Viral recombinant vectors for expression in muscle cells

Non-replicatable viral recombinant vectors which are recognizable by muscle cell receptors, and furthermore modified by an insertion nucleic acid coding for a polypeptide sequence to be expressed in said muscle cells, are used to obtain a drug for treating muscle cell diseases or diseases which, by virtue of their location in the body, are accessible to the products of the expression of the above-mentioned nucleotide sequence, as secreted by said muscle cells. A method for producing said vectors, vectors such as those described above, and their use in pharmaceutical compositions are also provided.

The invention relates to recombinant vectors of viral origin which contain 
a nucleotide sequence coding for a specific polypeptide, and their use for 
the expression of this polypeptide in muscle cells. The invention also 
relates to a procedure for producing these vectors, as well as to their 
uses, in particular as medicines in the field of muscle diseases. 
The hitherto unresolved problem of the direct diffusion of a gene towards a 
specific tissue is an obstacle to the development of gene therapy in the 
field of muscle diseases. 
The various attempts to modify muscle tissue performed hitherto consist 
mainly of that involving fusion of muscle cells with a host cell 
(Salminen, A. et al., Hum. Gene Ther. 2, 15-26 (1991); Partridge, T. A. et 
al., Nature 337, 176-179 (1989), and that involving direct injection of 
DNA into the muscles (Wolff, J. A. et al., Science 247, 1465-1468 (1991); 
Acsadi, G., New Biol. 3, 71-81 (1991)). 
The method proceeding by the fusion in mice of precursors of muscle cells 
derived from a normal donor with muscle fibers of a host (Partridge, T. A. 
et al., mentioned above) has been carried out with success and this 
cellular therapy has been the subject of preliminary trials in children. 
However, this approach seems to present too many disadvantages to be 
applicable to the treatment of muscle diseases. In fact, since the 
migratory capacities of the precursor cells are reduced to a few 
millimeters, the cellular implantation of these latter would necessitate 
millions of injections requiring hours of anaesthesia. Inevitably, there 
would be the risk of immunological problems leading to rejection phenomena 
occurring, as in the case of very many grafts. In addition, the treatment 
of Duchenne's muscular dystrophy (DMD) not only requires making contact 
with the skeletal muscles but also with the myocardial cells; the 
difficulties likely to be encountered in implanting precursors of muscle 
cells in the myocardium can easily be imagined. Consequently, cellular 
therapy hardly seems to be appropriate for the treatment of diseased cells 
showing such dissemination in the organism. 
Gene therapy by direct in vivo introduction of nucleic acids into the 
interior of organs is an attractive method on account of its simplicity, 
but its development is confronted with a number of obstacles. In 
particular, the expression of genes in the muscles remains localised at 
the site of injection (Wolff, J. A. et al., mentioned above) and seems to 
be of quite limited duration, particularly in cardiac muscle (Acsadi, G. 
et al., mentioned above). 
The aim of the present invention is precisely to make possible the 
introduction of a very large number of nucleic acids into a considerable 
number of muscle cells (up to 50% or more) of a human or animal organism, 
whether these muscle cells be those of skeletal muscle or those of the 
myocardium. 
The present invention relates more particularly to the transport of nucleic 
acids to target muscle cells by the blood, while protecting these nucleic 
acids against attack by various blood constituents. 
Another aim of the present invention is to make available to the public 
pharmaceutical compositions which make possible the treatment of muscle 
diseases, and more particularly genetic diseases of the muscle system, or 
also diseases, the localization of which in the organism makes them 
accessible to the expression products of the above-mentioned nucleic 
acids, these products being secreted by the said muscle cells. 
The present invention follows from the discovery made by the inventors of 
the fact that beta-galactosidase activity is found in many tissues after 
injection into mice of recombinant vectors of viral origin, more 
particularly of adenoviral origin, into the genome of which the gene 
coding for beta-galactosidase has been inserted. Such tissues include the 
lungs, liver, intestine, heart and the skeletal muscles. The expression of 
the gene for beta-galactosidase is constant with time, since the 
proportion of blue-coloured cells (colour obtained subsequent to gene 
expression) in the muscle tissue is more or less the same from one month 
to the next.

The subject of the present invention is the use of recombinant vectors of 
viral origin, incapable of replication and likely to be recognized by the 
receptors of human and animal muscle cell which can be infected by these 
viruses, these vectors being additionally modified by a nucleic acid 
insert containing a nucleotide sequence which codes for a polypeptide 
sequence, the expression of which in the said muscle cells is sought, this 
sequence being under the control of a promoter recognized by the 
polymerases of these cells, for the production of drug compositions which 
can be administered by the general route, particularly the intravenous or 
intraarterial route, and designed for the treatment of either diseases 
affecting muscle cells or diseases, the localization of which in the 
organism makes them accessible to the expression products of the 
above-mentioned nucleic acids and secreted by the said muscle cells. 
The adenoviruses, in particular the human adenoviruses type 2 or 5 
represent particularly preferred vectors in the framework of the present 
invention by virtue in particularly of the large size of the foreign DNA 
fragment which it is possible to insert into the genome of these viruses. 
Advantageously, the above-mentioned nucleic acid insert is included in a 
defective genome of an adenovirus, this genome lacking essential sequences 
necessary for the replication of these adenoviruses, and more particularly 
the EA and EB transactivators; nonetheless, this genome preferentially 
includes all of those essential sequences necessary for the encapsidation 
of these adenoviruses. 
The promoter used may be an endogenous promoter (for example, an early or 
late promoter of the adenovirus used) or an exogenous promoter. 
It will be advantageous to have recourse to strong promoters, for example 
one having about the same strength as the promoter contained in the LTR 
(Long Terminal Repeat) of RSV (Rous Sarcoma Virus). 
As examples of other promoters whose use may be considered, mention should 
be made of: 
the promoter of the IE gene of CMV (cytomegalovirus) 
the MMTV (Mouse Mammary umor Virus) inducible promoters or metallothionine 
promoters. 
The strength of the promoter which is used may be estimated in assays 
similar to those which are described in the examples which follow, for 
example by replacing the promoter under study in the vectors of these 
examples by the promoter contained in the LTR of RSV and by the evaluation 
of the intensity of expression of the marker obtained, an intensity which 
can then be compared with that obtained with the promoter of LTR of RSV. 
The amount of vectors administered to the organism is advantageously chosen 
so as to overwhelm the immune system of the organism into which they are 
injected 
Advantageously, the route of adminstration selected in the framework of the 
present invention is the intravenous or intraarterial route. 
Among the diseases affecting muscle cells mentioned above, mention may be 
made of genetic diseases such as muscular dystrophy. 
Consequently, the nucleic acid inserted into the genome of the viral vector 
and the diffusion of which into the muscle mass is desired, comprises a 
nucleotide sequence coding for a polypeptide capable of treating the 
disease in question, and more particularly of playing the role in the 
muscle cell of the polypeptide normally present in a healthy cell, but the 
deficiency of which is due either to an abnormally low level or the 
complete failure of the production of this polypeptide, or to an error in 
its amino acid sequence which results from genetic anomalies in its coding 
nucleotide sequence. 
Vectors according to the invention used to produce a medicine designed for 
the treatment of muscular dystrophy are more particularly characterized in 
that the nucleic acid insert is constituted by all or part of a healthy 
gene for dystrophin. The introduction of the entire gene for dystrophin or 
even of any part of this gene which codes for a polypeptide conserving an 
activity similar to that of the whole protein can be carried out in 
accordance with a method identical with that described hereafter for the 
introduction of the gene for beta-galactosidase. 
As examples of diseases other than muscle diseases, susceptible to 
treatment in the framework of the present invention, mention may be made 
of the thromboses originating from infarctuses or also phlebites. 
Vectors according to the invention used to produce a drug designed for the 
treatment of thromboses and for the prevention of infarcts and phlebites 
are more particularly characterized in that the nucleic acid insert 
comprises a nucleotide sequence coding for a thrombolytic substance. The 
latter sequence is advantageously preceded by a signal sequence which 
codes for a peptide signal which ensures the secretion of the thrombolytic 
substance outside the muscle cell. 
The invention also relates to any recombinant vector characterized in that 
it is constituted of the defective genome of an adenovirus comprising, 
nonetheless; all of the essential sequences necessary for the 
encapsidation of this adenovirus, and into which is inserted a recombinant 
nucleic acid, the diffusion of which is desired in the muscle mass, this 
nucleic acid being placed under the control of a promoter capable of being 
recognized by the polymerases of the muscle cells, in particular by the 
strong promoter of the ElA early region of the genome of the adenoviruses. 
A preferred recombinant vector of the invention is characterized in that 
this recombinant nucleic acid is constituted by all or part of the gene 
for dystrophin. 
The invention also relates to pharmaceutical compositions consisting one or 
more recombinant vectors such as those described above, in combination 
with a pharmaceutically acceptable vehicle. 
The subject of the invention is also a procedure for producing the 
recombinant vectors described above which comprises, after the 
construction stage itself of these vectors by the introduction of the 
nucleic acid insert into their genome, a transformation step of 
transformable cell lines of higher eukaryotes (particularly of human or 
animal origin), themselves carrying a distinct nucleotide sequence capable 
of complementing the part of the genome of the adenovirus essential for 
the replication thereof and which the above-mentioned vector lacks, the 
said distinct sequence being preferably incorporated into the genome of 
the cells of the said cell line. 
As a preferred example of such cell lines, mention should be made of line 
293, a human embryonic kidney line which contains, integrated into its 
genome, the first eleven percent of the lefthand end of the genome of an 
Ad5. This portion complements defective recombinant viruses which bear 
deletions in this region. Such a production procedure is more particularly 
described in the European patent application No. 0 185 573 of 20/11/85. 
After transformation of these cell lines, the vectors which are thus 
multiplied are recovered and purified. 
The present invention will be illustrated more particularly with the aid of 
the detailed description which follows of the construction of recombinant 
adenovirus vectors comprising the gene coding for beta-galactosidase, and 
of the properties of this adenovirus vector. 
1. Construction of the recombinant adenovirus, Ad-RSV-beta-gal, by means of 
in vivo recombination. 
This recombinant adenovirus was constructed by homologous recombination 
between a suitable plasmid and the genome of a type 5 (Ad5) adenovirus. In 
this construction, the gene for beta-galactosidase is placed under the 
control of the RSV (Rous Sarcoma Virus) promoter. The plasmid pAdRSV "Beta 
" ontains the PvuII segment of the lefthand end of the Ad5 (segment 
situated between the positions 0 and 1.3 of the plasmid in FIG. 1) 
comprising the inverted terminal repeat, the origin of replication, 
encapsidation signals and the amplifier Ela. This fragment is followed by 
a nlslacZ gene (described in Bonnerot, C. et al., Proc. Nati. Acad. Sci. 
USA, 1987, 84, 6795-6799) which codes for beta-galactosidase, and by a 
fragment of the adenovirus Ad5 situated between the positions 9.4 and 17 
of the plasmid of FIG. 1. 
The values of the positions 1.3, 9.4 and 17 indicated above are units 
indicating the number of base pairs included within these fragments, one 
unit representing 360 base pairs. 
The AdS sequence situated between the above-mentioned positions 9.4 and 17 
allows recombination with the adenovirus dl324 treated by the restriction 
enzyme ClaI (corresponding to a deletion mutant E3; the deletion being 
made between the positions 78.4 and 84.3 of the genome of the adenovirus 
shown in FIG. 1), after transfection of 293 cells (human embryonic kidney 
cells transformed by the adenovirus and mentioned above) in order to 
generate the recombinant vector Ad-RSV-betagal. The nlslacZ gene is 
controlled by the RSV LTR promoter and possesses the polyadenylation 
signal of the SV40 virus. The recombinant virus thus obtained is incapable 
of replicating on account of the deletion of the E1 genes. 
2. Study of the transfer of the gene to the organs of the mouse through the 
intermediary of the adenovirus. 
Four day old Balb/C mice are given an intravenous injection of 20-40 
microliters of highly purified recombinant adenovirus Ad-RSV-betagal 
(10.sup.9 plaque-forming units: PFU/ml), the organs were excised 15 days 
after the injection and treated with 4% paraformaldehyde in a phosphate 
buffer for 30 minutes. After being rinsed, the organs were incubated 
overnight at 30.degree. C. in a X-gal solution. The whole organs were then 
frozen and treated appropriately so that cryosections (10 micrometers 
thick) could be prepared and these sections were staned with the aid of 
hematoxylin and eosin. 
The demonstration by means of histochemical staining in the manner 
indicated above of beta-galactosidase activity in the sections prepared 
indicates the presence of the gene inserted into the adenovirus vector. in 
the cells of the excised organs. The macroscopic examination of the heart 
as well as the skeletal muscles excised from these treated mice reveals 
the great efficiency with which this gene transfer was made after only one 
injection of the recombinant adenovirus. The significance of the choice of 
the intravenous route resides in the fact that the viral vector is not 
concentrated in any particular zone of the muscle tissue but, conversely, 
it is favourably distributed throughout the muscle mass. The histochemical 
staining leads to estimates that the number of transformed cells in some 
zones attains as much as 50% of the number of muscle cells present in this 
zone. 
The expression of beta-galactosidase in the myocardium as well as in the 
skeletal muscles is perfectly stable. It was possible to observe positive 
stains 15, 33, 55, 66, 90, 127 and 150 days after the injection of the 
recombinant adenovirus. The expression of the gene seems to be constant as 
a function of time since the proportion of blue cells in the muscle 
tissues seem to be more or less the same from one month to the next. 
The analysis of isolated muscle fibers reveals that a single fiber is 
likely to offer many "centers of expression". 
Analyses by (Southern) immunoblot performed on the heart of a treated mouse 
have led to the demonstration of an intense and unique band at 35 kbp 
indicating that the viral DNA introduced into the muscle cells is 
essentially extrachromosomal.