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Title:  Muscle reinnervation and motor axon sprouting by administering DNA sequences encoding NT-3 and CNTF

United States Patent:  6,552,003

Issued:  April 22, 2003

Inventors:  Finiels; Fran.cedilla.oise (Tarne, FR); Gimenez-Ribotta; Minerva (Montpellier, FR); Mallet; Jacques (Paris, FR); Privat; Alain (Saint Clement de Riviere, FR); Revah; Frederic (Paris, FR)

Assignee:  Aventis Pharma S.A. (Antony, FR); Institut National de la Sante et de la Recherche Medicale ( INSERM) (Paris, FR)

Appl. No.:  356032

Filed:  July 16, 1999

Abstract

The present invention relates to methods and compositions for delivering nucleic acids to motor neurons by administering the nucleic acids to muscle tissue. The invention relates to methods for treating pathologies of the nervous system, such as trauma and neurodegenerative diseases.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a particularly efficient method for the selective transfer of genes into motor neurons. The invention demonstrates that it is possible to specifically transfer a gene into the motor neurons by administration into the muscle. Applicants describe herein that adenoviruses are advantageously absorbed at the level of the neuromuscular junctions (motor endplates), and transported up to the cellular bodies of the motor neurons (ventral horn of the spinal cord) by retrograde transport along the motoneuronal axons. Intramuscular injections of recombinant adenoviruses expressing a trophic factor provides a particularly attractive mode of administration. After intramuscular injection adenoviruses will infect myotubes, thus allowing the trophic factor to be produced at the synaptic end of motoneurons and to be released continuously in the circulation. Intramuscular injections lead to retrograde transport of injected recombinant adenoviruses, allowing high yield infection of the afferent motoneurons (Finiels et al., Specific and efficient gene transfer strategy offers new potentialities for the treatment of motor neurone diseases. Neuroreport, 7, 373-378, 1995). This leads to the production of the transgene within the spinal cord.

Intramuscular administration of a therapeutic gene constitutes a new and very specific method for infecting the motor neurons by retrograde transport. The present invention enables one to target precisely the medullary stage on which it is desired to act, according to the location of the trauma and/or of the degeneration. In particular, the present invention advantageously enables one to specifically and unilaterally infect the motor neurons of the different medullary functional stages by following the precise map of the neuromuscular junctions. The present invention has been found to be less traumatic and more specific than stereotaxic injection into the medullary parenchyma, which is more diffuse and not restricted to the motor neurons.

General Molecular Biology

The techniques of recombinant DNA technology are known to those of ordinary skill in the art. General methods for the cloning and expression of recombinant molecules are described in Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and in Ausubel (Current Protocols in Molecular Biology, Wiley and Sons, 1987), which are incorporated by reference.

Nucleic Acids

The present invention relates to the discovery that instramuscular administration provides a means for delivering a nucleic acid sequence to motor neurons. The nucleic acid of the invention preferably encodes a neuroactive substance; a substance capable of exerting a beneficial effect on nerve cells. It may be a substance capable of compensating for a deficiency in or of reducing an excess of an endogenous substance. Alternatively, it may be a substance conferring new properties on the cells.

The neuroactive substance may be an antisense sequence or a protein. Among the proteins suitable for practice of the invention are growth factors, neurotrophic factors, cytokines, neurotransmitter synthesizing enzymes, enzymes, neurotransmitter receptors and hormone receptors.

Preferably, the growth factor is a colony stimulating factor (G-CSF, GM-CSF, M-CSF, CSF, and the like), fibroblast growth factor (FGFa, FGFb) or vascular cell growth factor (VEGF). Among the neurotrophic factors, the preferred factors are ciliary neurotrophic factor (CNTF), glial cell maturation factors (GMFa, b), GDNF, BDNF, NT-3, NT-5 and the like.

The neurotrophic factor NT-3 is particularly preferred. The complete nucleotide sequence encoding NT-3 is disclosed in WO91/03569, the contents of which are incorporated herein by reference.

Preferred cytokines are the interleukins and interferons. Enzymes included within the scope of the invention are the enzymes for the biosynthesis of neuro transmitters (tyrosine hydroxylase, acetylcholine transferase, glutamic acid decarboxylase) and the lysosomal enzymes (hexosaminidases, arylsulphatase, glucocerebrosidase, HGPRT). The enzymes involved in the detoxification of free radicals (super oxide dismutase I, II or III, catalase, glutathione peroxidase) are preferred. Receptors include the androgen receptors (involved in Kennedy's disease).

These proteins may be used in native form, or in the form of a variant or fragment thereof.

The neuroactive substance may also be an antisense sequence. The down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with a nucleic acid encoding an endogenous neuroactive substance or the corresponding messenger RNA. These antisense nucleic acids can be synthetic oligonucleotides, optionally modified to improve their stability and selectivity. They can also be DNA sequences whose expression in the cell produces RNA complementary to all or part of the mRNA encoding an endogenous neuroactive substance. Antisense nucleic acids can be prepared by expression of all or part of a nucleic acid encoding an endogenous neuroactive substance, in the opposite orientation, as described in EP 140308. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of the endogenous neuroactive substance. Preferably, the antisense sequence is at least 20 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is disclosed in WO92/15680, the contents of which are incorporated herein by reference.

The nucleic acid may be of natural or artificial origin. It may be especially genomic DNA (gDNA), complementary DNA (cDNA), hybrid sequences or synthetic or semisynthetic sequences. It may be of human, animal, plant, bacterial or viral origin and the like. It may be obtained by any technique known to persons skilled in the art, and especially by screening libraries, by chemical synthesis, or alternatively by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries. It is preferably cDNA or gDNA.

Regulatory Regions

Generally, the nucleic acids of the present invention are linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art.

The regulatory regions may comprise a promoter region for functional transcription in the motor neurons, as well as a region situated in 3' of the gene of interest, and which specifies a signal for termination of transcription and a polyadenylation site. All these elements constitute an expression cassette.

Promoters that may be used in the present invention include both constituitive promoters and regulated (inducible) promoters. The promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source. In particular, it may be promoter sequences of eucaryotic or viral genes. For example, it may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, it may be promoter sequences derived from the genome of a virus, including the adenovirus used. In this regard, there may be mentioned, for example, the promoters of the E1A, MLP, CMV and RSV genes and the like.

In addition, the promoter may be modified by addition of activating or regulatory sequences or sequences allowing a tissue-specific or predominant expression (enolase and GFAP promoters and the like). Moreover, when the nucleic acid does not contain promoter sequences, it may be inserted, such as into the virus genome downstream of such a sequence.

Some promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, U.S Pat. Nos. 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

Vectors

As discussed above, a "vector" is any means for the transfer of a nucleic acid according to the invention into a host cell. Preferred vectors are viral vectors, such as retroviruses, herpes viruses, adenoviruses and adeno-associated viruses.

Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.

Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles.

The retroviruses are integrating viruses which infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant retroviral vectors has been described: see, in particular, EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, etc. In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are disclosed in WO95/02697.

In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP+envAm-12 cell line (WO89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al., J. Virol. 61 (1987) 1639). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions which carry the encapsidation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the said gene of interest in vitro (into cultured cells) or in vivo, (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced are then purified by standard techniques.

The invention also relates, therefore, to an AAV-derived recombinant virus whose genome encompasses a sequence encoding a nucleic acid encoding a neuroactive substance flanked by the AAV ITRs. The invention also relates to a plasmid encompassing a sequence encoding a nucleic acid encoding a neuroactive substance flanked by two ITRs from an AAV. Such a plasmid can be used as it is for transferring the nucleic acid sequence, with the plasmid, where appropriate, being incorporated into a liposomal vector (pseudo-virus).

In a preferred embodiment, the vector is an adenovirus vector.

Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.

Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94/26914). Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for example).

Preferably, the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the E1 region of the adenoviral vector is non-functional. The deletion in the E1 region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment). Other regions may also be modified, in particular the E3 region (WO95/02697), the E2 region (WO94/28938), the E4 region (WO94/28152, WO94/12649 and WO95/02697), or in any of the late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO95/02697 and WO96/22378, the contents of which are incorporated herein by reference. In still another preferred embodiment, the adenoviral vector has a deletion in the E1 region into which the E4 region and the nucleic acid sequence are inserted (see FR94 13355, the contents of which are incorporated herein by reference).

The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia, the DNA sequence of interest. The homologous recombination is effected following cotransfection of the said adenovirus and plasmid into an appropriate cell line. The cell line which is employed should preferably (i) be transformable by the said elements, and (ii) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the E1 and E4 functions, as described in applications WO94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

Pharmaceutical Administration

The process according to the present invention enables one to target precisely the motor neurons of each medullary functional stage. Thus, according to the site of the impairment to be treated, the administration is made into a muscle carrying a nerve linkage with the said site. According to the present invention, it is now possible, by a judicious choice of various injections, to infect specifically and unilaterally a large number of medullary motor neurons distributed over the various levels.

In a preferred embodiment, administration into the muscles of the upper limbs (biceps, triceps) makes it possible to transfer a gene into the motor neurons at the cervical level; administration into the muscles of the thorax (pectoral muscles) makes it possible to transfer a gene into the motor neurons at the thoracic level; or administration into the muscles of the lower limbs (gastrocnemial muscles) makes it possible to transfer a gene into the motor neurons at the lumbar and sacral levels.

Other muscles may of course be used for administration into these motor neurons, and other motor neurons may also be targeted. To this end, it is possible to use precise maps of the neuromuscular junctions in order to determine, depending on the medullar stage targeted, the most appropriate muscle(s) for the administration. Such maps are accessible to persons skilled in the art (see especially Nicholopoulos et al., J. Comp. Neurol. 217, 78-85; Peyronnard et Charon, Exp. Brain Res. 50, 125-132). Depending on the medullar stage which it will prove convenient to infect, one or more muscles known to be innervated by the stage in question can thus be chosen.

The intramuscular administration can be carried out in various ways. According to a first embodiment, it is performed by injection at several points of the same muscle so as to affect a very large number of motor endplates. This embodiment is particularly efficient when the point of insertion of the nerve into the muscle considered is not identifiable. When the point of insertion of the nerve can be located, the administration is advantageously carried out by one or more injections at or near the said point. According to this embodiment, the efficiency of the transfer is greater because a high proportion of vector administered is absorbed at the level of the neuromuscular junction.

In a preferred embodiment of the present invention, the intramuscular administration is carried out by injections at several points of the same muscle.

In another preferred embodiment of the present invention, the intramuscular administration is carried out by injection(s) at or near the point of insertion of the nerve.

A preferred subject of the present invention is a method for the transfer of nucleic acids into motor neurons comprising the muscular administration of an adenoviral vector incorporating the said nucleic acid into its genome. Preferably, the method according to the invention is carried out by injection(s) at several points of the same muscle, or when the point of insertion of the nerve can be located, by one or more injections at the level of or close to the said point.

Pharmaceutical Compositions

For their use according to the present invention, the nucleic acids, either in the form of a vector or naked DNA, are preferably combined with one or more pharmaceutically acceptable carriers for an injectable formulation. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, allow the constitution of injectable solutions.

The preferred sterile injectable preparations can be a solution or suspension in a nontoxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof. 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.

The virus doses used for the administration may be adapted as a function of various parameters, and in particular as a function of the site (muscle) of administration considered, the number of injections, the gene to be expressed or alternatively the desired duration of treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu, and preferably 106 to 1011 pfu. The term pfu (plaque forming unit) corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate cell culture and measuring, generally after 15 days, the number of plaques of infected cells. The technique for determining the pfu titre of a viral solution are well documented in the literature.

In a preferred embodiment, the composition comprises an adenovirus comprising the NT-3 gene (AdNT-3) in a concentration of about 1.times.109 pfu/100 .mu.l.

The nucleic acid can also be administered as a naked DNA. Methods for formulating and administering naked DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, the contents of which are incorporated herein by reference.

The compositions according to the invention are particularly useful for administration to motor neurons as described above.

Treatment of Motor Neuron Impairments

The process according to the present invention is particularly advantageous for the treatment of medullary traumas or of motoneuronal degeneration diseases. Medullary traumas correspond more particularly to sections at the level of the motor neurons which deprive them of their afferences coming from the higher centres and cause their degeneration. The transfer of genes encoding growth factors, for example, into the sublesional motor neurons by retrograde transport according to the invention now offers the possibility of reducing or even preventing this degeneration.

Neuropathies of the motor neuron include amyotrophic lateral sclerosis, spinal amyotrophias type I (Werdnig Hoffman disease), type II or III (Kugelberg-Welander disease), bulbar spinal amyotrophias (such as Kennedy's disease). The transfer of genes encoding growth factors or other molecules known to exert a neurotrophic effect on the motor neuron undergoing degeneration according to the present invention also offers a new route for the treatment of this type of pathology.

The efficacy of the process of the invention can be demonstrated on animal models, such as a model of partial or complete section of the spinal cord, (Wobbler mouse--animal model for studying amyotrophic lateral sclerosis (Leestma J. E., Am. J. Pathol., 100, 821-824)); the mnd mouse (motor neuron degeneration: animal model for studying amyotrophic lateral sclerosis (Messer et al., 1992, Genomics. 18, 797-802)); pmn mouse (progressive motor neuron neuropathy: animal model for studying motor neural degeneration during development), and SOD* mice: transgenic mice expressing mutated forms of Cu/Zn SOD responsible for familial forms of amyotrophic lateral sclerosis, as illustrated in the examples. The incorporation, tolerance and safety for man can be tested on in vitro models of culture of human embryonic medullary neurons.

Claim 1 of 45 Claims

What is claimed is:

1. A method of muscle reinnervation and of inducing peripheral or collateral sprouting of motor axon endings in a mammal comprising administering directly to muscle tissue at least one nucleic acid encoding at least one neurotrophic factor chosen from NT-3 and CNTF, wherein said at least one nucleic acid is operably linked to a promoter, and wherein expression of said nucleic acid sequence in said mammal results in increased innervation of muscle tissue of the same medullary level as the muscle tissue that was administered the nucleic acid, increased peripheral sprouting of motor axon endings, increase collateral sprouting of motor endings, or combinations thereof.
 


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