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Novel expression vectors and uses thereof

A technology of expressing vectors and vectors, applied in the field of new vectors, can solve the problems of reducing efficacy, human genome danger, etc.

Inactive Publication Date: 2004-09-15
FIT BIOTECH OY PLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These vectors also express E1 and E2 proteins other than the gene of interest, and contain sequences such as the rabbit β-globin sequence, which is partially homologous to the human sequence, causing a serious risk of integration into the human genome, which reduces the potential of these vectors as Efficacy of DNA Vaccines

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  • Novel expression vectors and uses thereof
  • Novel expression vectors and uses thereof
  • Novel expression vectors and uses thereof

Examples

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Effect test

example 2

[0263] Cloning and analysis of expression properties of plasmids in series 1 and NNV

[0264] Further modifications were made in these vectors in order to increase the copy number of the vectors super6 and super6wt in E. coli. The Tn903 kanamycin resistance gene, pMB1 replicon and ten E2 binding sites were removed by HindIII / Nhe1 digestion and then replaced with the HindIII / Nhe1 fragment of the retroviral vector pBabe Neo [Morgenstern, J.P. and Land, H. ., Nucteic Acids Research 18(1990) 3587-3596]. This fragment contains a modified pMB1 replicon and the Tn5 kanamycin resistance gene, allowing loose high copy number replication of the plasmid in bacteria. The new plasmids were named Product 1 (Figure 5) and Product 1wt, respectively. Reinsertion of the ten E2 binding sites back into the blunt Nhe1 site upstream of the CMV promoter in product 1 resulted in unsuccessful results in the generation of the new vector NNV, respectively, with only two binding sites integrated into t...

example 3

[0269] Analysis of expression characteristics of NNV-2wt

[0270] In order to analyze the expression characteristics of NNV-2wt, four different cell lines, namely Jurkat (human T cell lymphoblastoid cells), P815 (mouse mast cell tumor cells), CHO (Chinese hamster ovary cells) lines and RD (human embryonic striated muscle sarcoma cells), were transfected by electroporation, and their expression of Nef and E2 was analyzed. To demonstrate the transcriptional activation and maintenance properties mediated by E2 proteins and E2 oligomerization binding sites, the product 1wt lacking E2 binding sites (Figure 5) was used as a control. An additional control plasmid is the plasmid NNV-2wtFS, which differs from NNV-2wt by an introduced frameshift in the E2 coding sequence, so it does not express functional E2 protein.

[0271] Each cell line was essentially transfected by electroporation as described in Example 1 with different amounts of vector DNA. Time points were determined approxi...

example 7

[0299] Analysis of NNV-2wt replication in the presence of human papillomavirus replication factors

[0300]Papillomavirus proteins have previously been shown to initiate replication of heterologous ori-containing plasmids from many other human and animal papillomaviruses [Chiang, C.M. et al., Proc Natl Acad Sci USA 89 (1992) 5799-5803]. Although NNV-2wt does not contain a complete origin of replication, it was detected how replication is initiated in the presence of HPV type 1 E1 and E2 proteins. CHO cells were treated with 1 mg vector NNV-2wt, NNV-2wtFS or product 1 alone or with 4.5 μg HPV-11 E1 expression vector pMT / E1 HPV11 or with the same amount of pMT / E1 HPV11 and 4.5 μg HPV-11 E2 protein expression vector pMT / E2 HPV11 were transfected together, as shown in the upper part of Figure 15. Transfection was performed essentially as described in Example 1. El and E2 expression vectors have been described previously (Chiang, C.M. et al., supra). An equimolar amount of the ...

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Abstract

The present invention relates to novel vectors, to DNA vaccines and gene therapeutics containing said vectors, to methods for the preparation of the vectors and DNA vaccines and gene therapeutics containing the vectors, and to therapeutic uses of said vectors. More specifically, the present invention relates to novel vectors comprising (a) an expression cassette of a gene of a nuclear-anchoring protein, which contains (i) a DNA binding domain capable of binding to a specific DNA sequence and (ii) a functional domain capable of binding to a nuclear component and (b) a multimerized DNA sequence forming a binding site for the anchoring protein, and optionally (c) one or more expression cassettes of a DNA sequence of interest. In particular the invention relates to vectors that lack a papilloma virus origin of replication. The nuclear-anchoring protein might be the E2 protein of Bovine Papilloma Virus type 1 or Epstein-Barr Virus Nuclear Antigen 1. The invention also relates to vectors that lack an origin of replication functional in a mammalian cell. The invention further relates to methods for expressing a DNA sequence of interest in a subject.

Description

1. Field of Invention [0001] The present invention relates to a new carrier, a DNA vaccine and gene therapy containing the carrier, a method for preparing the carrier and a DNA vaccine and gene therapy containing the carrier, and the therapeutic application of the carrier. More particularly, the present invention relates to novel vectors comprising (a) expression cassettes for nuclear-anchored proteins containing (i) DNA binding domains capable of binding to specific DNA sequences and (ii) capable of binding to nuclear components and (b) a multimerized DNA sequence forming an ankyrin binding site of a nuclear-ankyrin, and optionally (c) one or more expression cassettes for a DNA sequence of interest. In particular, the invention relates to vectors lacking a papillomavirus origin of replication. The invention also relates to vectors lacking an origin of replication functional in mammalian cells. The invention further relates to methods of expressing a DNA sequence of interest...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K35/76A61K35/763A61K39/12A61K48/00A61P31/00A61P35/00C12N1/21C12N5/10C12N15/85
CPCC12N2830/42C12N2840/203C12N2800/108A61K2039/53C12N2840/20A61K48/00C12N15/85A61P31/00A61P31/12A61P31/18A61P35/00
Inventor K·克龙V·布拉热维奇M·塔蒂南M·阿斯特弗U·托茨A·麦尼克A·兰基E·阿斯特弗
Owner FIT BIOTECH OY PLC
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