Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing
Reexamination Certificate
1996-10-09
2004-02-03
Reynolds, Deborah J. (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
C435S325000, C435S235100
Reexamination Certificate
active
06685934
ABSTRACT:
The present invention relates to recombinant adenoviruses which encompass a DNA sequence encoding the basic fibroblast growth factors. The invention also relates to the preparation of these vectors, to the pharmaceutical compositions containing them, and to their therapeutic use, in particular in gene therapy for treating and/or preventing neurodegenerative diseases.
The increase in length of life in western countries is accompanied by a steady growth in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, etc. Thus Parkinson's disease, for example, affects 4% of people over the age of 65, and Alzheimer's disease affects 10% of those over the age of 70 and 30% of those over the age of 80. Generally speaking, all these diseases result from a progressive loss of neuronal cells in the central nervous system, or even within very localized structures as in the case of Parkinson's disease.
During recent years, numerous research projects have been developed with a view to understanding the mechanisms of this degeneration associated with ageing, in order to be able to develop means of treatment, and also means of prevention, using gene therapy.
The trophic factors are a class of molecules possessing properties which stimulate axonal growth or survival of nerve cells. The first factor possessing neurotrophic properties, i.e. NGF (“nerve growth factor”), was characterized some forty years ago (for review, see Levi-Montalcini and Angelleti, Physiol. Rev. 48 (1968) 534). Other neurotrophic factors have been identified, in particular the fibroblast growth factors (FGFs). The fibroblast growth factors (FGFs), the acidic and basic forms of which have been identified, are subjected to retrograde transport in several neuronal populations, including the nigrostriatal system (Fergusson and Johnson, 1991 J. Comp. Neurol. 313:693), and enable dopaminergic neurones of the mesencephalon to survive in vitro (Knusel et al, 1990, J. Neurosci. 10, 558). The main forms of human bFGFs possess molecular weights of 22.5 kd, 21 kd and 18 kd, respectively. (Prats H., Kagahd M., Parts A. C., Klagsbrun M., Lelias J. M., Liauzun P., Chalon P., Tauber J. P., Amalric F., Smith J. A. and Caput D., Proc. Natl. Acad. Sci. U.S.A. 86, 1836-1840 (1989)).
In their basic form, human fibroblast growth factors (bFGFs) are, in particular, recommended for preventing and/or treating hereditary or acquired retinopathies which are of a degenerative nature. Similarly, they are able to restore normal photoreceptor morphology.
For this purpose, bFGFs are usually injected directly by the intravitreous or subretinal route at the site to be treated. However, this form of administration is not completely satisfactory. Thus, in view of the tumorigenic character of bFGFs, it is important to ensure that they are essentially localised in the desired region of the body. When they are injected by a general route, it is not possible to be certain of preventing the bFGFs from diffusing into the blood circulation.
The particular object of the present invention is to propose a particularly advantageous solution to this problem.
The present invention relates to the development of particularly efficient vectors for delivering, in vivo and in a localized manner, therapeutically active quantities of bFGF, thereby enabling undesirable side-effects to be avoided.
The present invention is particularly advantageous for administering bFGF as a therapeutic agent.
More precisely, the present invention is directed towards developing particularly efficient vectors for delivering, in vivo and in a localized manner, therapeutically active quantities of the specific gene encoding bFGF into the nervous system.
In Application No. PCT/EP93/02519, which is pending concomitantly, it was demonstrated that it was possible to employ adenoviruses as vectors for transferring a foreign gene into the nervous system in vivo, and for expressing the corresponding protein.
More specifically, the present invention relates to novel constructs which are particularly suitable and efficient for transferring basic fibroblast growth factors (bFGFs).
More precisely, it relates to a recombinant adenovirus which encompasses a DNA sequence encoding bFGF or one of its derivatives, to its preparation and to its use for treating and/or preventing neurodegenerative diseases.
Thus, the Applicant has demonstrated that it is possible to construct recombinant adenoviruses which contain a sequence encoding bFGF, and to administer these recombinant adenoviruses in vivo, and that this administration makes it possible to achieve stable and localized expression of therapeutically active quantities of bFGF in vivo in the nervous system, in particular for the treatment of retinopathies, with no cytopathic effect.
An initial subject of the invention is, therefore, a defective recombinant adenovirus which encompasses at least one DNA sequence encoding all, or an active part, of basic fibroblast growth factor (bFGF) or of one of its derivatives.
The basic fibroblast growth factor (bFGF) which is produced within the scope of the present invention can be human bFGF or an animal bFGF. In particular, it can be rat bFGF.
The DNA sequence which encodes bFGF, and which is used within the scope of the present invention, can be a cDNA, a genomic DNA (gDNA), or a hybrid construct consisting, for example, of a cDNA in which one or more introns are inserted. The sequences involved can also be synthetic sequences or semisynthetic sequences.
Particularly advantageously, a cDNA or a gDNA is employed.
According to one preferred embodiment of the invention, the sequence is a gDNA sequence encoding bFGF. When this sequence is used, it is possible to achieve improved expression in human cells.
Naturally, prior to its incorporation into an adenovirus vector according to the invention, the DNA sequence is advantageously modified, for example by means of site-directed mutagenesis, particularly for the purpose of inserting appropriate restriction sites, because the sequences described in the prior art are not constructed to be used in accordance with the invention, and prior adaptations may prove to be necessary in order to obtain substantial levels of expression.
Within the meaning of the present invention, a derivative of bFGF is understood to mean any sequence which is obtained by modification and which encodes a product which retains at least one of the biological properties of bFGF (trophic effect and/or differentiating effect). Modification is understood to mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature. These modifications can be carried out using the techniques known to a person skilled in the art (see general molecular biological techniques below). Within the meaning of the invention, the derivatives can also be obtained by hybridization from nucleic acid libraries, using the native sequence or a fragment thereof as the probe.
These derivatives are, in particular, molecules which have a great affinity for their sites of attachment, sequences which result in improved expression in vivo, molecules which are more resistant to proteases, and molecules which have a greater therapeutic efficacy or side-effects which are less marked, or, perhaps, novel biological properties.
Those preferred derivatives which may more specifically be cited are natural variants, molecules in which one or more residues have been replaced, derivatives which have been obtained by deleting regions which are not involved, or are only involved to a limited extent, in interaction with the binding sites under consideration, or which express an undesirable activity, and derivatives which, as compared with the native sequence, include additional residues, such as, for example, a secretory signal and/or a junction peptide.
According to one favoured embodiment of the invention, the DNA sequence encoding bFGF or one of its derivatives also includes a secretory signal which enables the synthesized
Abitbol Marc
Mallet Jacques
Perricaudet Michel
Revah Frederic
Roustan Paul
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner LLP
Reynolds Deborah J.
Woitach Joseph
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