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Cell, Tissue, and Gene Therapies Elizabeth Read, MD May 11, 2011.

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Presentation on theme: "Cell, Tissue, and Gene Therapies Elizabeth Read, MD May 11, 2011."— Presentation transcript:

1 Cell, Tissue, and Gene 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 – Present Stem 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 Discovery & Early Translation Preclinical Development Clinical TrialsCommercialization 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 of HCT/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 for HCT/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 361 HCT/Ps 351 HCT/Ps (Tissue) Establishment registration√√ (Tissue) Donor eligibility√√ (Tissue) CGTP manufacturing√√ cGMP regulations√ IND / IDE regulations√ Premarket approval (BLA or PMA)√

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

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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 : 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 (α 2 γ 2 ), 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 CMC (Product) Preclinical Studies Clinical Protocol

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 β AS3 lentiviral vector

51 Quantitative targets Initial quantitative targets for product CD34+ : minimum 2 x 10 6 /kg Back up BM MNCs: 5 x 10 7 /kg Vector in cells: 1-3 copies/cell Based on estimates of Hb produced per VCN, and data showing benefit from Hb F of 10-20%

52 CMC: Beginning with the end in mind Cell Source Donor selection criteria Donor screening Donor manipulation, if any Collection methods Manufacturing and Storage Ex vivo manipulation, cryopreservation, and hold steps Full description of vector Ancillary reagents Assays: in-process & release Storage Product stability Administration Patient preparation (medical, surgical) Product transport to clinical site On site product preparation Product labeling & tracking

53 CMC Development: Basic Manufacturing Process Bone marrow collection CD34 Selection Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

54 CMC Development: Basic Manufacturing Process Bone marrow collection CD34 Selection Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

55 Bone Marrow Source Autologous - SCD Is cell content (MNC, CD34) of bone marrow of SCD patients comparable to normal BM? PILOT STUDIES SAY YES How much marrow to harvest? ENOUGH TO YIELD AT LEAST 1-2 x 10 6 CD34/kg IN FINAL PRODUCT, PLUS BACK UP OF 5 x 10 7 MNCs/kg

56 CMC Development: Basic Manufacturing Process Bone marrow collection CD34 Selection Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

57 CD34+ Selection Miltenyi CliniMacs CD34 Selection System High RBC content of bone marrow interferes with selection Need to reduce RBC content of bone marrow before CliniMacs selection Ficoll hypaque in tubes – open system, cumbersome, cell loss Automated closed processing: goal > 90% of MNCs Cobe 2991 cell washer

58 CMC Development: Basic Manufacturing Process Bone marrow collection CD34 Selection Culture/Transdu ce CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

59 Culture & Gene Transduction Small scale experiments Minimize differentiation of HSCs Cytokines (SCF, Flt-3L, IL-3, Tpo) Overall culture duration Optimize transduction efficiency Timing: pre-stimulation in culture improves transduction How many hits? Recombinant human fibronectin fragment Preserve vector Vector titers are not high Quantity will be limited

60 Culture & Gene Transduction Assays Vector sequence in HSCs qPCR (vector copy number per cell) Gene-modified HSCs are capable of erythroid differentiation In vitro erythroid differentiation model Fold expansion & flow phenotype RBC progeny have appropriate function Rheology and morphology Gene-modified HSCs still contain stem cells NOD/SCID/γc(null), primary/secondary transplants

61 CMC Development: Basic Manufacturing Process Bone marrow collection CD34 Selection Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

62 A hitch: Timing of product manufacturing vs clinical protocol Bone marrow harvest to obtain HSCs must occur BEFORE busulfan starts Busulfan schedule = 4 days + 2 days washout Final gene-modified CD34 cell product cannot be given until after busulfan washout Extended culture of cells is likely to result in differentiation of HSCs THEREFORE WILL NEED TO CRYOPRESERVE EITHER INTERMEDIATE PRODUCT (CD34+ CELLS) OR FINAL GENE- MODIFIED PRODUCT

63 CMC Development: Manufacturing Process – Option A Bone marrow collection CD34 Selection Cryopreservation Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Infuse Product into Patient

64 CMC Development: Manufacturing Process – Option B Bone marrow collection CD34 Selection Culture/Transduc e CD34+ Cells Harvest Transduced CD34+ Cells Cryopreservation Infuse Product into Patient

65 Cryopreservation & Thaw Evaluate effects of cryopreservation & thaw CD34+ cells vs final gene-modified CD34+ cells Optimal cryomedium Controlled rate device vs Mr. Frosty Readouts Recovery of viable cells In vitro clonigenic assays Vector in cells and expression of gene product

66 Assay development AssayFor preclinical studiesFor product in clinical Trial Cell counts, flow phenotype (CD34), viability Research lab methodsClinical lab methods Gene/vector in cellsqPCR for vector copy numberSame Gene expression product (Hb AS3) Isoelectric focusing (IEF): Hb AS3 migrates with Hb A, not with Hb S Need another assay – patients transfused (Hb A) Characterization & function of transduced cells (in vitro) Erythroid differentiation culture Currently being optimized Assess expansion, differentiation (flow phenotype), transduction Generate enucleated RBCs to evaluate rheology No Function of transduced cells (in vivo) SCID-repopulating cells by LDA in NOD/SCID/γc(null) mouse model Clinical endpoints Rheology of RBCs generated in vivo Safety/ToxicityAssess risk of insertional mutagenesis and clonal imbalance in vitro “clonal dominance” assay in vivo mouse transplants Micro cultures, endotoxin (RCL not needed if cells in culture < 4 days)

67 Project status Clinical protocol in draft CMC development in progress Preclinical studies in progress Pre-IND meeting this summer

68 FDA CMC guidances

69 Cell & tissue therapies approved by FDA to date ProductCompanyDescription; indicationYear approved FDA Center- Mechanism CarticelGenzyme Autologous cultured chondrocytes; repair of traumatic knee injury 2000CBER (BLA) ProvengeDendreon Autologous dendritic cell tumor vaccine; prostate cancer 2010CBER (BLA) TransCyteAdvanced Biohealing Human fibroblast-derived temporary skin substitute; severe burns (now off market) 1997CDRH (PMA) ApligrafOrganogenesis Human keratinocytes + human fibroblasts in bovine collagen matrix; venous stasis leg ulcers, diabetic foot ulcers) 1998CDRH (PMA) DermagraftAdvanced Biohealing Human fibroblasts + extracellular matrix + bioabsorbable scaffold; diabetic foot ulcers 2001CDRH (PMA) OrcelForticell Keratinocytes + dermal fibroblasts + bovine collagen; epidermolysis bullosa, burns 1998CDRH (HDE) EpicelGenzyme Autologous keratinocytes grown w/ murine fibroblasts; deep dermal or full thickness burns 2007CDRH (HDE)


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