THERAPEUTIC APPROACHES IN DEVELOPMENT FOR DUCHENNE MUSCULAR DYSTROPHY
Duchenne muscular dystrophy is a severe, progressive muscular disorder caused by mutations in the dystrophin encoding DMD gene. Dystrophin provides muscle ﬁbers with stability during exercise and lack of functional dystrophin leads to a progressive loss of muscle tissue and muscle function. Several therapeutic approaches are currently in diﬀerent stages of therapeutic development. We will here give an overview of gene, genetic and cell therapy approaches, utrophin upregulation and approaches aiming at improving muscle quality, focusing primarily on approaches currently in the clinical trial phase.
Duchenne and Becker muscular dystrophies (DMD and BMD, respectively) are caused by muta-tions in the dystrophin encoding DMD gene. DMD is the most severe and most common inherited neu-romuscular disorder, with an incidence of ~1 in 4000 newborn males. Boys generally show the initial signs of muscle weakness in the first years of life, which presents as frequent falling, difficulty with climbing stairs and the Gower’s sign . Most boys are wheel-chair-dependent by the age of 12 years, and need as-sisted ventilation before the age of 20 years. Death of-ten occurs in the 3rd or 4th decade due to respiratory or heart failure. BMD is a less severe and less frequent (incidence ~1 in 20000) neuromuscular disorder with a more varied severity than DMD. Diagnosis can oc-cur as early as 4 years or as late as 60 years or even older. Generally, BMD patients remain ambulant for at least 10 years after diagnosis. Life expectancy is slightly reduced to normal .
The discrepancy between the phenotypes lies in the function of the dystrophin protein . Dys-trophin provides muscle fibers with stability during contraction by linking the cytoskeletal actin to the extracellular matrix (Figure 1A). In DMD patients mutations disrupt the open reading frame and dystro-phin translation is truncated prematurely, rendering dystrophin completely non functional (Figure 1B). By contrast, in BMD patients the open reading frame is maintained and dystrophin translation continues to the end. Resulting dystrophins are internally deleted, but still partly functional (Figure 1C).
The pathology of DMD has not yet been com-pletely elucidated, but it has been proposed that loss of muscle fibers is the result of different interacting pathways in which dystrophin plays a role [3-6]. First, due to the lack of functional dystrophin, muscle fibers are damaged continuously during contractions. This damage and/or the increased activation of stretch ac-tivated calcium channels results in increased levels of Ca2+ in the fibers . This in turn activates calpains (proteases) and also leads to damage to mitochondria resulting in oxidative stress in muscle fibers. Both processes further increase the amount of muscle fiber damage. Due to the persistent nature of the damage, the muscles are chronically inflamed. The inflamma-tory cells produce cytokines and other toxins that further damage muscle cells and also impede muscle re-generation and enhance the formation of fibrosis . Consequently, muscle fibers are continuously lost, muscle regeneration is impaired and eventually, muscle is replaced by fibrotic and adipose tissue and muscle function is progressively lost. Due to the muscle fiber damage the muscle specific enzyme creatine ki-nase leaks into the blood stream and is much increased in younger patients. When patients grow older and muscle tissue is lost, levels go down due to the lower abundance of muscle tissue .
Although the gene and protein were identified over two decades ago, there is currently no cure for DMD. Patients are treated symptomatically. Due to improved care [8,9] life expectancy has increased from below two decades to over three decades in the Western world. Currently, most patients are treated with corticoste-roids, for which the exact mechanism of action is un-known, but which are thought to work by inhibiting the chronic inflammation. Although this is not with-out side effects, chronic treatment with corticosteroids leads to a delay in disease progression in most patients, reflected in prolonged ambulation, increased respira-tory function, improved upper limb strength, less sco-liosis and/or improved heart function .
In this review we will give an overview of new therapeutic approaches that either target the lack of dystro-phin directly or aim to improve muscle quality, focusing on those that are close to or tested in clinical trials.
A) In unaffected individuals the reading frame of the dystrophin mRNA is intact and a full length, functional, dystrophin protein can be produced. The protein provides a link between the actin cytoskeleton and the extracellular matrix. Thereby it provides the muscle fibers with stability during contractions and prevents muscle damage.
B) In DMD, mutations disrupt the open reading frame of the dystrophin mRNA. This causes the translation to stop prematurely. The resulting protein is not functional, as the link between actin and the extracellular is lost and muscle fibers are prone to exercise induced damage.
C) In BMD part of the DMD gene is deleted, but the open reading frame of the dystro-phin mRNA stays intact. Therefore a slightly shorter, internally deleted, protein can be formed. This protein is still largely functional and can provide the functional link between the actin cytoskeleton and the extracellular matrix.
DRUG THERAPIES TO IMPROVE MUSCLE QUALITY AND MASS
As the symptoms in DMD and BMD are caused by a loss of muscle fibers, the progression of the disease might be delayed by improving muscle quality or mass. Idebenone (Catena®) is a compound that reduces oxi-dative stress, by acting as a free radical scavenger, and improves mitochondrial function, by increasing ATP production and protecting the mitochondria against lipid peroxidation . In the mdx mouse model ide-benone treatment resulted in a reduction of inflam-mation and lower levels of fibrosis in heart, thereby normalizing heart function and improving voluntary running . In a small phase I/II clinical trial in DMD patients no significant difference in heart func-tion was observed . However, this study was not ideal due to the small number of patients and the fact that the treated group was significantly older than the placebo-treated group. Nevertheless, the forced vital capacity of treated patients was better than those of placebo-treated patients, suggesting a protective effect on respiratory function. Idebenone is currently tested in a larger group of DMD patients in two stages; first, in patients that have never been treated with cortico-steroids and then in combination with corticosteroids (http://clinicaltrials.gov/ct2/show/NCT01027884). Results are anticipated in the beginning of 2014.
Similarly, decaffeinated green tea extract is rich in antioxidants (polyphenols), and treatment with green tea extract reduced fibrosis and improved muscle function in mdx mice . This compound is currently tested in a small clinical trial in Germany in DMD patients (http://clinicaltrials.gov/ct2/show/ NCT01183767).
There is a plethora of other compounds that im-prove muscle quality in mdx mice, some of which are currently also tested in clinical trials in patients (see Table I). However, one has to realize that the lack of dystrophin is less disastrous in mdx mice than in hu-mans. Due to a very effective regeneration and up-regulation of utrophin, survival in mdx mice is near normal, muscle function is only slightly impaired compared to wild type mice and muscle quality is much better than in humans. Therefore, compounds that have an effect on muscle quality in mice, do not necessarily work in humans.
Attempts are also made to improve muscle mass, in order to compensate for the loss of muscle tissue. There are growth factors that enhance muscle mass and fac-tors that inhibit the formation of muscle mass. Of the latter, myostatin is the most well known, as mutations that lead to complete loss of myostatin are viable and have been described for several animals (e.g. Belgium Blue cattle, Texel sheep and dogs) and one human boy . In each case lack of myostatin results in a vast increase in muscle mass. Thus, by inhibiting myostatin one could perhaps compensate for the loss of muscle tissue observed in DMD patients and also other dis-eases where loss of muscle tissue is the main symptom (i.e. most neuromuscular disorders). Anti-myostatin antibody treatment in adult neuromuscular disorder patients (including BMD patients) did not lead to a significant increase in muscle mass in a first clinical trial . It should be noted however that patients were treated for only one month. Meanwhile, a new fusion protein joining a human antibody Fc receptor and the ligand binding domain of the activin type IIB receptor has been generated (ACE-031) . This receptor can bind myostatin, but also its family member TGF-P, which is implicated in fibrosis formation in many diseases, including DMD. Thus, ACE-031 may act through separate mechanisms simultaneously, both increasing muscle mass and inhibiting fibrosis. In mice treatment resulted in increased muscle mass and in mdx mice in a normalization of muscle force (www.acceleronpharma.com). In a phase I clinical trial in healthy volunteers (postmenopausal women) treat-ment resulted in a dose dependent increase in muscle mass at the cost of fatty tissue. Treatment was toler-ated well for both single and multiple doses (www. acceleronpharma.com). Recently, a dose escalation safety trial in DMD patients was suspended due to unexplained nose and gum bleeding and dilated small blood vessels observed in some treated patients (www. acceleronpharma.com). The mechanism of these un-expected side effects is currently under investigation.
As DMD is a mono-allelic and recessive disease, gene therapy is a logical therapeutic approach: add-ing a functional DMD gene would allow production of dystrophin protein in patients. Viruses are efficient at delivering genomic information to host tissues and viral vectors, where the viral genes have been replaced by a gene of choice, are generally used to deliver thera-peutic genes. There are however two major challenges for the development of gene therapy for DMD. The first is the abundance and accessibility of muscle tissue. Muscle tissue makes up 30-40% of the human body, and in DMD most of the skeletal muscles and heart are affected. Thus, it will be difficult to target each and every muscle fiber, or even the majority. This is further impeded by the fact that muscle fibers are surrounded by layers of connective tissues, which fil-ter out most of the larger viral particles. In fact there is only one viral vector that can efficiently infect the postmitotic muscle and heart fibers, the adeno associated viral (AAV) vector . AAV is a small virus and as such its capacity is limited to ~4.9 kb. Unfor-tunately, the DMD gene happens to be the largest gene in the genome, with 2.4 Mb it is close to 0.1% of the complete genome. Even the coding sequence is too large (11 kb) to be encompassed by AAV vectors. However, there are BMD patients lacking large parts of the central domain, implying that as long as both the actin and extracellular matrix connecting domains and some of the interspersing parts are present, dys-trophin will have some functionality . Thus, mi-crodystrophins were synthetically engineered, con-taining only the most crucial domains . Several of these microdystrophins have been generated, and when expressed as transgenes in a dystrophin negative background they improved muscle function and quality . One of the most used microdystrophins contains the N-terminal actin-binding domain, the cysteine-rich domain (that connects to the extracel-lular matrix), 4 of the 24 spectrin-like repeat domains and 3 of the 4 proline-rich hinge domains that are located between the N-terminal and the cysteine-rich domain. The gene encoding this microdystrophin is small enough to fit into an AAV vector and local and systemic treatment of mdx mice with an AAV vector containing this gene resulted in good transduction and improved muscle function and muscle quality . Treatment with AAV vectors containing micro-dystrophin and vasculature-permabilization factors has shown to induce some dystrophin expression in skeletal muscle and also heart, albeit at lower levels than observed in the skeletal muscles . There are many AAV serotypes, some of which have extreme tropism for muscle (e.g. AAV5, AAV8 and AAV9) and heart (e.g. AAV8) and are thus ideally suited for gene therapy for muscles.
The second challenge is potential immunity against the AVV and/or the microdystrophins, since unfortunately, despite early reports that the AAV vector was not immunogenic, these AAVs turned out to induce an antibody response in mice and a more full blown immune response (both antibodies and T-cells) in larger animal models and humans . A clinical trial with microdystrophin delivered by AAV through intramuscular injection in 6 patients has been conducted without immune repression . An immune response to the viral capsid was found in a muscle biopsy of each of the 6 injected patients, while only in two patients a few microdystrophin expressing muscle fibers were found in a muscle biopsy. Unex-pectedly, an immune response was also found to the newly expressed microdystrophin in 4 patients . It was always assumed dystrophin restoration would not result in an immune response in DMD patients, as most DMD patients make some dystrophin. This can be in the form of revertant fibers (i.e. dystrophin positive fibers due to either spontaneous exon skip-ping or a frame-restoring secondary mutation, which are present at low levels in most patients) and/or as in most patients at least some dystrophin isoforms are still expressed, since the promoter of the ubiquitous Dp71 isoform is located in intron 62, which is present in most patients. Interestingly, in two patients, dystro-phin-specific T-cells were already present before the injection with AAV containing microdystrophin . Recent research showed that more patients contain
autoreactive T-cells that recognize dystrophin in their circulation (Jerry Mendell, personal communication). Whether this will have negative consequences on dystrophin restoring therapies is not yet clear. The T-cells have only been found in the circulation and not in muscle tissue of the patients; furthermore these pa-tients have revertant fibers containing the dystrophin epitopes recognized by the T-cells, suggesting that these T-cells do not extravagate to target dystrophin expressing muscle fibers .
Unfortunately, whole body treatment with AAV is currently not feasible in humans and the immune response elicited by AAV will hamper repeated injec-tions with AAV. Current research is optimizing treat-ment of muscle groups or limbs and using transient immune suppression to allow repeated treatments.
Cell therapy is in fact a form of gene therapy, as cells from a healthy donor will contain the functional DMD gene. The added benefit is that these cells will also contribute to the regeneration of damaged muscle in patients. In theory cell therapy thus seems very appealing. However, again the abundance and acces-sibility of muscle tissue is impeding this approach. Furthermore, muscle tissue is primarily post-mitotic. Upon muscle damage, satellite cells are activated and proliferate and repair the damage . These satellite cells can be isolated from healthy donors and expand-ed ex vivo and the resulting cells (called myoblasts) can be transplanted into patients. Unfortunately, early clinical trials using this approach revealed that the majority of myoblasts died quickly and that none of them was able to leave the blood stream to migrate into muscle tissue . Even upon direct injection into muscle the migration of myoblasts was poor. To overcome this, multiple injections have been used and dystrophin restoration has been obtained by high-density injections of many (25-250) injections per cubic centimeter [26,27]. While this may be feasible for some small superficial muscles, it is not for larger and/or more difficult to reach muscles such as the dia-phragm and whole body treatment.
It has become clear that there are other stem cells that are able to migrate from the blood stream into muscle tissue and participate in muscle fiber regeneration. These include cells from the immune system, from blood vessel walls (mesangioblasts), fat stem cells (pericytes) and bone cells . For most of these cells the efficiency is very low and often dystrophin positive fibers in treated animal models are below 5%. Mesangioblasts are the most promising cell type so far. In the golden retriever muscular dystrophy dog (GRMD) model, mesangioblast transplant ation by intra-arterial injection resulted in up to 10% of dys-trophin positive fibers and functional improvement in these dogs . In March 2011 a clinical trial in which DMD patients will undergo several consecutive mesangioblast transplantations from healthy HLA-matched family members has been initiated in Italy (Giulio Cossu, personal communication).
Instead of trying to bring back dystrophin into muscle, an alternative strategy is to increase expres-sion of the utrophin, an autosomal dystrophin homo-logue, which can functionally compensate (to some extent) for the lack of dystrophin. Like dystrophin, utrophin forms a link between the cytoskeleton and the extracellular matrix. Structurally utrophin is very similar to dystrophin: the N-terminal, cysteine-rich and C-terminal domains show ~80% similarity and the spectrin-like repeat domain ~35%. Utrophin is ubiquitously present in muscle in early fetal stages, but levels decrease during development and in adult muscle it is solely found at the neuromuscular junc-tion, to retain its structural integrity. In developing and regenerating fibers it its present along the entire sarcolemma, where it is later replaced by dystrophin. In the absence of dystrophin, utrophin can be found along the entire muscle fiber membrane where it re-cruits most of the proteins normally associated with dystrophin [29-31]. In both DMD patients and the mdx mouse model, utrophin is upregulated , al-beit to a larger extent in mdx mice, which may in part underlie the fact that mdx mice are less severely af-fected than humans by lack of dystrophin. Indeed a mouse without both dystrophin and utrophin is very severely affected and dies at 2-3 months of age .
In the mdx mouse model it has been shown that transgenic overexpression of micro-utrophin ame-liorates the dystrophic phenotype . Full-length utrophin is even more efficient and could prevent pa-thology .
This started off high throughput screens for drugs that can increase utrophin expression. A cell model
stably expressing the utrophin promoter linked to luciferase has been used to assess the potential of thousands of drugs. One of these drugs, SMTC1100 was one of the most efficient hits . Its effect has been validated in the mdx mouse model, where oral treatment with this compound increased utrophin ex-pression up to twofold, resulted into improved muscle histology and function . Unfortunately, a phase I clinical trial in healthy volunteers revealed that the drug plasma levels needed to induce an effect could not be obtained in humans . Current research focuses on reformulation of this compound, and on the screening of other compounds that can increase utrophin expression.
Biglycan is an endogeneous protein that is present during development outside skeletal muscle fibers and cardiac muscle. It plays an important role in the regu-lation of signaling pathways and structural proteins, among which proteins that are part of the dystrophin associated glycoprotein . Biglycan injection (local and systemic) in mdx mice resulted in upregulation of utrophin and improved muscle function and resis-tance to exercise-induced damage . Clinical trials are being planned for 2012 or 2013.
MUTATION SPECIFIC THERAPIES – READTHROUGH OF STOP CODONS
Some aminoglycoside antibiotics were shown to have the potential to force the translational machin-ery to ignore stop codons and build in an amino acid instead. This would have therapeutic potential for an estimated 14% of patients who carry nonsense muta-tions somewhere in their DMD gene, while the rest of the transcript is unaffected . In vitro studies in cells derived from DMD/BMD-patients variable readthrough efficiencies (form 1 to 10%) were ob-served and showed that this is dependent on the type of stop codon mutation (UGA, UAA or UAG) and the nucleotides flanking the stop codon . Early studies with gentamicin in the mdx mouse model (which has a nonsense mutation in exon 23 of the mouse Dmd gene) resulted in dystrophin levels of up to 20% . Ensuing clinical trials in patients showed poor results. This turned out to be due to dif-ferent gentamicin isomers. Only one isomer has high readthrough activity, and gentamicin batches contain a mix of different isomers, which ratio varies between different batches . A recent clinical trial using the proper isomer showed a drop in serum creatine kinase levels in patients with stop mutations, while those in patients with frame shifting deletions remained unal-tered after one month of gentamicin treatment . A follow up study in which patients with stop mutations were treated for six months led to a clear increase in dystrophin levels (up to 15%) in 3 out of 16 patients . Unfortunately, chronic treatment with gentamicin is toxic, so, due to the risk of irreversible oto-and nephrotoxicity long-term treatment with this antibiotic is not possible.
A screening system in a cell system with a luciferase enzyme with a stop mutation identified a new compound with readthrough potential. This compound, PTC124 (ataluren), has a much better safety profile than genta-micin. It is more selective for premature stopcodons and reportedly does not induce readthrough of normal stop codons . Furthermore, unlike gentamicin, which needs to be given intravenously, this compound can be taken orally. Studies in the mdx mouse model showed dystrophin levels of up to 25%, which was accompanied by improved muscle strength and lower serum creatine kinase levels . A study in human volunteers con-firmed that ataluren was well tolerated, and in a study where patients were treated for 4 weeks with different doses of ataluren, a modest increase in dystrophin levels . In a placebo-controlled phase II/III trial patients were treated with two doses or placebo for 48 weeks and then all were treated with the high dose in an open label extension study . Unfortunately, the primary outcome (30 meter improvement in the six minute walk test) was not reached and the extension study was put on hold. What is interesting is that no difference could be found between the high dose and placebo groups, while the low dose group appeared to do better, though not significantly. This suggests that the drug has a “bell shaped curve”, meaning that there is an optimal concen-tration and that both lower but also higher concentra-tions perform less. No results on the analysis of dystrophin levels have been reported yet. Different analysis of subgroups of patients is currently ongoing, as well as studies to identify the most optimal dose. The open label extension study has been reopened in the USA, but not yet elsewhere in the world.
The approach that is currently closest to clini-cal application is antisense mediated exon skipping.Here, the aim is to restore the reading frame of the dystrophin transcripts through manipulation of the splicing process to allow production of a BMD-like, partially functional dystrophin. This can be achieved using antisense oligonucleotides, which are modified pieces of DNA or RNA, that hybridize to a target exon during pre-mRNA splicing, thereby hiding it from the splicing machinery, resulting in the skipping of the targeted exon . For most DMD patients, the reading frame can be restored through the skip-ping of one or two exons . This approach will not apply to all patients, as the domains binding to the cytoskeleton and the extracellular matrix are essential for protein functionality. Fortunately, most DMD patients have deletions clustering in the region between exon 45 and exon 55 in the redundant central domain of the protein. This clustering also means that, while exon skipping is a personalized medicine approach, the skipping of some exons applies to more patients. The largest group of patients would benefit from the skipping of exon 51 (13% of all patients) , which is why this is currently the focus of clinical develop-ment for this approach.
After initial experiments that showed that exon skipping and dystrophin restoration was feasible in patient-derived cell cultures and resulted also in functional improvement in the mdx mouse model, exon 51 skipping was tested in clinical trials in DMD patients . There are two different chemical modifi-cations that are currently explored in patients: Pro-sensa Therapeutics and GlaxoSmithKline (GSK)) use the 2′-O-methyl phosphorothioate modification (2OMePS, PRO051/ GSK2402968), while AVI-Biopharma uses phosphorodiamidate morpholino oligomers (PMO, AVI-4658) . Both showed comparable levels of dystrophin after intramuscu-lar injection with the respective compounds in the absence of serious side effects [48-50]. While these trials were crucial and showed significant dystrophin restoration in patients for the first time, whole body treatment is required. Antisense oligonucleotides are very small and as such will be filtered out by the kid-ney leading to a short serum half-life. For PMOs this is indeed the case, but 2OMePS antisense oligonucle-otides have a much improved serum half-life, as the sulfur atom in the phosphorithoate backbone allows low affinity binding to serum proteins, that act as a carrier and prevent clearance from the kidney . Nevertheless, uptake of antisense oligonucleotides by healthy muscle is very low, so body-wide treatment of muscle tissue with antisense oligonucleotides was long thought to be difficult. However, studies where wild type and mdx mice were systemically treated with 2OMePS antisense oligonucleotides revealed that uptake in dystrophic muscle is up to ten-fold higher than in healthy muscle . It is hypothesized that the antisense oligonucleotides (both PMOs and 2OMePS) enter the muscle fibers through the same holes that allow the creatine kinase to escape. Thus, in a strange twist of fortune, the disease state is helping with the delivery of the therapeutic compound.
After optimization of systemic treatment in animal models, systemic trials were initiated for both compounds. For the 2OMePS a trial with PRO051 was completed in 2009 where groups of patients received 5 weekly subcutaneous injections in doses varying from 0.5 mg/kg to 6 mg/kg. All patients then enrolled in an open label extension study where they received weekly subcutaneous injections of the highest dose. The trial resulted in exon 51 skipping and dystrophin restoration in the majority of muscle fibers (60-100%) in a dose dependent way in levels up to 16% in the absence of serious side effects . More importantly, after 3 months in the open label extension study, there was a clear improvement in the 6 minute walk test for most of the patients . Of course these results must be interpreted with a great deal of caution as a placebo group is lacking and a placebo effect is expected, since all patients know they receive the drug. In January 2011 GSK initiated a placebo-controlled trial where 180 patients will be treated either with GSK2402968 or placebo for 12 month to assess whether long term treatment with this 2OMePS antisense oligonucleotide is safe and leads to functional improvement. In parallel, a study where 2 different dosing regimes are compared and a study to assess the pharmacodynamic profile in non-ambulant patients with the same drug are ongoing and planned. Finally, Prosensa is conducting a dose escalation trial with PRO044, a 2OMePS targeting exon 44 (applying to 6% of patients) (see Table I).
A systemic trial with AVI-4658 has been complet-ed as well. Here, patients received weekly intravenous infusions with 0.5 mg/kg – 20 mg/kg of the PMO for 12 weeks. Again a dose dependent increase in dys-trophin was observed. Notably, 3 patients responded very well (one each in the 2 mg/kg, 10 mg/kg and 20 mg/kg group), showing dystrophin in up to 50% of fibers and levels of up to 21% and in the 2 highest dose groups (10 and 20 mg/kg) all patients clearly showed dystrophin restoration (Muntoni, personal communi-cation). A trial where higher doses (30 and 50 mg/kg) will be tested for 6 months will be initiated soon.
Although AON uptake is better in DMD muscle than in healthy muscle, research is undertaken to fur-ther improving uptake by the target tissues (muscle and heart). For PMOs conjugating them to a cell-penetrating peptide (pPMOs), markedly increased the exon skip efficiency (also in the cardiac tissue) in mice models [54,55] and could even rescue the very severely affected dystrophin/utrophin-deficient dou-ble-knockout mouse . However pPMOs turned out to be toxic in primates  and the peptide might evoke an immune response.
The mutation specificity of this approach will pose a major challenge as currently each antisense oligo-nucleotide is considered a new drug by the regulat-ing authorities, meaning that each will need to un-dergo preclinical toxicity tests and will need to go through the clinical trial phases . There is hope that perhaps less extensive clinical testing is required for future antisense oligonucleotides will be allowed by the regulatory authorities once safety and efficacy has been confirmed for 2 (or more) antisense oligonucleotides of the same chemistry. In either case the traditional development phases will not apply to exon skipping for the rarer mutations, as there are simply not sufficient patient numbers to conduct a large scale clinical trial .
Another challenge is that, due to turnover of antisense oligonucleotides and muscle fibers, patients need repeated treatment. There are human genes that consist of an RNA and a protein part and it is possible to replace this regular RNA part with an antisense sequence of interest. The most often used genes for this are the U1 and U7 small nuclear ribonucleopro-teins [60,61]. As these genes are very small they fit within an AAV vector. In mdx mice treatment with AAV containing U1 or U7 antisense genes resulted in exon 23 skipping, dystrophin restoration and func-tional improvement [60,61]. However, this approach faces the same problems as AAV-mediated delivery of microdystrophin.
Finally a word of caution is in order. While results obtained with exon skipping so far are very encourag-ing, the trials to validate whether exon 51 skipping re-ally works (i.e. leads to functional improvement) and is safe are still ongoing. Thus, exon skipping is still a potential therapeutic approach and not yet a treat-ment and, until the results of the placebo-controlled trials are reported, patients should not be treated with these compounds .
Table I:Compounds currently or recently in clinical trials for DMD
1 Using this unique identifi er more information about trials can be found on www.clinicaltrials.gov.
There are many different therapeutic approaches being developed and many have reached the clinical trial phase. However, one should not get too high ex-pectations, as in general less than 10% of compounds that enters the clinical trial phase makes it to a reg-istered drug. The application closest to clinical application (exon skipping) is mutation specific and only applies to a subset of patients. Furthermore, it will not cure patients, but improve their phenotype. While it is understandable that the main focus is a treatment or a cure, one should not forget that improved care has made a huge impact on survival (from below 20 to over 30) and quality of life [8,9].
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Annemieke Aartsma-Rus LUMC S4-P, Albinusdreef 2, 2333 ZA Leiden, Netherlands, Tel +31 71 5269436, Fax +31 71 5268285, Email: firstname.lastname@example.org