Hematopoietic Stem Cells as Vehicles for Therapeutic Gene Delivery in Individuals with Sickle Cell Disease

John F. Tisdale, MD Senior Investigator Molecular and Clinical Hematology Branch

Hematopoietic stem cells as vehicles for therapeutic gene delivery

Allogeneic stem cell transplantation
Transplantation using stem cells from a normal donor Bone marrow stem cells from a matched brother or sister carrying the normal gene

Autologous stem cell gene transfer

Transplantation using the patients own modified stem cells Bone marrow stem cells exposed to viral vectors carrying a normal or therapeutic gene

Hematopoietic stem cells as vehicles for therapeutic gene delivery

Autologous stem cell gene transfer
Murine High gene transfer rates easily achieved in vivo Early human clinical Equally high gene transfer rates estimated by in vitro assays In vivo levels of <1/100,000 cells Too low to expect clinical benefit Predictive human HSC assays needed Nonhuman primate competitive repopulation model developed

Rhesus competitive repopulation model

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

Immune reaction not limiting

(Human Gene Therapy, 2001)

Optimal cytokine support

( Blood, 1998)

Clinically feasible methods

(Molecular Therapy, 2000)

True stem cell transduction (Blood, 2000)

Southern blotting: RC501

FN

STR

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

Retroviral integration site analysis

Lineage specific integration site analysis

Alignment of flanking genomic DNA sequences

Multiple clones contribute to hematopoietic reconstitution

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

Rhesus competitive repopulation model

Steady state bone marrow comparable to G-CSF or G-CSF/SCF mobilized peripheral blood as stem cell source
(Stem Cells, 2004)

Neo not toxic to differentiation

(Human Gene Therapy, 1999)

Immune reaction not limiting

(Human Gene Therapy, 2001)

Clinical success feasible in simple disorders?

100 cGy TBI sufficient in mice

(Human Gene Therapy, 2001)

Optimal cytokine support

( Blood, 1998)

Low level engraftment in rhesus (Molecular Therapy, 2001) Low-dose busulfan promising
(Experimental Hematology, 2006)

Clinically feasible methods

(Molecular Therapy, 2000)

True stem cell transduction (Blood, 2000)

Rhesus competitive repopulation model

Steady state bone marrow comparable to G-CSF or G-CSF/SCF mobilized peripheral blood as stem cell source
(Stem Cells, 2004)

Neo not toxic to differentiation

(Human Gene Therapy, 1999)

Immune reaction not limiting

(Human Gene Therapy, 2001)

100 cGy TBI sufficient in mice

(Human Gene Therapy, 2001)

Retroviral globin vectors including LCR elements unstable and prone to rearrangement Optimal cytokine support
( Blood, 1998)

Low level engraftment in rhesus (Molecular Therapy, 2001) Low-dose busulfan promising alternative

Clinically feasible methods

(Molecular Therapy, 2000)

True stem cell transduction (Blood, 2000)

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

Preclinical testing needed to improve the odds of successful clinical application

Nonhuman primate model for therapeutic -globin gene transfer
b -globin gene Locus Control Region

LTR

SD

y RRE

HS2

HS3

HS4

dLTR

SA
4 bp Insertion (Xba1)

Modified vector developed to facilitate analysis and improve 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

In vivo expression of human b-globin at day 30 after transplantation

Collect mobilized CD34+ cells Transduce with TNS9 Infuse after lethal XRT Assess human bglobin expression

Confirmed by RNAse protection assay 32% and 13% of BM and PB cells positive, respectively, by genomic Southern blotting Clonogenic bone marrow progenitors positive for vector at 54%

In vivo expression of human -globin at extended follow up in both animals

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

In vivo persistence of genetically modified cells limited by poor HSC transduction

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

In vivo persistence of genetically modified rhesus cells limited by block to HSC transduction
Production of chimeric vectors to overcome restriction from TRIM5-alpha and APOBEC3G

HIV1-Gag/Pol + SIV-CA plasmid Gag

Promoter

Pol
PolyA

CA

SIV-Capsid (CA)

HIV1- Rev/Tat + SIV-Vif plasmid Rev Vif

Promoter Promoter PolyA

Tat

SIV-Vif & promoter

Screening of chimeric vector production by viral titer

Gag/Pol Normal HIV1 vector HIV1 Rev/Tat HIV1 Vif (-)

titers titier 5x106

9.0E+06

1x107

HIV with hVif

HIV with sGag/Pol HIV with sVIf HIV with sGag/Pol and sVif HIV with sVif-Rev/Tat HIV with sGag/Pol and sVif-Rev/Tat HIV with sRev/Tat

HIV1
SIV HIV1 SIV HIV1 SIV HIV1

HIV1
HIV1 HIV1 HIV1 SIV SIV SIV

HIV1
(-) SIV SIV SIV SIV (-)

1.0E+07
3.6E+04 * 9.5E+06 2.7E+04 * 3.0E+05 * <1.0E+05 * 7.4E+05 *

HIV with sGag/Pol and sRev/Tat

HIV with sCA HIV with sCA+sVif

SIV
HIV1+sCA HIV1+sCA

SIV
HIV1 HIV1

(-)
(-) SIV

<1.0E+05 *

* p<0.01

Dose escalation of chimeric vectors on human and rhesus cell lines

CEMx174 cells (Human Lymphoblast)
Transduction rate (%)

LCL8664 cells (Rhesus Lymphoblast)

MOI

MOI

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.

Competitive repopulation assay to compare HIV with HIV1 vectors

Transduction (MOI=50) Single 24 hr Chi-HIV-GFP vector Rhesus CD34+ cells mixture

HIV1-YFP vector G-CSF/SCF mobilized PBSCH

Transplantation

Rhesus macaques

Total Body Irradiation (2x5Gy)

competitive assay

The HIV vector achieves superior expression rates in vivo animal 1

% GFP / YFP

Days after transplantation Uchida, et al., J. Virol, 83(19):9854-62, 2009

HIV
HIV1

Competitive repopulation assay to compare HIV with SIV vectors

Transduction (MOI=50) Single 24 hr Chi-HIV-GFP vector Rhesus CD34+ cells mixture

SIV-YFP vector G-CSF/SCF mobilized PBSCH

Transplantation

Rhesus macaques

Total Body Irradiation (2x5Gy)

competitive assay

The HIV vector achieves equivalent expression rates in vivo animal 4

% GFP / YFP

Days after transplantation

HIV

The HIV vector achieves multilineage expression in vivo day 138

HIV SIV

% GFP / YFP

CD33+ Myeloid cells

CD20+ B cells

CD3+ T cells

CD4+ T cells

CD8+ CD56+ CD71+ RBC+ T cells NK cells Reticulo cells cytes

CD41a+ Platelets

In vivo GFP among red blood cells

Hematopoietic stem cells as vehicles for therapeutic gene delivery: Future efforts for human application
Autologous stem cell gene transfer
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)

Crew
Tisdale lab Matt Hsieh Courtney Fitzhugh Pat Weitzel Naoya Uchida O.J. Phang Kareem Washington Department of Transfusion Medicine Susan Leitman Dave Stoncek Elizabeth Kang Bob Donahue Mark Metzger Barrington Thompson Allen Krouse Michel Sadelain

Roger Kurlander

Beth Link Terri Wakefield Nona Coles Karen Kendrick Griffin Rodgers