WO1997036602A1 - Method of restoring a functional protein in a tissue by cell transplantation - Google Patents

Abstract

The present invention relates to a method for improving the success of a transplantation which at least addresses to one of the most serious problems: transplant survival. The method comprises the steps of transplanting donor cells in a patient in need for such a transplantation, the transplantation being made in the presence of an agent blocking the interaction between a ICAM-1 molecule located at the membrane surface of the donor cell and a LFA-1 molecule located at the membrane surface of the patient's leucocytes, which blocking increases the viability of said transplanted donor cells by at least about four fold. The agent blocking this interaction is an anti-LFA-1 antibody which is injected to the host at the time of transplantation or an anti-ICAM-1 antibody fragment which is not capable of fixing complement, such as a F(ab)2 fragment.

Description

THEE OF THE INVENTION

Method of restoring a functional protein in a tissue by

cell transplantation.

FIELD OF THE INVENTION

This invention relates to a method of restoring a

functional protein in a tissue by transplanting healthy

donor cells or genetically modified donor cells. More

particularly, the present method reduces the mortality

rate of the transplanted cells by at least about 4-fold.

BACKGROUND OF THE INVENTION

Cell transplantation is a potential treatment for

several diseases. Myoblast transplantation may indeed be

use to treat Duchenne Muscular Dystrophy (Partridge

1991) , nanism (Dhawan et al . 1991) , hemophilia (Dai et

al. 1992) and Parkinson (Jiao et al . 1993) ; neuron

transplantation may be used to treat Huntington disease

(Emerich, 1995) and Parkinson disease (Borlongan and

Sandberg, 1995) ; islet may be use for diabetic (Hering et al. 1993), hepatocytes for liver diseases (Raper and

Wilson, 1993) . The success of these transplantations has

been so far rather limited. This limited success has

been attributed in part to the specific immune responses

(Huard, 1992, Tremblay, 1993) . However, some research

groups have also reported high levels of cellular death

during the first three days following cellular transplantations (Beauchamp et al . 1994; Huard et al.

1994) . This time course is too rapid to be attributed to

a specific immune response in naive animals. All the

treatments used so far to improve the success of a

transplantation never overcome the problem of the early

mortality of transplanted cells.

The principal problems to overcome during cell

transplantation are the following:

a) a host immune reaction towards donor cells;

b) the number of cells expressing the non-

defective protein; c) the amount of non-defective protein expressed

in the cells participating to tissue function

recovery; and

d) the high donor cell death rate.

a) A host immune response towards donor cells:

It is now known that an adequate immunosuppressive

therapy should be undertaken in the case wherein host

and donor are histoincompatible, to avoid rejection of

the transplanted cells. Shortly after transplantation,

infiltrating cells may be observed at the site of

transplantation. Even in the case wherein

histocompatible host and donor are selected (matched for

MHC class I and class II antigens) , other non-MHC

antigens might be responsible for an observed immune

response. Although the cellular immunoresponse may

decrease with time, the humoral response starts building

after one week and may be largely responsible for the

lack of long-term success of transplantation, especially

in immunohistoincompatible subjects. Nonspecific immunosuppression has been found

successful for improving and maintaining the success of

transplantation. Immunosuppressors like FK506,

rapamycin, cyclosporin A and cyclophosphamide did

improve the residence time of the transplant . Among

these, FK506 and rapamycin have been found the most

potent. Since immunosuppressors provoke undesirable

side effects, it is highly desirable to match

histocompatible subjects and to use a more specific

immunotherapy, when needed.

Histocompatibility is not the only factor to

consider for increasing the success of transplantation.

When identical twins are matched, there is still a poor

regeneration of muscle function. It appears to be due

to the low spontaneous muscle regeneration or to

inflammation. b) The number of cells expressing the non defective

protein:

The number of cells expressing a non-defective

protein, e.g. fused donor cells or hybrid donor- recipient cells, are usually quite low in non-

immunosuppressed individuals. Beside the beneficial

effect of an adequate immunosuppressive therapy or of an

adequate match of donors and recipients on the number of

functional cells, other strategies have been used to

increase the production of non-defective proteins. It

has been found that pre-treating recipient mdx mice (an

animal model for Duchenne muscular dystrophy) with

notexin and radiation, which has for effect to destroy

the capability of the recipient muscle cells to

proliferate, gives a much better chance to the donor

cells to proliferate and to reconstruct a functional

tissue. Up to date, the best results have been obtained

by combining immunosuppressive and tissue treatment with

notexin and irradiation. However, notexin and

irradiation remain unacceptable from a clinical point of

view because they have severe undesirable side effects.

In my co-pending application which serial number is US

08/404,888, a pre-treatment step consisting of culturing

yoblasts in the presence of a trophic factor is described. Particularly, the obtention of primary

cultured myoblasts in the presence of bFGF (basic

fibroblast growth factor) is shown to increase by four¬

fold the number of dystrophin-positive cells. This

represents a clear progress to enhance the success of

transplantation in absence of notexin and irradiation

pre-conditioning step in dystrophic individuals. Even

with this progress, the level of cell mortality still

remains very high (more than 90%) . The high level of

early cell mortality has not yet been solved.

c) The amount of non-defective protein expression in

the cells participating £Ω tissue function

recovery;

Although a high percentage of cells which survive

after one week of transplantation expresses a non-

defective protein (about 90% expressing dystrophin in an

mdx mouse) , the level of expression of a functional

protein (dystrophin) per cell remains quite variable.

There is therefore room for improving the level of expression of a non-defective protein in transplanted or

fused cells. d) The hiσh donor death cell rate:

This problem remains one of the most serious to

overcome. Immunosuppressive treatment has been

successful in inhibiting the humoral immune response

which appears more lately than the cellular immune

response. However, there is still a very high early

mortality of the transplanted cells (more than 90%) .

Inflammation may be one of the major factors involved in

the important early mortality e.g. before the building

of a cell or humoral response. Inflammation may be also

partly responsible for the poor success of muscle

regeneration observed after the transplantation of

myoblasts between identical twins.

Despite all the attempts made to improve the

success of transplantation, there still a need for

increasing the number of cells which survive after the

first five days of transplantation, beyond which

immunosuppressive treatments as well as an enhanced proliferation of the cells which have survived may exert

their beneficial effects to improve long-term recovery

of tissue function.

U.S. Patent 5,130,141 to Peter Law describes and

claims a method for treating muscle degeneration and

weakness similar to the one that we are presenting. All

the scientific experiences which are the basis for this

patent have been made on a dystrophic mouse model (as

indicated column 2, line 35; column 3, line 25) . The

dystrophic mouse which was used was (C57BL/6J-dy² /dy^2J)

as indicated in column 6, line 2 and in column 7, line

40. The author of patent 5,130,141 indicated that the

dystrophy in that mouse model was treated by the

injection of myogenic cells (column 1, lines 17 and 18;

column 2, lines 34 and 35; column 3, line 26; column 4,

line 39; column 5, line 67; column 6, line 54; column 9,

line 5; column 10, line 64) . In fact, it is stated quite

clearly that myogenic cells are the "active ingredient"

(column 6, line 54) . It is also clearly stated that

other cell types such as fibroblasts interfere with the practice of the claimed invention and cause detrimental

effects (column 4, line 66) . The main problem with this

patent is that its scientific basis is not sound. Since

the experiments made by Dr. Law's group, it has been

established that in the dystrophic mouse dy is missing

a protein called merosin (Arahata et al . 1993; Sunada et

al. 1994, 1995; Xu et al. 1994a, b) . The dy mice have an

intact dystrophin. We clearly demonstrate in this

application that merosin is not restored by myoblast

transplantation but that merosin is, however, restored

by another type of cell present in primary muscle

culture. Therefore the interpretation of the experiments

of Dr. Law's group is erroneous and thus the scientific

basis of the patent is not sound,

Dr. Law's patent does not further describe any

method to solve the problem of early mortality of

transplanted cells. STATEMENT OF THE INVENTION

It is now provided a method for improving the

success of a transplantation which at least addresses to

one of the most serious problems: transplant survival.

It is therefore an object of the invention to

provide a method of restoring a functional protein in a

tissue which comprises the steps of transplanting donor cells in a patient in need for such a transplantation,

said transplantation being made in the presence of an

agent blocking the interaction between a ICAM-1 molecule

located at the membrane surface of the donor cell and a

LFA-l molecule located at the membrane surface of the

patient's leucocytes, which blocking increases the

viability of said transplanted donor cells.

In a preferred embodiment, the agent blocking this

interaction is an anti-LFA-l antibody which is injected

to the host at the time of transplantation.

In an alternatively preferred embodiment, the

blocking is effected by using an anti-ICAM-1 antibody

fragment which is not capable of fixing complement, such - li ¬

as a F(ab)₂ fragment. The advantage of using the latter

resides in the capability of treating with F(ab)₂ the

cells before their transplantation which would impede

the recognition of leucocytes towards the transplanted

cells.

In the above method, it is still preferable to

match the donors and the patients for their

histocompatibility (MHC class I and class II antigens) .

Concurrently, immunosuppressive therapy should be

undertaken particularly in the case wherein a MHC-

incompatible donor is used. The preferred

immunosuppressive agents are rapamycin or FK506,

although a combination or other suppressive agents may

achieve similar good results. When a MHC-

histocompatible donor is used, the immunosuppressors are

mostly selected amongst those inhibiting the building of

an immune humoral response towards antigens other than

the MHC antigens . The donor cells might be selected from the group

consisting of primary cultured donor cells, cloned donor

cells and genetically modified donor cells.

To increase the number of cells that may be

transplanted from a donor biopsy, these cells may be

grown in vi tro in the presence a trophic factor. When

primary cultures of myoblasts are contemplated, the use

of bFGF is preferred, for a short period to avoid

growing of fibroblasts. bFGF has been shown to increase

by four-fold the number of dystrophin-positive cells in

a mdx mouse model. When cloned cells are used, longer

time of growing in the presence of bFGF is contemplated.

Before transplantation, the proliferation of donor

cells may be alternatively encouraged by the use of

cloned cells which have been transfected with a

recombinant vector containing a gene encoding a molecule

encouraging cell proliferation, which gene is under the

control of a promoter compatible with the donor cells.

The used promoter might be an inducible promoter. In a preferred embodiment, the molecule encouraging

cell proliferation is SV40-T antigen. The promoter is

a promoter of a major histocompatibility gene. Preferably, this promoter is a MHC Class II gene

promoter. A MHC Class II promoter is inducible by γ-

interferon (γ-IFN) . To ascertain that the introduction

of SV4T0 antigen does not render the transplant

tumorigenic, thermosensitive mutants thereof may be used

to encourage the proliferation of cells in vi tro at

33°C, which cells are returned to 37 or 39°C, and tested

for cell differentiation prior to transplantation. At

37°C no functional SV40T antigen would be produced.

The above invention is particularly applicable to

treat myopathies, dystrophies, natural muscle weakness

due to aging, and motoneuron diseases which are due to

a lack of trophic substances.

Some of the muscle weaknesses may be

eventually treated by gene therapy when the gene

mutation responsible for each of these diseases is

known. These diseases can, however, be treated by the transplantation of cells present in normal muscle. For

some diseases, myogenic cells may be required while for

other diseases other non-myogenic cell may be necessary.

In the case of muscle weakness due to natural aging or

following mechanical or other type of physical injuries

to the muscles, gene therapy will not be effective since

new muscle fibers have to be formed by injecting new

myogenic cells. In these cases, it would be envisageable

that cells of an individual may be banked at a younger

age, proliferated in vi tro and transplanted when needed

as an autologous transplant.

DETAILED DESCRIPTION OF THE INVENTION;

The present invention provides for the first

time, methods to restore missing functional molecules in

affected individuals, which restoration is greatly

improved by solving the problem of the early mortality

of transplanted donor cells. In dystrophic patients,

transplantation of myoblasts (eg. restoration of

dystrophin) or the transplantation of non myogenic cells (eg. restoration of merosin) will achieve such

restoration. The invention is based on successful

demonstrated restoration of these molecules (Vilquin et

al., 1994a, 1995a, ; Huard et al. 1991a,b; 1992a, b;

1993, 1994a,b; Tremblay et al. 1991; 1993a,b; Roy et al .

1993; Kinoshita et al. 1994a, b,c; Asselin et al. 1994;

1995) .

The claimed methods have been demonstrated in mice

affected of various hereditary diseases, in monkeys and

in Duchenne Muscular Dystrophy patients (see references

above and Roy et al. 1991; Tremblay et al. 1991a;

Labrecque et al. 1992; Satoh et al . 1992, 1993; Albert

and Tremblay 1992; Meola et al. 1993; Sansone et al .

1993; Belles-Isles et al. 1993; Vilquin et al . 1995;

Kinoshita et al. 1995) .

Dystrophic muscle fibers degenerate because of the

lack of a normal gene product. Cell transplantation can

restore this missing gene product either in the case of

myoblasts by forming new muscle fibers or forming hybrid

muscle cells (i.e. by fusing with host myoblasts or muscle fibers) containing the missing gene product (eg.

dystrophin) . In the case of transplantation of

non-myogenic cells, the missing gene product (eg.

merosin) may be secreted in the extracellular matrix.

The myogenic or non myogenic cells to be

transplanted can be histocompatible or histoincompatible

with the host. In cases of histoincompatibility and in

cases where a new gene product is introduced by the

transplanted cells, adequate immunosuppression will be

required. This immunosuppression may be assured with

pharmacological agents such as cyclosporin-A, rapamycin

or FK506 (Kinoshita et al. 1994a, b; Vilquin et al .

1994, 1995a, b) . The immunosuppression may also be

induced by monoclonal antibodies against various

lymphocytes or antigen presenting cell determinants. A

suitable regimen therapy using a combination of CTLA4-Ig

combined with an anti-CD4 Mab or combination of anti-

CD4 , anti-CD8 and anti-LFA-1 Mabs were both capable of

increasing the number of positive cells temporarily in

MHC-incompatible subjects. It is suggested that the regimen therapy should be carefully monitored in such a

way that the dose is adjusted to still permit the

formation of suppressor T-cells which may be responsible

for the development of tolerance. These

immunosuppressive agents may maintain long-term graft

survival and induce tolerance to the myoblasts and

muscle fibers of donor origin.

The immunosuppressive treatment may therefore be

induced by non-specific suppressive agents or by more

specific ones.

The used of mAbs may eventually lead to permanent

tolerance for the new antigens introduced by the cell

transplantation procedure (see review by Waldman and

Cobbold 1993) .

The myogenic and the non myogenic cells may be

grown and cloned using several different methods.

Several examples of such methods have been described by

the inventor (for human myoblast culture technique see

Tremblay et al. 1991a, b; 1993a, b; Huard et al. 1992a,

b) . Experts in tissue culture may, however, modify these methods to reach the same purpose. The cloning of cells

may also be facilitated by introducing in the founder

cell a gene to induce a conditional immortalization.

The use of cloned cells may also prevent undesired

effects produced by other cell types present in primary

muscle cell cultures.

A mouse cell line MB3 has been produced which

contains the heat sensitive SV40T antigen under control

of the IFN-γ inducible H-2Kb promoter. The MB3 cells

were transfected using the retrovirus LNPOZ (kind gift

of Dr. D. Miller, Fred Hutchinson, Cancer Research

Center, Seattle, WA) which contains the LacZ reporter

gene and a neomycin resistance gene driven by retroviral

LTR (Adam et al . 1991) . The clone 3LN was obtained by

further selection for resistance to geneticine (200

μg/ml) and β-Gal expression, and expanded for use in

transplantation experiments. The survival of the β-Gal

labelled cells was assessed by dosing the β-Gal activity

(Sambrook et al. 1989) in homogenates of control muscles

(obtained 3 hours after the transplantation) and in muscle homogenates obtained three days after the

transplantation.

The method of increasing the proliferation of the

transplanted cells by introducing a γ-IFN inducible SV40 T antigen may be subject to variation, as will be

apparent to the skilled artisan, to reach the same goal.

Animal models are applicable to other mammals

including humans. To evaluate how a non-defective

protein is produced in a recipient individual, a PCR

reaction may be effected on a biopsy. The messenger

cDNAs so amplified may have a differential restriction

enzyme pattern on an electrophoretic gel. This has been

shown to be the case for the dystrophin cDNA which is

different in mdx mice and in C57BL/10SnJ donor mice,

(Asselin et al. (1994 and 1995)) . Furthermore, the fate

of transplanted myoblasts may be monitored by way of

fluorescent latex microspheres-labeled myoblasts (Satoh

et al. (1993) ) .

The amount of cells to be injected may depend of

the type of disease to be treated, of the severity and stage of the disease and of the type of cells to be

injected for the treatment. In the case of myoblasts it may be necessary to inject these cells throughout the

muscle because these cells do not diffuse very well in

the muscle. The distribution of the myoblasts throughout

the muscle may be facilitated by developing a device

controlled by a robot. This robotic device may receive

from an imaging system (eg. magnetic scanner) the

information describing the exact muscle shape and size,

and the position of bones, nerves and blood vessels. The

robot will use this information to plan injection

trajectories avoiding the major nerves and blood vessels

and thus avoiding damaging them.

The cells required to treat the muscle may also be

inserted by some other routes. Some type of cells may

even be injected in the blood vessels of the patients

and migrate inside the muscles. The secretion of these

cells may also diffuse in the muscles. The secretion of

these cells may also diffuse in the muscle. This could

be the case for merosin or for factor VIII for example. The solution to inject the cells may range from

simple sterile saline isotonic solution, to

sophisticated culture mediums containing growth factors

such as bFGF to facilitate the cell survival and

migration. The injection solution may also contains

pharmacological agents and monoclonal antibodies to

prevent death of the injected cells. These solutions

will have to be prepare using Good Laboratory Practice.

Since one of the principal objects of this

invention is to enhance the early survival rate of

transplanted cells, monoclonal antibodies at least

comprise an anti-LFA-I antibody (if administered

directly to the patient) or an anti-CAM-I antibody

fragment .

The claimed invention can be clarified by examples

of experimental treatments in animals and human

patients. The experiments described are examples and

should not be use to limit the scope of the invention. EXAMPLES

1) Myogenic cells transplanted in mdx dγ.q rophic mice

to restore the expression of dystrophin

The mdx mice are an animal model of Duchenne

muscular dystrophy (Bulfield et al . 1984) . As the human

patients, this animal model lacks dystrophin due to a

mutation of that gene located in the X-chromosome

(Sicinski et al . 1989) . The absence of this protein

under the sarcolemma lead to muscle fiber degeneration

and to eventual muscle weakness (Pastoret and Sebille,

1995) . The development of this disease may be prevented

by transplanting normal myoblasts which restore the

expression of dystrophin in the muscle fibers. The donor

myoblasts may be grown from a muscle biopsy of an

histocompatible or an histo-incompatible mouse. In the

cases where myoblasts are obtained from an

histoincompatible donors, the host may be im uno-

suppressed with cyclosporin, but more effective immuno¬

suppression may be obtained with rapamycin, FK506

(Kinoshita et al . 1994; Vilquin et al . 1994, 1995) or monoclonal antibodies against molecules located on the

membrane of lymphocytes or of antigen presenting cell,

as described above.

2) Myogenic cells transplanted in monkeys

Myogenic cells have been shown to be able to form

new or hybrid (containing donor and host nuclei) muscle

fibers in monkeys. The injected myoblasts can be

obtained either from the animal itself or from another

animal. In both cases, the host has to be adequately immunosuppressed. Immunosuppression is required at least

for a short term because foreign antigens are present in

the culture medium. This experiment demonstrated that

new muscle fibers can be formed in a primate. In this

experiment, the injected cells were labelled with a

retrovirus vector containing the β-galactosidase gene.

The muscle fibers formed partially or totally by the

injected myoblasts expressed that gene demonstrating

that new genes can be effectively introduced in muscle

cells of primates by myoblast transplantation. 3) Myogenic c_e_lls injected in Puchenne muscular

dystrophy patients

Duchenne muscular dystrophy is due to the absence

of dystrophin in the muscle fibers. The protein can be

restored in the muscle fibers of these patients by the

transplantation of myoblasts obtained preferentially but

not exclusively from an MHC-histocompatible donor. This

has been demonstrated by the inventor's research team

(Huard et al . 1992a,b; Tremblay et al . 1993a,b) .

4) Injection of non-myoαenic cells in dv/dγ dystrophic

mice

The muscular dystrophy in dy/dy mice is due to the

absence of an extracellular matrix protein called

merosin. This animal is an excellent model of Congenital

Muscular Dystrophy (Arahata et al . 1993; Sunada et al .

1994, 1995; Xu et al . 1994a,b) . The expression of

merosin can be restored by the transplantation in the

muscle fibers of non-myogenic cells. Since this protein

is present in the extracellular matrix it has to be

secreted by the injected cells to restore the expression of merosin in the extracellular matrix surrounding the

muscle fibers. The presence of merosin in the

extracellular matrix stabilizes the muscle fibers.

Animals

The C57BL/6J dy/dy mice (Jackson Lab.) were used as

recipients for myoblast transplantation. The C57BL/6J

+/+ normal newborn mice (Jackson) were used as

compatible donors for some myoblast transplantations.

The transgenic TnI-LacZV29 mice (Tn-LacZ, gift from

K. Hasting, McGill University, Montreal, Canada) contain

the LacZ gene under the control of the quail fast

troponin I promoter, thus, differentiated muscle cells

express a cytoplasmic β-galactosidase (β-gal) protein

(Hallauer et al . , 1993; Kinoshita et al . , 1994a,b) .

β-gal expression is not restricted to the nucleus in

this model . Both male and female parents are

heterozygous animals, and newborn mice were used as

donors for some immunologically noncompatible myoblast

transplantations. The H-2K^b-tsA58 transgenic mice carry the

thermolabile tsA58 mutant of SV40 large T antigen under

the control of the H-2K^D promoter (Jat et al . , 1991) .

Interferon Y (IFN-γ) increases the transcription of this

promoter. The thermolabile protein is functionally

active at 33°C but not at 39°C. These characteristics

facilitate the derivation of conditionally immortalized

cell lines (Morgan et al . , 1994) . An homozygous

H-2K -tsA58 male mouse (Charles River Lab., Wilmington,

MA) was crossed with a heterozygous transgenic Tn-LacZ

female mouse. Offsprings were all heterozygous for the

H-2K^b-tsA58 transgene and some were heterozygous for the

Tn-LacZ transgene. Thus, newborns were tested for β-gal

expression, and only β-gal positive animals were used

for the establishment of myogenic cell lines (see

below) . Primary cell cul tures

Mouse primary myoblast cultures were obtained from

biopsies from newborn skeletal muscle as previously

described (Vilquin et al . , 1995c) . The cell suspension was cultured in 199 medium (Gibco, Grand Island, NY)

supplemented with 15% FBS (Gibco) and antibiotics. Cells

were harvested at 70% confluence, that is, 2 days after

plating, either for immediate grafting or for freezing

until grafting. These cultures were not pure and

contained several cell types, with 30% to 40% being

commited myoblasts as assessed by desmin immunostaining

(personal results) .

Cultures were also obtained from newborn transgenic

Tn-LacZ. Because parents were both heterozygous,

newborns were individually tested for the expression of

β-gal using X-gal (see below) . Only β-gal-positive

newborns were used for primary myoblast cultures .

Different batches of primary cell cultures have

been used for this work. The mice transplanted with

these batches have been gathered under the letters A, B

(β-gal cells) , and H (histocompatible cells) . Establishment of permanent myogenic cell lines from

(TnI-LacZl/29J X (H-2tf^>- tsA68) offsprings .

Primary muscle cell cultures were started using the

β-gal-positive offsprings. Following preplating, the

cells were plated at 50 cells/cm² in gelatin-coated

wells and grown in DMEM (Gibco) supplemented with 20%

FCS and 2% chick embryo extract (Gibco) at 33°C in 10%

C0₂. Mouse recombinant IFN-γ (Genzy e Co., Cambridge,

MA) was added at the final concentration of 20 U/ml

(Morgan et al . , 1994) . Colonies of typical myogenic

morphology were subsequently cloned in 96-well plates at

the limit dilution of 1 cell per well. The myogenicity

of colonies and clones was assessed by desmin

immunostaining and by the ability to fuse and form

myotubes in vi tro . Fusion was obtained by reducing the

FCS concentration to 5% and growing the cells at 37°C in

5% C0₂ in the absence of IFN-γ. Cell transplantation

On the day of transplantation, the cells were

harvested by trypsinization or thawing, washed three

times in HBSS (Gibco) , and concentrated as pellets. Cell

viability was assessed using trypan blue staining. The

Tibialis anterior (TA) muscles were exposed and injected

with approximately 4 x IO⁶ (primary muscle cell

cultures) , or IO⁶ (myogenic cell lines # 24, MB7, MB27) or 3 x 10^s (myogenic cell line MB3) viable cells

suspended in 10 μl of HBSS. When the role of myoblasts

in LAMA2 restoration was explored by clonal cell

transplantation, the left TA of the mouse received only

myogenic clones, while the right TA received both

myogenic clones and a histocompatible primary muscle

cell culture (i.e. obtained from normal C57BL6J+/+

newborn mice) . Some protocols included γ~irradiation

and/or notexin treatment of the muscles before cell

transplantation: three days before transplantation, one

or both hind legs of the dy/dy mice were Cobalt-

irradiated (20 Gy) . This level of irradiation has been shown to block host myoblast proliferation and to favour

donor myoblast implantation (Wirtz et al . , 1982; Morgan

et al . , 1990; Wakeford et al . , 1991) ; one day before

transplantation, one or both TA were exposed and

injected with 10 μl of notexin venom (5 μg/ml) , which

has been shown to trigger muscle fiber degeneration

without damaging myoblasts (Harris et al . , 1975) . FK506

immunosuppression was started on the day of

transplantation in noncompatible grafting models (2.5

mg/kg/d i.m., Fujisawa Co, Osaka, Japan; Kinoshita et

al . , 1994b) . The transplantation schedules are presented

in Fig. 1. Muscle collection

At the times indicated, the TA muscles were

collected and immersed in a sucrose solution (Tremblay

et al . , 1993) . Muscles were embedded, frozen in liquid

nitrogen, and serially sectioned at 8 μm. Adjacent

serial sections were thus spaced by 8 μm, while the

series of sections were separated by 180 μm. -gal histochemis try

Differentiated conditionally immortalized myogenic

cells and muscle cryostat sections were fixed with 0.25%

glutaraldehyde for 3 min and incubated overnight at room

temperature with 0.4 mM X-gal (5-bromo-4-chloro-

3-indolyl-β-D-galactopyranoside, Boehringer Mannheim,

Laval, Canada) (Kinoshita et al . , 1994a) . Preliminary

experiments indicated that the X-gal enzymatic

histochemistry was as sensitive as three-step

immunohistochemistry using monoclonal

anti-β-galactosidase antibodies to localize muscle

fibers formed by the fusion of donor myoblasts (not

shown) . Newborns originating from Tn-LacZ mice were

tested for β-gal expression using the X-gal reagent,

except that no fixation was necessary and incubation of

a small muscle piece was performed at 37°C for 1 h.

LAMA2 immunohistochemistry

Muscle sections were fixed in acetone at -20°C for

10 min. then nonspecific Ig binding was blocked with 10%

FBS in PBS for 30 min. The rabbit polyclonal anti-mouse LAMA2 (Xu et al . , 1994a) was used 1/300 in PBS

containing 1% FBS for 2 h at 37°C. The second antibody

was a biotinylated goat anti-rabbit Ig (1/100 in PBS

containing 1% FBS for 1 h; Dako, Copenhagen, Denmark) .

The next step was incubation with streptavidin-HRP or

streptavidin- FITC in some cases (1/200 in PBS

containing 1% FBS; Dako) . Binding was revealed with DAB

(0.5 mg/ml, Sigma) and 0.015% hydrogen peroxide. Slides

were mounted in PBS-glycerol. Immunoperoxidase-positive

and -negative fibers were counted by microscopic

examination of each muscle on the section with the most

positive fibers. Desmin im unocytochemis try

Conditionally immortalized myogenic cells were

grown on 2% gelatin-coated plates and allowed to fuse in

5% FBS at 37°C. The cells were fixed and permeabilized

with methanol at -20°C . Nonspecific binding was blocked

using 10% FBS in PBS for 30 min. The cells were

incubated with a mouse anti-desmin antibody (1/50 in PBS

containing 1% FBS for 1 h; Dako) . The second antibody was FITC-conjugated rabbit anti-mouse IgG (1/100 in PBS

containing 1% FBS for 1 h; Dako) .

Primary muscle cel l cul ture transplanta tion under FK506

i mmunosuppre ssion β-gal expression

Primary muscle cell cultures from transgenic mice

expressing β-gal under the control of a muscle-specific

promoter were able to develop inside the TA muscles of

dy/dy mice immunosuppressed with FK506. Some myoblasts

fused together or with host muscle fibers to form new or

hybrid muscle fibers expressing β-gal. These β-gal

expressing fibers were not numerous and they were not

dispersed throughout muscles. They were presumably

located only near the injection sites. As seen in Table

I, the percentage of β-gal-positive fibers was rarely

higher than two. These low percentages were obtained

whatever the age of the recipient mice at the time of

transplantation, a slight but not significant increase

was observed when animals were kept longer after

transplantation. LAMA2 expression

As previously reported, LAMA2 was absent from

muscles of dy/dy mice (Arahata et al . , 1993; Sunada et

al . , 1994; Xu et al . , 1994a, b) and was never expressed

in muscles only injected with HBSS. LAMA2 surrounded

normal mouse muscles. LAMA2 was expressed around some muscle fibers following Tn-LacZ primary muscle cell

culture transplantation. The total and relative numbers

of LAMA2-positive fibers, however, depended on the age

of the mouse at transplantation and on the pretreatment

of the muscle (Table I) . The percentage of

LAMA2-positive fibers was low (mean ± SD, 6.4 ± 4.4)

when older animals (i.e., more than 3 months) , were used

as recipients. The percentage of LAMA2-positive fibers

was higher (15.9 ± 9.0) when younger animals (i.e., 6

week old) were used as recipients. Notexin alone, or γ~

irradiation alone, did not increase the number of

LAMA2-positive fibers. The combination use of notexin

and Y-irradiation increased the percentage of LAMA2-

positive fibers in transplanted muscles (27.8 ± 14.9) . There was an overall but not significant increase in the

percentage of LAMA2-positive fibers with the duration of

the experiment (Table I, the mice in group A all

received the same myoblast preparation) . Colncali7.at.ion of β-σal and LAMA2

The Tn-LacZ primary muscle cultures originate from

mice with normal LAMA2 expression. Thus, both the normal

gene for LAMA2 and the reporter gene coding for β-gal

are present in the donor cells. LAMA2 and β-gal were

characterized on serial sections only spaced by 8 μm. As

shown in Table I, the percentages of LAMA2-positive and

β-gal positive fibers were comparable when old animals

(more than 3 months) were used for transplantation.

LAMA2 and β-gal labeling were localised in the same

clusters of fibers. Some LAMA2-positive fibers, however,

were negative for β-gal expression, and some β-gal

positive fibers were negative for LAMA2 expression on

serial sections. Most of the smallest fibers were β-gal

positive. These results indicated that β-gal and LAMA2 expression were not regulated similarly, or that the same type of cells were not responsible for their

expression.

When experiments were designed in younger animals

(i.e. 6 weeks old) , the LAMA2-positive fibers were much

more abundant than β-gal positive fibers (Table I) .

Surprisingly, the pattern and intensity of β-gal and

LAMA2 expression greatly differed. Some LAMA2-positive

clusters of fibers were totally devoid of β-gal. The

β-gal positive fibers were restricted to smaller areas

than LAMA2-surrounded fibers. LAMA2-positive fibers were

observed over long distances throughout the muscle, i.e.

more than lOOOμm, whereas no β-gal was observed in most

of these fibers over all length of LAMA2 expression. Long-term histocompatible transplantations

Transplantation of histocompatible, isogenic

primary mouse muscle cells in 6 week-old male dy/dy mice

led to long-term LAMA2 expression (i.e. 11 weeks) in the

transplanted muscles of all the animals. The percentage

of LAMA2-positive muscles was greater when muscles were

treated using notexin and γ~irradiation before transplantation (14.75 ± 6.4 without pretreatment versus

41.2 ± 14. 3 with pretreatment) . Notexin alone (12.75 ±

1.2 positive fibers) , or irradiation alone (19.25 ± 6.9

positive fibers) , followed by transplantation, was not

sufficient to increase dramatically the percentage of

LAMA2-positive fibers (Table II) . Given the small number

of dy/dy mice available for these experiments, however,

no statistical analysis was performed.

LAMA2 seemed to have spread centrifugally from the

center of the injection site to its periphery. While

LAMA2 completely surrounded normal mouse muscle fibers,

its localization was frequently incomplete and disrupted

in transplanted dy/dy muscles, especially in case of the

largest fibers. Thus, LAMA2 deposition seemed to be

localized. LAMA2 deposition also presented important

variations in transplanted mice as compared to normal

mice. Most of the smallest fibers were totally

surrounded by LAMA2. Condi tional ly immortal ized myoblast cul ture

transplan ta tion under FK506 immunosuppression

Characteri ation of immortalized myogenic cell lines.

The problem of tumorigenicity was frequently

reported upon the use of the classical C2 mouse myoblast

cell line (Wernig et al . , 1991; Morgan et al . , 1992) .

Thus, Morgan et al . (1994) developed a technique to

obtain pure, immortalized myoblast clones with the

ability to form new muscle fibers after transplantation

into immunodeficient and dystrophin-deficient nu/mdx

mice, without inducing tumors. The transgenic myoblast

cultures developed in the present study have the

additional advantage that differentiated cells express

β-gal under the control of a specific muscle promoter.

This allows the rapid identification of new or hybrid

fibers formed by the fusion of these myoblasts in vi tro

or in vivo in any mouse model.

The muscle cells isolated as colonies or single

clones from newborn muscles of (Tn-LacZ) X (H-2K^b-tsA58)

mice were able to grow and proliferate at 33°C in a 10% C0₂ atmosphere when stimulated by murine IFN-γ. When

shifted to differentiation medium the muscle cells fused

together and formed giant myotubes . These cells, either

myotubes or myoblasts alone, expressed the intracellular

filament desmin, which is an early marker of myoblasts

(Lin et al . , 1994) . These myotubes and some myoblasts

expressed also β-gal. These cells were able to fuse and

to form new or hybrid muscle fibers expressing β-gal in

vivo in immunodeficient SCID mice without forming solid

tumors. The colony # 24 (two cells in the original

cloning well) and the clones MB3 , MB7 and MB27 were

selected based on their in vi tro myogenic

characteristics and were injected into dy/dy muscles.

Lack of LAMA2 expression despite muscle fiber formation

after myoblast clone transplantations

Two, 3 and 4 weeks after injection of conditionally

immortalized myoblast clones into left TA of dy/dy

mouse, some β-gal positive fibers were formed using

clones MB27 and MB3, and Table III) . The number of β-gal

positive fibers was low possibly because the muscles were not pretreated with notexin and irradiation. On

adjacent sections, however, no LAMA2 could be detected

in the same TA muscle, neither surrounding β-gal

positive fibers or β-gal negative fibers (using clone

MB27) . LAMA2 was also absent from the series of sections

preceding and succeeding β-gal-positive sections, thus

indicating that a putative difference in the restriction

domain of LAMA2 and β-gal was not responsible for the

lack of LAMA2 expression in this model. A very faint

labeling could be observed around some, but not all

muscle fibers (using clone MB3) and some of these

faintly-positive fibers were β-gal-negative. Pure

myoblasts were thus able to fuse together or with host

muscle fibers and to trigger β-gal expression, but in

most of the cases they did not trigger LAMA2 formation

or extracellular deposition at least at levels

detectable by immunohistochemistry or

immunohistofluorescence (Table III) . The right dy/dy TA

of the same mouse, which was injected with a classical

histocompatible primary cell culture together with the same myogenic clone MB27, however, contained many LAMA2-

surrounded muscle fibers and/or β-gal-positive fibers.

The β-gal and LAMA2 proteins were not always located in

and around the same fibers. Overall transplanta tion success

Three transplantation models were used in this

study. In the first one, nonhistocompatible primary

muscle cells expressing the β-gal under control of a

muscular promoter were transplanted into dy/dy mice.

This allogenic transplantation required an efficient

immunosuppression, which was obtained using FK506

(Kinoshita et al . , 1994b; Vilquin et al . , 1995b) . The

use of β-gal expressing cells showed the exogenous

origin of the labelled muscle fibers and allowed the

comparison between LAMA2 and β-gal localization after

transplantation. In the second model, histocompatible

syngeneic primary cultures from normal littermates were

transplanted into dy/dy mice. This model did not require

immunosuppression and allowed the study of muscle

regeneration over longer time. In the third model, pure but nonhistocompatible myoblasts were transplanted into

one leg of FK506-immuno-suppressed dy/dy mice, whereas

the other leg received histocompatible primary culture

as a control of LAMA2 expression. This model allowed the

direct comparison of the outcome of transplantation

between pure myoblasts and primary muscle cells and

allowed the investigation of the potential role of

myoblasts in LAMA2 deposition. Cell transplantation

allowed the restoration of a structural, extracellular

protein. In these immunologically controlled models the

transplantation of primary muscle cells lead to LAMA2

expression in variable amounts in all animals. The

number of LAMA2-surrounded fibers was shown to depend on

the age of the animal at the time of transplantation and

on the muscle pretreatment . The highest numbers of

LAMA2-surrounded fibers were obtained when young animals

(6 week old) received histocompatible cells after

Y-irradiation and notexin pretreatment of the muscle.

Irradiation was shown to hamper myoblast proliferation

and thus reduce normal muscle regeneration, and to favour extensive fibrosis in dy/dy (Wirtz et al . , 1982)

and mdx/mdx (Wakeford et al . , 1991) mice. This treatment

greatly increased donor myoblast permeation into host

degenerating fibers and the formation of new or hybrid

dystrophin-positive fibers in mdx/mdx mice (Morgan et

al . , 1990) . In our models, it is thus likely that

irradiation and notexin necrosis favour donor cell

development in vivo over the dy/dy recipient cells.

In the histocompatible model LAMA2 , which is the

only known difference between dy/dy host mice and +/?

donor mice, is not sufficient to trigger efficient acute

rejection of LAMA2- expressing cells. Chronic rejection

was not assessed in this study, but the overall highest

numbers and percentages of LAMA2-surrounded fibers were

observed 11 weeks after grafting. LAMA2 is undetectable

in immunohistochemistry but very low amounts of LAMA2

mRNA are detected in untreated dy/dy mice by RT-PCR

(Arahata et al . , 1993; Xu et al . , 1994a) . These small

amounts of LAMA2 would be sufficient to make the animals tolerant to their self antigens and to avoid

immunological rejection of the transplanted LAMA2.

The intensity of β-gal staining and the number of

β-gal positive fibers after transgenic Tn-LacZ culture

transplantation was low as compared to results obtained

in other, non dy/dy mice strains under the same FK506

immunosuppressive treatment. In a previous report, up to

90% of mdx/mdx TA muscle fibers could express β-gal only

one month after transplantation (Kinoshita et al . ,

1994b) . The present results suggest that muscle cell

transplantation is less efficient in dy/dy mouse than in

mdx. Actually, more than 1000 dystrophin-positive fibers

are frequently obtained in only 2 months following

histocompatible primary muscle culture transplantation

in mdx/mdx mice (Vilquin et al . , 1995c) , whereas it is

difficult to obtain more than 300 to 400

laminin-2-surrounded muscle fibers in the same time in

dy/dy mice. The dy/dy muscle fibers are smaller than

normal muscle fibers and they show important size

variation within a single muscle. This may reflect trophic problems in dy/dy muscles related to the absence

of LAMA2 in basal lamina that could explain the

variation in efficiency between the mdx/mdx and the

dy/dy models. First, the initial lack of LAMA2 could

hamper myoblast migration or alignment either with other

myoblasts or with host muscle fibers, because basal

lamina is known to play important roles in the control

of migration, proliferation and differentiation of

various cell types (Engvall et al . , 1992) . Second, an

impaired muscle innervation due to the absence of LAMA2

around peripheral nerve fibers or neuromuscular

junctions (Leivo and Engvall, 1988; Sunada et al . , 1994;

Xu et al . , 1994a) could induce a muscle atrophy. Third,

the myoblast proliferation and/or migration could be

impaired by the abundant connective tissues, present

especially in the oldest dy/dy mice. Taken together,

these problems may explain why muscle regeneration was

relatively poor following transplantation, whatever the

pretreatment used. LAMA2 and β-gral colocalization after transplantation of transgenic primary cells

In transplanted muscle, LAMA2 was generally more

widely distributed than β-gal. This could indicate

differences in the regulation of expression of these two

proteins, that LAMA2 but not β-gal is diffusible, or

that the cell type responsible for LAMA2 expression is

not the cell type responsible for β-gal expression,

β-gal expression is restricted to skeletal muscle cells

as an intracellular protein, and its nuclear domain is

about 1000 μm. In contrast, laminins and likely LAMA2

are secreted outside of the cells, and may have a

relatively long half-life in vivo (Engvall, 1993) as

components of muscle cells basal lamina. Thus, the fate

of β-gal and LAMA2 in vivo is probably different.

Whereas β-gal expression requires myoblast fusion and

formation of hybrid or new muscle fibers, the

developmental mechanisms of LAMA2 expression are still

unclear. β-gal and LAMA2 did not always colocalize in and

around the same segments of muscle fibers. On serial

sections β-gal positive segments of fibers could be

LAMA2-negative and vice-versa. Thus, either myoblasts

and muscle fibers do express both β-gal and LAMA2, or

these proteins are expressed by several or only

partially overlapping different cell types, or by cells

at different stages of differentiation. If muscle cells

express both LAMA2 and β-gal, then β-gal positive fibers

should be surrounded by LAMA2, even on short muscle

fiber segments. This was not always observed. The other

hypothesis implies that myoblasts and muscle fibers

should express β-gal, whereas other cells should secrete

LAMA2. Depending on the presence and development of

myoblasts, or fibroblast-like, or both cell types in the

vicinity of host muscle fibers after transplantation,

β-gal positive, or LAMA2-positive, or both β-gal and

LAMA2-positive fibers should be observed. LAMA2 expression pa t tern

LAMA2 generally deposited continuously around small

caliber muscle fibers, whereas this deposition was

frequently discontinuous around normal diameter muscle

fibers, even two months after transplantation (Fig. 5e,

f) . In contrast, dystrophin was expressed along the

complete inner circumference of muscle fibers by some

weeks after transplantation, and dystrophin disruption

was rarely observed one or several months after

transplantation in the mdx/mdx or SCID mouse models

(Partridge et al . , 1989; Morgan et al . , 1990, 1993,

1994; Huard et al . , 1994; Kinoshita et al . , 1994b;

Vilquin et al . , 1995c) . The expression of LAMA2 seemed

to be centrifuge relative to injection sites. This

observation suggests that competent cells could

proliferate and diffuse, or that the secretion product

may diffuse, thus producing a gradient of LAMA2

expression from center to periphery, that would

progressively accumulate around the most proximal muscle

fibers expressing the appropriate receptors and extracellular matrix components, allowing LAMA2

sequestration around these muscle fibers. This

hypothesis could explain the polarization of LAMA2

deposition around the biggest fibers in the particular

orientation from periphery to center. Small-diameter

fibers would be more completely surrounded by LAMA2

because they were located at the injection sites in

close contact with LAMA2-secreting cells. The surface to

be covered by LAMA2 is also less in small-diameter than

in normal-diameter fibers.

The role of myoblasts in LAMA2 restoration in dy/dy mice

Cloned myogenic cells formed new or hybrid

β-gal-positive muscle fibers following transplantation

into dy/dy mice. Small and normal caliber p-gal positive

fibers were obtained already 2 weeks after

transplantation. Small-caliber fibers were likely

entirely of donor origin. Normal-caliber fibers probably

originated from the fusion of donor myoblasts with host

muscle fibers undergoing a segmental degeneration-regeneration process at the site of injection. The β-gal positive fibers, however, were very

rarely surrounded by LAMA2, thus indicating that

myoblasts and muscle fibers formed by them were not able

to secrete LAMA2, at least to levels detectable by

immunohistochemistry and immunohistofluorescence. As a

control for LAMA2 expression, primary cells from mixed

cultures injected in the contralateral leg were able to

trigger LAMA2 deposition around new and hybrid muscle

fibers within 2 weeks. Thus, LAMA2 may be secreted by

some competent cells present in primary culture, but not

in cloned myoblasts. Another hypothesis which cannot be

ruled out yet, is that none of the myogenic clones used

in this study was able to produce LAMA2 in vivo, despite

their ability to express desmin, to express β-gal under

control of a muscular promoter, to form myotubes in

vi tro and to form muscle fibers in vivo . Some distinct

populations of satellite cells have been described in

regenerating rat skeletal muscle (Rantanen et al . ,

1995) . Last, the secretion of LAMA2 could be dependent

on cell dif erentiation status. Indeed, LAMA2 deposition was observed in vitro in cultures of myogenic clones and

colonies (personal results) . Some, but not all myoblasts

produced LAMA2 and small myotubes did not, as observed

on immunocytochemistry preparations (data not shown) .

This suggests that LAMA2 secretion could be regulated in

myogenic cells, as are other muscle proteins and

myogenic factors (Rantanen et al . , 1995) . This

hypothesis would be supported by the very faint labeling

observed around 2 or 3 fibers following clone MB3 and

clone MB7 transplantation, and is currently under

investigation.

The transplantation models and results presented

above should prove useful to study in vivo interactions

between muscle cells and extracellular matrix, and

interactions or coordinate regulations between nerve and

muscle cells. These models should allow to study the

regulations of extracellular matrix protein interactions

in vivo . The critical steps for normal and pathologic

muscle regeneration, that is, myoblast proliferation, alignment or fusion, or the stability of nerve-muscle

regulations, should be discriminated.

The above results suggest that the dy/dy mouse

develops a pathology of the extracellular matrix. Some

clinical signs of this matrix pathology are similar to

those observed in some neuromuscular diseases primarily

originating from muscle or nerve cell deficiencies.

These observations should prove useful in the

investigation of human CMD aetiology.

The identification of the cell type responsible for

LAMA2 secretion and its relation with other muscle

constituents are important future issues, and could help

to categorize clinical cases of CMD and develop new

strategies for the correction of muscular diseases by

gene complementation. The technologies will differ from

those used for myoblast culture and transplantation in other types of muscular dystrophies if the

LAMA2-secreting cell is a connective cell, with no

fusion ability but adhesion and migration properties.

Long-term experiments should also be designed to further explore the feasibility of myelination restoration in

the peripheral nervous system of the dy/dy mouse

(Bradley and Jenkison, 1973; Madrid et al . , 1975;

Montgomery and Swenarchuk, 1977) . Alternatively, the

transducibility of the LAMA2-secreting cell type by

different gene vectors (adenoviruses, retroviruses,

herpesviruses) should indicate if it could constitute a

good target candidate for in vivo or ex vivo gene

therapy of CMD. 5) Increasing the earlv survival of cells by blocking

the interaction of LFA-l and ICAM-1 molecules:

Cell transplantation is a potential treatment for

several diseases . Myoblast transplantation may indeed be

use to treat Duchenne Muscular Dystrophy (Partridge

1991) , nanism (Dhawan et al . 1991) , hemophilia (Dai et

al . 1992) and Parkinson (Jiao et al . 1993) ; neuron

transplantation may be used to treat Huntington disease

(E erich, 1995) and Parkinson disease (Borlongan and

Sandberg, 1995) ; islet may be use for diabetic (Hering

et al . 1993), hepatocytes for liver diseases (Raper and Wilson, 1993) . The success of these transplantations has

been so far rather limited. This limited success has

been attributed in part to the specific immune responses

(Huard, 1992, Tremblay, 1993) . However, some research

groups have also reported high levels of cellular death

during the first three days following cellular

transplantations (Beauchamps et al . 1994; Huard et al .

1994) . This time course is too rapid to be attributed to

a specific immune response in naive animals. Our group

now demonstrates that abundant neutrophils and

macrophages infiltrate within hours the sites of the

cellular transplantation. Respiratory burst of these

infiltrating cells could be responsible for the early

damage to the transplanted cells. We demonstrate that

the rapid death of transplanted cells is indeed due to

an inflammatory reaction which can be largely reduced by

treatments with a monoclonal antibody against LFA-l.

β-Galactosidase labelled myoblasts were initially

transplanted in the muscles of non-immunosuppressed

C57BL10J mice. The reporter gene expression was reduced by 74 ± 12.4% by the third day following the

transplantation. This was attributed to necrosis of the

transplanted cells containing this reporter gene. The

reduction of the β-Gal labelled cells in the injected

muscle was also confirmed by histochemistry. When

myoblasts were transplanted in a host previously

irradiated over their whole body, their death was

significantly reduced suggesting that some host cells

were contributing to their mortality. Two experiments

were therefore made to verify whether this cell death

could be due to a specific cellular immune reaction.

β-Gal labelled myoblasts were injected in mice

immunosuppressed with FK506 starting one day before the

transplantation. FK506 has already been shown to control

very effectively both the cellular and humoral reaction

following myoblast transplantation in mice and thus to

permit very successful myoblast transplantation as

indicated by a high percentage (95%) β-Gal positive

muscle fibers one month after the myoblast

transplantation (Kinoshita et al . 1994) . In the present experiment, the short term mortality of the injected

myoblasts was however not reduced by the FK506

immunosuppression. The mortality of the injected cells

was also not reduced when they were injected in

immunodeficient SCID mice. These two results rule out a

rapid cellular immune reaction by lymphocytes as the

cause of the rapid cell death. During the first

three days following myoblast transplantation, the

injected muscles were, however, infiltrated by Mac-l

positive cells (cells/section) (i.e. macrophages and

neutrophils) . The presence of neutrophils was also

confirmed by immunohistochemistry using a specific mAb

(RB6-8C5, Pharmingen, San Diego, Ca) and by dosage of

myeloperoxidase activity. This observation suggests that

the death of the transplanted cell is due to an

inflammatory reaction rather than to a specific immune

reaction. Attempts to reduce cell death by injecting the

host with a monoclonal antibody (mAb) against Mac-l

(CDllb) were not successful. The death of the

transplanted cells was, however, significantly reduced to only 18.2 ± 7.2% by injecting the host with a mAb

against LFA-l (CDlla) before the transplantation. This is

in sharp contrast with the mortality rate already

observed (more than 90%) . This mAb blocks the

interaction of LFA-l located on leukocytes with ICAM-1

located on target cells and thus blocks the attachment

of macrophages and neutrophils to their target cells

(ref ) . This binding is required for the infiltrating

cells to produce oxidative damage to these target cells

(ref ) . Thus the anti-LFA-l treatment reduced the

inflammatory damages and improved the myoblast survival .

When cells are transplanted into a tissue there is

always some damage to the transplanted cells and to the

host tissue by the mechanical process, i.e. the

injection. This initial cell death would trigger an

inflammatory process which leads to an infiltration of

the host tissue by macrophages and neutrophils. These

infiltrating cells would then release free radicals and

thus trigger much more damage to the transplanted cells

and the host tissue during the following three days. This situation is similar to reperfusion damages

previously reported following short ischemia of the

heart (Entman et al. , 1992) . The presence of such

inflammatory reactions could seriously limit the

effectiveness of different cellular transplantations.

They could be especially detrimental when the

transplanted cells are either not abundant, non or

poorly proliferating. This inflammatory damage is

largely reduced by blocking neutrophils and macrophages

attachment to the transplanted cell with an anti-LFA-l mAb. Although the importance of the inflammatory process

has been demonstrated in these experiments for myoblast

transplantation, inflammation may also limit the

transplantation success of other cell types. An anti-

LFA-I Mab reduced by at least about four fold the

mortality rate of transplanted myoblasts, and there is

no apparent reason why an anti-CAM-I antibody or

fragment thereof could not achieve similar results, and

this, in other types of cells to be transplanted. 6) Other defective proteins may b£ replaced by

transplanted corresponding non-defective proteins

The above examples described the recovery of

merosin and dystrophin in deficient animals or humans by

transplanting healthy donor cells expressing the

corresponding non-deficient proteins. This invention

should not be restricted in any way to these two

proteins. For example, the cytoplasmic restoration of

glucose-6-phosphate dehydrogenase has been described in

stable hybrid myotubes (Sansone et al. (1993) ; Meola et

al . (1993)) . Therefore, the present invention applies

at least to different types of myopathies of a genetic

origin or other origins. Since other types of cells

(neurons, islets, hepatocytes) may be transplanted to

cure diseases like Huntington, Parkinson diseases,

diabetes and liver diseases, it will be readily

appreciated that the present invention applies to all

types of diseases that may be cured by transplanting

healthy donor cells. Laminin α2 chain restoration in dy/dy mouse (Vilquin e t al . ⁾

Table I. Outcome of transplantation of transgenic primary muscle cell culture in FK506-immunosuppressed animals Cultured cells were injected in one or both TA at the ages indicated, with or without muscle pretreatment. At the times indicated, the mice were sacrificed. The β-gal- and LAMA2-positive fibers were counted on the best section " Tκ, transplantation ^b Notexin indicates notexin injection and Y, /ray irradiation

Mouseft Age at Tx^a Days post Tx Leg Muscle Total nb β-gal- LAMA2- % β-gal- % LAMA2- (days) treatment⁶ of fibers positive positive positive positive

Al 92 21 Left None 1655 53 85 3 2 5.1

A2 92 21 Left None 1187 88 97 7 4 8 2

A3 92 21 Left None 1165 28 45 2 4 3 9

Right Notexin 875 5 55 0 6 6 3

A4 92 21 Left None 774 15 24 1.9 3.1

A5 92 39 Left Notexin 920 58 128 6 3 13 7 ¹

A6 92 39 Left None 1125 103 165 9 1 14 7 o r

A7 92 39 Left None 1277 25 47 1 9 3 7

Bl 45 27 Left Y + Notexin 932 3 162 0 3 17 4

Right None 1454 70 204 4.8 14

B2 45 27 Left Y + Notexin 1161 5] 511 4 4 44

Right None 1375 58 355 4 2 25 8

B3 45 27 Left Y + Notexin 1505 85 285 5.6 18 9

Right None 1468 48 118 3 3 8

B4 45 27 Left Y 502 24 97 4 8 19 3

Right Notexin 895 8 115 0.9 12 8

B5 45 27 Left Y 651 2 101 0 3 15 5 Right Notexin 953 21 193 2 2 20 3

Laminin 2 chain restoration in dy/dy mouse (Vilquin et al . )

Table II. Outcome of long-term histocompatible primary muscle cell transplantation without jm unosupression. Cell cultures were injected in both legs after muscle pretreatment. The mice were killed 11 weeks after transplantation, and LAMA2-positive fibers were counted on the best section ^Λ : Tx, transplantation. ^b: Notexin indicates notexin injection, and Y, yray irradiation.

Mouse)) Age at Days at Leg Muscle Total inb LAMA2- %LAMA2-

_Tχa post Tx treatment¹³ of fibers positive posi ive

(days)

HI 45 77 Left Y 1238 178 14.4

Right Notexin 1655 225 13.6

H2 45 77 Left Y 1396 336 24.1

Right Notexin 1475 175 11.9

H3 45 77 Left Y + Notexin 1007 517 51.3

Right None 942 182 19.3

H4 45 77 Left Y + Notexin 711 221 31.3

Right None 1280 130 10.2

Laminin 2 chain restoration in dy/dy mouse (Vilquin et al . )

Table III. Characteristics of the conditionally immortalized myogenic cells. Myogenic cells were selected in vi tro based on desmin expression and the ability to form β-gal positive myotubes i differentiation medium. The cells were injected in FK506-immunosuppressed, 4 to 8 week=old dy mice with or without addition of primary muscle cell culture. 2, 3 or 4 weeks later the mice were sacrificed, β-gal and LAMA2 expression were investigated on adjacent sections. Typical β-gal and

LAMA2 expression are presented in Fig. 6. ^a: only the myogenic clone or colony injected. ^b: myogenic clone or colony injected together with primary muscle cell culture. ^c: number of mice expressing

LAMA2; (n) total number of mice in one experiment. ^d: number of mice expressing β-gal; (n) total number of mice in one experiment. ^e: 2 or 3 fibers were faintly surrounded. ND: not done.

In vi tro characterisation Injection in left Injection in right leg leg to

Cell Passage# Desmin β-gal LAMA2- β-gal LAMA2- β-gal culture expression expression positive positive positive positive __!_ _i_ _____ ^(n)d

MB3 9 pos (>95%) myotubes 0(1)^e 1(1} ND ND

MB7 7 pos (>95%) myotubes 0(3)^e 3(3) 2(2) 2(2)

Colony #24 4 pos (>90%) myotubes 0(1) 1(1) 1(1) l(l)

MB27 8 pos (>90%) myotubes 0(3) 3(3) 3(3) 3(3)

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Claims

WHAT IS CLAIMED IS:

1. A method of restoring a functional protein in a

tissue producing a corresponding defective protein, by

transplanting donor cells which express said functional

protein in a patient in need of such a transplantation

who expresses said defective protein, said method

comprises the step of blocking the interaction between

a ICAM-I molecule located at the membrane surface of the

donor cells and a LFA-I molecule located at the membrane

surface of the patient's leucocytes, which blocking

increases the viability of the transplanted donor cells.

2. A method according to claim 1, wherein said

blocking is effected by treating said patient with of an

anti-LFA-I antibody prior to transplantation.

3. A method according to claim 1, wherein said

blocking is effected by treating said patient with an

anti-ICAM-I antibody.

4. A method according to claim 3, wherein said

antibody is an antibody fragment which is non-capable of

fixing complement.

5. A method according to claim 4, wherein said

fragment is a F(ab)₂ fragment.

6. A method according to claim 1, which further

comprises matching the donor and the patient for their

MHC-histocompatibility.

7. A method according to claim 1, which further

comprises the step of concurrently immunosuppressing the

patient .

8. A method according to claim 7, wherein the

immunosuppressing is achieved by administrating

rapamycin or FK506.

9. A method according to claim 1, wherein said donor

cells are cultured as primary cultured cells in the

presence of a trophic factor prior to transplantation.

10. A method according to claim 1, wherein said donor

cells are cloned cells grown in the presence of a

trophic factor prior to transplantation.

11. A method according to claim 10, wherein said cloned

cells are transfected with a recombinant vector

comprising a gene encoding a molecule encouraging cell proliferation under the control of a promoter compatible

with the donor's cells, said promoter being inducible by

an activating factor, prior to or following

transplantation.

12. A method according to claim 11, wherein said

molecule encouraging cell proliferation is SV40 T

antigen.

13. A method according to claim 12, wherein said

promoter is a promoter of a major histocompatibility

gene.

14. A method according to claim 13 , wherein said

promoter is a MHC class II gene promoter.

15. A method according to claim 14, wherein said

activating factor is γ-IFN.

16. A method according to claim 1, wherein said patient

suffers of muscle cell degeneration or weakness.

17. A method according to claim 16, wherein said

patient suffers of a myopathy.

18. A method according to claim 17, wherein said

patient suffers of muscular dystrophy.

19. A method according to claim 18, wherein said

patient suffers of Duchenne muscular dystrophy.

20. A method according to claim 16, wherein said donor

cells are myogenic cells or non-myogenic cells.

21. A method according to claim 17, wherein said donor

cells are myogenic cells or non-myogenic cells.

22. A method according to claim 19, wherein said donor

cells are myogenic cells.