IVS7-ISS motif revealed overall similar binding of hnRNP A1 to the motif, compared to the human wild type IVS7-ISS, despite a putative increase in the strength of the distal hnRNP A1 core motif. The loss of this site is likely enough to effectively disrupt the function of the entire ISS in the context of pig Smn1 exon 7. This is in line with previous evidence that suggests a more central role of the proximal site compared to the distal site. In the context of human SMN2 exon 7, abrogation of this site improves the inclusion of exon 7, indicating that this site is a major contributor to the inefficient splicing of human SMN2 exon 7. Additionally, the IVS7-ISS has proved a valuable therapeutic target and has resulted in on-going clinical trials with an ASO specifically blocking this ISS motif. Together, these findings indicate that in pigs, the IVS7-ISS is inactive due to a mutation in the proximal hnRNP A1 site and this inactivation has relaxed the requirements for a strong ESE at the 39ss of exon 7. This indicates that insertion of an SMN2-like mutation in the endogenous porcine Smn1 gene would be insufficient to recapitulate the splicing of SMN2 exon 7 and that insertion of a larger fragment of the human SMN2 gene would be required. In the mouse, the SRSF1 ESE is identical to the human ESE but like the pig, the mouse IVS7-ISS is abrogated in the proximal site. Furthermore, the distal site does not appear to be strengthened indicating that the ISS is likely to be functionally weakened also in the mouse. These observations indicate that the human ISS would have to be inserted into the murine Smn1 gene if Smn1 exon 7 skipping was to be induced by an +6C.T mutation, but a mouse model containing a murine Smn1 to Smn2-like conversion has been published without insertion of the human ISS. This model exhibited a milder SMA phenotype similar to human SMA type III, indicating that even though the Smn2-like exon 7 was skipped, the level of inclusion was still higher than human SMN2 exon 7, resulting in a mild phenotype. One possible explanation for this, is that the murine ISS contains a 3 bp deletion which moves the distal site in closer proximity to the 59ss, although not as close as the proximal site of the human ISS. This indicates that the inhibitory function of this hnRNP A1 core motif may be highly dependent on its proximity to the 59ss and that it may function by directly blocking access of U1 snRNP to the 59ss, and not by inducing a polymerization of hnRNP A1 proteins along exon 7, or by looping out the exon through interaction with other ISS/ESS elements, such as the hnRNP A1 binding motif spanning the 39ss of exon 7 or the hnRNP A1 motif generated across the previous SRSF1 binding ESE in SMN2. In conclusion, we established the presence of a functional and likely SRSF1 binding ESE in porcine Smn1, that this ESE may be disrupted by a mutation similar.