1 / 24

Kristen J. Skvorak , Ph.D. Postdoctoral Fellow University of Pittsburgh

Hepatocyte transplantation improves blood and brain Phe and neurotransmitter imbalances in a mouse model of PKU. Kristen J. Skvorak , Ph.D. Postdoctoral Fellow University of Pittsburgh Mentors: Dr. Stephen Strom and Dr. Jerry Vockley NPKUA Conference Cherry Hill, NJ July 26-29, 2012 .

ash
Download Presentation

Kristen J. Skvorak , Ph.D. Postdoctoral Fellow University of Pittsburgh

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Hepatocyte transplantation improves blood and brain Phe and neurotransmitter imbalances in a mouse model of PKU Kristen J. Skvorak, Ph.D. Postdoctoral Fellow University of Pittsburgh Mentors: Dr. Stephen Strom and Dr. Jerry Vockley NPKUA Conference Cherry Hill, NJ July 26-29, 2012

  2. Outline • A mouse model of PKU and the human disease • Current Treatments • Liver Transplant vs. Liver Cell Transplant • Cell transplant and metabolic disease • Treatment Strategy • Results • Blood • Brain • Summary • Human amnion epithelial stem cells • What’s next? • Acknowledgements

  3. A mouse model of PKU +BH4 PAH Dopamine DOPAC HVA Phe Tyrosine • Missense mutation results in complete inactivity of phenylalanine hydroxylase (PAH) enzyme • High levels of Phe in the blood, tissue, and brain • Hypopigmented(fur changes from black to light brown) • Slight delay in growth compared to healthy siblings • Cognitive (memory, learning) impaired • Adult female mice are fertile, but offspring suffer early developmental defects similar to human maternal PKU • PKUenu2 mouse is a great model for human PKU. 3-MT

  4. Current Treatments • Dietary restriction • Complicated, expensive • Bad taste • Non-compliance • BH4 (Kuvan™) supplementation • Does not work for everyone • Liver Cell Transplantation (Tx) • Healthy liver cells contain 100% functional PAH to boost enzyme levels thus reducingPhe levels Harding, C. , Clin. Genet., 2008 Aug;74(2):97-104

  5. Benefits of Liver Cell Tx over Liver Tx • Less expensive (5-10% the cost of liver transplant) • Less invasive, faster recovery • Fewer incidents of serious complications or surgical related deaths • Multiple treatments are possible into one patient • Cells 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

  6. Liver Cell Tx and Metabolic Disease • Liver 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 clinically • Crigler-Najjar • Glycogen storage disease • OrnithineTranscarbamylase (OTC) deficiency • Factor VII deficiency • BiliaryAtresia • Additional Urea Cycle disorders • Liver failure

  7. Treatment Strategy • Liver cell transplant could increase enzyme activity thus helping to improve patient symptoms • <10% activity: more manageable disease; increased Phe tolerance • 10-20% activity: potential cure (Harding & Gibson, 2010) • Treatment would be most beneficial at birth • Clinically relevant • Avoid surgery – mouse livers are clearly visible through skin • By weaning (21 days old) mice are already showing symptoms of PKU • Neonatal mouse livers are rapidly expanding • possible growth advantage for transplanted cells

  8. 1 million cells transplanted directly into liver (1 liver  ~30 transplants) Liver Cell Tx 7 days PKU mice (birth) Treatment Strategy Isolate mouse liver cells 14 days 21 days (normal diet)

  9. 2 million cells transplanted into the spleen (1 liver  ~30 transplants) Liver Cell Tx 7 days PKU mice (birth) AA profiles (blood and brain) Neurotransmitter profiles Treatment Strategy Isolate mouse liver cells 14 days 21 days 28 days 35 days (young adult) (normal diet)

  10. Results – Phe was reduced in blood after cell transplant • Females had almost double blood Phe levels compared to males. • Phe was reduced 18% in Rosatx Females and 25% in C57 and AEtx 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 Cells Mouse liver cells: Rosa, C57 Human stem cells from placenta: AE

  11. Results – Phe was reduced in brain after cell transplant • 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. Statistics * = p<0.05 ** = p<0.01 *** = p<0.001 Transplanted Cells Mouse liver cells: Rosa, C57 Human stem cells from placenta: AE

  12. 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.

  13. 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

  14. Results – improvements in the Dopamine pathway after cell transplant Significantly Corrected Normalized Statistics * = p<0.05 ** = p<0.01 *** = p<0.001

  15. Summary • My 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 transplant • Observed 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 transplant • Metabolites along the Dopamine pathway were either normalized or significantly corrected after mouse liver cell transplant.

  16. Placental Amnion Epithelial (AE) Stem Cells Placental Tissue Amnion • 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 Chorion Decidua x100

  17. 1 million cells/mouse transplanted directly to liver (birth) hAETx 1 week Isolate human amnion epithelial cells (hAEC) 2 million cells/mouse transplanted directly to liver (3+ weeks) 3 weeks Long term study 14 weeks (100d) 5 weeks (35d) • 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 Liver analysis Enzyme activity AA and Neurotransmitters (Blood and Brain) Survival and Growth Hepatocyte Tx in a mouse model of MSUD Mol Ther. 2009; 17(7): 1266-73 BiochimBiophysActa. 2009; 1792(10):1004-10 KJ Skvorak, et al. 2012

  18. 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

  19. hAE Transplant doubled residual enzyme activity • BCKDH enzyme activity was increased from 6% to ~13% in AE transplanted animals. ~13% 6%

  20. 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

  21. 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 BiochimBiophysActa. 2009; 1792(10):1004-10) • Further studies involving human AE cell transplant in this mouse model improved: • Survival and growth • BCKDH enzyme activity • BCAA levels in blood and brain • Other relevant amino acid levels • Some 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.

  22. What’s next? • Continue testing cell therapies in the PKU mouse • Acquire patient cells and make induced pluripotent stem cells (iPSC) • Correct the mutation(s) with molecular means • May also provide alternate source of cells • Continue to work with the clinical hepatocyte (liver cell) transplant program at the Children’s Hospital of Pittsburgh

  23. Acknowledgements University of PittsburghFunding Stephen Strom NPKUA Roberto Gramignoli Ken Dorko Marc Hansel VeyselTahan Jerry Vockley Michigan Tech University K. Michael Gibson Baylor Research Institute ErlandArning Terry Bottiglieri KarolinskaInstitutet Stockholm, Sweden

  24. Thank you! Questions? The Golden Triangle Pittsburgh, PA

More Related