Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the deterioration of alpha motor neurons in the brainstem and spinal cord. Currently, there is no cure for SMA, which calls for an urgent need to explore affordable and effective therapies and to maximize patients’ independence and quality of life. Adeno-associated virus (AAV) vector, one of the most promising and well-investigated vehicles for delivering transgenes, is a compelling candidate for gene therapy. Some of the hallmarks of AAVs are their nonpathogenicity, inability to incur an immune response, potential to achieve robust transgene expression, and varied tropism for several tissues of the body. Recently, these features were harnessed in a clinical trial conducted by AveXis in SMA patients, where AAV9 was employed as a vehicle for one-time administration of the SMN gene, the causative gene in SMA. The trial demonstrated remarkable improvements in motor milestones and rates of survival in the patients. This review focuses on the advent of SMA gene therapy and summarizes different preclinical studies that were conducted leading up to the AAV9–SMA trial in SMA patients.
Spinal muscular atrophy (SMA) is a genetic neuromuscular disease that results in the degeneration of alpha motor neurons of the spinal cord and brainstem . It is the leading genetic cause of mortality in infants, affecting ~1 in 6000 to 1 in 11,000 live births [2, 3]. SMA is an autosomal recessive disorder with a carrier frequency of 1 in 40 to 1 in 60 [4, 5]. Alpha motor neurons innervate the skeletal muscles; thus, degeneration of these neurons results in a myriad of symptoms that affect voluntary muscle activities such as speaking, walking, breathing, and swallowing, ultimately resulting in paralysis and early death . Over 50% of SMA patients die before the age of 2, and management of the disease is achieved by treating the symptoms to prevent further deterioration of the condition. The severity of this devastating disease makes it imperative to find effective treatments for which important progress has been made recently and will be discussed in this review.
SMN gene and function of SMN protein
In 1995, Lefebvre et al. identified and characterized the disease-causing gene for SMA, the survival motor neuron 1 (SMN1) gene that is located on chromosome 5q13 . Over 95% of cases of SMA are caused by a homozygous deletion, conversion, or mutation in the SMN1 gene, which otherwise produces fully functional SMN protein in healthy humans . Missense, nonsense, or frameshift mutations in the SMN1 gene have been reported in the remaining 5% of patients . Apart from the telomeric SMN1 gene, humans are unique in also having another copy of the SMN gene, the SMN2 gene . As a result of genomic duplication, the centromeric SMN2 gene exists as multiple copies on chromosome 5q13 and is an almost identical homolog to the SMN1 gene except for a 5-nucleotides difference . Importantly, the presence of a critical substitution of cysteine with thymidine at position 6 in exon 7 of SMN2 disrupts the splicing of the SMN2 gene (Fig. 1). This disruption results in ~90% of transcripts lacking exon 7, giving rise to a truncated form of the SMN protein that is unable to oligomerize and undergoes rapid enzymatic degradation in the cell . Thus, in SMA patients, although the presence of the centromeric SMN2 gene results in the production of SMN delta 7 proteins, these proteins degrade rapidly and cannot compensate for the loss of the SMN1 gene, leading to subsequent degeneration of spinal motor neurons [12, 13] (Fig. 1).
The SMN gene encodes a 38 kDa SMN protein composed of a 294 amino-acid polypeptide, which is found in the nucleus as well as the cytoplasm [14,15,16]. The SMN protein is ubiquitously expressed in all tissues, and a particularly high level of this protein is found in the brain, spinal cord, kidney, and liver . This pattern of expression raises an important question as to why the loss of SMN protein, which is ubiquitously expressed in all cells, is particularly detrimental to motor neurons in SMA patients.
The SMN protein plays an important role in the biogenesis and assembly of small nuclear ribonucleic particles (snRNPs), which play a crucial role in spliceosome assembly as well as pre-mRNA splicing [18,19,20]. Therefore, one possibility is that reduction in the SMN protein results in the mis-splicing of essential genes that are critical for motor neurons. Furthermore, it has been demonstrated that a reduction in the assembly of snRNPs correlates with a slight reduction in their steady state levels. Interestingly, this reduction in the endogenous snRNP profile is variable across various tissues in the body, thus supporting the hypothesis that a reduction in functional SMN protein can have a particular effect on motor neurons [21,22,23,24]. In light of these findings, additional data illuminate that the SMN protein may play roles that are particularly important for the survival of motor neurons. Another possibility is that SMN affects the transport of RNAs along axons, thus affecting axonal function and maintenance. Studies have shown that the SMN protein plays crucial roles in multiple processes, such as axonal transport, maintenance of neuromuscular junctions, and axonal cone growth [25,26,27,28,29,30]. Recent studies have demonstrated that the SMN protein forms a complex with HuD, an RNA binding protein, and interacts with its target, cpg15, a protein important for axonal growth, motor neuron development, and maintenance of neuromuscular synapses [31, 32]. However, whether factors regulated by SMN are specifically altered in spinal motor neurons and the detailed mechanisms by which motor neurons are more vulnerable than other types of neurons await further investigation.
Treatment of SMA
SMA is classified as a monogenic disorder based on simple Mendelian genetics . Considering that SMA is caused by reduced levels of SMN proteins, most of its treatment strategies centre on improving functional SMN, including reexpression of the SMN1 gene, modification of SMN2 gene splicing, and stabilization of SMN proteins (Fig. 1). For example, several small molecule drugs, such as quinazolines, exert their effects by preventing the decapping of RNA in SMN2 transcripts, resulting in increased SMA expression levels. Although this drug has shown promising results in in vitro and in vivo studies, clinical trials with these molecules have not been as successful . Another attractive therapeutic intervention for SMA is the use of antisense oligonucleotides (ASOs), which are single-stranded oligonucleotides of 10–50 base pairs that work by binding to their target RNA and altering its function . As mentioned earlier, the SMN2 gene undergoes aberrant splicing resulting in the loss of exon 7, which then gives rise to nonfunctional SMN protein. One such antisense drug, nusinersen, is an 18 mer 2′-O-methoxyethyl phosphorothioate ASO that binds to the SMN2 pre-mRNA and mediates the inclusion of exon 7, resulting in the production of SMN full length (SMN-FL) protein . Various preclinical experiments using ASOs in SMA mice have demonstrated an increase in SMN-FL expression as well as an improvement in motor neuron defects, function, and survival [37,38,39]. These promising results propelled Ionis Pharmaceuticals to conduct the first human, open-label, single-escalating dose, phase 1 clinical trial to assess the safety and efficacy of nusinersen in SMA patients.
SMA is characterized by the degeneration of spinal motor neurons. Though other types of neurons and systems have also been reported to be affected [40, 41], the degeneration of spinal motor neurons is the major contributor to the impairment of motor function and the failure of respiratory function. Therefore, it is important that candidate drugs can effectively target spinal motor neurons. For this reason, nusinersen was administered intrathecally, since ASOs lack the ability to cross the blood–brain barrier (BBB) when delivered systemically. This study established the safety of nusinersen along with other benefits, such as increased SMN expression in the cerebrospinal fluid and improved motor functions in SMA patients . Approximately 40% of patients achieved an overall increase in motor milestones with nusinersen treatment . In the subsequent phase 2 and phase 3 clinical trials, SMA patients exhibited similar improvements in achieving motor milestones. This positive response to nusinersen was an impetus for its FDA approval in 2016. nusinersen was commercialized as spinraza in 2016 by Ionis Pharmaceuticals and Biogen .
Despite being the first exciting treatment option for SMA, several concerns were associated with this therapy, including the need for patients to undergo repeated invasive intrathecal injections. Another disadvantage of nusinersen is that the treatment is delivered as six loading doses in the first year followed by three doses per year for the patient’s entire lifetime. This regimen would cost the patient a staggering $750,000 for the first year of treatment and $375,000 for each year of subsequent treatments . Finally, studies [45, 46] have reported that apart from motor neurons, SMA may also involve defects in other tissues of the body, including the heart, which could be mitigated by treatment increasing systemic SMN levels. All of these concerns associated with previous SMA therapies have led researchers to look for alternative strategies that could have robust long-term effects that would improve the quality of life for SMA patients.
The advent of gene therapy was led by the ability to use next-generation sequencing to screen for various genes that are known to be mutated in diseases. At present, several studies are being meticulously designed to deliver genes that could compensate for either the underactivity or the overexpression of genes, which are the causative factors for these disorders. Gene therapy presents as an attractive option for monogenic disorders such as SMA that follow simple Mendelian inheritance. However, one of the biggest hurdles associated with the use of gene therapy for neuromuscular diseases is the therapeutic agent’s inability to gain access to specialized cells such as motor neurons that are often affected in these diseases.
Viral vectors are an appealing choice for transgene transfer due to their ability to transduce a wide variety of human cells. In 2004, Azzouz et al. demonstrated for the first time in the field how gene therapy using viral vectors as a modality can be employed to treat SMA . They achieved this feat in SMA mice by utilizing lentiviral vectors expressing the SMN gene. To determine their efficacy as well as their effect on the survival of SMA mice, the lentivectors were administered intramuscularly into regions that were important for breathing, feeding, and locomotion. In this study, the lentivectors were capable of transducing ~70% of motor neurons in the brainstem and spinal cord region of SMA mice by retrograde transport. Furthermore, it was also elucidated that the lentivector was capable of delaying motor neuron degeneration as well as slightly improving life expectancy in these mice. Although the lentivectors elicited a low immune response in this study, concerns associated with the use of retroviral vectors, such as impending immune responses or insertional mutagenesis, still exist . The need to be prudent with the use of viral vectors was emphasized in a clinical trial conducted with patients suffering from X-linked severe combined immunodeficiency disease. In this study, retroviral-mediated gene therapy resulted in the development of leukemia in two out of five patients, raising safety concerns surrounding its use [49, 50].
For gene therapy to be successful, it is important to  choose a vehicle that can deliver the transgene of choice as well as achieve a stable, robust, and extensive transduction of desired tissues with no pathogenicity or immunogenicity. Adeno-associated viruses (AAVs) fulfill all of these criteria and have been the preferred choice for virus-mediated gene therapy. The first few clinical trials that utilized AAVs for diseases such as cystic fibrosis and hemophilia B opened new avenues for them as a promising tool to disseminate gene therapy. In Europe, AAV vectors have been approved to treat lipoprotein lipase deficiency. As of February 2019, there were 180 clinical trials involving AAVs registered at ClinicalTrials.gov, showing the promise of using AAV for gene therapy.
AAVs are small viruses (25 nm) of the Parvoviridae family belonging to the Dependovirus genus, which is indicative of their dependence on a helper virus for productive infection . These viruses have a nonenveloped capsid that stores genetic material in the form of a single-stranded DNA . AAVs depend on helper viruses such as adenovirus and herpes simplex virus for replication and can infect dividing as well as nondividing cells. These characteristics make them potential agents for administering gene therapy to post-mitotic cells such as neurons. The 4.7 kb genome of the AAV consists of ORFs that contain rep genes coding for proteins involved in viral replication, cap genes encoding structural proteins required for capsid formation, and finally aap genes, which are needed for assembly of the capsid proteins. These ORFs are interspersed with two ITRs that are the cis-acting elements crucial for viral replication and packaging . Based on the presence or absence of helper viruses, these viruses undergo lytic or lysogenic cycles, respectively. In the case of a lytic cycle, the virus replicates its genome with the help of the host’s DNA polymerase followed by virion production. For lysogenic cycles, the virus integrates into the host genome in a site-specific manner and needs to be rescued by a helper virus to revert to its lytic state [54, 55]. For gene therapy, recombinant AAV vectors are constructed by replacing the ORFs with the gene of choice while maintaining the ITRs. The helper functions required to maintain a lytic cycle are provided by cotransfection with a plasmid containing the genes that confer these requirements .
Currently, based on the capsid protein composition, there are 13 serotypes of AAV (AAV1–AAV13), which show differences in their tropism and transduction efficiencies across various tissues . These differences in various AAV serotypes can be harnessed to selectively target particular specialized cells. Although different serotypes of AAV have demonstrated varied tropism for various tissues, out of all the serotypes that have been identified and characterized, AAV9 was found to have the highest tropism for the CNS .
One of the bottlenecks associated with using recombinant AAV is that it relies on the host system for the synthesis of the second strand . However, this step can be obviated by using self-complementary vectors. These vectors package inverted repeat genomes that are capable of folding and forming base pairs, ultimately giving rise to double-stranded DNA, sans the host polymerase. This modification results in a dramatic increase in the copy numbers of vector genomes as well as the early onset of gene expression, leading to an overall increased and faster viral transduction in a variety of tissues .
The use of self-complementary adeno-associated vectors (scAAVs) in gene therapy was explored by Duque et al. when they carried out peritoneal administration of scAAV9-GFP and scAAV9-murine secreted alkaline phosphatase in neonatal mice and saw an effective transduction of the CNS  (Table 1). To replicate similar results at the adult stage, they delivered the same vector intravenously into adult mice and discovered a resulting high transduction of the spinal cord. Another important finding was that the transgene was stably expressed for up to 5 months in the CNS as well as in nonnervous tissues. To translate these findings into clinical trials, it was paramount to evaluate the transduction capabilities of scAAV in higher animals. The same group went on to deliver scAAV intravenously in higher animals, such as neonatal and adult cats, and demonstrated a similar trend with the high transduction of spinal cord and motor neurons. The high spinal cord transduction efficiency could be attributed to the combined effect of the unique ability of AAV9 to traverse the BBB as well as the elimination of the second strand synthesis step by virtue of the scAAV9 .
In another study carried out in neonatal SMA mice, Foust et al. showed that intravascular administration of scAAV9 not only resulted in a high penetration of the BBB but also demonstrated high transduction of the dorsal root ganglia, lower motor neurons of the spinal cord, and the different neurons throughout the brain  (Table 1). However, in the case of adult mice, greater transduction of astrocytes but not neurons was observed. This observation suggested that there might exist a therapeutic window to achieve maximum transduction in neurons using AAV9, further bolstering the theory that SMN may have a crucial role in the early stages of development . This study led to AAV9 garnering much attention as an attractive noninvasive approach to treat disorders such as SMA.
AAV9 for SMA
Having accumulated substantial evidence that scAAV was capable of effectively crossing the BBB and transducing motor neurons in mice and higher animals, Foust et al. further investigated this approach for the delivery of the SMN gene to SMA animal models. They demonstrated that vascular administration of scAAV9 SMN into the facial vein of neonatal SMA mice increased SMN expression levels in the brain, spinal cord, and muscle tissues compared to control mice . Other highlights of the study included that the treated mice were able to right themselves and that the abnormal neuromuscular transmission, a pathological consequence of SMA, was also corrected. In corroboration with the previous study , it was observed that treating the SMA mice in the earlier stages of disease rescued the disease phenotype, thus establishing a therapeutic window for targeting motor neurons. These results raise important points regarding the time of intervention, the need for characterizing early changes that occur in motor neurons, and the reparability of targeting motor neurons after this particular timepoint  (Table 1).
During this period, other independent groups also carried out similar investigations using scAAV9. These groups modified the scAAV9-SMN vectors by codon optimization and delivered them to SMA mice. Encouraging results such as the reversal of phenotype, motor neuron survival, and increased lifespan were observed in these studies [63, 64]. Even though these studies fortified the potential use of gene therapy for SMA, it was important to investigate whether these findings could be applied to nonhuman primates. In addition to regulating the SMN gene, several studies using AAV9 to regulate other therapeutic target genes showed mitigated SMA symptoms and effectively extended lifespan of mouse models. The alternative targets include the actin-binding protein plastin 3 (PLS3) [65, 66], ubiquitin-like modifier activating enzyme 1 (UBA1) , and phosphatase and tensin homolog (PTEN) . In one of these studies, a combinatorial therapeutic involving coadministration of AAV9-PLS3 and splice-switching ASOs in an intermediate model of SMA mice significantly increased SMN production and improved the disease phenotype . These studies demonstrate that combinatorial therapeutics targeting SMN and SMN-independent pathways can be a potential option for SMA.
One of the previous problems associated with using AAVs in neonatal and adult mice was the existence of a therapeutic window for vector administration in these mouse models . Foust et al. went on to validate this result by systemically delivering an AAV9-GFP into 3-month-old cynomolgus macaques and observed transduction of motor neurons and glial cells  (Table 1). Interestingly, along with the CNS, skeletal muscles and other organs were also highly transduced by the AAV vectors. Notably, unlike what was elucidated in mouse studies, AAV9 also mediated the transduction of motor neurons in a 3-year-old macaque . This observation means that the window of opportunity seen previously in mice might be prolonged in nonhuman primates. The consistent transduction of neurons in all animals across different age groups also gave a proof-of-concept that SMA patients of different ages can be targeted for gene therapy. Thus, SMA type-2 and SMA type-3 patients, who have milder forms of the disease, can also undergo trials, albeit with a reduced viral load. The same group also went on to intrathecally administer the AAV9-GFP in 5-day-old pigs and found transgene expression in the brain and a high transduction of motor neurons but not peripheral organs, thus limiting the therapy to target only the CNS . Another team also tested the efficacy of scAAV9-GFP in cynomolgus macaques and found extensive transduction of the brain, thus providing evidence for the use of this powerful therapy to deliver genes to the CNS [69, 70]. All of these studies outline the unique ability of AAV9 viruses for transducing neurons in most animals across all ages, thus rendering it a potential mode of delivery for gene therapy for SMA.
Recently, studies were also carried out in both mouse and nonhuman primate models to find the lowest dose of AAV that would achieve the highest transduction of motor neurons in these animals. Surprisingly, the minimum effective dose required to achieve maximum transduction in nonhuman primates was lower than what was established in mouse studies . The lower levels of AAV dose required for gene therapy would also reduce production and treatment costs, making it more affordable. This result underscores the importance of testing putative therapies in nonhuman primates before translating them to clinical medicine.
All of the previously described preclinical studies (Table 1) set the stage for AveXis in 2014 to conduct the first phase 1 clinical trial in SMA patients using AAV9 vectors. This study was conducted to determine the safety and efficacy of scAAV9-SMN therapy administered intravenously as a single dose to 15 type-1 SMA patients  (Table 1). The patients were divided into two cohorts; one consisted of 12 patients who received a higher dose of the drug than the other cohort, comprising only 3 patients. As of now, this therapy boasts a 100% survival rate in both of the cohorts as opposed to a historical control that describes a meagre 8% overall survival rate. The patients in both cohorts demonstrated enhanced motor functions as indicated by increased points from baseline on the Children’s Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) scale. In the higher dose cohort, a greater percentage of patients (11 out of 12) also achieved major milestones in improved motor function, such as the ability to swallow, talk, gain head control, or sit unassisted, all of which were not previously reported in the historical group. A majority of the patients also ceased to require ventilatory and nutritional assistance. Two of the 12 patients in the higher dose group, who were subjected to early treatment, were able to crawl, stand, and walk without any support. This result was a remarkable discovery, since these two patients were diagnosed with SMA much earlier than the other patients, enabling early treatment. In this study, increased serum aminotransferase levels were the only serious adverse effect observed in two patients, which were eventually alleviated with the help of a drug—prednisolone . In spite of the myriad unprecedented benefits that were observed with this trial, further studies need to be carried out to check for any possible looming immune responses or other adverse effects, as well as the transgene’s long-term expression and robustness in SMA patients. After acquiring AveXis in 2018, Novartis plans to sell this gene therapy for SMA as AVXS-101 at a cost of $4–5 million, which is similar to the total cost of Spinraza .
Conclusion, therapeutic prospects, and challenges
AAVs have emerged as a viable tool to deliver sustainable transgene expression in most human cell types owing to their high transduction efficiency. These viruses are the choice for administering gene therapy due to their lack of pathogenicity and the achievement of a long-term stable expression in the cell . Despite these beneficial effects, one of the potential drawbacks associated with the use of AAVs for gene therapy is the small size of the AAV genome, which limits the ability to introduce larger transgenes into desired tissues . Another pitfall includes the impending possibility of eliciting an immune response. Therefore, genetic engineering of the AAV capsids and further studies on AAV transduction, replication, and gene expression are warranted to obviate immune response-related problems . Efforts also need to be taken during vector engineering to give rise to next-generation gene therapy vectors that would minimize or eliminate these problems. However, one of the major concerns regarding the testing of improved AAV9 vectors for SMA is the lack of SMA nonhuman primate models to help validate findings observed in SMA mouse models. The establishment of SMA higher animal models will facilitate the discovery of potential gene therapies for SMA.
Before employing AAV9 as a vector for administering a transgene, it is also important to conduct an appropriate screening for preexisting antibodies to AAVs. For example, in the study carried out by AveXis, 1 of the 16 SMA patients screened had to be excluded from the study due to the presence of anti-AAV9 antibody titres that may cause severe side effects . Another important take-home message from this study relates to early diagnosis and treatment for SMA patients. Two patients who were treated at an earlier stage due to early diagnosis showed significantly greater improvement in terms of motor function. Thus, new born screening, early diagnosis, and treatment would play an important role in improving the efficacy of gene therapy for SMA. In this study, AAV9 was administered intravenously, as opposed to preclinical studies in mice and nonhuman primates, where the AAV9 was delivered intrathecally. Given that SMA also involves defects of peripheral tissues such as cardiac abnormalities [45, 46], systemic administration would help in transducing the peripheral organs, which could potentially facilitate effective therapy. This information is invaluable in regard to targeting disorders that affect the levels of a ubiquitously expressed protein, which is seen in the case of many neurodegenerative disorders. Moreover, because the transgene is being delivered as a one-time administration, the use of AAV9 as a gene therapy for SMA eliminates the need for frequent and expensive drug delivery strategies, providing a powerful approach for the treatment of SMA. In addition, with its varied tropism for different tissues, AAV poses an ideal modality for delivering transgenes relatively noninvasively to different organs, making it a promising candidate for formulating targeted therapies for many types of disorders.
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This work was supported by the Blazer Foundation.
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Pattali, R., Mou, Y. & Li, X. AAV9 Vector: a Novel modality in gene therapy for spinal muscular atrophy. Gene Ther 26, 287–295 (2019). https://doi.org/10.1038/s41434-019-0085-4