Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1996-10-04
2001-06-12
Nguyen, Dave Trong (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C514S04400A, C435S320100, C435S455000
Reexamination Certificate
active
06245330
ABSTRACT:
The present invention relates to recombinant adenoviruses which contain a DNA sequence encoding the glial cell-derived neurotrophic factor. The invention also relates to the preparation of these vectors, to the pharmaceutical compositions which contain them, and to their therapeutic use, especially in gene therapy, for treating and/or preventing neurodegenerative diseases.
The increase in the length of life in Western countries is accompanied by a steady growth in neurode-generative 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 above the age of 65, and Alzheimer's disease affects 10% of those above the age of 70 and 30% of those above 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 programmes have been developed in order to understand the mechanisms of this degeneration associated with ageing, with a view to developing means for treating it, and also for preventing it, by gene therapy.
Since the neurodegenerative diseases are an expression of the progressive death of the neuronal cells, stimulation of the production of the growth factors involved in the development of these neuronal cells has in fact appeared to be a possible route for preventing and/or opposing this degeneration.
The object of the present invention is, in particular, to propose vectors which make it possible directly to promote the survival of the neuronal cells which are involved in these pathologies by means of expressing, in an efficient and localized manner, certain trophic factors.
The trophic factors are a class of molecules which possess properties of stimulating axonal growth or the survival of the nerve cells. The first factor possessing neurotrophic properties, NGF (“Nerve Growth Factor”), was characterized some 40 years ago (for review, see Levi-Montalcini and Angelleti, Physiol. Rev. 48 (1968) 534). Other neurotrophic factors, in particular the glial cell-derived neurotrophic factor (GDNF) (L.-F. Lin, D. Doherty, J. Lile, S. Besktesh, F. Collins, Science, 260, 1130-1132 (1993)) have only been identified recently. GDNF is a protein of 134 amino acids with a molecular weight of 16 kD. Its essential function is the in-vitro promotion of the survival of dopaminergic neurones.
The present invention is particularly advantageous for administering GDNF in the form of a therapeutic agent.
More precisely, the present invention is directed towards developing vectors which are particularly effective in delivering, in vivo and in a localized manner, therapeutically active quantities of the specific gene encoding GDNF in the nervous system.
In application No. PCT/EP93/02519, which is pending concomitantly, it was demonstrated that it was possible to use the adenoviruses as vectors for transferring a foreign gene in vivo into the nervous system and expressing the corresponding protein.
More specifically, the present invention relates to specially adapted and efficient novel constructs for transferring glial cell-derived neurotrophic factor (GDNF).
More precisely, it relates to a recombinant adenovirus which encompasses a DNA sequence encoding GDNF or one of its derivatives, to its preparation, and to its use for treating and/or preventing neurodegenerative diseases.
Thus, the Applicant has clearly demonstrated that it is possible to construct recombinant adenoviruses which contain a sequence encoding GDNF, and to administer these recombinant adenoviruses in vivo, and that this administration permits stable and localized expression of therapeutically active quantities of GDNF in vivo, in particular in the nervous system and without any cytopathic effect.
An initial subject of the invention is thus a defective recombinant adenovirus which encompasses at least one DNA sequence encoding all, or an active part, of the glial cell-derived neurotrophic factor (GDNF) or one of its derivatives.
The glial cell-derived neurotrophic factor (GDNF) which is produced within the scope of the present invention can either be human GDNF or an animal GDNF.
The cDNA sequences encoding human GDNF and rat GDNF have been cloned and sequenced (L.-F. Lin, D. Doherty, J. Lile, S. Besktesh, F. Collins, Science, 260, 1130-1132 (1993)).
The DNA sequence which encodes GDNF 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 could be inserted. The sequence may also consist of synthetic or semisynthetic sequences. Particularly advantageously, the sequence of the present invention encodes GDNF which is preceded by the native pro region (pro GDNF).
Particularly advantageously, a cDNA or a gDNA is employed. According to a preferred embodiment of the invention, the sequence is a gDNA sequence encoding GDNF. Use of this latter sequence can make it 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 site-directed mutagenesis, especially in order to insert appropriate restriction sites. Thus, the sequences described in the prior art are not constructed so that they can be used in accordance with the invention, and preliminary adaptations may prove to be necessary in order to obtain a substantial level of expression.
Within the meaning of the present invention, a derivative of GDNF 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 GDNF (trophic effect and/or differentiating effect). Modification should be understood to mean any mutation, substitution, deletion, addition or modification of a genetic and/or chemical nature. These modifications can be effected by techniques known to the person skilled in the art (see general molecular biological techniques below). The derivatives within the meaning of the invention 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 greater affinity for their sites of attachment, sequences which permit improved expression in vivo, molecules which are more resistant to proteases, and molecules which have greater therapeutic efficacy or less pronounced secondary effects, or, perhaps, novel biological properties.
The preferred derivatives which may most particularly 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 only involved to a limited extent, in the interaction with the binding sites under consideration, or which express an undesirable activity, and derivatives which include residues which are additional to those in the native sequence, such as, for example, a secretory signal and/or a junction peptide.
According to one preferred embodiment of the invention, the DNA sequence encoding GDNF or one of its derivatives also includes a secretory signal which makes it possible to direct the synthesized GDNF into the secretory paths of the infected cells. According to one preferred embodiment, the DNA sequence contains a secretory sequence in the 5′ position and in reading frame with the sequence encoding the GDNF. In this way, the synthesized GDNF is advantageously released into the extracellular compartments and can in this way activate its receptors. The secretory signal is advantageously the native secretory signal of the GDNF (referred to below by the term “pre”). However, the secretory signal can also be a secretory signal which is heterologous or even artificial. Advantageously, the DNA sequence encodes pre-GD
Horellou Philippe
Mallet Jacques
Perricaudet Michel
Revah Frederic
Vigne Emmanuelle
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Nguyen Dave Trong
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