evolved from the duplication and mutation of the ancestral telomeric SMN
gene, and it is found in all extant human populations (35
). Given that SMN2
is capable of making a small amount of SMN protein by alternative splicing, SMN2
serves as a viable target for therapeutic intervention in all patients with SMA. Empirical testing of a large number of MOE oligonucleotides harboring complementary sequences to SMN2
identified an ASO (ASO-10-27) that exhibited the appropriate splicing activity in vitro, and in a transgenic mouse model without appreciable SMA phenotypes (29, 36
). Thus, the current report is a critical proof-of-concept study to determine whether splice-switching antisense technology can ameliorate the hallmark molecular, cellular, and functional phenotypes of a mouse model that recapitulates severe SMA disease. We show that a single administration of ASO-10-27 into the cerebral lateral ventricles of mice with severe SMA was sufficient to increase SMN protein levels by altering the SMN2
splicing pattern, which correlated with increases in the number of motor neurons and improvements in behavior and survival. CNS-directed therapy improved muscle physiology by increasing the size of myofibers and improving the structure of the neuromuscular junction in affected skeletal muscles. The correction of muscle pathology by targeting motor neurons through intrathecal delivery of therapeutic oligonucleotides is a significant finding because it allows for a single route of administration (that is, intrathecal) to treat the disease.
A previous study that used an ASO against the ISS-N1 region of SMN2
with a different chemistry (2′-OMe) required injections on days 1, 3, 5, 7, and 10 to affect SMN protein levels in the same severe SMA mouse model (33
). Furthermore, the molecular mechanism that increased expression of SMN protein, the correction of cellular pathologies including muscle physiology, and the benefits on survival were not reported (33
). Because of the similarities of the ASO sequence in the two studies, the improvements in efficacy in the current study are likely attributable to the MOE chemistry. Indeed, a side-by-side comparison of both chemistries in the CNS of a mouse model of mild SMA showed that the MOE chemistry provided more efficient modulation of splicing, improved in vivo stability, and decreased cellular inflammation compared to the 2′-OMe chemistry (36
). The poor in vivo performance of 2′-OMe–based ASOs makes this chemistry less ideal for the clinic, as it would likely require a substantially greater number and frequency of administrations compared to the more active and better tolerated MOE chemistry. Furthermore, the in vivo efficacy of ASO-10-27 exceeded other more complex strategies that use nucleic acids to modulate SMN2
splicing, such as transplicing and bi-functional RNAs (37–39
The pharmacokinetic and pharmacodynamic data for ASO-10-27 showed that 8 μg/g was a pharmacologically active tissue concentration for increasing SMN protein levels in the spinal cord. Although there was a decrease in the ASO tissue concentration between 3 and 16 days, the amount of exon 7
inclusion and SMN protein peaked at 16 days. The inability to sustain elevated levels of SMN protein at 30 days suggests that the ASO tissue concentration at 16 days was not capable of modulating splicing; hence, the minimal concentration required for efficacy must be greater than 2 to 3 μg of ASO per gram tissue. The loss of SMN protein by 30 days is consistent with the Kaplan-Meier survival curve showing a median survival of 26 days. The pharmaco-dynamic profile in neonatal mice corroborated an earlier report in the adult SMA type III mouse model. Intracerebroventricular infusion of ASO-10-27 in adult SMA type III mice loaded the spinal cord with ASO (10 μg/g tissue), resulting in >90% of the SMN2
transcripts containing exon 7
). However, the ASO tissue concentration of 10 μg/g in the type III SMA mouse model was sustained for at least 2 months, and the effect on splicing was maintained for at least 6 months (36
). It is unclear why the pharmacokinetic data differ between the two models. It is possible that the developing and growing neonate may promote the cellular depletion of the ASO more rapidly than that in the adult. Regardless, it is reasonable to predict that the pharmaco-kinetic data in the current neonatal study might be a more relevant indicator of the kinetic profile to be encountered in infants and young children with SMA.
A necessary prerequisite for antisense product development is to demonstrate that the spinal cord of a large animal, such as a NHP, can achieve therapeutic concentrations of ASOs after a clinically viable route of delivery. There are multiple parameters predicted to influence the efficiency of ASOs to target the spinal cord parenchyma, including but not limited to the site of infusion, the dose and volume of the ASO to be infused, and the duration and rate of infusion. Our data showed that intrathecal infusion of a fixed dose of 3 mg was sufficient to load the spinal cord with therapeutic levels (>8 μg/g tissue
) of ASO-10-27, and that increasing the infusion times of this fixed dose did not improve spinal cord loading. A 24-hour intrathecal infusion of 3 mg was well tolerated in monkeys and provided ASO (~20 μg/g) in the spinal cord. This demonstrates that a uniform RNA-based ASO can efficiently load the NHP spinal cord, which had been previously demonstrated only with intracerebroventricular infusions of an RNA-DNA hybrid ASO against superoxide dismutase–1 (40
). Thus, intrathecal infusion is an effective delivery method for global distribution of splice-switching ASOs to the spinal cord of primates.
The eventual cause of death in ASO-10-27–treated SMA mice is not known. However, the sudden appearance of animals found dead, coupled with the observation of acute breathing abnormalities in end-stage SMA mice, suggests that cardiac and/or respiratory failure may be responsible. Cardiac failure (that is, bradycardia) thought to be caused by autonomic dysfunction was recently described in this severe mouse model and in SMA patients (41–44
). In this scenario, a drop in the ASO tissue concentration in the lower brainstem may lead to the loss of autonomic function in the cardiac system, which would explain the sudden and acute appearance of respiratory defects in ASO-10-27–treated SMA mice. A similar acute breathing aberration was observed in the long-lived scAAV8-hSMN–treated SMA mice (12
). Analysis of the intercostal muscles of these mice showed that 85 to 90% of the neuromuscular junctions had a normal structure. Similarly, the current study showed that >95% of the intercostal neuromuscular junctions were normal in the ASO-10-27–treated SMA mice that lived beyond weaning age. These data suggest that the respiratory defects in the scAAV-hSMN– and ASO-10-27–treated SMA mice occurred independently of the skeletal muscles involved in breathing (12
). However, additional studies are needed to confirm the role and relevance of the autonomic system in SMA disease.
In conclusion, delivery of ASO-10-27 to the CNS of mice with a severe form of SMA corrected the hallmark molecular, cellular, and functional phenotypes of this disease, and provided significant and reproducible survival benefits. The demonstration that a pharmacologically relevant tissue concentration—extrapolated from the mouse studies—could be attained throughout the NHP spinal cord indicates that intrathecal infusions may be an amenable route of delivery for ASO-10-27 in the clinic. We thus achieved our primary objectives of correcting SMA phenotypes in mice, identifying the tissue concentration required for efficacy, and verifying that this concentration can become loaded into the spinal cord using a clinically viable delivery strategy. Future studies in large-animal models that investigate the optimal metrics for delivery (for example, bolus versus slow infusion, catheter placement, and timing and frequency of repeat administration) and safety will be required to determine the feasibility of translating ASO-10-27 to the clinic.