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motor neuron dysfunction is still uncertain (60). In mice, SMN levels are higher in neonatal days (around postnatal day 5) and then are gradually reduced (around postnatal day 15), which underlines their importance during the neonatal period (61). Indeed, tamoxifen-induced SMN reduction confirmed the pivotal role of SMN protein in NMJ formation during the neonatal period (until postnatal day 17). Once the NMJs are matured, the amount of SMN protein required for maintenance is significantly lower (88). Together, these data narrow the time window for effective treatment, mainly corresponding to the NMJ developmental period. Identifying Disease-Modifying Genes and Pathways of Therapeutic Relevance Using Invertebrate Models The C. elegans SMA model shows delayed development and defects in motor functions such as motility and pharyngeal pumping. Genome-wide genetic screenings of C. elegans have been performed and revealed the genetic modifiers and affected pathways of SMA: grk-2 as a modifier of SMA pathology and endocytosis as an affected cellular function of SMA (40, 41). Genetic screens in C. elegans or Drosophila SMA models unraveled more than 300 genetic modifiers (156). Validating Therapeutic Tools for or During Preclinical Studies Splice-modifying ASOs were first tested in an SMA mouse model with SMN2 to confirm their effectiveness in vivo (78, 80). Recently, combinatorial therapy with two different ASOs—an SMN2 www.annualreviews.org • 25 Years of Spinal Muscular Atrophy Research 243 Annu. Rev. Genom. Hum. Genet. 2020.21:231-261. Downloaded from www.annualreviews.org Access provided by 2600:1700:3c30:a3d0:89dc:b3d1:ac73:5085 on 01/23/22. For personal use only. splice corrector and an inhibitor of the NCALD genetic modifier—was tested in an SMA mouse model and showed promising outcomes (169). Gene therapy with self-complementary adenoassociated virus (scAAV) delivery of SMN1 alone or with other genetic modifiers, including IGF, PLS3, and STMN1, has also been tested in SMA mouse models (45, 55, 87, 121, 134, 170, 176). The efficiency and safety of gene therapy were later confirmed in large animals, such as pigs and monkeys (121, 134). Pigs have been used as a model organism for preclinical trials because the size and morphology of their spinal cords are similar to those of humans (47). For example, the efficiency of scAAV vector serotypes for spinal motor neuron transduction has been tested in pigs (163). A porcine SMA model has been produced and used to optimize the time window of treatment and to test functional outcomes of scAAV9-mediated SMN gene therapy (44). This study showed that even after the disease manifests, SMN restoration could still ameliorate SMA phenotypes. However, restoring SMN levels in presymptomatic stages showed impressive effects on motor function, implying that neonatal screening is indeed crucial to protect newborns from the disease. Needless to say, monkeys are used as nonhuman primate models to validate the efficiency of gene therapy before clinical trials. The monkey models confirmed that intrathecal injection is the most efficient method of delivering scAAV9 to spinal cords (134). Reference 86 provides a nice overview of preclinical studies. THERAPY OF SPINAL MUSCULAR ATROPHY SMN-Dependent Therapies The unique genetic scenario of SMA patients (lack of functional SMN1 and variable SMN2 copy numbers, short cDNA length, and a uniform mutation spectrum) provided an avenue for therapy development and became the epicenter of SMA translational research. Over the past few years, efforts aiming to modulate both SMN genes led to the first effective therapies for SMA: the upregulation of full-length SMN2 transcripts (and consequently of functional SMN protein) and the replacement of the defective SMN1 gene. SMN2 modulators. New agents designed to correct the missplicing of the SMN2 gene product are at the forefront of SMA clinical trials (Table 2). Specifically, SMN2 splicing modulators target the SMN2 pre-mRNA and promote the retention of the exon 7 in SMN2 transcripts, thereby enhancing the translation of fully functional SMN2-encoded
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