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Hematopoietic Stem Cells as Vehicles for

Therapeutic Gene Delivery in Individuals

with Sickle Cell DiseaseJohn F. Tisdale, MD

Senior Investigator

Molecular and Clinical Hematology Branch


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Hematopoietic stem cells as vehicles for

therapeutic gene delivery

Allogeneic stem cell transplantation

Autologous stem cell gene transfer

Transplantation using the patients ownmodified stem cells

Bone marrow stem cells exposed to

viral vectors carrying a normal or

therapeutic gene

Transplantation using stem cells

from a normal donor

Bone marrow stem cells from a

matched brother or sistercarrying the normal gene


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Hematopoietic stem cells as vehicles for

therapeutic gene delivery


Murine

High gene transfer rates easilyachieved in vivo

Early human clinical

Equally high gene transfer ratesestimated by in vitro assays

In vivo levels of


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Rhesus competitive repopulation model

Optimal cytokine support( Blood, 1998)

Clinically feasible methods(Molecular Therapy, 2000)

True stem cell transduction(Blood, 2000)

Neo not toxic to differentiation(Human Gene Therapy, 1999)

Immune reaction not limiting

(Human Gene Therapy, 2001)


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Southern blotting: RC501

FN

STR


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Prior studies on in vivo hematopoiesis

Direct murine retroviral tagging studies

Oligoclonal hematopoiesis

Clonal succession

Deterministic model

Indirect large animal and human studies

Polyclonal hematopoiesis

Clones contribute randomly

Stochastic model


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Retroviral integration site analysis


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Lineage specific integration site analysis


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Alignment of flanking genomic DNA sequences


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Multiple clones contribute to hematopoietic

reconstitution


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Capture-recapture procedure estimates

polycolonal hematopoiesis in large animals

N=nD/X

(D=originally captured/tagged, n=newly captured total, X=newly captured tagged)

5-44 marked clones animal 1, 8-60 marked clones animal 2

contributing over the first year

1,000 total clones based upon 5% marking

5 stem cells per 107 bone marrow mononuclear cells

Data support stochastic model of human hematopoiesis


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Steady state bone marrow comparable

to G-CSF or G-CSF/SCF mobilized

peripheral blood as stem cell source

(Stem Cells, 2004)

100 cGy TBI sufficient in mice(Human Gene Therapy, 2001)

Low level engraftment in rhesus

(Molecular Therapy, 2001)

Low-dose busulfan promising(Experimental Hematology, 2006)

Clinical

success

feasible insimple

disorders?


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Steady state bone marrow comparable

to G-CSF or G-CSF/SCF mobilized

peripheral blood as stem cell source

(Stem Cells, 2004)

100 cGy TBI sufficient in mice(Human Gene Therapy, 2001)

Low level engraftment in rhesus

(Molecular Therapy, 2001)

Low-dose busulfan promising

alternative

Retroviral globin vectors

including LCR elements unstable

and

prone to rearrangement


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NATURE |VOL 406 | 6 JULY 2000 |www.nature.com

P li i l t ti d d t i th dd f


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Preclinical testing needed to improve the odds of

successful clinical applicationNonhuman primate model for therapeutic -globin gene transfer

Modified vector developed to facilitate analysis andimprove transduction rate in nonhuman primates

Vector produced at preclinical scale

Both SIV and HIV (with alternate cyclophillin binding domain)backbone compared

Developed human -globin specific detection assays

Optimized lentiviral transduction procedures

Initiated in vivo non-human primate studies

SA

RRE

SD

pe

Locus Control Region-globin gene

dLTRLTR HS2 HS3 HS4y

4 bp Insertion (Xba1)


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In vivo expression of human b-globin at day 30

after transplantation

Confirmed by RNAse protection assay

32% and 13% of BM and PB cells positive, respectively, by genomic Southern blottingClonogenic bone marrow progenitors positive for vector at 54%

Collect mobilized

CD34+ cells

Transduce

with TNS9

Assess human b-

globin expression

Infuse after

lethal XRT


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In vivo expression of human -globin at extended

follow up in both animals

Hayakawa et al., Hum Gene Ther., 20(6):563-72, 2009.


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In vivo persistence of genetically modified cells

limited by poor HSC transduction

Hayakawa et al., Hum Gene Ther., 20(6):563-72, 2009.


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In vivo persistence of genetically modified rhesus cells

limited by block to HSC transductionProduction of chimeric vectors to overcome restriction from TRIM5-alpha and APOBEC3G

HIV1- Rev/Tat + SIV-Vif plasmid

HIV1-Gag/Pol + SIV-CA plasmid

Promoter PolyA

Promoter

Rev

Tat

SIV-Vif & promoter

SIV-Capsid (CA)

Vif Promoter

CA

Gag Pol

PolyA


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Gag/Pol Rev/Tat Vif titier

Normal HIV1 vector HIV1 HIV1 (-) 9.0E+06

HIV with hVif HIV1 HIV1 HIV1 1.0E+07

HIV with sGag/Pol SIV HIV1 (-) 3.6E+04

HIV with sVIf HIV1 HIV1 SIV 9.5E+06

HIV with sGag/Pol and sVif SIV HIV1 SIV 2.7E+04

HIV with sVif-Rev/Tat HIV1 SIV SIV 3.0E+05

HIV with sGag/Pol and sVif-Rev/Tat SIV SIV SIV


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Dose escalation of chimeric vectors on human

and rhesus cell lines

The HIV1 vector with sCA (HIV) allowed efficient transduction of human and rhesus blood

cell lines. Addition of sVif reduced transduction efficiency for the human blood cell line.

CEMx174 cells

(Human Lymphoblast)

LCL8664 cells

(Rhesus Lymphoblast)

MOI

Transductio

nrate

(%)

MOI

C i i l i


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Competitive repopulation assay to compare

HIV with HIV1 vectors

Rhesus macaques

Rhesus CD34+ cells

Transplantation

Transduction

(MOI=50)

Single 24 hr

Chi-HIV-GFP vector

competitive assay

mixture

HIV1-YFP vector

G-CSF/SCF mobilized

PBSCH

Total Body Irradiation

(2x5Gy)

h hi i i


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The HIV vector achieves superior expression

rates in vivo

Days after transplantation

animal 1

HIV

%G

FP/YFP

HIV1Uchida, et al., J. Virol, 83(19):9854-62, 2009

C titi l ti t


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Rhesus macaques

Rhesus CD34+ cells

Transplantation

Transduction

(MOI=50)

Single 24 hr

Chi-HIV-GFP vector

competitive assay

mixture

SIV-YFP vector

G-CSF/SCF mobilized

PBSCH

Total Body Irradiation

(2x5Gy)

Competitive repopulation assay to compare

HIV with SIV vectors

Th HIV hi i l i


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animal 4

Days after transplantation

%G

FP/YFP

HIV

The HIV vector achieves equivalent expression

rates in vivo

Th HIV hi l ili i


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CD33+

Myeloid

cells

CD8+

T cells

CD20+

B cells

RBC+

cells

CD41a+

Platelets

CD71+

Reticulo

cytes

CD56+

NK cells

CD4+

T cells

CD3+

T cells

HIVSIV

day 138

%G

FP/YFP

The HIV vector achieves multilineage expression

in vivo

I i GFP d bl d ll


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In vivo GFP among red blood cells


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Hematopoietic stem cells as vehicles for therapeutic

gene delivery: Future efforts for human application

Optimize lentiviral vectors for use in non-human primate(Modified HIV or SIV)

Determine stem cell transduction efficiency(Test in myeloablated nonhuman primates)

Determine vector directed globin expression(Compare vector designs to maximize expression)

Determine integration pattern of optimized vector/transduction(Assess effects of additional safety measures including insulators)

Initiate human clinical trials

(Testing in thalassemia followed by SCD)



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Crew Tisdale lab

Matt Hsieh

Courtney Fitzhugh

Pat Weitzel

Naoya Uchida

O.J. Phang

Kareem Washington

Department of Transfusion Medicine

Susan Leitman

Dave Stoncek

Roger Kurlander

Elizabeth Kang

Bob Donahue

Mark Metzger

Barrington Thompson

Allen Krouse

Michel Sadelain

Beth Link

Terri Wakefield

Nona Coles

Karen Kendrick

Griffin Rodgers

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