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
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.
• 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
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 glycosaminoglycans results in damage to several bodily
Figure 1. Accumulation of
systems including central nervous system,
substrates in lysosomes.
muscoskeletal system, cardiorespiratory system, etc.
•There are 6 distinct forms that are each characterized by GAG
storage and type of enzyme deficiency.
http://media.photobucket.com/image/lysosomal%20storage/Yehia
/lysosome.jpg
Another mouse exhibited unusual facial and bodily
swelling
www.brc.riken.go.jp/lab/animal/images/00122.jpg
• Mice were euthanized according to Institutional
Animal Care and Use Committee (IACUC)
requirements.
Figure 3. Image of mouse from C57/BL 6 strain.
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
• As a result, the optimal bone marrow transplantation
parameters remained undefined.
•Level 1 and 2 behaviors depicted by the mice indicated
the success of providing a dosage of lethal irradiation.
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
•Analyzing BMT results, one of the transplanted mice
exhibited unusual facial and bodily swelling .
http://www.dpr.com/images/projects/large/Sharp%20Health_Lin
ear%20Accelerator_Interior.jpg
Figure 4. Linear accelerator used
for irradiation.
of Lethality
•Post-irradiation,
•It can be hypothesized that the facial swelling may have
been attributed to a bacterial infection.
the mice were monitored twice a day.
Assessment of Mouse Activity
What is Mucopolysaccharidoses Type IIIB?
•There
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.
•The failure of the Bone Marrow Transplantation could
be attributed to several factors:
Level of
Description of Activity
Activity
1 -Listless
-No mobility altogether
-Failure to eat or drink
-Severe hunching
2 -Hunched posture
- Lethargic
- Little to no movement
- Does not approach strangers
- Migrates to corners of cage
- Slight tremors
3 - Noticeable decrease in activity yet still active
- Approaches strangers
- Eating and drinking properly
4 - Demonstrating typical behavior:
Active
Approaches strangers
Eating and drinking appropriately
are four variant forms of MPS III:
http://commons.wikimedia.org/wiki/File:Chromosome_17.jpeg
Figure 2. Deficiency of
α-N-acetylglucosaminidase is
caused by gene mutation on
chromosome 17q21.
oMPS
IIIB is marked by progressive degeneration of the central
nervous system.
children exhibit progressive mental retardation, sleep
disorders, aggressive behavior, dementia, and hyperactivity.
• Mortality is common around the second decade of life.
•The definite cause of this atypical symptom in this
mouse was not elucidated.
Insufficient number and poor quality of graft tissues
Recipient specific engraftment problems
Table 1. Used to
assess the
effectiveness of
irradiation and
transplantation
in the mice by
looking at
mouse behavior.
Excessive dosage of irradiation
A weakened immune system and the lapse of time
before the BMT procedure (24 hours and 48 hours)
• 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
•Affected
Survivorship of MPS IIIB Mice Post-Irradiation
120%
(1)
100%
•There
Current Treatments Are Available?
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.
80%
% Alive
What
(2)
(3)
BMT
no BMT
60%
40%
Graph I. Indicates
percentage of
transplanted and nontransplanted mice
post-irradiation.
20%
0%
0.00
2.00
4.00
6.00
8.00
Days Post Irradiation
10.00
(4)
(5)
(6)
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.
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.
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