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. 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

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
(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

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 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)

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. Cellular gene therapy for sickle cell disease
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
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 βAS3 lentiviral vector

51. 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%

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/Transduce CD34+ Cells
Harvest Transduced CD34+ Cells
Infuse Product into Patient

54. CMC Development: Basic Manufacturing Process
Bone marrow collection
CD34 Selection
Culture/Transduce 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 106 CD34/kg IN FINAL PRODUCT, PLUS BACK UP OF 5 x 107 MNCs/kg

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

64. CMC Development: Manufacturing Process – Option B
Bone marrow collection
CD34 Selection
Culture/Transduce 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 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)

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
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