1. Researchers generated a knockout mouse model of Pompe disease by disrupting the murine acid α-glucosidase gene (Gaa). Mice with this gene knockout (Gaa-/-) represented both the infantile and adult phenotypes of the human lysosomal storage disorder. 2. Comparison of different mouse models found that mice with a disruption of exon 13 remained healthy while mice with disruptions of exon 6 and 14 showed weakness and increased glycogen accumulation, particularly in the heart muscle. 3. Emerging treatments for Pompe disease in mouse models include recombinant human acid α-glucosidase enzyme with optimized carbohydrate structures to enhance uptake in muscles and a "double knockout" mouse model suppressing both

1. Knock out mouse
model of Pompe
disease
Question: Discuss an example of knock out mouse model
used for disease modeling

2. Introduction
● Gene knockout is a genetically engineered organism that carries one or more gene in its chromosome (that have been
made inoperative).
● Also known as knockout organism that are used in a learning about a gene that has been sequenced.
● Purpose: to create a transgenic animal that has altered its gene.
A laboratory mouse that is genetically engineered mouse whose gene are made inoperable through a gene knockout.
1. Rats have genetic, biological and behavior characteristic closely resemble to humans and share many processes with
humans (Melina, 2010).
The characteristic of the knockout gene enable researchers to detect diseases, example: cancer, obesity, heart disease (NIH, n.d).
Example of the mouse model: p53 gene.
2. Convenient (The Jackson Laboratory, 2017).
3. Inexpensive (Melina, 2010).
WHY ME?WHY ME?

3. Gene targeting vs gene trapping

4. Disease model: GSDII Fatal disorder- characteristic progressive loss
of skeletal and/or heart muscle function.
Early onset (Infantile form) Late onset (Juvenile/Adult form)
- Most severe type
- Characterized by cardiomegaly,
generalized muscle weakness,
hypotonia, hepatomegaly.
- Death from respiratory failure before end
of 1st year of life.
- α-glucosidase activity: < 1% of normal
activity
- Lysosomal glycogen storage: massive
storage in many tissues
- Milder form
- Appears after the age of 1
- Affects skeletal muscle leading to
progressive muscle weakness &
respiratory impairment.
- Respiratory complications more severe.
- α-glucosidase activity: < 10% in juvenile
form and < 40% in adult form.
- Lysosomal glycogen storage: minimal to
no storage in tissues other than skeletal
muscles.
(Barba-Romero 2012)
Table 1.1

5. GSD11- Pompe disease
➔ Inherited in an autosomal recessive mode.
➔ Mutations in the acid α-glucosidase gene (GAA)- fully/partially inhibit
biosynthesis of acid α-glucosidase.
◆ Preventing degradation of lysosomal glycogen.
➔ Acid α-glucosidase: degrades lysosomal glycogen to glucose.
◆ Catabolic pathway essential for mobilization of glycogen in neonatal liver.
Later stages of life, acid α-glucosidase functions to prevent glycogen
storage in lysosomes. (Wisselaar 1992)
➔ Clinical phenotype: determined by mutant alleles combination & level of
residual acid α-glucosidase activity. (Table 1.1)

6. Generation of Knockout mouse model of Pompe disease

7. - Involved the targeted disruption of murine acid ⍺-glucosidase gene (Gaa)/
Gaa -/-
- Mouse Gaa cDNA obtained from mouse liver cDNA & human acid
⍺-glucosidase cDNA as probe
- 6.8 kb Asp718 fragment containing exon 5-14 of murine Gaa gene
- Positive control : neo cassette at EagI site in exon 13
- Negative control : tk cassette at 5’ end

8. Comparison & Challenges
Model Type Features Challenges
Disruption of exon 13 Represents infantile form. Remained phenotypically
normal.
6 neo/neo
) similar to
Δ14 neo
/ Δ14 neo
Represents infantile and
adult phenotype of disease.
Showed weakness at 3.5
weeks,
Δ6/ Δ6 Glycogen accumulation
(storage) was more
pronounced in the heart
tGaa -/-
Low level of GAA but retain
phenotype of disease
Increased enzyme activity;
decreased glycogen
accumulation
GAA-KO/SCID No anti-hGAA Ab
(Geel et al., 2007)

9. Emerging & Future Development
ATB200, a unique recombinant human
acid alpha-glucosidase (rhGAA)
enzyme with optimized carbohydrate
structures, particularly mannose-6
phosphate (M6P), to enhance uptake in
muscles, co-administered with AT2221
((Amicus Therapeutics, 2017))

10. Emerging & Future Development
The study's findings, published recently in
the Journal of Biological Chemistry, are
based on a "double knockout" mouse model
in which both acid alpha-glucosidase (GAA),
the enzyme that's deficient in Pompe
disease, and starch binding
domain-containing protein1 (Stbd1), a
cellular protein with previously unknown
function, were suppressed in mice.
(Researchers find conceivable alternative
way to treat Pompe disease, 2017)

11. Conclusion
● The mouse had an impact on the lives of human being in understanding
human biology and disease
● The mouse offers a close glimpse of humankind in the underlying tissue
structure, physiology and organization.
● The inbreeding of these mouse strain has been essential for identifying
genes with important implications.
● Best method for medical diagnosis and therapeutic development.
THANK YOU!

13. Reference
Amicus Therapeutics, I., 2017. Amicus Therapeutics Presents Important New Scientific Findings and Preclinical Data for Pompe Program at WORLDSymposium™ 2017. [online]
GlobeNewswire News Room. Available at:
<https://globenewswire.com/news-release/2017/02/15/917383/0/en/Amicus-Therapeutics-Presents-Important-New-Scientific-Findings-and-Preclinical-Data-for-Pompe-Program-
at-WORLDSymposium-2017.html> [Viewed 28 June. 2017].
Anon, 2017. “Gene Knockout” Available at: <http://www.biotec.uniba.it/area_docenti/documenti_docente/materiali_didattici/43_knockout.PDF> [Viewed 1 Jun. 2017].
Anon, 2017. “Knockout Mice Fact Sheet.” National Human Genome Research Institute (NHGRI). Available at: <https://www.genome.gov/12514551/> [Viewed 1 Jun. 2017].
Anon, 2017. Researchers find conceivable alternative way to treat Pompe disease. [online] News-Medical.net. Available at:
<http://www.news-medical.net/news/20160725/Researchers-find-conceivable-alternative-way-to-treat-Pompe-disease.aspx> [Accessed 1 Jun. 2017].
Barba-Romero, MA, Barrot, E, Bautista-Lorite, J, Gutiérrez-Rivas. E, Illa, I, Jiménez, LM, Ley-Martos, M, Munain LM, Pardo, J, Pascual-Pascual, SI, Pérez-López, J, Solera, J &
Vílchez-Padilla, JJ 2012, ‘Clinical guidelines for late-onset Pompe disease’, Rev Neurol, vol. 54, pp. 497-507.
Bijvoet, AGA, Van de Kamp, EHM, Kroos, MA, Ding, JH, Yang, BZ, Visser, P, Bakker, CE, Verbeet, MP, Oostra, BA, Reuser, AJJ & Van der Ploeg, AT 1998, ‘Generalized glycogen storage
and cardiomegaly in a knockout mouse model of Pompe disease’, Human Molecular Genetics, vol. 7, no. 1, pp. 53-62.
Geel, T., McLaughlin, P., de Leij, L., Ruiters, M. and Niezen-Koning, K., 2007. Pompe disease: Current state of treatment modalities and animal models. Molecular Genetics and
Metabolism, 92(4), pp.299-307.
Nguyen, D. and Xu, T., 2017. “The expanding role of mouse genetics for understanding human biology and disease.” Available at
<https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2561976/> [Viewed 1 Jun 2017].

14. Remy Melina., 2010. “Why Do Medical Researchers Use Mice?.” Live Science. Available at: <http://www.livescience.com/32860-why-do-medical-researchers-use-mice.html> [Viewed
1 Jun. 2017].
Skarnes, W., 2017. “Two ways to trap a gene in mice.” Available at : <http://www.pnas.org/content/102/37/13001.full> [viewed 1 June 2017]
The Jackson Laboratory, 2017. “Why mouse genetics” Available at: <https://www.jax.org/research-in-action/why-mouse-genetics#> [Viewed 1 Jun. 2017].
Wisselaar, HA, Kroos, MA, Hermans, MMP, Beeumen, JV & Reusers, AJJ 1992, ‘Structural and Functional Changes of Lysosomal Acid a-Glucosidase during Intracellular Transport
and Maturation’, The Journal of Biological Chemistry, vol. 268, no. 3, pp. 2223 – 2231.