Abstract: A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a “gutless” adenoviral vector that include cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins. Also provided are methods of using the subject systems to produce virions having the subject helper dependent adenoviral vectors encapsulated in an adenoviral capsid. In addition, kits for use in practicing the subject methods are provided.

Abstract: This invention provides transcriptionally-activated AAV ITRs (inverted terminal repeats) which are small and transcriptionally active and uses thereof to optimize the expression of relatively large transgenes packaged in recombinant AAV vectors.

Abstract: AAV vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantities of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. Stable AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and can negatively affect the host cell. This invention provides packaging systems and processes for packaging AAV vectors that effectively circumvent these problems and that allow for substantially increased packaging efficiency.

Abstract: The present invention generally relates to viral vectors and their use as expression vectors for transforming human cells, both in vitro and in vivo. More particularly, the present invention relates to adenoviral vectors containing propapoptotic genes and their use in cancer therapy.

Abstract: The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.

Abstract: The invention includes a viral vector method and composition comprising transcomplementary replication incompetent viral vectors, preferably adenoviral vectors, which are cotransformed to a recipient cell. The two vectors complement each other and thus allow viral replication, in a synergistic combination which enhances both gene delivery and gene expression of genetic sequences contained within the vector.

Abstract: The invention features non-transformant immortal avian cells, in particular derived from avian tissues, i.e., other than blood or hematopoietic cells, particularly fibroblasts and epithelial cells, for instance of embryos. The avian cells are immortalized by the SV40 T+t gene in the dependence of the MTI promoter. In particular they integrate the pDAMT vector.

Abstract: The invention provides novel replication deficient adenovirus vectors and methods for making and using these viruses. The invention also provides vector systems and kits using a serotype specific strategy for making adenoviral vector preparations substantially free of replication competent “helper” virus. The helper virus-free preparations provide novel pharmaceutical compositions substantially free of helper virus for use in gene transfer and gene therapy.

Abstract: A method of packaging a recombinant viral vector is carried out by: (a) providing a packaging cell, the packaging cell containing and expressing a nucleic acid encoding a mutant adenovirus E4orf6 protein, the E4orf6 protein containing at least one mutation that renders the protein non-toxic to the host cell; (b) transfecting or infecting the packaging cell with a nucleic acid that encodes a recombinant viral vector (e.g., an adenovirus vector or an adeno-associated virus vector), where the vector lacks a functional gene encoding E4orf6 protein; (c) culturing the transfected cells; and then (d) collecting packaged recombinant viral vector from the cultured cells. Nucleic acids, vectors and packaging cells used for carrying out the methods, as well as proteins utilized in the methods, are also described.

Abstract: The present invention relates to a genetically modified helper phage, named Ex-phage, for packaging phagemid vector. For the modification, amber codons are introduced at 5′region of the helper phage genome by site-directed mutagenesis. The resulted mutant helper phage produces wild-type pIII in suppressive strains but not in non-suppressive strains. Furthermore, this invention provides a method of preparing phage display library expressing various foreign proteins on the surfaces of the phages.

Abstract: A method for the production of adeno-associated virus stocks and recombinant adeno-associated virus stocks that are substantially free of contaminating helper virus is described. The method utilizes transfection with helper virus vectors to replace the infection with helper virus used in the conventional method.

Abstract: A method of screening for a target molecule from a group of potential target molecules is described. This method involves using a library of lentiviral vectors where the members encode the group of target molecules, then transducing a group of cells and screening the transduced cell for a desired phenotype. The cell(s) displaying the desired phenotype is selected and the target molecule is identified.

Abstract: The invention relates to AAV helper plasmids for the helper virus-free packaging of AAV vectors. These AAV helper plasmids comprise the following DNA sequences: (a1) the rep gene of AAV-2, and (a2) the cap gene of AAV-1, AAV-3, AAV-4, AAV-5 or AAV-6, or (b) the cap gene and the rep gene of AAV-1, AAV-3, AAV-4, AAV-5 or AAV-6 each, and (c) all of the other helper virus DNA sequences necessary for forming AAV particles. The invention also relates to the use of these AAV helper plasmids or AAV particles with a coat encoded by these AAV helper plasmids and an AAV expression vector for gene therapy.

Abstract: The present invention relates to a retroviral vector which is especially applicable as a safe gene transfer vehicle for targeted gene therapy. Said retroviral vector comprises one or more promoters inserted in antisense orientation within the 5′ LTR region and one or more coding sequences inserted in antisense orientation within the 3′ LTR region. Both, the promoter as well as the coding sequence, are additionally inserted in such a way as to ensure that the promoter and the coding sequence become duplicated during the process of reverse transcription in a target cell and thus appear in the 3′ as well as in the 5′ LTR region of the resulting provirus in a fashion where the promoter is located upstream of the coding sequence allowing it to drive gene expression. This system avoids any leakiness of gene expression in the packaging cells, and allows expression of transferred genes in the target cell without the necessity for external stimuli.

Abstract: A recombinant adenoviral vector which has multiple adenovirus packaging domains is provided. This vector has advantages over conventional adenoviral vectors in packaging plasmid vectors into adenoviral capsids. Methods of making and using this vector are described.

Abstract: A defective Cre-loxP based helper virus (HSV-1 LaL&Dgr;J), which genome is of reduced size and is free of the genes encoding ICP4 and ICP34.5 proteins from the helper genome, in addition to the native “a” signals. HSV-1 LaL&Dgr;J carries a single floxed “a” signal in gC locus. To produce HSV-1 LaL&Dgr;J and to prepare the amplicon vectors, two novel cell lines expressing the essential ICP4 protein, either alone or in combination to Cre recombinase, are also disclosed. These cell lines complement ICP4 while minimizing the probability of generating replication-competent particles. The novel helper system enables production of large amounts of high-titer amplicon vectors. Residual helper particles generated do not exceed 0.5% of the viral population and can grow only in cells expressing ICP4. Amplicon vectors produced with this method showed no cytotoxicity for infected cells.

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 relates to methods and materials for recombinant adeno-associated virus production. More particularly, the invention relates to use of recombinant adenovirus encoding adeno-associated virus protein in recombinant adeno-associated virus production methods.

Abstract: The invention is directed to a lentiviral vector system that can be used for episomal replication of a desired gene. The vector system contains a first vector containing a lentiviral gag gene encoding a lentiviral gag protein, a second vector containing an env gene encoding a functional envelope protein, and a lentiviral pol gene encoding a lentiviral pol protein. The pol protein is at least integrase, but that the integrase has been modified so that it is not capable of integration. This pol gene can be on the first or second vectors or on a third vector. The lentiviral gag and pol and the envelope protein are not on a single vector, and these vectors do not contain nucleotides to effectively package lentiviral RNA. The system also contains another vector having a nucleic acid sequence encoding a target molecule operably linked to a component of an episomal replicon and a lentiviral packaging sequence.

Abstract: The present invention relates to AAV vector packaging plasmids for the helper virus-free preparation of (pseudotyped) AAV particles by means of single transfection. The AAV vector packaging plasmids for the (pseudotyped) AAV particles comprise the following DNA sequences: (a) a rep gene of AAV, (b) a cap gene of AAV, (c) AAV expression vector DNA sequences, and (d) all further helper virus DNA sequences necessary for forming AAV particles. The AAV vector packaging plasmids for the preparation of wtAAV particles are characterized in that they (a) comprise the complete AAV genome and (b) all of the helper virus DNA sequences necessary for forming AAV particles. The invention also relates to the use of these AAV vector packaging plasmids for preparing wtAAV particles or pseudotyped AAV particles in particular for gene therapy or tumor therapy.

Abstract: Viral replicon selected nucleic acid expression libraries are useful for analyzing multiple antigens associated with a parasite, pathogen or neoplasia or for preparing immunogenic compositions for generating immune responses specific for the parasite, pathogen or neoplasia. Alphavirus replicon particles representative of the nucleic acid expression library are preferred. The nucleic acid library can be a random library, or it can be prepared after a selection step, for example, by differential hybridization prior to cloning into the replicon vector.

Abstract: The invention discloses hybrid vectors for delivering genes or other nucleic acids into mammalian cells. The hybrid vectors of the invention contain both a helper dependent adenoviral portion and a second portion derived from a transposon. Such vectors provide efficient transduction of quiescent cells and provide for stable integration of the gene to be delivered.

Abstract: The present invention relates to novel methods for the identification of antigens recognized by cytotoxic T cells (CTLs) and specific for human tumors, cancers, and infected cells, and the use of such antigens in immunogenic compositions or vaccines to induce regression of tumors, cancers, or infections in mammals, including humans. The invention encompasses methods for induction and isolation of cytotoxic T cells specific for human tumors, cancers and infected cells, and for improved selection of genes that encode the target antigens recognized by these specific T cells. The invention also relates to differential display methods that improve resolution of, and that reduce the frequency of false positives of DNA fragments that are differentially expressed in tumorous, cancerous, or infected tissues versus normal tissues. The invention further relates to the engineering of recombinant viruses as expression vectors for tumor, cancer, or infected cell-specific antigens.

Abstract: This invention relates to novel nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector (“carrier vector”) is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous preparation, compositions to produce the recombinant virus, and novel recombinant viruses.

Abstract: The present invention provides a helper cell for expressing an infectious, replication defective, alphavirus particle in an alphavirus-permissive cell. The helper cell includes (a) a first helper RNA encoding (i) at least one alphavirus structural protein, and (ii) not encoding at least one alphavirus structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding the alphavirus structural protein encoded by the first helper RNA, and (ii) encoding the at least one alphavirus structural protein not encoded by the first helper RNA. Preferably, the helper cell is co-transfected with a replicon RNA encoding an alphavirus packaging segment and an inserted heterogeneous RNA, such that all of the alphavirus structural proteins assemble together into alphavirus particles in the cell, with said replicon RNA packaged therein.

Abstract: The subject matters of this invention are the hormone-dependent forms of Rep 78 and Rep 68 proteins of the Adeno-associated virus (AAV), obtained by the fusion of their specific mutants with the hormone binding domain (HBD) of steroid hormone receptors, and the DNA sequences coding for them. The invention also refers to a method for the hormonal regulation of the activity of the fusion products Rep78/68-HBD, inserted into eucaryotic cells utilizing viral or non-viral systems, in order to direct the stable integration of DNA sequences in specific regions of the host human genome for therapeutic purposes. The fusion products Rep78/68-HBD are also utilized to generate viral hybrid vectors (i.e. adenovirus vectors AAV) and for generating recombinant vectors AAV.

Abstract: The present invention relates to the use of non-primate mammalian cell lines having substantially no endogenous retroviral sequences as producer and packaging lines for preparation of human serum-resistant retroviral vector particles with improved safety for use in gene therapy applications. In a preferred embodiment, the cell line used in the present invention is the &agr;-galactosyl (&agr;Gal)-positive cell ferret brain cell line designated as Mpf or a cell line having those identifying characteristics of the Mpf cell line suitable for the practice of the invention.

Abstract: The present invention provides a process for preparing a retrovirus to be expressed at a high titer by specifically transferring a desired foreign gene into target cells. A pseudotyped retrovirus vector having a high titer can be prepared by transferring a DNA construction wherein a promoter, an loxP sequence, a VSV-G gene and a polyA addition signal are arranged in this order is transferred into cells carrying the retrovirus gag and pol gene expression systems, and then transferring a retrovirus vector containing the desired foreign gene thereinto, followed by treatment with a recombinase.

Abstract: The present invention related to methods and compositions comprising recombinant vectors comprising chimeric capsids and recombinant pseudotyped vectors with non-native capsid protein(s). The recombinant vectors of the invention confer an altered tropism that permits selective targeting of desired cells.

Abstract: The present invention provides a chimeric protein IX (pIX). The chimeric pIX protein has an adenoviral pIX domain and also a non-native amino acid. Where the non-native amino acid is a ligand that binds to a substrate present on the surface cells, the chimeric pIX can be used to target vectors containing such proteins to desired cell types. Thus, the invention provides vector systems including such chimeric pIX proteins as well as methods of infecting cells using such vector systems.

Abstract: The invention relates to host cells for packing a recombinant adeno-associated virus (rAAV). Said cells contain at least one copy of a first auxiliary construct for expressing at least one AAV Rep protein, and at least one copy of another auxiliary construct for expressing at least one AAV Cap protein. The invention also relates to auxiliary constructs for expressing at least one AAV Rep protein and one AAV Cap protein in a host cell; vector constructs comprising at least one nucleic acid which is heterologous in relation to the AAV; a method for producing a host cell for packing a recombinant adeno-associated virus (rAAV); and the use of the host cell for producing an rAAV.

Abstract: The present invention relates to methods and compositions for the production of viral vectors. In particular, the present invention provides methods and compositions for faster, higher titer and higher purity production of viral vectors (e.g. adenoviral vectors). In some embodiments, the present invention provides gutted and helper viruses with identical or similar termini. In other embodiments, the present invention provides terminal protein linked adenoviral DNA. In certain embodiments, the present invention provides template extended adenoviral DNA.

Abstract: The vectors comprise a recombinant defective viral genome expressing at least one antigen suitable for the induction of systemic and secretory immune responses or an antibody conferring protection against an infectious agent. The defective viral genome comprises the genome of a parental virus having the viral replicase recognition signals located on ends 3′ and 5′, further comprising internal deletions, and wherein said defective viral genome depends on a helper virus for its replication and encapsidation. These vectors are suitable for the forming of a recombinant system comprising the aforesaid expression vector, and a helper virus. The system is suitable for the manufacture of mono- and polyvalent vaccines against infectious agents of different animal species, especially pigs, dogs and cats, and as expression vehicles for antibodies protective against infectious agents.

Abstract: The present invention relates to novel methods for the identification of antigens recognized by cytotoxic T cells (CTLs) and specific for human tumors, cancers, and infected cells, and the use of such antigens in immunogenic compositions or vaccines to induce regression of tumors, cancers, or infections in mammals, including humans. The invention encompasses methods for induction and isolation of cytotoxic T cells specific for human tumors, cancers and infected cells, and for improved selection of genes that encode the target antigens recognized by these specific T cells. The invention also relates to differential display methods that improve resolution of, and that reduce the frequency of false positives of DNA fragments that are differentially expressed in tumorous, cancerous, or infected tissues versus normal tissues. The invention further relates to the engineering of recombinant viruses as expression vectors for tumor, cancer, or infected cell-specific antigens.

Abstract: It is intended to establish a novel cell line for efficiently producing a virus vector without resort to any troublesome operations and provide a process for producing a virus vector having a high titer with the use of the cell line. Namely, cells to be used in producing a virus vector having an antisense gene been transferred thereinto are provided, wherein the gene expresses an antisense RNA against the whole or partial sequence of a sense RNA expressed by a gene encoding a cytotoxic polypeptide.

Abstract: Novel molecular chimaeras produced by recombinant DNA techniques are described. They comprise a target-tissue specific transcriptional regulatory sequence (TRS) linked and controlling the expression of a heterologous enzyme, for example Varicella Zoster Virus Thymidine Kinase (VZV TK) or non-mammaliam Cytosine Deaminase (CD). A molecular chimaera is packaged into a synthetic retroviral particle that is capable of infecting mammalian tissue. This, in turn, may be administered to a host, and the TRS will be selectively transcriptionally activated in the target tissue (for example cancerous cells). Administration of compounds that are selectively metabolised by the enzyme produce cytotoxic or cytostatic metabolites in situ thereby selectively killing or arresting the growth of the target cells.

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: The present invention is directed to alphavirus expression vectors comprising at least part of an alphavirus genome and heterologous RNA inserted downstream of an alphavirus base sequence having translation enhancing activity. Such vectors can be used to achieve enhanced levels of expression of DNA or cDNA coding for a desired product and being complementary to said heterologous RNA after introduction of said vector in eukaryotic cells in cell culture or in a living body. The expression product may have therapeutic or prophylactic activity.

Abstract: This invention provides novel methods and compositions for use in the efficient and large-scale production of recombinant adeno-associated virus (AAV). Described herein are new producer cell lines, recombinant adenovirus or herpes virus vectors and AAV constructs. Also disclosed are particularly advantageous methods of using such materials to produce recombinant AAV virions using only the efficient process of viral infection, without requiring transfection steps. The AAV produced may be used in a variety of embodiments including, for example, for transferring exogenous genes into human cell lines and for use in human gene therapy regimens.

Abstract: The invention provides cells and methods of using the cells for the propagation of replication-deficient adenoviral vectors. The cells comprise at least one heterologous nucleic acid sequence which upon expression produces at least one non-adenoviral gene product that complements in trans for a deficiency in at least one essential gene function of one or more regions of an adenoviral genome so as to propagate a replication-deficient adenoviral vector comprising an adenoviral genome deficient in the at least one essential gene function of the one or more regions when present in the cell.

Abstract: The present invention relates to a triple hybrid vector amplicon system comprising genetic elements derived from Herpes Simplex Virus (HSV), Epstein-Barr Virus (EBV) or Adeno-Associated Virus (AAV), and a retrovirus. The vector was developed to stably transform cells, both in culture or in vivo, into retrovirus packaging cells in a single step. This step can be accomplished both by transfection using liposomes, electroporation, calcium phosphate, or any other methodology used to transfer naked or complexed DNA into cells or by infection when the vector is packaged as an amplicon vector in HSV virions.

Abstract: The present invention provides methods for making and using chimeric AAV particles having broad tissue tropism. The AAV particles may be used to deliver nucleic acid sequences encoding a desired protein wherein infection by chimeric AAV particles provides means to transduce cells with the nucleic acid sequences. Such gene transduction results in production of the desired protein in a cell, and thus establishes or restores the activity of the protein in the cell. The present invention also provides pharmaceutical compositions comprising such chimeric AAV particles for use in the therapeutic treatment of patients.

Abstract: The invention provides improved methods and products based on adenoviral materials which can advantageously be used in for instance gene therapy. In one aspect an adenoviral vector is provided which has no overlap with a suitable packaging cell line which is another aspect of the invention. This combination excludes the possibility of homologous recombination, thereby excluding the possibility of the formation of replication competent adenovirus. In another aspect an adenovirus based helper construct which by its size is incapable of being encapsidated. This helper virus can be transferred into any suitable host cell making it a packaging cell. Further, a number of useful mutations to adenoviral based materials and combinations of such mutations are disclosed, which all have in common the safety of the methods and the products, in particular avoiding the production of replication competent adenovirus and/or interference with the immune system. Further, a method of intracellular amplification is provided.

Abstract: The subject invention concerns a recombinant adeno-associated virus vector characterized as being capable of delivering and expressing at least one mammalian gene into a genome of a mammalian host cell such that the expression of the gene is regulated in a tissue specific manner by cis-acting regulatory and promoter elements of the gene. A method of using this recombinant adeno-associated virus vector for therapeutic purposes is also provided.

Abstract: The present invention provides a helper cell for expressing an infectious, replication defective, alphavirus particle in an alphavirus-permissive cell. The helper cell includes (a) a first helper RNA encoding (i) at least one alphavirus structural protein, and (ii) not encoding at least one alphavirus structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding the alphavirus structural protein encoded by the first helper RNA, and (ii) encoding the at least alphavirus one structural protein not encoded by the first helper RNA, such that all of the alphavirus structural proteins assemble together into alphavirus particles in the cell. Preferably, the helper cell also includes a replicon RNA encoding an alphavirus packaging sequence and an inserted heterogeneous RNA.

Abstract: The present invention relates to methods for efficient and reliable construction of adenovirus vectors which contain and express foreign DNA and are useful for gene transfer into mammalian cells, for vaccines and for gene therapy. The invention provides for the growth and purification of adenovirus vectors (helper dependent vectors or HDVs) from which all or most of the viral genes have been removed. The vector system described herein is a new method designed to eliminate helper viruses from the final HDV preparation by cleavage of the helper virus DNA with an endonuclease, alone or in combination with other methods known to limit the level of helper virus contamination of helper dependent vector preparations. The disclosed methods and compositions also provide for regulated control of gene expression.

Abstract: Gene therapy can treat obesity in mammals. An obesity regulating gene is delivered to a mammal. Preferably, the gene encodes leptin or a leptin receptor. The protein which is delivered and expressed in vivo is more effective than protein which is injected into the animal.

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: The present invention is directed to methods for generating high titer, contaminant free, recombinant AAV vectors, methods and genetic constructs for producing recombinant AAV vectors conveniently and in large quantities, methods for the delivery of all essential viral proteins required in trans for high yields of recombinant AAV, recombinant AAV vectors for use in gene therapy, novel packaging cell lines which obviate the need for cotransfection of vector and helper plasmids, helper plasmids and vector plasmid backbone constructs, a reporter assay for determining AAV vector yield. Further provided are recombinant AAV vectors in a pharmaceutically acceptable carrier, methods of delivering a transgene of interest to a cell, compositions and methods for delivering a DNA sequence encoding a desired polypeptide to a cell, and transgenic non-human mammals that express a human chromosome 19 AAV integration locus.

Abstract: AAV vectors may have utility for gene therapy but heretofore a significant obstacle has been the inability to generate sufficient quantities of such recombinant vectors in amounts that would be clinically useful for human gene therapy application. Stable AAV packaging cell lines have been elusive, mainly due to the activities of Rep protein, which down-regulates its own expression and can negatively affect the host cell. This invention provides packaging systems and processes for packaging AAV vectors that effectively circumvent these problems and that allow for substantially increased packaging efficiency.