Últimas Notícias - Apresentação  e Anais de Congressos 

Seattle -  Em Congresso que está ocorrendo de 30 de maio a 3 de junho de 2001 são relatados pelo menos 19 trabalhos de um total de mais de 1150 abordando a terapia gênica com vetores virais no tratamento da distrofia muscular. Esta linha de tratamento é considerada internacionalmente como a mais promissora no tratamento das distrofias musculares. O sistema imune é considerado um obstáculo ao sucesso desta terapia. Em um estudo de Jeffrey Chamberlain e seu grupo da Universidade de Washington em  Seattle construiram um vetor sem as proteínas que podem desencadear as respostas imunológicas denominado "gutted" adenovirus. Usando camundongos portadores de distrofia muscular observaram que o vetor com o gene da distrofina provoca uma mínima resposta imune; os genes são liberados para os músculos e provocam melhora da força muscular. Em outro estudo os autores desenvolveram micro-distrofinas com cerca de um terço do tamanho da distrofina completa e observaram, em camundongos, melhora da força muscular inclusive do músculo diafragma. Outro estudo independente da Universidade de Pittsburgh realizado por Xiao-Xiao e colaboradores  eles demonstraram, em camundongos, que após sete meses da aplicação do minigene da distrofina  (a última notícia desta página) o músculo apresenta força significativa maior do que o que não recebeu o tratamento com expressão da distrofina entre 20 e 50%. Leia os resumos completos dos artigos relacionados a distrofia muscular

Today's Findings From the ASGT Annual Meeting: New Cardiovascular Gene Therapy And New Gene Therapy Research for Muscle and Connective Tissue Disease

http://www.mdausa.org/research/010602_progressgenetherapy.html

http://www.mdausa.org/research/010601_dmdminigene.html

Londres - nesta palestra,  Kay Davies da Universidade de Oxford aborda uma das formas possíveis de correção das distrofias musculares com aumento da expressão do gene da utrofina, uma protéina da membrana da célula que tem a capacidade de fazer as mesmas funções da distrofina. O aumento da utrofina em 3 a 4 vezes nos estudos experimentais não causou efeitos tóxicos às células. No momento a pesquisadora  já está testando moléculas que podem aumentar a expressão do gene da utrofina.

http://ex2.excerptamedica.com/01wcn/DetailSession.cfm?Abstract=11456

http://news.bmn.com/conferences/list/view?rp=2001-WCN-5-S4

Califórnia - mais de 40 trabalhos apresentados no Congresso abordam temas relacionados a diferentes formas de distrofia; alguns trabalhos foram produzidos no Brasil. A maioria deles tem interesse exclusivo para especialistas ou pesquisadores; de qualquer modo para ler os  resumos click aqui

http://www.faseb.org/genetics/ashg/meet-2001/2001meetmenu.htm

San Diego - este encontro, que se realizará de 10 a 15 de novembro de 2001, disponibiliza online os resumos a serem apresentados; alguns dos selecionados estão disponíveis  aqui; o mais significativo é sobre o transplante de medula óssea em um modelo experimental de distrofia em camungongos realizado na China.

http://sfn.scholarone.com/

USA - de 8 a 12 de dezembro de 2001 realizar-se-á em Washington está reunião de especialistas em pesquisa experimental. Alguns trabalhos envolvem pesquisas  com stem cells, distrofia muscular, etc. Com 20 dias de antecedência disponibilizamos 22 resumos do Congresso (em inglês).

Denver (USA) - no período de 14 a 20 de abril de 2002 realizar-se-á em Denver o Congresso da Academia  Americana de Neurologia, o mais conceituado encontro de neurologistas dos USA. Dos 1541 artigos selecionei os que se relacionam a distrofia muscular, sendo vários abordam a  terapia gênica das distrofias, alguns sobre o acompanhamento clínico (corticóides, função pulmonar), genética, etc. Para ler os resumos em inglês clique aqui

Strasbourg - Está sendo realizado de 25 a 28 de maio de 2002 o Congresso da Sociedade Européia de Genética Humana na cidade de Strasbourg . Os resumos do Congresso que se relacionam com as distrofias musculares você pode ler aqui . Há um resumo das pesquisas com transplantes de mioblastos na Rússia.

USA - Começa hoje em Boston, USA, e irá até 9 de junho, o Encontro da Sociedade Americana de Terapia Gênica. As mais recentes novidades em terapia gênica serão relatadas neste encontro. Dos 1374 resumos apresentados, 20 se referem a distrofia muscular. Leia os resumos aqui.  

USA - neste Congresso realizado em Baltimore de 15 a 19 de outubro de 2002 foram apresentados vários trabalhos relacionados a distrofia muscular. Os mais importantes relacionados com o tratamento da doença estão aqui. Em um deles é descrito o uso de bifosfonato no tratamento da osteoporose de portadores de distrofia muscular e outras doenças genéticas; nos outros três são descritas formas de terapia gênica em modelos experimentais de distrofia muscular.

USA - este encontro anual será realizado em São Francisco de 14 a 18/12/2002. Há poucos trabalhos relacionados a distrofia muscular; os mais importantes estão relacionados abaixo. No primeiro artigo é estudado o papel da TGF-beta no desenvolvimento da fibrose muscular; existem estudo sendo realizados tentando inibir esta proteína para retardar as alterações musculares da distrofia muscular. No segundo trabalho os pesquisadores observaram que as células tronco são atraidas em maior quantidade  para o músculo na lesão aguda do que nas lesões crônicas. O terceiro artigo fala do papel da inflamação no desenvolvimento das lesões musculares da distrofia muscular experimental; os pesquisadores reiteram a opinião que o tratamento desta inflamação no músculo pode retardar a evolução da doença. No último trabalho os pesquisadores discutem as dificuldades em fazer as células tronco se transformarem em células musculares em grande quantidade.

I) TGF-beta triggers the differentiation of myogenic cells toward myofibroblasts: Implication for muscle fibrosis

Y. Li, W. Foster, Y. Chan, B. M. Deasy, T. Payne, T. Horaguchi, N. Badlani, J. Cummins, J. Huard; Dept. of Orthopeadic Surgery, Children's Hospital of Pittsburgh, Pittsburgh, PA

Injured muscle can undergo regeneration; however, the healing process is often inefficient and hindered by progressive fibrosis, which is caused by stimulation of inflammatory factors. Transforming Growth Factor-beta1 (TGF-b1) is considered to be a central factor in fibrotic diseases of the liver, lung, heart, and kidney. Our previous research has found that muscle-derived stem cells are capable of differentiating into myofibroblasts following muscle laceration injury. We also have observed TGF-b1 over-expression in injured skeletal muscle. In this study, we determined that TGF-b1 stimulation can induce myoblasts (C2C12 cells) to express TGF-b1 in an autocrine manner, down-regulate the expression of myogenic proteins, and activate the production of fibrotic-related proteins in vitro. In addition, direct injection of human recombinant TGF-b1 into skeletal muscle in vivo was found to stimulate myogenic cells, including myofibers, to express TGF-b1 and subsequently induce scar tissue formation within the injected area. These results demonstrate that TGF-b1 is a key factor that can initiate the fibrotic cascades in skeletal muscle and induce a population of myogenic cells, which includes myofibers, to de-differentiate into myofibroblastic cells in injured muscle. These observations shed new light on the process of scar tissue formation, which commonly occurs in injured and diseased muscle.


II) Side population stem cells are recruited to sites of acute but not chronic muscle degeneration

K. A. Lapidos, E. M. McNally; Cardiology, University of Chicago, Chicago, IL

Muscular dystrophies are degenerative disorders in which skeletal muscle undergoes chronic cycles of degeneration and regeneration. Stem cells are a critical component of the regenerative process. Recently, a pluripotent population of stem cells has been isolated from bone marrow and muscle of adult mice, and these side population (SP) cells were shown to regenerate hematopoietic lineages and skeletal muscle. Based on their ability to differentiate into both cell types, we hypothesized that SP cells comprise an in vivo, stable stem cell compartment available for the regeneration of muscle tissue or the hematopoietic lineages. For example, in muscular dystrophy, a high demand for stem cells in the muscle promotes recruitment of SPs from the bone marrow. Over time, this may result in a quantifiable decrease in SP cells from bone marrow and a quantifiable, concomitant increase at the muscle. We tested this hypothesis by quantifying the percentage of SP cells obtained from bone marrow or muscle in a chronic model of muscular dystrophy, g-sarcoglycan null mice (gsg-/-). Whole bone marrow and lower limb skeletal muscle were isolated from wild type and gsg-/- mice at either 6 weeks or 6 months of age. Using the Hoechst dye exclusion technique, SPs from bone marrow (HSPs) and muscle (MSPs) were quantified. We found no statistically significant differences between the mutant and wild type mice. We also studied an acute model of muscle degeneration elicited by cardiotoxin injection of the crural muscles of normal mice. Three days post-injection, SP cells showed a five to nine-fold increase in MSPs in cardiotoxin-treated versus the control. In conclusion, we have shown that side population cells increase at the site of acute, but not chronic, muscle degeneration/regeneration.

III) Staging Interactive Pathogenic Mechanisms in mdx Mouse Dystrophinopathy by Temporal Gene Expression Signatures

J. D. Porter, A. P. Merriam, S. Khanna; Ophthalmology, Case Western Reserve University, Cleveland, OH

Although Duchenne muscular dystrophy (DMD) results from mutations of dystrophin, multiple interactive mechanisms likely contribute toward pathogenesis. We used Affymetrix oligonucleotide arrays to compare hindlimb muscle of the mdx mouse dystrophinopathy to wild type mice at postnatal (P) ages 7, 14, 18, 23, 28, 56, and 112 days; 3 independent replicates/age/strain. Extraocular muscle, which shows no signs of pathology in dystrophinopathy, also was studied at most ages. Patterned alterations in hindlimb gene expression between P7 and P14 (prenecrotic stage) included only 8 genes. Rapid accumulation of expression changes was observed thereafter, with a 20-fold increase in the number of up-regulated genes between P14 and P28, which coincided with appearance of histologic changes in muscle. Assessed at the expression level, disease peaked with 376 differentially regulated genes at P56, but a persistent response was evident at P112 (~130 genes). Correlation analyses provided insight into the interactive nature of inflammatory, fibrotic, and muscle regenerative events, with proinflammatory mechanisms particularly evident. By contrast, few changes were detected in extraocular muscle at any age. Immunocytochemistry and quantitative PCR for chemotaxic molecules confirmed their early role in recruitment of an inflammatory infiltrate to dystrophic muscle, along with a corresponding absence in extraocular muscle. These findings suggest that inflammation is a key contributor to, rather than a consequence of, the pathogenesis of dystrophin deficiency. The absence of changes in extraocular muscle suggest that this tissue does not adapt, but rather is constitutively protected in muscular dystrophy. Taken together, genome-wide expression profiling establishes an integrated profile of dystrophinopathy and the resultant mdx temporal expression signature for hindlimb suggests that blockade of inflammatory cell recruitment may be effective in ameliorating the disease, an attractive prospect in the absence of effective therapy for the primary mutation.

IV) Migration and Fusion of Muscle SP Cells into Dystrophic Myofibers

R. L. Sohn,1 E. Gussoni2 ; 1 Medicine/Cardiology, Brigham and Women's Hospital, Boston, MA, 2 Division of Genetics, Children's Hospital, Boston, MA

Cell therapy with muscle SP cells in animal models is being explored as a potential treatment for muscular dystrophy. However, though promising, stem cell therapy is plagued by its low efficiency: only about 1-5% of these stem cells fuse into pre-existing myotubes. In fact, how myoblasts, or even muscle SP cells, fuse to form multinucleate myotubes is a fascinating and highly regulated process. Recent work in Drosophila has elucidated some of the molecular components of this process. Our hope is to improve the efficiency of cell therapy with muscle SP cells by utilizing the tools and concepts of myoblast fusion found in Drosophila. As a first step, we have looked for cell surface markers that promote the migration of SP cells from the blood where they are injected to skeletal muscle where they then fuse into myotubes. Chemokines are known to play major roles in leukocyte migration. Therefore, we looked for the presence of chemokine receptors in skeletal muscle. By immunohistochemistry on human fetal skeletal muscle, we have found cells positive for the chemokine receptor CXCR-4 along the myofibers. These are not satellite cells as they are not positive for the transcription factor Pax-7. We have also found myotubes positive for CDO (CAM-regulated, down-regulated by oncogenes), a cell surface protein of the immunoglobulin/fibronectin type III repeat family that has been found to positively regulate myogenic differentiation in vitro (Kang, et al. 1998, JCB 143: 403-13). Work is currently underway to determine if these cell surface proteins are also present in muscle SP cells. If so, it is possible that by increasing the expression of these molecules, we will be able to also increase the efficiency of the SP cells to migrate to muscle and to fuse into the myotubes.

USA - Esta semana foram divulgados os mais de 1500 trabalhos que serão apresentados no Encontro Anual da Academia Americana de Neurologia que realizar-se-á de 29 de março a 5 de abril de 2003 no Hawaii. Destes artigos selecionamos 24 que se relacionam a distrofia muscular e estão relacionados aqui. Os principais são: 

- o artigo número 1 que mostra que os pacientes com Duchenne tratados com corticóides mantém a marcha por mais de 3 anos em relação aos que não usam e tem menor grau de escoliose. Os tratados com corticóides apresentaram maior incidência de fraturas inclusive com compressão vertebral, que pode ser explicada por maior atividade e maior incidência de osteoporose.

- o artigo 2 descreve o uso da ressonância nuclear magnética para avaliar o acometimento dos músculos que será um exame muito utilizado num futuro próximo.

- o artigo 3 demonstra que o ecocardiograma é o melhor exame para avaliar a função cardíaca em mulheres que são portadoras do gene da distrofina  e que a realização do ecocardiograma ao esforço demonstrou anormalidades em 83% das portadoras assintomáticas indicando que elas devam ser acompanhadas para o diagnóstico (as mulheres estudadas estavam com idade entre 37+ 10 anos)

- o artigo 4 descreve um novo vetor para levar o gene até o músculo com bons e prolongados resultados.

- o artigo 5 demonstra os bens resultados da terapia gênica em camundongos utilizando corrente elétrica mas há um declínio da expressão do gene ao longo do tempo que deverá ser estudada; pelo resultado exposto a terapia gênica se bem sucedida deveria ser repetida mais de uma vez.

- o artigo 6 estuda aspectos da lesão muscular da distrofia e o papel dos receptores e do fator de crescimento plaquetário nas lesões observadas.

- o artigo 14 e 15 descrevem alterações no sistema nervoso central de portadores de distrofia miotônica; no artigo 14 são analisadas alrterações na distrofia muscular miotônica congênita que apresentam alterações compatíveis com retardo mental e atraso do desenvolvimento. Não há adequada explicação para estas alterações.

USA - de 4 a 8 de junho de 2003 será realizado o Encontro Anual da Sociedade Americana em terapia gênica; neste encontro os maiores pesquisadores em terapia gênica irão apresentar seus trabalhos. Vinte e sete destes trabalhos se referem a pesquisas em terapia gênica na distrofia muscular. Os resumos destes trabalhos em inglês estão aqui. Todos eles demonstram avanços na terapia gênica experimental. Tentarei fazer um resumo das principais pesquisas nos próximos dias. O conteúdo dos primeiros resumos:

1) Sucesso com oligonucleotídeos para terapia gênica

2) Sucesso na correção da distrofia muscular experimental intra-útero

3) Uso da gentamicina na mutação de ponto: descrição de um teste que demonstra os prováveis pacientes que se beneficiarão do tratamento.

4) Sucesso na terapia gênica com vetor viral

5) Sucesso da terapia gênica no músculo diafragma utilizando vetor viral e gel.

6) Terapia gênica com sucesso em um modelo de distrofia tipo cinturas com gene liberado por via intraarterial

7) Uso de terapia gênica utilizando o Igf-1

8) Terapia gênica com um indutor de formação de vasos, o VEGF 165, induzindo  a regeneração dos músculos.

9) Descrição de um novo modelo de distrofia muscular em cães.

10) Sucesso com o uso do mini-gene da distrofina em cães com distrofia muscular

11) Explora o uso do vetor lentiviral em modelos de distrofia experimental; o vetor lentiviral é maior que o AAV e facilita a colocação do gene da distrofina

12) Discute as alterações da células musculares dos vasos dos músculos e a importância da introdução do gene da distrofina também nestas células.

13) Demonstra os resultados positivos do minigene da distrofina em modelos experimentais, com aumento da força muscular.

14) Estuda a relação entre o aumento da utrofina e a redução da calpaína em camundongos que receberam o gene da distrofina; a utrofina pode exercer o mesmo papel da distrofina.

15) Estudo com o uso da corrente elétrica para melhorar a passagem do gene para as células musculares.

16) Obteve maior sobrevida dos mioblastos com a utilização da interleucina 10 por terapia gênica em camundongos.

17)  Apresenta um estudo sobre as diferentes formas de célula tronco.

18) Demonstra que a superexpressão da matrilisina aumenta a migração dos mioblastos e reduz a necessidade de tantas injeções.

19) Estuda maneiras de modificar os mioblastos antes do transplante para evitar rejeição.

20) Analisa as possiblidades de terapia gênica na cardiomiopatia das distrofias.

21) Demonstra os bons resultados da terapia gênica intra-útero (antes do nascimento)

22) Estudo o uso de oligonucleotídeos  na  distrofia miotônica (estuodo in vitro,em laboratório) 

23) Observou que a injeção de células tronco por via intramuscular aumenta a expressão da distrofina e que o transplante de medula óssea não produziu efeito.

24) Estuda a manipulação prévia (em laboratório) das células tronco para terapia gênica

25) Utiliza na terapia gênica vetores com distrofina e proteínas imunossupressoras e obtem a expressão do gene por mais tempo.

26) Demonstra os efeitos do minigene da proteína agrin em camundongos com uma forma de distrofia muscular congênita

27) Estuda a terapia gênica nas células satélites dos músculos responsavéis pela regeneração muscular.

USA - O Encontro Anual da Sociedade de Neurociências será realizado de 8 a 12 de novembro de 2003, em New Orleans. Deste encontro selecionei três artigos relacionados com a distropia muscular: o mais importante é relacionado com a terapia gênica e foi realizado na China. Neste estudo os camundongos com distrofia muscular foram submetidos a transplante de células tronco da medula óssea. Os animais foram submetidos a radioterapia antes do tratamento. Após três meses do tratamento 10% dos animais apresentavam expressão da distrofina. Houve melhora da força muscular nos animais tratados e aumento da sobrevida em relação aos animais do grupo controle. No segundo artigo os autores demonstraram o aumento da expressão do receptor da transferrina em fibras de regeneração em pacientes com distrofia muscular. Este receptor esta relacionado a entrada do ferro e pode indicar um papel deste elemento no retardo da deterioração muscular. O terceiro artigo estuda o efeito dos corticóides na distrofia muscular experimental, tentando entender como esta droga atua.

1) C. Zhang, S. Chen, Y. Xie, W. Huang, S. Li, W. Zhang, Q. Li, X. Liu. THERAPEUTIC EFFECT OF DUCHENNE MUSCULAR DYSTROPHY MICE WITH BONE MARROW STEM CELLS TRANSPLANTATION. Program No. 413.9. 2003

Objective: To investigate the locomotive function, electronic physiology changes, and expression of dystrophin in Duchenne muscular dystrophy mice(mdx and dko) with bone marrow stem cells transplantation.
Methods: The bone marrow stem cells of C57BL/6
4-to-5 weeks age were isolated and cultured in vitro for 3 days, then the stem cells about 5.0X106 injected intravenously into the mdx mice and dko mic 7-to-8 weeks age , respectively. Before injecting, 10 mdx mice and 10 dko mice were preconditioned with 7 Gy gamma ray to decrease the immune reaction. The clinical features of the graft verse host disease (GVHD). in the transplanted mice were assessed and investigated after bone marrow stem cell transplatation..12 weeks after being transplanted, the locomotion function, muscle electrophysiologic features(EMG) and dystrophin expression of the mdx mice and dko mice were investigated..
Results: Three month after bone marrow stem cells transplanting, we find that (1) there were more than 10% of muscle cells expression dystrophin protein in each transplanted mice(mdx and dko); (2) the electromyography showed almost normal waves in the transplantation mdx mice and dko mice; (3)the locomotive function improved obviously in transplantation dko mice; (4)the life span of the transplantation dko mice were more than 200 days( the life span of control of dko mice were less than 140 days).
Conclusions: Allogenic bone marrow stem cells transplantation can improve the electromyographic items, dystrophin expression in muscles of mdx and dko mice. Further more, the stem cell transplantation can improve the locomotive function and prolong the life span of dko mice.

2) Y. Zhu, B. Chen, H. Gong, J. Pan, W. Zhang. EXPRESSION OF TRANSFERRIN RECEPTOR IN DUCHENNE MUSCULAR DYSTROPHY TISSUES. Program No. 80.2. 2003

Duchenne muscular dystrophy (DMD) is an X-linked, fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Skeletal muscle of DMD patients is characterized by an ongoing process of degeneration and regeneration. Transferrin receptor (TfR, CD71), an integral membrane glycoprotein, mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post-transcriptional level. To determine if TfR is involved in muscular regeneration in DMD patients, we examined the expression of TfR in muscle samples of 42 patients with DMD, and 10 from normal volunteers. Immunohistochemical staining of TfR was positive with variable intensity in normal control and DMD patients. In normal control, TfR was faintly expressed on the surface of muscle fibers. In DMD patients, strong TfR reaction was detected. In some muscular fibers from DMD patients, TfR was found strongly expressed inside the fibers. Further detection with Enzyme histochemistry and acridine orange (AO) fluorescence techniques showed that these fibers with strong TfR expression were regenerative IIc type fibers. The increase of TfR expression in regenerative fibers suggests that the high uptake of iron may promote the capacity of muscle regeneration and thus may delay the deterioration of DMD patients.

3) C.G. Carlson. STEADY STATE CALCIUM AND RESTING CALCIUM INFLUX IN NONDYSTROPHIC AND MDX MYOTUBES EXPOSED TO PREDNISONE OR PREDNISOLONE. Program No. 413.10. 2003

Certain glucorticoids, such as prednisone, prednisolone and deflazacort, increase muscle strength and slow the progress of Duchenne muscular dystrophy (Fenichel et al., Neurology, 41, 1874, 1991) by one or more mechanisms that have not yet been fully characterized. Evidence from 45calcium uptake studies in the C2C12 muscle cell line suggests that one mechanism may involve decreases in calcium influx following one or more days of exposure to these agents beginning at the period of myoblast fusion (Passaquin et al., Brit. J. of Pharmacol., 1998). Recent evidence from this laboratory obtained in FURA-loaded cultured lens epithelial cells indicates that some steroids, however, may induce very rapid (seconds to minutes) effects on resting calcium influx measured by the manganese quench technique (Samadi et al., Pfluegers Arch. Europ. J. Physiol.,444, 700-709, 2002). To determine whether glucorticoids exert a unique action on resting calcium influx in dystrophic muscle, studies have begun to characterize any acute, nongenomic effects of prednisone or prednisolone on steady state calcium levels and resting calcium influx in cultured nondystrophic and mdx myotubes. Initial results indicate that neither prednisone (10 microM, 100 microM) nor prednisolone (100 microM) has immediate effects on steady state calcium levels in nondystrophic myotubes, but that prednisolone may induce a slight decrease in manganese quench rate in mdx myotubes at relatively early stages in culture (culture day 12-14). These results further demonstrate the utility of the manganese quench technique in assessing whether short or long term exposure to glucorticoids induces a beneficial reduction in resting calcium influx in dystrophic skeletal muscle.

USA - em dezembro, de 13 a 17, será realizado na Califórnia o Encontro Anual da Sociedade Americana de Biologia Celular. Neste encontro serão apresentadas inúmeras pesquisas, das quais eu selecionei as duas que se relacionam com terapia na distrofia muscular. No primeiro artigo os pesquisadores utilizaram a negamicina, um antibiótico da mesma classe da gentamicina para tratamento da mutação de ponto na distrofia muscular de camundongos. Os resultados obtidos com o estudo das células dos camundongos demonstrou que a negamicina tem a mesma capacidade que a gentamicina em corrigir a mutação de ponto e é menos tóxica. No segundo artigo os pesquisadores criaram um camundongo com distrofia muscular e expressava a calcineurina. A calcineurina promoveu aumento da utrofina A, que pode substituir as funções da distrofina. Drogas que aumentem a expressão da calcineurina podem se constituir em uma alternativa para a terapia medicamentosa da distrofia muscular.

Abstracts from 43rd Annual Meeting The American Society for Cell Biology  - California,  13-17 december, 2003 

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice
M. SHIOZUKA,1 M. ARAKAWA,1 Y. NAKAYAMA,2 T. HARA,2 M. HAMADA,3 D. IKEDA,3 Y. TAKAHASHI,3 R. SAWA,3 Y. NONOMURA,3 K. SHEYKHOLESLAMI,4 K. KONDO,4 K. KAGA,4 S. TAKEDA,5 R. MATSUDA1 ; 1 Dept. of Life Sciences, The University of Tokyo, Tokyo, Japan, 2 Dept. of Tumor Biochemistry, Tokyo Metropolitan Institute of Medical Sciences, Tokyo, Japan, 3 The Institute of Microbial Chemistry, Tokyo, Japan, 4 Dept. of Otolaryngology, The University of Tokyo, Tokyo, Japan, 5 Dept. of Molecular Therapy, National Institute of Neuroscience, Tokyo, Japan

 The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interests in these drugs as potential therapeutic agents in genetic diseases. However, strong toxicity of aminoglycoside antibiotics may cause severe side effects during long-term treatment. In this study, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of mdx mouse; an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrated that negamycin bound to a partial sequence decording the eukaryotic rRNA A-site. We conclude that negamycin is a promising new candidate for chemotherapy for DMD and other genetic diseases caused by nonsense mutations.

 Calcineurin-Induced Upregulation of Utrophin Attenuates the Dystrophic Pathology in mdx Mouse Muscle
J. V. Chakkalakal,1 M. Harrison,2 E. R. Chin,3 R. N. Michel,2 B. J. Jasmin1 ; 1 Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada, 2 Neuromuscular Lab, Laurentian University, Sudbury, ON, Canada, 3 Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT

 We recently showed that mice expressing a constitutively active form of calcineurin (CnA*) have elevated levels of utrophin A in their muscles (Proc. Natl. Acad. Sci. USA, 100: 7791-7796, 2003). In the present study, we crossed these transgenic animals with mdx mice to determine whether mdx/CnA* mice would be less affected by the dystrophic process. Since expression of CnA* has been shown previously to stimulate the slow/oxidative myofiber program, we examined the expression of myosin heavy chain isoforms in mdx/CnA* and mdx mice. By immunofluorecence and RT-PCR assays, we observed in mdx/CnA* a shift in myosin heavy chain profile towards a slower, more oxidative phenotype compared to mdx muscles. In addition, we determined that expression of utrophin A and its transcript were increased by ~ 2 fold in mdx/CnA* mouse muscles. Immunodetection of IgM inside myofibers, used in this case as an index of sarcolemmal disruption, showed that the number of IgM-positive fibers was significantly reduced in muscles from mdx/CnA* mice. Consistent with these findings, we also observed that muscles from mdx/CnA* mice showed less variability in fiber size and contained fewer central nuclei. Together, these results demonstrate that enhanced calcineurin activity can have important beneficial effects on the dystrophic phenotype by stimulating the expression of utrophin A. Furthermore, these findings provide specific targets for which pharmacological strategies may be designed to enhance utrophin levels in muscles from patients with Duchenne muscular dystrophy. Funded by the MDA and CIHR.

USA - No encontro dos cardiologistas foram apresentados poucos artigos sobre o coração na distrofia muscular. Selecionei os dois mais importantes: no primeiro os pesquisadores franceses utilizaram o peridropril em portadores de Duchenne com função cardíaca adequada. O peridropril é uma droga inibidora da enzima de conversão da angiotensina. Os resultados demonstraram que o medicamento  foi bem tolerada pelos pacientes e retardaram o aparecimento da disfunção cardíaca nestes pacientes. No segundo artigo os autores ingleses apresentaram os resultados de um novo método de diagnóstico da doença cardíaca em portadores de distrofia muscular de Duchenne. Este método se demonstrou mais eficiente e permitiu o diagnóstico mais precoce que o ecocardiograma.

Perindopril Prevents the Onset and Progression of Left Ventricular Dysfunction in Children with Duchenne Muscular Dystrophy

Henri-Marc Becana, Pitie-Salpetriere Hospital, Paris, France; Christophe Meune, Cochin Hospital, Paris, France; Guy Lerebours, Pitie-Salpetriere Hospital, Paris, France; Jean-Yves Devaux, Denis Duboc, Cochin Hospital, Paris, France; The French Working Group on Heart Involvement in Myopathies

Background: Duchenne Muscular disease (DMD) is an inherited X-linked disease due to the absence of dystrophin and clinically characterized by progressive muscle weakness and constant myocardial involvement responsible for sudden death or end-stage heart failure in 40% of patients between the age of 15 and 30. The aim of this study was to evaluate the preventive effect of early ACEI treatment on left ventricular (LV) dysfunction in young patients with DMD and normal LVEF at inclusion. Methods: In phase I, children with genetically proven DMD and radionuclide ejection fraction (EF) superior than 55% were enrolled in a multicentric, controlled, randomized, double blind trial of perindopril 2-4 mg/day versus placebo for 3 years. In phase II, all patients then received open-label perindopril for 24 more months. Radionuclide LVEF was performed at 0, 36 and 60 months. Student's t-tests and chi-square analysis were used for comparisons. Results: 60 children asymptomatic for heart disease were included in the phase I study (10.6±1.2 years, EF 65.0±5.4%)(31 in the perindropil group and 29 in the placebo group) and 46 in the phase II study (n=23 in both groups). No adverse effect related to treatment was documented. At the end of phase I, LVEF remained stable in both groups (from 64.8±5.3% to 59.6±8.5% in the perindopril group and 65.5±5.4% to 64.5±9.9% in the placebo group, p=0.114). However, at 60 months, 6 patients in the control group (26%) had a LVEF of less than 45, versus one in the perindopril group (4%)(p=0.0319). Conclusions: Early treatment with ACEI perindopril is well tolerated in these young patients and delay onset of LVEF deterioration. This preventive efficacy of ACEI has to be evaluated in other groups of patients genetically exposed to develop LV dysfunction.

Impaired Strain Rate Measurements in Patients With Duchenne Muscular Dystrophy

Nickolaos Giatrakos, Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Maria Kinali, Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital, ICSM, London, UK, London, United Kingdom; George Koutroulis, Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom; Francesco Muntoni, Dubowitz Neuromuscular Centre, Department of Paediatrics, Hammersmith Hospital, ICSM, London, UK, London, United Kingdom; Petros Nihoyannopoulos; Cardiology dept, Hammersmith Hospital, NHLI, ICSM, London, United Kingdom

Background: Patients with Duchenne muscular dystrophy (DMD) develop dilated cardiomyopathy at the later stages of the disease. Strain rate (SR) has been used to study myocardial function in ischeamia and cardiomyopathies. The aim of this study was to investigate the usefulness of SR for the early detection of cardiac involvement in young, asymptomatic patients with DMD.
Methods: We studied 53 patients with genetically confirmed diagnosis of DMD (mean age 8.7±2.8 years) without clinical symptoms from the cardiovascular system and normal conventional echocardiographic studies, and compared with 22 normal controls matched for age (mean age 8.5±2.5 years). We used the HDI 5000 (Philips Medical Systems) to acquire from the parasternal long axis the colour M-mode tissue Doppler (TDI) of the posterior wall of the LV. Images were digitally stored for offline analysis with dedicated software HDI-lab (Philips Medical Systems). We calculated the SR using the formula SR=Ua-Ub/d where U the velocities of the endocardium a and epicardium b, and d the distance of a and b at each time point.
Results: There was no significant difference for the parameters from conventional echocardiographic studies between the two groups. The velocities derived from the TDI, mean velocity at systole (26,99±7,12mm/sec vs. 33,4±7,3mm/sec, p<0,000), at early diastole (-45,79±13,93mm/sec vs.-60,46±7,58mm/sec, p<0,000) and late diastole (-10,93±3,41mm/sec vs.-13,32±6,4mm/sec, p<0,02) were significantly different in patients with DMD when compared with controls. SR was found to be significantly lower at systole (1,78±0,75s-1 vs. 2,82±0,5s-1, p<0,000) and early diastole (-5,17±1,98s-1 vs.-9,02±1,25s-1, p<0,000) but not at late diastole (-1,52±0,84s-1vs.-1.6±0,46s-1, p<0,568) in patients with DMD.
Conclusions: Estimation of SR of the posterior wall of the LV showed systolic and diastolic dysfunction at early stages in asymptomatic patients with DMD and when the conventional echocardiography is still normal. Estimation of SR could be a sensitive method to investigate the pathophysiology of the disease and identify early impairment of the cardiac function.

USA - este congresso será realizado em São Francisco a partir de 24 de abril de 2004 e é considerado um dos maiores encontros de neurologistas do mundo. No entanto o número de artigos falando sobre distrofia é muito baixo, muito inferior ao de outras doenças como a esclerose múltipla e a doença de Parkinson. Além disso poucos destes resumos abordam aspectos da terapia da doença. A maioria descreve algumas formas de distrofia, características genéticas, etc. Isto demonstra um desinteresse por este grupo de doentes, em especial com relação aos aspectos importantes da doença como tratamento medicamentoso, fisioterapia, cirurgia, etc. Os resumos que falam sobre distrofia (todas as formas) podem ser lidos em inglês aqui.

USA - este é o congresso mundial  mais importante na área de doenças pulmonares. Neste ano o congresso ocorrerá em Orlando, em maio e lá serão apresentados 8 trabalhos relacionados à distrofia. Um dos artigos (o número 1) fala dos resultados positivos com terapia genética para o músculo diafragma de camundongos com distrofia. O artigo sete fala de uma nova forma de ventilação mecânica em pacientes com distrofia muscular.  O número 8 descreve os efeitos da associação entre corticóides e creatina em ratos normais. Os resumos em inglês podem ser lidos aqui. Encontrei também no site do congresso 22 artigos apresentados nos últimos anos (2001-2003) nestes encontros. Há importantes artigos falando na melhora da função pulmonar com o uso dos corticóides, nos benefícios da ventilação não invasiva (que já é usada no exterior há 10 anos), alterações do sono na distrofia, equipamentos de estimulação da tosse, experimentos com terapia gênica em animais entre outros. Os resumos em inglês podem ser lidos aqui.

USA - no Encontro que reunirá os principais pesquisadores na área de terapia gênica serão apresentados 20 artigos que falam sobre esta forma de tratamento na distrofia muscular; a maioria é experimental, em camundongos ou cães, e não trazem nenhum avanço significativo. O único trabalho em humanos (artigo 7) fala do uso de mioblastos na distrofia muscular, trabalho realizado no Canadá, e que já foi divulgado aqui mais de uma vez (estão ainda com poucos crianças com distrofia muscular de Duchenne e o tratamento está sendo realizado em apenas um músculo). Os resumos em inglês dos artigos pode ser lido aqui.

 

São Paulo - em 2003 eu decidi realizar pesquisas com camundongos com distrofia muscular na Faculdade de Medicina da Fundação do ABC, além de apoiar as pesquisas que estão sendo realizadas em Ribeirão Preto, no Centro de Pesquisas em Distrofia Muscular da AADM. O meu primeiro estudo experimental foi aprovado para apresentação no 44o Encontro Anual da Sociedade Americana em Biologia Celular. Neste trabalho eu estudei a droga tamoxifeno, que é utilizada para tratamento de mulheres com câncer de mama. Eu decidi estudar esta droga porque ela atua sobre as TGF beta, um grupo de proteínas que está alterado na distrofia muscular de Duchenne. Para este estudo eu utilizei a droga nos camundongos mdx, que tem uma mutação do gene da distrofina. Os animais que receberam a droga apresentaram aumento da força muscular após a quinta semana que se manteve até o final do experimento na 12a semana. Biópsias musculares foram realizadas e estão ainda sob análise (no Centro de Estudos do Genoma Humano). As primeiras análises demonstram que os animais tratados com tamoxifeno tem menor grau de degeneração muscular do que os não tratados. Há ainda outros estudos bioquímicos que estão sendo realizados nos músculos dos animais cujos resultados serão conhecidos em breve. Neste mês estamos iniciando o estudo de uma segunda droga no mesmo modelo experimental. O resumo do meu trabalho que será apresentado em Washington, USA, em dezembro de 2004 é o seguinte:

Tamoxifen increases muscular strength of the mdx dystrophic mice NEW

(Abstract from 44th Annual Meeting - American Society for Cell Biology (December, 4-8, 2004, Washington)

A. Cavalsan,1 R. F. Vasquez,1 R. C. Lin,1 S. B. Zyngier,1 F. J. Velloso,2 B. H. Santos,2 L. L. Fogaca,2 M. Vainzof,2 D. Feder1 ; 1 Pharmacology, ABC Faculty of Medicine, Santo Andre, Brazil, 2 Human Genome Research Center - IB USP, Sao Paulo, Brazil

The mdx mice is a well-known model of Xp21 dystrophin-deficient muscular dystrophy. Although a good genetic and biochemical model, the mdx shows no muscle weakness, but under physical exercise, a loss of muscular strength can be detected. Here we have tested the possible therapeutic beneficial effect of Tamoxifen in the degenerative process of the dystrophic muscle, analyzing muscle strength of tamoxifen treated mdx mice, under intensive physical exercise. A total of 22 mices aged 4 weeks were divided into 3 groups: control (n= 8) treated with 0.5 mL saline ip, Tamoxifen 5 mg/Kg body weight ip (n= 8) daily and Tamoxifen 10 mg/Kg ip (n=6) daily. The exercise protocol was done in a wheel revolving at 18 cm/s, for 10 minutes, twice a day, 5 days/week, up to 12 weeks. Whole-body strength was measured weekly using a force transducer coupled to a computer. Mice tails were attached via a non-flexibile nylon cord to the transducer, the animals were electricly stimulated to run, and the force to pull the cord was registered continuously. Results were analyzed by the Kruskal-Wallis test. The mice treated either with 5 mg/Kg (6.26+ 1.44 dynes/g body weight) or 10 mg/Kg (6.46+ 2.52) showed a significant increase of muscular strength (p<0.05) compared to the control group (3.66+ 0.77) starting in the 5th week, and maintaining significance up to the end of the experiment. Histological and histochemical analysis of the complex of the gastrocnemius muscle are under analysis, but preliminary results suggest a less degree of degeneration in the Tamoxifen treated groups.
Tamoxifen is an anti tumoral drug and act on TGF-beta. Its possible therapeutic effect in the degenerative process of the dystrophic muscle could ameliorates the clinical course of dystrophic patients.
FAPESP-CEPID, CNPq, PRONEX, NEPAS-FMABC.

USA - neste Congresso que se realizará em dezembro em Washington, além da minha pesquisa sobre tamoxifeno outros estudos serão apresentados:

1) um estudo fala dos resultados positivos com o uso do gene da distrofina retiniana humana utilizado em camundongos sem distrofina e utrofina que melhoram os sintomas musculares; de uma forma severa de distrofia eles passaram a apresentar poucos sintomas musculares;

2) o segundo estudo fala da relação entre calcificação ectópica de musculos com distrofia muscular pode ser um sinal de regeneração e auxiliar na avaliação do tratamento com medicamentos;

3) o terceiro estudo trata dos níveis de protoglicans em distrofia muscular de Duchenne em relação às outras formas da doença; há redução dos níveis de protoglicans em Duchenne pode estar relacionado aos altos níveis de TGF beta e TNF

4) O quarto estudo demonstra que as células tronco originadas de músculos de camundongos fêmeas com distrofia muscular são mais eficientes para a terapia gênica do que as células dos machos.

Os resumos em inglês destes estudos estão a seguir:

Human Retinal Dystrophin Transgene Converts Lethal Muscular Dystrophy into Viable Mild Myopathy in Dystrophin-Utrophin Null Mice
R. Gaedigk,1,2 D. J. Law,3 K. M. Fitzgerald-Gustafson,4,2 S. G. McNulty,1 N. N. Nsumu,1 A. C. Modrcin,5 R. J. Rinaldi,5 D. Pinson,6 S. C. Fowler,7 M. Bilgen,4,8 J. Burns,9 S. D. Hauschka,10 R. A. White1,2 ; 1 Medical Research, Children's Mercy Hospitals & Clinics, Kansas City, MO, 2 Pediatrics, UMKC School of Medicine, Kansas City, MO, 3 School of Biological Sciences, Univ. of Missouri-Kansas City, Kansas City, MO, 4 Hoglund Brain Imaging Center, Univ. of Kansas Medical Center, Kansas City, KS, 5 Rehabilitation Medicine, Children's Mercy Hospitals & Clinics, Kansas City, MO, 6 Pathology & Laboratory Medicine, Univ. of Kansas Medical Center, Kansas City, KS, 7 Pharmacology & Toxicology, Univ. of Kansas, Lawrence, KS, 8 Molecular & Integrative Physiology, Univ. of Kansas Medical Center, Kansas City, KS, 9 Veterinary Imaging Services, Topeka, KS, 10 Biochemistry, Univ. of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by severe dystrophin gene mutations that often result in death by the third decade. The mdx mouse is the most commonly used DMD model. Although they lack dystrophin and have underlying muscle disease, mdx mice appear physically normal. This may be partially due to compensatory expression of the dystrophin-related protein, utrophin. In contrast, double mutant mice (DM), deficient for both dystrophin and utrophin (mdx/+, utrn -/-), die by 3 months of age and suffer from severe muscle weakness, pronounced growth retardation, kyphosis, weight loss, slack posture, and immobility (Deconinck et al. (1997) Cell 90: 717-727; Grady et al. (1997) Cell 90: 729-738). These features make them an excellent physiological model for DMD research. The capacity of a naturally occurring isoform, human retinal dystrophin (Dp260), to compensate for the missing muscle isoform Dp427 was tested in DM mice. The expression of this transgene prevents premature death and reduces the severe muscular dystrophy phenotype to a mild myopathy. Electromyography (EMG), histology, radiography, magnetic resonance imaging, and behavior studies show that DM transgenic mice grow normally, have normal spinal curvature and locomotion, and have reduced muscle pathology. EMG and histologic data from transgenic DM mice are typical of mild myopathy, while the DM mice exhibit severe abnormalities commonly seen in human dystrophinopathies. The expression of human Dp260 in DM mice converts a severe and lethal muscular dystrophy into a non-lethal mild myopathy. Muscle-specific expression of Dp260 could have several advantages over other treatments of DMD

Visualization of Ectopic Calcification in mdx Mouse Skeletal Muscle
N. Kikkawa, T. Ohno, M. Shiozuka, R. Matsuda; The Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan

It has been demonstrated that osteogenic differentiation of skeletal muscle cells are induced by osteogenic factors, such as bone morphogenetic proteins, both in vitro and in vivo. Spontaneous ectopic bone formation in vivo has also been reported in, for instance, fibrodysplasia ossificans progressiva, which is a rare bone disorder. Another example of self-generating ectopic calcification has been found in skeletal muscle of mdx mouse, a model of Duchenne muscular dystrophy (DMD).
We observed the ectopic calcification in mdx mouse thigh muscle by using x-ray micro CT-scanner SkyScan-1074, which gives resolution of 22um. The x-ray images were reconstructed into three dimentional visions. Calcifications were found as spicular structures running parallel to the muscle fiber. Paraffin sections of the regions found by X-ray microtomography were Von Kossa stained to confirm calcium deposits. We also detected ectopic calcifications of living mice with mouse-whole-body x-ray CT-scanner of Aloka. No ectopic calcification was observed in skeletal muscle of B10 mouse, which possesses normal dystrophin gene and was used as a negative control.
Calcified regions of mdx mouse thigh did not overlap with the Evans blue-positive areas but corresponded to some of the regenerating areas. Thus, ectopic calcification can be a diagnostic marker of muscle regeneration in mdx mice, and be available to determine the effects of drug or gene therapies.

Muscle Proteoglycan Levels in Duchenne Muscular Dystrophy Differ from Other Muscular Dystrophies
S. Zanotti, E. Canioni, F. Cornelio, L. Morandi, M. Mora; Neuromuscular Diseases and Neuroimmunology, National Neurological Institute C. Besta, Milano, Italy

Biglycan and decorin are small leucine-rich extracellular proteoglycans that interact with several matrix proteins particularly collagens, and also with cytokines whose activity they may modulate. To better understand the role of these proteoglycans in muscle fibrosis in muscular dystrophies, we investigated the expression of their transcripts and proteins in several forms of muscular dystrophy compared to age-matched controls. mRNAs of both proteoglycans were significantly downregulated in Duchenne muscular dystrophy and significantly upregulated in Becker muscular dystrophy, sarcoglycanopathies and dysferlinopathy. By immunohistochemistry, decorin and biglycan were mainly localized in connective tissue and apparently increased with age. Their presence increased in relation to increased fibrosis in all types of dystrophic muscle.
The increase in biglycan and decorin in most muscle diseases indicates a role of these proteoglycans in the extracellular matrix organization. The significant decrease of decorin and biglycan mRNA in DMD distinguishes this disease from the other studied and may be related to the high levels of TGF-beta1 and TNF-alpha found in this disease.

Sex Differences in Muscle Stem Cell Transplantation Efficiency
B. Deasy,1 A. Lu,1 K. Urish,1 J. Tebbets,1 B. Gharaibeh,1 R. Rubin,2 J. Huard1 ; 1 University of Pittsburgh, Pittsburgh, PA, 2 Allegheny General Hospital, Pittsburgh, PA

We previously demonstrated that muscle-derived stem cells (MDSCs) efficiently regenerate skeletal muscle tissue after transplantation into dystrophic mdx mice, which model muscular dystrophy. Here we show that transplantation of female MDSC (F-MDSC) rather than male MDSCs (M-MDSC) significantly improves skeletal muscle regeneration.
We transplanted F-MDSCs into the gastrocnemius muscles of female mdx mice and the same number of M-MDSCs into the gastrocnemius muscles of male mdx mice. We used a previously described protocol to measure the cells’ regeneration index (RI)_the number of dystrophin-positive fibers in the host muscle per 105 donor cells_two weeks after transplantation. The F-MDSCs elicited significantly superior muscle regeneration equivalent to a 6-fold increase in efficiency (F-MDSC RI=686±120 versus M-MDSCs RI=105±25, P< 0.01). Sex-crossed transplantation also demonstrated the same trend for the donor cells. We hypothesize that this difference may be related to reduced ability of the M-MDSC to self-renew or to tolerate hypoxic stress. F-MDSCs maintained CD34 expression (>80% of total cells) for up to 200 population doublings, whereas M-MDSCs exhibited a rapid drop in CD34 expression (from >80% to <30% of total cells) after only 45 population doublings. To support the notion that the F-MDSC are more tolerant to stress, we also found that proliferation of F-MDSC was less affected by low O2 conditions as compared with M-MDSC.
Our discovery of sex-related differences in muscle stem cell biology reveals clear limitations to using male stem and progenitor cells for cell-tracking purposes. This finding also warrants careful consideration by researchers working to identify optimal populations of stem and progenitor cells for use in cell therapy and tissue engineering, clinicians who perform bone marrow stem cell transplantation, and basic scientists investigating cell and developmental biology.

Canadá - neste Congresso realizado em Toronto de 26 a 30 de outubro de 2004 foram apresentados apenas dois trabalhos relatando experimentos em terapia das distrofias musculares.

No primeiro trabalho realizado nos Estados Unidos os pesquisadores apresentam uma nova forma de terapia gênica em camundongos; eles associaram ao vetor viral uma proteína (fator de crescimento do endotélio vascular); com esta terapia os camundongos passaram a expressar distrofina em quantidade suficiente para reduzir os sintomas da doença. O vetor viral foi injetado em uma veia, que é um passo importante para poder tratar todos os músculos; a forma de administração do gene da distrofia muscular é um dos problemas ainda não resolvido na terapia gênica da doença.

No segundo trabalho os pesquisadores holandeses estudam os oligonucleotídeos para "consertar" o gene da distrofina. Estudando células musculares humanas em laboratório ou camundongos com distrofia muscular eles tem conseguido demonstrar que esta forma de tratamento é viável. Estes oligonucleotideos tem que ser construidos para cada forma de mutação do gene e eles tem desenvolvido vários destes oligonucleotideos para utilização em seres humanos.

Os resumos destes dois trabalhos em inglês:

1) Body-wide delivery of a microdystrophin gene via intravascular administration of rAAV6 vectors for treatment of muscular dystrophy. P. Gregorevic, M. Blankinship, J. Allen, L. Meuse, S. Abmayr, J. Han, J. Chamberlain. Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, Dept. of Neurology, University of Washington, Seattle, WA.

   Severe neuromuscular disorders, such as Duchenne muscular dystrophy (DMD), lead to reduced quality of life and premature mortality. Historically, genetic interventions for these diseases have been limited by an inability to achieve widespread gene transfer to the affected tissues. Here we demonstrate a method that, for the first time, enables transduction of the vast majority of both the cardiac and skeletal musculature of adult mammals via a single intravenous administration of pseudotype 6 recombinant adeno-associated virus (rAAV6) vectors. As a means to enhance gene transfer, we have determined that IV coadministration of vascular endothelium growth factor (VEGF) with rAAV6 vectors can significantly increase vector accumulation and transgene expression in striated muscles. Having established in reporter gene studies that this technique enables tolerable, high-level transgene expression, we sought to evaluate the potential of this technique for delivering a therapeutic transgene to the musculature of the mdx dystrophic mouse, a model of DMD. Using this technique, we have observed that treated mdx mice exhibit expression of microdystrophin throughout the striated musculature in a manner that is sufficient to reduce pathological features of the dystrophic phenotype. Compared with untreated mice, the muscles of treated animals exhibit reduced susceptibility to contraction-induced injury, and reduced serum creatine kinase levels, reflecting a global reduction in muscle degeneration. These data are the first to demonstrate body-wide amelioration of symptoms associated with dystrophin deficiency in an adult animal following a genetic intervention. In subsequent studies we have established that these techniques can be adapted to express alternate transgenes for the treatment of other muscle diseases. We are currently undertaking studies to determine whether the existing techniques are effective in animals more similar in size and immunology to humans, and present relevant data summarizing our progress

2) Therapeutic antisense-induced exon skipping for Duchenne muscular dystrophy. A. Aartsma-Rus1, A. Janson1, G. Platenburg2, M. Bremmer-Bout1, J.T. Den Dunnen1, J.C.T. Van Deutekom1, G-J.B. Van Ommen1. 1) LUMC, Leiden, the Netherlands; 2) Prosensa, Leiden, the Netherlands.

   The severe Duchenne muscular dystrophy (DMD) is mostly caused by frame disrupting mutations in the dystrophin gene, which result in non-functional proteins. Mutations that keep the reading frame intact give rise to internally deleted, semi-functional dystrophins and result in the milder Becker muscular dystrophy (BMD). Antisense oligonucleotides (AONs) have the potential to modulate the pre-mRNA splicing such that a specific exon is skipped. As a result, the reading frame can be restored, which allows the synthesis of a BMD-like dystrophin.
   We have recently demonstrated the skipping of 20 different human DMD exons using 2'-O-methyl RNA AONs with a full-length phosphorothioate backbone (2OMePS), and confirmed the therapeutic applicability of this strategy in cultures from 10 different DMD patients. We now compare the efficacy and efficiency of 2OMePS AONs to morpholino, locked nucleic acid (LNA) and peptide nucleic acid (PNA) AONs. While LNA was most efficient in inducing exon skipping, we find that it acts in a less sequence-specific manner, probably due to its extremely high RNA affinity. Awaiting further improvements in oligochemistry, we therefore consider 2OMePS AONs currently the most favourable compounds.
   To facilitate clinical application, multiple in vivo studies in animal models are ongoing to develop safe and efficient AON-delivery methods. However, as exon skipping is a sequence-specific therapy, it is desirable to directly target the human DMD gene. We have therefore set up sequence-specific human exon skipping in vivo in transgenic mice carrying the an intact copy of the full-length 2.5 Mb human gene (hDMD). We injected 2OMePS AONs targeting human exons 44, 46 and 49 into the m. gastrocnemicus of hDMD mice, and showed skipping of the human (but not the murine) exons, which persisted for at least 28 days. Based on pre-clinical data obtained by our group and others, we are currently setting up a clinical trial aiming at local dystrophin restoration following intramuscular injections of exon 46 and 51 specific AONs.

Espanha - realizou-se em junho de 2004 esta reunião de neurologistas europeus na qual foram apresentadas 4 pesquisas em distrofia muscular. Na primeira os autores descrevem a melhora da força e da fadiga muscular em camundongos com distrofia muscular utilizando duas drogas: a IRFI 042, antioxidante, e a PDTC, um inibidor da NF-kB, uma proteína que causa degeneração muscular. Na segunda os pesquisadores encontraram um pequeno resultado com o transplante de células da medula óssea em camundongos com distrofia muscular (baixa expressão da distrofina nos músculos). Na terceira pesquisa os autores encontraram déficits de linguagem em portadores de distrofia muscular de Duchenne (déficit em compressão de sintaxe e gramática, sem dificuldade de leitura). Na quarta pesquisa os autores discutem as causas de insuficiência respiratória na distrofia miotônica (apnéia do sono, fraqueza muscular). Os trabalhos como foram apresentados no Congresso podem ser lidos aqui:

1) Effect of treatment with PDTC and IRFI 042 on strength and fatigue in MDX mice

S. Messina, P. Seminara, M. Aguennouz, M.C. Monici, H. Marini, F. Squadrito, G. Vita (Messina, I)

Previous studies provided evidences that generation of reactive oxygen species and activation of transcription factor NF-kB may play important roles in the pathogenesis of Duchenne muscular dystrophy. We tested whether IRFI 042, a vitamin E-like antioxidant, and PDTC, a NF-kB inhibitor, could have an effect on muscle weakness in mdx mice. We treated 48 5/6-week old mdx and wild type mice with intraperitoneal injections of PDTC (50 mg/kg), IRFI 042 (20 mg/kg), or vehicle, three times a week for five weeks. Data regarding weight, survival and forelimb strength and fatigue were collected. Motor performance measurements were carried out using a grip meter attached to a force transducer which measures peak force generated. Mdx mice treated with IRFI 042 or PDTC showed at the end of treatment a significantly higher forelimb strength than vehicle controls (IRFI 042: +53.6%, p<0.001; PDTC: +53.1%, p<0.05) as well as higher strength normalised to weight (IRFI 042: +57.8%, p<0.001; PDTC: +54%, p<0.05). Furthermore PDTC-treated mdx mice had significantly less fatigue than vehicle animals (-120%, p<0.004).

Our results suggest that PDTC and IRFI 042 might have a beneficial effect on weakness and fatigue in mdx mice. Further studies are needed to investigate the morphological and biochemical substrates of such encouraging preliminary results.

2) Bone marrow transplantation poorly restores dystrophin expression in MDX mice: comparison of GFP and dystrophin expression

F. Chretien, P.A. Dreyfus, P. Caramelle, B. Chazaud, R.K. Gherardi (Créteil, F)

We have previously developed a murine model of bone marrow (BM) transplantation from B6-TgGFP transgenic mice to normal irradiated B6 mice, the cytoplasmic green fluorescent protein (GFP) being used as an unambiguous marker of donor-derived cells in host muscle. Using this model we were able to demonstrate that stem cell marker-expressing cells found in connective tissue and myogenic precursor cells (satellite cells) may be derived from bone-marrow in adulthood. To investigate the therapeutic potential of BM transplantation in muscle diseases such as dystrophinopathies, we compared the results obtained in C57Bl6 (B6) mice and mdx mice, a model for Duchenne muscular dystrophy in which muscle regeneration is dramatically increased. In mdx muscle compared to B6 muscle, we observed numerous GFP+ mononucleated cells corresponding to macrophages and numerous necrotic fibers filled by GFP+ macrophages and myoblasts. The number of GFP+ satellite cells number was similar in mdx and B6 mice 6 months post transplantation. However, GFP+ muscle fibers were more numerous in mdx mice. They were from month 1 post transplantation, a time point showing no GFP+ fiber in B6 mice. Six months post transplantation, 4 fold more GFP+ muscle fibers were found in mdx mice compared to B6 mice.
In contrast, dystrophin expression was only mildly increased (0,65% at month 6) and colocalizations between GFP and dystrophin was rarely found on serial cross sections. We conclude that the nuclear domain of dystrophin is probably much shorter than that of GFP.

3) Language disorders in Duchenne's muscular dystrophy

F. Civati, M. Guglieri, M.G. D' Angelo, A. Tavano, F. Fabbro, M.L. Lorusso, M. Sironi, A.C. Turconi, G.P. Comi, N. Bresolin (Bosisio Parini, San Vito, Milan, I)

Background: Duchenne Muscular Dystrophy (DMD) is a fatal recessive x-linked muscular disease caused by the absence of dystrophin. Dystrophin isoforms are also expressed in the cerebral neocortex and in the cerebellum. Cognitive impairment is described in 1/3 of DMD patients, particularly in patients carrying deletions in the distal part of the gene. Difficulties in verbal skills and reading abilities are more frequently described in English speaking patients. According to the literature children with DMD show linguistic deficits already in the early phases of language development which mainly consist of poor expressive verbal abilities and deficits in short-term memory. These observations suggest that in some cases dystrophinopathy may be associated to language disorders. Preliminary results in our group of Italian speaking children showed deficit in Grammatical and Syntax Comprehension.
Aims of the study are:
1) to describe the language and reading ability in a group of italian DMD children
2) to clarify the nature of the language and reading deficits
Patients: 13 children with DMD (mean age 8,3 years; standard deviation 1,7) were diagnosed according to international standard criteria. Full Intelligence Quotient(assessed using Wechsler Intelligence Scale) was >70.
Methods: Language and Reading abilities were determined through: “Test dello sviluppo Morfosintattico”, Battery of standardized tests, Battery to evaluate Dyslexia and Dysortography and Tasks of correctness and rapidity. To exclude additional cognitive deficits we evaluated attention/executive functions domain and memory and learning domain thought a Developmental Neuropsychological Assessment (NEPSY particularly: Auditory attention and response set - Visual attention - List learning - Memory for names).
Results: 8 patients out of 13 showed deficit in Syntax Comprehension. 7 patients out of 13 manifested deficits in Grammatical Comprehension. No DMD patients (except one) presented disabilities in reading. Most of the patients showed mild attention and memory deficits.
Discussion: An early identification of the language and eventually attention/memory difficulties, would be important for an early treatment, to support DMD children in their learning course.

4) Sleep apnoea does not simply attribute to respiratory failure in myotonic dystrophy: evaluation by somnography

H. Takada, S. Kon (Namioka, JP)


Sleep apnoea and hypopnoea have been reported in myotonic dystrophy (MD), but it is unclear whether those are simply attributable to respiratory impairment as well as respiratory muscle weakness. The aim of this study was to assess the correlation of the disrupted nocturnal sleep and awake respiratory function, and to deduce a mechanism of respiratory failure in MD. The study population comprised eighteen patients with MD and seventeen patients with other types of muscular dystrophy for a control group. Patients in the control group were chosen to represent a similar range of the results of routine spirometry examination, particularly the expected/observed forced vital capacity (%VC). Arterial blood gases were also examined early in the morning. Sleep apnoea and hypopnoea were evaluated in all patients using somnography (Sleep Tester LT-200, Fukuda Denshi Co., Japan). The number of apnoea and hyponoea during night (NA) was counted and types of apnoea were judged; the percentage of central type apnoea (%CSA) and obstructive apnoea (%OSA) among total apnoea. Transcutaneous recording of oxygen saturation during night were simultaneously performed. The mean nadir and minimal value of oxygen saturation were obtained. The apnoea index (AI) and the nocturnal hypoxia index (NHI) were calculated. The mean age of the MD group was significantly younger than that of the control group (42.9 +/- 11.7 vs. 51.6 +/- 12.8 years). There was no significant difference in the %VC between the two groups (63.0 +/- 15.4 vs. 65.7 +/- 23.4). The mean PaO2 in MD was significantly lower and the mean PaCO2 in MD was higher than those in the control (78.8 +/- 13.2 vs. 94.1 +/- 10.9 mmHg, 49.1 +/- 3.9 vs. 40.3 +/- 4.9 mmHg). The mean nadir (94.7 +/- 1.6 vs. 96.9 +/- 0.7 mm Hg) and minimal value (73.0 +/- 15.9 vs. 85.6 +/- 7.5 mm Hg) of oxygen saturation in MD were significantly lower than those in the control. The mean NHI in MD was significantly higher than that in the control (96.1 +/- 45.3 vs. 36.0 +/- 16.7). On the other hand, there were not significant differences in the NA, the %CSA, the %OSA, or the AI between the two groups. Moreover, all the parameters for apnoea did not correlate with the results of spirometry or arterial blood gases, although the AI correlated with the NHI or the %CSA in MD. Our results suggest that respiratory failure in MD is not simply owing to sleep apnoea or respiratory muscle weakness, but might be due to impaired chemosensitivity or ventiratory output. 

USA -  este congresso se realizará em Miami, em abril de 2005. É o mais importante encontro de neurologistas. Eu selecionei todos os resumos com temas em distrofia muscular. Os mais importantes estão relacionados com a droga PTC 124, que começou a ser testada em seres humanos para tratamento da mutação de ponto da distrofia muscular de Duchenne. Outro artigo inovador fala do uso de corticóides em mulheres portadoras do gene da distrofia muscular, com resultados positivos na melhora da fraqueza muscular progressiva. Outro artigo fala do efeito positivo do  uso da mexelitina para controle da miotonia na distrofia miotônica e o outro relata que a droga modafinil não melhora a sonolência diurna nesta forma de distrofia. Os resumos em inglês de todos os trabalhos pode ser lido aqui.

USA -  neste encontro que realizar-se-á em julho, de 1 a 5, serão apresentados 29 artigos sobre terapia gênica em distrofia muscular. A maioria das pesquisas é americana (12), seguida do Canadá (7) e França e Japão (4 cada). A grande maioria é experimental (cães e camundongos) e algumas são realizadas em células musculares humanas ou estudam a terapia gênica em um único músculo. Apesar de promissoras não há nenhuma pesquisa em andamento que permita a sua utilização.

Brasil - este Congresso reune importantes pesquisadores na área da distrofia muscular, tanto na parte clínica quanto experimental; inúmeros trabalhos serão apresentados neste encontro e eu selecionei os principais, que se relacionam com a parte de tratamento das doenças.  Há muitos artgios falando dos benefícios dos corticóides em distrofia muscular de Duchenne. Há também interessantes pesquisas na área de tratamento das distrofias, inclusive com experimentos com células tronco e terapia gênica. Os resumos são escritos em inglês e pelo volume de informação não é possível traduzí-los na integra. Esta é a versão mais completa dos resumos do que a colocada na página News in DMD. Os resumos podem ser lidos clickando abaixo:

Estudos clínicos

Estudos experimentais

USA - neste encontro serão apresentados poucos artigos importantes relacionados com distrofia muscular. Os principais resumos em inglês podem ser lidos aqui. Os mais importantes são dois artigos descrevendo mediadores da fibrose muscular em camundongos com distrofia muscular cuja inibição poderia retardar a evolução da doença; outro artigo interessante é a pesquisa das células tronco que tem maior capacidade de se diferenciar em células musculares.

USA - neste encontro de especialistas em terapia gênica serão apresentados 30 pesquisas sobre o uso da terapia gênica em diversas formas de distrofia muscular experimental. São estudos importantes mas ainda sob pesquisa, não havendo nenhum estudo de aplicação imediata.

Bélgica - este congresso que ocorrerá em outubro de 2006 é o maior encontro de especialistas em doenças neuromusculares. Dos trabalhos que serão apresentados eu selecionei os que se relacionam às distrofias musculares em especial com aspectos de tratamento (são 29 trabalhos que eu selecionei). Há várias pesquisas com terapia gênica, corticóide, osteoporose mas muitas poucas pesquisas com drogas para uso imediato. A maioria dos trabalhos é experimental mas há vários sobre Duchenne.

 

 

 

 

 

 

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