1. Genetic Modified Cell Therapy for Amyotrophic Lateral Sclerosis (ALS) Tianyi Cai PBIO-4500 12/02/2014

2. Overview and Futures of ALS A fatal neurodegenerative disorder causes death of motor neurons Patients develop paralysis rapidly and die due to respiratory insufficiency in 3-5 years after diagnosis 10% ALS are familial (fALS) 20% of fALS cases are linked to mutation for SOD1 (over-expressed) allele containing Gly93. Traditional pharmacological therapies have largely failed so far

3. Design and Mechanism of the Experiment The researchers found that glucagon-like peptide 1 (GLP-1) exhibits anti- oxidation and is neuroprotective against excitotoxicity. To avoid systemic administration, the researchers decided to deliver GLP-1 directly into the central neurvous system. Researchers investigative tested an encapsulated cell line which can produce GLP-1 to replace an osmotic minipump which may cause more complications. Scientists modified a mesenchymal stromal cell (MSC) line to produce GLP-1. An encapsulated GM MSC is intracerebroventricular injected in experimental animals to test the results.

4. Materials and Methods Experimental animals – G93A transgenetic familial ALS mice (Figure 1.) Cell Line and Alginate microcapsules – Cell lines: hMSC cell line was immortalized by transduction with the human Telomarase Reverse Transcriptase (hTERT) gene. Then, cells are transfected with a plasmid vector encoding GLP-1 fusion gene. – Microcapsules: Cells are embedded in a 160 μm diameter spherical alginate matrix. One matrix contains about 94 cells. (Figure 2.)

5. Figure 1. Analysis of post-mortem brain tissue at day 110 of SOD1 (G93A) mice. Knippenberg S, Thau N, Dengler R, Brinker T, et al. (2012) Intracerebroventricular Injection of Encapsulated Human Mesenchymal Cells Producing Glucagon-Like Peptide 1 Prolongs Survival in a Mouse Model of ALS. PLoS ONE 7(6): e36857. doi:10.1371/journal.pone.0036857 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036857

6. Figure 2. Alginate encapsulated GLP-1 producing mesenchymal cells. Knippenberg S, Thau N, Dengler R, Brinker T, et al. (2012) Intracerebroventricular Injection of Encapsulated Human Mesenchymal Cells Producing Glucagon-Like Peptide 1 Prolongs Survival in a Mouse Model of ALS. PLoS ONE 7(6): e36857. doi:10.1371/journal.pone.0036857 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036857

7. Materials and Methods Method – Drill a 1.4 mm hole on the head of each mouse. – Inject microcapsules which were treated by the cell solution into brain through the hole. Treatment group and vehicle control group – One group of mice was treated by microcapsules contains MSC which can produce GLP-1. – The other group mice was treated by empty microcapsules.

8. Result Survival Study (Figure 3, Figure 4) – General conditional – Survival percentage – Weight – Rotarod performance – Step length analysis – Runtime analysis Histological analysis

9. Figure 3. Effects of GLP-1 treatment on survival times, general condition and weight measurements. Knippenberg S, Thau N, Dengler R, Brinker T, et al. (2012) Intracerebroventricular Injection of Encapsulated Human Mesenchymal Cells Producing Glucagon-Like Peptide 1 Prolongs Survival in a Mouse Model of ALS. PLoS ONE 7(6): e36857. doi:10.1371/journal.pone.0036857 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036857

10. Figure 4. Effects of GLP-1 treatment on rotarod performance and footprint analyses. Knippenberg S, Thau N, Dengler R, Brinker T, et al. (2012) Intracerebroventricular Injection of Encapsulated Human Mesenchymal Cells Producing Glucagon-Like Peptide 1 Prolongs Survival in a Mouse Model of ALS. PLoS ONE 7(6): e36857. doi:10.1371/journal.pone.0036857 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036857

11. Figure 5. Immunhistological analysis of MAP2 in spinal cord tissue of SOD1 (G93A) mice. Staining against microtubule associated protein 2 was stronger in animals treated with GLP-1 MSC (right) compared to control group (Left ).

12. Conclusion According to the data, we can conclude that intracerebroventricular injection of encapsulated hMSC-GLP1 exhibits neuroprotective potential in SOD1- G93A mice.

13. Discussion Advantages – Compare to the only medicine, Riluzole, hMSC-GLP1 delays the disease onset and weight loss. – This cell therapy has less side effects compare to the other therapies. Limitation – Has to be treated before the disease onset. – Surgery may cause other systemic inflammation.

14. Reference Knippenberg S, Thau N, Dengler R, Brinker T, Petris; Intracerebroventricular injection of encapsulated human mesenchymal cells producing glucagon-like peptide 1 prolong survival in a mouse model of ALS. PLoS One, 2012. Lewis C, Suzuki M; Therapeutic applications of mesenchymal stem cells for amyotrophic lateral sclerosis. Stem Cell Research & Therapy, 2014, 5. Keifer O, O’Connor D, Boulis N; Gene and protein therapies utilizing VEGF for ALS. Pharmacology & Therapeutics, 2014, 141. Gao A, Peng Y, Deng Y, Qing H; Potential therapeutic applications of differentiated inducedpluripotent stem cells in the treatment of neurodegenerative diseases. Neuroscience, 2013, 228.

15. Thank you!