Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Canine cell – per se
Reexamination Certificate
1995-11-30
2002-08-13
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Canine cell, per se
C436S069000, C436S169000, C436S169000, C436S169000, C436S169000, C436S169000, C436S169000
Reexamination Certificate
active
06432709
ABSTRACT:
The object of the invention is novel cell lines so-called transcomplementation cell lines which enable the packaging of recombinant retroviral RNAs carrying nucleotide-sequences derived from genes which in some instances might be of therapeutic interest (called transgenes), aiming at transferring and expressing these transgenes in eucaryotic target cells. The invention also relates to expression vectors for the transcomplementation of defective retroviral vectors.
The transfer of genes for therapeutic purposes or somatic “gene therapy” consists in inserting a “repairer” gene (transgene) in the somatic cells of a constituted organism in order to compensate for the dysfunction of an endogenous gene, or even to add a novel function for a therapeutic purpose. The resulting genetic change is likely to be transmitted to the daughter cells of the manipulated cell but it will not be inherited. The normal counterpart of altered DNA sequences is thus transformed into a medicine.
Various approaches are currently being explored in order to introduce into target cells genes or more generally, nucleotide sequences, for therapeutic purposes. These target cells might be directly the cells benefiting from the therapeutic intervention or cell-intermediate between the vector carrying the transgene and the cells to be treated.
Vectors currently under use for gene transfer into target cells are derived either from inactivated viruses, like retroviruses or adenoviruses, or else from macromolecular conjugates. Retroviruses usually address a target tissue including a stem cell pool which can be manipulated ex vivo; whereas, when the target tissue is made of terminally differentiated cells or embedded into an organ where architectural constraints have major functional consequences, like the lung, gene transfer must be performed in vivo, by means of recombinant adenovirus for instance.
Gene therapy finds applications in diseases as diverse as hereditary disorders due to the alteration of a single gene, such as Duchenne's myopathy, lyososomal diseases, cystic fibrosis or acquired disorders such as AIDS, cancers, thrombo-embolic disease or degenerative neurological diseases and inherited hematological diseases.
The development of retroviral vectors more efficacious than the existing tools constitute a major objective. In fact, retroviral vectors have demonstrated their efficacy in stably and permanently transferring genes where classically the cell-targets for the transfer undergo mitoses and ideally include a contingent of stem cells.
Current limitations stem from still inadequate infectivity of the viruses used and/or too moderate a level of transcription. Additional potential limitations deal with the safety of the transfer which to date remain unsatisfactory. This is both related to the risk for generation of replication-competent viruses and to the concomitant transmission of endogenous sequences which are generated by packaging cell-lines and may be packaged together with the defective recombinant virus genome.
The development of more efficacious and safer vector systems than those currently available is a matter of great significance.
Whenever the transgene to be expressed is delivered by means of a retroviral vector, the manufacture of stocks of retrovirus vector particles carrying along the transgene is a compulsory step preceding infection of target cells.
In order to achieve this, one should proceed with transcomplementation of the defective retrovirus vector carrying the transgene by the viral proteins, the corresponding encoding sequences of which being first deleted to be replaced by the transgene. This transcomplementation step can be performed passing through packaging cells. These cells are genetically engineered cells comprising the genetic material required for synthesis of viral components, in particular ENV envelope proteins, GAG nucleoproteins and POL reverse transcriptase, which allows for viral replication. The gag, pol and env genes are transfected into packaging cells through the transfection of the nucleic acid sequences which contain them, by means of a transcomplementation vector defective for the Psi-sequence which is otherwise mandatory to achieve virus packaging.
Expression of gag, pol and env genes within the packaging cells allows for further packaging of retroviral recombinant RNA sequences recombined with the transgene carried by a retroviral vector including the Psi-packaging sequences. Packaging may proceed following transcomplementation by the viral proteins encoded by the gag, pol and env sequences.
Currently available packaging cell lines are derived from cells of murine origin and harbour the following drawbacks:
1 Co-packaging of endogenous retroviral sequences (MCF, VL30, and even retrotransposons in some instances), together with defective constructs. Sequences which are co-packaged may as well integrate into the host genome following infection of gene transfer target cells.
2-Within these packaging cell lines, expression of complementing proteins is driven off a retrovirus LTR. Although third generation cell-lines make use of complementing retrovirus constructs including several mutation or deletion sites, maintaining LTR sequences might result in potential disadvantage; these sequences might well be able to induce genetic recombination with defective constructs to be complemented.
This has already been observed even from so-called third generation cell-lines where GAG and POL proteins encoding sequences, are being transfected separately from envelope coding sequences in order to reduce the likelihood for competent virus-generating combinations.
Envelope-encoding sequences currently under use to complement defective retroviruses towards gene transfer into mammalian cells and in particular of primate and human origin are amphotropic envelopes. It remains unsure that those cell-types that one wishes to infect towards gene therapy purpose would harbour enough receptors to amphotropic viruses.
All these elements justify efforts towards improvement in the safety of retrovirus-mediated gene transfer.
The object of the invention is to provide means to engineer novel packaging cell-lines to solve at least part of the abovementioned issues.
The cell-lines of the invention allow for the improvement of gene transfer safety since they are developed from cell-lines originating from animal species devoid of endogenous retroviral sequences identified to date and using, in a preferred embodiment non-viral promoters, to drive the expression of at least part of the viral complementing proteins, in particular inducible promoters for the conditional synthesis of defective vectors complementing proteins, especially of the envelope, upon request, whenever an infectious virus supernatant is to be prepared.
In addition GAG and POL proteins complementation is mediated by nucleotide sequences of a Friend retrovirus from which the vector has been built.
In order to implement the gag, and pol sequences are being transfected into the packaging-dedicated cell-line separately from envelope encoding sequences.
These cell-lines also lead to improvement in transfer efficiency through the use of envelope sequences, of human origin in particular, in order to circumvent the potential insufficient expression of the receptor to an amphotropic virus at the surface of human cell-targets for gene therapy; in particular potentially in case haematopoietic stein cells represent the targets.
According to a first embodiment, the object of the invention is an expression vector for the transcomplementation of a retroviral vector, which permits the transfer and/or integration within the genome of a target cell of a chosen nucleotide sequence (termed “transgenic sequence”), characterized in that it comprises:
a) a nucleotide sequence termed env, coding for the polypeptides derived from the envelope (ENV polypeptides) of a retrovirus of the spumavirus family, the ENV polypeptides permitting the packaging of retroviral RNAs, and
b) transcription regulator signals controlling the expression of the env sequ
Cohen-Haguenauer Odile
Guzo David
Merchant & Gould P.C.
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