## 2 Materials and Methods
### Case Study--Synthesis of Spinal Motor Neuron Protein from Spinal Motor Neuron 2 Gene  
A mutation in _SMN1_ gene leads to the insufficient production or complete absence of SMN protein, causing spinal muscular atrophy (SMA), a debilitating motor neuron disease. Humans have a second gene copy, _SMN2_, which possesses the necessary genetic information to mitigate the effects of SMA. However, _SMN2_ cannot fully compensate for  
Figure 3: (**a**) The definition of a triangular fuzzy number, and (**b**) its adaptation to the rate of transcription. the loss of _SMN1_ because it only produces 10-15% of the full functional SMN protein, the remaining 85-90% being the truncated and dysfunctional SMNA7 protein. To address this, various candidate drugs have been proposed to treat SMA or reduce its severity by increasing the production of functional SMN protein from _SMN2_. As of now, there is no complete cure for SMA. The most effective therapies include Zolgensma [17; 18], a gene replacement therapy, and two medications: nusinersen (marketed as Spinraza) and risdiplam (known as Evrysdi) [19; 20]. Although these treatments represent major advances in SMA therapy, they are focused primarily on alleviating symptoms and preventing complications rather than offering a complete cure. However, understanding the genetic basis of SMA has allowed the development of targeted treatment options that fall into four main categories:  
* Inhibition of the inhibitor: Targeting pathways that suppress _SMN2_ expression. * Regulation of pre-mRNA splicing: Enhancing the correct splicing of the _SMN2_ transcript. * Upregulation of promoter activity: Boost promoter activity to increase _SMN2_ expression. * Targeting DNA methylation: Modifying DNA methylation patterns to enhance _SMN2_ activity. In the present study, we combine various treatment options to evaluate the comprehensive effectiveness of the following 7 potential drugs or chemicals with existing qPCR data and all possible combinations of these drugs. Thus, we conducted dry lab experiments for 7 drug candidates and their 120 possible combinations (combinations of 2 drugs, combinations of 3 drugs, etc.). A possible SMA treatment strategy, referred to as strategy A, focuses on inhibiting histone deacetylase (HDAC) activity, which suppresses _SMN2_ gene expression. Our research has identified Valproic Acid (VPA) [21], Trichostatin A (TSA) [22], Dacinostat, and Resveratrol as the only HDAC inhibitors documented in the biological literature that present qPCR and protein data indicating an increase in SMN protein levels derived from _SMN2_. According to [21], treating fibroblast cultures from SMA patients with 0.5-500 uM of VPA led to a boost in SMN levels up to four times. In [22], it was observed that the application of TSA to an SMA mouse model resulted in a twofold increase in SMN levels in the brain, liver, and spinal cord. Furthermore, in [23], a 1.3-fold increase in SMN levels was reported in cell cultures treated with 100\(\mu\) resveratrol, compared to the untreated control. In [24], it was demonstrated that in a type III SMA mouse model, treatment with the tetracycline derivative PTMK-SMA1 significantly increases the inclusion of exon 7 in _SMN2_ mRNA during splicing. This is in accordance with strategy B. This leads to a nearly five-fold increase in SMN protein levels compared to untreated mice. Notably, PTMK-SMA1 is the only compound identified so far that directly alters the splicing process to favor the inclusion of exon 7, promoting the production of functional SMN protein. In [25], according to strategy C, the authors suggest enhancing SMN transcription from _SMN2_ by targeting modulation of the _SMN2_ promoter's activity. The research demonstrated that Indole treatment in patient-derived cells significantly impacts _SMN2_ promoter activity, leading to a three-fold increase in SMN transcription compared to untreated controls. In line with strategy D, it was discovered that the _SMN2_ gene is subject to silencing through DNA methylation [26].