**6. Clinical trials with antisense-mediated exon skipping**

#### **6.1 Local treatment with AONs**

68 Neuromuscular Disorders

AONs are sequence-, and therefore species-, specific. So, to be able to test human-specific AONs, a mouse containing the full-length human *DMD* gene was generated (*hDMD*). These mice have a fully functional *hDMD* transgene integrated on mouse chromosome 5. The functionality of the transgene was proven by rescuing the severe dystrophic phenotype of the *mdx/utrn-/-* mouse after crossing of both models ('t Hoen et al., 2008). Intramuscular injection (gastrocnemius) of 2OMePS AONs against exon 44, 46 or 49, induced specific skipping of the targeted human exons. It also highlighted the sequence-specificity of the AONs, since in the corresponding mouse sequences, with only 2 or 3 mismatches, no detectable skipping was observed (Bremmer-Bout et al., 2004). As described before, PMOs were more efficient in the *mdx* mouse than 2OMePS AONs. However in the *hDMD* mouse, AONs targeting human exon 44, 45, 46 or 51 were comparably effective or only marginally different between both chemistries. This indicated that the differences between PMO and 2OMePS AONs are probably more due to sequence differences than to chemistry differences. Furthermore, it also suggested important differences in sequence-specificity. 2OMePS AONs with 2 mismatches had a greatly reduced efficiency, whereas PMO AONs remained equally effective. This can increase the risk of off-target side effects (Heemskerk et

Studies in these *hDMD* mice revealed that the uptake of AON by the healthy *hDMD* muscle fibres is much lower than by dystrophic *mdx* fibres. This can probably be explained by the dystrophic nature of the *mdx* fibres: the lack of dystrophin results in damage to the muscle fibres, leading to leakage of the muscle enzyme creatine kinase into the bloodstream. It has been proposed that the AONs migrate into the muscle fibres through these same holes (Hoffman, 2007). In this way the disease is facilitating delivery of the potential therapeutic compound. Indeed AON uptake and skipping in the *hDMD* mouse is more difficult. The exon skipping levels observed after intramuscular injection with either 2OMePS or PMO AONs were lower than previously observed in the *mdx* mouse and in cell cultures (Heemskerk et al., 2009a). A pilot experiment with systemic (intravenous) injection of 2OMePS AONs targeting exon 51 in the *hDMD* mouse resulted in very low or undetectable exon skipping in the muscles (Heemskerk et al., 2010). Recently, vivo-morpholinos against exon 50 were shown to be able to achieve high levels of exon skipping after systemic (intravenous) injection in the healthy skeletal muscles of the *hDMD* mouse and even low levels in the cardiac muscle. There were no large signs of toxicity or adverse effects, only a small increase in serum CK levels, which could reflect a bit of membrane integrity disturbance (Wu et al., 2011). The influence of the nature of the muscle fibres on AON delivery efficiency might also explain why targeting of the heart is so difficult. The heart muscle is structurally and pathologically different from skeletal muscle, since it is made up

First AON experiments with the canine model have been performed *in vitro* in myoblast cell cultures of the GRMD dog. The nature of the mutation requires the skipping of 2 exons (exon 6 and exon 8) to restore the reading frame, thereby making it more challenging. *In vitro*, 2OMePS AONs induced higher exon skipping levels than the PMOs, but only for a short term and without induction of detectable dystrophin protein. PMOs could restore a low level of dystrophin production, but only at very high concentrations. pPMOs could

of individual cardiomyocytes, which do not become 'leaky'.

**5.2 AONs in the canine models** 

al., 2009b).

After the promising preclinical results *in vitro* and *in vivo*, the first clinical trials were initiated. These trials used local (intramuscular) injections to obtain proof-of-principle in humans and examine possible adverse effects. Normally, the first human trials are done in healthy volunteers (phase I). However, this is not possible in this case, since exon skipping in healthy persons would result in disruption of the reading frame. Therefore this phase was skipped and AONs were tested immediately in DMD patients (phase I). These first trials focused on skipping of exon 51 for both 2OMePS (in 2006) and PMO AONs (in 2008), since this would be applicable to the relatively largest group of known mutations (13%) (Aartsma-Rus et al., 2009a).

A single injection in the tibialis anterior with 0.8 mg of a 2OMePS AON (called PRO051) in 4 patients resulted in specific exon 51 skipping without adverse effects. It restored dystrophin expression at the sarcolemma in 64-97% of the myofibres and restored protein levels till 17- 35% of control levels. However, it also clearly indicated the importance of muscle quality since the target of AONs, the dystrophin transcript, is only expressed in muscle fibres and not in adipose and fibrotic tissue, which replaces the muscle tissue when the disease progresses. The patient with the lowest dystrophin levels had the most advanced disease state and relatively little muscle tissue left (van Deutekom et al., 2007).

For PMO AONs a placebo-controlled, single-blinded study was performed. Seven patients received an injection with a PMO AON (called AVI-4658) into their extensor digitorum brevis (EDB) and saline into the contralateral muscle. In 2 patients receiving the lowest dose (0.09 mg) this resulted in low levels of exon 51 skipping, but no observed increase in dystrophin expression. However, a clear dystrophin restoration was observed in the higher dose (0.9 mg) group. As for the PRO051 study no adverse events, like an inflammatory response, were observed. Immunofluorescent staining for dystrophin indicated 11-21% higher intensity levels in the AON-treated muscle compared to the contralateral saline-

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 71

The next steps are larger randomised, placebo-controlled studies and targeting of other exons. For GSK2402968 a phase III study was initiated in January 2011. 180 ambulant patients will receive 6 mg/kg bodyweight AON once weekly for 1 year or placebo (http://clinicaltrials.gov/ct2/show/NCT01254019?term=GSK2402968&rank=1). This study will tell us whether long-term treatment is safe and leads to functional improvement or slowing down of disease progression (compared to placebo-treated patients). In parallel, a study in non-ambulant patients with different AON doses, primarily to determine the pharmacokinetical profile in older patients, and a study in ambulant patients where different treatment regimes are compared, are conducted (http://clinicaltrials.gov/ct2/show/NCT01128855?term=GSK2402968&rank=3 and http://clinicaltrials.gov/ct2/show/NCT01153932?term=GSK2402968&rank=2). In addition a clinical trial for AVI-4658 (eteplirsen) with higher doses (30 mg/kg and 50 mg/kg bodyweight) for 24 weeks has been initiated to assess its efficacy and safety (http://clinicaltrials.gov/ct2/show/NCT01396239?term=eteplirsen&rank=1). These trials focus on skipping of exon 51, applicable to the relative largest group of patients. Skipping of exon 44 would be useful for another large group of patients (6.2%) (Aartsma-Rus et al., 2006b). A phase I/IIa study with PRO044 (2OMePS AON against exon 44) with the same set-up as the phase I/IIa study for PRO051 is currently ongoing (http://clinicaltrials.gov/ct2/show/NCT01037309?term=PRO044&rank1). Furthermore, preclinical studies with other 2OMePS AONs (against exon 45, 52, 53 and 55) are performed by Prosensa Therapeutics. In addition to this, preclinical tests with AVI-5038 (pPMO AON against exon 50) are ongoing, although toxicity issues with this pPMO have been reported

(http://investorrelations.avibio.com/phoenix.zhtml?c=64231%20&p=irol-

The efficacy of AONs depends partly on the amount of AON that reaches its target, i.e. the muscle fibre nuclei. Several strategies to improve muscle-specific uptake are under investigation, like muscle-homing peptides and cell-penetrating peptides (see above). Due to AON clearance and turnover, the effect of AONs is only temporarily, thus repeated, life-long, injections are required, should this approach prove to be efficacious. The first clinical trials showed that the average serum half-life was 29 days for 2OMePS AONs and around 1.5 hours for PMOs. A way to allow a more prolonged effect is the use of viral vectors stably expressing modified small nuclear ribonucleoprotein (snRNP) genes, in which the normal antisense sequence is replaced by an antisense sequence of choice. snRNPs are small protein-RNA hybrids that are amongst others involved in pre-mRNA splicing and histone processing. The U1 and U7 snRNPs have been used most in splicing modulation experiments (Brun et al., 2003). Exon 51 targeting U1 snRNPs induced effective skipping of exon 51 and rescue of dystrophin synthesis in a patient-derived cell line (De Angelis et al., 2002). Adeno-associated viruses (AAVs) are very efficient at transferring genes into skeletal muscles. Injection of AAV vectors expressing U7 or U1 snRNPs targeting mouse exon 23 resulted in sustained production of functional dystrophin in the *mdx* mouse after intramuscular injection and body-wide dystrophin expression and reduced muscle wasting after systemic treatment (Denti et al., 2008; Goyenvalle et al., 2004). However serious problems with the use of AAV vectors are the possibility of an immune response against the viral capsid and the difficulty to produce them on a large scale under good manufacturing practice (GMP), necessary for implementation in

newsArticle&ID=1406001&highlight=).

**7. Improvement of AON delivery and efficiency** 

treated muscle, and levels of 22-32% of control dystrophin levels (Kinali et al., 2009). Since both studies studied different muscles and used different techniques for quantifying immunocytochemistry the results are not directly comparable (Aartsma-Rus & van Ommen, 2009). However both studies showed unequivocal effectiveness of the used compound in the absence of side effects.

#### **6.2 Systemic treatment with AONs**

The next step towards clinical application of exon skipping are systemic clinical trials. The first pilot experiment has been conducted in Japan. Takeshima et al. treated 1 DMD patient intravenously with a weekly dose of 0.5 mg/kg bodyweight of a PS AON against exon 19 for 4 weeks. Only very low levels of exon skipping and dystrophin protein were observed in a muscle biopsy (Takeshima et al., 2006). This is not surprising, as the dose used was very low and the PS backbone chemistry is not ideal for exon skipping purposes (see above). Furthermore, this was only 1 single patient, so no real, reliable conclusions can be drawn from this experiment.

More extensive, open-label, dose-escalation, phase I/IIa studies have recently been completed for both 2OMePS and PMO AONs. The first was a study with abdominal subcutaneous injections of PRO051 (2OMePS AON, now called GSK2402968)) in 12 patients testing 5 weekly doses (0.5, 2, 4 and 6 mg/kg bodyweight) in groups of 3 patients. Doses of 2 mg/kg bodyweight or higher resulted in specific exon 51 skipping. In 10 out of 12 patients dystrophin expression in a tibialis anterior biopsy could be observed in 60-100% of the muscle fibres at levels up to 15.6% of healthy levels in a dose-dependent manner. After analysis of this first phase (6 to 15 months later), all patients entered an open-label extension study in which they received weekly injections of the highest dose. After 12 weeks, this resulted in functional improvement as measured by the 6-minute walk test. Since a placebo group is lacking, interpretation of this improvement must be done with caution. Nevertheless, the overall results are encouraging and only mild adverse events, like irritation at the injection side and mild proteinuria, were observed (Goemans et al., 2011).

AVI-4658 (PMO AON, also called eteplirsen) was tested by 12 weekly intravenous infusions of different doses (0.5, 1, 2, 4, 10 and 20 mg/kg bodyweight) in a total of 19 patients, without serious adverse events. In a biceps biopsy, exon 51 skipping and restoration of protein expression was observed starting at a dose of 2 mg/kg bodyweight, albeit variable between individual patients. The responding patients showed dystrophin levels of 8-16% of healthy controls by immunofluorescent staining. Notably, there were 3 patients who responded very well, with up to 55% of dystrophin-positive fibres by immunofluorescent staining and dystrophin levels up to 18% by western blot. In 4 other patients some improvement was observed. Furthermore, the functionality of the newly formed proteins was confirmed by the restoration of DGC-associated proteins at the sarcolemma. In addition, a reduction of inflammatory infiltrates was observed in the highest dose group, which probably indicates a reduction in necrosis and an increased resistance to mechanical load (Cirak et al., 2011). Not all patients responded equally well, which may be explained by the short serum half-life of PMOs. Since PMOs do not bind to plasma proteins (see above), they are rapidly filtered out by the kidney (accounting for 40-60% of total plasma clearance). Thus, the amount available for uptake by other tissues (e.g. muscles) is low. Therefore further optimisation (e.g. higher doses) is needed.

treated muscle, and levels of 22-32% of control dystrophin levels (Kinali et al., 2009). Since both studies studied different muscles and used different techniques for quantifying immunocytochemistry the results are not directly comparable (Aartsma-Rus & van Ommen, 2009). However both studies showed unequivocal effectiveness of the used compound in the

The next step towards clinical application of exon skipping are systemic clinical trials. The first pilot experiment has been conducted in Japan. Takeshima et al. treated 1 DMD patient intravenously with a weekly dose of 0.5 mg/kg bodyweight of a PS AON against exon 19 for 4 weeks. Only very low levels of exon skipping and dystrophin protein were observed in a muscle biopsy (Takeshima et al., 2006). This is not surprising, as the dose used was very low and the PS backbone chemistry is not ideal for exon skipping purposes (see above). Furthermore, this was only 1 single patient, so no real, reliable conclusions can be drawn

More extensive, open-label, dose-escalation, phase I/IIa studies have recently been completed for both 2OMePS and PMO AONs. The first was a study with abdominal subcutaneous injections of PRO051 (2OMePS AON, now called GSK2402968)) in 12 patients testing 5 weekly doses (0.5, 2, 4 and 6 mg/kg bodyweight) in groups of 3 patients. Doses of 2 mg/kg bodyweight or higher resulted in specific exon 51 skipping. In 10 out of 12 patients dystrophin expression in a tibialis anterior biopsy could be observed in 60-100% of the muscle fibres at levels up to 15.6% of healthy levels in a dose-dependent manner. After analysis of this first phase (6 to 15 months later), all patients entered an open-label extension study in which they received weekly injections of the highest dose. After 12 weeks, this resulted in functional improvement as measured by the 6-minute walk test. Since a placebo group is lacking, interpretation of this improvement must be done with caution. Nevertheless, the overall results are encouraging and only mild adverse events, like irritation at the injection side and mild proteinuria, were observed (Goemans et al., 2011). AVI-4658 (PMO AON, also called eteplirsen) was tested by 12 weekly intravenous infusions of different doses (0.5, 1, 2, 4, 10 and 20 mg/kg bodyweight) in a total of 19 patients, without serious adverse events. In a biceps biopsy, exon 51 skipping and restoration of protein expression was observed starting at a dose of 2 mg/kg bodyweight, albeit variable between individual patients. The responding patients showed dystrophin levels of 8-16% of healthy controls by immunofluorescent staining. Notably, there were 3 patients who responded very well, with up to 55% of dystrophin-positive fibres by immunofluorescent staining and dystrophin levels up to 18% by western blot. In 4 other patients some improvement was observed. Furthermore, the functionality of the newly formed proteins was confirmed by the restoration of DGC-associated proteins at the sarcolemma. In addition, a reduction of inflammatory infiltrates was observed in the highest dose group, which probably indicates a reduction in necrosis and an increased resistance to mechanical load (Cirak et al., 2011). Not all patients responded equally well, which may be explained by the short serum half-life of PMOs. Since PMOs do not bind to plasma proteins (see above), they are rapidly filtered out by the kidney (accounting for 40-60% of total plasma clearance). Thus, the amount available for uptake by other tissues (e.g. muscles) is low. Therefore further optimisation (e.g. higher

absence of side effects.

from this experiment.

doses) is needed.

**6.2 Systemic treatment with AONs** 

The next steps are larger randomised, placebo-controlled studies and targeting of other exons. For GSK2402968 a phase III study was initiated in January 2011. 180 ambulant patients will receive 6 mg/kg bodyweight AON once weekly for 1 year or placebo (http://clinicaltrials.gov/ct2/show/NCT01254019?term=GSK2402968&rank=1). This study will tell us whether long-term treatment is safe and leads to functional improvement or slowing down of disease progression (compared to placebo-treated patients). In parallel, a study in non-ambulant patients with different AON doses, primarily to determine the pharmacokinetical profile in older patients, and a study in ambulant patients where different treatment regimes are compared, are conducted (http://clinicaltrials.gov/ct2/show/NCT01128855?term=GSK2402968&rank=3 and http://clinicaltrials.gov/ct2/show/NCT01153932?term=GSK2402968&rank=2). In addition a clinical trial for AVI-4658 (eteplirsen) with higher doses (30 mg/kg and 50 mg/kg bodyweight) for 24 weeks has been initiated to assess its efficacy and safety (http://clinicaltrials.gov/ct2/show/NCT01396239?term=eteplirsen&rank=1). These trials focus on skipping of exon 51, applicable to the relative largest group of patients. Skipping of exon 44 would be useful for another large group of patients (6.2%) (Aartsma-Rus et al., 2006b). A phase I/IIa study with PRO044 (2OMePS AON against exon 44) with the same set-up as the phase I/IIa study for PRO051 is currently ongoing (http://clinicaltrials.gov/ct2/show/NCT01037309?term=PRO044&rank1). Furthermore, preclinical studies with other 2OMePS AONs (against exon 45, 52, 53 and 55) are performed by Prosensa Therapeutics. In addition to this, preclinical tests with AVI-5038 (pPMO AON against exon 50) are ongoing, although toxicity issues with this pPMO have been reported (http://investorrelations.avibio.com/phoenix.zhtml?c=64231%20&p=irolnewsArticle&ID=1406001&highlight=).
