Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in 50 percent of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10 percent of full-length (FL) protein identical to SMN1, whereas the majority of SMN2 transcripts are aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta1. In the present work, it is demonstrated that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-50 microM) of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) the level of SMN protein increased about 3fold. Augmented SMN protein levels could be attributed to elevated FL-SMN2 transcript levels which were triggered by two different mechanisms: a transcriptional activation of the SMN2 gene, and a preferred exon 7 inclusion in SMN2 transcripts. The latter observation was most likely due to increased levels of the splicing factor Htra2-beta1. In addition to Htra2-beta1, VPA increased the expression of further SR proteins which may have important implications for other disorders affected by alternative splicing. Importantly, the drug was able to elevate rSmn transcript and protein levels ex vivo through transcriptional activation in organotypic hippocampal brain slices from rats. This demonstrated that VPA also exerts an effect on neuronal tissue, the target for a potential SMA therapy. Since VPA is a drug highly successfully used in long-time epilepsy therapy, these findings opened the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing. The evaluation of two second-generation HDAC inhibitors in SMA fibroblasts in vitro revealed that SAHA, a drug that belongs to the hydroxamic acids, also efficiently elevated SMN protein levels 2.4fold to 3fold. Therefore, SAHA was identified as another attractive candidate for SMA therapy. In contrast, the data obtained for MS-275, an HDAC inhibitor of the benzamide class, demonstrated that the drug does not possess enough potency to substantially elevate SMN protein levels in vitro. Thus, MS-275 will not have a chance to move forward to SMA clinical trials. Based on the promising data for VPA in vitro and ex vivo and given that VPA is already approved for application to humans, a first pilot trial with VPA was carried out in ten SMA carriers (parents of patients with SMA) aiming to evaluate drug potency to increase SMN transcript and protein levels in vivo. In order to further validate the outcome of the study, SMN2 gene expression was analyzed in peripheral whole blood derived from 20 patients with SMA (5x type I, 11x type II, 4x type III) treated with VPA in individual experimental curative approaches all over Germany according to section 41 of the German Drug Act (AMG). Moreover, the value of these screenings was determined for the development of a clinical biomarker to monitor the response to VPA and other HDAC inhibitors in treated individuals. Such a biomarker would be highly useful for clinical trials and future therapies in SMA patients. Drug treatment revealed elevated full-length SMN RNA and protein levels in blood from 7/10 SMA carriers. Importantly, SMN protein levels increased far more substantially (up to 13.8fold) than the levels of the intermediate product, FL-SMN RNA, that showed an increase of up to 3.4fold. These observations provided first proof of the in vivo activation of a therapeutic target gene by VPA in an inherited disease. Among the investigated SMA patients, FL-SMN2 RNA levels were increased 1.5fold to 1.9fold in seven subjects, whereas 13 patients presented unchanged or even decreased transcript levels. This data suggested that some individuals are responders to VPA, while others are most likely nonresponders or even negative-responders. However, so far it is unknown whether SMN expression in blood reflects SMN expression in alpha-motor neurons and correlates with muscle strength. Therefore, systematic long-term clinical trials in SMA patients that correlate SMN expression in blood with individual motor function tests are required in the future to address the question whether SMN transcript and protein levels in blood may serve as biomarker, and to study the effect of VPA on motor function
