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Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof

A technology of sequences and vectors, applied in the field of design and development of new systems, which can solve problems such as low activity levels

Inactive Publication Date: 2004-03-17
GENCELL SA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the level of activity obtained was still extremely low and it was therefore found necessary to isolate the promoter sequence located between the two inverted terminal repeats (AAV-ITR) of the adeno-associated virus in order to examine the activity in vivo

Method used

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  • Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof
  • Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof
  • Heart ankyrin repeat protein gene upstream sequence, carrier containing the same sequence and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Characterization of upstream polynucleotides of CARP gene

[0087] The 2.3Kb BamHI-XhoI fragment of the 5' sequence of the mouse gene encoding the CARP protein was cloned by the chain termination method (Sanger et al., 1977, PNAS, 74, 5463) using the Sequenase Kit (United States Biochemical, Cleveland, Ohio) Both strands are sequenced. The sequence is shown in Figure 1 and thus contains between nucleotides -2266 and +92 (relative to the transcription site +1) a part upstream of the gene encoding the mouse CARP protein (SEQ ID NO: 1).

Embodiment 2

[0088] Embodiment 2: Construction of CARP plasmid vector

[0089] 2.1 Plasmid pXL3634

[0090] The 2.3 Kb BamHI-XhoI fragment characterized in Example 1 was cloned into plasmid pGL3-Basic (Promega) previously digested with XhoI and SmaI after filling in the BamHI site to obtain plasmid pXL3634. Figure 3 shows a schematic representation of the plasmid.

[0091] 2.2 Plasmid pXL3728

[0092] Plasmid pXL3728 was obtained from plasmid pXL3179, a vector derived from plasmid pXL2774 (WO 97 / 10343), into which a fusion of cDNA encoding human fibroblast interferon signal peptide and FGF1 (fibroblast growth factor 1) was introduced The gene (sp-FGF1, Jouanneau et al., PNAS 88 (1991), 2893-2897), placed in the promoter obtained from the human giant cell early region (hCMV IE) and the polyadenylation of the SV40 virus late region (GenBankSV4CG) under the control of acidification signaling.

[0093] The 2.3 Kb BamHI-XhoI fragment characterized in Example 1 was cloned into the plasmid pX...

Embodiment 3

[0096] Example 3: Comparison of plasmids

[0097] 3.1 Plasmids pXL3130 and pXL3153

[0098] Plasmids pXL3130 and pXL3153 contain human smooth muscle α-actin promoter (-680, +30) and mouse SM22 promoter (-436, +43) coupled to CMV enhancer (-522, -63), respectively, As described in application WO 00 / 18908.

[0099] 3.2 Plasmid pXL3635

[0100]RSV-229 was cloned by PCR from a construct containing a longer form of the RSV promoter (contained in Ad1.0RSVLAcZ, Stratford-Perricaudet et al., J Clin Invest 90 (1992) 626-30), the +34 promoter, The primers 5'-GGC GAT TTAAAT AAT GTA GTC TTA TGC AAT-3' and 5'-GGG GTC TAG AAGGTG CAC ACC AAT GTG GTG A-3' were used in the PCR to introduce a SwaI and XbaI sites. The promoter fragment was then introduced into pGL3-basic using these two restriction sites to generate pXL3635.

[0101] 3.2 Plasmid pXL3031

[0102] Plasmid pXL3031 was described by Soubrier et al., Gene Ther. 6 (1999), 1482-8. It is a vector derived from the plasmid pXL2774 (...

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Abstract

The invention relates to novel promoter sequences derived from a portion upstream of the coding sequence of the gene for the CARP protein (Cardiac Ankyrin Repeat Protein), and which are capable of controlling the level and the specificity of expression of a transgene in vivo in cardiac muscle cells. The invention thus describes novel compositions, constructs, vectors and their uses in vivo for the transfer and expression of a nucleic acid in vivo in cardiac muscle cells. The subject of the present invention is also the use of the promoter sequences for generating transgenic animals which constitute models for studying certain cardiac pathologies.

Description

field of invention [0001] The present invention relates to the field of biology. Specifically in the field of gene expression targeting, and more specifically in the design and development of new systems for the specific expression of transgenes. The subject of the invention is in particular a new promoter sequence capable of controlling the level and specificity of the in vivo expression of the transgene in cardiomyocytes. The present invention describes novel compositions, constructs and vectors capable of controlling and directing the expression of nucleic acids in cardiomyocytes. The uses deriving from the present invention are numerous, for example in the experimental, clinical, therapeutic and diagnostic fields, more particularly for the treatment and / or prevention of certain heart diseases. Background of the invention [0002] Controlling the level and targeting of transgene expression is necessary for many uses. Thus, for gene therapy, therapeutic success may requ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/76A01K67/027A61K38/00A61K48/00A61P9/00A61P9/04A61P37/06C07K14/47C12N5/10C12N15/09C12N15/11C12N15/67C12N15/85
CPCC12N2830/15C07K14/47C12N2830/00A01K2217/05C12N2830/85C12N2830/60C12N2830/008C12N15/85A61P37/06A61P9/00A61P9/04C12N15/11
Inventor B·施沃茨D·布拉乃尔莱克K·奇恩陈炬P·贝诺伊特
Owner GENCELL SA
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