Reversible immortalisation, human artificial chromosomes, and induced pluripotency: new gene and cell therapy technologies for Duchenne muscular dystrophy

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• 作者：Sara Benedetti ; PhD^a ; Hidetoshi Hoshiya ; PhD^a ; Narumi Uno ; PhD^b ; Giulia Ferrari ; MD^a ; Yasuhiro Kazuki ; PhD^b ; Sara Maffioletti ; MSc^a ; Tamara Casteels^a ; Martina Ragazzi ; MSc^a ; Graziella Messina ; PhD^c ; Prof Francesco Muntoni ; MD^a ; ^d ; Prof Mitsuo Oshimura ; PhD^b ; Prof Giulio Cossu ; FMedSci^e ; Dr Francesco Saverio Tedesco ; PhD^a ; ^{f.s.tedesco@ucl.ac.uk" class="auth_mail" title="E-mail the corresponding author}
• 刊名：The Lancet
• 出版年：2016
• 出版时间：25 February 2016
• 年：2016
• 卷：387
• 期：supp_S1
• 页码：S98
• 全文大小：54 K

文摘

Duchenne muscular dystrophy is caused by mutations in the gene that encodes dystrophin, a major component of muscle fibres. The combination of gene therapy with cell therapy to treat the disorder is encouraging, but it is challenging because dystrophin is the largest human gene, and skeletal muscle the most abundant human tissue. We aimed to assess use of autologous stem cells with human artificial chromosomes (HACs) containing the entire dystrophin locus (DYS-HACs) as a solution to these challenges.

Methods

We describe two complementary strategies for the generation of genetically corrected myogenic stem cells from patients with Duchenne muscular dystrophy, one from muscle biopsy samples and the other from induced pluripotent stem (iPS) cells. We also describe the generation of a multifunctional DYS-HAC applicable in both strategies. Notably, these technologies are also being developed in genome-integration-free platforms.

Findings

Reversible immortalisation with lentivirally delivered excisable telomerase and BMI1 complementary DNAs allowed bypassing of replicative senescence and DYS-HAC transfer in muscle-derived cells. When isolation of tissue-derived progenitors proved challenging, we successfully derived skeletal myogenic cells from Duchenne muscular dystrophy iPS cells reprogrammed with non-integrating technologies and corrected them with the DYS-HAC. Finally we describe the engineering of a next-generation, multifunctional DYS-HAC that can provide complete genetic correction, enhanced proliferation, controllable cell death (as a safety system), and inducible myogenesis in both tissue-derived or iPS cell-derived stem cells.

Interpretation

This project provides the foundation for preclinical and clinical development of an autologous ex-vivo gene therapy protocol for Duchenne muscular dystrophy based on HACs and myogenic cells.

Funding

National Institute for Health Research, Medical Research Council, Takeda New Frontier Science, Muscular Dystrophy UK, Duchenne Children's Trust, Duchenne Research Fund, Duchenne Parent Project Onlus.