Kristen J. Skvorak, Ph.D. Postdoctoral Fellow University of Pittsburgh Mentors: Dr. Stephen Strom and Dr.
Download ReportTranscript Kristen J. Skvorak, Ph.D. Postdoctoral Fellow University of Pittsburgh Mentors: Dr. Stephen Strom and Dr.
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 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 A mouse model of PKU Phe +BH4 PAH Tyrosine Dopamine DOPAC HVA 3-MT 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. 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 reducing Phe levels Harding, C. , Clin. Genet., 2008 Aug;74(2):97-104 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 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 Ornithine Transcarbamylase (OTC) deficiency Factor VII deficiency Biliary Atresia Additional Urea Cycle disorders Liver failure 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 Treatment Strategy 1 million cells transplanted directly into liver (1 liver ~30 transplants) Liver Cell Tx 7 days PKU mice (birth) (normal diet) 14 days 21 days Isolate mouse liver cells Treatment Strategy 2 million cells transplanted into the spleen (1 liver ~30 transplants) Isolate mouse liver cells Liver Cell Tx 7 days 14 days 21 days 28 days 35 days (young adult) PKU mice (birth) (normal diet) AA profiles (blood and brain) Neurotransmitter profiles 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. Transplanted Cells Mouse liver cells: Rosa, C57 Human stem cells from placenta: AE A combination of early + late tx is most beneficial because it will maximize cells engrafted in the liver. 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 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 Phe 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. Normalized Significantly Corrected Results – improvements in the Dopamine pathway after cell transplant Statistics * = p<0.05 ** = p<0.01 *** = p<0.001 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. Placental Amnion Epithelial (AE) Placental Tissue Stem Cells Amnion – thin membrane surrounding the Amnion 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 csections/year in the US Easy to isolate, easy to maintain in culture Non-controversial source of stem cells Chorion 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 Decidua x100 Isolate human amnion epithelial cells (hAEC) 1 million cells/mouse transplanted directly to liver (birth) 2 million cells/mouse transplanted directly to liver (3+ weeks) hAE Tx 1 week 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 Hepatocyte Tx in a mouse model of MSUD Mol Ther. 2009; 17(7): 1266-73 Biochim Biophys Acta. 2009; 1792(10):1004-10 Long term study 5 weeks (35d) 14 weeks (100d) Liver analysis Enzyme activity AA and Neurotransmitters (Blood and Brain) Survival and Growth KJ Skvorak, et al. 2012 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 ~13% 6% BCKDH enzyme activity was increased from 6% to ~13% in AE transplanted animals. 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 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. 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 Acknowledgements University of Pittsburgh Stephen Strom Roberto Gramignoli Ken Dorko Marc Hansel Veysel Tahan Jerry Vockley Michigan Tech University K. Michael Gibson Baylor Research Institute Erland Arning Terry Bottiglieri Karolinska Institutet Stockholm, Sweden Funding NPKUA Thank you! The Golden Triangle Pittsburgh, PA Questions?