Myoblast transplantation between monozygotic twin girl carriers of Duchenne muscular dystrophy

Neuromusc. Disord., Vol. 3, No. 5/6, pp. 583-592, 1993

Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0960-8966/93 $6.00+ .00

Pergamon

MYOBLAST GIRL

TRANSPLANTATION CARRIERS

OF

BETWEEN

DUCHENNE

MONOZYGOTIC

MUSCULAR

TWIN

DYSTROPHY

J. P. TREMBLAY,* J. P. BOUCHARD,* F. MALOUIN,* D. THEAU,t F. COTTRELL,~H. COLLIN,~ A. ROUCHE,~ S. GILGENKRANTZ,§ N. ABBADI,§ M. TREMBLAY,* F. M. S. T O M I ~ and M. FARDEAU~ * Centre de recherche en Neurobiologie, Universit6 Laval, 1401 18e Rue, QuObec,Canada G1J 1Z4; tP6diatre, 18 rue Aiguillon, 29200 Brest, France; :~ Unit6 INSERM 153 and Consultation Risler, Htpital de la Salp~tritre, 47 Blvd de l'HOpital, 75013 Paris, France; § Centre R6gional de Transfusion Sanguine et d'Htmatologie de Nancy, Avenue de Bourgogne, 54511 Vandoeuvre-les-Nancycedex, France

Abstract--Monozygotic twin girls, both carriers of Duchenne muscular dystrophy, only one a severe symptomatic carrier and the other asymptomatic due to opposite lyonization, were studied. Myoblast clones were obtained from a muscle biopsy of the asymptomatic carrier. PCR amplification showed that most (94%) of these clones produced normal dystrophin mRNA. Roughly 704 million myoblasts were produced from 119 clones. These myoblasts were transplanted into the extensor carpi radialis (ECR) and in the biceps of one arm of the manifesting carrier while the other arm acted as the control. The strength of the patient was evaluated in a series of pre- and post-tests and a biopsy was obtained about 1 yr after the transplantation. The myoblast injections produced a significant force gain (12%-31%) in wrist extension but no force gain for elbow flexion. Muscle biopsies on the injected and control muscles obtained 1 yr after the injections showed only a small increase in the number of dystrophin positive fibers and the presence of numerous small type II fibers. The small beneficial effect of this transplantation cannot be attributed to immune problems, the donor and the recipient being identical twins, but may be due to a low level of spontaneous muscle regeneration.

Key words: Duchenne muscular dystrophy, myoblast transplantation, manifesting carrier, monozygotic twin, dystrophin, muscle strength.

INTRODUCTION Duchenne muscular dystrophy ( D M D ) is a hereditary disease linked to the X-chromosome. It is characterized by the lack of dystrophin under the sarcolemmal membrane [1-3], a defect that may be alleviated by the transplantation of normal myoblasts into the affected muscles. Transplanted myoblasts fuse together and/or with those of the patient to form new muscle fibers expressing dystrophin [4-10]. The expression of a truncated form of dystrophin that is missing part of the rod domain, has also recently been obtained using an adenoviral [11] or a retroviral [12] vector. Myoblast transplantation permits, however, not only the expression of dystrophin but may also lead to fiber hypertrophy and to an increase in the number of muscle fibers so that strength is improved. Although positive results have been reported following myoblast transplantation in mice [5, 7], the results of the initial clinical trials have been rather disappointing [13-16]. Nevertheless, the

number of dystrophin positive fibers is apparently higher following transplantation [14, 15, 17] and the donor's dystrophin m R N A has been detected by P C R in two out of eight patients [18]. So far only two laboratories have reported permanent or transient strength increases in some patients, while the other research groups report no significant gain [13, 14, 18, 19]. A possible mechanism responsible for the limited success of myoblast transplants is rejection by the immune system [20, 21]. Indeed, our group has observed the presence of antibodies against the donor's myoblasts and myotubes in the serum of several patients even though they were M H C compatible with their donor [14, 15, 20]. These antibodies were directed against dystrophin and other unknown minor antigens. D M D usually affects males while females are asymptomatic carriers. A few cases of D M D , however, have been reported in women [22-24]. These cases m a y be due to a translocation of part of one X-chromosome into an autosome [22]. In 583

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J.P. TREMBLAYel al.

other cases, the clinical manifestation has been attributed to a more frequent inactivation during lyonization of the X-chromosome containing the normal dystrophin gene [23, 24]. In female monozygotic twins, a discordant manifestation of X-linked diseases occurs frequently. This is apparently due to unequal lyonization; the paternal X-chromosome being preferentially inactivated in one twin and the maternal Xchromosome preferentially inactivated in the other twin. Unequal severity of phenotype in identical twin Duchenne carriers has been reported several times [25 28]. Recently, Abbadi et al. [29] identified such a pair of female monozygotic twins who were D M D carriers. In that family, the mutation, identified in an affected brother, is a deletion of the promoter and of the dystrophin exon 1. Owing to unequal inactivation of the paternal chromosome during lyonization, one of these twins became a severe symptomatic carrier while the other remained asymptomatic. This pair of identical twins described by Abbadi et al. [29] thus represents an ideal situation for transplantation of myoblasts without immunological problems. The results of transplantation of myoblast clones obtained from a biopsy of the asymptomatic twin into two muscles of the symptomatic twin are reported in this article. MATERIALS A N D M E T H O D S

by conventional histology, histochemistry and immunocytochemistry on frozen cryostat sections. Indirect immunofluorescence microscopy using anti-dystrophin antibodies (DYS2 and DYS3 of Novocastra, anti-dystrophin of Torpedo marmorata [30]) was performed as described previously [30]. Photographs were taken from all the sections of both right and left extensor carpi radialis muscles stained with the anti-dystrophin anti-torpedo antibody and all dystrophin labelled fibers were counted in montages made from these photographs. Myoblast clones

From the 1 g muscle biopsy obtained from the asymptomatic twin, cells were dissociated enzymatically with 0.1% trypsin and with 0.1% collagenase following a modification of Yasin et al. [31, 32]. Clones were obtained by placing about 1 cell per well in a 96 well plate. Roughly 1500 wells were initially started. Following proliferation, the cells were trypsinized and for each clone a 100,000 cell sample was tested by flowcytometry to verify the presence of the NCAM antigen using the CD56 antibody (Coulter Electronic, Hialeah, FL, U.S.A.). The myoblast clones were identified by the presence of NCAM. Only those that displayed over 95% NCAM-positive cells were kept for further proliferation. About 6 million myoblasts were then produced by each clone.

Patients

P C R ampl(fication o f dystrophin m R N A exon 1

The twins were 14 yr old when the study was initiated. The symptomatic carrier had severe weakness of the upper and lower extremities and was confined to a wheelchair but was still able to walk a few steps. The non-manifesting twin had no clinical symptoms; she was able to walk, swim and participate in sports. Permission for the myoblast transplantations and biopsies was granted by the Human Ethics Committee at Laval University. The parents were required to sign an informed consent.

Exon I of the dystrophin transcript was amplified by PCR in 54 different myoblast clones. The RNA was extracted following the method of Chomczynski and Sacchi [33]. A sample of 500 ng of RNA was used for PCR amplification following the method described by Roberts et al. [34]. The primers used were D M D la and D M D lb which amplify exons 1-10.

Muscle biopsies

Biopsies were obtained from: (1) the deltoid muscle (one arm) of both twins at study initiation: and (2) the extensor carpi radialis (ECR, symmetrical areas of right and left arms) muscle of the affected twin, 1 yr after transplantation of the myoblasts. Muscle samples were frozen in isopentane cooled in liquid nitrogen and stored at - 80°C. Both biopsies were studied

Myoblast transplantation

A total of 119 different myoblast clones were identified and used to produce 704 million cells. Each day, about 90 million myoblasts were trypsinized, washed in HBSS, and suspended at 30 million cells ml -~ in HBSS. Then, the myoblasts were placed in a syringe labelled A, and for control purposes a syringe containing only HBSS was labelled B. The injections were made bilaterally into two or three different sites of the biceps brachii and extensor carpi radialis by a neurologist (J.P.B.) who was blind to the

585

Myoblast Transplantation syringe contents. He randomly decided to inject the contents of syringe A into the muscles of the right arm at the first session and thereafter continued to inject the contents of the syringe labelled A into the right arm muscles during two daily sessions over a period of 7 consecutive days. At each session, an equal volume of the contents of the syringe labelled B were injected into the muscles of the left arm. After each injection, a small sample left in the myoblast containing syringe was tested for viability by extrusion of 0.4% of trypan blue. Another sample was placed in culture in low serum (5%) to test the capacity of the myoblasts to fuse.

Strength measurements Static voluntary strength for elbow flexion and wrist extension was evaluated bilaterally four times prior (pre-test) and six times after (posttest) the injections. The pre-tests were made at 1 month intervals over the 4 month period preceding the injections, while the post-tests were made over a 9 month period: monthly for the first 3 months and, then at months 6, 7 and 9. The evaluations were carried out by the same evaluator who was blind to the side of the myoblast injections. Measurements were made with a Penny & Giles myometer following standardized procedures [21]. Three isometric maximal voluntary contractions (MVC), separated by a 1 rain rest period were repeated at each testing session.

Data analysis At each testing session, a mean force value (in Newtons) was derived from three MVCs. A global mean representing all the pre-tests was then calculated by averaging the four pre-tests to obtain a baseline mean and its confidence limits ( + 2 S.E.). Subsequently, mean force values for each post-test were compared to this baseline value. Force changes were judged to be significant (p < 0.05) if they fell beyond the confidence limit interval represented by the mean force value ( + 2 S.E.) at the baseline.

control serums. After incubation at room temperature for 30 min, the cells were washed in 4 ml of PBS and the cell pellet was resuspended in 1 0 0 / A o f 1:20 dilution of fluorescein-isothiocyanate (FITC) conjugated anti-human IgG F(ab)2 fragment. The cells were incubated for an additional 30 min at 4"C and washed in PBS. They were immediately analyzed for the percentage of fluorescence with an EPICs cytofluorometer operated at 488 nm.

RESULTS Figure 1 illustrates the genealogical tree of the family of these twin girls. The brother is a Duchenne patient confined to a wheel chair since the age of 12. Abbadi et al. [29] have demonstrated, by D N A analysis, that the girls are really monozygotic twins.

Muscle biopsies of the deltoid muscle The histological and histochemical study of the biopsy of the affected twin showed the characteristic features of an X-linked recessive type muscular dystrophy. By immunofluorescence, there was a mosaic pattern of the sarcolemmal labelling of the muscle fibers. Thirty two per cent of the fibers showed labelling of the sarcolemma. While in many fibers the labelling was seen all around the periphery, in others it was incomplete, only a more or less long portion of the sarcolemma being labelled (Fig. 2a). In the unaffected twin all anti-dystrophin antibodies showed normal staining of the sarcolemma (Fig. 2b).

Probes

pERT 87.1 2

The presence of antibodies in the recipient's serum was evaluated by cytofluorometric analysis. Donor myoblast suspensions (2 x 105 cells) were washed in a phosphate buffer and resuspended in 40/11 of PBS and 20 ;tl of the recipient serum or appropriate negative and positive

1

del

1

754

1969

Detection of the humoral reaction by flowcytometry

1

2

II

(~ 2

1

del

1

1977

11972

:l 21 212 del

2

1

2

1

del

1

del

1

Fig. 1. Genealogicaltree of the family of the monozygotic twins. There was only one other case of DMD in the family, an older brother of the twins.

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J.P. TREMBt.AY et ul.

Fig. 2. Indirect immunofluorescence localization of dystrophin in unfixed cryostat transverse sections of muscle fibers using an antibody raised against the dystrophin of Torpedo marmorata. (a) Mosaic pattern of the sarcolemmal labelling of the muscle fibers (affected twin). Note the incomplete labelling in some fibers. (b) Normal distribution of dystrophin in the sarcolemma of all fibers (unaffected twin) x 275.

587

Myoblast Transplantation

Myoblast clones expressing normal dystrophin mRNA RNA was extracted from a sample of 3 x 105 ceils of 54 different myoblast clones. Since the mutation in this family is a deletion of the dystrophin promoter and of exon 1, the presence of the normal transcript was detected by PCR amplification using one primer within exon 1 and one primer within exon 10. These primers (DMDIa and DMDlb) were developed by Roberts et al. [34]. They amplify exons 1-10 of dystrophin mRNA producing a 1207 bp product. When there is a deletion of exon 1 there is no amplification. When a clone has the Xchromosome containing the normal dystrophin gene active, there is amplification of the dystrophin mRNA (Fig. 3). The majority of these clones (51 clones of 54 tested) produced a normal amplified product.

Myoblast transplantation Given that the majority of the clones that were tested for the presence of the normal dystrophin transcript were found to express this normal transcript, this test was not done on all the 119 clones used to perform the myoblast transplantation. A total of 704 million myoblasts were produced and transplanted in the biceps (roughly 400 million) and in the ECR (roughly 300 million). About 45-50 million myoblasts were transplanted in the morning and in the afternoon over a period of 7 days. All myoblast clones used for our transplantation spontaneously fused during the last few days in culture. Moreover, myoblasts in the sample left in the injection syringes were 95% viable and when placed in culture in low serum they were still able to proliferate and fuse. A marked oedema in both muscles injected with myoblasts was observed 7 days after the onset of transplantation. No oedema was observed in the contralateral muscles injected only with HBSS, although the same volume was injected. For this reason, the last injections planned for the 8th day were not done.

Muscle strength measurements Figure 4 gives the mean static force recorded at each pre- and post-test; the horizontal lines delineate the confidence limit intervals. On the myoblast injected side, the force values for wrist extension increased 31, 19, 12 and 26% in posttests 1-4 ( 1 ~ months), respectively, and in all cases the increases exceeded the confidence

limits. Although wrist extension force on the control side also increased in post-tests, these changes were generally within the variation of the measurements. Thus, although wrist extension force increased on both sides, the increase was more consistently significant over time on the myoblast injected side, suggesting some effect from the myoblast injections in addition to a natural strength gain. No significant force increase was observed for elbow flexion on either side; on the contrary, there was a gradual strength decline bilaterally.

Post-transplantation muscle biopsies In the biopsies of both ECR muscles made 1 yr after the myoblast transplantation, the percentage of fibers containing dystrophin was slightly higher in the myoblast-injected muscle than in the control muscle (Table 1). In addition, the myoblast injected muscle had more (34.8 %) type II muscle fibers than the non-injected muscle (18.6%). Moreover, there was a shift in the distribution of muscle fiber size (Fig. 5). The frequency of small muscle fibers was higher in the myoblast-injected muscle.

Absence of immune reaction against donor myoblasts No antibodies against the donor's myoblasts were found by flowcytometry in the recipient's serum, 10 days and 1 yr after the myoblast transplantation. DISCUSSION

Myoblast transplantation, as with any other transplantation, can lead to immune reactions. To avoid these immune reactions some research groups have used immunosuppressive treatments such as cyclosporine [18, 19] or cyclophosphamide [16]. Instead, our group chose to use donors and recipients who were perfectly compatible for MHC class IA, B, C and class IIDr. The success of our transplantations, however, has been rather limited [14, 15]. We have shown that some of our patients developed humoral immune reactions directed against minor antigens, including dystrophin [15, 20, 21]. More recently, we have also obtained evidence of a cellular immune reaction following xenotransplantation [35] as well as in allotransplantation in mice. A DMD-manifesting carrier with an asymptomatic identical twin sister seemed like an ideal

588

J.P. TREMBLAYet

al.

Fig. 3. RNA was extracted from a sample of about 300,000 cells from 3 different clones obtained from the asymptomatic twin's muscle biopsy. Dystrophin mRNA was amplified by PCR using primers DMD la and DMD lb of [34]. The amplified segment has 1207 bp which is the expected size for a normal mRNA. No amplification is obtained when no RNA is added ( - RNA) or when no reverse transcriptase is added to the RNA ( - RT). case to e v a l u a t e m y o b l a s t t r a n s p l a n t a t i o n , since n o r e j e c t i o n p r o b l e m s w e r e foreseen. Because, the d o n o r a n d the r e c i p i e n t are identical twins, the r e c i p i e n t s h o u l d h a v e the s a m e a n t i g e n i c

d e t e r m i n a n t s as the d o n o r . M o r e o v e r , the r e c i p i e n t is a m a n i f e s t i n g carrier, w h o has r o u g h l y 3 0 % o f h e r m u s c l e fibers c o n t a i n i n g normal dystrophin and, therefore, should not

589

Myoblast Transplantation ELBOW FLEXION MYOBLAST INJECTED

ELBOW FLEXION CONTROL 40. 35.

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IS, lO,

'1'2'3'4'

BEFORE

'

'

'

'1~2'3'4'5'6

'

AFTER

WRIST EXTENSION CONTROL

BEFORE

AFTER

WRIST EXTENSION MYOBLAST INJECTED

BEFORE AFFER BEFORE AFTER Fig. 4. This figure illustrates elbow flexion and wrist extension strength of the control and myoblast-injected side. Strength was evaluated four times before (dates of evaluation were 10January 1992,7 February 1992,6 March 1992, 8 April 1992)and six times after myoblast injection over a 9 month period (dates of evaluation were 27 May 1992, 26 June 1992, 31 July 1992, 2 October 1992, 20 November 1992, 22 January 1993). Horizontal lines give mean + 2 S.E. (confidence limits) of pre-test force values.

Table 1. Detection of dystrophin positive fibers Myoblast injected side

Dystrophin positive fibers Dystrophin negative fibers

520 990

Total

1510

Dystrophin positive fibers Dystrophin negative fibers

310 730

Total

1050

34.43% 65.57%

dyst.pos./dyst.neg. = 0.5252

30.47% 69.53%

dyst.pos./dyst.neg. = 0.4383

Control side

develop any i m m u n e reaction against dystrophin. The only sign o f a possible i m m u n e response was oedema in the muscles injected with myoblasts. This could have been due to the presence o f foreign proteins from the culture medium still attached to the myoblasts or simply to the presence o f d a m a g e d cells following myoblast injection. However, no sign o f a h u m o r a l i m m u n e reaction against the d o n o r ' s myoblasts was detected by flowcytometry.

However, despite this predicted absence o f a manifest immune reaction against the d o n o r ' s myoblast, only a small beneficial effect was produced by the myoblast transplantation. Wrist extension strength only increased slightly after transplantation o f the E C R . The presence o f a higher percentage o f dystrophin positive fibers in the injected E C R and o f n u m e r o u s small diameter type II fibers are also possible consequences o f the myoblast transplantion. These

590

J. P. TREMBLAY et al.

(A)

Control ECR

I

(B) I

ECR myoblast injected J

Frequency (%) 3O

3O

25

FIBRES I 81,4%

25

20

20

15

15

10

10

5

5

0

FIBRES I 65,2%

0 0

10

20 30

40 50 60

70 80 90 100 120

(c)

(D)

3O

3O

FIBRES II 34,8%

FIBRES II 18,6% 25

25

2O

2O

15

15

l°/lj,

10

5

5

0

0 0

10

20 30

40 50 60

70 80 90 100 120

0102030405060708090100

Fig. 5. Distribution of the size ofmascle fibers (type I and type If) in the control ECR (A and C) and in myoblast-injected ECR (B and D). There is an increase of small size type ]I muscle fibers in the myoblast-injected muscle.

small muscle fibers may be due to still ongoing regeneration or perhaps to aborted regeneration. The significance of these observations is difficult to assess, however, because only one biopsy was obtained per muscle. It is possible that the small differences observed are the result of the natural variability of biopsy sampling. One can question why the myoblast transplantation produced such a limited beneficial effect despite this apparently ideal immune situation. Help in interpreting this result comes from our recent transplantation experiments in SCID mice [36]. Normal human myoblasts were injected into the tibialis anterior muscle of these mice, known to have a deficient immune system (i.e. no lymphocytes B and T). Despite the absence of immune reactions, however, the transplantation

of human myoblasts did not produce muscle fibers containing human dystrophin. Fibers containing human dystrophin were only obtained when the injected muscle was irradiated and destroyed with notexin. These manipulations triggered muscle regeneration and prevented the host myoblasts from participating in the muscle regeneration, thus favouring a major participation of the donor's myoblasts to regeneration. Therefore, a possible explanation of the limited success of the myoblast transplantation in the present study is that there is not only a low rate of muscle regeneration but also a major participation of the host myoblasts. This may account for the very small increase of dystrophin positive fibers observed in this patient. To obtain a significant increase of strength,

Myoblast Transplantation

myoblast transplantation must not only increase the expression of dystrophin but also lead to an increase of either the size and/or the number of muscle fibers. It is possible that the preponderance of small muscle fibers revealed in the biopsy 1 yr after transplantation was because the new fibers formed by the transplanted myoblasts lacked additional myoblasts to encourage their hypertrophy? Did we inject enough myoblasts to produce sufficient muscle regeneration? At least two barriers for successful myoblast transplantation must be circumvented. These concern: (1) the host immune system; and (2) participation of the donor's myoblasts to muscle regeneration. The first barrier may be countered by identifying an appropriate immunosuppressive treatment. The second barrier may be less important in Duchenne patients more than 6 yr old, since their own myoblasts progressively lose their myogenic potential and should not compete with the donor's myoblasts. On the other hand, given the advanced stage of muscle wasting in these patients, a very large number of donor myoblasts will be required. Acknowledgernents--The authors wish to thank J. Cartaud

for the gift of the polyclonal antibody against dystrophin. This work was supported by grants from AFM, MDAC and Lyon's Gate Foundation. The authors also thank Dr J. Beckmann and O. Broux from G~n&hon for their collaboration in the genetic investigation of the monozygotic twins.

9.

10.

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12.

13. 14. 15.

16. 17.

18. 19.

REFERENCES

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mammalian muscle into mononucleated cells. Exp Cell Res 1976; 102: 405~,08. 32. Yasin R, Shebic G, Beers V, Thompson E J. A quantitative comparison of the dissociation of adult mammalian muscle by various enzymes. Biochem Soc Trans 1976; 4:53 55. 33. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 1987; 162: 15(~ 159. 34. Roberts R G, Barby T F M, Manners E, Bobbrow M, Bentley D R. Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Genet 1991: 49: 298 310. 35. Gu~rette B, Asselin I, Roy R, Tremblay J P. Evidence of cellular immune reaction following myoblast transplantation in mdx mice. Muscle Nerve (submitted). 36. Huard J, Verreault S, Roy R, Tremblay M, Tremblay J P. High efficiency of muscle regeneration following human myoblast clone transplantation in SCID mice. J Clin Invest (in press).