J.C.T. van Deutekom

Dystrophin levels and clinical severity in Becker muscular dystrophy patients

Objective Becker muscular dystrophy (BMD) is characterised by broad clinical variability. Ongoing studies exploring dystrophin restoration in Duchenne muscular dystrophy ask for better understanding of the relation between dystrophin levels and disease severity. We studied this relation in BMD patients with varying mutations, including a large subset with an exon 45–47 deletion. Methods Dystrophin was quantified by western blot analyses in a fresh muscle biopsy of the anterior tibial muscle. Disease severity was assessed using quantitative muscle strength measurements and functional disability scoring. MRI of the leg was performed in a subgroup to detect fatty infiltration. Results 33 BMD patients participated. No linear relation was found between dystrophin levels (range 3%–78%) and muscle strength or age at different disease milestones, in both the whole group and the subgroup of exon 45–47 deleted patients. However, patients with less than 10% dystrophin all showed a severe disease course. No relation was found between disease severity and age when analysing the whole group. By contrast, in the exon 45–47 deleted subgroup, muscle strength and levels of fatty infiltration were significantly correlated with patients’ age. Conclusions Our study shows that dystrophin levels appear not to be a major determinant of disease severity in BMD, as long as it is above approximately 10%. A significant relation between age and disease course was only found in the exon 45–47 deletion subgroup. This suggests that at higher dystrophin levels, the disease course depends more on the mutation site than on the amount of the dystrophin protein produced.

Identification of a novel beta-tubulin subfamily with one member (TUBB4Q) located near the telomere of chromosome region 4q35.

The human β-tubulin supergene family consists of several isotypes with many associated pseudogenes. Here we report the identification of yet another β-tubulin sequence designated TUBB4Q. This tubulin maps 80 kb proximal to the facioscapulohumeral muscular dystrophy (FSHD1) associated D4Z4 repeats on chromosome 4q35. The genomic structure contains four exons encoding a putative protein of 434 amino acids. The TUBB4Q nucleotide and protein sequence show 87% and 86% homology to β2-tubulin, respectively. Although the genomic structure shows all functional aspects of a genuine gene, no transcript could be detected. TUBB4Q-related sequences were identified on multiple chromosomes. Since these sequences mutually exhibit a high nucleotide sequence homology, they presumably belong to a novel subfamily of β-tubulin genes. Although the chromosome 4q35 tubulin-member probably represents a pseudogene, ectopic expression due to a postulated position effect variegation (PEV), makes TUBB4Q an ideal dominant-negative candidate gene for FSHD1.

Hereditary spastic paraparesis: Clinical and genetic data from a large Dutch family

A large Dutch family is presented with pure hereditary spastic paraparesis. Pedigree analysis indicates autosomal dominant inheritance with complete penetrance and possibly anticipation in successive generations. These observations may have implications for genetic studies. Linkage studies in this family have excluded more than 40% of the autosomal genome.

P.13.12 An objective method for immunofluorescence analysis of dystrophin levels in muscle from DMD patients in clinical studies

Duchenne muscular dystrophy (DMD) is characterized by absence or very low (trace) expression of dystrophin at the sarcolemmal membrane of the muscle fibers. In clinical studies aiming to restore dystrophin expression, dystrophin levels are measured in muscle biopsies by immunofluorescence analysis of cross-sections or western blot analysis of total protein extracts. However, appropriate quantification poses a technical challenge as dystrophin levels may be low and/or variable between fibers in the same biopsy. We have developed an immunofluorescence method and automated image analysis that measures the dystrophin intensity per individual fiber in a biopsy. It reproducibly detects even small differences in dystrophin levels. Muscle cross-sections co-stained for dystrophin and spectrin are imaged by confocal microscopy and image analysis is performed using Definiens software. Using a customized algorithm, and the sarcolemmal spectrin signal as a mask, the software automatically segments each image into individual muscle fibers, measures the varying dystrophin intensity per individual fiber, and objectively produces a histogram of the distribution in the fiber population, including revertant fibers. Duplicated analysis of the biopsies on the same or multiple days and by different operators was shown to be reproducible, objective and able to distinguish between different low dystrophin levels in Becker muscular dystrophy and DMD samples. Moreover, in DMD patients treated with antisense oligonucleotides to restore dystrophin expression, comparisons of the dystrophin intensity distribution histograms of pre- and post-treatment muscle biopsies showed increases in dystrophin intensities of entire muscle fiber populations post-treatment.

G.P.76 Dystrophin levels do not influence disease progression in Becker muscular dystrophy patients with an exon 45–47 deletion

Becker Muscular Dystrophy (BMD) shows a highly variable disease course. The cause of this diversity is mostly unknown. Dystrophin levels in muscle tissue are thought to be an important factor. We investigated the relationship between the amount of dystrophin in muscle biopsies and clinical severity in BMD patients with an exon 45–47 deletion. Thirteen patients with an exon 45–47 deletion were included, ranging in age from 20 to 63 years. Muscle strength was bilaterally measured using the Quantitative Muscle Testing (QMT) system in nine muscle groups: shoulder abduction, elbow flexion/extension, handgrip, hip flexion/extension, knee flexion/extension and ankle flexion. To assess muscle quality 3T MRI of the lower leg was performed. A muscle biopsy was taken from the anterior tibial muscle. Dystrophin quantification was performed by Western Blot analysis, using two antibodies: DYS1 (rod domain) and AB15277 (C-terminus). Statistical analysis was performed using Pearson’s correlation test. Dystrophin levels ranged from 15% to 71% compared to healthy control muscle. The correlation between the two antibodies was excellent (R 0.88; p < 0.001). No relationship was present between dystrophin levels and QMT-sum score or fatty infiltration on MRI. In contrast, the QMT-sum score and fatty infiltration on MRI correlated significantly with patients’ age. The current study shows that the dystrophin level in itself does not explain the variation in disease severity in BMD patients with an exon 45–47 deletion. The strong relationship between strength or MRI abnormalities and age suggests that in this subgroup of Becker patients the disease course is determined by the mutation rather than the quantity of the internally deleted dystrophin protein.

G.P.132

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disorder caused by an expanded CTG repeat in the 3′ UTR of the DM protein kinase (DMPK) gene, resulting in formation of toxic RNA aggregates that interfere with RNA splicing and dysregulate normal cell function. RNA-modulation by antisense oligonucleotides (AONs) represents an interesting therapeutic approach for DM1 and is currently in pre-clinical development. We have previously obtained molecular proof-of-concept for a panel of AONs that target the CUG expansion and result in efficient knockdown of toxic RNA in DM1 patient myoblasts with different repeat expansion lengths and in cultured muscle cells derived from DM500 mice. To facilitate systemic AON delivery and uptake, we used a 7-amino acid linear muscle targeting peptide (PP08) and conjugated this to different CUG-targeting AONs. We have shown that these peptide-conjugated AONs are well tolerated and do not result in complement activation in human and monkey plasma or induce cytokine release in human whole blood in vitro safety assays. Subcutaneous administration of peptide-conjugated AONs in different DM1 mouse models (DM500, HSALR) resulted in enhanced tissue PK levels in several tissues of relevance for DM1 such as skeletal muscle and brain, indicating that the peptide-AON displayed efficient tissue uptake and was even able to pass the blood–brain-barrier. The higher tissue levels were accompanied by an increased knockdown of mutant RNA compared to non-conjugated AONs. Besides inducing a significant RNA knockdown, peptide-conjugated AONs resulted in a mild but statistically significant reduction of myotonia in gastrocnemius and tibialis muscles of HSALR mice, as determined by EMG analysis. These data demonstrate that peptide-AONs targeting the CUG-repeat have potential in a multisystemic therapeutic approach for DM1.

Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG∗CAG) n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding of this RNA to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and muscle and brain cell membranes. It has been reported that DM1 patients show elevated levels of muscular markers in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface distribution of dystrophin was normal. Combined, our findings show that cells in DM1 tissues have a normal functional membrane, which forms a barrier that must be overcome in future work towards effective drug delivery.

G.P.87

Antisense oligonucleotides (AONs) for Duchenne muscular dystrophy (DMD) are designed to induce skipping of a single exon during pre-mRNA splicing, thus restoring the transcript’s open reading frame and consequently synthesis of the deficient dystrophin protein. Although this approach is mutation-dependent, the majority of mutations cluster in a hotspot region from exon 45 to 53, allowing application of one AON to a subpopulation of patients with grouped mutations. Some mutations however are ultra-rare with only few patients worldwide, and require the skipping of an exon outside the hotspot region. Combining these ultra-rare mutations by applying multiple exon skipping is therefore attractive. The feasibility of multiple exon skipping using a cocktail of AONs has been demonstrated, but a drawback is the increased complexity of the development pathway and manufacturing costs. We therefore studied the possibility of designing a single AON capable of inducing simultaneous skipping of multiple exons. The repetitive nature of the central rod domain in dystrophin implies the occurrence of homologous stretches within the transcript. Indeed, in the exon 10 to 40 region several exon pairs were identified sharing such a homologous stretch, which we explored as a potential dual target for a single AON. In-frame exon combinations that would mimic a Becker-like mutation associated with a relatively mild phenotype and that would apply to a considerable patient subpopulation (up to ∼15%) were selected. In vitro proof-of-concept was obtained for healthy donor as well as for DMD patient-derived muscle cells and resulted in novel dystrophin expression. Results demonstrating the applicability in the mdx mouse model in vitro and in vivo will be presented. The data support further (pre) clinical development of such multiple exon skipping AONs, not only for more rare mutations that are not targeted by current single skip programs but also for mutations in the hotspot region.

G.P.86

Antisense oligonucleotide (AON) therapies for Duchenne muscular dystrophy (DMD) are aimed at inducing skipping of specific exons during pre-mRNA splicing. This results in restoration of the reading frame and consequently synthesis of a shorter yet functional version of the dystrophin protein, as has been demonstrated in healthy control and DMD patient-derived muscle cell cultures, various DMD mouse and dog models, and recently in DMD patients treated with eteplirsen or drisapersen. As in many DMD patients a low level of spontaneous, rescuing background exon skipping occurs, accurate quantification of exon skip levels before and after AON treatment is required to monitor molecular AON drug effect in clinical studies. So far exon skip levels have generally been assessed using conventional 1st generation RT-PCR, in which non-skipped and skipped transcripts are co-amplified followed by visual comparison or densitometry of exon skip levels by gel electrophoresis or lab-on-chip. As dystrophin is not a highly abundant protein, underlying DMD transcript levels are relatively low and two rounds of PCR amplification (primary and nested) are required, often resulting in more efficient amplification of the shorter ‘skipped’ fragment compared to the longer ‘non-skipped’ fragment and end-stage saturation of the amplified products. With the recent introduction of 3rd generation digital droplet RT-PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of copy numbers of transcripts with and without exon skip in a single PCR reaction, with high precision, sensitivity and reproducibility. We have developed ddPCR TaqMan probes targeting specific exon–exon junctions in control, BMD, DMD and mdx samples and will present data demonstrating its suitability for assessing DMD transcript copy numbers and exon skip levels. We recommend ddPCR for quantifying exon skip efficiencies in the various stages of (pre-) clinical development of AONs.

M02 Therapeutic Benefit Of A Htt-lowering Antisense Oligonucleotide Targeting The Cag-repeat In The R6/2 Huntington’s Disease Mouse Model

Background Different therapeutic approaches using antisense oligonucleotides (AONs) are currently under development for Huntington’s disease (HD). Ideally, an AON-based therapeutic approach for HD would selectively lower levels of the transcript carrying the disease-causing CAG-repeat expansion, but not affect the non-expanded transcript. We have previously demonstrated preferential knockdown of the mutant transcript in vitroin fibroblasts derived from HD patients using a CAG-repeat targeting AON consisting of a (CUG)7 sequence. Aims Aim of this study was to gain in vivoproof-of-concept with our (CUG)7 AON in a relevant HD animal model. Methods/techniques R6/2 mice transgenic for an N-terminal fragment of human HTT containing ˜150 CAG repeats (n = 30) received a total of 6 weekly ICV infusions with the (CUG)7 AON or vehicle. Bodyweight was monitored and a battery of motor tests (grip strength, rotarod, open field, 3D kinematic analysis) were performed. In addition, brain volume and striatal metabolites were quantified using MRI and MRS respectively. Two weeks after the last infusion at 12 weeks of age RNA was isolated from 7 different brain regions, followed by RT-qPCR analysis of HTT transcript levels. Results/outcome A highly signficant and strong (˜90%) reduction of mutant HTT mRNA levels was observed throughout the R6/2 brain. In addition, there was a clear positive treatment effect on motor performance, indicated by an increase in rotarod latency and vertical activity in the open field. 3D kinematic analysis of fine motor skills showed a significant improvement of several parameters impaired in R6/2 mice. A trend towards an increased whole brain and cortical volume was observed using MRI volumetry as well as a significant decrease in striatal levels of the astrocytic marker inositol, known to be elevated in the striatum of R6/2 mice and HD patients. Conclusions These data demonstrate our AON targeting the CAG-repeat has therapeutic potential for HD.