Dr. Kristen Skvorak's Keynote presentation at the 2014 PKU Illinois Annual Meeting

1. Placental Stem Cell Transplant
Improves PKU Symptoms in Mice
Kristen J. Skvorak, Ph.D.
Postdoctoral Fellow
University of Pittsburgh Med Center, Pediatrics
Mentors: Dr. Stephen Strom and Dr. Jerry Vockley
PKU Organization of IL Meeting
Skokie, IL
Nov. 8, 2014

2. Current Treatments
• Physiological / non-physiological
amino acid
therapy
• Phe-restricted diet
– Lifelong strict compliance
– Expensive
– Taste?
• BH4 (Kuvan™)
supplementation
– Expensive
– Does not work for everyone
• PEG-PAL
– Phase 2 clinical trials
• Gene Therapy
• Cell Transplant
– Liver cells
– Placental cells
Harding, C. , Clin. Genet., 2008 Aug;74(2):97-104

3. WHY Cell Transplant?
Previous Studies –
Liver Cell Transplant

4. Benefits of Liver Cell Transplant
1. Less expensive (5-10% the cost of liver transplant)
2. Less invasive, fast recovery (several infusions over 24-48h)
Picture of a patient receiving liver cell transplant at the Children’s
Hospital of Pittsburgh. A catheter is inserted into the umbilical vein,
which can directly access the liver, and cells are slowly infused.

5. Benefits of Liver Cell Transplant
1. Less expensive (5-10% the cost of liver transplant)
2. Less invasive, fast recovery (several infusions over 24-48h)
3. Fewer incidents of serious complications
4. Transplanted cells are from “recycled” livers
- Still relying on donor livers (limited source)
- lifelong(?) immunosuppression
5. Transplanted cells only have ONE JOB
 If cells fail, the patient would only go back to the condition he/she was
in before undergoing cell transplant. (Failure of a transplanted liver
would mean an immediate and serious threat to the patient’s life.)

6. Liver Cell Transplant and Disease
• Animal models of disease
– Crigler-Najjar
– Maple Syrup Urine Disease (K. Skvorak)
– PKU (C. Harding, K. Skvorak)
– Glycogen storage disease
– Wilson’s Disease
• Patients – bridge therapy and beyond
– PKU (ongoing clinical trial, Children’s Hospital of Pittsburgh)
– Crigler-Najjar (1998)
– Glycogen storage disease
– Ornithine Transcarbamylase (OTC) deficiency
– Factor VII deficiency
– Biliary Atresia
– Additional Urea Cycle disorders
– Liver failure (1997)

7. MSUD and PKU
• Both are caused by a nonfunctional or dysfunctional liver enzyme that
breaks down essential amino acids.
PKU MSUD
 IEM detected by
newborn screening?
 Mutation in liver
enzyme?
 Incidence in the
population?
 Toxic build up of
essential amino acids?
 Primarily brain toxicity?
 Commonly treated with
strict diet and ‘formula’?
 Premature death is
common?
 Other treatments
available?
Yes
PAH
1 in 10,000-14,000 births
(general)
Phenylalanine
Yes
Yes
No
Biopterin (Kuvan®)
Yes
BCKDH
1 in 290,000 (general)
1 in 176 (Mennonite)
BCAA (Leucine, Valine,
Isoleucine)
YES
Yes
YES
Liver Transplant!
Hi, I have
MSUD!

8. Procedure and Rationale
• Healthy donor cells have normal enzyme activity
– <10% PAH activity: more manageable disease; increased protein tolerance
– 10-20% activity: potential cure for PKU (Harding & Gibson, 2010)
– Classic MSUD patients have <2% enzyme activity – poor outcome
• Non-Classic MSUD is often missed by newborn screening
• Liver transplant (only active enzyme is in liver) = cure
• Treatment at birth – clinically relevant to treat this way
• No surgery required (baby mice are TINY!)
– Can clearly see the liver through the skin.
– Cells are loaded into a syringe and are injected
directly into the liver
• Newborn mouse livers are rapidly growing
Liver
Tx
Cell
Time
Newborn Adult
PKU
MSUD

9. Previous Results – MSUD mouse
After liver cell transplant:
• Survival was significantly lengthened
70% survived to
35 days of age
after transplanting
liver cells.
Untreated
animals all died
within days of
weaning (~21
Skvorak, et al., 2009, Mol Ther 17:1266 days of age).

10. Previous Results – MSUD mouse
After liver cell transplant:
• Survival was significantly lengthened
• Blood BCAA (Leucine) levels were reduced 75% compared to
untreated animals.
75%
Skvorak, et al., 2009, Mol Ther 17:1266

11. Previous Results – MSUD mouse
6%
13%
Skvorak, et al., 2009, Mol Ther 17:1266
After liver cell transplant:
• Survival was significantly lengthened
• Blood BCAA levels were reduced 75%
This was achieved despite the fact that:
• Enzyme activity was only increased from 6% to 13%

12. Summary – MSUD mouse
After liver cell transplant:
• Survival was significantly lengthened
• Blood BCAA levels were reduced 75%
• Enzyme activity was increased from 6% to 13%
• Neurotransmitters were also improved
• BUT at the time of sacrifice, blood and brain amino
acids level improvements had begun to reverse…
WHY?
Skvorak, et al., 2009, Mol Ther 17:1266;
2009, B Acta 1792:1004

13. We needed to find a cell that would not be targeted for rejection.
Placental Stem Cells:
Amnion Epithelial (AE) Cells

14. Amnion – thin membrane surrounding the fetus during pregnancy
Epithelium – cells that line the inner and outer body surfaces
• Acquired after full term birth
– Plentiful – C-sections account for a third of
all births in the USA
• Easy to isolate, easy to grow in the lab
• Non-controversial source of stem cells
• Not cord blood cells
• Anti-fibrotic, anti-inflammatory, and anti-microbial
characteristics
• Can “hide” from the immune system
• Freeze/thaw well
– Cell banking potential x100
Amnion
Chorion
Decidua
Placental Tissue

15. Placenta cross section
AA Analysis (blood/brain)
Neurotransmitters
Enzyme activity
Human DNA in mouse liver
KJ Skvorak, et al. 2012
7 days
Isolate human
amnion epithelial
(hAE) cells
hAEC Tx
1 million cells/mouse
transplanted directly
to liver (birth)
Birth
On normal diet
No immunosuppression
x100
Amnion
Chorion
Dedidua
1 month post-tx
(young adult)
14 days 21 days 28 days
100 days
Post-tx
Skvorak, et al. 2013 Hepatology. 57(3):1017-23

16. Previous Results – MSUD mouse
After AE cell transplant:
A. Growth rate was normalized to healthy litter mates (WT)
B. Survival was significantly lengthened (80% at 100 days)
Skvorak, et al. 2013 Hepatology. 57(3):1017-23;
A. B.

17. Summary of Previous Results – MSUD
After AE cell transplant:
• Growth rate was normalized to healthy litter mates (WT)
• Survival was significantly lengthened (80% at 100 days)
• Leucine was normalized,
• Neurotransmitters (dopamine, serotonin) were
significantly improved.
mouse
This was achieved despite the fact that:
• Enzyme activity was increased from 6% to 13% (the same
amount as with liver cell transplant)
WHY then are we seeing better results?
NO EVIDENCE OF REJECTION!
Skvorak, et al. 2013 Hepatology. 57(3):1017-23;
2013 Mol Genet Metab.;109(2):132-8.

18. Now we can move on to…
A Mouse Model of PKU
Hi, I have
PKU!

19. A mouse model of PKU
PKU
PKU on Diet
Normal
PAH
Phe Tyrosine
Dopamine
HVA
DOPAC
3-MT
Melanin
• No enzyme activity
•  Phe in the blood, organs, and brain
• Disruptions in brain amino acids / neurotransmitters
• Hypopigmented (fur changes from black to light brown)

20. A mouse model of PKU
PAH
Phe Tyrosine
Dopamine
• No enzyme activity
•  Phe in the blood, organs, and brain
• Disruptions in brain amino acids / neurotransmitters
• Hypopigmented (fur changes from black to light brown)
• Smaller than healthy siblings
• Cognitive (memory, learning) problems
• Offspring of PKU moms suffer defects similar to human
maternal PKU

PKUenu2 mouse is a great model for human PKU.
HVA
DOPAC
3-MT
Melanin

21. Placenta cross section
AA Analysis (blood/brain)
Neurotransmitters
PAH activity
Human DNA in mouse liver
KJ Skvorak, et al. 2012
7 days
Isolate human
amnion epithelial
cells (hAE)
hAEC Tx
1 million cells/mouse
transplanted directly
to liver (birth)
PKU MOUSE
On normal diet
No immunosuppression
x100
Amnion
Chorion
Dedidua
1 month post-tx
(young adult)
14 days 21 days 28 days
100 days
Post-tx

22. Results – Blood Phe Improvement
Male vs Female mice
• Female blood Phe
levels were on
average 40% higher
than males.
• This difference has
not been reported in
patients, likely
because people are
all genetically
different.
Statistics
* = p<0.05
** = p<0.01
*** = p<0.001
Skvorak, 2014

23. Results – Blood Phe Improvement
After liver cell
transplant, PKU
mouse Phe was
reduced ~25%.
Statistics
* = p<0.05
** = p<0.01
*** = p<0.001
25%
Skvorak, 2014

24. Results – Blood Phe Improvement
Statistics
* = p<0.05
** = p<0.01
*** = p<0.001
After AE cell
transplant, PKU
mouse Phe was
reduced ~60%.
60%
Skvorak, 2014

25. Results – Brain Phe Improvement
There was no difference between male and female Phe levels in the brain
Statistics
* = p<0.05
** = p<0.01
*** = p<0.001
After liver cell
transplant, PKU
mouse Phe was
reduced ~60%.
Skvorak, 2014

26. Results – Brain Phe Improvement
There was no difference between male and female Phe levels in the brain
Statistics
* = p<0.05
** = p<0.01
*** = p<0.001
After AE cell
transplant, PKU
mouse Phe was
normalized!
Skvorak, 2014

27. Results - Neurotransmitter Improvement
Dopamine is one of the most important
neurotransmitters in the brain.
Phe Tyrosine Dopamine
20% 50%
NS = not statistically different
from control mice
HVA
DOPAC
3-MT
PAH
The dopamine pathway is begins with phenylalanine,
and is therefore disrupted in PKU.

28. Results - Neurotransmitter Improvement
Also
Normalized:
Taurine
Glycine
Aspartate
NS = not
statistically
different
from control
mice

29. Summary
1. Cell therapy was tested in a mouse model of PKU
– Mouse liver cells and human placental stem cells
2.  Blood and Brain Phe with cell transplant
– Brain Phe was normalized with placental stem cells
3. Additional corrections in brain (Neurotransmitters!)
4. Viable alternative therapy for PKU (and other liver-based
metabolic diseases)
– Placental stem cells are a non-controversial source of
cells, which has immunomodulatory properties

30. Acknowledgements
University of Pittsburgh, PA
Jerry Vockley, MD, PhD
Kansas University Med. Center, KS
Kenneth Dorko
University of Cagliari, Cagliari, Italy
Fabio Marongiu, PhD
Yale University Medical School, CT
Marc Hansel, PhD
University of Iowa Health Care
Veysel Tahan, MD
Washington State University, WA
K. Michael Gibson, PhD
Baylor Research Institute, TX
Erland Arning, PhD
Teodoro Bottiglieri, PhD
Karolinska Institutet, Stockholm, Sweden
Stephen Strom, PhD
Roberto Gramignoli, PhD
Funding
National PKU Alliance
The Strom Lab in Pittsburgh

31. Thank you!

32. Questions?