Abstract: A retroviral delivery system capable of transducing a target site is described. The retroviral delivery system comprises a first nucleotide sequence coding for at least a part of an envelope protein; and one or more other nucleotide sequences derivable from a retrovirus that ensure transduction of the target site by the retroviral delivery system; wherein the first nucleotide sequence is heterologous with respect to at least one of the other nucleotide sequences; and wherein the first nucleotide sequence codes for at least a part of a rabies G protein or a mutant, variant, derivative or fragment thereof that is capable or recognising the target site.

Abstract: The present invention provides random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in sense orientation. Also provided are random cDNA expression vector libraries, comprising expression vectors which comprise random cDNAs positioned in antisense orientation. Methods for producing these libraries through directional cloning of random cDNAs are also provided. Also provided herein are methods of using these libraries to screen for agents capable of modulating cell phenotype in desirable ways.

Abstract: This invention provides for a chamber and system designed for use in assaying drug effects on heart rate. The chamber consists of a series of wells, each 3 mm by 3 mm in inner diameter. Cardiac myocytes disaggregated from neonatal animals are plated onto the bottom of each well and grown under standard tissue culture conditions. The chamber holds from 24-96 such wells. When drugs are to be assayed, the cells in each well are loaded with a calcium sensitive dye and the beating rate in each is monitored with a photodiode. Drug is added in graded concentrations to each well, and equilibrated and effects on rate are observed. This construct permits use of a cell based bioassay for the study of drugs or agents that may alter cardiac rate. This invention can be used in high throughput screening of drugs to evaluate/predict their effects on cardiac rate and rhythm. Further provided for by this invention is a vector which comprises a compound which encodes an ion channel.

Abstract: The present invention relates to (a) methods for improving a genetic stability of an insert nucleotide sequence in a recombinant single-stranded RNA virus vector, which comprises performing a mutagenesis of the foreign insert nucleotide sequence to provide even distribution of G/C content throughout the overall foreign insert nucleotide sequence and/or to increase G/C content of the foreign insert without substantially causing amino acids substitutions (b) a recombinant single-stranded RNA virus comprising an insert nucleotide sequence with improved genetic stability and (c) a recombinant poliovirus comprising an insert nucleotide sequence with improved genetic stability.

Abstract: Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus.

Abstract: This invention relates to a method for producing a zinc finger nucleic acid binding protein comprising preparing a zinc finger protein according design rules, varying the protein at one or more positions, and selecting variants which bind to a target nucleic acid sequence by polysome display.

Abstract: A phagemid has been constructed that expresses an antibody fused to coliphage pIII protein. The phagemid is suitable for selecting specific antibodies from large gene libraries with small quantities of antigen. The antibody-pIII gene can be strongly repressed, so that it allows antibody libraries to be amplified without the danger of deletion mutants predominating. After induction, large quantities of the fusion protein may be expressed.

Abstract: Provided are compositions and methods useful for modulating the activity of autoinducer synthase catalysts. A method for identifing modulators of the autoinducer synthesis reaction is also provided. Such modulators are useful for controlling bacterial growth and can be used for therapeutic treatment of bacterial infections particularly in immunocompromised subjects. They are also useful in treating disease states associated with autoinducer synthesis and biofilm development.

Abstract: The present invention relates to a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest. Also, the invention provides a method of selecting a DNA sequence of interest from an environmental pool of organisms. Further, the invention relates to a gene library prepared from an enriched environmental pool of organisms enriched in DNA encoding a polypeptide with an activity of interest.

Abstract: The invention provides novel yeast promoters useful for controlling the expression of homologous and heterologous nucleic acid molecules in yeast cells. The yeast promoters are induced by a fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both. Therefore, expression of nucleic acid molecules encoding a polypeptide under the control of the novel yeast promoters may be regulated by varying the level of a fermentable carbon source, or a non-fermentable carbon source, or both.

Abstract: Disclosed herein are improved plasmid shuttle vectors, vaccines based on them, and methods related to their construction and use. Particular arrangements of functional elements of such plasmids, namely origins of replication and eukaryotic transcription/translation control elements, which give rise to generally undesirable side-products upon propagation of the plasmids in bacterial culture are disclosed. These side-products apparently arise as terminated replication intermediates. Strategies both to eliminate accumulation of these side-products, and to make them useful as a vaccine adjuvant, are described.

Abstract: The present invention provides a method of transfer of a gene of interest from a first vector to a product vector comprising contacting a first and second vector in vitro with a site-specific recombinase so as to generate a co-integrate vector comprising the components of the first and second vector, and introducing the co-integrate vector to a prokaryotic host cell so as to generate a product vector by rolling circle replication, comprising the gene of interest.

Abstract: Disclosed herein are replication-competent adenovirus vectors comprising co-transcribed first and second genes under transcriptional control of a heterologous, target cell-specific transcriptional regulatory element (TRE), wherein the second gene is under translational control of an internal ribosome entry site. Methods for the preparation and use of such vectors are also provided. The vectors provide target cell-specific virus replication in applications such as cancer therapy and gene therapy.

Abstract: A recombinant baculovirus is provided with a genetic coding sequence for the production of a foreign protein that is toxic to insects. Preferred are nuclear polyhedrosis viruses, with preferred embodiments having been constructed and expressed in insect cells: BmNPV.AaIT and AcNPV.AaIT. Both embodiments function in insect cells infected therewith so as to have the toxin AaIT (originating from the venom of the scorpion A. australis) secreted. A clone designated (pCIB4223), which contains the Bombyx mori signal sequence fused to the AaIT sequence, has been deposited with the American type culture collection and designated “ATCC 40906.

Abstract: The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions.

Abstract: The invention provides for methods of modifying immunodominant epitopes on polypeptides, preferably polypeptides intended for therapeutic use. Knowledge of immunodominant epitopes prior to clinical use of polypeptides would be useful to design and engineer less immunogenic molecules. The invention provides for methods of identifying immunodominant epitopes and modifying an immunodominant epitope to reduce the immune response to the polypeptide while still retaining a substantial therapeutic activity of the polypeptide. The modified polypeptides are useful therapeutically.

Abstract: The present invention relates to pharmaceutical compositions, kits, and methods of use thereof, comprising, a mutant human herpes simplex-type 1 virus, which is cytopathic to susceptible hyperproliferative cells, such as neoplastic cells. Preferably, the virus does not produce a fully functionally active wild-type ICP0 polypeptide coded for the IE gene 1.

Abstract: The present invention provides a method for covalently joining a DNA strand to an RNA strand using a topoisomerase enzyme. This invention also provides a method for obtaining a cDNA corresponding to a gene using a topoisomerase enzyme.

Abstract: An adenoviral vector including at least one DNA sequence encoding a clotting factor, such as, for example, Factor VIII, or Factor IX. Such vectors may be administered to a host in an amount effective to treat hemophilia in the host. The vectors infect hepatocytes very efficiently, whereby the hepatocytes express the DNA sequence encoding the clotting factor.

Abstract: High-efficiency AAV packaging constructs and methods for their use are provided. in the present invention. These high-efficiency packaging constructs comprise an activating element (such as the P1 sequence located within the AAV S1 integration site of human chromosome 19) amplifiably linked to one or more AAV packaging genes. The constructs may be either integrated into a mammalian cell genome or maintained episomally. Use of the high-efficiency AAV packaging vectors of the invention provides for controlled amplifiable production of rAAV vector constructs.

Abstract: Adenoviral mutants are described that have single amino acid mutations in the E1B-55K protein which mutations effect the p53 binding/inactivation and the late functions of the E1B-55K protein in a manner that enhances the efficacy of such viruses for treating cancer when compared to adenoviral mutants that have the E1B-55K region deleted.

Abstract: A method of directional cloning using the 5′ ends of RNAs for use, for example, in cloning and cDNA library construction is provided.

Abstract: The present invention provides an adenoviral gene transfer vector comprising a first fiber gene and a second fiber gene, wherein the fiber genes are different. The present invention also provides related recombinant adenoviral gene transfer vectors and methods of propagating an adenovirus with a fiber protein that does not bind to a native adenoviral fiber receptor.

Abstract: Disclosed are a recombinant vector for use in gene therapy for insulin-dependent diabetes mellitus and a therapeutic composition thereof. Following the injection of a &bgr;-galactosidase expression vector having a K14 promoter gene, along with a Drosophola's P transposase expression helper vector, into murine skin in a liposome-mediated manner, the &bgr;-galactosidase gene is expressed in the keratinocyte layer from 24 hours to 20 weeks after injection as measured by X-gal staining. With the enhancement effect and tissue specificity, the K14 promoter is applied for the expression of a human insulin gene in keratinocytes, thereby suggesting a new gene therapy method for treating insulin-dependent diabetes mellitus. When, in combination with the P-element expression helper vector, a human insulin expression vector with the K14 promoter is injected into the skin of diabetic mice, which lack insulin-producing &bgr;-cells of the pancreas, their blood glucose levels are maintained in a normal range.

Abstract: The present invention provides polynucleotide sequences of the genome of Staphylococcus aureus, polypeptide sequences encoded by the polynucleotide sequences, corresponding polynucleotides and polypeptides, vectors and hosts comprising the polynucleotides, and assays and other uses thereof. The present invention further provides polynucleotide and polypeptide sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use.

Abstract: The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions.

Abstract: The inventive method of producing a eukaryotic viral vector comprises contacting a eukaryotic cell, which comprises a unique enzyme that nicks or cleaves a DNA molecule, with a recombinant phage vector, or contacting a eukaryotic cell, which does not comprise a unique enzyme that nicks or cleaves a DNA molecule, simultaneously or sequentially, in either order, with (i) a unique enzyme that nicks or cleaves a DNA molecule, and (ii) a recombinant phage vector. The recombinant phage vector comprises the DNA molecule comprising (a) a eukaryotic viral vector genome comprising a coding sequence, (b) a phage packaging site that is not contained within the eukaryotic viral vector genome, and (c) a promoter that is operably linked to the coding sequence.

Abstract: Non-infectious, non-replicating immunogenic HIV-like particles are produced by stable long-term constitutive expression in mammalian cells by eliminating elements toxic to the mammalian cells. An expression vector contains a nucleic acid molecule comprising a modified HIV genome devoid of long terminal repeats and wherein Tat and vpr sequences are functionally disabled and a constitutive promoter operatively connected to the modified HIV genome for constitutive expression of the modified genome to produce the HIV-like particles.

Abstract: Provided herein are methods and compositions relating to the attachment of water soluble polymers to proteins. Provided are novel methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel chemically modified G-CSF compositions and related methods of preparation.

Abstract: The present invention provides a composition of matter, comprising: DNA encoding a viral Vpx protein fused to DNA encoding a protein. In another embodiment of the present invention, there is provided a composition of matter, comprising: DNA encoding a viral Vpr protein fused to DNA encoding a protein. The present invention further provides DNA, vectors and methods for expressing a lentiviral pol gene in trans, independent of the lentiviral gag-pol. A gene transduction element is optionally delivered to a lentiviral vector according to the present invention. Also provided are various methods of delivering a virus inhibitory molecule to a target in an animal. Further provided is a pharmaceutical composition.

Abstract: cDNA including the 5′-terminal sequence of full-length mRNA with a cap structure is synthesized from a mRNA sample containing the full-length mRNA with the cap structure and non-full-length mRNA without any cap structure in mixture. At the first step, the phosphate group at 5′-terminus of the non-full-length mRNA in the mRNA sample is removed. At the second step, the cap structure at the 5′-terminus of the full-length mRNA in the mRNA sample is removed. At the third step, an oligoribonucleotide is ligated to the phosphate group at 5′-terminus of mRNA generated through the first and second steps. At the fourth step, mRNA with the oligoribonucleotide ligated at the 5′-terminus thereof at the third step is subjected to a reverse transcriptase process using a short-chain oligonucleotide capable of being annealed to an intermediate sequence within the mRNA as primer, to synthesize a first-strand cDNA.

Abstract: This invention provides methods for constructing a normalized cDNA library, constructing a low copy gene library, preparing a probe from a biological sample and a kit for constructing a normalized cDNA library.

Abstract: The present invention is directed to improved helper vectors and cell lines for the production of pseudoadenoviral (PAV) vectors containing substantially reduced levels of contaminating helper vector. The invention provides for helper vectors for the production of substantially helper vector-free PAV stocks comprising phag C31 recombinase recognition sequences which, depending upon their arrangement within the helper vector, can prevent helper vector packaging. The invention also provides for improved cell lines for the production of substantially helper vector-free PAV stocks comprising a stably introduced novel circular PAV genome into the cell.

Abstract: The present invention is directed to a bacterial delivery system for delivering alphavirus replicon DNA into an animal or animal cells with the replicon encoding one or more heterologous genes to be expressed in the animal or the animal cells. The bacteria are invasive bacteria or attenuated invasive bacteria engineered to contain a DNA vector that encodes the alphavirus replicon in a eukaryotic expression cassette. The heterologous gene can encode an antigen, a therapeutic agent, an immunoregulatory agent, an anti-sense RNA, a catalytic RNA, a protein, a peptide, an antibody or an antigen-binding fragment of an antibody. In a preferred embodiment, the heterologous gene encodes one or more antigens useful as a vaccine for HIV. In addition to the bacterial delivery system, the invention provides methods of introducing and expressing the heterologous gene(s) in animal or animal cells and methods of stimulating or inducing an immune response.

Abstract: The present invention provides the sequencing of the entire genome of Haemophilus influenzae Rd, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies over 1700 protein encoding fragments of the genome and identifies, by position relative to a unique Not I restriction endonuclease site, any regulatory elements which modulate the expression of the protein encoding fragments of the Haemophilus genome.

Abstract: The present invention provides the sequencing of the entire genome of Haemophilus influenzae Rd, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies over 1700 protein encoding fragments of the genome and identifies, by position relative to a unique Not I restriction endonuclease site, any regulatory elements which modulate the expression of the protein encoding fragments of the Haemophilus genome.

Abstract: Provided are lacZ&agr; gene fragments which have been modified to introduce multiple restriction enzyme sites. Vectors according to the present invention include at least one promoter operatively linked to a DNA sequence encoding lacZ&agr;(&agr;-peptide); multiple cloning sites cleavable by distinct restriction enzymes which have been introduced within a lacZ coding sequence from and including the codon for amino acid 8, and in the lacZ coding sequence downstream of the codon for amino acid 8, in forming the modified lacZ&agr; coding sequence; and a replicon. Also provided are methods of using the vectors wherein a DNA molecule is cloned into at least one restriction enzyme site in the modified lacZ&agr; coding sequence, in forming recombinant vectors; introducing the recombinant vectors into competent host cells; growing the host cells in the presence of a chromogenic substrate cleavable by &bgr;-galactosidase; and screening for indicia of lac operon marker inactivation.

Abstract: The invention provides in one embodiment a composition which is a linear DNA molecule having a desired replacement sequence, and second and third sequences substantially homologous to non-identical portions of the gene and having proximal and distal ends, the proximal ends flanking the desired replacement sequence and the distal ends having a terminating nucleotide analog at each end of the molecule. Another embodiment of the invention provides methods, by blocking the 3′ ends of transforming DNA with 2′3′ dideoxynucleotides, to reduce the frequency of end-mediated DNA insertion. These methods introduce only one copy of the selectable gene at the target locus to achieve a precise gene disruption, reducing or eliminating undesirable and multiple insertions that occur both non-homologously and at the targeted locus.

Abstract: The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

Abstract: Proteins from the merozoite stage of the malaria parasite, fragments and derivatives thereof, DNA coding for the said proteins, and processes for the preparation of the proteins and plasmid and viral vectors useful in said processes. The invention also provides antibodies to the proteins and immunological compositions containing the proteins.

Abstract: A synthetic polynucleotide and a method are disclosed for selectively expressing a protein in a target cell or tissue of a mammal. Selective expression is effected by replacing at least one existing codon of a parent polynucleotide encoding a protein of interest with a synonymous codon to produce a synthetic polynucleotide having altered translational kinetics compared to the parent polynucleotide. The synonymous codon is selected such that it has a higher translational efficiency in the target cell or tissue relative to one or more other cells or tissues of the mammal.

Abstract: Novel adenovirus-derived viral vectors, the preparation thereof, and their use in gene therapy, are disclosed. In particular, recombinant adenoviruses including an adenovirus genome wherein (i) the E1 region is inactivated, (ii) the genomic organization is modified, and (iii) optional recombination with the producing line genome generates non-viable viral particles, are disclosed.

Abstract: The invention relates to a nucleic acid construct which constitutes a self-enhancing expression system and which comprises the following components: at least one first structural gene that encodes an active compound; at least one second structural gene that encodes a transcription factor protein; and at least one activation sequence comprised of at least one sequence that binds the transcription factor protein and at least one promoter sequence; wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein; and to the use of the nucleic acid construct for preparing a drug for treating diseases.

Abstract: The present invention relates to mammalian cell lines which efficiently support the growth and productive infection of Marek's Disease Virus at high titers. The present invention also relates to mammalian cell lines which have been engineered to support the growth and productive infection of recombinant Marek's Disease Virus at high titers. The present invention relates a process for the preparation of Marek's Disease Virus in quantities suitable for vaccine purposes.

Abstract: The invention features two methods for making eucaryotic trans-dominant suppressor genes encoding polypeptide translation products capable of suppressing the activity of a growth factor that requires an oligomeric state for function, suppressor genes made by the methods, protein products of the suppressor genes, and methods for using these genes and protein products.

Abstract: The present invention provides a dual selection cassette (DSC) comprising first and second DNA segments having homology to a eukaryotic viral vector, positive and negative selection genes, each operably linked to their own promoter, and one or more unique restriction enzyme sites (URES) or sitey-directed homologous recombination sites. The present invention also provides a plasmid, pN/P, comprising an independent positive selection marker gene, an origin of replication, and a dual selection cassette. The dual selection cassette and pN/P plasmid can be used to produce eukaryotic gene transfer vectors without requiring temporally-linked double recombination events or the use of specialized bacterial strains that allow the replication of plasmids comprising defective origins of replication. This method usefully increases the ratio of desired to undesired plasmid and vector constructs. Additionally, this invention provides a method for the creation of eukaryotic viral vector libraries.

Abstract: A DNA vector comprises a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus DNA genome replication regions, and a second DNA sequence encoding a paramyxovirus protein, particularly a respiratory syncytial virus fusion (RSV F) protein or a RSV F protein fragment that generates antibodies that specifically react with RSV F protein, the first and second DNA sequences being under the transcriptional control of a promoter, preferably a cytomegalovirus promoter, which may include Intron A. Such vectors also contain a further nucleotide sequence located between the promoter sequence and the alphavirus sequence to enhance the immunoprotective ability of the RSV F protein when expressed in vivo.

Abstract: Disclosed is a vaccine against Lyme Disease or its causative agent Borrelia burgdorferi (sensu stricto or sensu lato) containing a plasmid a DNA encoding a promoter for driving expression in a mammalian cell, DNA encoding a leader peptide for facilitating secretion/release of a prokaryotic protein sequence from a mammalian cell, a DNA encoding Borrelia OspA or OspB, and a DNA encoding a terminator. Disclosed too is an immunogenic composition against Lyme Disease or its causative agent Borrelia burgdorferi (sensu stricto or sensu lato) containing a plasmid comprising a DNA encoding a promoter for driving expression in a mammalian cell, DNA encoding a leader peptide for facilitating secretion/release of a prokaryotic protein sequence from a mammalian cell, a DNA encoding a Borrelia OspC, and a DNA encoding a terminator. And, methods for making and using such vaccines and the immunogenic composition are also disclosed.

Abstract: A method for performing site-directed mutagenesis characterized in that the method includes the step of carrying out PCR by the use of a double-stranded DNA vector having one or more amber codons, the vector resulting from insertion of a target DNA fragment for site-directed mutagenesis, and at least two kinds of selection primers; and a kit for site-directed mutagenesis for use in the above method, characterized in that the kit includes amber codon reversion primers. According to the present invention, there can be provided a method for performing site-directed mutagenesis and a kit, which is useful for genetic engineering or protein engineering, more simply and rapidly. By using the method and the kit of the present invention, it is possible to efficiently obtain a mutation-introduced gene at the desired position by simply transforming a host with a PCR product obtained by PCR.

Abstract: A human tissue inhibitor of metalloproteinases-4 polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for the treatment of diseases, including arthritis and cancer. Antagonists against such polypeptides and their use as a therapeutic to resorb scar tissue are also disclosed. Diagnostic assays for detecting levels of human TIMP-4 protein and mutations in human TIMP-4 nucleic acid sequence are also disclosed.