Abstract
Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is a fatal neurodegenerative disease that results in respiratory failure usually 2-5 years after diagnosis. The cause of ALS is enigmatic since pathogenesis of the disease possibly results from mutations and/or dysfunction of proteins found within the cell's many important signaling pathways such as a mutation in the superoxide dismutase-1 gene (SOD1). Fortunately, there is a mouse model for ALS with a mutation in the SOD gene, S0DG93 \\ which can be used to study the disease. Our overall goal is to utilize ALS mice to develop in vivo therapeutic options to rescue their neurons and neuromuscular junctions by using MS Cs as a type of delivery system to deliver an anti-apoptotic factor such as IGF-1. However, fail-safes need to be incorporated into the approach since the delivery of allogeneic sources of MS Cs could possibly cause problems such as tumor formation. Therefore, an approach that utilized a "suicide gene" incorporated into the vector for transduction into MSCs with the IGF-1 gene was examined in this study. The "suicide gene" that was explored in this study was the Herpes Simplex Virus- 1 Thymidine Kinase (HSVl-TK). HSVl- TK functions in the recycling of deoxythymidine ( dT) into various nucleotides, which are incorporated into nascent DNA during replication and repair. When Ganciclovir, an anti-viral nucleoside analog drug, is present it will be phosphorylated by the HSV 1-TK into its toxic tri-phosphorylated form, which will terminate the IGF-1 transduced MSCs. However, care needs to be taken since surrounding tissue could also be toxified by a bystander effect. Within our retroviral constructs, HSVl-TK was constitutively expressed, human IGF-1 and eGFP (included for detection) was regulated by doxycycline. The goal of this project was to determine the effectiveness of HSV 1-TK as a suicide gene in two different environments, in vitro and in vivo, and in the absence and presence of the anti-apoptotic protein, IGF-1. The last goal of my project was to determine whether there is a toxic bystander effect of the construct to non-transduced cells both in vitro and in vivo. Two MSC cell lines from different bone marrow samples, named 17 and 18, were transduced with HSVl-TK lentiviral vectors (IGFl-eGFP-TK or eGFP-TK), and were further characterized after transduction. Confirmation and efficiency checks of completed transductions performed in cell lines 17 and 18 were accomplished by measuring the eGFP signal using a fluorescent activated cell sorter (F ACS) cell counting method. Cell line 17 MSCs had transduction efficiencies greater than 80%, but cell line 18 had much less (23% and 63% for the IGFleGFP- TK or eGFP-TK vectors respectively). Thus, cell line 17 transduced MSCs were used for both the in vitro and in vivo experiments. The initial HSV 1-TK cytotoxicity assay provided confirmation that the thymidine kinase gene worked efficiently in the absence of the IGF-1 protein and in the presence ofIGF-1, although the presence ofIGF- 1 caused a reduction in the rate of ablation ( cell death). Two additional assays, the MTT method and a trypan blue exclusion manual counting method, were completed to support the initial findings but the data was inconclusive. Subsequently, a MTT assay was attempted to create a kill curve over a period of 7 days, using a fixed Ganciclovir concentration, 25 μM, so as to determine the time point, which had the greatest decrease in the cell count or the time point in vitro. Unfortunately, the assay was unsuccessful and provided no useful information. An in vivo retention study of transduced MS Cs was then performed using 16 immunosuppressed mice (strain NOD/SCID/ILR2RG-/-). MSCs were transduced with four vector constructs: 1) luciferase reporter gene only, 2) IGFl-eGFP-TK co-transduced with luciferase with induction in vitro, 3) IGFl-eGFP-TK co-transduced with luciferase with induction in vivo, and 4) eGFP-TK co-transduced with luciferase. After intramuscular injection, mice were IVIS imaged to detect luciferase after receiving Ganciclovir injections for up to 50 days. After termination, tissues were removed for analyses such as quantitative PCR and morphological staining using H&E to identify necrosis caused by the activity of the HSV 1-TK. The in vitro data revealed that increasing Ganciclovir concentrations led to a reduction in the rate of cell proliferation, thereby confirming the cytotoxic effect of HSV 1-TK. In addition, it was determined that the cytotoxic effect of HSV 1-TK also worked in the presence ofIGF-1, but at a lower level. It was found that at least 1 % of transplanted MSCs were retained within the mice even in the presence of Ganciclovir. Further studies are needed to support our findings that the HSY 1-TK suicide gene strategy is a viable fail-safe method in the delivery of anti-apoptotic genes such as IGF-1 for prevention of neuronal death using transduced MSCs.