WO2002058627A2 - A cystic fibrosis transmembrane-conductance regulator (cftr)-membrane translocation sequence fusion protein (cftr-mts) as a therapeutic agent - Google Patents
A cystic fibrosis transmembrane-conductance regulator (cftr)-membrane translocation sequence fusion protein (cftr-mts) as a therapeutic agent Download PDFInfo
- Publication number
- WO2002058627A2 WO2002058627A2 PCT/US2001/049958 US0149958W WO02058627A2 WO 2002058627 A2 WO2002058627 A2 WO 2002058627A2 US 0149958 W US0149958 W US 0149958W WO 02058627 A2 WO02058627 A2 WO 02058627A2
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- Prior art keywords
- cftr
- fusion protein
- mts
- cystic fibrosis
- protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4712—Cystic fibrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- the present invention relates to the field of the therapy of cystic fibrosis in human subjects.
- the present invention relates to a method of treating cystic fibrosis comprising administering a fusion protein to affected cells of the subject.
- Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) which is a chloride channel expressed in epithelial tissues.
- CFTR cystic fibrosis transmembrane conductance regulator
- the mutations in CF patients result in abnormally rapid intracellular degradation of the CFTR protein.
- This defect in intracellular CFTR protein results in abnormal chloride transfer across epithelial membranes, causing excessive thickening of the mucus lining the airways.
- the viscid mucus allows for invasion by Staphylococcus aureus, Hemophilus influenzae and Pseudomonas aeruginosa which provoke a vigorous and excessive neutrophilic inflammatory response that over years causes lung destruction manifested as bilateral bronchiectasis.
- CF is the most common lethal inherited disease in Caucasians. Over 25,000 people in the US have CF and death occurs, on average, in the third decade of life due progressive lung disease. No curative therapy is available and current medications only slow the progression of the lung disease. Health care costs are tremendous in these patients: on average a typical patient with CF with take 40 to 50 pills per day and 3 to 5 different inhalers. Most require hospitalization on at least a yearly basis. This level of care often begins in early childhood and extends through the life of the patient.
- the present addresses these needs by pro aiding a method of introducing a functional CFTR protein into affected cells of patients diagnosed with cystic fibrosis (CF).
- CF cystic fibrosis
- Figure 1A is a fluorescent micrograph of an indirect immuno-fluorescence assay (IIFA) of cells contacted with unmodified FGF-R.
- IIFA indirect immuno-fluorescence assay
- Figure IB is a fluorescent micrograph of an indirect immuno-fluorescence assay (UFA) showing the insertion of FGF-R-MTS fusion protein on the cell surface.
- UFA indirect immuno-fluorescence assay
- nucleic acid includes multiple copies of the nucleic acid and can also include more than one particular species of nucleic acid molecule.
- the present invention provides a fusion protein comprising a cystic fibrosis transmembrane conductance regulator (CFTR) and a membrane translocation sequence (MTS).
- CFTR cystic fibrosis transmembrane conductance regulator
- MTS membrane translocation sequence
- the fusion protein can further comprise a tag, such as a His tag or GST among others.
- the MTS can be either C-terminal or N-terminal to the CFTR.
- the CFTR of the present fusion protein comprises, for example, the entire normal human CFTR sequence.
- the term "CFTR" as used herein can include any domain of the normal CFTR protein having the chloride channel activity of the nonnal CFTR, including additions, substitutions and/or deletions in amino acids that retain the activity. Examples of other CFTRs include those described below and in the cited publications, which have been incorporated by reference.
- the fusion protein also comprises a membrane translocation sequence (MTS), comprising, for example, the amino acid sequence AAVLLPVLLAAP (SEQ ID NO:l).
- MTS membrane translocation sequence
- the MTS can be a sequence of amino acids generally of a length of about 10 to about 50 or more amino acid residues, many (typically about 55-60%) residues of which are hydrophobic such that they have a hydrophobic, lipid-soluble portion.
- the hydrophobic portion is a common, major motif of the signal peptide, and it is often a central part of the signal peptide of protein secreted from cells.
- a signal peptide is a peptide capable of penetrating through the cell membrane to allow the export of cellular proteins.
- the signal peptides of this invention are also MTSs, i.e., capable of penetrating through the cell membrane from outside the cell to the interior of the cell.
- the amino acid residues can be mutated and/or modified (i.e., to form mimetics) so long as the modifications do not affect the translocation-mediating function of the peptide.
- the word "peptide” includes mimetics and the word “amino acid” includes modified amino acids, as used herein, unusual amino acids, and D-form amino acids. All MTS peptides encompassed by this invention have the function of mediating translocation across a cell membrane from outside the cell to the interior of the cell.
- MTS peptides could potentially be modified such that they lose the ability to export a protein but maintain the ability to import molecules into the cell.
- a putative signal peptide can easily be tested for this importation activity following the teachings provided herein, including testing for specificity for any selected cell type.
- fusion proteins of the invention include the following: CFTR-MTS, GST-CFTR-MTS and HIS-CFTR-MTS. Additional fusion proteins taught herein include FGFR-MTS, GST-FGFR-MTS and HIS-FGFR-MTS.
- nucleic acids which encodes fusion proteins of the invention, more specifically the fusion proteins comprising CFTR-MTS.
- Nucleic acid as used herein includes single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T), C, and G.
- the nucleic acid may represent a coding strand or its complement.
- Nucleic acids may be identical in sequence to the portion of the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence.
- nucleic acids can include codons which represent conservative substitutions of amino acids as are well known in the art.
- nucleic acid can selectively hybridize under stringent conditions.
- Stringent conditions refers to the hybridization conditions used in a hybridization protocol or in the primer/template hybridization in a PCR reaction. In general, these conditions should be a combination of temperature and salt concentration for washing chosen so that the denaturation temperature is approximately 5-20 °C below the calculated T m (melting/denaturation temperature) of the hybrid under study. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference nucleic acid are hybridized to the primer nucleic acid of interest and then amplified under conditions of different stringencies.
- MgCl 2 concentrations used in PCR buffer can be altered to increase the specificity with which the primer binds to the template, but the concentration range of this compound used in hybridization reactions is narrow, and therefore, the proper stringency level is easily determined.
- hybridizations with oligonucleotide probes 18 nucleotides in length can be done at 5-10 °C below the estimated T m in 6X SSPE, then washed at the same temperature in 2X SSPE.
- the T m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C.
- An 18 nucleotide probe of 50% G+C would, therefore, have an approximate T m of 54 °C.
- the starting salt concentration of an 18 nucleotide primer or probe would be about 100-200 mM.
- stringent conditions for such an 18 nucleotide primer or probe would be a T m of about 54°C and a starting salt concentration of about 150 mM and modified accordingly by preliminary experiments.
- T m values can also be calculated for a variety of conditions utilizing commercially available computer software (e.g., OLIGO ® ).
- the nucleic acid encoding a protein of this invention can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations.
- the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding a protein of this invention.
- the nucleic acid encoding a protein of this invention can be any nucleic acid that functionally encodes a protein of this invention.
- a nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.
- Preferred expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc.
- a nucleic acid encoding a selected protein can readily be determined based upon the genetic code for the amino acid sequence of a selected protein and many nucleic acids will encode any selected protein. Modifications in the nucleic acid sequence encoding a protein are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression of a protein to make production of a protein inducible or repressible as controlled by the appropriate inducer or repressor. Such methods are standard in the art.
- the nucleic acid of this invention can be generated by means standard in the art, such as by recombinant nucleic acid techniques and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
- the present invention provides a vector comprising the nucleic acid which encodes a CFTR-MTS fusion protein.
- the vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art.
- the expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. Specific vectors and expression systems that exemplify the types of vectors and expression systems that can be used to express the CFTR-MTS fusion proteins are provided in the Examples.
- a fusion protein of the present invention can be in a pharmaceutically acceptable carrier.
- pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the a protein without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
- the present invention also provides a method of treating cystic fibrosis in a human subject diagnosed with cystic fibrosis comprising administering to the subject, in a pharmaceutically acceptable carrier, an effective amount of the fusion protein of the present invention, whereby the fusion protein can be taken up by affected cells in the subject.
- the principal route of administration of the CFTR/MTS fusion protein is by aerosol in order to deliver the protein to the lung airway epithelium.
- other routes of administration for example, intravenous, intramuscular, and intraperitoneal delivery and infusion into the afferent blood supply to the liver.
- Oral administration can also be used in view of the availability of methods to protect the protein from gastric secretions, e.g., by microencapsulation, etc.
- the exact amount of the protein required will vary from subject to subject, depending on the age, weight and general condition of the subject, the particular protein used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every protein. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation. For example, a dose of about l ⁇ g to about lOO ⁇ g of the protein can be administered in an aerosol to the lungs of a subject. The amount of protein administered can be adapted to the specific mode of administration by the skilled clinician. The protein can be administered from about once per week to about 3 times per day.
- a membrane translocation sequence can be made as described in U.S. Patent No. 5,807,746 (incorporated herein in its entirety); PCT Publication WO 01/37821 (incorporated herein in its entirety); in "Regulation of NF- ⁇ B, AP-1, NFAT, and STAT 1 Nuclear Import in T Lymphocytes by Noninvasive Delivery of Peptide Carrying the Nuclear Localization Sequence of NF- ⁇ B p50V Torgerson et al, The Journal of Immunology, pages 6084-6092, 1998 (incorporated herein in its entirety) ; and “Genetic Engineering of Proteins with Cell Membrane Permeability," Rojas et al, Nature Biotechnology, Volume 16, pages 370-375, April, 1998 (incorporated herein in its entirety).
- the MTS used in this procedure has the following sequence AAVLLPVLLAAP (SEQ ID NO:l).
- the CFTR gene is known (Riordan et al, 1989, Science 245:1066-1073; Rommens et al, 1989, Science 245: 1059-1065).
- the CFTR cDNA clone Due to the size of the CFTR cDNA clone we designed a new MTS vector and used a double vector expression system.
- the present vector expresses the MTS fusion protein associated to the HIS tag sequence instead of GST.
- the CFTR cDNA was introduced into the QIA-expressionist (pQE) from Qiagen, a commercially available vector that contains the HIS-tag.
- the cDNA was introduced into the pQE vector, which permits the expression of a high level of His-tag fusion proteins under the control of lac Z.
- a second vector, pREP4 is used to regulate the expression of the fusion proteins in the pQE vector.
- the pREP4 vector constitutively express the lac repressor, and its function can be inhibited by the addition of IPTG which stimulates the expression of pBR22 (represses the repressor). Once the repressor is inactivated the bacteria begin the transcription of the fusion proteins.
- the bacteria can be kept at refrigeration temperature for several hours to allow for better folding of the expressed fusion proteins.
- the primers designed to introduce MTS into the pQE vectors were: Primer pQEPst-mtsl(50-mer)
- Vstl digestion released the DNA insert that conesponds to CFTR.
- the CFTR fragment was gel purified following the protocol of QIAGEN QIAEX II gel extraction kit. The purified fragment was ligated into Vstl digested pQE32 and pQE32- MTS vectors (from QIAGEN) and introduced into a bacteria strain containing pREP4 plasmid. Selection of positive clones by in-situ hybridization
- oligonucleotide probe was designed from the C- terminal sequence of CFTR cDNA. This probe was label by T4 kinase using ⁇ 32 P, and used to screen CFTR- pQE32 and CFTR-pQE32-MTS transformations. Approximately 400 colonies were screened and 32 positive colonies were identified.
- the PCR product was purified by gel extraction using QIAGEN QIAEX II kit following the protocol recommended by the manufacture. After purification, the PCR product was inserted into pGEMT-easy (Promega). This vector is modified to insert PCR products.
- FGFR1 cDNA was released from the pGEM-T by BamHl digestion and subcloned into MTS-2 vector (pGEX-3X expression vector (Promega) containing MTS sequence).
- MTS-2 vector pGEX-3X expression vector (Promega) containing MTS sequence.
- the FGFR1 -MTS-2 construct was introduced into BL-21 bacteria strain (high efficiency in protein expression) and positive clones were determined by isolation of the plasmids and digestion with different restriction enzymes
- 2x10 6 BaF3 (T cells without FGFR) cells were incubated with 35 g/ml of the fusion proteins: GST, GST-FGFR1 and GST-FGFR1-MTS, during 2 hours at 37°C. Cells were washed four times with cold-PBS, transferred to a new tube and washed one time more. The pellet was resuspended in SDS-loading buffer and 20 ⁇ l were used in a 7.5 % SDS-PAGE. The presence of the fusion protein was detected using an anti-GST and anti-FGFR antibodies. Protein was detected only in the samples in which the cells were incubated with GST-FGFR1-MTS.
- IIFA Indirect Immuno-Fluorescence Assay
- Cells were washed with PBS, and treated or not with 0.25% Triton X-100 for 10 min to permeabilize the cell membrane.
- Cells were incubated with blocking solution (PBS+ 1% goat serum + 1% BSA) for 30 min at 37°C. After blocking, cells were incubated with anti-GST in PBS+ 1% BSA for 2 hours. Cells were washed 3 times with PBS and blocked for 30 min. Protein-antibody complexes were incubated during 1 hour with goat anti-rabbit IgG labeled with Texas Red (Southern Biotech, AL). Cells were washed 3 times.
- the CFTR protein is a transmembrane cyclic AMP (cAMP) activated chloride channel, and one of the standard means to assess appropriate functioning of this channel is through the patch clamp assay.
- cAMP transmembrane cyclic AMP
- the delivery of an active CFTR-MTS fusion protein into cells is measured using a patch clamp assay on cells to which the fusion protein has been administered.
- the control bath solution contains (in mM): 140 NMDG-C1 (N-methyl-D-glucamine-chloride), 2.0 CaCl 2 , 2.0 MgCl 2 , 5 HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanosulfonic acid], pH 7.4, -275 mosmol/kg.
- the pipette solution contains (in mM): 140 NMDG-Cl, 2.0 MgCl 2 , 5 HEPES, 5 MgATP, 1 EGTA (ethylene glycol-bis-[ ⁇ -aminoethyl ether], pH 7.4, -270 mosmol/kg.
- the pipette solution is diluted 9-13% with distilled water to prevent activation of swelling-activated Cl " currents.
- As a reference electrode an agar- bridge with composition similar to the control bath solution are utilized.
- the holding potential is 0 mV in all experiments, and the whole-cell currents are measured from -100 to +100 mV (in 20 mV steps) 50 ms after the start of the voltage pulse. Pulse generation, data collection and analyzes are done with Clampex 7.0 (Axon Instruments, Inc.).
- the anionic selectivity of the cAMP-activated cunent is determined in NaCl bath solution (in mM): 140 NaCl, 2.0 CaCl 2 , 2.0 MgCl 2 , 5 HEPES, pH 7.4, -275 mosmol/kg) by replacing NaCl with equimolar NaBr, Nal, or Na-gluconate.
- the liquid-junction potentials generated upon changing the extracellular anion are calculated using the Junction Potential Calculator in Clampex 7.0.
Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU2002246793A AU2002246793A1 (en) | 2000-11-09 | 2001-11-09 | A cystic fibrosis transmembrane-conductance regulator (cftr)-membrane translocation sequence fusion protein (cftr-mts) as a therapeutic agent |
US10/416,285 US20040047808A1 (en) | 2001-11-09 | 2001-11-09 | Cystic fibrosis transmembrane-conductance regulator (cftr)-membrane translocation sequence fusion protein (cftr-mts) as a therapeutic agent |
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US24749400P | 2000-11-09 | 2000-11-09 | |
US60/247,494 | 2000-11-09 |
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WO2002058627A2 true WO2002058627A2 (en) | 2002-08-01 |
WO2002058627A3 WO2002058627A3 (en) | 2003-05-22 |
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PCT/US2001/049958 WO2002058627A2 (en) | 2000-11-09 | 2001-11-09 | A cystic fibrosis transmembrane-conductance regulator (cftr)-membrane translocation sequence fusion protein (cftr-mts) as a therapeutic agent |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5639661A (en) * | 1994-03-23 | 1997-06-17 | The University Of Iowa Research Foundation | Genes and proteins for treating cystic fibrosis |
US5807746A (en) * | 1994-06-13 | 1998-09-15 | Vanderbilt University | Method for importing biologically active molecules into cells |
-
2001
- 2001-11-09 AU AU2002246793A patent/AU2002246793A1/en not_active Abandoned
- 2001-11-09 WO PCT/US2001/049958 patent/WO2002058627A2/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5639661A (en) * | 1994-03-23 | 1997-06-17 | The University Of Iowa Research Foundation | Genes and proteins for treating cystic fibrosis |
US5807746A (en) * | 1994-06-13 | 1998-09-15 | Vanderbilt University | Method for importing biologically active molecules into cells |
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AU2002246793A1 (en) | 2002-08-06 |
WO2002058627A3 (en) | 2003-05-22 |
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