Abstract: The invention relates to methods and reagents for influencing alternative RNA splicing in living cells. More particularly, the invention relates to novel means for influencing RNA splice choice in living cells using polynucleotide-based reagents that compete for binding sites in nucleotide binding proteins, and novel methods for using these reagents as therapeutics.

Abstract: Provided, for example, is a method of transforming a cell comprising the steps of: applying a transformation effective amount of a nucleic acid to the cell; applying a fibrin gel to the cell so as to entrap a transformation effective amount of the nucleic acid; and transforming the cell with the nucleic acid. In one aspect, the nucleic acid is applied in admixture with a fibrin or fibrinogen composition that forms the fibrin gel.

Abstract: Unmethylated plasmid DNA vectors are a major contributor to the inflammatory response associated with gene delivery. Results of clinical studies where CF subjects were subjected to either aerosolized liposomes alone or cationic lipid:DNA complexes indicated that bacterial derived plasmid DNA may be inflammatory. Additionally, unmethylated CpG dinucleotides have been shown to be immunostimulatory and are present at a much higher frequency in bacterially-derived plasmid DNA compared to vertebrate DNA. The invention provides for methods of modulating the immunostimulatory response to gene delivery by modifying the plasmid delivered to the cell. The plasmid is modified to reduce or eliminate the immunostimulatory response in order to preserve the efficacy of gene transfer but reduce the associated toxicity.

Abstract: The invention provides improved vectors for cell-specific gene delivery to a target cell. The vectors according to the instant invention comprise a recombinant core containing the genetic materials to be delivered and an artificially reconstituted surface encompassing the core. The surface facilitates targeting and cell fusion of the vector, and also provides an immunoprotection function for the vector. Methods for preparing the sectors and for transfecting eukaryotic cells using the vectors also are disclosed.

Abstract: The present invention is directed to a method of separating cells of interest which method comprises: determining cells of interest; selecting a promoter specific for the cells of interest; introducing a nucleic acid molecule encoding green fluorescent protein under control of the promoter into a plurality of cells; and separating cells of the plurality of cells that are expressing said green fluorescent protein, wherein the separated cells are the cells of interest.

Abstract: A composition containing a plurality of multicellular vesicles and at least one nucleic acid, at least one part of which is found included inside the multilamellar vesicles. Each of the vesicles is formed of a succession of lamellar bi-layers extending from each vesicle center to its periphery and including at least one surfactant agent. The bi-layers are concentric and are separated by a liquid medium. Such compositions can be used in pharmaceutics, particularly gene therapy, and in in vitro and in vivo transfection.

Abstract: Liposomes containing therapeutic genes are conjugated to multiple targeting agents to provide transport of the encapsulated gene across the blood-retinal barrier and the plasma membrane of ocular cells. Once across the blood-retinal barrier and ocular cell membrane, the encapsulated gene expresses the encoded therapeutic agent within the ocular cells to provide diagnosis and/or treatment of disease.

Abstract: Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, or from alkyl or acyl groups; and cationic groups, protonatable at physiological pH, derived from amines, alkylamines or polyalkylamines. There are provided also therapeutic compositions prepared typically by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA, and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile.

Abstract: An polyampholyte is utilized in a condensed polynucleotide complex for purposes of nucleic acid delivery to a cell. The complex can be formed with an appropriate amount of positive and/or negative charge such that the resulting complex can be delivered to the extravascular space and may be further delivered to a cell.

Abstract: The invention relates to a system for expressing a transgene in a target cell or a human or animal cell, characterized in that it consists of a eukaryotic cell established as a line, into which there have been transfected:

Abstract: Novel lipid compounds are provided that may be termed “pro-cationic” in that they are neutral or negatively charged until they are either brought into contact with cellular membranes or are internalized by cells. The lipids have a hydrophobic tail group and a hydrophilic head group, the head group incorporating both a positively and negatively charged region at physiological pH. The hydrophobic tail group is stably connected to the positive region of the head group which in turn is connected to the negative region by a disulfide bond that is susceptible to cleavage by membrane-bound and intracellular factors. Cleavage of the disulfide bond removes the negatively charged region from the head group resulting in a lipid that is cationic and therefor fusogenic with negatively charged cell membranes. Consequently, lipids of the invention are useful as components of liposomes that serve as vehicles for delivering pharmaceutical agents into cells with reduced toxicity.

Abstract: The present invention provides compositions useful for transfecting eukaryotic cells comprising nucleic acid complexes with peptides, wherein the peptide is optionally covalently coupled to a nucleic acid-binding group, and cationic lipids or dendrimers as transfection agents. The invention also provides transfection compositions in which a peptide is covalently linked to the transfection agent (lipid, cationic lipid or dendrimer). Inclusion of peptides or modified-peptides in transfection compositions or covalent attachment of peptides to transfection agents results in enhanced transfection efficiency. Methods for the preparation of transfection compositions and methods of using these transfection compositions as intracellular delivery agents and extracellular targeting agents are also disclosed.

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

Abstract: A method is provided for inducing DNA synthesis in differentiated neurons. According to certain embodiments of the invention, a method for inducing DNA synthesis in a differentiated neuron is provided that includes obtaining a vector comprising nucleic acid encoding an E2F regulator and/or an E1A regulator, wherein the vector can be used to express the nucleic acid in a differentiated neuron, and transfecting a differentiated neuron with the vector.

Abstract: The present invention is directed to a composition for gene transfer which composition contains a quaternary ammonium salt represented by formula (1): wherein A represents (wherein each of R1, R2, R3, R4 and R5, which are identical to or different from one another, represents a C9-C17 aliphatic group); X1 represents a halogen atom; and n is an integer from 1 to 10 inclusive; and a method for introducing a gene into a cell by use of the composition. The composition enables effective delivery and expression of a gene which previously could not be effectively expressed in cells due to the low ratio at which the gene is delivered into cells. Therefore, the composition is advantageously used as a gene transfer reagent or a pharmaceutical.

Abstract: Liposomes containing therapeutic genes are conjugated to multiple blood-brain barrier and brain cell membrane targeting agents to provide transport of the encapsulated gene across the blood-brain barrier and brain cell membrane. Once across the blood-brain barrier and brain cell membrane, the encapsulated gene expresses the encoded therapeutic agent within the brain to provide treatment and diagnosis of disease.

Abstract: Described herein are liposome complexes and the individual components thereof for intracellular and/or intranuclear delivery of substances. Methods of use of the provided liposome complexes and components are also described. Generally, the liposome complexes described herein include a non-cationic lipid, a fusogenic peptide and a substance to be delivered to the cell and/or nucleus. In some of the liposome complexes described herein, the fusogenic peptide does not contain multiple positive charges at neutral pH and above. In these liposome complexes, two additional components are used in assembling the liposome complex with DNA.

Abstract: Unmethylated plasmid DNA vectors are a major contributor to the inflammatory response associated with gene delivery. Results of clinical studies where CF subjects were subjected to either aerosolized liposomes alone or cationic lipid:DNA complexes indicated that bacterial derived plasmid DNA may be inflammatory. Additionally, unmethylated CpG dinucleotides have been shown to be immunostimulatory and are present at a much higher frequency in bacterially derived plasmid DNA compared to vertebrate DNA. The invention provides for methods of modulating the immunostimulatory response to gene delivery by modifying the plasmid delivered to the cell. The plasmid is modified to reduce or eliminate the immunostimulatory response in order to preserve the efficacy of gene transfer but reduce the associated toxicity.

Abstract: Target cell-specific, non-viral vectors for inserting genes into cells, pharmaceuticals composition comprising such vectors, and methods of their use. Target cell-specific, non-viral vectors for inserting at least one gene into cells of an organism, comprising a complex comprising the following components: a) a non-viral carrier for the gene to be inserted, b) a ligand which can bind specifically to the desired target cell, c) a fusion protein for the penetration of the vector into the cytoplasm of the target cell, and d) the gene to be introduced are disclosed. Vectors of this nature are used, for example, in gene therapy.

Abstract: The present invention relates to a synergistic method of gene transfection, including gene therapy of human diseases. The invention provides use and preparation of specific combinations of cationic lipids and cationic polymers for the transfection of nucleic acids into host cells. This invention includes also the use of described combinations in specific systems for gene transfection, expression, repair, activation, inhibition and regulation.

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: A hematopoietic growth factor delivery composition includes a hematopoietic growth factor, a liquid vehicle, a first biocompatible polymer and a second biocompatible polymer. The composition exhibits reverse-thermal viscosity behavior, due to interaction between the first biocompatible polymer and the liquid vehicle. The second biocompatible polymer helps to protect the first biocompatible polymer from being dissolved in vivo following administration to a host.

Abstract: The present invention relates to the discovery in eukaryotic cells, particularly mammalian cells, of a novel family of cell-cycle regulatory proteins (“CCR-proteins”). As described herein, this family of proteins includes a polypeptide having an apparent molecular weight of 16 kDa, and a polypeptide having an apparent molecular weight of approximately 15 kDa, each of which can function as an inhibitor of cell-cycle progression, and therefore ultimately of cell growth. Thus, similar to the role of p21 to the p53 checkpoint, the subject CCR-proteins may function coordinately with the cell-cycle regulatory protein, retinoblastoma (RB). Furthermore, the CCR-protein family includes a protein having an apparent molecular weight of 13.5 kDa (hereinafter “p13.5”). The presumptive role of p13.5, like p16 and p15, is in the regulation of the cell-cycle.

Abstract: A transposition assembly for the transfer of a DNA fragment of interest into the ribosomal nuclear DNA of an eukaryotic cell. An insertion means, an eukaryotic cell and a pharmaceutical composition comprising said transposition assembly, as well as a method for the in vitro transfer of said DNA fragment, are also disclosed.

Abstract: The cationic liposomal formulations of the present invention provide nucleic acid and gene product delivery devices having a glycosaminoglycan covalently attached to the liposome surface. The glycosaminoglycan can be any glycosaminoglycan, including but not limited to hyaluronic acid, the chondroitin sulfates, keratan sulfate, chitin and heparin. Preferably, the glycosaminoglycan is hyaluronic acid. The present invention also provides methods of preparing the nucleic acid-liposome formulations.

Abstract: A gene transfection particle includes a polymer, a support particle conjugated with the dendritic polymer, and genetic material conjugated with the dendritic polymer. The gene transfection particles are highly efficient and are capable of delivering higher quantities of genetic materials to cells, with reduced cell damage. A gene transfection method involves bombarding cells with conjugates of polymers and genetic material, with or without a support particle.

Abstract: A composition for delivering at least one DNA sequence encoding a desired protein or polypeptide (such as a therapeutic agent) to a cell. The composition comprises an adeno-associated virus rep protein (or a nucleic acid sequence encoding an adeno-associated virus rep protein) and a genetic construct including at least one DNA sequence encoding a protein or polypeptide or genetic transcript of interest and a promoter controlling the at least one DNA sequence. The genetic construct also includes a first adeno-associated virus ITR or protein or derivative thereof and a second adeno-associated virus ITR or a portion or derivative thereof. The first and second adeno-associated virus ITRs or portions or derivatives thereof flank the at least one DNA sequence encoding the protein or polypeptide or genetic transcript of interest and the promoter controlling the at least one DNA sequence encoding the protein or polypeptide or genetic transcript of interest.

Abstract: An integrative DNA vector and one or more viral proteins having affinity for DNA are packaged in cochleate precipitates. The integrative DNA vector contains one or more therapeutic nucleotide sequences that are preferably positioned between DNA substrates for the proteins. Upon contact with a lipid bilayer of a target cell, the cochleate vector structure delivers one or more of the therapeutic nucleotide sequences and one or more proteins to the interior of the target cell. Upon entry into the cell, the proteins facilitate the integration of the therapeutic nucleotide sequence into the genome of the host cell.

Abstract: The invention provides a gene construct encoding a cell targeting moiety and a heterologous prodrug activating enzyme for use as a medicament in a mammalian host wherein the gene construct is capable of expressing the cell targeting moiety and enzyme as a conjugate within a target cell in the mammalian host and wherein the conjugate is directed to leave the cell thereafter for selective localisation at a cell surface antigen recognised by the cell targeting moiety.

Abstract: Peptide-lipid conjugates are incorporated into liposomes so as to selectively destabilize the liposomes in the vicinity of target peptidase-secreting cells, and hence, to deliver the liposomes to the vicinity of the target cells, or directly into the cells. The liposomes can thus be used to treat mammals for diseases, disorders or conditions, e.g., tumors, microbial infection and inflammations, characterized by the occurrence of peptidase-secreting cells.

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: The present invention relates to lipid compounds of formula I, wherein said compounds are optionally in a cationic form and are optionally combined with one or more biologically acceptable anions. The present invention also relates to complexes comprising at least one cationic lipid compound of the formula I and an active substance comprising negative charges. The present invention further relates to methods of gene therapy using the complexes of the present invention.

Abstract: The present invention relates generally to the fields of cancer therapy and gene therapy. More particularly, the invention demonstrates methods for repressing or preventing transformation and proliferation of tumor cells. The method comprises contacting a cell with a p202 polypeptide in an amount effective to inhibit a transformed phenotype or cell proliferation. Inhibition of transformation may be indicated by a reduction in a transforming, tumorigenic or metastatic potential of a cell.

Abstract: Novel cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, or from alkyl or acyl groups; and cationic groups, protonatable at physiological pH, derived from amines, alkylamines or polyalkylamines. There are provided also therapeutic compositions prepared typically by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA, and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile.

Abstract: The present invention relates to a method of transforming cells in which an appropriate quantity of nucleic acid fragments is introduced into the cells. The nucleic acid fragments are introduced in the form of a nucleic acid composition comprising a nitrogen-containing silicone, useful for compacting the nucleic acid fragments; the compositions comprise aggregates of nucleic acid fragments and silicones according to the invention.

Abstract: An expression vector is provided for use in the treatment of neoplasms which vector comprises nucleic acid including at least one which encodes in expressible form a polypeptide which is a heat shock polypeptide (hsp) or a chaperone.

Abstract: The effective introduction of foreign genes and other biologically active molecules into targeted mammalian cells is a challenge still facing those skilled in the art. Gene therapy, for example, requires successful transfection of target cells in a patient. The present invention relates to novel micellar complexes of cationic amphiphilic compounds that facilitate delivery of biologically active molecules to the targeted cells of a mammal. The novel micellar complexes are comprised of a cationic amphiphile, a biologically active molecule, a derivative of polyethylene glycol (PEG), and optionally, a co-lipid. A further aspect of the invention is the use of targeting agents in any of the methods that effectuate the delivery of biologically active molecules into the cells of mammals. A targeting agent is usually any molecule, peptide sequence, or large protein that preferentially targets or binds to specific mammalian cells.

Abstract: Disclosed are methods for producing recombinant cells and especially recombinant mammalian cell lines with enhanced expression of an amino acid sequence. Also disclosed are recombinant mammalian cell lines producing high levels of the amino acid sequence. The methods and recombinant cell lines of the invention have a number of useful applications including use in the efficient and large-scale production of recombinant proteins and polypeptides.

Abstract: Antisense compounds, compositions and methods are provided for modulating the expression of caspase 3. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding caspase 3. Methods of using these compounds for modulation of caspase 3 expression and for treatment of diseases associated with expression of caspase 3 are provided.

Abstract: The invention relates generally to compositions of and methods for obtaining and using a polypeptide other than BCL-2 that affects programmed vertebrate cell death. The invention relates as well to polynucleotides encoding those polypeptides, recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant polypeptides. The invention further provides methods for using the isolated, recombinant polypeptides in assays designed to select and improve substances capable of altering programmed cell death for use in diagnostic, drug design and therapeutic applications.

Abstract: A dry powder composition comprises nucleic acid constructs dispersed within with a hydrophilic excipient material, where the powder particles have an average size in the range from 0.5 &mgr;m to 50 &mgr;m. Nucleic acid constructs may comprise bare nucleic acid molecules, viral vectors, or vesicle structures. The hydrophilic excipient material will be selected to stabilize the nucleic acid molecules in the constructs, enhance dispersion of the nucleic acid in dry powder aerosols, and enhance wetting of the nucleic acid constructs as they are delivered to moist target locations within the body.

Abstract: This invention provides a self-assembling polynucleotide delivery system comprising components aiding in the delivery of the polynucleotide to the desired address which are associated via noncovalent interactions with the polynucleotide. The components of this system include DNA-masking components, cell recognition components, charge-neutralization and membrane-permeabilization components, and subcellular localization components. Specific compounds useful in this system are also provided.

Abstract: The invention concerns compounds for transferring nucleic acids into cells. The compounds are more particularly related to the lipopolyamine family, and comprise amidine functions. The compounds are useful for transferring nucleic acids of interest into various cellular types, in vitro, in vivo, or ex vivo.

Abstract: The invention concerns a complex comprising at least a lipid and at least a therapeutically active substance useful for transferring said substance into a target cell, characterised in that said lipid is of formula (I): in which: n1, n2, identical or different are whole numbers between 0 and 1; R1, R2, identical or different are: a) selected among the group consisting of a hydrogen atom and alkyl radicals with 1 to 6 carbon atoms optionally substituted, independently of one another, by a hydroxyl radical; or b) in one particular case for which n1=n2=1, R1 and R2 can form together a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3); R3, R4, identical or different are alkyl radicals of 1 to 6 carbon atoms or can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3); m, p, identical or different are whole numbers between 1 and 10; R5, R6, identical or different are selected in the group consisting of radicals of formula: 1) R7 C(═O)—X— in which: X=NH, O

Abstract: The invention relates to pharmaceutical compositions suitable for treating or curing clinical complications mediated by endotoxin, including sepsis. The compositions contain components suitable for detoxifying endotoxin rendering it less deleterious to mammals such as humans, in particular to patients with reduced host-defence resistance. The invention also relates to pharmaceutical compositions suitable for stimulating bone formation, e.g. for mending broken bone or for prophylaxis or therapy of metabolic bone diseases such as osteoporosis and osteomalacia and pharmaceutical compositions for decreasing or inhibiting undesired bone formation. The pharmaceutical compositions according to the invention are directed at modulating phosphatase activity in vivo.

Abstract: Lipid-therapeutic agent particles are prepared containing a charged therapeutic agent encapsulated in lipid portion containing at least two lipid components including a protonatable or deprotonatable lipid such as an amino lipid and a lipid that prevents particle aggregation during lipid-therapeutic agent particle formation such as a PEG-modified or polyamide oligomer-modified lipid. Other lipid components may also be present and these include a neutral lipid such as DSPC, POPC, DOPE or SM, and a sterol such as Chol. The therapeutic agent is encapsulated by combining a mixture of the lipids with a buffered aqueous solution of a charged therapeutic agent to form an intermediate mixture containing lipid-encapsulated therapeutic agent particles, and changing the pH of the intermediate mixture to neutralize at least some surface charges on the particles. The method permits high ratios of therapeutic agent to lipid and encapsulation efficiencies in excess of 50%.

Abstract: A neurogenesis inducing gene coding for the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a protein which consists of the amino acid sequence shown in SEQ ID NO: 2 having deletion, substitution or addition of at least one amino acid and which has neurogenesis inducing activity.

Abstract: The present invention provides for improved methods of gene transfer, both in vitro and in vivo. By treating neoplastic cells with a DNA-damaging agent prior to transduction with a transgene, the expression of the transgene is improved up to about 3-fold over the expression seen in the absence of the DNA-damaging agent treatment. This effect is not dependent on the tumor cell type, the method of DNA transduction or type of DNA-damaging agent. The effect is most dramatic when the transduction is performed about 1-3 days following treatment with the DNA-damaging agent.

Abstract: Compositions and methods are provided for gene delivery to the respiratory tract. In particular, compositions comprising a nebulized nucleic acid/stabilizing agent complex, and methods employing such complexes for pulmonary gene delivery, are provided. Such complexes are preferably sonic nebulized.

Abstract: Methods of transfecting cells in vivo, including tumor cells in the peritoneal cavity are provided. Related lipid:nucleic acid formulations adapted to transfecting cells in the peritoneal cavity are provided. Assays, including high-throughput assays for screening lipid:nucleic acids are also provided.