Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-12-29
2003-12-02
Wehbe', Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C514S04400A, C435S455000, C435S456000
Reexamination Certificate
active
06656727
ABSTRACT:
BACKGROUND OF THE INVENTION
Retroviruses infect a wide variety of cells and are ideal tools for the delivery of genes to target cells. They are furthermore an ideal tool to stably integrate a heterologous sequence in the genome of a target cell, since the infecting retrovirus is able to integrate the DNA form of its RNA genome into the genome of the target cell. Thus, all daughter cells of a retroviral infected cell carry the retroviral vector DNA possibly comprising a heterologous gene.
A retroviral genome consists of a RNA molecule with the structure R-U5-gag-pol-env-U3-R. For the development of a retroviral vector (RV) said retroviral genome can be modified by replacing the genes gag-pol-env-encoding viral proteins—with one or more genes of interest such as marker genes or therapeutic genes. To generate a recombinant retroviral particle and a packaged RV, respectively, the principle of a retroviral vector system is used. This system consists of two components: the RV itself in which the genes encoding the viral proteins have been replaced, and a packaging cell which provides the modified RV with the missing viral proteins. This packaging cell has been transfected with one or more plasmids carrying the genes enabling the modified RV to be packaged, but lacks the ability to produce replication competent viruses.
After introduction of the vector into the packaging cell line, the RV is transcribed into RNA. This RNA which represents the recombinant retroviral genome is packaged by the viral proteins produced by the packaging cell to form retroviral particles which bud from the packaging cell. These particles are further used to infect a target cell. In the target cell the RNA genome is released again from the particle, reverse transcribed and stably integrated into the cellular genome.
Therefore, RVs are currently the method of choice for a stable transfer of therapeutic genes into a target cell in a variety of approved protocols both in the USA and in Europe. However, most of the protocols require that the infecting of target cells with the RV carrying the therapeutic gene occurs in vitro. Subsequently, successful infected cells are returned to the affected individual. Advantageously, such ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use. Furthermore, the ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated cultured and then reintroduced to a patient. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the RV, or cells producing the RV, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all safety considerations have to be addressed. One serious safety risk is that virus will be produced, possibly form an implantation of virus producing cells. Thus, there will be no opportunity to precheck said produced virus. Another problem is that the proviral form of the retroviral genome integrates randomly in the genome of infected cells. This random integration can result in an integration directly into a cellular gene or into the vicinity of a cellular gene, leading to new genomic arrangements. As a result of this function of the cellular gene can be altered or lost. In the case that the cellular gene is involved in the regulation of growth control, uncontrolled proliferation of the cell may result. Therefore, using RV in gene therapeutic applications there is a potential risk that simultaneously to the repair of one genetic defect with retroviral vectors, a second defect can be established resulting in uncontrolled proliferation, and thus, in tumor development.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a safe retroviral vector which prevents random integration of the recombinant viral genome into genes or into the vicinity of genes of a target cell genome, thus, preventing genomic rearrangements of the target cell genome.
The invention inter alia comprises the following, alone or in combination:
A retroviral vector comprising one or more heterologous nucleic acid sequence(s) as well as at least one sequence allowing site-specific integration of said heterologous sequence(s) into a non-coding region of a genome;
the retroviral vector as above, wherein the sequence(s) allowing site specific integration is inserted at the U3 region(s) and/or the U5 region(s) of the retroviral Long Terminal Repeat (LTR);
the retroviral vector as above, wherein the sequence allowing site specific integration is an Inverted Terminal Repeat (ITR) sequence of Adeno-associated virus (AAV); the retroviral vector as any above, wherein the genome is a chromosome of a mammal, including human;
the retroviral vector as above, wherein the chromosome is chromosome 19;
the retroviral vector as any above, wherein at least one of the heterologous nucleic acid sequence(s) is a heterologous gene relevant for the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
the retroviral vector as any above, wherein at least one of the heterologous nucleic acid sequence(s) is a sequence encoding an integration-mediating protein;
the retroviral vector as above, wherein the integration-mediating protein is the AAV Rep protein;
the retroviral vector as above, wherein the sequence encoding for the integration-mediating protein is under transcriptional control of an inducible promoter;
a retroviral vector system comprising the vector as any above as a first component, and a packaging cell harboring at least one DNA construct encoding for proteins required for said vector to be packaged;
the retroviral vector system as above, wherein the packaging cell synthesizes a mutated or a completely or partially deleted retroviral integrase (IN);
a retroviral particle comprising a retroviral vector as any above;
the retroviral particle as above obtainable by transfecting a packaging cell of a retroviral vector system as above with the retroviral vector as above;
a retroviral provirus produced by infection of target cells with the retroviral particle as above;
mRNA of a retroviral provirus as above;
RNA of the retroviral vector as any above;
cDNA of the RNA as above;
a host cell infected with the retroviral particles as above;
a method for introducing homologous and/or heterologous nucleotide sequences into target cells comprising infecting the target cells with retroviral particles as above;
the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above for the use in the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
use of the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above for producing a pharmaceutical composition for the treatment of a viral infection or the treatment of a genetic, metabolic, proliferative or any other relevant disorder or disease;
a pharmaceutical composition containing a therapeutically effective amount of the retroviral vector as any above and/or the retroviral particle as above and/or the retroviral vector system as above;
a method of treating a viral infection or a genetic, metabolic, proliferative or any other relevant disorder or disease comprising administering to a subject in need thereof a therapeutically effective amount of the retroviral particle as above and/or the retroviral vector system as above.
DETAILED DESCRIPTION OF THE INVENTION
The basic idea underlying the
Goller Sabine
Günzburg Walter H.
Klein Dieter
Salmons Brian
Baker & Botts L.L.P.
Institut Fur Virologie
Li Qian J
Wehbe' Anne M.
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