530 lentiviral gene therapy of murine hematopoietic stem cells using codon optimized IL2RG cDNA: a comparison of multiple promoter elements and transplant conditions

530 Lentiviral Gene Therapy of Murine Hematopoietic Stem Cells Using Codon Optimized IL2RG cDNA A Comparison of Multiple Promoter Elements and Transplant Conditions Molecular Therapy Volume 18, Supple[.]

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HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II

528 Transgenic Expression of Cytidine

Deaminase (CDD) for Myeloprotective Purposes

Nico Lachmann,1 Nils Pfaff,1 Sebastian Brennig,1 Doreen Lüttge,1

Tobias Cantz,2 Christopher Baum,3 Axel Schambach,3 Thomas

Moritz.1

1 REBIRTH Cluster of Excellence RG Reprogramming, Medical

School Hannover, Hannover, Germany; 2 REBIRTH Cluster of

Excellence JRG Stem Cell Biology, Medical School Hannover,

Hannover, Germany; 3 Department of Experimental Hematology,

Medical School Hannover, Hannover, Germany.

Introduction: Hematopoietic stem cells genetically modi ed

to overexpress drug resistance genes such as

O6-methylguanine-DNA methyltransferase (MGMT) or multidrug-resistance gene 1

(MDR1) have been advocated to overcome chemotherapy induced

myelosuppression Cytidine deaminase (CDD) represents another

drug resistance gene which deaminates cytosine nucleosides and

their analogs such as cytarabine (Ara-C) and gemcitabine This

prevents the intracellular accumulation of the triphosphate derivates,

which act as the active metabolites of these cytotoxic drugs In this

context we have recently shown in a murine in vivo model that CDD

overexpression profoundly protects hematopoiesis from Ara-C and

Gemcitabine toxicity (Rattmann, Blood, 2006) As these studies

also revealed lymphoid toxicity and the lack of long term in vivo

selection following CDD gene transfer and drug therapy, we now

have generated novel and improved SIN lentiviral vector constructs

for CDD expression in the hematopoietic system Methods: Vectors

express the human CDD either from a spleen focus forming virus

(SFFV) or a truncated elongation factor 1α (EFS) promoter/enhancer

element In addition, inducible CDD expression based on a TET-on

system was established CDD-mediated drug resistance was evaluated

in murine myeloid (32D) and lymphoid (BWα-β-) cell lines as well as

in primary murine (lineage negative) and human (cord blood CD34+)

hematopoietic cells In these experiments cells transduced with CDD

expressing lentiviral constructs (SFFV-CDD, EFS-CDD, TET-CDD)

were sorted (GFP+) and treated with different concentrations of Ara-C

or gemcitabine for 48h Subsequently, cell viability was determined

utilizing propidium iodide and primary hematopoietic cells were

analyzed in clonogenic assays

Results: Irrespective of the lentiviral construct used, CDD

transduced murine myeloid and lymphoid cell lines showed

profound protection against Ara-C levels of up to 5000 nM, whereas

untransduced control cells died at concentration of 200 nM With the

TET-CDD construct protection in 32D cells was achieved within

24h of Doxycyclin treatment and upon Doxycyclin cessation CDD

expression returned to baseline within three to  ve days Similar

protection was observed in primary clonogenic hematopoietic

cells Here, cells transduced to yield EFS- or SFFV-driven CDD

expression were protected from Ara-C levels of up to 300 and 600

nM, respectively, with untransduced controls dying at 50 nM Ara-C

Conclusion: These data demonstrate profound protection from

Ara-C toxicity in both, hematopoietic cell lines as well as primary

hematopoietic cells by lentiviral vectors employing SFFV- or

EFS-driven as well as TET-inducible CDD expression Next, the in vivo

selection potential of these promising new vector constructs will be

investigated in murine model systems employing optimized Ara-C

application schedules

529 Development of a Pre-Clinical Model for the Gene Therapy Treatment of Wiskott-Aldrich Syndrome (WAS)

Alexander Astrakhan,1 Byoung Ryu,2 Brigid Stirling,2 Blythe Sather,2 Jit Khim,2 Mikhail Garibov,2 Stephanie Humblet-Baron,2

Hans Ochs,2 David J Rawlings.1,2

1 Department of Immunology, University of Washington, Seattle, WA; 2 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA.

Wiskott-Aldrich Syndrome is an immunode ciency characterized

by recurrent infections, thrombocytopenia, eczema, and increased susceptibility to autoimmunity and malignancy The disease is caused by defective function of the WAS protein (WASp), a crucial intermediary in receptor-mediated actin cytoskeletal rearrangement

While prognosis is relatively good for patients receiving matched bone marrow transplants, overall outlook is poor for patients lacking suitable donors Lentiviral-mediated gene therapy is a promising therapeutic approach for the treatment of primary immunode ciencies

We used WASp-de cient mice to analyze the ef cacy of lentiviral gene therapy in rescuing WAS-associated hematopoietic defects In initial experiments we utilized the strong, ubiquitous, retrovirally-derived MND promoter to drive WASp expression in vivo Lentiviral transduction of hematopoietic stem cells (HSCs) resulted in stable WASp expression in all hematopoietic lineages, including the myeloid, lymphoid and platelet compartments We observed selective outgrowth of WASp+ cells within T cell subsets, marginal zone (MZ)

B cells and platelets Gene therapy treated mice demonstrated at least partial correction of WAS-associated defects, with normal numbers

of marginal zone B cells and functional T cell proliferation and IL-2 production following T cell receptor (TCR) engagement Due to the strong oncogenic potential of the MND promoter in vivo, we next compared MND to mammalian promoters, including the minimal WAS promoter (WS1.6) and the short elongation factor 1 alpha (sEF1a) promoter Surprisingly, the WS1.6 promoter was poorly active in all hematopoietic lineages, and was particularly ineffective

in driving WASp expression in B cells The reduced expression levels was speci c to the WAS promoter, as the sEF-1a promoter exhibited robust activity in all hematopoietic lineages Combined, these  ndings suggest that lentiviral-mediated gene therapy can lead

to successful correction of WAS-associated hematopoietic defects in the murine model of WAS Our observations argue against the use of the endogenous WAS promoter in human clinical trials; rather, we propose that the EF-1a promoter is likely to be more effective and potentially safer promoter for transition into clinical testing

530 Lentiviral Gene Therapy of Murine Hematopoietic Stem Cells Using Codon Optimized IL2RG cDNA: A Comparison of Multiple Promoter Elements and Transplant Conditions

Marshall W Huston,1 Niek P van Til,1 Trudi P Visser,1 Roya Sawari,1 Shazia Arshad,1 Gerard Wagemaker.1

1 Hematology, Erasmus University Medical Center, Rotterdam, Netherlands.

Gene therapy for X-linked severe combined immunode ciency (X-SCID) by gammaretroviral vectors to deliver a functional copy

of the IL2RG gene into the host genome has been very effective in the clinical setting, but carries the risk of insertional oncogenesis (Hacein-Bey-Abina, JCI 2008) HIV-1 derived lentiviral vectors have advantages over gammaretroviruses in transducing quiescent cells, such as long-term repopulating hematopoietic stem cells (HSC), are less likely to integrate near transcription start sites, and are thought

to have lower genotoxicity (Montini, Nat Biotechnol 2006) To test ef cacy and safety of lentiviral IL2RG gene therapy for X-SCID,

a self-inactivating lentiviral vector was constructed containing a

codon optimized human IL2RG cDNA (coγc) Coγc expression was driven by the SFFV viral promoter, PGK cellular promoter or a native human IL2RG promoter region (γcPROM) Lineage negative HSC

of Il2rg-/- mice were transduced with these vectors or a GFP control vector, resulting in 1 to 5 transgene copies per transduced cell The transduced cells were transplanted into Il2rg-/- mice and compared

to Il2rg-/- recipients of healthy wild type cells

Blood collected monthly and analyzed by differential cell counting and immunophenotyping demonstrated that mice transplanted with cells transduced with the coγc transgene reconstituted mature T and

B cell populations, whereas recipients of cells transduced with GFP vectors did not TCRβ repertoire, IL-2/Con-A spleen cell proliferation assays, basal IgM/IgG1 serum levels and T-cell dependent immune responses in coγc treated mice yielded results similar to wild type controls Polyclonal integration patterns were con rmed by LAM-PCR eight months post-transplant

Further detailed studies with the γcPROM-coγc vector demonstrated that reducing the viral MOI to reach an average copy number/cell

of 1 and the number of cells transplanted to 5x107/kg body weight had a negligible effect on the ef cacy of the gene therapy treatment

Reducing pre-transplant irradiation conditioning from 6 Gy to

2 Gy likewise had little effect, but mice given no pre-transplant conditioning had a more protracted immune reconstitution in both coγc-treated and wild type groups

We conclude that lentiviral-based coγc gene therapy is an effective alternative to gammaretroviral gene delivery even with low levels of conditioning and cell dosage and that the PGK promoter and native IL2RG promoter region are ef cacious candidates for future clinical gene therapy development Current experiments focus on integration analyses of treated mice and long-term follow-up monitoring of potential adverse effects

531 Human Fetal Liver HSCs Demonstrate High Transduction Rates and Sustained Transgene Expression Following Implantation

Niraja Dighe,1 Maroun Khoury,4 Mark Chong,1 Citra Mattar,1

Michael N Antoniou,5 Jianzhu Chen,4 Mahesh Choolani,1 Jerry Chan.1,2,3

1 Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; 2 Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore; 3 Cancer and Stem Cell Program, Duke-NUS Graduate Medical School, Singapore, Singapore;

4 Interdisciplinary Research Group in Infectious Diseases, Singapore-Massachusetts Institute of Technology Alliance in Research and Technology, Singapore, Singapore; 5 Department of Medical and Molecular Genetics, King’s College London School of Medicine, Guys Hospital, London, United Kingdom.

Introduction: Hematopoietic Stem Cells (HSC) targeted gene

transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects Recently, it has been shown that fetal stem cells demonstrate a higher propensity for transduction compared to adult cells We hypothesized that fetal tissue-derived HSC is more amenable to transduction and hence a suitable candidate for targeted gene transfer Here, we report the ef cient transduction

of primitive human CD34+ fetal liver cells with a lentiviral vector encoding an A2UCOE-eGFP cassette, compared to cord

blood-derived HSC Methods: CD34+ cells were isolated from second

trimester human liver ( HSC) and umbilical cord blood (UCB-HSC) by MACS, and subsequently infected with UCOE-GFP at a multiplicity of infection (MOI) from 1 to 20 FACS, CFU assays and vector copy number analyses were done to evaluate the effects

of transduction on HSC character and ef ciency of transduction respectively Subsequently, cells infected at MOI 20 were transplanted

into sublethally-irradiated neonatal NOD/SCID/Il2rg-/- mice via intracardiac injection, to study multilineage engraftment and secondary transplantation capacity of transduced HSC In parallel, some cells were expanded in culture to study the continued expression

of GFP over 21 days Results:  HSC were transduced with a maximal

transduction rate of 85% at MOI 10 Increasing MOI beyond 10 failed to raise the transduction rates appreciably (87% at MOI 20)

In contrast, transduction of UCB-HSC was poor, with only 2% transduced at MOI of 20 CFU assays demonstrated that transduced

 HSC were able to differentiate toward multiple lineages Finally, transduced  HSC were found to be capable of long term multi-lineage engraftment of sublethally-irradiated mice, and retained expression

of eGFP for more than 8 weeks post-transplantation This correlates

with in vitro data showing sustained expression of eGFP (64% at day

3, 61% at day 21) during continuous culture Conclusions: Our data

suggest that CD34+ cells from human fetal liver represent a suitable source of HSC for targeted gene transfer, capable of ef cient lentiviral transduction rates compared to UCB-HSC, These encouraging results lay the foundation for the use of lentiviral vectors containing β-globin locus control region (βLCR)-driven therapeutic gene cassettes to effect lineage-restricted expression for the treatment of various hematopoietic disorders, including the hemoglobinopathies

532 FANG Autologous Tumor Cell Vaccine Development and Manufacturing

Phillip B Maples,1 Padmasini Kumar,1 Yang Yu,1 Beena O Pappen,1 Chris M Jay,1 Zhaohui Wang,1 Donald D Rao,1 Joseph Kuhn,2 John Nemunaitis,1,3,4,5 Neil Senzer.1,3,4,5

1 Gradalis, Inc., Dallas, TX; 2 General and Oncology Surgery Associates, Dallas, TX; 3 Mary Crowley Cancer Research Centers, Dallas, TX; 4 Baylor Sammons Cancer Center, Dallas, TX; 5 Texas Oncology, P.A., Dallas, TX.

Gene modi ed cell-based cancer vaccines have demonstrated durable responses in selected patients We have developed the FANG expression vector which we believe, when transfected into tumor cells, will evoke an enhanced immune recognition /stimulation versus our previous TAG vaccine vector The FANG nonviral vector system expresses both GM-CSF and a proprietary bifunctional shRNA to furin Preclinical data demonstrated that blocking furin protein expression in turn blocked the activation of both TGFβ1 and TGFβ2

antisense Data from our TAG Phase I autologous vaccine clinical trial and others indicate that TFG β1 overexpression is present in a wide range of cancers In fact our data suggest that TGFβ1 tends to

be about tenfold higher than TGFβ2 expression in the more than thirty tumors we examined in that study So while the TAG vector blocked TGFβ2 expression, there was no effect on TGFβ1 expression The FANG expression vector is identical to the TAG expression vector except that the TGFβ2 antisense coding sequence has been replaced with the furin shRNA sequence FANG plasmid DNA was GMP-S manufactured We generated 2 nonclinical and 8 clinical vaccines under cGMP as part of our IND submission data (4 melanoma, 3 colorectal, 1 gall bladder, 1 NSCLC and 1 breast cancer) All vaccine manufacturing processes met speci cations (no contamination or failure to meet  nal dose or quality requirements) Average cell viability is 91.5+5.3%, median 93.5% and range 78-96% (values taken on Day 2 of manufacturing) Average GM-CSF expression is 657+550pg/1x106 cells/ml, median 602pg and range 80-1870pg The mean pretransfection TGFβ1 is 1241+1115pg/1x106 cells/ml, median 1039pg The mean posttransfection TGFβ1 is 211+421pg/1x106 cells/

89+20%, median 97% and range 36-100% The mean pretransfection TGFβ2 is 293+189pg/1x106 cells/ml, median 257pg The mean posttransfection TGFβ2 is 9.1+12pg/1x106 cells/ml, median 4pg The average percent knockdown of TGFβ2 was 94+12 %, median 99% and

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