Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1994-10-13
1996-08-06
Fleisher, Mindy
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
514 8, 514 12, 514851, 530350, 530415, 530417, 424450, A61K 3817, A61K 9127, C07K 136, C07K 14435
Patent
active
055433995
DESCRIPTION:
BRIEF SUMMARY
This invention relates to purified and functionally reconstituted preparations of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and to pharmaceutical compositions and methods of use employing these preparations.
The discovery of the gene which is mutated in patients with cystic fibrosis (CF) and the principal disease-causing mutation (Rommens et al., 1989; Riordan et al., 1989; Kerem et al., 1989) has given rise to the possibility of the development of molecular therapies. These can be considered in at least three broad categories: A.) The creation or identification of drugs to appropriately modify CFTR function or biosynthesis; B.) gene therapy by the delivery of the CFTR DNA sequence in an appropriate vector to affected epithelial cells; C.) protein replacement therapy in which the CFTR protein in an appropriate vehicle is delivered to the same cells.
The steps to be accomplished for the effective application of the third strategy 1) the production of large quantities of functional CFTR protein; 2) the solubilization and purification of the CFTR protein; 3) the reconstitution of the homogeneous purified protein into a lipid environment in which it can function; 4) demonstration that the purified and reconstituted CFTR molecule has the same functional properties as it had in the epithelial cells to which it is native; 5) fusion of proteoliposomes containing functional purified CFTR with the apical surfaces of CF epithelial cells expressing nonfunctional mutant CFTR or no CFTR at all in order to restore regulated chloride channel activity.
The original CFTR cDNAs which we had isolated and cloned (Riordan et al., 1989) and deposited with ATCC have been used for expression of CFTR in a number of different heterologous mammalian cell systems (Tabcharani et al., 1991; Anderson et al., 1991a; Cheng et al, 1990; Dalemans et al., 1991). However, because of limitations on the amount of CFTR which can be synthesized in human and other mammalian cells (Cheng et al., 1990; Gregory et al., 1991), it was necessary to utilize an alternative system to generate adequate amounts for purification. We employed the baculovirus expression vector system (BEVS; Lucknow and Summers, 1988) to produce large quantities of functional human CFTR in insect Sf9 cells (Kartner et al., 1991). More recently, others have produced CFTR protein in the milk of transgenic mice (DiTullio et al., 1992) as another potential means of producing sufficient protein for purification. However, in that work no evidence of functionality was demonstrated, nor were any attempts at purification made.
The present invention involves the fulfilment of steps 2.), 3.) and 4.) resulting in the production of highly purified CFTR protein as judged by stringent criteria of homogeneity. The purified protein is further demonstrated to exhibit the same functional properties of a regulated chloride ion channel as it does in its native location in vivo. In addition, as expected, structural features including N-terminal amino acid sequence (6 residues), overall amino acid composition and isoelectric point are identical to those predicted from the translated DNA sequence of the coding region of the cloned CFTR gene. The only feature of the protein produced in the insect cell expression system which differs from that produced in human epithelial cells in the type of carbohydrate added when the protein is glycosylated during synthesis. However, we have already demonstrated that this difference is without influence on the function of the glycoprotein (Kartner et al., 1991). The glycosylation of the protein in any other of the alternate expression systems which may be used such as milk of transgenic animals (DiTullio et al., 1992) will also differ from that in the human lung which will be the principal site of delivery for therapeutic purposes.
The invention also teaches that the proteoliposomes of the type known to be capable of fusing with the membranes of cells, can be fused to planar lipid bilayers in which the generation of electrical currents carried by chlor
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Bear Christine E.
Li Canhui
Ramjeesingh Mohabir
Riordan John R.
Degen Nancy J.
Fleisher Mindy
HSC Research & Development Limited Partnership
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