Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-01-27
2002-11-12
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S235100, C435S320100, C435S091330, C435S091400, C435S455000, C536S023100
Reexamination Certificate
active
06479290
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to novel adenoviral vectors which have the characteristic of containing a region essential for encapsidation which is heterologous with respect to the adenoviral genome from which they are derived. These vectors may be used as helper or recombinant vectors, the former allowing propagation of the latter. The subject of the invention is also a method for preparing a viral preparation containing said adenoviral vectors, a cell, a pharmaceutical composition or a composition of material comprising them as well as their use for therapeutic or prophylactic purposes. Finally, the present invention also relates to an adenoviral genome of animal origin having attenuated encapsidation capacities compared with the native genome from which it is derived. The ainvention is of especial interest in the perspectives of gene therapy especially in humans.
DESCRIPTION OF THE RELATED ART
Gene therapy is defined as the transfer of genetic information into a host cell or organism. The first protocol applied to humans was initiated in the United States in September 1990 on a patient who was genetically immunodeficient because of a mutation affecting the gene encoding Adenine Deaminase (ADA). The relative success of this first experiment encouraged the development of this technology for various diseases including both genetic (with the aim of correcting the dysfunction of a defective gene) and acquired (cancers, infectious diseases such as AIDS and the like) diseases. Most of the current strategies use vectors to carry the therapeutic gene to its cellular target. Many vectors including viral and synthetic vectors have been developed during the past few years and have been the subject of many publications accessible to persons skilled in the art.
The importance of adenoviruses as gene therapy vectors has already been mentioned in many prior art documents. They infect many cell types, both dividing and quiescent cells, are nonintegrative and not very pathogenic. In addition, they possess a natural tropism for the respiratory tracts. These specific properties make adenoviruses vectors of choice for many therapeutic and even vaccine applications. As a guide, their genome consists of a linear and double-stranded DNA molecule of about 36 kb which carries about thirty genes involved in the viral cycle. The early genes (E1 to E4; E for early) are divided into 4 regions dispersed in the genome. The E1, E2 and E4 regions are essential for viral replication whereas the E3 region, which is involved in modulating the anti-adenovirus immune response in the host, is not. The late genes (L1 to L5; L for late) encode predominantly the structural proteins and partially cover the early transcription units. They are for the most part transcribed from the Major Late Promoter MLP. In addition, the adenoviral genome carries, at its ends, cis-acting regions which are essential for encapsidation, consisting of inverted terminal repeats (ITR) situated at the 5′ and 3′ ends and an encapsidation region which follows the 5′ ITR.
The adenoviral vectors which are currently used in gene therapy protocols lack the major part of the E1 region in order to avoid their dissemination in the environment and in the host organism. Additional deletions in the E3 region make it possible to increase the cloning capacities. The genes of interest are introduced into the viral DNA in place of one or other of the deleted regions. While the feasibility of transferring genes using these so-called first-generation vectors is now well established, the question of their safety remains. In addition to the risk of generating replication-competent particles, the potential immunogenecity of the viral proteins still expressed can, in some specific applications, prevent the persistence of the transduced cells and the stable expression of the transgene. These disadvantages have justified the construction of new-generation vectors. They conserve the regions in cis (ITRs and encapsidation sequences) which are essential for encapsidation but comprise additional genetic modifications aimed at suppressing the in vivo expression of most of the viral genes (see for example International Application Wo 94/28152). In this regard, a so-called minimal vector, which is deficient for all the adenoviral functions, represents an alternative of choice.
The techniques for preparing adenoviral vectors are widely described in the literature. In a first instance, the genome is prepared by homologous recombination in the 293 line (see in particular Graham and Prevect, 1991, Methods in Molecular Biology, Vol 7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The Human Press Inc, Clinton, N.J.) or in
Escherichia coli
(see for example International Application WO 96/17070). It is then necessary to propagate the vector in order to constitute a stock of viral particles containing it. This production step is critical and should make it possible to obtain high infectious particle titers to be able to envisage a large-scale development for the purpose of the preparation of clinical batches. Complementation lines providing in trans the viral products of expression for which the vector is defective are used to this effect. For example, the viruses deleted for E1 can be propagated in the 293 line which is established from human embryonic kidney cells (Graham et al., 1977, J. Gen. Virol. 36, 59-72). As regards the secondgeneration vectors, it is possible to use lines complementing two essential viral functions, such as those described by Yeh et al. (1996, J. Virol. 70, 559-565), Krougliak and Graham (1995, Human Gene Therapy 6, 1575-1586), Wang et al. (1995 Gene Therapy 2, 775-783), Lusky et al. (1998, J. Virol. 72, 2022-2033) and in International Applications WO 94/28152 and WO 97/04119. Because of the potential toxicity of the viral products of expression, these lines need to be optimized in terms of growth capacity and viral particle yield before envisaging their use in an industrial process. Furthermore, a line complementing all the adenoviral functions, suitable for the propagation of the minimal vectors is currently not yet available.
Another alternative is based on the use of an additional viral element designated “helper virus” to complement, at least in part, the defective functions of a recombinant adenoviral vector. The helper viruses of the prior art consist of an adenoviral genome, optionally deleted for an essential region for which the recombinant vector does not require complementation. By way of example, cotransfection into the 293 line of an E1
−
helper virus and of an E1
−
E4
−
recombinant adenoviral vector leads to the formation of viral particles of recombinant vector. The E1 function is provided by the 293 line and the E4 function by the helper virus.
However, a major disadvantage of this method is that the cells produce a mixed population of viral particles, some comprising the recombinant vector and others the helper vector. In practice, the preparations predominantly contain helper viral particles, these having a selective advantage, such that the contamination may reach and even exceed 90% . The presence of the helper virus is not desirable in the context of a therapy applied to humans and, because of this, requires the use of physical separation techniques, such as ultracentrifugation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention proposes exploiting the respective growth properties of the human and animal adenoviruses. The inability of bovine BAV3 adenoviruses to be propagated in a human line has now been demonstrated whereas Ad5 can be propagated in bovine cells. Indeed, the infection by BAV3 adenoviruses, alone or in the presence of Ad5, in the human 293 line does not lead to the formation of infectious BAV3 viral particles. On the other hand, Ad5 virions are obtained by infecting a bovine line. In addition, there is no expression of BAV3 viral proteins in human cells.
On the basis of these observations, the present invention proposes in parti
Lusky Monika
Mehtali Majid
Winter Arend Jan
Burns Doane Swecker & Mathis L.L.P.
Guzo David
Leffers, Jr. Gerald G.
Transgene S. A.
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