OCTGT Site visit, September 29, 2005

1. OCTGT Site visit, September 29, 2005 Research Management, Priorities, and Accomplishments Suzanne Epstein, Ph.D. Associate Director for Research, OCTGT

2. The Challenge • OCTGT formed to regulate cell therapies, gene therapies, tissues, other novel products Key issues include: • Vector safety, efficacy • Survival and function of cellular products • Immune responses: efficacy of tumor vaccines, barrier to therapies • Characterization of complex products

3. Roadmap of this presentation • Research management • Communication tools • Identifying research priorities • Assessment tools • Management of resources • Research, selected accomplishments related to: • Gene therapy • Cell therapy • Tissue engineering • Xenotransplantation • Tumor vaccines • New technologies

4. Communication within OCTGT • Purpose: • Keep all staff informed as to research expertise available in-house for consultation in relation to regulatory work • Encourage flow of Critical Path ideas among all review scientists

5. Tools for Communication within OCTGT • Research work-in-progress seminars • Short research vignettes at staff meetings • Brief abstracts of research programs circulated to staff • Web-based, searchable research annual reports • Dialogue of research managers with leaders of all review divisions • Discussions initiated by review scientists

6. Communication Beyond OCTGT • Briefings of Center and Agency leadership, Grand Rounds • Information exchange with stakeholders: • Scientific conferences • Publications • Advisory Committee meetings • October 2004 Critical Path Workshop, FDA Science Forum, this site visit

7. How OCTGT Identifies Research Priorities • Receive input about new products on the horizon from pre-submission inquiries, conferences, literature • Identify anticipated areas of major product activity, and Critical Path issues • Monitor for gaps and weaknesses in expertise or redundancies, and address them • Example: A few years ago, we identified the need for adenovirus expertise and recruited an adenovirus research expert to fill the gap.

8. How We Identify Research Priorities Priorities/ Agenda Center Director/ADR Office Director/ADR Division Director/ Branch Chief Staff Project ideas

9. OCTGT Future Research Priorities Identified Expertise identified as needed for future product review: • Tissue engineering: partnering/leveraging, adapting by existing staff • Cancer biology models for surrogate endpoints: Build on existing programs • Bioinformatics: enhance/leverage FDA capabilities, collaborate outside FDA • Proteomics: recruitment

10. Assessment Tools for Research Productivity • Quantitative metric for peer-reviewed publications, uses impact factors and authorship weighting (limitations of impact scores vs. quality, relevance). • Investigators describe the contributions of their research to Agency mission and other outputs (policy, guidance, advisory committee meetings).

11. Management of Resources: Outside Funding • Eligible for limited set of grant resources • Grant application prescreening: reviewed for conflicts of interest, alignment with OCTGT mission, possible recusals • Tracking of outside funds • Other leveraging (collaborations and partnering with other FDA centers, other government agencies, academic institutions)

12. OCTGT Research Strategies Anticipate needs, as well as addressing current problems: • Stay ahead of the curve as products and technologies change • Perform studies relevant to entire product classes, not only individual products • Make results public, and thus accessible to all sponsors, to advance the entire field • Some OCTGT research uses current product systems, some of it addresses underlying issues we must understand to move products forward.

13. Critical Path Challenges in Gene Therapy • Major issues: vector safety and characteristics, • patient immune responses. • Strategies to address them - Projects: • Adenoviral vector safety and biodistribution. • Retroviral vector safety and detection. • Herpesvirus vector safety and characterization. • Host immune responses induced by viral and plasmid vectors.

14. Adenovirus Vector Gene Therapy Public Health Issues and Regulatory Challenges: • Adenovirus vectors being tested in many clinical trials • They efficiently deliver transgenes to the liver • Toxicity has been seen, leading to the death of one patient • Animal models are needed for predicting adverse events and understanding their mechanisms

15. Shift in vector biodistribution Fatal pulmonary edema Smith et al. (2004). Molecular Therapy 9:932 Smith et al. (2004). Gene Therapy 11:431 What happens in animals with liver disease? Cirrhotic rats have a severe pulmonary reaction after i.v. adenovirus vector

16. Adenovirus Vector Gene Therapy Outcomes: • Insight into how adenovirus vectors cause toxicity • Animal model for gene therapy in the context of pre-existing liver disease – influence on clinical trial design • Model can be used for safety testing of new vectors

17. National Toxicology Program • NTP is administered by NIEHS, federally funded. • Goal: Evaluate the safety of regulated products products nominated by FDA Centers • In 2004, CBER proposed studies of toxicity and pharmacokinetics of gene therapy vectors – the first NTP study of complex biologicals. • Collaborations of FDA, NIH and academic investigators to do large-scale, long term studies.

18. Goals of NTP Study: Retroviral Vectors • Establish a preclinical model for assessing risk of retroviral vector-mediated insertional tumorigenesis. • Assess the effect of vector dose, of deleting the viral enhancer, of using an insulator element • Compare risk of using enhancer-deleted lentivirus vectors

19. Goals of NTP Study: Plasmid Biodistribution • Establish a method for quantitative assessment of vector biodistribution. • Assess the effects of route of administration and of formulation on gene expression, DNA persistence. • Provide baseline information bridging to other vectors and formulations.

20. Cell Therapy Products: Living and Changing Cell Product Safe, effective cell therapy tumor inappropriate differentiation or localization cell death

21. Critical Path Challenges in Cell Therapy Major issues: controlling growth and differentiation of cells, product characterization, immune rejection • Strategies to address them - Projects: • Key signaling pathways determining cell fate, cell death, and development of anatomic structures • Cell-cell interactions controlling differentiation of cells derived from bone marrow precursors • Immune cell activation and immune responses to cellular therapy products

22. Cell Therapy: Interacting Signals That Promote Product Efficacy • Public Health Issue: • For many cell products, only a small fraction survive after administration to the patient. Thus, cell survival is a Critical Path efficacy question • Experimental approach: High throughput whole genome screening to evaluate interacting genes and identify new predictors of cell survival and product efficacy • Model: eye progenitor cells

23. Identify Markers Supporting BMP Signaling and Predicting Cell Product Survival • Markers predictive of • cell survival identified • in screen: • Signaling molecules • Transcription factors • Cell cycle regulators • Cell adhesion molecules Normal BMPsignaling Low BMP signaling Apoptosis markers: caspase 3, activated JNK pathway Unpublished

24. Cell Therapy: Interacting Signals That Promote Product Efficacy • Outcomes: • Identification of biomarkers that predict survival of cell therapy products, and can serve as manufacturing process controls • Functional biological interactions, providing link to clinical outcomes • Suggests approaches for improving survival of cellular products following administration to the patient

25. Cell Therapy: Requirements for Product Efficacy • Public Health Issues and Regulatory Challenges: • Cellular products being developed for treatment of myocardial infarction, neurodegenerative diseases other diseases. • Once potential biomarker identified, explore its role in vivo to permit characterization tests predictive of product efficacy and safety • A highly conserved signaling pathway which is crucial during development and reactivated during repair of injury requires Notch2

26. vs lv lv rv rv Inactivation of Notch2 Results in Loss of Notch2+ Myocardial Cells and Hypoplastic Hearts Model: mice in which Notch2 can be turned on or off in specific tissue Heart Notch2- Wild type Notch2- Wild type Notch2+ cells are blue vs = ventricular septation defect Correct tissue formation requires Notch2 signaling Unpublished

27. Cell Therapy: Requirements for Product Efficacy • Outcomes: • Identification of markers on cellular products that predict their function and efficacy in vivo • Markers for process controls to characterize cellular products made from different precursors or under different culture conditions

28. Critical Path Challenges in Tissue Engineering Major issues: interactions yielding proper tissue structure and function. Strategies to address them - Projects: • Tissue anatomy and factors controlling joint development • Molecular signals determining liver development

29. Tissue Engineering: Joint Development – Repair • Public Health Issues and Regulatory Challenges: • Joint damage is common, and inadequately treated • Products for repair of joint surfaces have given mixed results. Need to identify factors influencing successful joint formation.

30. Spatial Patterns of Gene Expression CDMP1/GDF5 Key Finding: Expression of growth factors and the enzymes that activate them overlaps only at anatomic boundaries Unpublished

31. Tissue Engineering: Joint Development – Repair • Outcomes: • Identified novel molecular mechanisms contributing to development of the joints. • Micro-environment in vivo influences how cells differentiate and tissues develop. Related future work relevant also to cell therapies: • Phosphoproteomic profiling of chondrocytes to refine cell product characterization • Correlation of molecular markers with in vivo outcomes to identify sound potency assays

32. Critical Path Challenges in Xenotransplantation Major issues: transmission of infectious agents between species, immune rejection. • Strategies to address them - Projects: • Porcine endogenous retrovirus (PERV) detection and species tropism • Transplantation immunology - approaches to avoiding rejection

33. Xenotransplantation:Porcine Endogenous Retrovirus (PERV) • Public Health Issues and Regulatory Challenges: • Many more patients waiting for transplants than there are organs available • Risk of cross-species transmission of infectious agents, especially in immune-suppressed patients • Some PERV’s can infect human cells

34. Porcine PBMC Release Retrovirus that Can Infect Human Cells Pig Human Wilson, C., et al., J. Virol., 1998. 72(4):3082.

35. PERV-A PERV-C PERV-A/C Infection of Human Cells Determined by Envelope Portion of PERV-C envelope differing from PERV-A by 9 amino acids greatly reduces infection of human cells Gemeniano, et al, Virology, In Press

36. Xenotransplantation: Porcine Endogenous Retrovirus Outcomes: • Product Testing: Technical and scientific advice concerning assays for detection of PERV. • Product safety: Determinants of human tropism may reveal mechanisms to block infection and reduce risk of PERV transmission to recipients.

37. Critical Path Challenges in Tumor Vaccines MAJOR ISSUES: Product characterization, including tests for identity, purity, potency; animal models, markers for monitoring and immunogenicity. • Strategies to address them - Projects: • Animal models of targeted intervention. • Markers of tumor growth for monitoring. • Immune response assays (used for potency tests)

38. Tumor Vaccines Public Health Impact and Regulatory Challenge: • More than 1.2 million Americans are diagnosed with cancer and half of them die each year • No tumor vaccine is currently available for general clinical use, but many are under development for cancer therapy

39. Identification of Interleukin-13 Receptor 2 as Tumor-Associated Biomarker • Overexpressed in variety of human tumors, compared to normal tissues Expression of receptor in astrocytoma • Overexpression sensitizes tumor cells to killing by receptor-targeted agents • Extracellular domain of receptor is cleaved and secreted into serum

40. Soluble IL-13 Receptor 2 as a Serum Biomarker Mouse model of metastatic ovarian cancer • Level of soluble receptor increased with tumor growth • Treatment with receptor targeted toxin decreases tumor burden as well as serum level of soluble receptor in mice • Survival improved Unpublished

41. Tumor-Associated Biomarkers Outcomes: • Cell surface receptor expression as a marker of identity for tumor vaccines • Serum biomarker as a candidate for monitoring. • Animal models for study of interventions.

42. Critical Path Challenges: New Technologies • MAJOR ISSUES: Complex products require • state-of-the-art analytical methods. • Gene expression microarray • Quantitative flow cytometry • Transgenic animal production • Proteomics

43. New Technologies Uses of gene expression microarray and flow cytometry High throughput screening provides detailed information. Can be used for characterization of: • Cellular products • Cell substrates • Patient samples

44. Quality Assessment of Stem Cells by Gene Expression Profile Microarray Identify markers of stem cell state CD24 GTCM-1 Outcome: CBER/NIH/Industry scientists identified and characterized common “stemness” genes in 6 stem cell lines. Bhattacharya, Blood: 103, 2956-2964, 2004

45. Quantitative Flow Cytometry: Fluorescence Intensity Standardization Calibration curve Intensity, cell subsets Microbead standards • Use fluorescence standardization to permit longitudinal clinical studies and comparison of data from different labs

46. Fluorescence Technology for the 21st Century • Flow Cytometry and Microarrays Need Standards • Federal Standardization Initiative: NIST – FDA – CDC • Standard Fluorescein Solution Developed • Standard Microbeads Developed NIST Microbead Standard

47. Reference Materials: Needed to Assure Sensitivity and Comparability of Test Methods Retroviral reference material Adenovirus reference material LTR gag pol env LTR • Available from ATCC. • Used to show RCR assay sensitivity, reduce testing without compromising product safety. External RNA spike-in controls for microarray and RT-PCR ERCC • Available from ATCC. • Allows precise titers: • Viral particle • Infectious titer Outcome: Sensitive, consistent testing facilitates progress, provides savings

48. Summary: Research Prioritizationas an Ongoing Process • New product classes present novel scientific and regulatory challenges and opportunities • We identify scientific questions of regulatory importance and address them. • Solutions to key problems facilitate product development, inform regulatory decisions and policy. We welcome questions and comments from the Committee.

49. Questions for the Committee • Please comment on the contributions OCTGT • research makes to the Critical Path development of biologics product and their availability. • 2. Please recommend opportunities for research expansion and redirection, and new collaborations or leveraging. • 3. Suggest research management strategies for anticipating future biological products and related scientific and product issues. • 4. Provide recommendations for attracting and retaining • high quality scientific staff.