己糖对杜兴肌肉萎缩症核酸药物Pip5e-PMO跳读活性研究
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摘要
目的:在杜兴肌肉萎缩症模型上探究己糖混合物对核酸药物Pip5e-PMO介导的外显子跳读活性的促进作用。方法:选择成年mdx小鼠为测试动物模型,通过局部肌肉和系统尾静脉注射,比较Pip5e-PMO与己糖和生理盐水联用的作用效果,利用免疫荧光(IHC)和蛋白印迹法(Western blot)检测dystrophin阳性肌纤维的数量和分布及dystrophin蛋白的表达水平;利用逆转录-聚合酶链式反应(RT-PCR)检测外显子跳读。结果:在局部肌肉给药试验中,己糖能够促进Pip5e-PMO诱导dystrophin蛋白表达水平(P<0.05)。在系统试验中,对己糖和生理盐水组比较发现:按15 mg/kg的剂量单次系统尾静脉注射Pip5e-PMO,骨骼肌中dystrophin蛋白表达恢复至正常水平的50%;在心脏中,也检测到明显的dystrophin恢复表达和外显子跳读。但两组无显著性差别(P>0.05)。结论:Pip5e-PMO可诱导有效的外显子跳读和dystrophin蛋白表达,但己糖对其促进效果有限。
Objective: To explore the potential of hexose(the mixture of glucose and fructose-GF) in enhancing the delivery of peptidemodified PMO(Pip5 e-PMO) in the Duchenne Muscular Dystrophy(DMD) mouse model(mdx mouse). Methods: Pip5 e-PMO in GF or saline was intramuscularly or intravenously administered into adult mdx mice. Immunohistochemistry(IHC) and Western blot were used to measure the distribution and expression of dystrophin. Reverse transcription-Polymerase chain reaction(RT-PCR) was utilized to examine the level of exon skipping. Results: Local intramuscular results showed that GF enhanced the activities of Pip5 e-PMO demonstrated by the increased number of dystrophin-positive fibers and level of dystrophin expression(P<0.05). Although single intravenous injection of 15 mg/kg Pip5 e-PMO induced substantial number of dystrophin-positive fibers and therapeutic levels of dystrophin restoration in peripheral muscles and heart of mdx mice treated with either GF or saline, the difference between GF and saline groups was not significant(P>0.05).Conclusion: GF could enhance the activities of Pip5 e-PMO intramuscularly but may fail to have any impact on dystrophin-deficient mdx mice.
Objective: To explore the potential of hexose(the mixture of glucose and fructose-GF) in enhancing the delivery of peptidemodified PMO(Pip5 e-PMO) in the Duchenne Muscular Dystrophy(DMD) mouse model(mdx mouse). Methods: Pip5 e-PMO in GF or saline was intramuscularly or intravenously administered into adult mdx mice. Immunohistochemistry(IHC) and Western blot were used to measure the distribution and expression of dystrophin. Reverse transcription-Polymerase chain reaction(RT-PCR) was utilized to examine the level of exon skipping. Results: Local intramuscular results showed that GF enhanced the activities of Pip5 e-PMO demonstrated by the increased number of dystrophin-positive fibers and level of dystrophin expression(P<0.05). Although single intravenous injection of 15 mg/kg Pip5 e-PMO induced substantial number of dystrophin-positive fibers and therapeutic levels of dystrophin restoration in peripheral muscles and heart of mdx mice treated with either GF or saline, the difference between GF and saline groups was not significant(P>0.05).Conclusion: GF could enhance the activities of Pip5 e-PMO intramuscularly but may fail to have any impact on dystrophin-deficient mdx mice.
引文
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[12] Fairclough R J, Bareja A, Davies K E. Progress in therapy for Duchenne muscular dystrophy[J]. Exp Physiol, 2011, 96(11):1101
[13] Lim K R, Maruyama R, Yokota T. Eteplirsen in the treatment of Duchenne muscular dystrophy[J]. Drug Des Devel Ther, 2017, 11:533
[14] Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment:an open-label, phase 2, dose-escalation study[J]. The Lancet, 2011,378(9791):595
[15] Gao X, Ran N, Dong X, et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy[J]. Sci Transl Med, 2018, 10(444). pii:eaat 0195.doi:10.1126/scitrans-lmed.aat0195
[16] Yin H, Saleh A F, Betts C, et al. Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice[J]. Mol Ther, 2011,19(7):1295
[17] Han G, Gu B, Cao L, et al. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice[J]. Nat Commun,2016, 7:10981
[18] Korinthenberg R. A new era in the management of Duchenne muscular dystrophy[J]. Dev Med Child Neurol, 2019, 61(3):292
[19] Klein S M, Prantl L, Geis S, et al. Circulating serum CK level vs.muscle impairment for in situ monitoring burden of disease in Mdxmice[J]. Clin Hemorheol Microcirc, 2017, 65(4):327
[20] Lewon M, Peters C M, Van Ry P M, et al. Evaluation of the behavioral characteristics of the mdx mouse model of duchenne muscular dystrophy through operant conditioning procedures[J].Behav Processes, 2017, 142:8
[21] Aartsma-Rus A, Straub V, Hemmings R, et al. Development of exon skipping therapies for duchenne muscular dystrophy:a critical review and a perspective on the outstanding issues[J]. Nucleic Acid Ther, 2017, 27(5):251
[22] Burns D P, Canavan L, Rowland J, et al. Recovery of respiratory function in mdx mice co-treated with neutralizing interleukin-6 receptor antibodies and urocortin-2[J]. J Physiol, 2018, 596(21):5175
[23] Mosqueira M, Zeiger U, Forderer M, et al. Cardiac and respiratory dysfunction in Duchenne muscular dystrophy and the role of second messengers[J]. Med Res Rev, 2013, 33(5):1174
[24] Malerba A, Thorogood F C, Dickson G, et al. Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice[J]. Hum Gene Ther, 2009, 20(9):955
[2] Van Der Pijl E M, Van Putten M, Niks E H, et al. Low dystrophin levels are insufficient to normalize the neuromuscular synaptic abnormalities of mdx mice[J]. Neuromuscul Disord, 2018, 28(5):427
[3] Tuffery-Giraud S, Miro J, Koenig M, et al. Normal and altered prem RNA processing in the DMD gene[J]. Hum Genet, 2017, 136(9):1155
[4] G uiraud S, Aartsma-Rus A, Vieira N M, et al. The pathogenesis and therapy of muscular dystrophies[J]. Annu Rev Genomics Hum Genet,2015, 16:281
[5] Aminzadeh M A, Rogers R G, Fournier M, et al. Exosome-mediated benefits of cell therapy in mouse and human models of duchenne muscular dystrophy[J]. Stem Cell Reports, 2018, 10(3):942
[6] Xu R, Jia Y, Zygmunt D A, et al. r AAVrh74.MCK.GALGT2 protects against loss of hemodynamic function in the aging mdx mouse heart[J]. Mol Ther, 2019, 27(3):636
[7] Ho P P, Lahey L J, Mourkioti F, et al. Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy[J]. Proc Natl Acad Sci U S A, 2018, 115(39):E9182
[8] Mcdonald A A, Hebert S L, Mcloon L K. Sparing of the extraocular muscles in mdx mice with absent or reduced utrophin expression:A life span analysis[J]. Neuromuscul Disord, 2015, 25(11):873
[9] Pisani C, Strimpakos G, Gabanella F, et al. Utrophin up-regulation by artificial transcription factors induces muscle rescue and impacts the neuromuscular junction in mdx mice[J]. Biochim Biophys Acta Mol Basis Dis, 2018, 1864(4 Pt A):1172
[10] Kole R, Krieg A M. Exon skipping therapy for Duchenne muscular dystrophy[J]. Adv Drug Deliv Rev, 2015, 87:104
[11] Welch E M, Barton E R, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations[J]. Nature, 2007, 447(7140):87
[12] Fairclough R J, Bareja A, Davies K E. Progress in therapy for Duchenne muscular dystrophy[J]. Exp Physiol, 2011, 96(11):1101
[13] Lim K R, Maruyama R, Yokota T. Eteplirsen in the treatment of Duchenne muscular dystrophy[J]. Drug Des Devel Ther, 2017, 11:533
[14] Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment:an open-label, phase 2, dose-escalation study[J]. The Lancet, 2011,378(9791):595
[15] Gao X, Ran N, Dong X, et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy[J]. Sci Transl Med, 2018, 10(444). pii:eaat 0195.doi:10.1126/scitrans-lmed.aat0195
[16] Yin H, Saleh A F, Betts C, et al. Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice[J]. Mol Ther, 2011,19(7):1295
[17] Han G, Gu B, Cao L, et al. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice[J]. Nat Commun,2016, 7:10981
[18] Korinthenberg R. A new era in the management of Duchenne muscular dystrophy[J]. Dev Med Child Neurol, 2019, 61(3):292
[19] Klein S M, Prantl L, Geis S, et al. Circulating serum CK level vs.muscle impairment for in situ monitoring burden of disease in Mdxmice[J]. Clin Hemorheol Microcirc, 2017, 65(4):327
[20] Lewon M, Peters C M, Van Ry P M, et al. Evaluation of the behavioral characteristics of the mdx mouse model of duchenne muscular dystrophy through operant conditioning procedures[J].Behav Processes, 2017, 142:8
[21] Aartsma-Rus A, Straub V, Hemmings R, et al. Development of exon skipping therapies for duchenne muscular dystrophy:a critical review and a perspective on the outstanding issues[J]. Nucleic Acid Ther, 2017, 27(5):251
[22] Burns D P, Canavan L, Rowland J, et al. Recovery of respiratory function in mdx mice co-treated with neutralizing interleukin-6 receptor antibodies and urocortin-2[J]. J Physiol, 2018, 596(21):5175
[23] Mosqueira M, Zeiger U, Forderer M, et al. Cardiac and respiratory dysfunction in Duchenne muscular dystrophy and the role of second messengers[J]. Med Res Rev, 2013, 33(5):1174
[24] Malerba A, Thorogood F C, Dickson G, et al. Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice[J]. Hum Gene Ther, 2009, 20(9):955