy-inosine-, deoxycytidine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytidine-, deoxypseudouridine-, deoxyribosepurine-, 2-aminodeoxyribosepurine-, 6-S-deoxyguanosine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine monophosphate or monothiophosphate; NUC represents 2'-deoxynucleoside chosen from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytidine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytidine-, deoxypseudouridine, -deoxyribosepurine-, 2-aminodeoxyribosepurine-, 6-S-deoxyguanosine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine; a and b are whole numbers from 0 to 100 under condition that a + b = 4-150; B and E are groups that are usual for 5'- and 3'-end of these molecules. Also, invention relates to immunostimulating pharmaceutical compositions containing indicated an immunostimulating agent or indicated an immunostimulating agent and antigen.

EFFECT: valuable medicinal property of oligodeoxynucleotides.

15 cl, 9 dwg, 6 ex

The invention relates to new methods for the production of medicines and new ways to treat infections of hepatitis b virus

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to immunostimulating agents representing oligodeoxynucleotides of the formula (I): wherein any X represents oxygen (O) or sulfur (S) atom and wherein NMP represents deoxynucleoside monophosphate or monothiophosphate chosen from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxy-inosine-, deoxycytidine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytidine-, deoxypseudouridine-, deoxyribosepurine-, 2-aminodeoxyribosepurine-, 6-S-deoxyguanosine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine monophosphate or monothiophosphate; NUC represents 2'-deoxynucleoside chosen from the group consisting of deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytidine-, deoxyuridine-, deoxythymidine-, 2-methyldeoxyinosine-, 5-methyldeoxycytidine-, deoxypseudouridine, -deoxyribosepurine-, 2-aminodeoxyribosepurine-, 6-S-deoxyguanosine-, 2-dimethyldeoxyguanosine- or N-isopentenyldeoxyadenosine; a and b are whole numbers from 0 to 100 under condition that a + b = 4-150; B and E are groups that are usual for 5'- and 3'-end of these molecules. Also, invention relates to immunostimulating pharmaceutical compositions containing indicated an immunostimulating agent or indicated an immunostimulating agent and antigen.

EFFECT: valuable medicinal property of oligodeoxynucleotides.

15 cl, 9 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to polymer conjugates of formula (I) comprising nucleotide or oligonucleotide residue, which can be applied for treatment of cancer and methods for their obtaining. where R1 and R2 independently represent H or polyalkylenoxyde, not necessarily having capping group selected from OH, NH2, SH, CO2N, C1-6 alkyls, compounds of formula (II) X2-(L2)n-(L1)o- and the formula (III) -(L4)p-(L3)m-X3, and when (o+n)≥2 each of n and o is a positive integer, each p and m are equal to zero, and R2 represents H, and when (p+m)≥2 each p and m is a positive integer, each n and o are equal to zero and R1 represents H; X1, X2, X3 are independently selected from a single-stranded or double-stranded oligonucleotide residue; L1 and L4 independently represent released link fragments; L2 and L3 are independently selected from bifunctional spacer groups.

EFFECT: developing of new nucleotide conjugates with antitumor activity.

21 cl, 25 ex, 7 tbl, 12 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. Offered is a vaccine composition containing immune-stimulating oligodeoxynucleic acid containing deoxyinosine residues (I - ODN) and antigens.

EFFECT: vaccine compositions elicit a more effective immune response in comparison with CpG sequence ODN and reduce induction of adverse reactions.

13 cl, 9 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and deals with anti-sense oligonucleotides for treatment and/or prevention of, at least, one of the following diseases: asthma, hypereosinophilia. Essence of the invention includes oligonucleotides aimed against sequences of nucleic acids, coding receptor, selected from group, which consists of receptor CCR3 and common subunit of receptors IL-3, IL-5 and GM-CSF with sequences SEQ ID NO:1 and SEQ ID NO:14.

EFFECT: reduction of toxicity in comparison with hormonal medications.

39 cl, 8 ex, 11 tbl, 21 dwg

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