本帖最後由 lsc0019 於 2009-9-13 00:37 編輯
作者：Deborah Brauser
出處：WebMD醫學新聞
August 26, 2009 — 根據8月26日線上發表於The Lancet Neurology期刊的一篇劑量漸增試驗研究結果，一種反義分子肌肉注射劑在增加肌萎縮蛋白方面安全且有效，缺乏此一蛋白會引起裘馨氏肌肉萎縮症(Duchenne muscular dystrophy，DMD)。
英國倫敦大學院、兒童健康研究中心、Dubowitz神經肌肉中心的Maria Kinali醫師等人寫道，我們在體內試驗中發現，磷醯二胺嗎啉代寡核苷酸[phosphorodiamidate morpholino oligomer] AVI-4658引起特定的外顯子51跳脫，調整肌纖維膜處的肌萎縮蛋白生產。此外，此治療和任何全身或局部副作用無關，也與肌萎縮蛋白的免疫反應無關。
DMD影響約3500分之一的新生男嬰，引起漸進式的肌肉虛弱、心肌病變、呼吸衰竭。病患一般在嬰兒時即被診斷，10多歲時即需仰賴輪椅行動，通常在30歲左右死亡。
此病是因為缺乏肌萎縮蛋白所引起，作者們解釋，肌萎縮蛋白會減少肌纖維膜的穩定度，增加細胞內鈣流入，之後演變成肌肉纖維退化。
【沒有疾病修飾治療】
雖然目前沒有可以修飾病程的治療方法，反義寡聚核苷酸顯示可以達到不錯的結果，可以用來跳過一些阻斷產生肌萎縮蛋白的外顯子。
在13%的DMD病患中，肌萎縮蛋白基因突變直接影響了外顯子51前後的基因。反義寡聚核苷酸和外顯子51結合後，可以防止受影響的區域停止生產，表示仍然可以製造某種的肌萎縮蛋白。
為了檢驗此一理論，研究者進行一個單盲、安慰劑控制、劑量漸增試驗，以評估AVI-4658的安全性(此為主要終點)以及生化效果(次級終點)，AVI-4658是一種磷醯二胺嗎啉代寡核苷酸(一種反義寡聚核苷酸)，可跳過肌萎縮蛋白mRNA上的外顯子51區。
主要研究者Francesco Muntoni醫師向Medscape Neurology表示，此研究的創新之處在於，它是首次使用嗎啉反義代寡核苷酸(morpholino antisense oligomers)來誘導人類肌萎縮蛋白基因上的外顯子跳躍。嗎啉反義代寡核苷酸之前曾在動物肌肉病變研究中顯示相當有效，以肌肉或靜脈注射給藥，不過未曾在DMD男童進行過研究。
研究者納入7個臨床診斷有DMD且有突變的10到18歲男孩， 理論上可以透過跳躍外顯子51區而救治。
其中2名病患接受0.09-mg的AVI-4658，注射到他們腳上的伸趾短肌(extensor digitorum brevis，EDB)，而以900 μL生理食鹽水注射到另一腳。其他5名病患以相同劑量的生理食鹽水注射到一腳的EDB處，以0.9 mg的研究藥物注射到另一腳的EDB。兩種劑量的AVI-4658都以0.9%生理食鹽水稀釋到900 μL。
注射之後3到4週對所有研究的EDB肌肉進行切片。從Dubowitz神經肌肉中心生物銀行獲得健康肌肉切片。以理學檢查和血液學與尿液檢查確認安全性，定期加以評估。也監測注射部位的局部反應和疼痛反應。全部病患在治療後以120天為間隔進行追蹤。
【較高劑量可增加肌萎縮蛋白】
研究結束時，所有的安全評估都顯示沒有藥物相關副作用。
雖然以低劑量AVI-4658治療的病患有顯示些微的肌萎縮蛋白表現，較高劑量組在所治療的全部EDB肌肉都增加了肌萎縮蛋白表現。
在給予AVI-4658之部位鄰近位置進行的免疫分析中，44%到79%的肌纖維增加了肌萎縮蛋白表現。接受治療之EDB肌肉的隨機篩選部位中，健康控制組的平均肌萎縮蛋白染色密度為22%到32%(平均26.4%)，且此平均密度比注射生理食鹽水的另一腳的密度多17% (範圍11% – 21%)。(單一樣本配對t檢定；P = .002)。
在肌萎縮蛋白陽性纖維中，健康肌肉的肌萎縮蛋白染色密度高達42%。
Muntoni醫師表示，使用高劑量嗎啉反義代寡核苷酸的所有病患，肌萎縮蛋白生產明顯增加的這個發現，而且沒有任何有害的反應，相當令人開心。
他指出，希望醫師們首要記住的是，嗎啉反義代寡核苷酸可有效誘導肌萎縮蛋白的外顯子跳躍。因為這些反義代寡核苷酸在給藥後有極佳的追蹤紀錄，它們相當適合用於身體所有肌肉的肌萎縮蛋白重建研究。
【呼籲進行更多研究】
荷蘭Leiden大學醫學中心人類基因部的Annemike Aartsma-Rus醫師與Gert-Jan va Ommen醫師在編輯評論中寫道，需進行後續研究，以確認這些發現。
他們寫道，只有系統性的試驗才可以發現此方法的真實效果，需要有後續研究以確認這些功能面、或至少延緩疾病惡化的幫助。
他們指出，雖然有13%的DMD病患可以跳過外顯子51，其他的87%並未能獲得幫助。不過，跳過其他10個外顯子可能可以讓超過70%有類似刪除突變的DMD病患或40%的整體病患獲益。
他們結論表示，我們或許需瞭解更多有關這個大基因(2.5 Mbp、79個外顯子)的複雜剪接，以使這個治療方法更妥當。
英國健康部支持本研究，皇家倫敦學院資助本研究。研究作者之一為AVI Biopharma的顧問，另一名作者是該公司的全職員工。其他人宣告沒有相關財務關係。編輯作者是外顯子跳躍技術與反義序列運用專利的共同發明人。Ommen醫師是Prosensa公司外部科學諮詢委員會的無給職委員。
Lancet Neurol. 線上發表於2009年8月26日。
Antisense Molecule Shows Promise in Duchenne Muscular Dystrophy
By Deborah Brauser
Medscape Medical News
August 26, 2009 — Intramuscular injection treatment of an antisense molecule is safe and effective in increasing dystrophin, the absence of which causes Duchenne muscular dystrophy (DMD), according to results of a dose-escalation study reported online August 26 in The Lancet Neurology.
"We showed, in vivo, that the [phosphorodiamidate morpholino oligomer] AVI-4658 induced specific skipping of exon 51 and the production of dystrophin that was correctly localized at the sarcolemma," write Maria Kinali, MD, from the Dubowitz Neuromuscular Centre at University College London Institute of Child Health, United Kingdom, and colleagues. In addition, "the treatment was not associated with any systemic or local adverse events or with any immune response against dystrophin."
DMD affects 1 in 3500 newborn boys, causing eventually progressive muscle weakness, cardiomyopathy, and respiratory failure. Patients are usually diagnosed when they are toddlers, become wheelchair-dependent in their early teens, and often die by age 30 years.
The disease is caused by the absence of the protein dystrophin, which "reduces the stability of the sarcolemma and increases intracellular calcium influx, which is followed by degeneration of the muscle [fibers]," the authors explain.
No Disease-Modifying Treatment
Although there is currently no treatment available to modify the progression of this disease, promising results have been achieved with antisense olignucleotides, which can be used to skip some of the exons that block the effective creation of dystrophin.
In 13% of people with DMD, mutations in the dystrophin gene affect parts of the gene immediately before or after the region known as exon 51. By binding to exon 51, the antisense obligonucleotide prevents the affected regions from stopping production, which means that a version of dystrophin can still be made.
To test this theory, the investigators conducted a single-blind, placebo-controlled, dose-escalation trial to assess the safety (primary endpoint) and biochemical efficacy (secondary endpoint) of AVI-4658, a phosphorodiamidate morpholino oligomer (a type of antisense oligonucleotide), to skip exon 51 in dystrophin mRNA.
"The novelty of this study is that it is the first one [that] uses morpholino antisense oligomers to induce exon skipping of the dystrophin gene in the human. Morpholino antisense [oligomers] have been previously shown to be very effective in animal models of muscular dystrophy following intramuscular or intravenous administration but have never been administered before to a boy with DMD," principal investigator Francesco Muntoni, MD, told Medscape Neurology.
The investigators enrolled 7 boys between the ages of 10 and 17 years old who were clinically diagnosed with DMD and had a mutation that could in theory be rescued by the skipping of exon 51.
Two of the patients received a 0.09-mg dose of AVI-4658 injected into their extensor digitorum brevis (EDB) foot muscle plus 900 μL saline injected into the EDB of their other foot. The other 5 patients received the same dose of saline injected into the EDB of 1 foot and 0.9 mg of the study drug injected into the EDB of the other. Both doses of AVI-4658 were diluted in 900 μL normal saline (0.9%).
A biopsy of all study EDB muscles was done 3 to 4 weeks after injection. Healthy muscle biopsies were obtained from the Dubowitz Neuromuscular Centre biobank. Safety was determined by physical examination and hematological and urinary parameters, which were assessed periodically. The injection sites were also monitored for local reaction and reactive pain. All patients were followed up at timed intervals for 120 days after treatment.
Higher Dose Increased Dystrophin
At the end of the study, all safety assessments showed no adverse events related to the study drug.
Although the patients treated with the lower dose of AVI-4658 showed little expression of dystrophin, the higher dose resulted in increased dystrophin expression in all treated EDB muscles.
In the areas of the immunostained sections adjacent to the spot where AVI-4658 was given, 44% to 79% of myofibers had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in the healthy control muscles (mean, 26.4%), and the mean intensity was 17% (range, 11% – 21%) greater than the intensity in the contralateral saline-treated muscles (1-sample paired t-test; P = .002).
In the dystrophin-positive fibers, the dystrophin staining intensity was up to 42% of that in the healthy muscles.
"The finding of robust dystrophin protein production in all patients receiving the high dose of the morpholino antisense [oligomers], and not accompanied by any deleterious effect, was very pleasing," said Dr. Muntoni.
He added that the number one takeaway message for clinicians is that "morpholino antisense [oligomers] are effective in inducing dystrophin exon skipping. As these antisense [oligomers] have an excellent track record following systemic administration, they are very well placed for studies aimed at restoring dystrophin expression in all muscles of the body."
Call for More Research
In an accompanying editorial, Annemike Aartsma-Rus, MD, and Gert-Jan va Ommen, MD, from the Department of Human Genetics, Leiden University Medical Center in the Netherlands, write that further research is needed to validate these findings.
"Only systemic trials will reveal the true promise of this approach, and further trials are needed to validate the functional benefit, or at least the decline in disease progression," they write.
They add that although the skipping of exon 51 is applicable to 13% of DMD patients, it will not benefit the other 87%. However, "the skipping of another 10 exons might be beneficial for more than 70% of patients with a deletion mutation in DMD, or 40% of all patients."
They conclude, "We might need to learn much more about the complex splicing of this large gene (2.5 Mbp and 79 exons) to optimize its therapeutic correction."
This study was supported by the UK Department of Health and sponsored by Imperial College London. One of the study authors is a consultant to AVI Biopharma, and another is a full-time employee of the company. The rest have disclosed no relevant financial relationships. The editorial authors are coinventors on patent applications for antisense sequences and exon-skipping technology, and Dr. van Ommen is an unpaid member of the external scientific advisory board of Prosensa.
Lancet Neurol. Published online August 26, 2009. |
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