Introgene B.v.

Crucell Holland B.v.

Other

Het Nederlands Kanker Instituut

Introgene

Domenico Valerio

Abraham Bout

Robert Cornelis Hoeben

Alex Jan Van Der Eb

Frits Jacobus Fallaux

Govert Johan Schouten

Victor Willem Van Beusechem

Markus Peter Einerhand

Markus Peter Wilhelmus Einerhand

Ronald Hans Plasterk

Govert Schoutten

Dirk W Van Bekkum

Jan C Vos

Hendricus Gerhard Van Luenen

Cells capable of at least, in part, complementing adenovirus an adenovirus defective in E2A function. Such cells include a nucleic acid-encoding adenovirus E2A or a functional part, derivative, temperature-sensitive mutation and/or analogue thereof, integrated into the cell's genome. Methods for producing an adenovirus particle/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 particles. The functional deletion may comprise a deletion in E2A. The nucleic acid-encoding E2A in the cell's genome may lack sequence overlap with the vector, preventing formation of a replication-competent adenovirus or restoration of E2A function. The adenovirus vector may further include a functional deletion in the E1-region. Methods are disclosed for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication-competent adenovirus vector, comprising administering the individual an adenovirus vector described herein.

Means and methods for nucleic acid delivery vehicle design and nucleic acid transfer

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. Due to the specificity of the ligand, the binding pair's high affinity, and the gene delivery vehicle's inability to be targeted when used alone, the universality of the method for gene delivery, together with its high cell type selectively can be achieved by using various targeting conjugates.

Methods and means for targeted gene delivery

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.

Packaging systems for human recombinant adenovirus to be used in gene therapy

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.

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. 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.

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.

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.

Methods and compositions for genetically modifying primate bone marrow cells

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.

Adeno-associated virus and adenovirus chimeric recombinant viruses useful for the integration of foreign genetic information into the chromosomal DNA of target cells

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. In such a method, the nucleic acid encoding adenovirus E2A in the genome of the cell preferably has no sequence overlap with the vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function. In the method, the adenovirus vector preferably further comprises a functional deletion of E1-region encoding nucleic acid. Adenovirus vectors comprising a functional deletion of adenovirus E2A, preferably a deletion of at least part of the nucleic acid encoding E2A are also disclosed. Preparations of adenovirus vector containing adenovirus particles wherein the adenovirus vector comprises a functional deletion of E2A are also disclosed. Such an adenovirus vector preferably further includes a deletion of nucleic acid encoding E1-region proteins, and may be free of adenovirus vectors comprising E2A function. Methods for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication competent adenovirus vector, comprising administering the individual one of the previously described preparations are also disclosed.

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.

Genetic modification of primate hemopoietic repopulating stem cells

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. 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.

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.

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. Due to the specificity of the ligand, the high affinity of the binding pair and to the inability of the gene delivery vehicle to be targeted when used alone, the universality of the method for gene delivery, together with its high cell type selectively can easily be achieved by the use of various adequate targeting conjugates.

Growing community of inventors

Oegstgeest, Netherlands

Domenico Valerio