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
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
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
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
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.
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
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
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
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.
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
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
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
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
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
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.
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
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
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
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.
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
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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