USRE39220E1 - Peptide-mediated gene transfer - Google Patents
Peptide-mediated gene transfer Download PDFInfo
- Publication number
- USRE39220E1 USRE39220E1 US09/404,979 US40497999A USRE39220E US RE39220 E1 USRE39220 E1 US RE39220E1 US 40497999 A US40497999 A US 40497999A US RE39220 E USRE39220 E US RE39220E
- Authority
- US
- United States
- Prior art keywords
- cells
- dna
- gene
- vector
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Definitions
- the present invention is directed to a methodology for highly efficient, stable integration of DNA into a eukaryotic genome. More specifically, the present invention is directed to the use of a synthetic polypeptide, containing a nuclear localization signal to complex with a DNA molecule and to facilitate its transportation and integration into the nuclear genome of a mammalian or other eukaryotic cell, for example, in the context of producing cell lines with an extended life.
- DNA-CaPO 4 co-precipitation was the first method developed to introduce genes into mammalian cells.
- Gene in this regard denotes a structural DNA segment, i.e., a DNA that codes for a polypeptide, and comprehends oncogenes as well as DNAs coding for a known expression product.
- the co-precipitation method was applicable only to certain cell types, however, and could not be used to introduce genes into a wide variety of cell lines, especially those of hematopoietic origin. Moreover, the stable gene transfer efficiency was rather low, on the order of 10 ⁇ 4 to 10 ⁇ 6 .
- transient expression The production of a gene product for only a short time period after transfection, usually from 48 to 72 hours, is called “transient expression.” Many of the DNA-complexing agents reported heretofore, while useful in transferring a gene into mammalian cells, resulted in only transient expression of the introduced gene in a small fraction of the transfected cells. See, for example, Miller et. al., Proc. Nat'l Acad. Sci., USA, 76: 949 (1979); Oi et al., loc. cit. 80: 825 (1983).
- Stable gene transfer is the ability of cells to maintain and express transfected DNAs in a stable manner, through integration of the transfected DNA into cell chromosomes.
- Retrovital vectors which were under development at about the same time seemed to be quite effective in transferring genes into different cell types. The use of such vectors was prompted by the elucidation of gene regulation in various murine and avian retroviruses. Two other developments led to the development of retrovirus-based gene transfer vehicles. The first development was the identification of minimal sequences required for efficient packaging of viral particles in a cell line which produced the coat proteins and other structural components of the viral particle in trans. The cell lines that provided the structural components for virus development are called “packaging” cell lines. The second significant step in the establishment of retroviral vectors was the development of both ecotropic and amphotropic packaging cell lines, which aided the design of recombinant retroviral particles which could infect both murine and human cell lines.
- retroviral vectors were deemed necessary to address concerns that retroviral vectors could recombine in vivo to generate wild-type virus. Developments in this regard yielded a number of safe retroviral vectors which have been used to transfer genes into a variety of established mammalian cell lines, as well as into certain primary cells in a few instances.
- Gilboa et al. BioTechniques 4: 504 (1986); A. D. Miller et al., Mol. Cell. Biol. 6: 2895 (1986); H. Guatemalamann et al., loc. cit. 9: 100 (1989); A. D. Miller et al., BioTechniques 7: 980 (1989); J. A. Zwiebel et al., Science 243: 220 (1989).
- viruses have been used to generate recombinant viral vectors for gene transfer studies.
- Adenovirus, adeno-associated virus, herpes simplex virus, and even HIV have been employed as vectors to introduce genes into both established cell lines and primary cells.
- Some of these viral vectors are capable of transferring genes into non-dividing cells.
- Viral vectors capable of transferring genes into non-dividing cells usually require the generation of high-titer viral stock in order to achieve high efficiency gene transfer into different cell types.
- a new viral stock must to be made and tittered for every modification. All these involve very time-consuming experimental manipulations.
- Still another concern relates to the application of viral vectors in human gene therapy.
- a number of studies have been carried out in primates to test the safety of retroviral vectors for introducing cells transduced with retroviral vectors into animals. Some of these animals have developed various forms of lymphoma. R. E. Donahue, et al., J. Exp. Med. 176: 1125 (1992). Additional safety features have been introduced into some of the newer versions of retroviral vectors, yet are not available for all types of viral vectors.
- a transfection vector comprising a synthetic polypeptide linked electrostatically to a DNA structural sequence, forming a polypeptide-DNA complex, where the polypeptide is comprised of (A) a polymeric chain of basic amino acid residues, (B) an NLS peptide and (C) a hinge region of neutral amino acids that connects the polymeric chain and the NLS peptide.
- the polymeric chain preferably is comprised of between 10 and 50 residues, which can selected from lysine, arginine and ornithine, for example, while the hinge region is comprised of between 6 and 50 amino acid residues selected, for example, from glycine, alanine, leucine and isoleucine.
- the NLS peptide preferably is located at the amino terminus of said polypeptide and the polymeric basic amino acid chain at the carboxyl terminus.
- exemplary NLS peptides are the SV40 large T antigen NLS sequence, the polyoma large T antigen NLS sequence, the adenovirus E1a NLS sequence, and the adenovirus E1b NLS sequence.
- an extended life cell line is provided that is the product of transfecting a mammalian cell with a vector as described above.
- the mammalian cell thus transfected can be selected, for example, from the group consisting of a human umbilical vein endothelial cell, a human dermal microvascular endothelial cell, a human peripheral blood monocyte/macrophage cell, a human aortic smooth muscle cell, and a rabbit liver non-parenchymal cell.
- the present invention allows for the highly efficient transfer and stable integration of DNA into eukaryotic cells, such as cells from established mammalian cell lines, primary cells from mammalian tissues, and plant cells.
- eukaryotic cells such as cells from established mammalian cell lines, primary cells from mammalian tissues, and plant cells.
- the present invention also can be applied to developing cell lines from non-dividing cells, such as human peripheral blood monocytes and macrophages.
- a synthetic polypeptide that can complex with a DNA molecule very efficiently by taking advantage of the high negative charge density on the polynucleotide.
- a synthetic polypeptide of the present invention comprises a DNA-binding sequence that is rich in basic amino acids, such as lysine, arginine and ornithine, and that is typically ten to fifty residues long. D-isomers of these basic amino acids are suitable so long as the length of the stretch of basic amino acids is within the prescribed length.
- the DNA-binding sequence can be a homopolymer of a basic amino acid, or it can comprise more than one kind of basic residue.
- the DNA binding sequence must be of adequate length to bind DNA, yet not so long that it precipitates out of the solutions employed in the present methodology, as discussed below.
- a synthetic polypeptide of the present invention also contains an amino acid sequence corresponding to a nuclear localization signal (NLS) sequence.
- NLS nuclear localization signal
- an NLS peptide which typically is six to fifteen amino acids in length, facilitates transport of the associated DNA into the nucleus. Because the synthetic polypeptide promotes the transport of the transfected gene into the nucleus of the host cell, this method provides both highly efficient stable and transient gene expression. Once inside the nucleus, the introduced DNA is immediately available to the transcription machinery, and can be expressed transiently. Simultaneously, the introduced DNA is also in the process of getting integrated into the host chromosome to give rise to stable expression. Thus, the method of the instant invention can achieve both transient and stable expression of introduced DNA.
- Transient gene expression results when the method of gene transfer results in the introduction of the DNA sequences into the nucleus in an non-integrated form. Transient transfection is measured 24 to 72 hours after transfection by assays that measure gene expression of the transfected gene(s). In contrast, stable expression of the encoded protein results when the transferred DNA sequences are stably integrated into the chromosomal DNA of the target cell. Stable transfectants remain capable of expressing the transfected DNA after two weeks or greater following the method of the invention.
- CAT chloramphenicol acetyltransferase
- LAC-Z ⁇ -galactosidase
- GUS ⁇ -glucuronidase
- luciferase or human growth hormone, each of which may be contained in the present invention.
- the NLS domain of the synthetic peptide is based on known endogenous peptide sequences that were identified by reference to two criteria: (1) sufficient to redirect a cytoplasmic protein to the nucleus and (2) necessary for directing a nuclear protein to the nucleus.
- Methods for assessing an NLS peptide's ability to direct protein to the nucleus are known in the art. See Garcia-Bustos, et al., supra, Sandler et al., J. Cell Biol. 109: 2665 (1989), and Citovsky et al., supra, the respective contents of which are hereby incorporated by reference.
- an NLS peptide or a natural protein containing an NLS is fused to an otherwise non-nuclear protein, by either synthetic or recombinant production.
- the hybrid protein is then assessed for its ability to target the non-nuclear protein to the nucleus.
- the presence of the non-nuclear protein in the nucleus can be determined by a functional assay or immunofluorescence.
- An illustrative assay entails the histochemical determination of a product produced by the non-nuclear protein, such as a colorimetric marker produced by ⁇ -gal or GUS.
- a “colorimetric marker” includes an enzyme that can catalyze a reaction with a substrate to elicit a colored product which can be detected or measured by a variety of means, such as standard fluorescence microscopy, flow cytometry, spectrophotometry or colorimetry.
- Immunofluorescence relates to detecting the presence of the non-nuclear protein in the nucleus by means of an antibody specific for the targeted protein.
- NLS peptides have been studied to assess their ability to target reporter proteins to the nucleus.
- endogenous proteins containing an NLS such as the VirD2 and VirE2 of Agrobacterium, have been shown to mediate the transfer of the Agrobacterium single-stranded DNA intermediate T-strand to the plant cell nucleus endogenously. See Citovsky, et al., supra. There has been no suggestion heretofore, however, to use an NLS peptide to target a polynucleotide to the nucleus of a eukaryotic cell.
- a preferred NLS domain contains a short stretch of basic amino acids like the NLS of the SV40 virus large T antigen (PKKKRKV) (SEQ ID NO:3), which is an NLS that has been shown to be effective in mammalian cells (basic residues are highlighted).
- Another preferred NLS domain consists essentially of short hydrophobic regions that contain one or more basic amino acids (KIPIK) (SEQ ID NO:1), which is like the NLS of mating type ⁇ 2.
- NLSs that transport DNA into the plant cell nucleus often are bipartite, which means that they are usually comprised of a combination of two regions of basic amino acids (identified as stippled segments in Table I) separated by a spacer of more than four residues (see stippled segments in Table I) , such as the Xenopus nucleoplasmin (KRPAATKKAGQAKKKK) (SEQ ID NO:55).
- the NLS peptide of the present invention can be designed to accommodate different host cells, both mammalian and plant cell hosts.
- the method described here can suitably be modified to introduce genes into plant protoplasts using plant NLSs, such as those described by Raikhel (1989), supra.
- the present gene transfer system is also capable of transferring foreign DNA into gymnosperms and angiosperms.
- Procedures for assessing the introduction of foreign DNA in plants are known to the art, such as those disclosed by Miki, B. L., et al., in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, B. R. Glick et al., eds. (CRC Press, 1993), and Gruber, M. Y. et al., id.
- a synthetic polypeptide of the present invention thus is comprised of a DNA binding domain and an NLS peptide domain which are separated by a third element, a hinge region of neutral amino acid, to minimize stearic steric interference between the two domains.
- the hinge region ranges in length from about six to twenty-five amino acids, and contains a stretch of neutral small amino acids without any bulky hydrophobic or ionic side chains.
- the NLS sequence can be located at either the amino terminus or the carboy-terminus of the synthetic peptide.
- the arrangement of the two domains, basic amino acid sequence and NLS sequence can be interchanged without affecting the high gene transfer efficiency.
- such a synthetic polypeptide binds electrostatically to the DNA that is to be introduced into the target cell.
- the weight ratio of polypeptide to DNA in the resulting complex generally is in the range of 1:1 to 1:10; for example, 1 ⁇ g polypeptide to 1 to 10 ⁇ g of polynucleotide.
- entry of the DNA-polypeptide complex into cells can be promoted by treating target cells with a hypertonic solution, followed by hypotonic treatment of cells in the presence of gene-peptide complex.
- a hypertonic solution can contain both polyethylene glycol (PEG) and sucrose, preferably in the concentration of 0.3M-0.6M and 10% to 25%, respectively, and is referred to as “primer” hereinafter.
- the methodology of the present invention has been used to develop stable transfectants of different established cell lines. It also has been employed to transfer genes into primary cells from different mammalian species, thereby to obtain cell lines that retain many of the characteristics of the cognate primary cells.
- Cell lines developed from primary cells via the methodology of the present invention are called “extended life” cell lines in this description, because the cell lines so developed retain almost all of the characteristics of their cognate primary cells even in their late passage.
- the range of cell types that can be converted to extended life cell lines, according to the present invention is based on the availability of primary cells or the ability to isolate a primary cell from the organ in question.
- the inventive methodology is not limited to cell types amenable to transformation.
- the present invention can be applied to pancreatic beta cells, human liver and kidney cells, and human hematopoietic stem cells, among others.
- the methodology of the present invention has been used to develop an extended life cell line from human monocyte/macrophage cells, which are normally non-dividing. In all these instances, stable cell lines were obtained with a very high efficiency, either comparable to or better than the efficiency using retroviral vectors.
- the present invention finds application as well in both ex vivo and in vivo gene therapies, where genetic material is transferred into specific cells of a patient.
- Ex vivo gene therapy entails the removal of the relevant target cells from the body, transduction of the cells in vitro, and subsequent reintroduction of the modified cells into the patient.
- a gene therapy pursuant to the present invention could involve an ex vivo introduction, into a particular cell type from the patient, of a polynucleotide coding for a correcting protein which can be produced in functional form by the targeted cell type.
- Genes suitable for expression in this regard include an adenosine deaminase gene, a globin gene, an LDL receptor gene, and a glucose cerebrosidase gene.
- the method of the present invention is advantageous in that it can be used in gene therapy requiring either stable gene expression or transient gene expression.
- Transient expression of a foreign gene is preferred when expression of the exogenous product is needed only for a short period of time; thereafter, rapid clearance of the gene product and its vector is desirable.
- Transient expression is also desirable when the prolonged effects of the exogenous protein's expression are unknown.
- Stable expression in gene therapy is needed when the patient has a genetic defect that is incompatible with life. Such genetic defects include but are not limited to cystic fibrosis, Tay Sachs and cancer. Mulligan, Science, 260: 926 (1993).
- a gene therapy pursuant to the present invention also could involve an in vivo introduction of a structural DNA into cells of a patent's body.
- the ligand to the target receptor will be conjugated to the synthetic polypeptide.
- the polypeptide-ligand combination can be complexed to a polynucleotide coding for the needed protein and then introduced into the host organism through blood circulation. When this complex reaches the target tissue, the whole complex will be taken up by cells containing the corresponding receptor for the ligand through receptor mediated process.
- the complex will enter into the nucleus, resulting in a stable integration of the introduced gene into the host chromosome and, thereby, a correction of the genetic defect in the host.
- Cell-specific receptors are well known to those of skill in the art, as are their ligands which can be used in complexes for receptor-mediated gene transfer. Michael, S. I, et al., J. Biol. Chem. 268: 6866 (1993).
- the DNA encoding corrective protein is complexed to a synthetic neoglycoprotein that will target the complex to the asialoglycoprotein receptor on hepatocytes.
- a cell type specific receptor ligand such as asialoglycoprotein can be chemically linked to the transfection vector at the carboxyl terminal of the synthetic polypeptide molecule to deliver the foreign gene directly into liver cells.
- An additional hinge region can be incorporated into the molecule before chemically linking the polypeptide molecule to a cell-type specific ligand molecule, such as asialoglycoprotein or a cell-specific monoclonal antibody.
- a carrier useful for receptor-mediated gene transfer to liver is a synthetic glycoprotein in which bovine serum albumin (BSA) is covalently bound to poly L-lysine using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ECD). Ferkol et al., FASEB 7: 1081 (1993).
- BSA bovine serum albumin
- EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
- a reaction mixture that contains about 170 mM galactose, 4 mM poly (L-lysine), 160 mM BSA and 10 mM EDC (pH 7.5) can be incubated for 48 hours at 22° C.
- DNA is complexed to the neoglycoprotein carrier in a 360:1 molar ratio.
- the carrier-DNA complexes are dialyzed against 150 mM sodium chloride before transfection.
- the method of the present invention greatly improves the cell-specific targeting of receptor-mediated transfection by providing stable expression by increasing stable integration of a foreign DNA in the host cell using a synthetic polypeptide molecule of the present invention.
- a variation of receptor-mediated gene transfer employs coupling a synthetic polypeptide as described above to monoclonal antibodies which recognize a cell surface antigen on the target cells. Maruyama et al., Proc. Nat'l Acad. Sci. USA 87: 5744 (1990).
- the coupled monoclonal antibody and synthetic polypeptide then are complexed with a DNA encoding the required or desired protein. This complex will target the DNA to the cells expressing the corresponding cell surface antigen.
- Any tissue of the human body can be targeted for the gene therapy of the present invention using the disclosed methods.
- a target tissue is suitable in this context so long as it is susceptible to genetic modification according to the present invention.
- the DNA or other polynucleotide to be transfected is complexed to a synthetic polypeptide molecule in different weight ratios in an isotonic buffer solution.
- an isotonic buffer solution such as Hanks buffered salt solution or HEPES buffered saline may be used for complexing DNA to polypeptide.
- the cells that are to be transfected either remain attached to a substratum, such as a tissue culture dish, or are pelleted (for cells that grow in suspension).
- the cells are treated with a hypertonic primer solution, such as a concentration of 0.3M-0.6M sucrose and 10% PEG in either Tris-HCl or HEPES (pH 7.2) buffered solution, for 3-5 mins at room temperature.
- the primer solution then is removed.
- the complex solution is hypotonic when it has a lesser osmotic pressure than a 0.15M or 0.9% solution of NaCl.
- the complex in isotonic buffer can be made 40-55% hypotonic or 0.075M simply by adding an amount of distilled water that is equal to the volume of the complex in isotonic buffer.
- the hypotonic complex solution then is added to the cells that have been treated with the primer solution. Cells remain in the hypotonic DNA-polypeptide solution for 3-4 minutes. Fresh medium then is added to the cells to rinse away excess DNA-polypeptide solution. Thereafter, the cells are grown normally.
- An example of an synthetic polypeptide molecule of the present invention is one consisting of the amino acid sequence PKKKRKVSGGGGGKKKKKKKKKKKK(SEQ ID NO:56).
- Such a peptide can be synthesized, using standard methods of peptide production, and purified by standard methods using high pressure liquid chromatography (HPLC).
- Transfected cells are grown in regular growth medium for 48 hours, and then plated in selective medium containing 400 ⁇ g/ml of G418. Cells were plated at a density of 100-1000 cells per 60 cm 2 dish. The number of G418-resistant colonies was determined two weeks after the initiation of selection. Other selectable markers, such as pHyg, may be used to achieve the results of the instant invention. K. Blochlinger, et al., Mol. Cell. Biol. 4: 2929 (1984).
- transfectants of the instant invention are stable is shown by the following example.
- G418 resistant colonies were grown without selection for variable period of times, and then tested for resistance to the antibiotic by plating the cells under clonal conditions, the same number of colonies were obtained both with and without G418. This result indicates that, once the cells are selected for the expression of the Neo gene, the resistance gene was retained stably in the chromosome.
- Mouse fibroblast cell line L cells
- mouse erythroleukemia cell line C19TK
- COS cell line was used to establish conditions for transient gene expression.
- the eukaryotic expression vector, CH110 contains bacterial ⁇ -gal and was employed in these studies.
- the ⁇ -gal gene in CH110 is under the control of SV40 virus early promoter.
- the COS cells were treated with primer and then exposed to DNA-polypeptide (2.5-5.0 ⁇ g) complex under hypotonic conditions. After this treatment, cells were returned to the normal growth condition. Transfected cells were grown at 37° C. for 48 hrs, and stained for the expression of the ⁇ -gal reporter gene. Forty to fifty percent of the cells were positive for the expression of the reporter gene.
- Mouse L cells were transfected with eukaryotic expression vector containing the Neo gene, which codes for the antibiotic G418 resistance gene. L cells are sensitive to G418 at 400 ⁇ g/ml. Cells plated in 24-well tissue culture plates were then transfected with synthetic polypeptide complexed to the plasmid pRSV-Neo via the methodology of the present invention.
- a mouse erythroleukemia cell line, C19TK also was used as a representative cell line for testing the transfection efficiency of the present invention with respect to hematopoietic cells.
- the expression vector, pDR2 which carries a hygromycin-resistance gene, was used for these studies, C19TK cells are extraordinarly sensitive for the antibiotic hygromycin. This cell line grows in suspension and, hence, was transfected in suspension.
- the gene transfer method of the present invention was used to generate extended life cell lines from different human primary cells. Most of the primary cells have a limited in vitro life span. The following cell types were employed to test the efficacy of the inventive method to generate extended-life cell lines by transfer of various oncogenes, either singly, in pairs of combinations, or combinations of more than two oncogenes. Rhim, J. S., et al., Oncogene 4: 1403 (1989).
- the method of introducing genes into primary cells is the same as that described above for introducing genes into established cell lines, such as the mouse fibroblast cell line L cells and the mouse erythroleukemia cell line C19TK.
- the main difference is that the host cell is a primary cell isolated from different species, human or other mammalian species, and the primary cells have only a limited in vitro life span.
- the isolation of primary cells from various tissue sources are well known to those of skill in the art.
- the cells are transfected with different oncogenes, such as SV40 large T antigen, polyoma large T antigen, adenovirus E1A and E1B, v-fms, Bc12, myc and ras.
- oncogenes such as SV40 large T antigen, polyoma large T antigen, adenovirus E1A and E1B, v-fms, Bc12, myc and ras.
- the oncogenes can be used either alone, in pairs of various combinations, or in combinations of more than two oncogenes.
- genes that do not come under the category of oncogenes may be used.
- genes that are important for DNA synthesis and normally active during the S phase of the cell cycle such as the dihydrofolate reductase gene (DHFR), thymidine kinase gene, thymidylate synthetase gene, a DRTF1/E2F transcription factor encoding DNA, or DNA encoding the E2F transcription factor can be complexed to synthetic polypeptide and used to extend the life of primary cells.
- the human DHFR gene complexed to synthetic polypeptide can be introduced into primary cells to produce extended life cell lines.
- DNA encoding a transcription factor that is active during the S phase of the cell cycle are particularly useful in the method of the instant invention.
- untreated primary cells have only a limited life span in vitro, their ability to grow continuously in culture after treatment with the present invention served to select for extended life cell lines. No other drug selection markers need to be used to select for extended life cell lines derived from primary cells.
- Extended life cell lines containing the oncogene are identified by restriction cleavage. Southern analysis and/or Northern analysis using appropriate DNA probes.
- each transformed extended life cell line is analyzed by Southern hybridization to determine whether the cell lines carry the oncogenes used to establish such extended life cell lines.
- DNA is extracted from the cell lines and the nucleic acid pellet is re-suspended in 200 ⁇ l of 10 mM Tris-Cl pH 7.4, 0.1 mM EDTA, and 10 ⁇ g is digested with a specific restriction enzyme, electrophoresed through 1.0% agarose, and transferred to nitrocellulose.
- Each transformed extended life cell line is analyzed by Northern hybridization to determine whether the cell lines transcribe the oncogenes.
- Cells not containing the oncogene of interest will not demonstrate transcripts in a Northern analysis whereas cells containing the DNA of interest will demonstrate a detectable transcript.
- an ELISA method was used to detect the presence of oncogene products in some of the extended life cell lines, using publicly available antibodies that recognize the corresponding oncogene protein.
- SV40 large T antigen and adenovirus E1A gene products in the HUVEC extended life cell line are shown in t Table 2 III .
- the cell line grown in a 96 well tissue culture plate is fixed with glutaraldehyde and paraformaldehyde.
- the cells are then treated with antibodies to the corresponding oncogenes. Thereafter, the cells are washed and then treated with a secondary antibody linked with to ⁇ -galactosidase.
- the cells are washed and then treated with a substrate for ⁇ -galactosidase.
- the reaction develops a product which is then measured using a microplate reader.
- Extended life cells lines containing the oncogene are assessed by Northern analysis using a DNA probes encoding a cell-specific protein.
- the cell-specific DNA probe is labeled with 32 P-dCTP by nick translation pursuant, for example, to Rigby et al., J. Mol. Biol. 113: 237 (1977).
- Northern hybridization indicates that the extended life cell line is capable of transcribing the cell-specific protein.
- the maintenance of the parental phenotype in cells lines established according to the present invention can be determined by a number of biochemical methods, such as ELISA and enzyme assays, that determine the presence or function of a protein specific to the parental cell line.
- An antibody recognizing a protein produced only bythe by the parental cell line can be used in an ELISA or immunofluorescence assay.
- Cell-specific markers are well known to those of skill in the art. For example, albumin is a marker for hepatocytes, insulin is a marker for pancreatic beta islet cells, factor VIII is a marker for endothelial cells, actin and myosin are markers for smooth muscle cells, and non-specific esterass esterase is a marker for brain microglial cells.
- the parental phenotype of the extended life endothelial cells produced by the present method of the invention was verified by several ELISAs to determine the expression of cell-specific endothelial markers.
- the parental phenotype of the monocyte/macrophage extended life cell lines produced by the present method was verified using a lysozyme enzyme assay to measure macrophage specific markers.
- Endothelial cells isolated from the human umbilical vein can only be cultured for a limited of passages, usually five to six. These cells were transfected with a combination of oncogenes, SV40 large T antigen and adenovirus E1A, or with another combination of genes. At least two oncogenes are needed to develop a truly transformed cell line. Ruley, H. E., et al., Nature 304: 602 (1983). For the instant invention, SV40 large T antigen combined with v-myc or ras or some an other oncogene can be used.
- extended life cell lines may be produced from human umbilical vein endothelial cells.
- E1A or E1B or SV40 large T antigen alone did not give rise to established cell line with the high frequency obtained from using SV40 large T antigen in combination with E1A or E1B.
- Synthetic polypeptide complexed to DNA encoding either the SV40 large T antigen or polyoma large T antigen combined with the E2F1 transcription factor genes also produces extended life HUVEC cells lines.
- the non-transfected primary cells normally grow in vitro only for a limited population doublings, cells that have taken up the oncogenes capable of generating extended life span cell lines were selected simply by repeated passage of the cells.
- the transfected population of cells grows continuously, as compared to a control population of parental cells, it is reasonable to conclude that the oncogenes used are capable of generating extended life cells from a given cell type.
- HUVEC for example, SV40 large T antigen and adenovirus E1A or E1B were effective in giving rise to a cell line.
- This cell line has now been growing in culture for 40 passages.
- normal HUVECs stop growing by passage 7 or 8.
- Such cell lines arose with a high efficiency. It also is possible to generate cell lines using as few as a couple of hundred cells, grown either in a 24- or 48-well plate. These cells also have the same morphological appearance as the primary HUVEC and also display many of the biochemical properties characteristic of normal HUVEC.
- Adherent cells from human cord blood cells were transfected with different combinations of oncogenes in suspension using the method of the present invention.
- the resulting cells are selected in Granulocyte-Macrophage Colony Stimulating Factor (G-CSF).
- G-CSF Granulocyte-Macrophage Colony Stimulating Factor
- Control cells did not grow in culture, whereas growing populations of monocytes were obtained with several combinations of oncogenes.
- One preferred combination of polyoma large T antigen and adenovirus E1B encoding DNA produced extended life monocyte cells lines with somewhat higher efficiency than other combinations.
- Another preferred combination of SV40 large T or polyoma large T antigen and the E2F1 transcription factor gene produces monocyte extended life cells with high efficiency.
- the monocyte extended life cells also display many of the properties of normal monocytes, which illustrates the utility of the present invention in generating cell lines of hematopoietic origin.
- the method of the instant invention has also been used to generate extended life cell lines using a specific combination of oncogenes.
- Human aortic smooth muscle cells were obtained from Clonetics Corporation (San Diego, Calif. U.S.A.) and transfected with several combination of oncogenes.
- the combination of polyoma large T antigen and EiB gave rise to a continuously growing population of smooth muscle cells.
- Another preferred combination of SV40 large T or polyoma large T antigen and the E2F1 transcription factor gene produces extended life human aortic smooth muscle cells with high efficiency.
- This cell line resembles the early passage primary aortic smooth muscle cells morphologically.
- the extended life human aortic smooth muscle cells also express smooth cell actin and myosin well beyond passage 20.
- Primary cells from other species such as rabbit and monkey, also have been used to generate cell lines. Transfection methods employed for primary cells from non-human species are similar to those used for human primary cells.
- several different combinations of available oncogenes should be tried. For example, at least five or six pairs of combinations of SV40 large T antigen, adenovirus E1A, adenovirus E1B, polyoma virus large T antigen or others available to those in the art. That combination of genes that gives rise to an extended life cell lines from a given primary cell type is determined as described in the above examples.
- extended life cells lines can be produced from a variety of primary cell types, such as HUVEC, dermal microvascular endothelial cells, human aortic smooth muscle cells, and bone marrow monocyte/macrophage cells.
- primary cell types such as HUVEC, dermal microvascular endothelial cells, human aortic smooth muscle cells, and bone marrow monocyte/macrophage cells.
- the method of the present invention can identify a combination of oncogene DNAs that is highly efficient in producing extended life cells lines from the primary cells of various species.
- the present invention also comprehends a combination of an oncogene and an S-phase transcription factor genes which likewise is highly efficient in producing extended life cells lines from different types of primary cells.
- the present invention provides a screening system for identifying sequences that influence the expression of cloned genes in various primary cell types from different species.
- the instant invention can identify cell type specific transcription and translational regulatory sequences.
- the sequence in question typically will be cloned into a vector containing a reporter gens, such as chloramphenicol acetyl transferass or luciferass, and then transfected into various cell types using the method described herein. Expression of the reporter genes determines the tissue specificity of the regulatory sequence.
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
A methodology that allows for highly efficient transfer and stable integration of DNA into both established eukaryotic cell lines and primary cells, including non-dividing cells such as human peripheral blood monocytes and macrophages, entails the use of a synthetic polypeptide comprised of a peptide domain which corresponds to a nuclear localization signal sequence and a DNA binding domain which is rich in basic amino acids, separated by a hinge region of neutral acid which prevents stearic interference between the two domains.A synthetic polypeptide that allows for highly efficient transfer of DNA into eukaryotic cells, including, for example, non-dividing cells such as human peripheral blood monocytes and macrophages.
Description
This application was filed as a reissue of Ser. No. 08/240,514, filed May 14, 1994, now U.S. Pat. No. 5,670,347.
The present invention is directed to a methodology for highly efficient, stable integration of DNA into a eukaryotic genome. More specifically, the present invention is directed to the use of a synthetic polypeptide, containing a nuclear localization signal to complex with a DNA molecule and to facilitate its transportation and integration into the nuclear genome of a mammalian or other eukaryotic cell, for example, in the context of producing cell lines with an extended life.
DNA-CaPO4 co-precipitation was the first method developed to introduce genes into mammalian cells. (“Gene” in this regard denotes a structural DNA segment, i.e., a DNA that codes for a polypeptide, and comprehends oncogenes as well as DNAs coding for a known expression product.) The co-precipitation method was applicable only to certain cell types, however, and could not be used to introduce genes into a wide variety of cell lines, especially those of hematopoietic origin. Moreover, the stable gene transfer efficiency was rather low, on the order of 10−4 to 10−6. McNally, M. A., et. al., BioTechniques 6: 8826 (1988); Yen, T. S. B., et. al., loc. cit. 6: 413 (1988).
Limits on introducing and expressing genes in cultured mammalian cells motivated a search for other, more efficient approaches to gene transfer. Methods were developed, for example, that utilized chemical agents which were positively charged and, hence, able to complex with negatively charged DNA molecules. Examples of such agents include DEAE dextran and various cationic lipid molecules. Cells treated with DNA complexes comprised of such an agent can lead to the introduction of the DNA into different mammalian cell lines. Mannino, R. J. et al., BioTechniques 6: 682 (1988); Felgner, P. et al., Proc. Nat'l Acad. Sci. USA 84: 7413 (1987); Fraley, R. et. al., Trend Biochem. Sci. 6: 77 (1981); Holter, W. et. al., Exp. Cell Res. 184: 546 (1989); McCutchan, J. H. et al., J. Nat'l Cancer Inst. 41: 351 (1986); Chaney, W. C. et al., Somatic Cell & Mol. Genet. 12: 237 (1986).
The production of a gene product for only a short time period after transfection, usually from 48 to 72 hours, is called “transient expression.” Many of the DNA-complexing agents reported heretofore, while useful in transferring a gene into mammalian cells, resulted in only transient expression of the introduced gene in a small fraction of the transfected cells. See, for example, Miller et. al., Proc. Nat'l Acad. Sci., USA, 76: 949 (1979); Oi et al., loc. cit. 80: 825 (1983).
In addition to giving poor results with respect to stable gene expression, transfer methods based on such DNA-complexing agents often were effective only with established cell lines, and did not work very well with primary cells isolated from various mammalian species. Other techniques therefore were needed to enhance gene transfer efficiency, to increase the variety of cell types capable of being transfected, and to effect stable gene transfer. Stable gene transfer is the ability of cells to maintain and express transfected DNAs in a stable manner, through integration of the transfected DNA into cell chromosomes.
Retrovital vectors, which were under development at about the same time seemed to be quite effective in transferring genes into different cell types. The use of such vectors was prompted by the elucidation of gene regulation in various murine and avian retroviruses. Two other developments led to the development of retrovirus-based gene transfer vehicles. The first development was the identification of minimal sequences required for efficient packaging of viral particles in a cell line which produced the coat proteins and other structural components of the viral particle in trans. The cell lines that provided the structural components for virus development are called “packaging” cell lines. The second significant step in the establishment of retroviral vectors was the development of both ecotropic and amphotropic packaging cell lines, which aided the design of recombinant retroviral particles which could infect both murine and human cell lines.
Additional modifications of retroviruses were deemed necessary to address concerns that retroviral vectors could recombine in vivo to generate wild-type virus. Developments in this regard yielded a number of safe retroviral vectors which have been used to transfer genes into a variety of established mammalian cell lines, as well as into certain primary cells in a few instances. E. Gilboa et al., BioTechniques 4: 504 (1986); A. D. Miller et al., Mol. Cell. Biol. 6: 2895 (1986); H. Stuhlmann et al., loc. cit. 9: 100 (1989); A. D. Miller et al., BioTechniques 7: 980 (1989); J. A. Zwiebel et al., Science 243: 220 (1989).
Even though these vectors were effective with respect to various mammalian cells, there were many restrictions on a wider application of the retroviral gene-transfer technique. These limitations included (1) the size of exogenous DNA that can be inserted into a retroviral vector and (2) the use of only dividing cells for retroviral gene transfer. E. Gilboa, BioTechniques, supra (1986); A. D. Miller, supra (1986); H. Stuhlmann, et al., Mol. Cell. Biol. supra, (1986); A. D. Miller et al., BioTechniques, supra (1986); J. A. Zwiebel et al., supra (1989).
Other viruses have been used to generate recombinant viral vectors for gene transfer studies. Adenovirus, adeno-associated virus, herpes simplex virus, and even HIV have been employed as vectors to introduce genes into both established cell lines and primary cells. Some of these viral vectors are capable of transferring genes into non-dividing cells. R. J. Samulski, et al., EMBO J. 10: 3941 (1981); J. D. Tratschin, et al., Mol. Cell. Biol. 5: 3251 (1985); P. L. Hermonat, et al., Proc. Nat'l Acad. Sci. (USA) 81: 6466 (1984); D. J. Fink, et al., Human Gene Therapy 3: 11 (1992).
Viral vectors capable of transferring genes into non-dividing cells usually require the generation of high-titer viral stock in order to achieve high efficiency gene transfer into different cell types. In addition, whenever a different regulatory sequence is to be tested for optimal level of gene expression into primary cells, a new viral stock must to be made and tittered for every modification. All these involve very time-consuming experimental manipulations.
Still another concern relates to the application of viral vectors in human gene therapy. A number of studies have been carried out in primates to test the safety of retroviral vectors for introducing cells transduced with retroviral vectors into animals. Some of these animals have developed various forms of lymphoma. R. E. Donahue, et al., J. Exp. Med. 176: 1125 (1992). Additional safety features have been introduced into some of the newer versions of retroviral vectors, yet are not available for all types of viral vectors.
It therefore is an object of the present invention to provide a method for high efficiency gene transfer to achieve expression, stable as well as transient, in a wide spectrum of cell types, including primary cells from various mammalian species.
It is also an object of the present invention to provide cell lines which, even if derived from primary mammalian cells, are characterized by an extended life in culture.
It is another object of the present invention to provide a readily implemented screening system for identifying sequences that influence in the expression of cloned genes in various primary cell types from different species.
In accomplishing these and other objectives, there has been provided, in accordance with one aspect of the present invention, a transfection vector comprising a synthetic polypeptide linked electrostatically to a DNA structural sequence, forming a polypeptide-DNA complex, where the polypeptide is comprised of (A) a polymeric chain of basic amino acid residues, (B) an NLS peptide and (C) a hinge region of neutral amino acids that connects the polymeric chain and the NLS peptide. The polymeric chain preferably is comprised of between 10 and 50 residues, which can selected from lysine, arginine and ornithine, for example, while the hinge region is comprised of between 6 and 50 amino acid residues selected, for example, from glycine, alanine, leucine and isoleucine. The NLS peptide preferably is located at the amino terminus of said polypeptide and the polymeric basic amino acid chain at the carboxyl terminus. Among exemplary NLS peptides are the SV40 large T antigen NLS sequence, the polyoma large T antigen NLS sequence, the adenovirus E1a NLS sequence, and the adenovirus E1b NLS sequence.
In accordance with another aspect of the present invention an extended life cell line is provided that is the product of transfecting a mammalian cell with a vector as described above. The mammalian cell thus transfected can be selected, for example, from the group consisting of a human umbilical vein endothelial cell, a human dermal microvascular endothelial cell, a human peripheral blood monocyte/macrophage cell, a human aortic smooth muscle cell, and a rabbit liver non-parenchymal cell.
Other object, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The present invention allows for the highly efficient transfer and stable integration of DNA into eukaryotic cells, such as cells from established mammalian cell lines, primary cells from mammalian tissues, and plant cells. The present invention also can be applied to developing cell lines from non-dividing cells, such as human peripheral blood monocytes and macrophages.
In accordance with the present invention, a synthetic polypeptide is provided that can complex with a DNA molecule very efficiently by taking advantage of the high negative charge density on the polynucleotide. To this end, a synthetic polypeptide of the present invention comprises a DNA-binding sequence that is rich in basic amino acids, such as lysine, arginine and ornithine, and that is typically ten to fifty residues long. D-isomers of these basic amino acids are suitable so long as the length of the stretch of basic amino acids is within the prescribed length. The DNA-binding sequence can be a homopolymer of a basic amino acid, or it can comprise more than one kind of basic residue. The DNA binding sequence must be of adequate length to bind DNA, yet not so long that it precipitates out of the solutions employed in the present methodology, as discussed below.
A synthetic polypeptide of the present invention also contains an amino acid sequence corresponding to a nuclear localization signal (NLS) sequence. A representative sample from the diverse range of nuclear localization signals which have been identified are listed in Table I below. (SEQ ID NOS:1-54).
TABLE 1 | ||
Source | Nuclear Protein | Deduced Signal Sequeuce |
Yeast | MATα2 | (SEQ ID NO: 1) K--I--P--I--K |
(SEQ ID NO: 2) V--R--I--L--E--S--W--F--A--K-- | ||
N--I | ||
SV40 | Large T | (SEQ ID NO: 3) P--K--K--K--R--K--V |
Influenza Virus | Nucleoprotein | (SEQ ID NO: 4) A--A--F--E--D--L--R--V--R--S |
Yeast | Ribosomal protein L3 | (SEQ ID NO: 5) P--R--K--R |
Polyoma virus | Large T | (SEQ ID NO: 6) V--S--R--K--R--P--R--P--A |
SV40 | VP1 | (SEQ ID NO: 7) A--P--T--K--R--K |
Adenovirus | E1a | (SEQ ID NO: 8) K--R--P--R--P |
SV40 | VP2 (VP3) | (SEQ ID NO: 9) P--N--K--K--K--R--K |
Frog | Nucleoplasmin | (SEQ ID NO: 10) R--P--A--A--T--K--K--A--G-- |
Q--A--K--K--K--K--L--D | ||
Rat | Glucocorticoid receptor | (SEQ ID NO: 11) K--K--K--I--K |
Monkey | v-sis (PDGF B) | (SEQ ID NO: 12) R--V--T--I--R--T--V--R--V--R- |
-R--P--P--K--G--K--H--R--K | ||
Yeast | Histone 2B | (SEQ ID NO: 13) G--K--K--R--S--K--A D--G-- |
K--K--W-S | ||
Chicken | v-re1 | (SEQ ID NO: 14) K--A--K--R--S--K--A Q--R |
Influenza | NS1 | (SEQ ID NO: 15) D--R--L--R--R |
(SEQ ID NO: 16) P--K--Q--K--R--K | ||
Frog | N1 | (SEQ ID NO: 17) V--R--K--K--R--K--T |
(SEQ ID NO: 18) A--K--K--S--K--Q--E | ||
Human | c-myc | (SEQ ID NO: 19) P--A--A--K--R--V--K--L--D |
(SEQ ID NO: 20) R--Q--R--R-N--E--L--K--4 R- | ||
-S--F | ||
Human | lamin A | (SEQ ID NO: 21) T--K--K--R--K--L--E |
HTLV-1 | Rex(p27x-III) | (SEQ ID NO: 22) P--K--T--R--R--R--P |
(SEQ ID NO: 23) S--Q--R--K--R--P--P | ||
Adenovirus | FTP | (SEQ ID NO: 24) R--L--P--V--R--R--R--R--R-- |
V--P | ||
HIV-1 | Tat | (SEQ ID NO: 25) G--R--K--K--R |
Frog | Lamin L1 | (SEQ ID NO: 26) V--W--T--T--K--G--K--R--K-- |
R--I--D--V | ||
Rabbit | Progesterone receptor | (SEQ ID NO: 27) R--K--F--K--K |
HIV-1 | Rev | (SEQ ID NO: 28) R--R--N--R--R--R--R--W |
Human | PDGF A-chain | (SEQ ID NO: 29) P--R--3 E--S--G--K-K--R--K- |
-R--K--R--L--K--P--T | ||
Mouse | c-ab1 | (SEQ ID NO: 30) K-K--K--K--K S--A--L--I-- |
K--K--K--K--K--M--A--P | ||
Adenovirus | DBP | (SEQ ID NO: 31) P--P--K--K--R |
(SEQ ID NO: 32)P--K--K--K--K--K | ||
Chicken | c-erb-A | (SEQ ID NO: 33) S--K--R--V--A--K--R--K--L |
Human | c-myb | (SEQ ID NO: 34) P--L--L--K--K--I--I K--Q |
Human | N-myc | (SEQ ID NO: 35) P--P--Q--K--K--I--K--S |
Human | p53 | (SEQ ID NO: 36) P--Q--P--K--K--K--P |
Human | Hsp 70 | (SEQ ID NO: 37) F--K--R--K--H--K--K--D--I--S- |
-Q--N--K--R--A--V--R--R | ||
Hepatitis B virus | Core protein | (SEQ ID NO: 38) S--K--C--L--G--W--L--W--G |
Chicken | Ets1 | (SEQ ID NO: 39) G--K--R--K--N--K--P--K |
Yeast | Ribosomal protein L29 | (SEQ ID NO: 40) K--T--R--K--H--R--G |
(SEQ ID NO: 41) K--H--R--K--H--P--G | ||
Protein | Nuclear Localization Signals |
TGA-1A (tobacco) | (SEQ ID NO: 42) R--R--L--A--Q--N--R--E--A--A--R--K--S--R- |
-L--R--K--K | |
TGA-1B (tobacco) | (SEQ ID NO: 43) K--K--R--A--R--L--V--R--N--R--E--S--A-- |
Q--L--S | |
(SEQ ID NO: 44) R--Q--R--K--K | |
O2 NLS B (maize) | (SEQ ID NO: 45) R--K--R--K--E--S--N--R--E--S--A--R--R--S-- |
R--Y--R--K | |
NIa (Polyvirus) | (SEQ ID NO: 46) K--K--N--Q--K--H--K--L--K--M-32aa-K--R- |
-K | |
VirD2 (Agrobacterium) | (SEQ ID NO: 47) K--R--P--R--E--D--D--D-G--E--P--S--E--R-- |
K--R--E--R | |
VirE2 NSE1 (Agrobacterium) | (SEQ ID NO: 48) K--L--R--P--E--D--R--Y--I--Q--T--E--K--Y-- |
G--R--R | |
VirE2 NSE2 (Agrobacterium) | (SEQ ID NO: 49) K--T--K--Y--G--S--D--T--E--I--K--L--L K-- |
S--K | |
O2 NLS A (maize) | (SEQ ID NO: 50) M--E--E--A--V--T--M--A--P--A--A--V--S-- |
S--A--V--V--G--D--P | |
(SEQ ID NO: 51) M--3 E--Y--N--A--I--L--R--R--K--L--E--E-- | |
D--L--E | |
R NLS A (maize) | (SEQ ID NO: 52) G--D--R--R--A--A--P--A--R--P |
R NLS M (maize) | (SEQ ID NO: 53) M--S--E--R--K--R--R--E--K--L |
RNLS C (maize) | (SEQ ID NO: 54) M--I--S--E--A--L--R--K--A--I--G--K--R |
See Garcia-Bustos et al., Biochem. Biophys. Acta 1071: 83 (1991). Raikhel, N., Plant Physiol. 100: 1627 (1992), and Citovsky, V. et al., Science 256: 1802 (1992), the contents of each of which are hereby incorporated by reference.
In the present invention, an NLS peptide, which typically is six to fifteen amino acids in length, facilitates transport of the associated DNA into the nucleus. Because the synthetic polypeptide promotes the transport of the transfected gene into the nucleus of the host cell, this method provides both highly efficient stable and transient gene expression. Once inside the nucleus, the introduced DNA is immediately available to the transcription machinery, and can be expressed transiently. Simultaneously, the introduced DNA is also in the process of getting integrated into the host chromosome to give rise to stable expression. Thus, the method of the instant invention can achieve both transient and stable expression of introduced DNA.
Transient gene expression results when the method of gene transfer results in the introduction of the DNA sequences into the nucleus in an non-integrated form. Transient transfection is measured 24 to 72 hours after transfection by assays that measure gene expression of the transfected gene(s). In contrast, stable expression of the encoded protein results when the transferred DNA sequences are stably integrated into the chromosomal DNA of the target cell. Stable transfectants remain capable of expressing the transfected DNA after two weeks or greater following the method of the invention. Commonly used assays monitor enzyme activities of chloramphenicol acetyltransferase (CAT), LAC-Z, β-galactosidase (β-gal), β-glucuronidase (GUS), luciferase, or human growth hormone, each of which may be contained in the present invention.
The NLS domain of the synthetic peptide is based on known endogenous peptide sequences that were identified by reference to two criteria: (1) sufficient to redirect a cytoplasmic protein to the nucleus and (2) necessary for directing a nuclear protein to the nucleus. Methods for assessing an NLS peptide's ability to direct protein to the nucleus are known in the art. See Garcia-Bustos, et al., supra, Sandler et al., J. Cell Biol. 109: 2665 (1989), and Citovsky et al., supra, the respective contents of which are hereby incorporated by reference. For example, an NLS peptide or a natural protein containing an NLS is fused to an otherwise non-nuclear protein, by either synthetic or recombinant production. The hybrid protein is then assessed for its ability to target the non-nuclear protein to the nucleus.
The presence of the non-nuclear protein in the nucleus can be determined by a functional assay or immunofluorescence. An illustrative assay entails the histochemical determination of a product produced by the non-nuclear protein, such as a colorimetric marker produced by β-gal or GUS. (A “colorimetric marker” includes an enzyme that can catalyze a reaction with a substrate to elicit a colored product which can be detected or measured by a variety of means, such as standard fluorescence microscopy, flow cytometry, spectrophotometry or colorimetry. “Immunofluorescence” relates to detecting the presence of the non-nuclear protein in the nucleus by means of an antibody specific for the targeted protein.)
In the past NLS peptides have been studied to assess their ability to target reporter proteins to the nucleus. Also, endogenous proteins containing an NLS, such as the VirD2 and VirE2 of Agrobacterium, have been shown to mediate the transfer of the Agrobacterium single-stranded DNA intermediate T-strand to the plant cell nucleus endogenously. See Citovsky, et al., supra. There has been no suggestion heretofore, however, to use an NLS peptide to target a polynucleotide to the nucleus of a eukaryotic cell.
A preferred NLS domain contains a short stretch of basic amino acids like the NLS of the SV40 virus large T antigen (PKKKRKV) (SEQ ID NO:3), which is an NLS that has been shown to be effective in mammalian cells (basic residues are highlighted). Another preferred NLS domain consists essentially of short hydrophobic regions that contain one or more basic amino acids (KIPIK) (SEQ ID NO:1), which is like the NLS of mating type α2. The NLSs that transport DNA into the plant cell nucleus often are bipartite, which means that they are usually comprised of a combination of two regions of basic amino acids (identified as stippled segments in Table I) separated by a spacer of more than four residues (see stippled segments in Table I) , such as the Xenopus nucleoplasmin (KRPAATKKAGQAKKKK) (SEQ ID NO:55).
The NLS peptide of the present invention can be designed to accommodate different host cells, both mammalian and plant cell hosts.
The method described here can suitably be modified to introduce genes into plant protoplasts using plant NLSs, such as those described by Raikhel (1989), supra.
The present gene transfer system is also capable of transferring foreign DNA into gymnosperms and angiosperms. Procedures for assessing the introduction of foreign DNA in plants are known to the art, such as those disclosed by Miki, B. L., et al., in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, B. R. Glick et al., eds. (CRC Press, 1993), and Gruber, M. Y. et al., id.
A synthetic polypeptide of the present invention thus is comprised of a DNA binding domain and an NLS peptide domain which are separated by a third element, a hinge region of neutral amino acid, to minimize stearic steric interference between the two domains. For this purpose, the hinge region ranges in length from about six to twenty-five amino acids, and contains a stretch of neutral small amino acids without any bulky hydrophobic or ionic side chains.
The NLS sequence can be located at either the amino terminus or the carboy-terminus of the synthetic peptide. The arrangement of the two domains, basic amino acid sequence and NLS sequence can be interchanged without affecting the high gene transfer efficiency. As indicated previously, such a synthetic polypeptide binds electrostatically to the DNA that is to be introduced into the target cell. The weight ratio of polypeptide to DNA in the resulting complex generally is in the range of 1:1 to 1:10; for example, 1 μg polypeptide to 1 to 10 μg of polynucleotide.
In accordance with the present invention, entry of the DNA-polypeptide complex into cells can be promoted by treating target cells with a hypertonic solution, followed by hypotonic treatment of cells in the presence of gene-peptide complex. See, for example, Okada and Rechsteiner, Cell 29: 33 (1982). A suitable hypertonic solution can contain both polyethylene glycol (PEG) and sucrose, preferably in the concentration of 0.3M-0.6M and 10% to 25%, respectively, and is referred to as “primer” hereinafter. Okada et al., supra, and T. Takai, et al., Biochem. Biophys. Acta 1048: 105 (1990).
The methodology of the present invention has been used to develop stable transfectants of different established cell lines. It also has been employed to transfer genes into primary cells from different mammalian species, thereby to obtain cell lines that retain many of the characteristics of the cognate primary cells. Cell lines developed from primary cells via the methodology of the present invention are called “extended life” cell lines in this description, because the cell lines so developed retain almost all of the characteristics of their cognate primary cells even in their late passage. The range of cell types that can be converted to extended life cell lines, according to the present invention, is based on the availability of primary cells or the ability to isolate a primary cell from the organ in question. In this regard, the inventive methodology is not limited to cell types amenable to transformation. In addition to the cell types already mentioned, the present invention can be applied to pancreatic beta cells, human liver and kidney cells, and human hematopoietic stem cells, among others.
The methodology of the present invention has been used to develop an extended life cell line from human monocyte/macrophage cells, which are normally non-dividing. In all these instances, stable cell lines were obtained with a very high efficiency, either comparable to or better than the efficiency using retroviral vectors.
The present invention finds application as well in both ex vivo and in vivo gene therapies, where genetic material is transferred into specific cells of a patient. Ex vivo gene therapy entails the removal of the relevant target cells from the body, transduction of the cells in vitro, and subsequent reintroduction of the modified cells into the patient.
A gene therapy pursuant to the present invention could involve an ex vivo introduction, into a particular cell type from the patient, of a polynucleotide coding for a correcting protein which can be produced in functional form by the targeted cell type. Genes suitable for expression in this regard include an adenosine deaminase gene, a globin gene, an LDL receptor gene, and a glucose cerebrosidase gene.
Different kinds of gene-therapy applications require either stable or transient gene expression. The method of the present invention is advantageous in that it can be used in gene therapy requiring either stable gene expression or transient gene expression. Transient expression of a foreign gene is preferred when expression of the exogenous product is needed only for a short period of time; thereafter, rapid clearance of the gene product and its vector is desirable. Transient expression is also desirable when the prolonged effects of the exogenous protein's expression are unknown. Stable expression in gene therapy is needed when the patient has a genetic defect that is incompatible with life. Such genetic defects include but are not limited to cystic fibrosis, Tay Sachs and cancer. Mulligan, Science, 260: 926 (1993).
A gene therapy pursuant to the present invention also could involve an in vivo introduction of a structural DNA into cells of a patent's body. For stable transfer of genes into a target tissue using this method, the ligand to the target receptor will be conjugated to the synthetic polypeptide. The polypeptide-ligand combination can be complexed to a polynucleotide coding for the needed protein and then introduced into the host organism through blood circulation. When this complex reaches the target tissue, the whole complex will be taken up by cells containing the corresponding receptor for the ligand through receptor mediated process. Because of the NLS in the polypeptide-ligand complex, the complex will enter into the nucleus, resulting in a stable integration of the introduced gene into the host chromosome and, thereby, a correction of the genetic defect in the host. Cell-specific receptors are well known to those of skill in the art, as are their ligands which can be used in complexes for receptor-mediated gene transfer. Michael, S. I, et al., J. Biol. Chem. 268: 6866 (1993). For example, when the liver is the tissue targeted for gene therapy, the DNA encoding corrective protein is complexed to a synthetic neoglycoprotein that will target the complex to the asialoglycoprotein receptor on hepatocytes. For example, a cell type specific receptor ligand such as asialoglycoprotein can be chemically linked to the transfection vector at the carboxyl terminal of the synthetic polypeptide molecule to deliver the foreign gene directly into liver cells. An additional hinge region can be incorporated into the molecule before chemically linking the polypeptide molecule to a cell-type specific ligand molecule, such as asialoglycoprotein or a cell-specific monoclonal antibody.
An example of a carrier useful for receptor-mediated gene transfer to liver is a synthetic glycoprotein in which bovine serum albumin (BSA) is covalently bound to poly L-lysine using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (ECD). Ferkol et al., FASEB 7: 1081 (1993). To produce a neoglycoprotein conjugate for use in targeting DNA to liver, a reaction mixture that contains about 170 mM galactose, 4 mM poly (L-lysine), 160 mM BSA and 10 mM EDC (pH 7.5) can be incubated for 48 hours at 22° C. DNA is complexed to the neoglycoprotein carrier in a 360:1 molar ratio. The carrier-DNA complexes are dialyzed against 150 mM sodium chloride before transfection.
Expression of a functional protein after transfection with DNA complexed to ligand alone is often transient. Ferkol et al., supra. The method of the present invention greatly improves the cell-specific targeting of receptor-mediated transfection by providing stable expression by increasing stable integration of a foreign DNA in the host cell using a synthetic polypeptide molecule of the present invention.
A variation of receptor-mediated gene transfer employs coupling a synthetic polypeptide as described above to monoclonal antibodies which recognize a cell surface antigen on the target cells. Maruyama et al., Proc. Nat'l Acad. Sci. USA 87: 5744 (1990). The coupled monoclonal antibody and synthetic polypeptide then are complexed with a DNA encoding the required or desired protein. This complex will target the DNA to the cells expressing the corresponding cell surface antigen. Any tissue of the human body can be targeted for the gene therapy of the present invention using the disclosed methods. A target tissue is suitable in this context so long as it is susceptible to genetic modification according to the present invention.
The present invention is further described with reference to the following examples, which are only illustrative and not limiting of the invention.
The DNA or other polynucleotide to be transfected, such as a plasmid containing a gene for a drug resistance marker or coding a protein needed for expression in the host cell, is complexed to a synthetic polypeptide molecule in different weight ratios in an isotonic buffer solution. For example the weight ratio of DNA:polypeptide can be between 1:1 and 10:1, although ratios outside of this range may be evaluated empirically for achieving the objects of the present invention. An isotonic buffer solution such as Hanks buffered salt solution or HEPES buffered saline may be used for complexing DNA to polypeptide.
While the complex is formed, the cells that are to be transfected either remain attached to a substratum, such as a tissue culture dish, or are pelleted (for cells that grow in suspension). The cells are treated with a hypertonic primer solution, such as a concentration of 0.3M-0.6M sucrose and 10% PEG in either Tris-HCl or HEPES (pH 7.2) buffered solution, for 3-5 mins at room temperature. The primer solution then is removed.
After the DNA-polypeptide complex is formed, it is made hypotonic. The complex solution is hypotonic when it has a lesser osmotic pressure than a 0.15M or 0.9% solution of NaCl. For example, the complex in isotonic buffer can be made 40-55% hypotonic or 0.075M simply by adding an amount of distilled water that is equal to the volume of the complex in isotonic buffer. The hypotonic complex solution then is added to the cells that have been treated with the primer solution. Cells remain in the hypotonic DNA-polypeptide solution for 3-4 minutes. Fresh medium then is added to the cells to rinse away excess DNA-polypeptide solution. Thereafter, the cells are grown normally.
An example of an synthetic polypeptide molecule of the present invention is one consisting of the amino acid sequence PKKKRKVSGGGGGKKKKKKKKKKKK(SEQ ID NO:56). Such a peptide can be synthesized, using standard methods of peptide production, and purified by standard methods using high pressure liquid chromatography (HPLC).
Transfected cells are grown in regular growth medium for 48 hours, and then plated in selective medium containing 400 μg/ml of G418. Cells were plated at a density of 100-1000 cells per 60 cm2 dish. The number of G418-resistant colonies was determined two weeks after the initiation of selection. Other selectable markers, such as pHyg, may be used to achieve the results of the instant invention. K. Blochlinger, et al., Mol. Cell. Biol. 4: 2929 (1984).
This method gave a stable-transfection efficiency of 5-10%. Similar results were obtained using either G418 or hygromycin selection. In general the stable transfection efficiency achieved by the method of the instant invention is a few orders of magnitude greater than prior art methods. The instant invention's5-10% efficiency is several orders of magnitude better than the efficiency of the DNA-CaPO4 co-precipitation method and at least equal or 5 times greater than the fairly high 1-10% level of stable transfection efficiency achieved by viral based methods. See Table II.
TABLE II | |||
Method | Stable Transfection Efficiency | ||
Peptide-Mediated | 5-10% | ||
Gene Transfer | |||
Retroviral vectors | 1-10% | ||
Non-viral methods | <2% | ||
(e.g., CaPO4) | |||
That the transfectants of the instant invention are stable is shown by the following example. When G418 resistant colonies were grown without selection for variable period of times, and then tested for resistance to the antibiotic by plating the cells under clonal conditions, the same number of colonies were obtained both with and without G418. This result indicates that, once the cells are selected for the expression of the Neo gene, the resistance gene was retained stably in the chromosome.
Three different cell lines were used to test the efficiency of gene transfer of the new method. Mouse fibroblast cell line (L cells), mouse erythroleukemia cell line (C19TK), and COS cells. The COS cell line was used to establish conditions for transient gene expression. The eukaryotic expression vector, CH110, contains bacterial β-gal and was employed in these studies. The β-gal gene in CH110 is under the control of SV40 virus early promoter.
The COS cells were treated with primer and then exposed to DNA-polypeptide (2.5-5.0 μg) complex under hypotonic conditions. After this treatment, cells were returned to the normal growth condition. Transfected cells were grown at 37° C. for 48 hrs, and stained for the expression of the β-gal reporter gene. Forty to fifty percent of the cells were positive for the expression of the reporter gene.
Mouse L cells were transfected with eukaryotic expression vector containing the Neo gene, which codes for the antibiotic G418 resistance gene. L cells are sensitive to G418 at 400 μg/ml. Cells plated in 24-well tissue culture plates were then transfected with synthetic polypeptide complexed to the plasmid pRSV-Neo via the methodology of the present invention.
A mouse erythroleukemia cell line, C19TK, also was used as a representative cell line for testing the transfection efficiency of the present invention with respect to hematopoietic cells. The expression vector, pDR2, which carries a hygromycin-resistance gene, was used for these studies, C19TK cells are exquisitely sensitive for the antibiotic hygromycin. This cell line grows in suspension and, hence, was transfected in suspension.
Briefly, about million cells are spun down and the cell pellet is treated with primer. The cells are then exposed to DNA-polypeptide complex under hypotonic condition. Forty-eight hours after transfection, a known number of cells are plated in microtiter plates with hygromycin. The number of wells with growing population of cells was enumerated to determine the transfection efficiency. The stable transfection efficiency was about 1-5%, as compared to most of the other non-virus-based methods that are very poor. Thus, the method described herein is very efficient for stable transfection efficiency both for hematopoietic and non hematopoietic cell lines. Only some retrovirus based vectors give a transfection efficiency comparable to the efficiency obtained with the current method for hematopoietic cell lines. See Gilboa, et al. (1986), Miller, et al. (1986), Stuhlmann, et al. (1989), Miller, et al. (1989), and Zwiebel, et al. (1989), each cited above.
The gene transfer method of the present invention was used to generate extended life cell lines from different human primary cells. Most of the primary cells have a limited in vitro life span. The following cell types were employed to test the efficacy of the inventive method to generate extended-life cell lines by transfer of various oncogenes, either singly, in pairs of combinations, or combinations of more than two oncogenes. Rhim, J. S., et al., Oncogene 4: 1403 (1989).
The method of introducing genes into primary cells is the same as that described above for introducing genes into established cell lines, such as the mouse fibroblast cell line L cells and the mouse erythroleukemia cell line C19TK. The main difference is that the host cell is a primary cell isolated from different species, human or other mammalian species, and the primary cells have only a limited in vitro life span. The isolation of primary cells from various tissue sources are well known to those of skill in the art.
In order to extend the life of primary cells that are endogenously incapable of extended growth in vitro, the cells are transfected with different oncogenes, such as SV40 large T antigen, polyoma large T antigen, adenovirus E1A and E1B, v-fms, Bc12, myc and ras. The oncogenes can be used either alone, in pairs of various combinations, or in combinations of more than two oncogenes.
In addition, other genes that do not come under the category of oncogenes may be used. For example, genes that are important for DNA synthesis and normally active during the S phase of the cell cycle, such as the dihydrofolate reductase gene (DHFR), thymidine kinase gene, thymidylate synthetase gene, a DRTF1/E2F transcription factor encoding DNA, or DNA encoding the E2F transcription factor can be complexed to synthetic polypeptide and used to extend the life of primary cells. The human DHFR gene complexed to synthetic polypeptide can be introduced into primary cells to produce extended life cell lines. DNA encoding a transcription factor that is active during the S phase of the cell cycle are particularly useful in the method of the instant invention. La Thangue, N. B. Trends in Biochemical Sciences 19: 108 (1994); Johnson, D. G. et al., Nature 365: 349 (1993), the respective contents of which are hereby incorporated by reference.
Because untreated primary cells have only a limited life span in vitro, their ability to grow continuously in culture after treatment with the present invention served to select for extended life cell lines. No other drug selection markers need to be used to select for extended life cell lines derived from primary cells.
To produce extended life cells lines from primary cells, newly cultured primary cells were treated by the method of the present invention employing synthetic polypeptide conjugated with various oncogenes, such as SV40 large T antigen and/or Adeno E1A. The treated cells were plated in their appropriate growth media and passed after the cells reached confluency. A parallel set of a control untreated primary cells were cultured under the same growth conditions. Typically, control primary cells stop growing after about 4-10 passages, depending upon the cell type (cell split ratio was usually 1:4 by surface area). In contrast, continuously growing cell lines were obtained from different primary cell types described in the following examples.
Extended life cell lines containing the oncogene are identified by restriction cleavage. Southern analysis and/or Northern analysis using appropriate DNA probes.
The DNA of each transformed extended life cell line is analyzed by Southern hybridization to determine whether the cell lines carry the oncogenes used to establish such extended life cell lines. DNA is extracted from the cell lines and the nucleic acid pellet is re-suspended in 200 μl of 10 mM Tris-Cl pH 7.4, 0.1 mM EDTA, and 10 μg is digested with a specific restriction enzyme, electrophoresed through 1.0% agarose, and transferred to nitrocellulose. Southern, J. Mol. Biol. 98: 503 (1975). Filters are hybridized to a radioactively labelled DNA, encoding each of the oncogenes that gave rise to the corresponding extended life cell line, in the presence of 10% dextran sulfate. After overnight hybridization, the filters were washed twice in 2×SSC, 0.1% SDS at 64° C.
Each transformed extended life cell line is analyzed by Northern hybridization to determine whether the cell lines transcribe the oncogenes. Cells not containing the oncogene of interest will not demonstrate transcripts in a Northern analysis whereas cells containing the DNA of interest will demonstrate a detectable transcript. Also, an ELISA method was used to detect the presence of oncogene products in some of the extended life cell lines, using publicly available antibodies that recognize the corresponding oncogene protein.
The presence of SV40 large T antigen and adenovirus E1A gene products in the HUVEC extended life cell line, as detected by ELISA, are shown in t Table 2 III. Briefly, the cell line grown in a 96 well tissue culture plate is fixed with glutaraldehyde and paraformaldehyde. The cells are then treated with antibodies to the corresponding oncogenes. Thereafter, the cells are washed and then treated with a secondary antibody linked with to β-galactosidase. The cells are washed and then treated with a substrate for β-galactosidase. The reaction develops a product which is then measured using a microplate reader.
To determine whether the extended life cell line has maintained the parental cell line phenotype may be determined by a number of ways. Extended life cells lines containing the oncogene are assessed by Northern analysis using a DNA probes encoding a cell-specific protein. The cell-specific DNA probe is labeled with 32P-dCTP by nick translation pursuant, for example, to Rigby et al., J. Mol. Biol. 113: 237 (1977). Northern hybridization indicates that the extended life cell line is capable of transcribing the cell-specific protein.
Also, the maintenance of the parental phenotype in cells lines established according to the present invention can be determined by a number of biochemical methods, such as ELISA and enzyme assays, that determine the presence or function of a protein specific to the parental cell line. An antibody recognizing a protein produced only bythe by the parental cell line can be used in an ELISA or immunofluorescence assay. Cell-specific markers are well known to those of skill in the art. For example, albumin is a marker for hepatocytes, insulin is a marker for pancreatic beta islet cells, factor VIII is a marker for endothelial cells, actin and myosin are markers for smooth muscle cells, and non-specific esterass esterase is a marker for brain microglial cells. In Table II III, the parental phenotype of the extended life endothelial cells produced by the present method of the invention was verified by several ELISAs to determine the expression of cell-specific endothelial markers. The parental phenotype of the monocyte/macrophage extended life cell lines produced by the present method was verified using a lysozyme enzyme assay to measure macrophage specific markers.
Endothelial cells isolated from the human umbilical vein can only be cultured for a limited of passages, usually five to six. These cells were transfected with a combination of oncogenes, SV40 large T antigen and adenovirus E1A, or with another combination of genes. At least two oncogenes are needed to develop a truly transformed cell line. Ruley, H. E., et al., Nature 304: 602 (1983). For the instant invention, SV40 large T antigen combined with v-myc or ras or some an other oncogene can be used. When the gens encoding SV40 large T antigen is combined with either adenovirus E1A or E1B genes in the method of the instant invention, extended life cell lines may be produced from human umbilical vein endothelial cells. E1A or E1B or SV40 large T antigen alone did not give rise to established cell line with the high frequency obtained from using SV40 large T antigen in combination with E1A or E1B. Synthetic polypeptide complexed to DNA encoding either the SV40 large T antigen or polyoma large T antigen combined with the E2F1 transcription factor gens also produces extended life HUVEC cells lines.
Since the non-transfected primary cells normally grow in vitro only for a limited population doublings, cells that have taken up the oncogenes capable of generating extended life span cell lines were selected simply by repeated passage of the cells. When the transfected population of cells grows continuously, as compared to a control population of parental cells, it is reasonable to conclude that the oncogenes used are capable of generating extended life cells from a given cell type.
In HUVEC, for example, SV40 large T antigen and adenovirus E1A or E1B were effective in giving rise to a cell line. This cell line has now been growing in culture for 40 passages. In contrast, normal HUVECs stop growing by passage 7 or 8. Such cell lines arose with a high efficiency. It also is possible to generate cell lines using as few as a couple of hundred cells, grown either in a 24- or 48-well plate. These cells also have the same morphological appearance as the primary HUVEC and also display many of the biochemical properties characteristic of normal HUVEC.
Some of the properties that are characteristic of endothelial cells that were measured in the HUVEC extended life cell line are also listed in Table 2 III. These properties were also measured by ELISA using specific antibodies listed in the Table 2 III.
TABLE III |
ELISA assay for the expression of ELAM-12, VCAM-1, |
ICAM-1, SV40 large T antigen and |
adenovirus EIA by extended life HUVEC line |
O.D.mm |
Antibody | −IL-1 | +IL-1 | ||
Control | 0.071 | 0.069 | ||
Anti ELAM-1 | 0.212 | 1.016 | ||
Anti VCAM-1 | 0.146 | 0.520 | ||
Anti ICAM-1 | 0.422 | 1.524 | ||
Anti SV40 large T | 0.618 | — | ||
Anti EIA | 0.725 | — | ||
Adherent cells from human cord blood cells were transfected with different combinations of oncogenes in suspension using the method of the present invention. The resulting cells are selected in Granulocyte-Macrophage Colony Stimulating Factor (G-CSF). Control cells did not grow in culture, whereas growing populations of monocytes were obtained with several combinations of oncogenes. One preferred combination of polyoma large T antigen and adenovirus E1B encoding DNA produced extended life monocyte cells lines with somewhat higher efficiency than other combinations. Another preferred combination of SV40 large T or polyoma large T antigen and the E2F1 transcription factor gene produces monocyte extended life cells with high efficiency. The monocyte extended life cells also display many of the properties of normal monocytes, which illustrates the utility of the present invention in generating cell lines of hematopoietic origin.
The method of the instant invention has also been used to generate extended life cell lines using a specific combination of oncogenes. Human aortic smooth muscle cells were obtained from Clonetics Corporation (San Diego, Calif. U.S.A.) and transfected with several combination of oncogenes. The combination of polyoma large T antigen and EiB gave rise to a continuously growing population of smooth muscle cells. Another preferred combination of SV40 large T or polyoma large T antigen and the E2F1 transcription factor gene produces extended life human aortic smooth muscle cells with high efficiency. This cell line resembles the early passage primary aortic smooth muscle cells morphologically. The extended life human aortic smooth muscle cells also express smooth cell actin and myosin well beyond passage 20.
Primary cells from other species, such as rabbit and monkey, also have been used to generate cell lines. Transfection methods employed for primary cells from non-human species are similar to those used for human primary cells. When developing an extended life cell line from a new primary cell, several different combinations of available oncogenes should be tried. For example, at least five or six pairs of combinations of SV40 large T antigen, adenovirus E1A, adenovirus E1B, polyoma virus large T antigen or others available to those in the art. That combination of genes that gives rise to an extended life cell lines from a given primary cell type is determined as described in the above examples.
When the E2F1 transcription factor gens is complexed to synthetic polypeptide in combination with DNA encoding either the SV40 large T antigen or polyoma large T antigen, extended life cells lines can be produced from a variety of primary cell types, such as HUVEC, dermal microvascular endothelial cells, human aortic smooth muscle cells, and bone marrow monocyte/macrophage cells. Thus, the method of the present invention can identify a combination of oncogene DNAs that is highly efficient in producing extended life cells lines from the primary cells of various species. The present invention also comprehends a combination of an oncogene and an S-phase transcription factor gens which likewise is highly efficient in producing extended life cells lines from different types of primary cells.
The present invention provides a screening system for identifying sequences that influence the expression of cloned genes in various primary cell types from different species. The instant invention can identify cell type specific transcription and translational regulatory sequences. The sequence in question typically will be cloned into a vector containing a reporter gens, such as chloramphenicol acetyl transferass or luciferass, and then transfected into various cell types using the method described herein. Expression of the reporter gens determines the tissue specificity of the regulatory sequence.
Claims (14)
1. A transfection vector comprising a synthetic polypeptide linked electrostatically to a DNA structural sequence, forming a polypeptide-DNA complex, wherein said polypeptide is comprised of (A) a polymeric chain of basic amino acid residues, (B) a nuclear localization signal (NLS) peptide and (C) a hinge region of neutral amino acids that connects said polymeric chain and said NLS peptide.
2. The vector of claim 1 , wherein said polymeric chain is comprised of between 10 and 50 residues.
3. The vector of claim 1 , wherein said basic amino acid residues are selected from the group consisting of lysine, arginine and ornithine.
4. The vector of claim 1 , wherein said NLS peptide is selected from the group consisting of a Simian Virus 40 (SV40) large T antigen nuclear localization signal sequence, a polyoma large T antigen nuclear localization signal sequence, an adenovirus E1a nuclear localization signal sequence, and an adenovirus E1b nuclear localization signal sequence.
5. The vector of claim 1 , wherein said hinge region is comprised of between 6 and 50 amino acid residues.
6. The vector of claim 1 , wherein said neutral amino acids are selected from the group consisting of glycine, alanine, leucine and isoleucine.
7. The vector of claim 1 , wherein said NLS peptide is located at the amino terminus of said polypeptide and said polymeric basic amino acid chain is located at the carboxyl terminus.
8. The transfection vector of claim 1 , further comprising (D) a cell type-specific ligand molecule.
9. The transfection vector of claim 1 , wherein said DNA structural sequence comprises (a) a segment coding for SV40 large T antigen or polyoma large T antigen and (b) a transcription factor gene.
10. A vector according to claim 1 , wherein said DNA structural sequence comprises an oncogene.
11. A vector according to claim 10 , wherein said oncogene is selected from the group consisting of SV40 large T antigen, polyoma large T antigen, adenovirus E1A, adenovirus E1B, v-fms, BC12, myc, and ras.
12. A vector according to claim 1 , wherein said DNA structural sequence comprises a DNA sequence selected from the group consisting of a dihydrofolate reductase gens gene (DHFR), a thymidine kinase gens gene, a thymidylate synthetase gene gene, a DRTF1/E2F transcription factor-encoding DNA sequence, and an E2F transcription factor-encoding DNA sequence.
13. A process for producing a transformed mammalian cell line, comprising the step of transfecting a mammalian cell with a vector according to claim 1 , wherein said DNA structural sequence comprises a DNA sequence selected from the group consisting of a dihydrofolate reductase gene (DHFR), a thymidine kinase gene, a thymidylate synthetase gene gene, a DRTF1/E2F transcription factor-encoding DNA sequence, and an E2F transcription factor-encoding DNA sequence.
14. A process for producing a transformed mammalian cell line, comprising the step of transfecting a mammalian cell with a vector according to claim 1 , wherein said DNA structural sequence comprises an oncogene.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/404,979 USRE39220E1 (en) | 1994-05-11 | 1999-09-22 | Peptide-mediated gene transfer |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/240,514 US5670347A (en) | 1994-05-11 | 1994-05-11 | Peptide-mediated gene transfer |
US09/404,979 USRE39220E1 (en) | 1994-05-11 | 1999-09-22 | Peptide-mediated gene transfer |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/240,514 Reissue US5670347A (en) | 1994-05-11 | 1994-05-11 | Peptide-mediated gene transfer |
Publications (1)
Publication Number | Publication Date |
---|---|
USRE39220E1 true USRE39220E1 (en) | 2006-08-01 |
Family
ID=22906839
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/240,514 Ceased US5670347A (en) | 1994-05-11 | 1994-05-11 | Peptide-mediated gene transfer |
US09/404,979 Expired - Lifetime USRE39220E1 (en) | 1994-05-11 | 1999-09-22 | Peptide-mediated gene transfer |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/240,514 Ceased US5670347A (en) | 1994-05-11 | 1994-05-11 | Peptide-mediated gene transfer |
Country Status (5)
Country | Link |
---|---|
US (2) | US5670347A (en) |
EP (1) | EP0759996A1 (en) |
JP (1) | JPH10500022A (en) |
CA (1) | CA2189975A1 (en) |
WO (1) | WO1995031557A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030144230A1 (en) * | 1995-06-07 | 2003-07-31 | Pamela Hawley-Nelson | Peptide-enhanced transfections |
US20030212031A1 (en) * | 1995-01-23 | 2003-11-13 | Leaf Huang | Stable lipid-comprising durg delivery complexesand methods for their production |
US20040152098A1 (en) * | 2001-05-17 | 2004-08-05 | Yoshiki Katayama | Cellular signal-responsive gene transcriptional regulation system |
US20080153166A1 (en) * | 1995-01-23 | 2008-06-26 | Leaf Huang | Stable lipid-comprising drug delivery complexes and methods for their production |
AU2018201356B2 (en) * | 2009-11-06 | 2020-05-28 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive diagnosis of graft rejection in organ transplant patients |
Families Citing this family (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2715847B1 (en) * | 1994-02-08 | 1996-04-12 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
US5910487A (en) * | 1994-12-09 | 1999-06-08 | Genzyme Corporation | Cationic amphiphiles and plasmids for intracellular delivery of therapeutic molecules |
US5948767A (en) * | 1994-12-09 | 1999-09-07 | Genzyme Corporation | Cationic amphiphile/DNA complexes |
US6383814B1 (en) | 1994-12-09 | 2002-05-07 | Genzyme Corporation | Cationic amphiphiles for intracellular delivery of therapeutic molecules |
US5650096A (en) * | 1994-12-09 | 1997-07-22 | Genzyme Corporation | Cationic amphiphiles for intracellular delivery of therapeutic molecules |
US5840710A (en) * | 1994-12-09 | 1998-11-24 | Genzyme Corporation | Cationic amphiphiles containing ester or ether-linked lipophilic groups for intracellular delivery of therapeutic molecules |
US5747471A (en) * | 1994-12-09 | 1998-05-05 | Genzyme Corporation | Cationic amphiphiles containing steroid lipophilic groups for intracellular delivery of therapeutic molecules |
US5719131A (en) * | 1994-12-09 | 1998-02-17 | Genzyme Corporation | Cationic amphiphiles containing dialkylamine lipophilic groups for intracellular delivery of therapeutic molecules |
US5767099A (en) * | 1994-12-09 | 1998-06-16 | Genzyme Corporation | Cationic amphiphiles containing amino acid or dervatized amino acid groups for intracellular delivery of therapeutic molecules |
US6331524B1 (en) | 1994-12-09 | 2001-12-18 | Genzyme Corporation | Organ-specific targeting of cationic amphiphile / DNA complexes for gene therapy |
US5645837A (en) * | 1995-01-17 | 1997-07-08 | Thomas Jefferson University | Peptides that inhibit T cell activation and methods of using the same |
US6531455B1 (en) * | 1995-03-24 | 2003-03-11 | The Regents Of The University Of California | Delivery of polynucleotides by secretory gland expression |
US6051429A (en) | 1995-06-07 | 2000-04-18 | Life Technologies, Inc. | Peptide-enhanced cationic lipid transfections |
EP0874910A4 (en) * | 1995-06-07 | 1999-04-21 | Life Technologies Inc | Peptide-enhanced cationic lipid transfections |
US5811297A (en) * | 1996-03-07 | 1998-09-22 | Amba Biosciences, Llc | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, & in vitro generation of dendritic cells and macrophages |
US5935936A (en) * | 1996-06-03 | 1999-08-10 | Genzyme Corporation | Compositions comprising cationic amphiphiles and co-lipids for intracellular delivery of therapeutic molecules |
CA2264012C (en) | 1996-08-13 | 2011-04-26 | Chiron Corporation | Compositions and methods for polynucleotide delivery |
US5925628A (en) * | 1997-03-31 | 1999-07-20 | Genzyme Corporation | Cationic amphiphiles for intracellular delivery of therapeutic molecules |
US5912239A (en) * | 1997-04-04 | 1999-06-15 | Genzyme Corporation | Imidazole-containing cationic amphiphiles for intracellular delivery of therapeutic molecules |
US5948925A (en) * | 1997-05-06 | 1999-09-07 | Genzyme Corporation | Cationic amphiphiles containing linkers derived from neutral or positively charged amino acids |
US5952516A (en) * | 1997-05-08 | 1999-09-14 | Genzyme Corporation | Cationic amphiphiles containing multiplesteroid lipophilic groups |
US5942634A (en) * | 1997-05-09 | 1999-08-24 | Genzyme Corporation | Cationic amphiphiles for cell transfections |
US6635623B1 (en) | 1997-06-13 | 2003-10-21 | Baylor College Of Medicine | Lipoproteins as nucleic acid vectors |
US6153729A (en) * | 1997-06-20 | 2000-11-28 | St. Jude's Children's Research Hospital | Isolated nuclear matrix targeting peptide |
US6197332B1 (en) | 1997-08-13 | 2001-03-06 | Chiron Corporation | Lipid-conjugated polyamide compounds and related compositions and methods thereof |
IL138624A0 (en) * | 1998-03-24 | 2001-10-31 | Aventis Pharma Sa | Nucleic acid transfer vectors, compositions containing same and uses |
FR2776669B1 (en) * | 1998-03-24 | 2001-06-15 | Rhone Poulenc Rorer Sa | NUCLEIC ACID TRANSFER VECTORS, COMPOSITIONS CONTAINING SAME, AND USES THEREOF |
JP4854853B2 (en) | 1998-11-12 | 2012-01-18 | ライフ テクノロジーズ コーポレーション | Transfection drug |
EP1013770A1 (en) * | 1998-12-23 | 2000-06-28 | Université Louis Pasteur de Strasbourg | Non-viral transfection vector |
AU764660B2 (en) * | 1999-01-08 | 2003-08-28 | Lonza Cologne Ag | Use of the cell's own transport system for transferring nucleic acids across the nuclear membrane |
US6790948B1 (en) * | 1999-02-26 | 2004-09-14 | The United States Of America As Represented By The Department Of Health And Human Services | Tumor suppressor gene p33ING2 |
DE19933492B4 (en) * | 1999-07-16 | 2008-01-10 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Conjugate for the mediation of a cell-, compartment- or membrane-specific transport of active substances, process for its preparation and its use |
WO2001047496A1 (en) * | 1999-12-29 | 2001-07-05 | Mixson A James | Histidine copolymer and methods for using same |
US7070807B2 (en) * | 1999-12-29 | 2006-07-04 | Mixson A James | Branched histidine copolymers and methods for using same |
CN1433478A (en) * | 1999-12-30 | 2003-07-30 | 诺瓦提斯公司 | Novel colloid synthetic vectors for gene therapy |
AU2002348163A1 (en) * | 2001-11-02 | 2003-05-19 | Intradigm Corporation | Therapeutic methods for nucleic acid delivery vehicles |
AU2003205380A1 (en) * | 2002-02-01 | 2003-09-02 | Intradigm Corporation | Cationic polymers for use in therapeutic agent delivery |
US20060014695A1 (en) * | 2002-02-01 | 2006-01-19 | Hamidreza Ghandehari | Hpma-polyamine conjugates and uses therefore |
US7465708B2 (en) | 2002-11-25 | 2008-12-16 | Mixson A James | Branched cationic copolymers and methods for antimicrobial use |
EP1900820A1 (en) * | 2006-09-13 | 2008-03-19 | Amaxa AG | Nuclear transport agent and method for producing said agent |
WO2016011203A1 (en) | 2014-07-15 | 2016-01-21 | Life Technologies Corporation | Compositions with lipid aggregates and methods for efficient delivery of molecules to cells |
AU2019269066B2 (en) | 2018-05-18 | 2022-10-06 | F. Hoffmann-La Roche Ag | Targeted intracellular delivery of large nucleic acids |
CA3118146A1 (en) * | 2018-11-08 | 2020-05-14 | Summation Bio, Inc. | Mini-nucleosome core proteins and use in nucleic acid delivery |
CN115109801B (en) * | 2022-08-25 | 2022-11-22 | 深圳市先康达生命科学有限公司 | Slow virus transfection transfer aid and application thereof |
Citations (93)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4046722A (en) | 1975-02-04 | 1977-09-06 | G. D. Searle & Co. Limited | Immunological materials |
US4671958A (en) | 1982-03-09 | 1987-06-09 | Cytogen Corporation | Antibody conjugates for the delivery of compounds to target sites |
US4691006A (en) | 1983-03-04 | 1987-09-01 | Ohio State University | Antigenic modification of polypeptides |
US4847240A (en) | 1978-01-16 | 1989-07-11 | The Trustees Of Boston University | Method of effecting cellular uptake of molecules |
US4867973A (en) | 1984-08-31 | 1989-09-19 | Cytogen Corporation | Antibody-therapeutic agent conjugates |
US4870023A (en) | 1987-03-16 | 1989-09-26 | American Biogenetic Sciences, Inc. | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
US4897355A (en) | 1985-01-07 | 1990-01-30 | Syntex (U.S.A.) Inc. | N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
US4904582A (en) | 1987-06-11 | 1990-02-27 | Synthetic Genetics | Novel amphiphilic nucleic acid conjugates |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US4946787A (en) | 1985-01-07 | 1990-08-07 | Syntex (U.S.A.) Inc. | N-(ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
US4950599A (en) | 1987-01-29 | 1990-08-21 | Wolf Bertling | Method for exchanging homologous DNA sequences in a cell using polyoma encapsulated DNA fragments |
US5024939A (en) * | 1987-07-09 | 1991-06-18 | Genentech, Inc. | Transient expression system for producing recombinant protein |
US5047227A (en) | 1988-02-08 | 1991-09-10 | Cytogen Corporation | Novel and improved antibodies for site specific attachment of compounds |
US5071651A (en) | 1986-09-03 | 1991-12-10 | University Of Saskatchewan | Rotavirus nucleocapsid protein VP6 as a carrier in vaccine compositions |
US5084441A (en) | 1987-03-04 | 1992-01-28 | Shaw Jack M | Acetylated low density lipoproteins: a delivery system to phagocytic cells for stimulating immunologic response and host resistance |
US5108921A (en) | 1989-04-03 | 1992-04-28 | Purdue Research Foundation | Method for enhanced transmembrane transport of exogenous molecules |
US5135736A (en) | 1988-08-15 | 1992-08-04 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
US5156840A (en) | 1982-03-09 | 1992-10-20 | Cytogen Corporation | Amine-containing porphyrin derivatives |
US5166320A (en) | 1987-04-22 | 1992-11-24 | University Of Connecticut | Carrier system and method for the introduction of genes into mammalian cells |
US5169933A (en) | 1988-08-15 | 1992-12-08 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
US5171563A (en) | 1988-09-30 | 1992-12-15 | Neorx Corporation | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates |
US5182107A (en) | 1989-09-07 | 1993-01-26 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
US5196510A (en) | 1988-12-29 | 1993-03-23 | Cytogen Corporation | Molecular recognition units |
US5229490A (en) | 1987-05-06 | 1993-07-20 | The Rockefeller University | Multiple antigen peptide system |
WO1993018759A1 (en) * | 1992-03-20 | 1993-09-30 | Baylor College Of Medicine | A dna transporter system and method of use |
US5283342A (en) | 1992-06-09 | 1994-02-01 | Neorx Corporation | Biotinylated small molecules |
US5298422A (en) | 1991-11-06 | 1994-03-29 | Baylor College Of Medicine | Myogenic vector systems |
US5326856A (en) | 1992-04-09 | 1994-07-05 | Cytogen Corporation | Bifunctional isothiocyanate derived thiocarbonyls as ligands for metal binding |
US5354844A (en) * | 1989-03-16 | 1994-10-11 | Boehringer Ingelheim International Gmbh | Protein-polycation conjugates |
US5420105A (en) | 1988-09-23 | 1995-05-30 | Gustavson; Linda M. | Polymeric carriers for non-covalent drug conjugation |
US5449761A (en) | 1993-09-28 | 1995-09-12 | Cytogen Corporation | Metal-binding targeted polypeptide constructs |
US5482858A (en) | 1987-05-21 | 1996-01-09 | Creative Biomolecules, Inc. | Polypeptide linkers for production of biosynthetic proteins |
US5502037A (en) | 1993-07-09 | 1996-03-26 | Neuromed Technologies, Inc. | Pro-cytotoxic drug conjugates for anticancer therapy |
US5503833A (en) | 1991-02-04 | 1996-04-02 | University Of Saskatchewan | VP6 encapsulated drug delivery |
US5514546A (en) | 1993-09-01 | 1996-05-07 | Research Corporation Technologies, Inc. | Stem-loop oligonucleotides containing parallel and antiparallel binding domains |
US5521290A (en) | 1988-09-30 | 1996-05-28 | Neorx Corporation | Targeting substance-diagnostic/therapeutic agent conjugates having schiff base linkages and methods for their preparation |
US5521291A (en) | 1991-09-30 | 1996-05-28 | Boehringer Ingelheim International, Gmbh | Conjugates for introducing nucleic acid into higher eucaryotic cells |
US5541287A (en) | 1992-06-09 | 1996-07-30 | Neorx Corporation | Pretargeting methods and compounds |
US5547932A (en) | 1991-09-30 | 1996-08-20 | Boehringer Ingelheim International Gmbh | Composition for introducing nucleic acid complexes into higher eucaryotic cells |
US5574142A (en) | 1992-12-15 | 1996-11-12 | Microprobe Corporation | Peptide linkers for improved oligonucleotide delivery |
US5576201A (en) | 1994-01-14 | 1996-11-19 | Alexion Pharmaceuticals, Inc. | Retroviral vector particles for transducing non-proliferating cells |
US5578287A (en) | 1992-06-09 | 1996-11-26 | Neorx Corporation | Three-step pretargeting methods using improved biotin-active agent |
US5583020A (en) | 1992-11-24 | 1996-12-10 | Ribozyme Pharmaceuticals, Inc. | Permeability enhancers for negatively charged polynucleotides |
US5589392A (en) * | 1991-01-14 | 1996-12-31 | Stratagene | Nucleic acid construct encoding a nuclear transport peptide operatively linked to an inducible promoter |
US5593866A (en) | 1992-08-21 | 1997-01-14 | The University Of British Columbia | Cationic peptides and method for production |
US5599704A (en) | 1992-08-26 | 1997-02-04 | Ribozyme Pharmaceuticals, Inc. | ErbB2/neu targeted ribozymes |
US5607691A (en) | 1992-06-12 | 1997-03-04 | Affymax Technologies N.V. | Compositions and methods for enhanced drug delivery |
US5608060A (en) | 1992-06-09 | 1997-03-04 | Neorx Corporation | Biotinidase-resistant biotin-DOTA conjugates |
US5610052A (en) | 1992-08-26 | 1997-03-11 | Ribozyme Pharmaceuticals Inc. | Enzymatic RNA with activity to ras |
US5616490A (en) | 1992-12-07 | 1997-04-01 | Ribozyme Pharmaceuticals, Inc. | Ribozymes targeted to TNF-α RNA |
US5622854A (en) | 1992-05-14 | 1997-04-22 | Ribozyme Pharmaceuticals Inc. | Method and reagent for inhibiting T-cell leukemia virus replication |
US5631236A (en) | 1993-08-26 | 1997-05-20 | Baylor College Of Medicine | Gene therapy for solid tumors, using a DNA sequence encoding HSV-Tk or VZV-Tk |
US5631237A (en) | 1992-12-22 | 1997-05-20 | Dzau; Victor J. | Method for producing in vivo delivery of therapeutic agents via liposomes |
US5635380A (en) | 1994-01-18 | 1997-06-03 | Vanderbilt University | Enhancement of nucleic acid transfer by coupling virus to nucleic acid via lipids |
US5637481A (en) | 1993-02-01 | 1997-06-10 | Bristol-Myers Squibb Company | Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell |
US5637471A (en) | 1992-09-30 | 1997-06-10 | Yale University | Therapeutic and diagnostic methods and compositions based on transducin-like enhancer of split proteins and nucleic acids |
US5639275A (en) | 1993-08-12 | 1997-06-17 | Cytotherapeutics, Inc. | Delivery of biologically active molecules using cells contained in biocompatible immunoisolatory capsules |
US5639655A (en) | 1993-01-19 | 1997-06-17 | Ribozyme Pharmaceuticals, Inc. | PML-RARA targeted ribozymes |
US5641662A (en) | 1991-12-17 | 1997-06-24 | The Regents Of The University Of California | Transfection of lung via aerosolized transgene delivery |
US5648248A (en) | 1994-12-30 | 1997-07-15 | Boehringer Ingelheim International Gmbh | Methods for producing differentiated cells from immature hematopoietic cells |
US5652122A (en) | 1989-12-21 | 1997-07-29 | Frankel; Alan | Nucleic acids encoding and methods of making tat-derived transport polypeptides |
US5654006A (en) | 1993-02-12 | 1997-08-05 | Mayo Foundation For Medical Education And Research | Condensed-phase microparticle composition and method |
US5661025A (en) | 1992-04-03 | 1997-08-26 | Univ California | Self-assembling polynucleotide delivery system comprising dendrimer polycations |
US5668255A (en) | 1984-06-07 | 1997-09-16 | Seragen, Inc. | Hybrid molecules having translocation region and cell-binding region |
US5670483A (en) | 1992-12-28 | 1997-09-23 | Massachusetts Insititute Of Technology | Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor |
US5672479A (en) | 1992-08-28 | 1997-09-30 | Mount Sinai School Of Medicine | Methods for identifying compounds that bind to PUR protein |
US5674835A (en) | 1987-08-07 | 1997-10-07 | New England Medical Center Hospitals, Inc. | Papillomaviral expression inhibitors |
US5674704A (en) | 1993-05-07 | 1997-10-07 | Immunex Corporation | Cytokine designated 4-IBB ligand |
US5674977A (en) | 1993-02-05 | 1997-10-07 | The Ontario Cancer Institute | Branched synthetic peptide conjugate |
US5674703A (en) | 1992-12-02 | 1997-10-07 | Woo; Savio L. C. | Episomal vector systems and related methods |
US5683874A (en) | 1991-03-27 | 1997-11-04 | Research Corporation Technologies, Inc. | Single-stranded circular oligonucleotides capable of forming a triplex with a target sequence |
US5686264A (en) | 1991-11-05 | 1997-11-11 | Board Of Regents, The University Of Texas Sys | Compositions and methods relating to transdominant Tat mutants |
US5693531A (en) | 1993-11-24 | 1997-12-02 | The United States Of America As Represented By The Department Of Health And Human Services | Vector systems for the generation of adeno-associated virus particles |
US5714166A (en) | 1986-08-18 | 1998-02-03 | The Dow Chemical Company | Bioactive and/or targeted dendrimer conjugates |
US5717058A (en) | 1993-02-23 | 1998-02-10 | Somatogen, Inc. | Peptide inhibitors of tax-dependent transcription |
US5750390A (en) | 1992-08-26 | 1998-05-12 | Ribozyme Pharmaceuticals, Inc. | Method and reagent for treatment of diseases caused by expression of the bcl-2 gene |
US5750367A (en) | 1993-11-08 | 1998-05-12 | Baylor College Of Medicine | Human and mouse very low density lipoprotein receptors and methods for use of such receptors |
US5756353A (en) | 1991-12-17 | 1998-05-26 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol-and liposome-based delivery |
US5759517A (en) | 1991-11-08 | 1998-06-02 | Somatogen, Inc. | Hemoglobins as drug delivery agents |
US5763209A (en) | 1988-09-26 | 1998-06-09 | Arch Development Corporation | Methods and materials relating to the functional domains of DNA binding proteins |
US5770580A (en) | 1992-04-13 | 1998-06-23 | Baylor College Of Medicine | Somatic gene therapy to cells associated with fluid spaces |
US5773227A (en) | 1993-06-23 | 1998-06-30 | Molecular Probes, Inc. | Bifunctional chelating polysaccharides |
US5780444A (en) | 1991-12-13 | 1998-07-14 | Trustees Of Princeton University | Compositions and methods for cell transformation |
US5795870A (en) | 1991-12-13 | 1998-08-18 | Trustees Of Princeton University | Compositions and methods for cell transformation |
US5804604A (en) | 1989-12-21 | 1998-09-08 | Biogen, Inc. | Tat-derived transport polypeptides and fusion proteins |
US5807746A (en) | 1994-06-13 | 1998-09-15 | Vanderbilt University | Method for importing biologically active molecules into cells |
US5808036A (en) | 1993-09-01 | 1998-09-15 | Research Corporation Technologies Inc. | Stem-loop oligonucleotides containing parallel and antiparallel binding domains |
US5811297A (en) * | 1996-03-07 | 1998-09-22 | Amba Biosciences, Llc | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, & in vitro generation of dendritic cells and macrophages |
US5821234A (en) | 1992-09-10 | 1998-10-13 | The Board Of Trustees Of The Leland Stanford Junior University | Inhibition of proliferation of vascular smooth muscle cell |
US5827703A (en) | 1992-06-04 | 1998-10-27 | The Regents Of The University Of California | Methods and composition for in vivo gene therapy |
US5837533A (en) | 1994-09-28 | 1998-11-17 | American Home Products Corporation | Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent |
US5840674A (en) | 1990-11-01 | 1998-11-24 | Oregon Health Sciences University | Covalent microparticle-drug conjugates for biological targeting |
US5843643A (en) | 1992-02-21 | 1998-12-01 | Ratner; Paul L. | Site-specific transfection of eukaryotic cells using polypeptide-linked recombinant nucleic acid |
-
1994
- 1994-05-11 US US08/240,514 patent/US5670347A/en not_active Ceased
-
1995
- 1995-05-05 JP JP7529672A patent/JPH10500022A/en active Pending
- 1995-05-05 WO PCT/US1995/005224 patent/WO1995031557A1/en not_active Application Discontinuation
- 1995-05-05 EP EP95917721A patent/EP0759996A1/en not_active Withdrawn
- 1995-05-05 CA CA002189975A patent/CA2189975A1/en not_active Abandoned
-
1999
- 1999-09-22 US US09/404,979 patent/USRE39220E1/en not_active Expired - Lifetime
Patent Citations (104)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4046722A (en) | 1975-02-04 | 1977-09-06 | G. D. Searle & Co. Limited | Immunological materials |
US4847240A (en) | 1978-01-16 | 1989-07-11 | The Trustees Of Boston University | Method of effecting cellular uptake of molecules |
US4671958A (en) | 1982-03-09 | 1987-06-09 | Cytogen Corporation | Antibody conjugates for the delivery of compounds to target sites |
US5156840A (en) | 1982-03-09 | 1992-10-20 | Cytogen Corporation | Amine-containing porphyrin derivatives |
US4691006A (en) | 1983-03-04 | 1987-09-01 | Ohio State University | Antigenic modification of polypeptides |
US5668255A (en) | 1984-06-07 | 1997-09-16 | Seragen, Inc. | Hybrid molecules having translocation region and cell-binding region |
US4867973A (en) | 1984-08-31 | 1989-09-19 | Cytogen Corporation | Antibody-therapeutic agent conjugates |
US4897355A (en) | 1985-01-07 | 1990-01-30 | Syntex (U.S.A.) Inc. | N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
US4946787A (en) | 1985-01-07 | 1990-08-07 | Syntex (U.S.A.) Inc. | N-(ω,(ω-1)-dialkyloxy)- and N-(ω,(ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
US5714166A (en) | 1986-08-18 | 1998-02-03 | The Dow Chemical Company | Bioactive and/or targeted dendrimer conjugates |
US5071651A (en) | 1986-09-03 | 1991-12-10 | University Of Saskatchewan | Rotavirus nucleocapsid protein VP6 as a carrier in vaccine compositions |
US4950599A (en) | 1987-01-29 | 1990-08-21 | Wolf Bertling | Method for exchanging homologous DNA sequences in a cell using polyoma encapsulated DNA fragments |
US5084441A (en) | 1987-03-04 | 1992-01-28 | Shaw Jack M | Acetylated low density lipoproteins: a delivery system to phagocytic cells for stimulating immunologic response and host resistance |
US4870023A (en) | 1987-03-16 | 1989-09-26 | American Biogenetic Sciences, Inc. | Recombinant baculovirus occlusion bodies in vaccines and biological insecticides |
US5166320A (en) | 1987-04-22 | 1992-11-24 | University Of Connecticut | Carrier system and method for the introduction of genes into mammalian cells |
US5229490A (en) | 1987-05-06 | 1993-07-20 | The Rockefeller University | Multiple antigen peptide system |
US5482858A (en) | 1987-05-21 | 1996-01-09 | Creative Biomolecules, Inc. | Polypeptide linkers for production of biosynthetic proteins |
US4904582A (en) | 1987-06-11 | 1990-02-27 | Synthetic Genetics | Novel amphiphilic nucleic acid conjugates |
US5024939A (en) * | 1987-07-09 | 1991-06-18 | Genentech, Inc. | Transient expression system for producing recombinant protein |
US5674835A (en) | 1987-08-07 | 1997-10-07 | New England Medical Center Hospitals, Inc. | Papillomaviral expression inhibitors |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US5047227A (en) | 1988-02-08 | 1991-09-10 | Cytogen Corporation | Novel and improved antibodies for site specific attachment of compounds |
US5773583A (en) | 1988-04-08 | 1998-06-30 | Arch Development Corporation | Methods and materials relating to the functional domains of DNA binding proteins |
US5135736A (en) | 1988-08-15 | 1992-08-04 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
US5169933A (en) | 1988-08-15 | 1992-12-08 | Neorx Corporation | Covalently-linked complexes and methods for enhanced cytotoxicity and imaging |
US5420105A (en) | 1988-09-23 | 1995-05-30 | Gustavson; Linda M. | Polymeric carriers for non-covalent drug conjugation |
US5763209A (en) | 1988-09-26 | 1998-06-09 | Arch Development Corporation | Methods and materials relating to the functional domains of DNA binding proteins |
US5521290A (en) | 1988-09-30 | 1996-05-28 | Neorx Corporation | Targeting substance-diagnostic/therapeutic agent conjugates having schiff base linkages and methods for their preparation |
US5171563A (en) | 1988-09-30 | 1992-12-15 | Neorx Corporation | Cleavable linkers for the reduction of non-target organ retention of immunoconjugates |
US5196510A (en) | 1988-12-29 | 1993-03-23 | Cytogen Corporation | Molecular recognition units |
US5354844A (en) * | 1989-03-16 | 1994-10-11 | Boehringer Ingelheim International Gmbh | Protein-polycation conjugates |
US5108921A (en) | 1989-04-03 | 1992-04-28 | Purdue Research Foundation | Method for enhanced transmembrane transport of exogenous molecules |
US5182107A (en) | 1989-09-07 | 1993-01-26 | Alkermes, Inc. | Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates |
US5804604A (en) | 1989-12-21 | 1998-09-08 | Biogen, Inc. | Tat-derived transport polypeptides and fusion proteins |
US5670617A (en) | 1989-12-21 | 1997-09-23 | Biogen Inc | Nucleic acid conjugates of tat-derived transport polypeptides |
US5652122A (en) | 1989-12-21 | 1997-07-29 | Frankel; Alan | Nucleic acids encoding and methods of making tat-derived transport polypeptides |
US5674980A (en) | 1989-12-21 | 1997-10-07 | Biogen Inc | Fusion protein comprising tat-derived transport moiety |
US5747641A (en) | 1989-12-21 | 1998-05-05 | Biogen Inc | Tat-derived transport polypeptide conjugates |
US5840674A (en) | 1990-11-01 | 1998-11-24 | Oregon Health Sciences University | Covalent microparticle-drug conjugates for biological targeting |
US5589392A (en) * | 1991-01-14 | 1996-12-31 | Stratagene | Nucleic acid construct encoding a nuclear transport peptide operatively linked to an inducible promoter |
US5503833A (en) | 1991-02-04 | 1996-04-02 | University Of Saskatchewan | VP6 encapsulated drug delivery |
US5683874A (en) | 1991-03-27 | 1997-11-04 | Research Corporation Technologies, Inc. | Single-stranded circular oligonucleotides capable of forming a triplex with a target sequence |
US5547932A (en) | 1991-09-30 | 1996-08-20 | Boehringer Ingelheim International Gmbh | Composition for introducing nucleic acid complexes into higher eucaryotic cells |
US5521291A (en) | 1991-09-30 | 1996-05-28 | Boehringer Ingelheim International, Gmbh | Conjugates for introducing nucleic acid into higher eucaryotic cells |
US5686264A (en) | 1991-11-05 | 1997-11-11 | Board Of Regents, The University Of Texas Sys | Compositions and methods relating to transdominant Tat mutants |
US5298422A (en) | 1991-11-06 | 1994-03-29 | Baylor College Of Medicine | Myogenic vector systems |
US5756264A (en) | 1991-11-06 | 1998-05-26 | Baylor College Of Medicine | Expression vector systems and method of use |
US5759517A (en) | 1991-11-08 | 1998-06-02 | Somatogen, Inc. | Hemoglobins as drug delivery agents |
US5795870A (en) | 1991-12-13 | 1998-08-18 | Trustees Of Princeton University | Compositions and methods for cell transformation |
US5780444A (en) | 1991-12-13 | 1998-07-14 | Trustees Of Princeton University | Compositions and methods for cell transformation |
US5756353A (en) | 1991-12-17 | 1998-05-26 | The Regents Of The University Of California | Expression of cloned genes in the lung by aerosol-and liposome-based delivery |
US5641662A (en) | 1991-12-17 | 1997-06-24 | The Regents Of The University Of California | Transfection of lung via aerosolized transgene delivery |
US5843643A (en) | 1992-02-21 | 1998-12-01 | Ratner; Paul L. | Site-specific transfection of eukaryotic cells using polypeptide-linked recombinant nucleic acid |
WO1993018759A1 (en) * | 1992-03-20 | 1993-09-30 | Baylor College Of Medicine | A dna transporter system and method of use |
US5661025A (en) | 1992-04-03 | 1997-08-26 | Univ California | Self-assembling polynucleotide delivery system comprising dendrimer polycations |
US5527885A (en) | 1992-04-09 | 1996-06-18 | Cytogen Corporation | Bifunctional isothiocyanate derived thiocarbonyls as ligands for metal binding |
US5326856A (en) | 1992-04-09 | 1994-07-05 | Cytogen Corporation | Bifunctional isothiocyanate derived thiocarbonyls as ligands for metal binding |
US5585468A (en) | 1992-04-09 | 1996-12-17 | Cytogen Corporation | Substituted thioureas as bifunctional chelators |
US5792751A (en) | 1992-04-13 | 1998-08-11 | Baylor College Of Medicine | Tranformation of cells associated with fluid spaces |
US5770580A (en) | 1992-04-13 | 1998-06-23 | Baylor College Of Medicine | Somatic gene therapy to cells associated with fluid spaces |
US5622854A (en) | 1992-05-14 | 1997-04-22 | Ribozyme Pharmaceuticals Inc. | Method and reagent for inhibiting T-cell leukemia virus replication |
US5827703A (en) | 1992-06-04 | 1998-10-27 | The Regents Of The University Of California | Methods and composition for in vivo gene therapy |
US5630996A (en) | 1992-06-09 | 1997-05-20 | Neorx Corporation | Two-step pretargeting methods using improved biotin-active agent conjugates |
US5541287A (en) | 1992-06-09 | 1996-07-30 | Neorx Corporation | Pretargeting methods and compounds |
US5578287A (en) | 1992-06-09 | 1996-11-26 | Neorx Corporation | Three-step pretargeting methods using improved biotin-active agent |
US5283342A (en) | 1992-06-09 | 1994-02-01 | Neorx Corporation | Biotinylated small molecules |
US5608060A (en) | 1992-06-09 | 1997-03-04 | Neorx Corporation | Biotinidase-resistant biotin-DOTA conjugates |
US5607691A (en) | 1992-06-12 | 1997-03-04 | Affymax Technologies N.V. | Compositions and methods for enhanced drug delivery |
US5593866A (en) | 1992-08-21 | 1997-01-14 | The University Of British Columbia | Cationic peptides and method for production |
US5750390A (en) | 1992-08-26 | 1998-05-12 | Ribozyme Pharmaceuticals, Inc. | Method and reagent for treatment of diseases caused by expression of the bcl-2 gene |
US5801158A (en) | 1992-08-26 | 1998-09-01 | Ribozyme Pharmaceuticals, Inc. | Enzymatic RNA with activity to RAS |
US5610052A (en) | 1992-08-26 | 1997-03-11 | Ribozyme Pharmaceuticals Inc. | Enzymatic RNA with activity to ras |
US5599704A (en) | 1992-08-26 | 1997-02-04 | Ribozyme Pharmaceuticals, Inc. | ErbB2/neu targeted ribozymes |
US5672479A (en) | 1992-08-28 | 1997-09-30 | Mount Sinai School Of Medicine | Methods for identifying compounds that bind to PUR protein |
US5821234A (en) | 1992-09-10 | 1998-10-13 | The Board Of Trustees Of The Leland Stanford Junior University | Inhibition of proliferation of vascular smooth muscle cell |
US5637471A (en) | 1992-09-30 | 1997-06-10 | Yale University | Therapeutic and diagnostic methods and compositions based on transducin-like enhancer of split proteins and nucleic acids |
US5583020A (en) | 1992-11-24 | 1996-12-10 | Ribozyme Pharmaceuticals, Inc. | Permeability enhancers for negatively charged polynucleotides |
US5674703A (en) | 1992-12-02 | 1997-10-07 | Woo; Savio L. C. | Episomal vector systems and related methods |
US5616490A (en) | 1992-12-07 | 1997-04-01 | Ribozyme Pharmaceuticals, Inc. | Ribozymes targeted to TNF-α RNA |
US5574142A (en) | 1992-12-15 | 1996-11-12 | Microprobe Corporation | Peptide linkers for improved oligonucleotide delivery |
US5631237A (en) | 1992-12-22 | 1997-05-20 | Dzau; Victor J. | Method for producing in vivo delivery of therapeutic agents via liposomes |
US5670483A (en) | 1992-12-28 | 1997-09-23 | Massachusetts Insititute Of Technology | Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor |
US5639655A (en) | 1993-01-19 | 1997-06-17 | Ribozyme Pharmaceuticals, Inc. | PML-RARA targeted ribozymes |
US5637481A (en) | 1993-02-01 | 1997-06-10 | Bristol-Myers Squibb Company | Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell |
US5674977A (en) | 1993-02-05 | 1997-10-07 | The Ontario Cancer Institute | Branched synthetic peptide conjugate |
US5654006A (en) | 1993-02-12 | 1997-08-05 | Mayo Foundation For Medical Education And Research | Condensed-phase microparticle composition and method |
US5717058A (en) | 1993-02-23 | 1998-02-10 | Somatogen, Inc. | Peptide inhibitors of tax-dependent transcription |
US5674704A (en) | 1993-05-07 | 1997-10-07 | Immunex Corporation | Cytokine designated 4-IBB ligand |
US5773227A (en) | 1993-06-23 | 1998-06-30 | Molecular Probes, Inc. | Bifunctional chelating polysaccharides |
US5502037A (en) | 1993-07-09 | 1996-03-26 | Neuromed Technologies, Inc. | Pro-cytotoxic drug conjugates for anticancer therapy |
US5639275A (en) | 1993-08-12 | 1997-06-17 | Cytotherapeutics, Inc. | Delivery of biologically active molecules using cells contained in biocompatible immunoisolatory capsules |
US5631236A (en) | 1993-08-26 | 1997-05-20 | Baylor College Of Medicine | Gene therapy for solid tumors, using a DNA sequence encoding HSV-Tk or VZV-Tk |
US5514546A (en) | 1993-09-01 | 1996-05-07 | Research Corporation Technologies, Inc. | Stem-loop oligonucleotides containing parallel and antiparallel binding domains |
US5808036A (en) | 1993-09-01 | 1998-09-15 | Research Corporation Technologies Inc. | Stem-loop oligonucleotides containing parallel and antiparallel binding domains |
US5449761A (en) | 1993-09-28 | 1995-09-12 | Cytogen Corporation | Metal-binding targeted polypeptide constructs |
US5798209A (en) | 1993-11-08 | 1998-08-25 | Baylor College Of Medicine | Human and mouse very low density lipoprotein receptors and methods for use of such receptors |
US5750367A (en) | 1993-11-08 | 1998-05-12 | Baylor College Of Medicine | Human and mouse very low density lipoprotein receptors and methods for use of such receptors |
US5693531A (en) | 1993-11-24 | 1997-12-02 | The United States Of America As Represented By The Department Of Health And Human Services | Vector systems for the generation of adeno-associated virus particles |
US5576201A (en) | 1994-01-14 | 1996-11-19 | Alexion Pharmaceuticals, Inc. | Retroviral vector particles for transducing non-proliferating cells |
US5635380A (en) | 1994-01-18 | 1997-06-03 | Vanderbilt University | Enhancement of nucleic acid transfer by coupling virus to nucleic acid via lipids |
US5807746A (en) | 1994-06-13 | 1998-09-15 | Vanderbilt University | Method for importing biologically active molecules into cells |
US5837533A (en) | 1994-09-28 | 1998-11-17 | American Home Products Corporation | Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent |
US5648248A (en) | 1994-12-30 | 1997-07-15 | Boehringer Ingelheim International Gmbh | Methods for producing differentiated cells from immature hematopoietic cells |
US5811297A (en) * | 1996-03-07 | 1998-09-22 | Amba Biosciences, Llc | Immortalized hematopoietic cell lines, cell system thereof with stromal cells, in vitro, ex vivo and in vivo uses, & in vitro generation of dendritic cells and macrophages |
Non-Patent Citations (116)
Title |
---|
"Nicholas B. La Thangue, "DRTF1/E2F: An Expanding Family Of Heterodimeric Transcription Factors Implicated In Cell-Cycle Control", [Clonexpress, Inc., pp. 108-114, (1994)."] Trends Biohem Sci., pp. 108-114, (1994). |
Aumailley, M., et al. (1989) Cell attachment properties of collagen type VI and Arg-Gly-dependent binding to its alpha2(VI) and alpha3(VI) chains. Experimental Cell Research 181:463-474. |
Berardi, A.C., et al. (1995) Functional isolation and characterization of human hematopoietic stem cells. Science 267:104-108. |
Cameron, P.U., et al. (1992) Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to DC4 T cells. Science 257:383-387. |
Carrasco, L., et al. (1982) Modification of membrane permeability in vaccinia virus-infected cells. Virology 117:62-69. |
Caux, C., et al. (1992) GM-CSF and TNF-alpha cooperate in the generation of dendritic langerhans cells. Nature 360:258-261. |
Caux, C., et al. (1994) Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263-1272. |
Chaney, et al. (1986) High-frequency transfection of CHO cells using polybrene. Somatic Cell and Molecular Genetics 12:237-244. * |
Chilinase A, accession #P29022, NCBI database, entered Dec. 1, 1992. |
Citosky. V., et al. (1992) Nuclear localization of agrobacterium VirE2 protein in plant cells. Science 256:1802-1805. |
Cotten, M., et al. (1990) Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. 87:4033-4037. |
Cotten, M., et al. (1992) High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles. Proc. Natl. Acad. Sci. 89:6094-6098. |
Coulombel, L., et al. (1983) Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoictic progenitors in the adherent layer. Blood 62:291-297. |
Curiel, D., et al. (1991) Adenovirus enhancement of transferrin-polylysine-mediated gene delivery. Proc. Natl. Acad. Sci. 88:8850-8854. |
Dedhar, S., et al. (1987) A cell surface receptor complex for collagen type 1 recognizes the Arg-Gly-Asp sequence. J. Cell. Biol. 104:585-593. |
DeRobertis, E., et al. (1978) Intracellular migration of nuclear proteins in xenopus oocytes. Nature 272:254-256. |
Epand. R., et al. (1992) Peptide models for the membrane destabilizing actions of viral fusion proteins. Biopolymers 32:309-314. |
Essentials of Molecular Biology (4<SUP>th </SUP>Ed.) Malacinski, Jones and Barlett, Eds. (2003). |
Eytan, G. (1982) Use of liposomes for reconstitution of biological functions. Biochimica et Biophysica Acta 694: 185-202. |
Fink, D., et al. (1992) In vivo expression of B-galactosidase in hippocampal neurons by HSV-mediated gene transfer. Human Gene Therapy 3:11-19. * |
Friedlander, D., et al. (1988) Functional mapping of cytotactin: proteolytic fragments active in cell-substrate adhesion. The Journal of Cell Biology 107(6):2329-2340. |
Garcia-Bustos, J., et al. (1991) Nuclear protein localization. Biochimica et BioPhysica Acta 1071:83-101. * |
Gardner, J., et al. (1985) Interaction of fibronectin with its receptor on platelets. Cell 42:439-448. |
Gilboa et al. (1986) Transfer and expression of cloned genes using retroviral vectors. Biotechniques 4:504-512. * |
Gould-Fogerite, S., et al. (1989) Chimerasome-mediated gene transfer in vitro and in vivo. Gene 84:429-438. |
Grant, D., et al. (1989) Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58:933-943. |
Haensler, J., and Szoka, Jr., F.C. (1993) Synthesis and Characterization of a Trigalactosylated Bisacridine Compound to Target DNA to Hepatocytes. Bioconjugate Chem. 4, 85-93. |
Haverstick, D., et al. (1985) Inhibition of platelet adhesion to fibronectin, fibrinogen, and von willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin. Blood 66(4):946-952. |
Hermonat, P., et al. (1984) Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Prod. Natl. Acad. Sci. 81:6466-6470. * |
Holter, W., et al. (1989) Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid. Experimental Cell Research 184:546-551. * |
Humphries M., et al. (1986) Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. The Journal of Cell Biology 103(6):2637-2647. |
Humphries, M., et al. (1987) Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. The Journal of Biological Chemistry 262(14):6886-6892. |
Isaka, Y., et al. (1993) Glomerulosclerosis induced by in vivo transfection of transforming growth factor-B or platelet-derived growth factor gene into the rat kidney, J. Clin. Invest. 92:2597-2601. |
J. S. Huston et al, Protein Engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichai coli; 1988, Proc. Natl. Acad. Sci USA, 85: 5879-5883. |
J. S. Huston et al, Protein Engineering of Single-Chain Fv Analogs and Fusion Proteins, Methods in Enzymology, 1991, 203:46-88. |
Jenster, G., et al. (1993) Nuclear import of the human androgen receptor. Biochem. J. 293:761-768. * |
Johnson, D., et al. (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365:349-352. * |
Kalderson, D., et al. (1984) A short amino acid sequence able to specify nuclear location. Cell 39:499-509. |
Kamata, H., et al. (1994) Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Research 22(3):536-537. |
Kaneda, Y., et al. (1987) The improved efficient moethod for introducing macromolecules into cells using HVJ (sendai virus) liposomes with gangliosides. Experimental Cell Research 173:56-69. |
Kaneda, Y., et al. (1989) Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science 243:375-378. |
Kaneda, Y., et al. (1989) Introduction and expression of the human insulin gene in adult rat liver. The Journal of Biological Chemistry 264(21):12126-12129. |
Kato, K., et al. (1991) Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats. The Journal of Biological Chemistry 266:22071-22074. |
Kato, K., et al. (1991) Expression of hepatitis B virus surface antigen in adult rat liver. The Journal of Biological Chemistry 266:3361-3364. |
Klappe, K., et al. (1986) Parameters affecting fusion between sendai virus and liposomes. Role of viral protein, liposome composition, and pH. Biochemistry 25: 8252-8260. |
L. Stryer, Biochemistry, 3<SUP>rd </SUP>Ed. Freeman & Co., NY, 1988 (p. 18-20, and 28-29). |
La Thanque, N. (1994) DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends in Biochem. Sci. 19:108-114. * |
Lanford, R., et al. (1986) Indeuction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell 46:575-582. |
Lapidot, M., et al. (1990) Fusion-mediated microinjection of liposome-enclosed DNA into cultured cells with the aid of influenza virus glycoproteins. Experimental Cell Research 189:241-246. |
Lau, Y., et al. (1984) Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector. Proc. Natl. Acad. Sci. 81:414-418. * |
Lawler, J., et al. (1988) Cell attachment to thrombospondin: the role of ARG-GLY-ASP calcium and integrin receptors. J. Cell. Biol. 107:2351-2361. |
Liljestrom, P., et al. (1991) A new generation of animal cell expression vectors based on the semliki forest virus replicon. Bio/Technology 9:1356-1361. |
Mannino, R., et al. (1988) Liposome mediated gene transfer. BioTechniques 6:682-690. * |
Marsh, M., et al. (1983) Interactions of semliki forest virus spike glycoprotein rosettes and vesicles with cultured cells. The Journal of Cell Biology 96:455-461. |
Maruyama, K., et al. (1990) Lipid composition is important for highly efficient target binding and retention of immunoliposomes. Proc. Natl. Acad. Sci. 87:5744-5748. |
Mason, P., et al. (1994) RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway. Proc. Natl. Acad. Sci. 91:1932-1936. |
Matheny, C., et al. (1984) The nuclear localization signal of NGFI-A is located within the zinc finger DNA binding domain. The Journal of Biological Chemistry 269:8176-8181. * |
McNally, M., et al. (1988) Optimizing electroporation parameters for a variety of human hematopoietic cell lines. Biotechniques 6:882-886. * |
Michael, S., et al. (1993) Binding-incompetent adenovirus facilitates molecular conjugate-mediated gene transfer by the receptor-mediated endocytosis pathway. The Journal of Biological Chemistry 268:6866-6869. * |
Miller et al. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7:980-988. * |
Miller, D., et al. (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Molecular and Cellular Biology 6:2895-2902. * |
Moreland, R.B. et al. (1987) Amino Acid Sequences That Determine the Nuclear Localization of Yeast Histone 2B, Mol. Cell Biol. 7, 4048-4057. |
Morin, N., et al. (1989) Nuclear localization of the adenovirus DNA-binding protein: requirement for two signals and complementation during viral infection. Molecular and Cellular Biology 9(10):4372-4380. * |
Mulligan, R.C. (1993) The basic science of gene therapy. Science 260:926-932. |
Neugebauer, J. (1990) Detergents: an overview. Methods in Enzymology 182:239-253. |
Okada, C., et al. (1982) Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29:33-41. * |
Otero, J., et al. (1987) Proteins are cointernalized with virion particles during early infection. Virology 160:75-80. |
P. A. Silver, How Proteins Enter the Nucleus, 1991, Cell, 64:489-497. |
P. Argos, An Investigation of Oligopeptides Linking Domains in Protein Tertiary Structures and Possible Candidates for General Gene Fusion, 1990, Mol. Biol., 211: 943-958. |
Phalen, T., et al. (1991) Cholesterol is required for inection by semliki forest virus. The Journal of Cell Biology 112(4):615-623. |
Pierschbacher, M., et al. (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309:30-33. |
Pierschbacher, M., et al. (1987) Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. The Journal of Biological Chemistry 262:17294-17298. |
Rafii, S., et al. (1994) Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 84:10-19. |
Rafii, S., et al. (1995) Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 85:3353-3363. |
Raikhel, N. (1992) Nuclear targeting in plants. Plant Physiol 100:1627-1632. * |
Remy, J., et al. (1995) Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: A stage toward artifical viruses. Proc. Natl. Acad. Sci. 92:1744-1748. |
Rhim, J., et al. (1989) Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene. Oncogene 4:1403-1409. * |
Roecklein, B.A., et al. (1995) Functionally distinct human marrow stromal cell lines immortalized by transductioin with the human papilloma virus E6/E7 genes. Blood 85:997-1005. |
Roget A to Z (1994). Harper Collins Pub. |
Rosenkranz, A.A., et al., (19920 Experimental Cell Res. 199, 323-329. |
Rothstein, L., et al. (1985) Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures. Blood 65(3):744-752. * |
Roux, et al. (1991) Oncogene 6(11):2155-2160. |
Ruoslahti, E., et al. (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491-496. |
Sadler, et al. (1989) A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, and Escherichia coli heat shock protein. J. Cell. Biol. 109:2665-2675. |
Sands, J. (1986) Virucidal activity of cetyltrimethylammonium bromide below the critical micelle concentration. FEMS Microbiology Letters 36:261-263. |
SC35, accession #Q01130, NCBI database, entered Apr. 1, 1993. |
Scheule, R. (1986) Novel preparation of functional sindbis virosomes. Biochemistry 25:4223-4232. |
Schlegel, R., et al. (1983) Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site. Cell 32:639-646. |
Schlegel, R., et al. (1985) Biologically active peptides of the vesicular stomatitis virus glycoprotein. Journal of Virology 53(1):319-323. |
Schreiber, V., et al. (1992) The human poly(ADP-ribose)polymerase nuclear localization signal in a bipartite element functionally separate from DNA binding and catalytic activity. The EMBO Journal (9):3263-3269. * |
Schwarzbaum, S., et al. (1984) The generation macrophage-like cell lines by transfection with SV40 origin defective DNA. J. Immunol. 132:1158-1162. |
Singh, P., et al. (1994) Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic trnasformation. EMBO Journal 13:3329-3338. |
Steff, A-M., et al. (1996) Isolation and characterization of c-fos-expressing murine bone marrow stromal cell lines supporting myeloid differentiation. Leukemia 10:505-513. |
Stegmann, T., et al. (1989) Protein-mediated membrane fusion. Annu. Rev. Biophys. Chem. 18:187-211. |
Stuhlmann, H., et al. (1989) Construction and properties of replication-competent murine retrovial vectors encoding methotrexate resistance. Molecular and Cellular Biology 9(3):100-108. * |
Suzuki, S., et al. (1985) Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and firbonectin. The EMBO Journal 4:2519-2524. |
Takai, T., et al. (1990) DNA transfection of mouse lymphoid cells by the combination of DEAE-dextran-mediated DNA uptake and osmotic shock procedure. Biochimica et Biophysica Acta 1048:105-109. * |
Tikchonenko, T., et al. (1988) Transfer of condensed viral DNA into eukaryotic cells using proteoliposomes. Gene 63:321-330. |
Tomita, N., et al. (1992) Direct in vivo gene introduction into rat kidney. Biochemical and Biophysical Reserach Communications. 186:129-134. |
Tratschin, J., et al. (1985) Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells. Molecular and Cellular Biology 5:3251-3260. * |
Vaananen, P., et al. (1980) Fusion and haemolysis of erythrocytes caused by three togaviruses: semliki forest, sindbis and rubella. J. Gen. Virol. 46:467-475. |
van der Krol, A., et al. (1991) The basic domain of planit B-ZIP proteins facilitates import of a reporter protein into plant nuclei. The Plant Cell 3:667-675. * |
Verfaillie, C.M. (1993) Soluble factor(s) produced by human bone marros stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood 82:2045-2053. |
Wagner, E., et al. (1991) Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc. Natl. Acad. Sci. 88:4255-4259. |
Wagner, E., et al. (1992) Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. 89:6099-6103. |
Wagner, E., et al. (1992) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. 89:7934-7938. |
Walker, C., et al. (1992) Cationic lipids direct a viral glycoprotein into the class I major histocompatibility complex antigen-presentation pathway. Proc. Natl. Acad. Sci. 89:7915-7918. |
Wayner, E., et al. (1989) Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. The Journal of Cell Biology 109:1321-1330. |
Wickham, TJ., et al. (1995) Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs. Gene Therapy 2:750-756. |
Wu, G., et al. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. The Journal of Biological chemistry 262:4429-4432. |
Wu, G., et al. (1988) Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro. Biochemistry 27:887-892. |
Wu, G., et al. (1988) Receptor-mediated gene delivery and expression in vivo. The Journal of Biological Chemistry 263:14621-14624. |
Yoshimura, K., et al. (1993) Adenovirus-mediated augmentation of cell transfection with unmodified plasmid vectors. The Journal of Biological Chemistry 268:2300-2303. |
Young, J., et al. (1983) Interaction of enveloped viruses with planar bilayer membranes: observations on sendai, influenza, vesicular stomatitis, and semlike forest viruses. Virology 128:186-194. |
Zhou, X., et al. (1994) DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochimica et Biophysica Acta 1189:195-203. |
Zwiebel, J., et al. (1989) High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors. Science 243:220-222. * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7655468B2 (en) | 1995-01-23 | 2010-02-02 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US20030212031A1 (en) * | 1995-01-23 | 2003-11-13 | Leaf Huang | Stable lipid-comprising durg delivery complexesand methods for their production |
US20050152964A1 (en) * | 1995-01-23 | 2005-07-14 | Leaf Huang | Stable lipid-comprising drug delivery complexes and methods for their production |
US7361640B2 (en) | 1995-01-23 | 2008-04-22 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US20080153166A1 (en) * | 1995-01-23 | 2008-06-26 | Leaf Huang | Stable lipid-comprising drug delivery complexes and methods for their production |
US20100184953A1 (en) * | 1995-01-23 | 2010-07-22 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US7993672B2 (en) | 1995-01-23 | 2011-08-09 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US8771728B2 (en) | 1995-01-23 | 2014-07-08 | University of Pittsburgh—of the Commonwealth System of Higher Education | Stable lipid-comprising drug delivery complexes and methods for their production |
US20030144230A1 (en) * | 1995-06-07 | 2003-07-31 | Pamela Hawley-Nelson | Peptide-enhanced transfections |
US8058068B2 (en) | 1995-06-07 | 2011-11-15 | Life Technologies Corporation | Peptide-enhanced transfections |
US20040152098A1 (en) * | 2001-05-17 | 2004-08-05 | Yoshiki Katayama | Cellular signal-responsive gene transcriptional regulation system |
US7892834B2 (en) * | 2001-05-17 | 2011-02-22 | Japan Science And Technology Agency | Cellular signal-responsive gene transcriptional regulation system |
AU2018201356B2 (en) * | 2009-11-06 | 2020-05-28 | The Board Of Trustees Of The Leland Stanford Junior University | Non-invasive diagnosis of graft rejection in organ transplant patients |
Also Published As
Publication number | Publication date |
---|---|
EP0759996A1 (en) | 1997-03-05 |
WO1995031557A1 (en) | 1995-11-23 |
CA2189975A1 (en) | 1995-11-23 |
JPH10500022A (en) | 1998-01-06 |
US5670347A (en) | 1997-09-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
USRE39220E1 (en) | Peptide-mediated gene transfer | |
Zhao et al. | Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein | |
Bos et al. | v-jun encodes a nuclear protein with enhancer binding properties of AP-1 | |
Brodsky | Living with clathrin: its role in intracellular membrane traffic | |
US5843643A (en) | Site-specific transfection of eukaryotic cells using polypeptide-linked recombinant nucleic acid | |
US5686278A (en) | Methods for enhanced retrovirus-mediated gene transfer | |
RU2098487C1 (en) | Method of nucleic acid transfer in animal cells | |
Giudice et al. | The transfection of epidermal keratin genes into fibroblasts and simple epithelial cells: evidence for inducing a type I keratin by a type II gene | |
Stove et al. | Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8αβ | |
EP3712272A1 (en) | Method for modulating rna splicing by inducing base mutation at splice site or base substitution in polypyrimidine region | |
JP2002525066A (en) | Transcriptional methods for specific cellular localization of nucleic acids | |
Krauzewicz et al. | Sustained ex vivo and in vivo transfer of a reporter gene using polyoma virus pseudocapsids | |
US5717058A (en) | Peptide inhibitors of tax-dependent transcription | |
CN113106094B (en) | Enhanced skeletal muscle cell efficient specific promoter, screening method and application | |
Welsh et al. | A second domain of simian virus 40 T antigen in which mutations can alter the cellular localization of the antigen | |
US20010010907A1 (en) | Isolation and identification of control sequences and genes modulated by transcription factors | |
US6503499B1 (en) | Viral vector complexes having adapters of predefined valence | |
LOBANENKOV et al. | Sequence‐specific DNA‐binding proteins which interact with (G+ C)‐rich sequences flanking the chicken c‐myc gene | |
Affara et al. | Analysis of chromatin changes associated with the expression of globin and non-globin genes in cell hybrids between erythroid and other cells | |
CN112111490B (en) | Method for visualizing endogenous low-abundance single-molecule RNA in living cells and application | |
Rehberger et al. | The activation domain of a hormone inducible HTLV-1 Rex protein determines colocalization with the nuclear pore | |
US5364761A (en) | Method for isolating a DNA encoding an autonomously replicating sequence | |
Fu et al. | Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo. | |
KR100236470B1 (en) | Rat2 fibroblast cell line producing ras protein used in evaluating the efficacy of farnesyltrasferase inhibitor and the preparation method thereof | |
Strair et al. | Retroviral mediated transfer and expression of exogenous genes in primary lymphoid cells: assaying for a viral transactivator activity in normal and malignant cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
RR | Request for reexamination filed |
Effective date: 20070323 |
|
B1 | Reexamination certificate first reexamination |
Free format text: THE PATENTABILITY OF CLAIMS 1-14 IS CONFIRMED. |
|
FPAY | Fee payment |
Year of fee payment: 12 |
|
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
SULP | Surcharge for late payment |