Abstract: The invention concerns the introduction of predetermined genetic changes in target genes of a living cell by introducing an oligodeoxynucleotide encoding the predetermined change. The oligodeoxynucleotides are effective in mammalian, avian, plant and bacterial cells. Specific end modifications that greatly increase the effectiveness of the oligodeoxynucleotides in bacteria are described. Surprisingly, unmodified oligodeoxynucleotides can be as effective in mammaliancells, including in vivo hepatocytes, as the modified nucleotides and can be as effective or more effective than chimeric oligonucleotides that consist of a mixture of deoxynucleotides and 2′-O-methyl ribonucleotides.

Abstract: The invention relates to the stable transfection of neurons with DNA encoding proliferating cell nuclear antigen (PCNA) and replication factor C (RFC). Also, co-transfection of a functional gene along with the DNA encoding PCNA and RFC causes stable integration of the functional gene.

Abstract: The present invention provides packaging cell lines and recombinant lentiviral or retroviral particles produced therefrom, particularly pseudotyped retroviral particles. The packaging cell lines of the invention are produced by inducibly expressing an envelope protein by methods described herein. Also described is a screening assay for compounds that affect integration of viral nucleic acid into target (e.g., host) nucleic acid. Such compounds are identified based on their effect on viral integrase.

Abstract: Antisense compounds, compositions and methods are provided for modulating the expression of PTP1B. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PTP1B. Methods of using these compounds for modulation of PTP1B expression and for treatment of diseases associated with expression of PTP1B are provided.

Abstract: Intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene which expresses a protein which has a desired effect. The method of the invention comprises administration of a formulation containing DNA to the gastrointestinal tract, preferably by an oral route. The expressed recombinant protein is secreted directly into the bloodstream. Of particular interest is the use of the method of the invention to provide for short term delivery of gene products to the bloodstream.

Abstract: The present invention provides improved cell delivery compositions. In particular, the invention provides biocompatible endosomolytic agents. In a preferred embodiment, the endosomolytic agents are also biodegradable and can be broken down within cells into components that the cells can either reuse or dispose of. Preferred endosomolytic agents include cationic polymers, particularly those comprised of biomolecules, such as histidine, polyhistidine, polylysine or any combination thereof. Other exemplary endosomolytic agents include, but are not limited to, other imidazole containing compounds such as vinylimidazole and histamine. More particularly preferred are those agents having multiple proton acceptor sites and acting as a “proton sponge”, disrupting the endosome by osmolytic action. In preferred embodiments, the endosomolytic agent comprises a plurality of proton acceptor sites having pKas within the range of 4 to 7, which endosomal lysing component is polycationic at pH 4.

Abstract: Secretory gland cells, particularly pancreatic and salivary gland cells, are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subject. The expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein. The transformed secretory gland cells provide long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

Abstract: The invention relates to methods for targeting an exogenous polynucleotide or exogenous complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a eukaryotic cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active eukaryotic cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active eukaryotic cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell.

Abstract: Complexes of nucleic acid and polyethyleneimine (PEI), wherein PEI is modified with a hydrophilic polymer covalently coupled thereto, such as polyethyleneglycol, and processes for preparing them. A cellular ligand such as transferrin is optionally coupled to PEI. The complexes may be used for preparing pharmaceutical compositions for transferring therapeutically effective genes into mammalian cells.

Abstract: The present invention relates to recombinant eukaryotic plasmids comprising an eukaryotic expression vector and an allergen gene for the prevention and/or treatment of allergic diseases. When the recombinant vector containing allergen-gene administrate to an individual in need of such prevention and/or treatment by intramuscular injection, intranasal delivery or intratracheal delivery, the production of allergen-specific IgE synthesis can be inhibited. The invention also relates to the pharmaceutical compositions comprising the the recombinant vector for use in the the prevention and/or treatment of allergic diseases and the production of allergen-specific IgE synthesis. A method for the prevention and/or treatment of allergic diseases is also provided.

Abstract: Transdominant repressors of viral gene phenotypic expression derived from the rev gene product of HIV-1 or the rex gene product of HTLV-1 and corresponding mutated genes are described, having the capability of repressing the Rev function in HIV-1 and/or the Rex function in HTLV-I and HTLV-II. Transient gene expression analysis of a series of missense and deletion mutants has been used. Sane of the mutants found repress both the Rev and the Rex function and are thus active in more than one viral species. Transdominant viral mutants represent a promising new class of anti-viral agents. Cellular expression of these transdominant inhibitors may be used in such therapeutic approaches as intracellular immunization in order to protect cells against the deleterious effects of viral, e.g. HIV-1 infection.

Abstract: The present invention discloses compositions and methods of using intracellular delivery vehicles for delivery and transfection of DNA, RNA, polypeptides, genes, proteins, drugs and biologically active agents into cells in vitro and in vivo. The vehicle comprises a mixture of a liposome and a polypeptide lacking specificity for cellular receptors. In another embodiment, a method for intracellular delivery of biologically active agents comprising combining a non-receptor-binding protein and a liposome, incubating the mixture for a period of time, adding the biologically active agent, incubating again, and finally, introducing the resulting mixture to the cell. Preferably, the liposome is a cationic liposome. The charge ratio of cationic liposome to DNA can effectively be varied from 2:1 to 1:2. Preferably, the non-receptor-binding protein is the serum albumin of the animal source of the cell to be transfected.

Abstract: The present invention provides an isolated nucleic acid covalently linked to a photolabile caging group which reversibly prevents expression of the nucleic acid. The present invention further provides a method of selectively expressing a nucleic acid in a cell, comprising: a) covalently linking the nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell.

Abstract: The present invention provides a conditionally replicating viral vector, methods of making, modifying, propagating and selectively packaging, and using such a vector, isolated molecules of specified nucleotide and amino acid sequences relevant to such vectors, a pharmaceutical composition and a host cell comprising such a vector, the use of such a host cell to screen drugs. The methods include the prophylactic and therapeutic treatment of viral infection, in particular HIV infection, and, thus, are also directed to viral vaccines and the treatment of cancer, in particular cancer of viral etiology. Other methods include the use of such conditionally replicating viral vectors in gene therapy and other applications.

Abstract: Antisense compounds, compositions and methods are provided for modulating the expression of ADAM10. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding ADAM10. Methods of using these compounds for modulation of ADAM10 expression and for treatment of diseases associated with expression of ADAM10 are provided.

Abstract: Intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect. Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA, and is preferably administered orally. Oral or other intragastrointestinal routes of administration provide a simple method of administration, while the use of naked nucleic acid avoids the complications associated with use of viral vectors to accomplish gene therapy. The expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein. The transformed intestinal epithelial cells provide short or long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

Abstract: Methods of screening for peptides useful in a gene delivery system to provide optimal transfection of cells based on kinetic parameters of the peptide-nucleic acid bimolecular interaction are described.

Abstract: The invention relates to an ionic conjugate, which is stable in a biological medium, and which is comprised of a particle vector with at least one cationic, nonliquid, hydrophilic nucleus and of polyanionic oligonucleotides. The invention further concerns the pharmaceutical compositions containing these conjugates and the use of a particle vector to carry the oligonucleotides to the cells.

Abstract: The present invention relates to a positively charged virosome for efficient delivery of genetic material to resting or proliferating mammalian cells in vitro and in vivo. The virosome membrane contains cationic and/or polycationic lipids, at least one viral fusion peptide and preferably at least one cell-specific marker, advantageously selected from the group consisting of monoclonal antibodies, antibody fragments F(ab′)2 and Fab′, cytokines, and growth factors, for a selective detection and binding of target cells. The invention further relates to a method for the manufacture of the novel virosomes and to applications thereof, particularly for the manufacture of pharmaceutical compositions to treat cancer or leukemia.

Abstract: A composition includes a liposome which has a polynucleic acid and a peptide capable of disrupting membranes under acidic conditions encapsulated within it. The composition is used for efficient transfer of nucleic acids into cells both in vitro as well as in vivo.

Abstract: Novel stem cell factors, oligonucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders involving blood cells are also disclosed.

Abstract: 4-Phenylbutyrate exerts many beneficial biological effects. It appears to induce the transcription of certain promoters, as well as having a remedial effect on proteins which are aberrantly localized within the cell. In addition, it appears to cause cells to developmentally differentiate. The present invention provides nanosphere formulations of 4-phenylbutyrate and other drugs which remediate defective protein localization intracellularly. These formulations permit lower concentrations of drugs to be administered, providing both cost and safety benefits.

Abstract: The present method provides a method for inhibiting restenosis associated with mechanical injury of a blood vessel. Human heme oxygenase I (HO1) is directly administered at the site of injury. The present inventors have discovered that carbon monoxide generated by HO1 is involved in the molecular pathogenesis of vascular proliferative disorders. By using adenoviral-mediated expression of inducible heme oxygenase 1 in primary vascular smooth muscle cells (vsmc) in vivo, the present inventors demonstrate that in vivo expression of HO1 can be used to treat restenosis.

Abstract: The present invention provides ortho ester lipids and their derivatives that, upon certain pH conditions, undergo hydrolysis with concomitant or subsequent headgroup cleavage. These ortho ester lipids can advantageously be formulated into liposomes. The liposome formulations are useful in nucleic acid transfection and entrapment/delivery of conventional small molecules and therapeutic agents. Moreover, the liposomes comprising compounds of the present invention are useful as drug delivery vehicles.

Abstract: Oligonucleotides containing unthylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response in a subject are disclosed. Also disclosed are therapies for treating diseases associated with immune system activation that are initiated by unthylated CpG dinucleotides in a subject comprising administering to the subject oligonucleotides that do not contain unmethylated CpG sequences (i.e. methylated CpG sequences or no CpG sequence) to outcompete unmethylated CpG nucleic acids for binding. Further disclosed are methylated CpG containing dinucleotides for use antisense therapies or as in vivo hybridization probes, and immunoinhibitory oligonucleotides for use as antiviral therapeutics.

Abstract: The present invention relates to compositions comprising at least one nucleic acid and one lipopolyamine, and their utilisation in gene therapy, particularly for the transfert in vivo of nucleic acids.

Abstract: Transfection of host cells cultured in the presence of serum is increased by adding VSV-G or polybrene to a nucleic acid-lipid complex or culture medium prior to transfection.

Abstract: The subject invention finds utility in the area of gene therapy of diseases. More specifically, the invention concerns the making of a novel non-viral vector which can bind to desired DNA to form a combination useful to transfect diseased mitochondria of human or animal cells. The non-viral vector comprises a dequalinium salt subjected to standard liposome production procedures to obtain the vector names DQAsomes.

Abstract: The subject invention concerns novel materials and methods for the delivery of substances into cells. In a specific embodiment substances are delivered into cells using a novel class of lipid compounds. These compounds, cationic lipid compounds having a disulfide bond, can be complexed with DNA to be inserted into a cell in gene therapy. Once inside the cell, enzymes present within the cell cleave the disulfide bond and the DNA is released.

Abstract: The present invention discloses methods for the treatment of colon cancer. The expression of gastrin by colon cancers is inhibited by the use of antisense gastrin expression. Methods are disclosed for the preparation of expression constructs and the use of such constructs to inhibit colon cancer growth.

Abstract: The invention concerns a method for preparing a composition for transferring nucleic acids consisting in contacting a nucleic acid with a cationic lipid, whereby, previous to contacting, the cationic lipid is subjected to a heating step. The invention also concerns the resulting compositions and their use.

Abstract: The subject invention concerns novel materials and methods for the delivery of substances, such as DNA or polypeptides, into cells. In a specific embodiment, substances are delivered into cells using a novel class of lipid compounds. These compounds, cationic lipid compounds having a disulfide bond, can be complexed with DNA to be inserted into a cell in gene therapy. Once inside the cell, enzymes present within the cell cleave the disulfide bond and the DNA is released.

Abstract: A pharmaceutical composition useful for nucleic acid transfection is disclosed. The composition contains, in addition to a nucleic acid and at least one transfection agent, at least one compound that combines DNA binding properties with a nuclear DNA vectorisation capability, and preferably belongs to the HMG ("High mobility group") protein family. The use of said composition for in vitro, ex vivo and/or in vivo nucleic acid transfer is also disclosed.

Abstract: A pharmaceutical agent which inhibits a replication of virus by increasing the specific enzymatic activity of liver and/or other tissue is offered.A viral replication inhibitor which contains N-acetyl-glucosaminyltransferase III (GnT-III) or gene thereof as an effective component. Examples of the gene are that which contains a sequence represented by SEQ ID NO:1 (length: 1,608) or by SEQ ID NO:2 (length: 1,593) in the Sequence List, that which is prepared by hybridization of it and codes for a polypeptide having a GnT-III activity or a functionally same activity and that in which the above is further integrated in vector.

Abstract: Genetic analysis of familial breast and ovarian cancer indicates that BRCA1 is a tumor suppressor gene. The BRCA1 gene encodes a 190 kDa protein with sequence homology and biochemical analogy to the granin family of proteins. Granins are secreted from endocrine cells via the regulated secretory pathway and are proteolytically cleaved to yield biologically active peptides. BRCA1 protein localizes to secretory vesicles, and was demonstrated to be secreted. Gene transfer of BRCA1 inhibits growth and tumorigenesis of breast and ovarian cancer cells, but not colon or lung cancer cells or fibroblasts, suggesting that BRCA1 encodes a tissue-specific growth inhibitor. Thus, BRCA1 is a secreted growth inhibitor and functions by a mechanism not previously described for tumor suppressor genes. The BRCA2 breast and ovarian cancer gene encodes a protein that also includes a granin region, indicating that the BRCA2 protein is also a secreted tumor suppressor.

Abstract: This invention provides methods and chemical agents for enhancing transient expression in eukaryotic cells. Also provided are a model system for achieving prolonged transient expression in solid tumors, a means for culturing hepatocytes without feeder cells or an extracellular matrix bonded to the substratum, a method for manipulating cellular metabolism to reduce the consumption of glucose and a means for inducing the secretion of an endogenous phosphatase activity.

Abstract: The invention provides a cell-specific nuclear targeting molecule having a nucleic acid sequence which includes a binding site for a nuclear DNA binding protein expressed only in a specific cell type. The invention further provides a plasmid for targeting a DNA molecule into the nuclei of a specific cell type. The plasmid comprises the cell-specific nuclear targeting molecule and a DNA molecule to be targeted to the nuclei of the specific cell type. This plasmid of the subject invention can be introduced into various host cells, and the cell-specific nuclear targeting molecule will target the DNA molecule to the nuclei of the specific cell type. Thus, the invention further provides a method of targeting a DNA molecule into the nuclei of a specific cell type. The method comprises providing a plasmid (the plasmid comprising the cell-specific nuclear targeting molecule and the DNA molecule to be targeted) and introducing the plasmid into the cytoplasm of the specific cell type.

Abstract: The subject invention provides methods for improving the efficiency of gene transfer to a target cell. The methods are particularly advantageous in that they facilitate the transduction of quiescent cells. In a preferred embodiment the efficiency of gene transfer using a viral vector is enhanced by transiently loading the dytosol of the target cell with dNTP and by transiently adding viral receptors to the surface of the target cell.

Abstract: A method of forming polymers in the presence of nucleic acid using template polymerization. Also, a method of having the polymerization occur in heterophase systems. These methods can be used for the delivery of nucleic acids, for condensing the nucleic acid, for forming nucleic acid binding polymers, for forming supramolecular complexes containing nucleic acid and polymer, and for forming an interpolyelectrolyte complex.

Abstract: A liposome composition for administration of a polynucleotide and a method of preparing the composition are described. The liposomes in the suspension are composed predominantly of liposomes having a bilayer membrane formed of cationic vesicle-forming lipids and neutral vesicle forming lipids. The polynucleotide is entrapped in the central core of the liposomes and is localized predominantly on the inner surface of the core.

Abstract: The present invention is directed to compositions and methods for a genetic system of detecting protein--protein interactions in a mammalian host cell. Two fusion proteins are made in the host cell. The first fusion protein contains a DNA binding domain which is fused to a so-called bait protein. The second fusion protein consists of a transcriptional activation domain fused to a so-called test protein. The transcriptional activation domain is recruited to the promoter through the functional interaction between the bait protein and the test protein. Subsequently the transcriptional activation domain interacts with the basal transcription machinery to activate expression of one or more reporter genes which can be identified and characterized. The individual compositions are useful for analyzing protein--protein interactions between known proteins and to isolate, clone and characterize unknown proteins. The individual compositions can be used to express the fusion proteins either transiently or stably.

Abstract: This invention relates to a novel Solvent Extraction and Direct Hydration (SEDH) method for preparing lipid-nucleic acid particles which are useful for the introduction of nucleic acids (e.g., plasmid DNA, antisense molecules, ribozymes, etc.) into cells. The lipid-nucleic acid particles prepared using the methods of the present invention have enhanced circulation characteristics and serum stability and, thus, they are extremely effective as nucleic acid delivery vehicles.

Abstract: The present invention provides a liposomal aerosol composition, comprising a pharmaceutical compound, a cationic lipid, (c) a neutral co-lipid; and (d) tryptone. Also provided is a nebulized cationic lipid:DNA suspension useful for lipid-DNA transfections, wherein said cationic lipid is bis(guanidinium)-tren-cholesterol.

Abstract: The present invention is directed to methods of sensitizing a human tumor cell with adenovirus E1A. The methods involve treating a human tumor cell by, first, introducing into the tumor cell nucleic acid encoding a polypeptide having adenovirus E1A activity, expressing the E1A active polypeptide in the cell, and then either contacting the E1A expressing tumor cell with a chemotherapeutic agent or irradiating the E1A-expressing tumor cell. The invention also provides methods of enhancing a subject's response to chemotherapy or irradiation by introducing into a subject's tumor cells nucleic acid encoding a polypeptide having adenovirus E1A activity, expressing the E1A active polypeptide in the cells and finally, administering either a chemotherapeutic agent or irradiation. The invention also provides a method of treating cancer.

Abstract: The present invention relates to transgene expression systems, related pharmaceutical compositions, and methods of making and using them. Preferred systems employ an adenovirus transgene expression vector comprising DNA sequence encoding a transgene which codes for a desired product, expressibly contained within an adenovirus vector containing at least a portion of the E3 region and certain portions of the E4 region. The E4 portions comprise the open reading frame sequence known as E4ORF3 and at least one other portion of E4. Preferably the E4 portion of the vector (or "E4 cassette") includes E4ORF3 and at least one other portion selected from E4ORF4, E4ORF6/7 and E4ORF3/4. The invention has a number of important features including improving persistency of transgene expression in a desired host cell. The transgene expression systems of the present invention are useful for a variety of applications including providing persistent cellular expression of the transgene in vitro and in vivo.

Abstract: This invention concerns stable particulate complexes with a neutral or negative global charge of lamellar structure made consisting of at least one globally anionic biologically active substance and mixture of a cationic constituent and an anionic constituent. The invention also concerns unilamellar vesicles for preparation of these complexes as well as their preparation and utilisation.

Abstract: The invention relates to a process for preparing clonogenic fibroblasts, with tissue being removed from the donor and the individual cells being isolated from the tissue, the resulting cell suspension being strained, the cells which are contained in the cell suspension being washed and the cells being converted into a tissue culture, with the exception of the isolation of individual cells by mechanical comminution, followed by an enzymic treatment with collagenase alone, and with at least one gene being inserted into the fibroblasts by means of the transfection, which gene encodes a biologically active protein, preferably a therapeutically active protein, for example a growth factor, a hormone, an enzyme, a coagulation factor or a coagulation inhibitor.

Abstract: The invention provides compositions and methods for introducing a nucleic acid fragment into the genome of a cell. Suitable compositions comprise an expression vector having first and second inverted repeated sequences from an adeno associated virus, a rep gene from an adeno associated virus and the nucleic acid fragment. The expression vector is complexed with lipids.

Abstract: Disclosed is a delivery system for biologically active molecules or agents which must enter cells to exert their effect. The delivery system comprises a mixture of cationic lipid in combination with a receptor ligand and is particularly suited for intracellular delivery of polynucleotides.

Abstract: Nucleic acids are compacted, substantially without aggregation, to facilitate their uptake by target cells of an organism to which the compacted material is administered. The nucleic acids may achieve a clinical effect as a result of gene expression, hybridization to endogenous nucleic acids whose expression is undesired, or site-specific integration so that a target gene is replaced, modified or deleted. The targeting may be enhanced by means of a target cell-binding moiety. The nucleic acid is preferably compacted to a condensed state.