Cell, Tissue, and Gene Therapies

1. Cell, Tissue, andGene Therapies Elizabeth Read, MD May 11, 2011

2. Cell, Tissue & Gene Therapies • Heterogeneous group of (potential) products • Very few products on the market • Regulatory framework has evolved relatively recently (over past 20 years) • Special development considerations

3. Cell-based therapies originated with hematopoietic transplantation in 1970s • Bone marrow harvested, filtered, and transferred to blood bags in operating room • BM product carried directly to patient unit for infusion • Minimal donor & product testing, graft manipulation, quality systems • To date, FDA considers conventional autologous and allogeneic family-related BMT as “Practice of Medicine”

4. 1980s – 2000s • Advances in science & technology spurred novel approaches for development of cell-based therapies • Hematopoietic transplants with “engineered” grafts starting with bone marrow, peripheral blood, or cord blood sources • Immunotherapies • T cells & subpopulations • Dendritic cell tumor vaccines • NK cells • Cellular gene therapies • Cell therapies derived from bone marrow, other tissues, and organs (e.g. mesenchymal stem cells, pancreatic islets)

5. During this period, clinical translation was facilitated by development of technologies for collecting & handling cells in closed systems (often with single-use disposables)…

6. And also by development of automated, large scale systems for cell collection, separation & isolation

7. 2000s – PresentStem Cells & Regenerative Medicine • Explosion in stem cell science led to interest in use of stem cell-based therapies for many diseases and conditions, from cosmetic to life-threatening • Multipotent • Adult stem cells from bone marrow, fat & other tissues/organs • Fetal stem cells & placental stem cells are usually considered “adult” • Pluripotent • Embryonic stem (ES) cells • Induced pluripotent stem (iPS) cells

8. Scope of cell & tissue therapies • Bone marrow and other hematopoietic stem cell transplantation • Cellular immunotherapies (dendritic cell vaccines, NK cells, T cells, etc) • Cell therapies derived from stem cells • Adult (including fetal) stem cells • Induced pluripotent stem cells • Embryonic stem cells • Cellular gene therapies • Conventional organ transplantation (e.g., kidney, heart, liver) • Conventional tissue transplantation (e.g., tendons, bone) • Reproductive tissue (sperm, oocytes, embryos) • Tissue engineering (autologous, allogeneic) – may include synthetic or natural biomaterials, or decellularized tissues • Xenotransplantation

9. How does FDA regulate these products?

10. Development pathway for cell & tissue therapies is similar to drugs & conventional biologics But with many important exceptions

11. Exception #1 • Transplantation of vascularized whole organs is regulated by HRSA, not FDA

12. Exception #2 • Xenotransplantation is regulated by its own separate set of FDA regulations

13. Exception #3 • Bone marrow transplantation using autologous or family-related allogeneic donors is not regulated at all (practice of medicine)

14. Exception #4 • Bone marrow transplantation from unrelated donors is regulated by HRSA, not FDA

15. Exception #5 • Some tissue products have been regulated by CDRH as devices, with less stringent requirements and minimal involvement of CBER • This is historical – CBER will be involved going forward

16. What’s left in scope? • Bone marrow and other hematopoietic stem cell transplantation • Cellular immunotherapies (dendritic cell vaccines, NK cells, T cells, etc) • Cell therapies derived from stem cells • Adult (including fetal) stem cells • Induced pluripotent stem cells • Embryonic stem cells • Cellular gene therapies • Conventional organ transplantation (e.g., kidney, heart, liver) • Conventional tissue transplantation (e.g., tendons, bone) • Reproductive tissue (sperm, oocytes, embryos) • Tissue engineering (autologous, allogeneic) – may include synthetic or natural biomaterials, or decellularized tissues • Xenotransplantation

17. What’s left falls into FDA definition ofHCT/Ps • Human cells, tissues, and cellular and tissue-based products (HCT/Ps) are articles containing human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient

18. FDA’s Risk-Based Approach forHCT/Ps • Lower risk “361” • Autologous or family related donors and minimally manipulated and homologous use • Regulated under section 361 of Public Health Service Act • Higher risk “351” • Allogeneic unrelated donors and/or more than minimally manipulated and/or non-homologous use • Regulated under section 351 of Public Health Service Act, and subject to same rules as drugs & other biologics for IND and premarket approval

19. FDA regulations for HCT/Ps

20. What about stem cells? Cellular products derived from multipotent or pluripotent stem cells are regulated as HCT/Ps

22. HCT/Ps derived from pluripotent stem cells: FDA concerns • CMC • Donor source • Consistency of differentiation & expansion process • Detection of residual pluripotent stem cells • Genetic and epigenetic stability • Preclinical studies • Case-by-case approach • “hybrid” efficacy/safety studies – much attention to modeling ROA and biodistribution • Tumorigenicity

23. HCT/Ps derived from pluripotent stem cells: FDA concerns • Clinical Protocol: for novel stem cell products, the risk : benefit assessment is difficult; therefore: • Rationale for clinical trial must be justified by especially strong proof of concept • Greater emphasis placed on product characterization and preclinical testing

24. Gene Therapies

25. Gene therapy approaches • IN VIVO: Vector administered directly to patient, and transfers genetic information to patient cells in vivo • Intravenously administered vector delivers gene for factor IX to patient with hemophilia B • EX VIVO: Vector used to transfer genetic information to cells ex vivo, then cells are administered to patient • Vector that delivers gene for enzyme adenosine deaminase is incubated ex vivo with autologous lymphocytes of patient with ADA-deficient form of SCID (severe combined immunodeficiency), and genetically modified cells are infused to patient

26. Gene therapy: history • 1974: NIH established Recombinant DNA Advisory Committee (RAC) • NIH Guidelines on recombinant DNA research • 1980s: New subcommittee of RAC to oversee clinical gene therapy • Appendix M to NIH Guidelines – covered design of preclinical & clinical research, consent issues, AE reporting • PUBLIC review of gene transfer protocols • 1989: First clinical gene transfer study (gene marking) using retroviral vector • 1990: First clinical gene transfer study (therapeutic intent) using retroviral vector

27. Gene therapy: history • 1995: No real clinical efficacy demonstrated, and NIH report concluded that enthusiasm had outstripped knowledge • Back to the bench for research on improved gene delivery methods (e.g., higher titer vectors, use of stromal feeder layer or fibronectin for HSC transductions) • By 1995, NIH RAC • Had approved 149 GT clinical protocols • No dire consequences • Policy change: public review & approval only for GT protocols that presented novel or unresolved issues • 1997: Role of NIH RAC modified – still required public review, but not “approval” of novel GT protocols

28. Jessie Gelsinger (1999) • 18 y.o. with clinically mild form of ornithine transcarbamlase defiency • Volunteered for clinical trial of gene therapy at U of Pennsylvania • Adenoviral vector caused massive immune response, muti-organ failure, and death within 4 days • All gene therapy trials placed on hold • Multiple ethical issues raised • Adverse events in primate studies • Adverse events in 2 previous human subjects • Informed consent • Principal investigator conflict of interest

29. Insertional Oncogenesis • 2000-2007: X-linked SCID trials, using gamma retroviral vectors to deliver the corrective gene (IL2RG) to autologous hematopoietic progenitor cells • 5 of 20 pts developed T cell leukemia-like proliferative disorder, caused by INSERTIONAL ONCOGENESIS • Retroviral vector integrated adjacent to one or more cellular proto-oncogenes (LMO-2 in 4 of the cases), which increased their expression, leading to malignant transformation and outgrowth of clonal population of T cells

30. Gene delivery methods • Vector = an agent used to introduce genetic material into cells • Vectors can be • Viral • Non-viral • Plasmid DNA • Liposomes or other agents that facilitate entry into cell

31. Viral vectors • Retrovirus and lentivirus (developed to overcome inability of γ-retroviral vectors to infect non-dividing cells) • Adenovirus • Parvovirus (adeno-associated virus or AAV) • Herpes simplex virus • Poxvirus • Togavirus

32. Vector selection depends on… • Disease state • Route of administration • Size of payload • genetic sequences, regulatory elements • Cell cycling • Lentivirus, adenovirus, AAV do not require cycling cells • Intended duration of expression • Retrovirus and lentivirus give stable integration • Plasmid used for transient expression • Target cells • Poor expression of adenoviral CAR receptor on hematopoietic cells

33. More advanced vector design features • Conditional replication-competence • Control of gene expression • Tissue-specific promoters • Drug-responsive promoters • To reduce risk of insertional oncogenesis ofγ-retroviral and lentiviral vectors • Self-inactivating (SIN design) • Insulators • Suicide genes • Ganciclovir administered to patient will kill cells with thymidine kinase gene

34. Safety issues • Observed to date • Insertional mutagenesis/oncogenesis • Immunogenicity • Vector • Transgene • FBS (bovine protein used to manufacture vector) • Potential • Inadvertent transmission & expression in non-target cells (including germline, transplacental)

35. FDA regulations & guidance for gene therapies • Overall similar to biotechnology products • ICH guidances • Gene therapy CMC guidance 2008 • Vector description, map, sequence analysis • Cell banks, viral banks, cell lines (packaging, producer, feeder) • Vector production/purification • Documentation of RAC review • For ex vivo gene therapy, cell requirements same as HCT/Ps (i.e. CMC guidance, tissue rules)

36. FDA guidance on GT delayed AEs • Recommends preclinical study designs to assess clinical risk • Requires long term clinical follow up, based on preclinical studies, for • In vivo gene therapy with persistence of vector sequences, when sequences are integrated • Ex vivo gene therapy with sequences integrated, or not integrated but have potential for latency & reactivation • Specific follow up observations yearly for at least 10 years, and reporting to FDA • Informed consent for long term follow up, and for use of retroviral vectors

37. RCR/RCL testing(FDA 2006 supplemental guidance)

38. Case Study Cellular gene therapy for sickle cell disease PI - Donald Kohn MD (UCLA) Funded by CIRM

39. Sickle Cell Disease (SCD) • Autosomal recessive disorder • Approx 8% of African Americans have mutation • Approx 1 in 500 African Americans is homozygous and has SCD • Clinical course • hemolytic anemia • vaso-occlusive episodes (pain), strokes, acute chest syndrome, progressive organ dysfunction

40. Molecular basis of SCD • Substitution of T for A in 6th codon of human β-globin gene • Results in non-polar valine instead of polar glutamic acid on the surface of HbS tetramer (α2βS2)

41. Molecular basis of SCD • During partial deoxygenation, valine creates hydrophobic pocket that fits into natural hydrophilic pocket on HbS tetramers, leading to HbS polymerization • This causes red blood cells to become rigid and poorly deformable, leading to hemolysis and impaired blood flow through microcirculation

42. Treatment of SCD • Supportive for vaso-occlusive crisis • Pain medication, hydration, oxygen • Blood transfusions • For some acute complications • Prophylaxis for stroke and other complications • Complications: iron overload, alloimmunization • Hydroxyurea • Key mechanism: raises Hb F, which has anti-sickling effect • Complications: pancytopenia • Allogenic bone marrow transplantation • Potential for cure, but only 14% have HLA-matched sibling donor

43. Potential Gene Therapy Strategies for SCD • Correct HbS mutation • But sickle β-globin acts in a dominant manner, and you would need very high levels of expression to achieve a state similar to sickle trait • Insert genes for normal HbF γ-globin into HSCs, in order to increase expression of HbF (α2γ2), to inhibit Hb S polymerization and sickling • But fetal γ-globin gene is poorly expressed in adult RBCs, due to absence of fetal-specific positive regulatory factors in adult cells • Modify HbS β-globin gene to have anti-sickling properties of γ-globin while retaining the adult HSC expression pattern inherent in the β-globin gene

44. Townes βAS3 vector • Self-inactivating (SIN) lentiviral vector • Carries and expressesβAS3, a β-globin gene with 3 amino acid substitutions • Expression product has biophysical anti-sickling properties equivalent to fetal γ-globin AND advantage over βS–globin for dimerization with α-globin • Incorporates β-globin transcriptional regulatory elements

45. Preclinical Proof of Concept(Levasseur 2003) • In murine model of SCD, transduction of HSC with the lenti/βAS3 vector • Expression: 2-3 gm Hb/dl/vector copy • Correction of hematological and clinical manifestations of SCD

46. IND development for SCD gene therapy

47. Clinical protocol considerations • Phase 1 trial • Risks: known and unknown • Benefits: unlikely in first trial • SCD patient population • Adults (ethical considerations for children) • Should not be candidates for allo BMT (i.e., matched sibling donor available) • Severity of disease may impact • feasibility of cell collection • endpoint assessment • Myeloablation with busulfan to create “space” in marrow

48. Product considerations • Vector: based on Townes SIN lentiviral vector • Additional engineering underway to further reduce risk of insertional oncogenesis • TAT independent backbone, insulators, etc. • Cell source • Autologous • Ideally want most primitive hematopoietic stem cells (HSCs) that will differentiate into erthyroid cells • HSCs vs iPS cells • iPS cells not quite ready for prime time • HSCs have track record, CD34+ selection isolates stem & progenitor cells

49. Product considerations • HSC options • Placental/umbilical cord blood • most proliferative source, but not useful for autologous protocol in adults • G-CSF mobilized peripheral blood HSCs • SCD patients have had serious adverse events, including death, associated with G-CSF • Bone marrow • Will require general anesthesia • Available cell dose will be an issue

50. Initial definition of product candidate • The investigational product is autologous human CD34+ hematopoietic stem cells (HSC) from the bone marrow of patients with sickle cell disease (SCD) modified by ex vivo transduction using the βAS3 lentiviral vector