Report of NPKUA Scientific Exchange Session

Transcription

1 Report of NPKUA Scientific Exchange Session July 10, 2014 Cary O. Harding, MD Oregon Health & Science University Portland, OR

2 Topics Effects of Phe upon the brain Liver-directed cell transplantation for PKU Gene therapy for PKU

3 Participants Desiree White, PhD St. Louis, MO Vincenzo Leuzzi, MD Rome, Italy Shawn Crist, PhD Columbia, MO K. Michael Gibson, PhD Spokane, WA Cary O. Harding, MD Portland, OR Stephen Strom, PhD Stockholm, Sweden Kristen Skvorak, PhD Pittsburgh, PA

4 Participants Rod Monroy, PhD Cytonet Ira Fox, MD Pittsburgh, PA James Wilson, MD, PhD Philadelphia, PA Ian Alexander, MD Sydney, Australia Hiu Man Viecelli, PhD Zurich, Switzerland Beat Thöny, PhD Zurich, Switzerland

5 Neuropathology White Matter Abnormalities

6 Clinical and white matter alteration outcome in early treated PKU Vincenzo Leuzzi Department of Pediatrics, Child Neurology and Psychiatry

7 EUROPEAN GUIDELINES PKU GROUP WM MRI alterations and PKU Q1: Are MRI abnormalities present in early treated PKU patients? Q2: What are the findings reported in the brain through MRI analysis? Q3 : Is there a link between the amount of brain abnormalities and lifetime Phe levels? Q4: Is there any association between WM alteration on MRI and clinical outcome/features in early treated PKU patients?

8 The clinical significance of WMA No association between WMA and IQ (Pearsen 1990, Bick 1993, Leuzzi 1993, Cleary 1994, Wegleage 1995, Leuzzi 1995, Pietz 1996, Leuzzi 2007; Wegleage 2013 ) A weak association between WMA and/or neurocognitive dysfunctions and/or high blood Phe levels (Lou et al 1991; Pietz et al 1996; Anderson et al 2004; Brumm et al 2004; Anderson et al, 2007; Sundermann et al 2011) No alterations of the central WM conduction (Cleary et al, 1994)

9 White Matter Integrity, Cognition, & Phe Control in Children with PKU Desiree A. White, Ph.D. Professor of Psychology Disclosure: Consultation & research funding from BioMarin Pharmaceutical Inc.

10 Correlations between DTI & Lifetime Phe skeleton p <.01 p <.05 Mean Phe SD Phe Mean Exposure SD Exposure

11 Phe Control & Cognition Variable Mean Phe SD Phe IQ ns -.33 Vocabulary ns ns Matrix Reasoning ns -.39 N-back # responses N-back # nonresponses ns -.39

12 Functional Connectivity in PKU Shawn Christ, Ph.D. Assoc Director, Brain Imaging Center Assoc Professor, Dept of Psych Sciences University of Missouri Disclosure: On occasion, I also serve as an outside consultant for BioMarin Pharmaceutical Inc., and they are funding some of our current research on PKU and BH4.

13 Task Positive vs. Negative Networks from Fox et al. (2005)

14 Christ et al. (JIMD, 2012)

15 Novel Chemical and Pharmacological Therapies for PKU Blood-Brain Transport Inhibition using NPAAs Mike Gibson, Chair Experimental and Systems Pharmacology Washington State University College of Pharmacy

16 Pilot Studies-Effect on Phe Dietary Approach (casein control) Significant Phe Reduction Effects on Tyr, BCAA Scale down, combinatorial Vogel et al 2012, 2013

17 I m a PKU mouse Liver-directed therapy Therapeutic liver repopulation Hepatocytes Stem cells

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19 Serum Phe vs. liver PAH activity

20 20 Liver Cell Therapy in Newborns and Infants with Severe Urea Cycle Disorders Rod Monroy, PhD Senior Director, NA Clinical Operations Cytonet LLC

21 Clinical Experience with Hepatocyte Transplantation for PKU National PKU Alliance 2014 International Scientific Exchange Ira Fox, MD

22 HEPATOCYTE TRANSPLANTATION CRIGLER-NAJJAR SYNDROME TYPE 1 Long-term engraftment and immune suppression

23 Hepatocyte Transplantation Problems..! Adequacy of engraftment and function to correct disease Long term engraftment, graft monitoring and immune suppression

24 Hepatocyte Transplantation Clinical Transplantation Access to the portal (hepatic) circulation for hepatocyte transplantation via a recannalized umbilical vein, with infusion of cells guided by IR contrast studies.

25 HEPATOCYTE TRANSPLANTATION FOR PKU Successful Conditioning Regimen Liver Biopsy 3 months post-ht: PAH immunohistochemistry shows engraftment of donor hepatocytes

26 Stem Cell Therapy of Liver Disease Stephen Strom, Ph.D. Torsten och Ragnar Söderberg Professor Regenerative Medicine Department of Laboratory Medicine, Division of Pathology Karolinska Institutet

27 Hepatocyte Transplantation Bridge patients to whole organ transplant. Support function and regeneration of liver in ALF. Correction of metabolic liver diseases monogenetic, pediatric

28 Human Amnion Epithelial Cells NOT cord blood OR amniotic fluid cells Not Mesenchymal Stem Cells (MSC) Express genes also found in pluripotent stem cells, such as ES or ips (Oct4, Nanog, Sox2) Express surface markers also found in pluripotent stem cells, such as ES or ips (SSEA3, SSEA4, TRA1-60, TRA1-81) Non-tumorigenic (mice, rats, pig, human) Plentiful Non-controversial Miki et al., Stem Cells,

29 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 NPKUA Conference Salt Lake City, UT July 10-13, 2014

30 Isolate human amnion epithelial cells (haec) Placenta cross section Amnion Chorion 1 million cells/mouse transplanted directly to liver (birth) Dedidua haec Tx x100 7 days 14 days 21 days 28 days PAHenu 2 (birth) 1 month post-tx (young adult) 100 days Post-tx normal diet Compare FRESH cells to PLATED AA Analysis (blood/brain) Neurotransmitters Frozen immediately post isolation Cultured hours Allowed to attach to plate Adhered cells isolated and frozen KJ Skvorak, et al. 2012

31 I m a PKU mouse Liver-directed Gene therapy

32 AAV for Liver Metabolic Diseases James M. Wilson, M.D., Ph.D. Departments of Pathology & Laboratory Medicine and Medicine National PKU Alliance July 10, 2014

33 Liver Transduction following IV Administration of AAV8 Vectors Mouse Liver Day 3 Day 90 AAV2 AAV5 AAV8 mouse: 5x10 12 GC/kg all other animals: 3x10 12 GC/kg 33

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35 6 adults with Hemophilia B (Factor IX) Single PIV administration, escalating doses 1.5-5% serum Factor IX activity No exogenous clotting factors required No acute toxicity Transient transaminitis 2-3 weeks after injection

36 AAV-mediated liver-directed gene therapy for OTC deficiency Ian Alexander

37 Liver-wide GFP expression following vector delivery to an adult mouse raav2/8-gfp PBS control 100 mm 100 mm Single low dose of vector injected (intra-peritoneal) Close to 100% of hepatocytes transduced

38 Impact of hepatocellular proliferation Cunningham et al, Molecular Therapy (2008) Essentially the same phenomenon reported in infant macaques Wang et al., Molecular Therapy (2012)

39 Key lessons Therapeutic benefit declines rapidly after perinatal treatment Following delivery in the newborn period the stably integrated fraction of AAV genomes is insufficient to control hyperammonaemia (~ 8-10% of hepatocytes) Vector re-delivery may be required for long-term benefit

40 LSPmPAH raav2/8 Portal vein injection 5 X vg/mouse 8 weeks post injection vg/haploid genome % PAH activity

41 Serum phenylalanine (µm) Temporary PAH expression F G H Time (weeks) LSPmPAH raav2/8 4 X vg/mouse Portal vein injection Loss of vector No PAH activity No vector DNA detected No evidence of immune-mediated destruction Normal histology No lymphocytic infiltration Normal serial serum ALT No anti-pah Ab No selective advantage for PAH+ cells

42 AAV8 readministration

43 Treatment of Phenylketonuria Using Minicircle-Based Naked-DNA Gene Transfer to Murine Liver Hiu Man Viecelli, PhD Division of Metabolism University Children s Hospital Zurich NPKUA July 10, 2014

44 Endo P Comparison of minicircle and parental plasmid expression in PKU mice P Pah BGHpA attb attp ColE1 Kanamycin R Pah attr BGHpA Parental Plasmid (PP) 6.1 kb 3.3 x copies 219 µg Minicircle (MC) 2.1 kb 3.3 x copies 77 µg

45 Minicircles containing mpah lower blood Phe level in a dose-dependent manner 219 µg PP, n = 7 <1% PAH activity 13 ± 7 copies/ hepatocytes 0.77 µg MC, n = 1 Blood Phe µm µg MC, n = 4 77 µg MC, n = 10 14% PAH activity 16 copies/ hepatocytes 9% ± 8 PAH activity 13 ± 7 copies/ hepatocytes Viecelli et al., Hepatology (2014)

46 CHILDREN S RESEARCH CENTER (CRC) Long-term correction of PKU by raav gene transfer: Liver vs. Muscle Beat Thöny, PhD Division of Metabolism, University Children s Hospital Zürich, Switzerland 2014 NPKUA Conference Salt Lake City, UT, July 10, 2014

47 Blood L-Phe (mm) Kinetics of L-Phe concentration after raav2/1-pku3 i.m. injection ( three-gene AAV-vector ) AAV2/1-PKU3 ITR CMV PAH EMCV-IRES GTPCH FMDV-IRES PTPS WPRE ITR AAV2/1-PKU3 AAV2/1-PKU3 3.5 x v.g. 1.5 x v.g. injection into M. gastrocnemius I m a PKU mouse weeks after muscle-injection

48 Conclusion Gene transfer into skeletal muscle using raav2 serotype 1 AAV2/1-PKU3: P CMV -PAH-IRES-GTPCH-IRES-PTPS A single intra-muscular (M. gastrocnemius) injection of PKU AAV2/1-PKU3 PKU Wild-type raav serotype 1 expressing the three transgenes PAH, GTPCH and PTPS resulted in long-term therapeutic correction of PKU in mice (> 1 year)

49 The Holy Grail A cell-directed therapy that: Or: Is safe Yields a physiologically-relevant permanent population of PAH+ cells Selective growth advantage for PAH+ cells Increased integration frequency of AAV genomes Stable episomes in other tissues Can be repetitively administered

50 Critical needs Neurocognitive outcomes Systematic neuropsychological evaluation of many individuals with PKU over the lifespan Application of novel functional neuroimaging techniques in more individuals with PKU International longitudinal natural history study in PKU

51 Critical needs Cell transplantation Recruitment of additional individuals for the hepatocyte transplantation trial Clinical trial of human amniotic epithelial cell transplantation Acute liver failure in adults Liver-directed gene therapy Further clinical trials in other IEM Familial hypercholesterolemia or OTC deficiency Further optimization of the gene therapy vector for PKU

52 Critical needs Funding Government: only three NIH funded grants on PKU currently Industry: need to develop a business case for investment Philanthropy: Thank you!!!! Awareness Contemporary therapy is good but not good enough! Prove the need for novel treatments

53 Action plan Consensus statement on need for novel treatments targeted to: Medical professionals who don t provide care for individuals with PKU Government officials Insurance industry Pharma companies Investors

54 Action plan Develop educational program for medical professionals Optimal dietary therapy Novel treatments Neurocognitive outcomes Research needs

55 Action plan Initiate conversations with leaders at NIH and EMA regarding the funding of an international longitudinal study on PKU Survey NPKUA membership What are your most critical needs? What are your biggest personal concerns?