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Plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to hivRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to hiv description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070190031, Plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to hiv. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] This invention relates to plasmids, immunogenic compositions and methods to improve prophylactic and therapeutic immune responses to antigens. BACKGROUND OF THE INVENTION [0002] Immunization using plasmid DNA-based immunogenic compositions is a powerful tool that is useful for developing approaches to prevent or treat infectious diseases or in the treatment of ongoing disease processes. Plasmid DNA immunization has been extensively tested in animal models where it has been found to be effective in inducing both cellular and humoral immune responses against a wide variety of infectious agents and tumor antigens. See Donnelly J J, et al., Ann. Rev. Immunol.; 15: 617-48 (1997); Iwasaki A, et al., J Immunol 158 (10): 4591-601 (1997); Wayne, C. L. and Bennett M., Crit. Rev. Immunol., 18: 449-484 (1998). [0003] An important advantage of plasmid DNA immunization is that genes can be cloned, modified and positioned into a potential plasmid DNA expression vector in such a way as to allow for relevant post-transcriptional modifications, expression levels, appropriate intracellular trafficking and antigen presentation. Plasmid DNA vectors useful for DNA immunization are similar to those employed for delivery of reporter or therapeutic genes. Plasmid DNA-based immunization uses the subject's cellular machinery to generate the foreign protein and stimulates the subject's immune system to mount an immune response to the protein antigen. Such plasmid DNA vectors generally contain eukaryotic transcriptional regulatory elements that are strong viral promoter/enhancer elements to direct high levels of gene expression in a wide host cell range and a polyadenylation sequence to ensure appropriate termination of the expressed mRNA. While, viral regulatory elements are advantageous for use in plasmid DNA vectors, the use of unmodified viral vectors to express the relevant genes may raise safety and technical issues not encountered with plasmid DNA. [0004] Current plasmid DNA designs, however, limit the expression of multiple genes from one vector backbone in a single target cell. Therefore, to transfer and express multiple genes, co-transfection of the target cells with separate plasmids is required. When cells must be co-transfected with multiple plasmids, it is difficult to achieve optimal expression of all encoded genes, especially when the plasmid is being used in vivo. Previous attempts to overcome these limitations and express two or more genes include the use of the following: viral vectors, multiple alternatively spliced transcripts from proviral DNA, fusion of genes, bicistronic vectors containing IRES sequences (Internal ribosome entry site) from viruses and dual expression plasmids. See Conry R. M. et al., Gene Therapy. 3(1):67-74, (1996); Chen T T. et al., Journal of Immunology. 153(10):4775-87, (1994); Ayyavoo V. et al., AIDS. 14(1):1-9, (2000); Amara R. R. et al., Vaccine. 20(15):1949-55, (2002); Singh G, et al.,. Vaccine 20: 1400-1411 (2002). [0005] None of the existing plasmid designs have solved the problem of providing a DNA plasmid suitable for expressing more than two independent open reading frames in human immunogenic compositions. In the case of bicistronic vectors, in many instances, only the first gene transcribed upstream of the IRES is expressed strongly from either a plasmid or a retroviral vector. See Sugimoto Y., et al., Hum. Gen. Ther. 6: 905-915 (1995); Mizoguchi H, et al., Mol. Ther. 1:376-382 (2000). Dual expression cassettes on the other hand have performed better. For example, it was found that co-delivery of cDNA for B7-1 and human carcinoembryonic antigen (CEA) with a single plasmid having two independent cassettes resulted in far superior immune responses, when compared to separate plasmids. See Conry R. M. et al., Gene Therapy. 3(1):67-74, (1996). However, in this case the two independent cassettes involved both consisted of homologous HCMV promoter and bovine growth hormone (BGH) poly-adenylation sequences. The presence of homologous sequences within a plasmid renders that plasmid unsuitable for use in DNA immunogenic compositions, because the presence of homologous sequences within the plasmid backbone increases the possibility of recombination between the repeated sequences and results in vector instability. [0006] Another constraint one confronts when designing a plasmid DNA vector for use in a human immunogenic composition involves size and organization of the plasmid. As transcriptional units are added to a plasmid, interference between transcriptional units can arise, for example in the form of steric hindrance. The cell's RNA transcription complex must be able to bind to the multiple sites on a polytranscriptional unit plasmid, uncoil the DNA and effectively transcribe the genes. Simply making the plasmid bigger is not necessarily the best solution for several reasons including plasmid instability, difficulty in plasmid manufacture and, most importantly, dosing considerations. To design an improved plasmid DNA multiple transcriptional unit vector, one must consider placement of genes, spacing and direction of transcription of open reading frames, level of expression, ease of manufacture, safety and the ultimate dose of the vector necessary to immunize the subject. [0007] Therefore, there remains a need for innovative plasmid DNA, non-viral vector designs for use in expressing multiple proteins from complex pathogens like HIV, where a broad immune response to many proteins is required. In addition, a need exists for polyvalent DNA-based immunogenic compositions that can direct expression of high levels of multiple HIV genes within a single cell. SUMMARY OF THE INVENTION [0008] In one embodiment, the present invention provides a DNA plasmid comprising: (a) a first transcriptional unit comprising a nucleotide sequence that encodes a first polypeptide operably linked to regulatory elements including a first promoter and a first polyadenylation signal; (b) a second transcriptional unit comprising a nucleotide sequence that encodes a second polypeptide operably linked to regulatory elements including a second promoter and a second polyadenylation signal; (c) a third transcriptional unit comprising a nucleotide sequence that encodes a third polypeptide operably linked to regulatory elements including a third promoter and a third polyadenylation signal; wherein said first, said second and said third promoters are each derived from different transcriptional units; and wherein said first, said second and said third polyadenylation signals are each derived from different transcriptional units. In another embodiment of the invention, the first, second and third polypeptides are expressed in a eukaryotic cell. [0009] In another embodiment, the present invention provides an immunogenic composition for inducing an immune response to selected antigens in a vertebrate host, the immunogenic composition comprising: (a) a DNA plasmid comprising a (i) a first transcriptional unit comprising a nucleotide sequence that encodes a first polypeptide operably linked to regulatory elements including a first promoter and a first polyadenylation signal; (ii) a second transcriptional unit comprising a nucleotide sequence that encodes a second polypeptide operably linked to regulatory elements including a second promoter and a second polyadenylation signal; (iii) a third transcriptional unit comprising a nucleotide sequence that encodes a third polypeptide operably linked to regulatory elements including a third promoter and a third polyadenylation signal; wherein the first, second and third promoters are each derived from different transcriptional units; wherein said first, second and third polyadenylation signals are each derived from different transcriptional units; and (b) at least one of a pharmaceutically acceptable diluent, adjuvant, carrier or transfection facilitating agent. In a particular embodiment of the invention, the transfection facilitating agent is bupivacaine. In another embodiment of the invention, the first, second and third polypeptides are expressed in a eukaryotic cell. [0010] In certain embodiments of the invention, the immunogenic composition is administered to a mammal using in vivo electroporation. In a particular embodiment, electroporation involves electrically stimulating the muscle with an electrical current having a field strength in the range of from about 25 V/cm to about 800 V/cm. [0011] In still another embodiment, the present invention provides a method of immunizing a vertebrate host against selected antigens comprising administering to the vertebrate host an immunogenic composition comprising: (a) a DNA plasmid comprising a (i) a first transcriptional unit comprising a nucleotide sequence that encodes a first polypeptide operably linked to regulatory elements including a first promoter and a first polyadenylation signal; (ii) a second transcriptional unit comprising a nucleotide sequence that encodes a second polypeptide operably linked to regulatory elements including a second promoter and a second polyadenylation signal; (iii) a third transcriptional unit comprising a nucleotide sequence that encodes a third polypeptide operably linked to regulatory elements including a third promoter and a third polyadenylation signal; wherein said first, second and third promoters are each derived from different transcriptional units; wherein the first, second and third polyadenylation signals are each derived from different transcriptional units; and (b) at least one of a pharmaceutically acceptable diluent, adjuvant, carrier or transfection facilitating agent. In another embodiment of the invention, the first, second and third polypeptides are expressed in a eukaryotic cell. [0012] In another embodiment of the invention, the selected antigens are derived from the group consisting of a bacterium, a virus, an allergen and a tumor. In a particular embodiment, the selected antigens are viral antigens derived from a virus selected from the group consisting of Human immunodeficiency virus, Simian immunodeficiency virus, Respiratory syncytial virus, Parainfluenza virus type 1, Parainfluenza virus type 2, Parainfluenza virus type 3, Influenza virus, Herpes simplex virus, Human cytomegalovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human papillomavirus, Poliovirus, rotavirus and coronavirus (SARS). [0013] In still another embodiment of the invention, the selected antigens are bacterial antigens derived from a bacterium selected from the group consisting of Haemophilus influenzae (both typable and nontypable), Haemophilus somnus, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetella pertussis, Alloiococcus otiditis, Salmonella typhi, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium tetani, Leptospira interrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum. [0014] In one embodiment of the invention, the vertebrate host is selected from the group consisting of mammals, birds and fish. In a certain embodiment of the invention, the vertebrate host is a mammal selected from the group consisting human, bovine, ovine, porcine, equine, canine and feline species. [0015] In one embodiment of the invention, the first, second and third promoters are active in eukaryotic cells. In other embodiments of the invention, the first, second and third promoters are selected from the group consisting of human cytomegalovirus (HCMV) immediate early promoter, the simian cytomegalovirus (SCMV) promoter, the murine cytomegalovirus (MCMV) promoter, the herpes simplex virus (HSV) latency-associated promoter-1 (LAP1), Simian virus 40 promoter, human elongation factor 1 alpha promoter, and the human muscle cell specific desmin promoter. [0016] In certain embodiments of the invention, the first, second and third polyadenylation signals are selected from the group consisting of rabbit beta-globin poly(A) signal, synthetic polyA, HSV Thymidine kinase poly A, Human alpha globin poly A, SV40 poly A, human beta globin poly A, polyomavirus poly A, and Bovine growth hormone poly A. [0017] In a particular embodiment of the invention, the first transcriptional unit expresses a gag-pol fusion protein from a fusion of the gag and pol genes of HIV. In one embodiment of the invention, the fusion of the gag and pol genes of HIV or gag-pol gene is derived from the HXB2 isolate of HIV. [0018] In a certain embodiment of the invention, the second transcriptional unit expresses an envelope protein from the envelope gene of HIV. In a particular embodiment of the invention, the envelope gene is derived from a primary isolate 6101 of HIV. [0019] In a specific embodiment of the invention, the third transcriptional unit expresses a nef, tat, and vif (NTV) fusion protein from a fusion of the nef, tat, and vif (ntv) genes of HIV. In a particular embodiment of the invention, the fusion of the nef, tat, and vif genes of HIV or ntv gene is derived from the NL4-3 isolate of HIV. [0020] In a specific embodiment of the invention, in a three transcriptional unit plasmid, the direction of transcription for the first transcriptional unit is in the opposite direction from the direction of transcription of the second transcriptional unit. In another embodiment of the invention, the direction of transcription for first transcriptional unit is in the opposite direction from the direction of transcription of the third transcriptional unit. [0021] In a certain embodiment of the invention, the invention provides a three transcriptional unit plasmid, which further comprises a nucleotide sequence that encodes a selectable marker operably linked to regulatory elements including a promoter and a polyadenylation signal. In one embodiment, the selectable marker is selected from the group consisting of kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene, hygromycin resistance gene and chloroamphenicol resistance gene. In another embodiment, the location of the selectable marker is selected from the group consisting of spacer region 1, spacer region 2 and spacer region 3. In a specific embodiment, the location of the selectable marker is spacer region 2. Continue reading about Plasmid having three complete transcriptional units and immunogenic compositions for inducing an immune response to hiv... 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