Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-11-03
2002-08-20
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S239000, C435S320100, C435S325000, C435S366000
Reexamination Certificate
active
06436693
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the Invention
The present invention relates to a method for helper virus-free packaging of gene vector DNA into the viral particles of a DNA virus as well as to eukaryotic helper cells for helper virus-free packaging of gene vector DNA into the viral particles of a DNA helper virus wherein a DNA virus having a genome ≧100 kbp is used.
(ii) Description of the Related Art
Epstein-Barr-virus (EBV) is one of eight known herpes viruses in humans. The DNA sequence of the EBV B95.8 isolate has been determined (Baer et al., 1984), and detailed scientific evidence has been worked out mainly with respect to DNA elements which play important roles in the two EBV phases. In the so-called ‘latent phase’ the virus establishes a stable host cell relation during which the vitality of the cell remains unaffected, however, the viral DNA genome is replicated in the form of an extrachromosomal plasmid in the host cells and passed on into the daughter cells. The latent phase may be associated with a transformation or immortalization, respectively, of the cells infected in latent manner. The replication origin of the plasmid, oriP, is the DNA element essential for maintenance and replication of the EBV genome in the latent phase. (Yates et al., 1985). This DNA element is also active in recombinant plasmids and has been used as such in several ways.
In the so-called ‘lytic or productive phase’ the virus is released which involves the expression of almost all of the viral proteins necessary for gene regulation or production of structural viral components, respectively. The lytic phase also requires two other DNA elements of the virus: the lytic origin of replication, oriLyt, is responsible for viral DNA amplification (Hammerschmidt and Sugden, 1988), the terminal repeats, TR, which represent packaging signals indispensable for encapsidation of the amplified EBV DNA (Hammerschmidt and Sugden, 1989; Zimmermann and Hammerschmidt, 1995).
There is a broad interest in the genetic analysis of EBV functions, construction of recombinant EBV genomes, as well as in EBV gene vectors. There is particular interest in the gene vectors because additional therapeutically relevant genes may be introduced into suitable recipient cells. In this respect, however, the co-transfer of undesired EBV genes into the target cells must be excluded. This problem has to be solved to comply with the requirements for saftey of this novel form of therapy and to be able to use herpes virus gene vectors in gene therapy in humans.
The strategy in the production of gene vectors is directed to provide only the gene vectors themselves with a viral capsid while the release of so-called helper viruses is prevented. This is achieved by removing or mutating cis-acting DNA elements of the helper virus genome necessary for the production of the helper virus itself. To date it was impossible to genetically manipulate the portions of the EBV genome which are indispensable for the maturation of viral particles. Thus, it was impossible to switch-off functions of EBV or other herpes viruses which are for example absolutely required for the packaging of the viral genome. Up to now, cell lines have been conventionally used which were infected by herpes viruses in a targeted manner to utilize all of the viral functions for gene vector amplification. The simultaneous amplification of the infectious virus and the packaging of its genome into a viral capsid was unavoidable. Similarly up to now also we used cell lines which were infected by EBV in a latent manner. In this case also the lytic functions of the virus are necessary for packaging of suitable gene vectors so that an amplification of the helper virus genome and subsequent packaging was unavoidable (Hammerschmidt and Sugden, 1989; Kempkes et al., 1995b).
DISADVANTAGES OF THE RELATED ART
Viral gene vectors are an important instrument in gene therapy. However, it must be ensured that in their preparation and therapeutical use viral gene vectors are prepared which do not contain any virus. Since certain viral gene functions are indispensable for the production of viral gene vectors it is impossible to avoid the use of so-called helper viruses or the functional segments thereof. This particularly applies to complex viral systems relying on numerous virus-encoded functions. Thus, EBV for example encodes more than 80 genes, of which about 50 are indispensable for viral synthesis. Since these genes in their entirety are present on the viral genome in a single DNA molecule certain properties have to be missing from the genome to prevent its packaging into virus particles and its release together with the gene vector. One possiblity to prevent this release of the helper virus is to delete the cis-acting portions of the genome which are essential for the packaging of the helper virus genome itself. In EBV, these genomic regions are referred to as TR (TR=terminal repeats) sequences, in other herpes viruses as pac sequences (pac=packaging). Alternatively, also other regions may be deleted or their function may be abolished which are indispensable for helper virus genome replication in cis. In EBV, this would be the lytic origin of replication, oriLyt, the functional equivalent of which is named oriS or oriL in other herpes viruses.
The deletion of these regions prevents helper virus release; however it also prevents the possibility to generate or even to introduce the helper virus itself via infection into the cells considered for packaging of the viral gene vector. Since these properties mutually exclude each other it has been impossible to date to establish cell lines capable of producing herpes virus gene vectors without releasing helper viruses. The same is true for gene vectors derived from other viral systems which have viral genomes of a size of more than 100 kb. In addition, it is also impossible to avoid this disadvantage by biochemical separation of helper virus and gene vector because both particles differ only with respect to their genetic but not their external or physical composition. Our invention permits for the first time to overcome said disadvantage.
SUMMARY AND OBJECTS OF THE INVENTION
It is an object of the present invention to provide a method of packaging of a gene vector DNA into the virus particles of a DNA virus using on the one hand the proteins for the production of viral particles of a DNA virus having a genome ≧100 kbp and on the other hand avoiding the packaging of helper virus DNA into the viral particles. This object has been solved by the method having the following steps of:
a) Introduction of a DNA helper virus vector DNA on one or more molecules having at least the following features:
(&agr;) a total size of ≧100 kbp;
(&bgr;) having at least one mutation designed to render one or more of the cis-acting signal sequences for packaging of the DNA helper virus vector DNA non-functional;
(&ggr;) having the information for the production of DNA virus particles of the DNA helper virus which do not contain a helper virus genome; into an eukaryotic cell;
(b) introduction of a gene vector DNA to be packaged having at least
(&agr;) a cis-acting signal sequence for packaging of the gene vector DNA into a viral particle of the DNA helper virus;
(&bgr;) a gene of interest; into the eukaryotic cell;
(c) induction of the lytic phase of the DNA helper virus and production of the proteins important for packaging of the DNA helper virus;
(d) packaging of the gene vector DNA into the viral particles of the DNA helper virus; optionally
(e) releasing the viral particles containing the gene vector DNA; and/or optionally
(f) purifying the viral particles.
According to the invention the DNA helper virus vector DNA may be present not only on one molecule but for example also on two or more molecules, the total size of the molecules being ≧100 kbp.
The DNA helper virus vector DNA is mutated so that one or more of the cis-acting signal sequences for packaging of the DNA are non-functional, i.e. the helper virus DNA may provide the inf
Delecluse Henri-Jacques
Hammerschmidt Wolfgang
Pich Dagmar
GSF Forschungszentrum fur Umwelt und Gesundheit GmbH
Mosher Mary E.
Nixon & Vanderhye P.C.
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