Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next‐generation human artificial chromosomes for Duchenne muscular dystrophy

Abstract

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large‐capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS‐HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS‐HAC into DMD satellite cell‐derived myoblasts and perivascular cell‐derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next‐generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.

Diltiazem improves contractile properties of skeletal muscle in dysferlin-deficient BLAJ mice, but does not reduce contraction-induced muscle damage

“…; Benedetti et al. ). In the context of our current set of studies, it appears that, DTZ is not likely to reduce the damage that is induced by a moderately intense bout of repetitive eccentric loading, in dysferlin‐deficient mammalian skeletal muscle.…”

“…The fact that dysferlinopathies are progressive conditions, should encourage the focus of rehabilitative interventions, to be more on slowing down the natural progression of muscle weakness and wasting, rather than increasing muscle strength and walking capacity at the risk of causing irreversible muscle damage. Preserving functional muscle mass for as long as possible, preventing joint contractures, and maintaining the central nervous system's synaptic connections for functional movement, might help optimize patient-readiness for promising gene therapies that are under intense investigation (Lostal et al 2010;Long et al 2014;Sondergaard et al 2015;Nelson et al 2016;Robinson-Hamm and Gersbach 2016;Tabebordbar et al 2016;Benedetti et al 2017). In the context of our current set of studies, it appears that, DTZ is not likely to reduce the damage that is induced by a moderately intense bout of repetitive eccentric loading, in dysferlin-deficient mammalian skeletal muscle.…”

Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders

“…27,33 However, HACs are sometimes silenced and/or are lost from the cells, 20 although other studies did show appropriate transgene maintenance and expression. 34,35 Correction of mutations in DMD cells could be achieved by CT followed by differentiation to muscle cells and their injection in the muscles, an option that has been used by several researchers in muscle pathology. 27,33,[36][37][38] The only needed additional step to the protocol used herein is the inactivation, before retro-MMCT fusion, of the HPRT gene on the endogenous X chromosome by CRISPR technology, allowing the use of HAT selection after retro-MMCT.…”

Molekulare Therapien von Muskeldystrophien

“…B. Human Artificial Chromosomes (HACs) mit Stamm-/Progenitorzellen könnte diese Limitierung überwinden, und damit eine neue Plattform für einen komplexen Gentransfer zu klinisch relevanten humanen Muskelprogenitoren für DMD Gentherapie bieten [36].…”