**5. Antisense-mediated exon skipping** *in vivo* **in animal models**

#### **5.1 AONs in mouse models for DMD**

#### **5.1.1 AONs in the** *mdx* **mouse model**

After the promising *in vitro* results, AONs have been tested *in vivo* in animal models. As mentioned before, the *mdx* mouse is most widely used. The target site for exon 23 was first optimised in *mdx* myotube cell cultures. This resulted in a 5' splice site targeting AON with a 2'-*O*-methoxyethyl phosphorothioate (2OMePS) backbone, which was tested locally in the *mdx* mouse. A single intramuscular injection of this compound in the tibialis anterior of both young (2 or 4 weeks old) and aged (6 months old) mice resulted in marked dystrophin expression 2 weeks after injection, which persisted up to 3 months after injection. The functionality of the dystrophin protein was suggested by the re-expression of dystroglycans, sarcoglycans and nNOS at the membrane. It also resulted in partial restoration of physiological function, maximum isometric titanic force, of the treated muscles. Importantly no auto-immune response against the newly formed dystrophin protein was observed (Lu et al., 2003). Of course, since DMD affects body-wide musculature, including heart and diaphragm, injection of every muscle separately is not feasible and systemic treatment is required. Three intravenous injections at weekly intervals resulted in dystrophin expression, highest in gastrocnemius, intercostal muscles and the diaphragm, without signs of toxicity or damage to other organs. However dystrophin could not be detected in the cardiac muscle (Lu et al., 2005). To optimise delivery and efficiency, different administration routes have

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 67

A PMO conjugated to a cell-penetrating peptide (pPMO) showed to be more effective than the naked PMO AON. Systemic (intravenous) treatment of *mdx* mice was very potent in both skeletal muscle, diaphragm and, importantly, heart. pPMOs lead to a decrease in CK levels (Jearawiriyapaisarn et al., 2008). Another study confirmed that the long term systemic treatment with pPMOs was effective in restoring dystrophin expression in skeletal muscle, improving muscle function and preventing heart failure (Wu et al., 2008). These pPMOs were also able to rescue the severe *mdx/utrn-/--* mouse model by systemic (intraperitoneal) treatment. Considerable improvement of muscle function was observed, combined with dystrophin expression in almost all muscles, except for the heart (Goyenvalle et al., 2010). Peptides might elicit an immune response, but no signs of such a response or toxicity were found in the mouse models so far. Unfortunately, when a pPMO compound was tested in primates, there were toxicity concerns. In cynomolgus monkeys pPMO doses equivalent to the ones used in mice, were not toxic, but also had little exon skipping effect. Higher doses were effective, but also caused tubular degeneration in the kidneys, a sign of renal toxicity (Moulton & Moulton, 2010). Yin et al. generated a chimeric fusion peptide consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (Bpeptide), which they conjugated to a PMO oligomer (B-MSP-PMO). These B-MSP-PMOs were already efficient at very low doses in restoring high levels of dystrophin expression body-wide (Yin et al., 2009). Novel cell-penetrating peptides have been discovered by inducing modifications to a *Drosophila melanogaster*-derived R6-Penetratin peptide. These peptides are called PNA or PMO internalisation peptides (Pips). A conjugate of Pip2b and a PNA AON (Pip2b-PNA) resulted in approximately threefold higher dystrophin-positive fibres compared to the naked AON after local injection in the tibialis anterior of *mdx* mice (Ivanova et al., 2008). More and improved Pips have been developed. Pip5e fused with a PMO (Pip5e-PMO) showed high exon skipping efficiency after a single intravenous injection in the *mdx* mouse. Most importantly it also efficiently targeted the heart, leading to

dystrophin levels of more than 50% of wild-type levels (Yin et al., 2011).

were observed (Wu et al., 2009).

(Mitrpant et al., 2009).

**5.1.2 AONs in the other mouse models** 

Another modification of the PMO is conjugation to a dendrimeric octaguanidine polymer (vivo-morpholino). This modification also significantly improved the delivery and dystrophin production in *mdx* mice after intravenous injection. Repeated treatment resulted in dystrophin expression in almost 100% of the skeletal muscle fibres and levels of protein up to 50% of wild-type levels. Importantly, levels of ~10% of those found in healthy hearts were found in the cardiac muscle. In these mice no signs of an immune response or toxicity

Both 2OMePS and PMO AONs have also been tested in the *mdx4Cv* mouse. These mice require skipping of both exon 52 and 53 to remove the mutation and maintain the reading frame. Immortalised myoblast cell cultures from these mice were used to design the most effective AONs against exon 52 and 53, which were then tested *in vivo* in the *mdx4Cv* mice. 2OMePS AONs induced exon skipping in these cell cultures, but no dystrophin protein was observed. Intramuscular injection of the cocktail of AONs in the tibialis anterior resulted in sporadic exon skipping in this muscle, but no detection of dystrophin protein. A combination of PMO AONs against both exons resulted both *in vitro* and *in vivo* (after injection in the tibialis anterior) in exon skipping and restoration of dystrophin expression

been compared. Intravenous injection resulted rapidly in high plasma levels, which were quickly cleared. Peak plasma levels were twofold lower after subcutaneous and intraperitoneal injection, but clearance was much slower. Furthermore, intravenous injection resulted in very high AON levels in the kidney and liver, which might induce toxicity after long term treatment. Skipping levels were highest after intravenous injection and slightly lower for both subcutaneous and intraperitoneal injection. Dystrophin expression followed a similar pattern. Importantly, all 3 routes resulted in exon skipping and dystrophin expression in the heart, albeit at low levels. Due to the better pharmacokinetic profile of subcutaneous versus intravenous injection and slightly higher exon skipping compared to intraperitoneal administration, subcutaneous injection seemed to be the delivery method of choice. After subcutaneous treatment also a decrease in serum creatine kinase (CK) levels was observed. Creatine kinase is an enzyme that leaks out of the muscles into the blood stream when muscles are damaged, so a decrease indicates an improvement of muscle integrity (Heemskerk et al., 2010).

Morpholino (PMO) AONs have been shown to be effective *in vivo* as well. Intramuscular injection in the tibialis anterior elicited a dose-dependent increase in dystrophin expression in the majority of muscle fibres and dystrophin protein levels up to 60% of levels found in healthy muscle. Efficiency was comparable in both young (3 weeks old) and aged (6 months old) *mdx* mice. Repeated systemic (intravenous) injections induced exon skipping and expression of dystrophin protein body-wide, albeit with large variations between individual muscles. Highest levels were found in the quadriceps, abdominal and intercostal muscles. Lower levels were found in the tibialis anterior and diaphragm. CK levels were decreased and muscle function was improved as well. As with 2OMePS AONs, targeting of the cardiac muscle appeared difficult, since exon skipping and dystrophin expression were undetectable (Alter et al., 2006). Wu et al. showed that dystrophin restoration could be achieved (up to 30% of healthy levels) by systemic PMO treatment, although extremely high doses (up to 3 g/kg bodyweight) were required (Wu et al., 2010). Furthermore, a dosing regime of multiple low doses seems to be preferable above a few high doses to reduce the risk of toxicity and increase the efficiency, since both AONs and dystrophin protein show an accumulation over time (Malerba et al., 2009).

In the *mdx* mouse model PMOs appeared more effective and at lower doses compared to 2OMePS AONs. A direct comparison revealed that this was indeed the case for mouse exon 23 in the *mdx* mouse. Intramuscular injection of both AONs in the gastrocnemius, resulted in much higher skipping levels for PMOs than for 2OMePS AONs at the same molar amount. Systemic (intravenous) comparison in the *mdx* mouse showed, as had been noticed before, that most of the 2OMePS AONs are taken up by the liver and kidney. However the PMOs were almost exclusively taken up by the kidney. A possible explanation is that 2OMePS AONs bind to serum proteins, which prevents renal clearance (Geary et al., 2001), whereas PMOs do not, which explains their high renal clearance (Oberbauer et al., 1995). 2OMePS AON uptake was higher for all skeletal muscles, diaphragm and heart. In contrast to the biodistribution, exon skipping efficiency was much higher for the PMO AONs in skeletal muscle and diaphragm (approximately 40% versus 10%). Skipping levels in the heart were much lower and almost comparable between both compounds (2.5% for the PMOs versus 1.5% for the 2OMePS AONs). Protein levels followed the same pattern (Heemskerk et al., 2009b).

been compared. Intravenous injection resulted rapidly in high plasma levels, which were quickly cleared. Peak plasma levels were twofold lower after subcutaneous and intraperitoneal injection, but clearance was much slower. Furthermore, intravenous injection resulted in very high AON levels in the kidney and liver, which might induce toxicity after long term treatment. Skipping levels were highest after intravenous injection and slightly lower for both subcutaneous and intraperitoneal injection. Dystrophin expression followed a similar pattern. Importantly, all 3 routes resulted in exon skipping and dystrophin expression in the heart, albeit at low levels. Due to the better pharmacokinetic profile of subcutaneous versus intravenous injection and slightly higher exon skipping compared to intraperitoneal administration, subcutaneous injection seemed to be the delivery method of choice. After subcutaneous treatment also a decrease in serum creatine kinase (CK) levels was observed. Creatine kinase is an enzyme that leaks out of the muscles into the blood stream when muscles are damaged, so a decrease indicates an improvement of muscle

Morpholino (PMO) AONs have been shown to be effective *in vivo* as well. Intramuscular injection in the tibialis anterior elicited a dose-dependent increase in dystrophin expression in the majority of muscle fibres and dystrophin protein levels up to 60% of levels found in healthy muscle. Efficiency was comparable in both young (3 weeks old) and aged (6 months old) *mdx* mice. Repeated systemic (intravenous) injections induced exon skipping and expression of dystrophin protein body-wide, albeit with large variations between individual muscles. Highest levels were found in the quadriceps, abdominal and intercostal muscles. Lower levels were found in the tibialis anterior and diaphragm. CK levels were decreased and muscle function was improved as well. As with 2OMePS AONs, targeting of the cardiac muscle appeared difficult, since exon skipping and dystrophin expression were undetectable (Alter et al., 2006). Wu et al. showed that dystrophin restoration could be achieved (up to 30% of healthy levels) by systemic PMO treatment, although extremely high doses (up to 3 g/kg bodyweight) were required (Wu et al., 2010). Furthermore, a dosing regime of multiple low doses seems to be preferable above a few high doses to reduce the risk of toxicity and increase the efficiency, since both AONs and dystrophin protein show an

In the *mdx* mouse model PMOs appeared more effective and at lower doses compared to 2OMePS AONs. A direct comparison revealed that this was indeed the case for mouse exon 23 in the *mdx* mouse. Intramuscular injection of both AONs in the gastrocnemius, resulted in much higher skipping levels for PMOs than for 2OMePS AONs at the same molar amount. Systemic (intravenous) comparison in the *mdx* mouse showed, as had been noticed before, that most of the 2OMePS AONs are taken up by the liver and kidney. However the PMOs were almost exclusively taken up by the kidney. A possible explanation is that 2OMePS AONs bind to serum proteins, which prevents renal clearance (Geary et al., 2001), whereas PMOs do not, which explains their high renal clearance (Oberbauer et al., 1995). 2OMePS AON uptake was higher for all skeletal muscles, diaphragm and heart. In contrast to the biodistribution, exon skipping efficiency was much higher for the PMO AONs in skeletal muscle and diaphragm (approximately 40% versus 10%). Skipping levels in the heart were much lower and almost comparable between both compounds (2.5% for the PMOs versus 1.5% for the 2OMePS AONs). Protein levels followed the same pattern (Heemskerk et al.,

integrity (Heemskerk et al., 2010).

accumulation over time (Malerba et al., 2009).

2009b).

A PMO conjugated to a cell-penetrating peptide (pPMO) showed to be more effective than the naked PMO AON. Systemic (intravenous) treatment of *mdx* mice was very potent in both skeletal muscle, diaphragm and, importantly, heart. pPMOs lead to a decrease in CK levels (Jearawiriyapaisarn et al., 2008). Another study confirmed that the long term systemic treatment with pPMOs was effective in restoring dystrophin expression in skeletal muscle, improving muscle function and preventing heart failure (Wu et al., 2008). These pPMOs were also able to rescue the severe *mdx/utrn-/--* mouse model by systemic (intraperitoneal) treatment. Considerable improvement of muscle function was observed, combined with dystrophin expression in almost all muscles, except for the heart (Goyenvalle et al., 2010). Peptides might elicit an immune response, but no signs of such a response or toxicity were found in the mouse models so far. Unfortunately, when a pPMO compound was tested in primates, there were toxicity concerns. In cynomolgus monkeys pPMO doses equivalent to the ones used in mice, were not toxic, but also had little exon skipping effect. Higher doses were effective, but also caused tubular degeneration in the kidneys, a sign of renal toxicity (Moulton & Moulton, 2010). Yin et al. generated a chimeric fusion peptide consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-

peptide), which they conjugated to a PMO oligomer (B-MSP-PMO). These B-MSP-PMOs were already efficient at very low doses in restoring high levels of dystrophin expression body-wide (Yin et al., 2009). Novel cell-penetrating peptides have been discovered by inducing modifications to a *Drosophila melanogaster*-derived R6-Penetratin peptide. These peptides are called PNA or PMO internalisation peptides (Pips). A conjugate of Pip2b and a PNA AON (Pip2b-PNA) resulted in approximately threefold higher dystrophin-positive fibres compared to the naked AON after local injection in the tibialis anterior of *mdx* mice (Ivanova et al., 2008). More and improved Pips have been developed. Pip5e fused with a PMO (Pip5e-PMO) showed high exon skipping efficiency after a single intravenous injection in the *mdx* mouse. Most importantly it also efficiently targeted the heart, leading to dystrophin levels of more than 50% of wild-type levels (Yin et al., 2011).

Another modification of the PMO is conjugation to a dendrimeric octaguanidine polymer (vivo-morpholino). This modification also significantly improved the delivery and dystrophin production in *mdx* mice after intravenous injection. Repeated treatment resulted in dystrophin expression in almost 100% of the skeletal muscle fibres and levels of protein up to 50% of wild-type levels. Importantly, levels of ~10% of those found in healthy hearts were found in the cardiac muscle. In these mice no signs of an immune response or toxicity were observed (Wu et al., 2009).

#### **5.1.2 AONs in the other mouse models**

Both 2OMePS and PMO AONs have also been tested in the *mdx4Cv* mouse. These mice require skipping of both exon 52 and 53 to remove the mutation and maintain the reading frame. Immortalised myoblast cell cultures from these mice were used to design the most effective AONs against exon 52 and 53, which were then tested *in vivo* in the *mdx4Cv* mice. 2OMePS AONs induced exon skipping in these cell cultures, but no dystrophin protein was observed. Intramuscular injection of the cocktail of AONs in the tibialis anterior resulted in sporadic exon skipping in this muscle, but no detection of dystrophin protein. A combination of PMO AONs against both exons resulted both *in vitro* and *in vivo* (after injection in the tibialis anterior) in exon skipping and restoration of dystrophin expression (Mitrpant et al., 2009).

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 69

induce slightly higher exon skipping levels and restored dystrophin expression (McClorey et al., 2006). Further testing of these AON cocktails *in vivo* by intramuscular injections, revealed that the AONs targeting exon 8 were effective, but the AONs targeting exon 6, which showed effectiveness *in vitro*, were not (Partridge, 2010). Another small experiment (in a 6 months old and a 5 years old dog) with cocktails of 2OMePS AONs or PMOs, resulted in high skipping levels of the desired exons and restoration of dystrophin protein to near normal levels after a single injection in the tibialis anterior with the highest test dose. The structure of the dystrophin-positive cells was reported to be improved. Furthermore, both backbone chemistries showed comparable results and results were better in the

Systemic (intravenous) treatment of CXMDj dogs with a cocktail of 3 PMO AONs targeting exon 6 (2 PMOs) and exon 8 (1 PMO), generated body-wide production of functional dystrophin. In the heart there was only modest production of dystrophin, as observed in mice. Furthermore, an interindividual variation between dogs and intra-individual variation between different muscles of the same dog was seen. Functional improvement could be

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-

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

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-

younger dog than in the older dog (Scheuerbrandt, 2009).

shown too and no signs of toxicity were observed (Yokota et al., 2009).

state and relatively little muscle tissue left (van Deutekom et al., 2007).

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

**6.1 Local treatment with AONs** 

Rus et al., 2009a).

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 al., 2009b).

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

#### **5.2 AONs in the canine models**

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 induce slightly higher exon skipping levels and restored dystrophin expression (McClorey et al., 2006). Further testing of these AON cocktails *in vivo* by intramuscular injections, revealed that the AONs targeting exon 8 were effective, but the AONs targeting exon 6, which showed effectiveness *in vitro*, were not (Partridge, 2010). Another small experiment (in a 6 months old and a 5 years old dog) with cocktails of 2OMePS AONs or PMOs, resulted in high skipping levels of the desired exons and restoration of dystrophin protein to near normal levels after a single injection in the tibialis anterior with the highest test dose. The structure of the dystrophin-positive cells was reported to be improved. Furthermore, both backbone chemistries showed comparable results and results were better in the younger dog than in the older dog (Scheuerbrandt, 2009).

Systemic (intravenous) treatment of CXMDj dogs with a cocktail of 3 PMO AONs targeting exon 6 (2 PMOs) and exon 8 (1 PMO), generated body-wide production of functional dystrophin. In the heart there was only modest production of dystrophin, as observed in mice. Furthermore, an interindividual variation between dogs and intra-individual variation between different muscles of the same dog was seen. Functional improvement could be shown too and no signs of toxicity were observed (Yokota et al., 2009).
