Kristen J. Skvorak, Ph.D. Postdoctoral Fellow University of Pittsburgh University of Pittsburgh Mentors: Dr. Stephen Strom and Dr. Jerry Vockley NPKUA.

Hepatocyte transplantation improves blood and brain Phe and neurotransmitter imbalances in a

mouse model of PKUKristen J. Skvorak, Ph.D.

Postdoctoral Fellow University of Pittsburgh

Mentors: Dr. Stephen Strom and Dr. Jerry Vockley

NPKUA ConferenceCherry Hill, NJ

July 26-29, 2012

OutlineA mouse model of PKU and the human diseaseCurrent TreatmentsLiver Transplant vs. Liver Cell Transplant

Cell transplant and metabolic diseaseTreatment StrategyResults

BloodBrain

SummaryHuman amnion epithelial stem cellsWhat’s next?Acknowledgements

A mouse model of PKU

Missense mutation results in complete inactivity of phenylalanine hydroxylase (PAH) enzyme

High levels of Phe in the blood, tissue, and brainHypopigmented (fur changes from black to light

brown)Slight delay in growth compared to healthy siblingsCognitive (memory, learning) impairedAdult female mice are fertile, but offspring suffer early

developmental defects similar to human maternal PKU

PKUenu2 mouse is a great model for human PKU.

Phe TyrosinePAH

+BH4

Dopamine HVADOPAC

3-MT

Current Treatments

Harding, C. , Clin. Genet., 2008 Aug;74(2):97-104

Dietary restriction Complicated, expensive Bad taste Non-compliance

BH4 (Kuvan™) supplementation Does not work for

everyoneLiver Cell Transplantation

(Tx) Healthy liver cells contain

100% functional PAH to boost enzyme levels thus reducing Phe levels

Benefits of Liver Cell Tx over Liver TxLess expensive (5-10% the cost of liver transplant)Less invasive, faster recoveryFewer incidents of serious complications or

surgical related deathsMultiple treatments are possible into one patientCells are harvested from donor livers rejected for

whole liver transplant Still limited by the availability of donor livers

Transplanted cells do not need to support all liver functions, only that of the missing enzyme (PAH) If cells fail, the patient would only revert to the condition

he/she was in before undergoing liver cell transplant

Liver Cell Tx and Metabolic DiseaseLiver cell transplant has cured many preclinical animal

models of metabolic disease Crigler-Najjar Maple Syrup Urine Disease (K. Skvorak) PKU (C. Harding) Glycogen storage disease Wilson’s Disease

Liver cell transplant has already been used clinicallyCrigler-NajjarGlycogen storage diseaseOrnithine Transcarbamylase (OTC) deficiencyFactor VII deficiencyBiliary AtresiaAdditional Urea Cycle disordersLiver failure

Treatment StrategyLiver cell transplant could increase enzyme activity

thus helping to improve patient symptoms<10% activity: more manageable disease; increased Phe

tolerance10-20% activity: potential cure (Harding & Gibson, 2010)

Treatment would be most beneficial at birthClinically relevantAvoid surgery – mouse livers are clearly visible through

skinBy weaning (21 days old) mice are already showing

symptoms of PKUNeonatal mouse livers are rapidly expanding

possible growth advantage for transplanted cells

Treatment Strategy

PKU mice (birth)

7 days

Liver Cell Tx

1 million cells transplanted directly into liver

(1 liver ~30 transplants)

Isolate mouse liver

cells

14 days 21 days

(normal diet)

Treatment Strategy

AA profiles (blood and brain)Neurotransmitter profiles

PKU mice (birth)

7 days

Liver Cell Tx

2 million cells transplanted into the spleen

(1 liver ~30 transplants)

Isolate mouse liver

cells

35 days14 days 21 days

(young adult)

28 days

(normal diet)

Results – Phe was reduced in blood after cell transplant

Females had almost double blood Phe levels compared to males.

Phe was reduced 18% in Rosa tx Females and 25% in C57 and AE tx Females.

Interestingly, human placental stem cells (AE) were just as effective as mouse liver cells.

A combination of early + late tx is most beneficial because it will maximize cells engrafted in the liver.

Transplanted CellsMouse liver cells: Rosa, C57Human stem cells from placenta: AE

Results – Phe was reduced in brain after cell transplant

Statistics* = p<0.05** = p<0.01*** = p<0.001

Phe was reduced ~50% in the brains of PKU mice tx with Rosa mouse liver cells.

Phe was reduced ~75% in the brains of PKU mice tx with C57 mouse liver cells.

Phe was normalized in the brains of PKU mice tx with human AE cells.

There was no difference between males and females.

Transplanted CellsMouse liver cells: Rosa, C57Human stem cells from placenta: AE

Results – Many other amino acids were normalized in brain

Statistics* = p<0.05** = p<0.01*** = p<0.001 High Phe concentrations in the brain disturb the healthy levels of

other important chemicals such as neurotransmitters, which are important brain messengers that carry information from one cell to another.

Neurotransmitter Pathways

Tryptophan and Serotonin were normal in the PKU mouse, but 5-HIAA was significantly reduced. 5-HIAA was not improved with cell transplant.

Metabolites along the Dopamine (Phe) pathway were improved after cell transplant.

Phe

Results – improvements in the Dopamine pathway after cell transplant

Sig

nifi

can

tly

Corr

ect

ed

Norm

ali

zed

Statistics* = p<0.05** = p<0.01*** = p<0.001

SummaryMy research involves testing cell therapy in a mouse

model of PKU that closely resembles the human disease.Cell therapy consists of a combination of several early

transplants immediately after birth and one transplant in older mice.

Blood Phe was improved 18-25% after cell transplantObserved difference between male and female

Unique to this mouse model , this has not been reported in the human disease

Brain Phe was improved 50-75% after mouse liver cell transplant while many other amino acids were normalized.

Brain Phe was normalized after human AE cell transplantMetabolites along the Dopamine pathway were either

normalized or significantly corrected after mouse liver cell transplant.

Placental Amnion Epithelial (AE) Stem Cells Amnion – thin membrane surrounding the

fetus during pregnancy Epithelium – cells that make up the outer

layer of the body

Acquired from human placenta following full term birth Plentiful – more than 1.2 million c-

sections/year in the US Easy to isolate, easy to maintain in culture Non-controversial source of stem cells

Not cord blood cells AE does not primarily function to produce blood

cellular components

Documented anti-fibrotic, anti-inflammatory, and anti-microbial characteristics

Can evade immune detection Freeze/thaw well

x100

Amnion

Chorion

Decidua

Placental Tissue

KJ Skvorak, et al. 2012

Liver analysisEnzyme activityAA and Neurotransmitters (Blood and Brain)Survival and Growth

1 week

hAE Tx

1 million cells/mouse transplanted directly

to liver (birth)

2 million cells/mouse transplanted directly

to liver (3+ weeks)

Long term study

14 weeks(100d)

5 weeks(35d)

3 weeks

MSUD mice • 16-fold increase BCAA/ala VS wildtype• 5-6% of normal BCKDH activity• Survive to ~3-4 weeks of age•Fed a Normal Protein Diet Throughout Study

Isolate human amnion epithelial

cells (hAEC)

Hepatocyte Tx in a mouse model of MSUD

Mol Ther. 2009; 17(7): 1266-73Biochim Biophys Acta. 2009;

1792(10):1004-10

Improvements in Growth and Survival

Growth was normalized in MSUD animals after AE cell transplant

All untreated MSUD animals consistently lost weight and died prior to 28 days.

Survival was significantly improved after AE cell transplant

100% survival at 35 days 82% survival at 100 days 0% survival post-28 days

in untreated animals

hAE Transplant doubled residual enzyme activity

BCKDH enzyme activity was increased from 6% to ~13% in AE transplanted animals.

6%

~13%

Amino Acid Improvements

At 100 days of age, hAE improved (in brain): leucine, isoleucine, and valine (the BCAA) by >60% BCAA/ Alanine ratio by >50% Alloisoleucine (biophysical marker of MSUD) >80% Normalized other Large Neutral Amino Acids and GABA (neurotransmitter) All improvements were also seen in blood, and at both timepoints at 35 days

hAE Cell Summary A mouse model of MSUD was partially corrected

after liver cell transplant (Skvorak et al. Mol Ther. 2009 17(7): 1266-73 and Biochim Biophys Acta. 2009; 1792(10):1004-10)

Further studies involving human AE cell transplant in this mouse model improved:Survival and growthBCKDH enzyme activityBCAA levels in blood and brainOther relevant amino acid levelsSome neurotransmitters (GABA, serotonin, dopamine

metabolites, serotonin and dopamine turnover)If clinical hepatocyte transplantation proves

successful for PKU, placental derived AE “stem” cells may provide an alternate source of cells for cell transplant to treat metabolic disease.

What’s next?Continue testing cell therapies in the PKU

mouseAcquire patient cells and make induced

pluripotent stem cells (iPSC)Correct the mutation(s) with molecular meansMay also provide alternate source of cells

Continue to work with the clinical hepatocyte (liver cell) transplant program at the Children’s Hospital of Pittsburgh

AcknowledgementsUniversity of Pittsburgh FundingStephen Strom NPKUARoberto GramignoliKen DorkoMarc HanselVeysel TahanJerry Vockley

Michigan Tech UniversityK. Michael Gibson

Baylor Research InstituteErland ArningTerry Bottiglieri

Karolinska InstitutetStockholm, Sweden

Thank you!

Questions?The Golden Triangle

Pittsburgh, PA