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  Sequences upstream of the carp gene, vectors containing them and uses thereofUSPTO Application #: 20060110362Title: Sequences upstream of the carp gene, vectors containing them and uses thereof 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. (end of abstract) Agent: Finnegan, Henderson, Farabow, Garrett & Dunner LLP - Washington, DC, US Inventors: Patrick Benoit, Bertrand Schwartz, Didier Branellec, Kenneth R. Chien, Ju Chen USPTO Applicaton #: 20060110362 - Class: 424093200 (USPTO) Related 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.) The Patent Description & Claims data below is from USPTO Patent Application 20060110362. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of U.S. Provisional Application No. 60/251,582, filed Dec. 7, 2000, which is incorporated herein in its entirety. [0002] The present invention relates to the field of biology. It relates in particular to the field of the targeting of the expression of genes, and more particularly the design and the development of a novel system for the specific expression of transgenes. The subject of the invention is, in particular, novel promoter sequences 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 and vectors that make it possible to control and to direct the expression of a nucleic acid in cardiac muscle cells. The applications stemming from the present invention are numerous, for example in the experimental, clinical, therapeutic and diagnostic fields, and more particularly for the treatment and/or prevention of certain cardiac pathologies. [0003] The control of the level and of the targeting of the expression of transgenes is necessary for many applications. For example, in gene therapy the success of the therapy may require targeting of the protein synthesized from the transgene and thus make it possible to limit the spread of side effects. The construction of transgenic animals and the study of the effects of a gene are additional examples in which an appropriate control of the specificity of expression of a protein can be used and can provide improvements. [0004] In this regard, many promoters have been tested for their capacity to direct a cardiospecific expression. They are in particular the promoters of the gene encoding the cardiac myosin light chain (MLC-2) in rats (Henderson S. A. et al., J Biol Chem, 264 (1989) 18142-8; Lee K. J. et al., J Biol Chem, 126 (1992) 15875-85), cardiac .alpha.-actin in mice (Biben C. et al., Dev Biol, 173 (1996) 200-12), atrial natriuretic factor (ANF) (Harris A. N. et al., J Mol Cell Cardiol, 29 (1997) 515-25), .alpha.- or .beta.-myosin heavy chain (.alpha.- or .beta.-MHC) (Colbert M C. et al., J Clin Invest, 100 (1997) 1958-68), muscle creatine kinase (MCK) in rabbits (Vincent C. K. et al., Mol Cell Biol, 13 (1993) 567-74), or cardiac troponin T (U.S. Pat. No. 5,266,488). [0005] While these promoters are known to confer a degree of tissue specificity, it is also known that their levels of activity remain well below those of so-called strong promoters, generally by a factor of between 10 and 100, such that a therapeutic use cannot really be envisaged. [0006] By way of example, Franz W. M. et al., (Cardiovasc Res, 35 (1997) 560-6) and Griscelli F. et al., (C R Acad Sci III, 320 (1997) 103-12) have shown that the levels of activity of the sequences upstream of the genes encoding rat .alpha.-MHC and MLC-2 in adenoviral constructs remain substantially lower than those of the RSV (Rous sarcoma virus) promoter, by a factor of about 10. [0007] The present application, therefore, relates to a novel promoter sequence derived from the region upstream of the CARP (Cardiac Ankyrin Repeat Protein) gene. This sequence is capable not only of directing a cardiospecific expression, but also exhibits a high level of expression in vivo, comparable to that of a strong promoter such as the CMV (cytomegalovirus) promoter. [0008] The CARP protein, which constitutes one of the first markers for differentiation of cardiomyocytes acting downstream of the homeobox gene Nbx2.5 in the regulation of the expression of the MLC-2v gene, has been studied and the coding portion of its gene has been sequenced in mice (Zou Y. et al., Development, 24 (1997) 793-804), in rabbits (Aihara Y. et al., Biochim Biophys Acta, 28 (1999) 318-24), and in humans (Chu W. et al., J Biol Chem, 270 (1995) 10236-45). [0009] Kuo H. et al. (Development, 126 (1999) 4223-34) have cloned a 10 Kb fragment and sequenced a 2.5 Kb fragment upstream of the coding sequence of the mouse CARP gene. Deletions from the 5'-end of the fragment were made and showed that a region of 213 bp of the promoter between nucleotides -166 and +47, relative to the transcription start position +1, was sufficient to confer cardiospecific expression in vitro, which suggested the presence, at the 5'-end, of an element for controlling the specificity of the promoter. Kuo et al. also generated transgenic mouse lines comprising a fragment of 2.5 Kb upstream of the CARP gene, showing specific expression of a transgene in cardiac and skeletal muscle cells at an early stage of embryonic development, this expression then being inhibited during development. [0010] Application WO 00/15821 describes a portion 5' of the coding sequence of the mouse CARP gene, situated between nucleotides -2285 and +62, relative to the transcription start position +1. This sequence was evaluated in particular for its in vivo activity in adenoviral vectors. The levels of activity obtained remain very low, however, such that it was found to be necessary, in order to detect an activity in vivo, to isolate the promoter sequence between two inverted terminal repeats of an adeno-associated virus (AAV-ITR). [0011] The Applicants focused on better characterizing the region upstream of the CARP gene protein-coding region. We were thus able to identify a novel sequence upstream of the CARP gene and demonstrate unexpected and advantageous properties of this novel sequence, in particular, a significant improvement in the level of activity in vivo. [0012] The Applicants have discovered, surprisingly, that while this newly identified sequence conferred no significant expression in vitro, it was, on the contrary, possible to obtain very good levels of activity in vivo, equivalent to those of so-called strong promoters, while preserving a high selectivity for expression in cardiac tissue. [0013] The subject of the present invention is therefore a polynucleotide comprising a portion upstream of the coding sequence of the gene for the CARP protein, or of a polynucleotide hybridizing under highly stringent conditions with said upstream sequence, the polynucleotide being capable of inducing specific expression in cardiac tissue of a transgene placed under its control. [0014] The invention also relates to any polynucleotide of natural origin or which is obtained by chemical synthesis, exhibiting at least 93%, preferably at least 95%, identity with SEQ ID NO: 1. In a further embodiment of the invention, the polynucleotide exhibits at least 98% identity with SEQ ID NO: 1. [0015] The term "polynucleotide of natural origin" is understood to mean a genomic DNA fragment obtained by cleaving cellular DNA with the aid of a restriction enzyme. [0016] The term "polynucleotide obtained by chemical synthesis" is understood to mean a DNA fragment generated by automated or manual synthesis, for example, with the aid of a suitable automated apparatus. [0017] For the present invention, the term "highly stringent conditions" is used in the sense given by Maniatis et al. 1982 (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor CSH, N.Y., USA) or one of its more recent editions. By way of example, the hybridization conditions are such that three washes at 65.degree. C. in the presence of 0.2.times.SSC, and 0.1% SDS are necessary in order to eliminate the nonhybridized fragments. [0018] The "specific" character of transgene expression means that the activity of the promoter is significantly higher in cells of cardiac tissue. Although nonspecific expression can be observed in other cells, the corresponding level of activity remains very low (negligible) compared with that observed in cardiac cells, in general lower by a factor of at least 10. [0019] The results presented in the examples show, in this regard, a difference in expression that may reach a factor of 1000, which reflects the high selectivity of the polynucleotides according to the invention for cardiac cells in vivo. [0020] Moreover, the results presented in the examples below clearly show that the use of the polynucleotides of the invention offers a system for high levels of expression, above those for other promoters known to be specific for cardiac tissue, it being possible for the difference to exceed a factor of 100. These elements, therefore, illustrate the advantages and unexpected properties of the polynucleotide according to the invention, in terms of promoter strength and specificity, for the expression of nucleic acids of interest in the cardiac tissue. [0021] Advantageously, the polynucleotide according to the invention comprises a portion of the sequence between -2266 and +92 (SEQ ID NO: 1), relative to transcription start position +1 of the CARP gene. [0022] The subject of the present invention is therefore the sequences hybridizing, under high stringency conditions, with the sequence SEQ ID NO: 1. [0023] The present invention is nevertheless not restricted to the polynucleotides containing fragments upstream of the mouse gene but relates to any functional variant or any other sequence of any other species having the same properties, namely being capable of specifically inducing expression in vivo of a transgene in cardiac tissue. [0024] Thus, persons skilled in the art will be able to refer to the sequence upstream of the human gene deposited in GenBank under the reference AF131884 (SEQ ID NO: 2). The present invention thus encompasses any sequence comprising fragments of the sequences upstream of the gene for the CARP protein, modified, for example, by deletion of certain structures and which preserve identical or similar functions to that of the sequence SEQ ID NO: 1. Continue reading... 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