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

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

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”

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)

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

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

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

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

How does FDA regulate these products?

But with many important exceptions Discovery & Early Translation Preclinical Development Clinical Trials Commercialization Development pathway for cell & tissue therapies is similar to drugs & conventional biologics But with many important exceptions

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

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

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

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

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

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

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

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

FDA regulations for HCT/Ps 361 HCT/Ps 351 (Tissue) Establishment registration √ (Tissue) Donor eligibility (Tissue) CGTP manufacturing cGMP regulations IND / IDE regulations Premarket approval (BLA or PMA)

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

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

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

Gene Therapies

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

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

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

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

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

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

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

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

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

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

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)

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

RCR/RCL testing (FDA 2006 supplemental guidance)

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

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

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)

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

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

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

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

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

IND development for SCD gene therapy CMC (Product) Preclinical Studies Clinical Protocol

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

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

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

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

Quantitative targets Initial quantitative targets for product CD34+ : minimum 2 x 106/kg Back up BM MNCs: 5 x 107/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%

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

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

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

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 106 CD34/kg IN FINAL PRODUCT, PLUS BACK UP OF 5 x 107 MNCs/kg

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

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

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

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

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

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

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

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

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

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

Assay development Assay For preclinical studies For product in clinical Trial Cell counts, flow phenotype (CD34), viability Research lab methods Clinical lab methods Gene/vector in cells qPCR for vector copy number Same 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/Toxicity Assess 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)

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

FDA CMC guidances

Cell & tissue therapies approved by FDA to date Product Company Description; indication Year approved FDA Center-Mechanism Carticel Genzyme Autologous cultured chondrocytes; repair of traumatic knee injury 2000 CBER (BLA) Provenge Dendreon Autologous dendritic cell tumor vaccine; prostate cancer 2010 TransCyte Advanced Biohealing Human fibroblast-derived temporary skin substitute; severe burns (now off market) 1997 CDRH (PMA) Apligraf Organogenesis Human keratinocytes + human fibroblasts in bovine collagen matrix; venous stasis leg ulcers, diabetic foot ulcers) 1998 Dermagraft Human fibroblasts + extracellular matrix + bioabsorbable scaffold; diabetic foot ulcers 2001 Orcel Forticell Keratinocytes + dermal fibroblasts + bovine collagen; epidermolysis bullosa, burns (HDE) Epicel Autologous keratinocytes grown w/ murine fibroblasts; deep dermal or full thickness burns 2007