Mouse regenerating myofibers detected as false-positive donor myofibers with anti-human spectrin.

Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1(null)mdx(5cv), NOD/LtSz-scid IL2Rγ(null) mice, or mdx nude mice, irrespective of whether they were injected with human cells. These "reactive" clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies.

Optimizing splice-switching oligomer sequences using 2'-O-methyl phosphorothioate chemistry.

We have taken an empirical approach in designing splice-switching oligomers to induce targeted dystrophin exon skipping. The nucleotide sequence of the exon under examination is first analyzed for potential exon recognition motifs and then a set of oligomers complementary to the acceptor and donor splice sites, as well as intra-exonic regions predicted to contain exon splice enhancers, are designed and synthesized as 2'-O-methyl-modified bases on a phosphorothioate backbone (2OMeAOs). The 2OMeAOs can be readily transfected into cultured normal myogenic cells as cationic lipoplexes, and are incubated for 24 h before total RNA extraction and subsequent analysis by semi-quantitative RT-PCR. The amplification conditions used for each dystrophin transcript region under investigation minimize preferential production of shorter amplicons and do not exaggerate the level of induced RT-PCR products, compared to the endogenous dystrophin transcript product. It is imperative that the test oligomers are transfected over a range of concentrations and that the target exon is excised in a reproducible and dose-dependent manner.Once it has been demonstrated that an oligomer can induce some degree of exon skipping, that target region of the pre-mRNA is assumed to be involved in splicing of the exon. A series of overlapping oligomers are prepared and evaluated by transfection into normal myogenic cells at lower concentrations to identify the more effective compounds. Clinical application requires antisense compounds that efficiently modulate splicing at low dosages, delivering the greatest benefits in terms of efficacy, safety, and cost.

Targeted exon skipping to address "leaky" mutations in the dystrophin gene.

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear "leaky", and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known "leaky" region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.Molecular Therapy - Nucleic Acids (2012) 1, e48; doi:10.1038/mtna.2012.40; published online 16 October 2012.

Multiple exon skipping strategies to by-pass dystrophin mutations.

Manipulation of dystrophin pre-mRNA processing offers the potential to overcome mutations in the dystrophin gene that would otherwise lead to Duchenne muscular dystrophy. Dystrophin mutations will require the removal of one or more exons to restore the reading frame and in some cases, multiple exon skipping strategies exist to restore dystrophin expression. However, for some small intra-exonic mutations, a third strategy, not applicable to whole exon deletions, may be possible. The removal of only one frame-shifting exon flanking the mutation-carrying exon may restore the reading frame and allow synthesis of a functional dystrophin isoform, providing that no premature termination codons are encountered. For these mutations, the removal of only one exon offers a simpler, cheaper and more feasible alternative approach to the dual exon skipping that would otherwise be considered. We present strategies to by-pass intra-exonic dystrophin mutations that clearly demonstrate the importance of tailoring exon skipping strategies to specific patient mutations.

Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice.

BACKGROUND: Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patient's skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings. METHODOLOGY/PRINCIPAL FINDINGS: Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts. CONCLUSIONS/SIGNIFICANCE: Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.

Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials.

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.

The contribution of human synovial stem cells to skeletal muscle regeneration.

Stem cell therapy holds promise for treating muscle diseases. Although satellite cells regenerate skeletal muscle, they only have a local effect after intra-muscular transplantation. Alternative cell types, more easily obtainable and systemically-deliverable, were therefore sought. Human synovial stem cells (hSSCs) have been reported to regenerate muscle fibres and reconstitute the satellite cell pool. We therefore determined if these cells are able to regenerate skeletal muscle after intra-muscular injection into cryodamaged muscles of Rag2-/gamma chain-/C5-mice. We found that hSSCs possess only limited capacity to undergo myogenic differentiation in vitro or to contribute to muscle regeneration in vivo. However, this is enhanced by over-expression of human MyoD1. Interestingly, hSSCs express extracellular matrix components laminin alpha2 and collagen VI within grafted muscles. Therefore, despite their limited capacity to regenerate skeletal muscle, hSSCs could play a role in treating muscular dystrophies secondary to defects in extracellular matrix proteins.

Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study.

BACKGROUND: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. METHODS: We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. FINDINGS: Two patients received 0.09 mg AVI-4658 in 900 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44-79% of myofibres 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 healthy control muscles (mean 26.4%), and the mean intensity was 17% (range 11-21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0.002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. INTERPRETATION: Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. FUNDING: UK Department of Health.

Human muscle precursor cells give rise to functional satellite cells in vivo.

Mouse satellite cells have been shown to be functional muscle stem cells, in that they are able to regenerate skeletal muscle and to reconstitute the satellite cell pool. Although human muscle precursor cells are able to contribute to skeletal muscle regeneration following transplantation into host mouse muscles, it is uncertain whether they also give rise to functional satellite cells. Here, we transplant human fetal muscle precursor cells into cryodamaged muscles in C(5)-/gamma-chain-/Rag2-host mice. The donor cells gave rise to muscle fibres that persisted for up to 6 months after grafting. Isolated muscle fibres, bearing satellite cells, were prepared from muscles 4 weeks after grafting. When placed in culture, a small proportion of these fibres gave rise to muscle precursor cells of human origin, indicating that the originally grafted cells had formed satellite cells as well as regenerated muscle fibres. These satellite cell-derived human muscle precursor cells were expanded in culture and formed muscle following their transplantation into a second series of host mice. This provides evidence that human, as well as mouse, muscle precursor cells, are capable of forming functional satellite cells in vivo.

VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection.

Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.