Abstract: Chimeric viral vectors having both the capacity to infect host cells efficiently and the capacity to integrate their genomic material into the host cell's genome. A chimeric viral vector having a functional packaging signal derived from a first virus and an integration means derived from a second virus. Typically, viruses capable of integrating their material into a host cell genome, having additional genetic material introduced therein recombinantly, do not have much room for insertion of such additional genetic material or are not very well capable of infecting every wanted host cell. Infecting viruses also lack a high insertion capacity or integration into the host cell's genome. The present invention provides integration of large inserts into a host cell's genome at an efficient infection rate by combining the properties of efficiently infecting viruses with efficiently integrating viruses.

Abstract: The problem of replication competent adenovirus in virus production is solved in that we have developed packaging cells that have no overlapping sequences with a new basic vector and thus are suited for safe large scale production of recombinant adenoviruses one of the additional problems associated with the use of recombinant adenovirus vectors is the host-defense reaction against treatment with adenovirus. Another aspect of the invention involves screening recombinant adenovirus vector lots, especially those intended for clinical use, for the presence of adenovirus E1 sequences, as this will reveal replication competent adenovirus, as well as revertant E1 adenoviruses. It is also an aspect of the present invention to molecularly characterize the revertants that are generated in the newer helper/vector combinations.

Abstract: Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces replication defective adenovirus. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, has at least one functional encapsidation signal and at least one functional Inverted Terminal Repeat, and lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

Abstract: Cells capable of at least, in part, complementing adenovirus E2A function of an adenovirus defective in E2A function. Such cells include a nucleic acid encoding adenovirus E2A or a functional part, derivative and/or analogue thereof, integrated into the cell's genome. The cell may have E2A nucleic acid derived from a temperature sensitive adenovirus. Methods for producing an adenovirus particle containing an adenovirus vector with a functional deletion of E2A are also disclosed. Such methods involve providing a cell with the functionally deleted adenovirus vector, culturing the cell, and harvesting viral particle. The functional deletion can comprise a deletion of nucleic acid encoding E2A. In such a method, the nucleic acid encoding adenovirus E2A in the cell's genome has no sequence overlap with the vector leading to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function.

Abstract: A method is provided for genetically modifying primate bone marrow cells, comprising isolating bone marrow cells from a primate and, by means which enhance the local concentration of retroviral contacting the isolated bone marrow cells to cells that produce a recombinant amphotropic retrovirus with a genome based on a retroviral vector that contains the genetic information to be introduced into the bone marrow cells. Recombinant amphotropic retrovirus-producing cells, suitable for use in this method also are provided, as are genetically modified primate bone marrow cells with the capacity for regeneration in vivo.

Abstract: The present invention provides chimeric viral vectors which have both the capacity to infect host cells efficiently and the capacity to integrate their genomic material into the host cell's genome. The invention provides a chimeric viral vector which comprises a functional packaging signal derived from a first virus and an integration derived from a second virus. Typically, viruses capable of integrating their material into a host cell genome, having additional genetic material introduced therein by recombinant process, do not have much room for insertion of such additional genetic material or are not very well capable of infecting every wanted host cell. Infecting viruses also lack a high insertion capacity or integration into the host cell's genome. The present invention provides integration of large inserts into a host cell's genome at an efficient infection rate by combining the properties of efficiently infecting viruses with efficiently integrating viruses.

Abstract: Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces adenovirus incapable of replicating. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell.

Abstract: Genetic modification of pluripotent hemopoietic stem cells of primates (P-PHSC) by transduction of P-PHSC with a recombinant adeno-associated virus (AAV). The genome of the recombinant AAV comprises a DNA sequence flanked by the inverted terminal repeats (ITR) of AAV. The DNA sequence will normally comprise regulatory sequences that are functional in hemopoietic cells and, controlled by these regulatory sequences, a sequence coding for a protein or RNA with a therapeutic property when introduced into hemopoietic cells. Preferred examples of DNA sequences are the human lysosomal glucocerebrosidase gene, a globin gene from the human &bgr;-globin gene cluster, a DNA sequence encoding an RNA or protein with anti-viral activity, the &agr;1-antitrypsin gene and the human multidrug resistance gene I (MDRI). The invention provides for effective gene therapy with PHSC of primates, particularly humans.

Abstract: Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces adenovirus incapable of replicating. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell.

Abstract: The present invention relates to the utilization of conditionally replicating recombinant nucleic acid molecules rescued from the integrated state for the expression of foreign proteins. The usefulness of the system is illustrated with a conditionally replicating recombinant nucleic acid molecule encoding the adeno-associated virus (AAV) capsid proteins. The present invention also relates to methods employing and conditionally replicating recombinant nucleic acid molecules for the packaging of recombinant AAV nucleic acid molecule into AAV capsids. The present invention also relates to packaging cell lines for recombinant AAV, expressing both the AAV rep and cap-genes.

Abstract: Cells capable of at least, in part, complementing adenovirus E2A function of an adenovirus defective in E2A function. Such cells include a nucleic acid encoding adenovirus E2A or a functional part, derivative and/or analogue thereof, integrated into the genome of the cell. Preferably, the cell has E2A nucleic acid derived from a temperature sensitive adenovirus such as adenovirus ts125. Methods for producing an adenovirus particle containing an adenovirus vector with a finctional deletion of E2A are also disclosed. Such a method involves providing a cell as previously described with the functionally deleted adenovirus vector, culturing the cell, and harvesting the virus particle. The functional deletion can comprise a deletion of at least part of the nucleic acid encoding E2A.

Abstract: A method for producing viral gene delivery vehicles which can be transferred to pre-selected cell types by using targeting conjugates. The gene delivery vehicles comprise: 1) the gene of interest; and 2) a viral capsid or envelope carrying a member of a specific binding pair, the counterpart of which is not directly associated with the surface of the target cell. These vehicles can be rendered unable to bind to their natural cell receptor. The targeting conjugates include the counterpart member of the specific binding pair, linked to a targeting moiety which is a cell-type specific ligand (or fragments thereof). The number of the specific binding pair present on the viral vehicles can be, for example, an immunoglobulin binding moiety (e.g., capable of binding to a Fc fragment, protein A, protein G, FcR or an anti-Ig antibody), or biotin, avidin or streptavidin. The virus' outer membrane or capsid may contain a substance which mediates entrance of the gene delivery vehicle into the target cell.

Abstract: Genetic modification of pluripotent hemopoietic stem cells of primates (P-PHSC) by transduction of P-PHSC with a recombinant adeno-associated virus (AAV). Tile genomc of the recombinant AAV comprises a DNA sequence flanked by the inverted terminal repeats (ITR) of AAV. The DNA sequence will normally comprise regulatory sequences which are functional in hemopoictic cells and, controlled by these regulatory sequences, a sequence coding for a protein or RNA with a therapeutic property when introduced into hemopoietic cells. Preferred examples of DNA sequences are the human lysosomal glococerebrosidase gene, a globin gene from the human &bgr;-globin gene cluster, a DNA sequence encoding an RNA or protein with anti-viral activity, the &agr;1-antitrypsin gene and the human multidrug resistance gene I (MDRI). The invention provides for effective gene therapy with PHSC of primates, in particular humans.

Abstract: Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A method of the invention produces adenovirus incapable of replicating. The method includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell.

Abstract: The problem of replication competent adenovirus in virus production is solved in that we have developed packaging cells that have no overlapping sequences with a new basic vector and, thus, are suited for safe large scale production of recombinant adenoviruses. One of the additional problems associated with the use of recombinant adenovirus vectors is the host-defense reaction against treatment with adenovirus. Another aspect of the invention involves screening recombinant adenovirus vector lots, especially those intended for clinical use, for the presence of adenovirus E1 sequences, as this will reveal replication competent adenovirus, as well as revertant E1 adenoviruses. It is also an aspect of the present invention to molecularly characterize the revertants that are generated in the newer helper/vector combinations.

Abstract: A method of producing viral gene delivery vehicles which can be transferred to pre-selected cell types by using targeting conjugates. The gene delivery vehicles comprise: 1) the gene of interest; 2) a viral capsid or envelope carrying a member of a specific binding pair, the counterpart of which is not directly associated with the surface of the target cell. These vehicles can be made unable to bind to their natural receptor on the cell. The targeting conjugates are composed of the counterpart member of the specific binding pair linked to a targeting moiety which is a cell-type specific ligand. The number of the specific binding pair present on the viral vehicles can be, for example, an immunoglobulin binding moiety, biotin, avidin, or streptavidin. The outer membrane or capsid of the virus may contain a substance which mediates entrance of the gene delivery vehicle into the target cell.

Abstract: The present invention relates to the utilization of conditionally replicating recombinant nucleic acid molecules rescued from the integrated state for the expression of foreign proteins. The usefulness of the system is illustrated with a conditionally replicating recombinant nucleic acid molecule encoding the adeno-associated virus (AAV) capsid proteins. The present invention also relates to methods employing and conditionally replicating recombinant nucleic acid molecules for the packaging of recombinant AAV nucleic acid molecule into AAV capsids. The present invention also relates to packaging cell lines for recombinant AAV, expressing both the AAV rep and cap-genes.

Abstract: Adenoviral vectors with a deletion of the E3 region such that the remaining E3 region reduces the TNF response of a host mammalian cell infected with the virus. The portion of the E3 region remaining in these vectors encodes the 14.7 kD protein, and may also contain a deletion of at least the E1a promoter. These partially deleted E3 vectors will inhibit the host cell's immune response so that the infected cell will live longer. Any non-adenoviral gene expressed from these vectors in the infected cell will be produced for a longer length of time and achieve a higher concentration than when adenoviral vectors not expressing function 14.7 kD protein are used. These 14.7 kD expressing adenoviral vectors will be useful for gene therapy and especially in cancer gene therapy where the non-adenoviral DNA sequence being expressed are preferably cytokine genes, such as IL-1, and suicide genes, such as HSV-tk. The vectors also are useful for antisense therapy.

Abstract: The invention is in the field of recombinant genetic materials, especially for use in gene therapy. Vectors used for transferring additional genetic information to cells in the field of gene therapy are often based on viruses. A group of viruses which has been proposed to use for transfection is the group of parvoviruses, in particular the use of adeno associated virus has been proposed. The invention provides improved methods and means for gene therapy and for preparing products to be used in gene therapy using parvovirus based materials. The invention particularly provides regulated expression of genes under control of the combination of a repressor moiety and an activator moiety, particularly for expression of products which are toxic to the host cell in which they are expressed.

Abstract: The present invention provides novel elements for improving genetic engineering techniques for producing recombinant nucleic acid molecules and/or recombinant cells. The novel elements are capable of integrating desired nucleic acid material into other nucleic acid materials, specifically into the genome of a host cell. The novel elements are derived from or based on transposons, in particular from the Tc/Mariner superfamily. In particular, the essential elements of Tc1 enabling excision and pasting of the desired nucleic acid material are provided, together with the relevant transposase activity in cis or in trans.