Cell, Tissue, andGene Therapies

Elizabeth Read, MDMay 11, 2011

Cell, Tissue & Gene TherapiesHeterogeneous 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 – 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-threateningMultipotent

Adult stem cells from bone marrow, fat & other tissues/organs

Fetal stem cells & placental stem cells are usually considered “adult”

PluripotentEmbryonic stem (ES) cellsInduced 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?

Development pathway for cell & tissue therapies is similar to drugs & conventional

biologics

But with many important exceptions

Exception #1Transplantation of vascularized whole organs is

regulated by HRSA, not FDA

Exception #2Xenotransplantation is regulated by its own

separate set of FDA regulations

Exception #3Bone marrow transplantation using autologous

or family-related allogeneic donors is not regulated at all (practice of medicine)

Exception #4Bone marrow transplantation from unrelated

donors is regulated by HRSA, not FDA

Exception #5Some 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?

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

FDA’s Risk-Based Approach forHCT/Ps

Lower risk “361”Autologous or family related donors and minimally

manipulated and homologous useRegulated 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/Ps361

HCT/Ps

351

HCT/Ps

(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 conceptGreater 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: history1974: 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: history1995: 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 Oncogenesis2000-2007: X-linked SCID trials, using gamma

retroviral vectors to deliver the corrective gene (IL2RG) to autologous hematopoietic progenitor cells5 of 20 pts developed T cell leukemia-like

proliferative disorder, caused by INSERTIONAL ONCOGENESISRetroviral 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 methodsVector = an agent used to introduce genetic

material into cells

Vectors can beViralNon-viral

Plasmid DNALiposomes or other agents that facilitate entry into

cell

Viral vectorsRetrovirus 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 issuesObserved to date

Insertional mutagenesis/oncogenesis Immunogenicity

VectorTransgeneFBS (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 2008Vector description, map, sequence analysisCell banks, viral banks, cell lines (packaging,

producer, feeder) Vector production/purificationDocumentation of RAC reviewFor 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)

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 coursehemolytic anemiavaso-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 vectorSelf-inactivating (SIN) lentiviral vector

Carries and expressesβAS3, a β-globin gene with 3 amino acid substitutionsExpression 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 vectorExpression: 2-3 gm Hb/dl/vector copy Correction of hematological and clinical

manifestations of SCD

IND development for SCD gene therapy

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 oncogenesisTAT 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 timeHSCs have track record, CD34+ selection isolates stem &

progenitor cells

Product considerationsHSC options

Placental/umbilical cord bloodmost proliferative source, but not useful for

autologous protocol in adultsG-CSF mobilized peripheral blood HSCs

SCD patients have had serious adverse events, including death, associated with G-CSF

Bone marrowWill require general anesthesiaAvailable cell dose will be an issue

Initial definition of product candidateThe 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 targetsInitial quantitative targets for product

CD34+ : minimum 2 x 106/kgBack up BM MNCs: 5 x 107/kgVector 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

CMC Development:Basic Manufacturing Process

CMC Development:Basic Manufacturing Process

Bone Marrow SourceAutologous - 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

CD34+ Selection

Miltenyi CliniMacs CD34 Selection SystemHigh RBC content of bone marrow

interferes with selection

Need to reduce RBC content of bone marrow before CliniMacs selectionFicoll hypaque in tubes – open

system, cumbersome, cell lossAutomated closed processing: goal >

90% of MNCsCobe 2991 cell washer

CMC Development:Basic Manufacturing Process

Culture & Gene TransductionSmall scale experiments

Minimize differentiation of HSCsCytokines (SCF, Flt-3L, IL-3, Tpo)Overall culture duration

Optimize transduction efficiencyTiming: pre-stimulation in culture improves

transductionHow many hits?Recombinant human fibronectin fragment

Preserve vectorVector titers are not highQuantity will be limited

Culture & Gene TransductionAssays

Vector sequence in HSCsqPCR (vector copy number per cell)

Gene-modified HSCs are capable of erythroid differentiationIn vitro erythroid differentiation modelFold expansion & flow phenotype

RBC progeny have appropriate functionRheology and morphology

Gene-modified HSCs still contain stem cellsNOD/SCID/γc(null), primary/secondary transplants

CMC Development:Basic Manufacturing Process

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

CMC Development:Manufacturing Process – Option B

Cryopreservation & Thaw

Evaluate effects of cryopreservation & thawCD34+ cells vs final gene-modified CD34+

cellsOptimal cryomediumControlled rate device vs Mr. FrostyReadouts

Recovery of viable cellsIn vitro clonigenic assaysVector in cells and expression of gene product

Assay developmentAssay For preclinical studies For product in

clinical TrialCell 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 statusClinical protocol in draft

CMC development in progress

Preclinical studies in progress

Pre-IND meeting this summer

FDA CMC guidances

Cell & tissue therapiesapproved 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 CBER(BLA)

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 CDRH(PMA)

Dermagraft

Advanced Biohealing

Human fibroblasts + extracellular matrix + bioabsorbable scaffold; diabetic foot ulcers

2001 CDRH(PMA)

Orcel Forticell Keratinocytes + dermal fibroblasts + bovine collagen; epidermolysis bullosa, burns

1998 CDRH(HDE)

Epicel Genzyme Autologous keratinocytes grown w/ murine fibroblasts; deep dermal or full thickness burns

2007 CDRH(HDE)