Animal Model Sixteen mice of the strain C57/BL6 were used in this experiment.

1. Research Experience in Molecular Biotechnology & Genomics • Summer 2010 Center for Integrated Animal Genomics Lethal Irradiation and Bone Marrow Transplantation in the Mouse Model of Mucopolysaccharidosis Type IIIB Angela A. Mensah1, Elizabeth Snella2, Eman Mohammed2, Michelle Rutz-Mendicino2, N. Matthew Ellinwood2 1Department of Biology, University of Maryland, Baltimore County, 2Department of Animal Science, Iowa State University Purpose Materials and Methods Results • Overall objective is to identify a curative therapy to treat neuopathic lysosomal disorders including MPS IIIB. • The purpose of this study is to determine the effectiveness of using a single 8-gray dose to lethally irradiate MPS IIIB mice, and to successfully rescue irradiated mice using BMT transplant. • Animal Model • Sixteen mice of the strain C57/BL6 were used in this experiment. • Ten mice were affected with MPS IIIB. • Six mice were heterozygote carriers of MPS IIIB. • Mice were housed in accordance with IACUC and NIH guidelines. • Irradiation and Bone Marrow Transplantation • Ten affected mice were irradiated using a • linear accelerator. • Four out of the ten mice were used as • bone marrow recipients. • Six mice were used as bone marrow • donors. • Bone Marrow Harvesting • The femurs and tibias of mice were • flushed with Phosphated Buffer Saline. • Trypsin was used to remove the bone • marrow from the Petri dish. • ACK was used to lyse red blood cells. • Assessment of Lethality • Post-irradiation, the mice were monitored twice a day. • On Day 7 of experimentation, mice began exhibiting signs of distress. • Day 9 of experimentation, irradiated mice were demonstrating Level 1 and 2 behaviors (see Table I). • Transplanted mice: • One mouse died unexpectedly • Another mouse exhibited unusual facial and bodily swelling • Mice were euthanized according to Institutional Animal Care and Use Committee (IACUC) requirements. • As a result, the optimal bone marrow transplantation parameters remained undefined. Introduction • What is a Lysosomal Storage Disease? • Caused by single-gene defects that affect lysosomal function. • Results in cellular accumulation of undigested substrates • Currently 40 known lysosomal storage diseases. • The incidence of lysosomal storage diseases is 1 in 7,700 births. • What are Mucopolysaccharidoses? • A group of inherited lysosomal storage disorders. • Characterized by deficiency of lysosomal enzymes to degrade glycosaminoglycans. • Cellular accumulation of glycosamino-glycans results in damage to several bodily systems including central nervous system, • muscoskeletal system, cardiorespiratory system, etc. • There are 6 distinct forms that are each characterized by GAG storage and type of enzyme deficiency. • What is Mucopolysaccharidoses Type IIIB? • There are four variant forms of MPS III: • Type A, B, C, and D. • Each form is caused by a deficiency of one • of the four enzymes that degrades heparan • sulfate. • MPS IIIB is caused by a mutation on • the gene located on chromosome 17q21. • Caused by the deficient enzyme • α-N-acetylglucosaminidase, which cannot • degrade heparan sulfate. • MPS IIIB is marked by progressive degeneration of the central nervous system. • Affected children exhibit progressive mental retardation, sleep disorders, aggressive behavior, dementia, and hyperactivity. • Mortality is common around the second decade of life. • What Current Treatments Are Available? • There is no available treatment to cure this rare disorder. • Therapies focus on decreasing substrate storage and increasing catalytic activity. • Enzyme Replacement Therapy: infusion of exogenous enzymes. • Gene Therapy: genetically-modified, enzyme producing cells. • Substrate Deprivation Therapy: inhibit substrate synthesis. • Bone Marrow Transplantation: provision of donor-derived, enzyme producing cells. www.brc.riken.go.jp/lab/animal/images/00122.jpg Figure 3. Image of mouse from C57/BL 6 strain. Discussion • Level 1 and 2 behaviors depicted by the mice indicated the success of providing a dosage of lethal irradiation. • Analyzing BMT results, one of the transplanted mice exhibited unusual facial and bodily swelling . • The definite cause of this atypical symptom in this mouse was not elucidated. • It can be hypothesized that the facial swelling may have been attributed to a bacterial infection. • The failure of the Bone Marrow Transplantation could be attributed to several factors: • Insufficient number and poor quality of graft tissues • Recipient specific engraftment problems • Excessive dosage of irradiation • A weakened immune system and the lapse of time before the BMT procedure (24 hours and 48 hours) http://media.photobucket.com/image/lysosomal%20storage/Yehia/lysosome.jpg http://www.dpr.com/images/projects/large/Sharp%20Health_Linear%20Accelerator_Interior.jpg Figure 1. Accumulation of substrates in lysosomes. Figure 4. Linear accelerator used for irradiation. Assessment of Mouse Activity • Table 1. Used to assess the effectiveness of irradiation and transplantation in the mice by looking at mouse behavior. Future Studies • Future studies involve: • Determining an accurate dosage of lethal irradiation • Making an irradiation gradient ranging from 4 gray to 8 gray • Utilizing five groups of three transplanted mice and one non-transplanted mouse http://commons.wikimedia.org/wiki/File:Chromosome_17.jpeg Figure 2. Deficiency of α-N-acetylglucosaminidase is caused by gene mutation on chromosome 17q21. Results Selected References • Champe, P.C, Harvey, R.A., Ferrier, D.R. “Glycosaminoglycans and Glycoproteins.” Biochemistry: Third Edition. Baltimore: Lipincott Williams and Wilkins, 2005. • Cressan,t A., Desmaris, N., Verot, L., et al. 2004. “Improved Behavior and Neuropathology in the Mouse Model of Sanfilippo Type IIIB Disease after Adeno-Associated Virus-Mediated Gene Transfer in the Striatum.” The Journal of Neuroscience. 45, 10229-10239. • Heard, Jean. 2003. “Gene Therapy for Mucopolysaccharidosis.” International Review of Neurobiology. 55, 271-296. • (4) Meikle, P.J, Hopwood, J.J, Clague, A.E, Carey, W.F. 1999. “Prevalence of lysosomal storage disorders. JAMA. 281, 249-254. • Neufeld, Elizabeth F. and Muenzer, Joseph. “The Mucopolysaccharidoses.” • Prasad, Vinod K. and Kurtzberg, Joanne. 2010. “Transplant Outcomes in Mucopolysaccharidoses.” Seminars in Hematology. 47, 59-69. Graph I. Indicates percentage of transplanted and non-transplanted mice post-irradiation. Acknowledgements Gratitude and appreciation are extended to Dr. Ellinwood, Elizabeth Snella, Eman Mohammed, and Michelle Rutz-Mendicino for their help, guidance, and knowledge during the course of this experiment. In addition, the author would like to thank Dr. Rothschild for providing such an opportunity to contribute to the world of academia. This study was funded by the National Science Foundation. The author would also like to extend thanks to the Lauren’s Hope Foundation. Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371