342 correction of murine hemophilia a following nonmyeloablative transplantation of hematopoietic stem cells engineered to express a bioimproved human FVIII using a safety augmented, b lymphoid lineage restricted lentiviral vector

342 Correction of Murine Hemophilia A Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Express a Bioimproved Human FVIII Using a Safety Augmented, B Lymphoid Lineag[.]

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HEMATOLOGIC/IMMUNOLOGIC: BASIC

340 Development of an S/MAR Based Episomal

Vector of a Speci c Zinc-Finger Activator That

Mediates Gamma-Globin Gene Activation, for the

Gene Therapy of Hemoglobinopathies

Eleana Stavrou,1 Eleni Lagadinou,2 Nikolas Zoumbos,2 Alexandros

Spyridonidis,2 Carlos Barbas, III,3 Kenneth Peterson,4 Aglaia

Athanassiadou.1

1 General Biology, School of Medicine, University of Patras,

Patras, Rion, Greece; 2 Internal Medicine, Hematology Unit,

School of Medicine, University of Patras, Patras, Rion, Greece;

3 Molecular Biology, Skaggs Institute for Chemical Biology, Scripps

Research Institute, La Jolla, CA; 4 Biochemistry and Molecular

Biology, University of Kansas Medical Center, Kansas City.

The increase of HbF through activation of gamma-globin gene is a

valid strategy for the treatment of hemoglobinopathies Zif-VP64 is a

selective, synthetic gamma-globin activator, containing a zinc- nger

DNA protein that binds the gamma-globin promoter -117HPFH area

and a transcription inducer that induces gamma globin gene in K562

cells, after viral transfer We report the study of an episomal vector of

this activator, which is based on a Scaffold/Matrix attachment region

(S/MAR) that supports retention of episomes in the nucleus of the host

cell We constructed an episomal vector, Zif-VP64-Ep1, containing

the activator Zif-VP64, the reporter gene cassette CMV-eGFP and the

S/MAR element Gene transfer into cells was done by electroporation

or nucleofection Expression of eGFP was documented by Florescent

Microscopy and Flow Cytometry, while the fate of vector molecules

in the cells was studied by Southern Blot and plasmid rescue

experiments Real time PCR, Western blotting and Intracellular Flow

Cytometry were used to investigate globin mRNA,

gamma-globin protein and HbF protein levels respectively Binding speci city

of the activator was determined by ChIP Gene transfer was done in

K562 cells producing long term stable cell lines; murine beta-YAC

cells, where the YAC contains the complete human, beta-globin gene

locus; and human progenitor hemopoietic CD34+ cells from healthy,

mobilized individuals, with transfection ef ciencies of 65%, 25%

and 23% respectively In K562 cells, gamma-globin mRNA levels

showed an increase of 250%, gamma-globin protein of 350% and

HbF protein of 165% as compared to the corresponding levels in the

untransfected K562 cells, at least 200 generations post-transfection

In murine beta-YAC cells, vector Zif-VP64-Ep1 was able to mediate

the activation of expression of the silent, human gamma-globin gene

at a level matching the (active) human beta-globin gene of the YAC

as well as the murine beta-globin gene, showing that it can ef ciently

activate the gamma-globin gene from within a heterochromatic region

Signi cantly, vector Zif-VP64-Ep1 was able to transfect the human,

hemopoietic progenitor CD34+ cells and to mediate a 3.0±1 fold

increase of gamma globin mRNA, compared to untrasnfected CD34+

cells, as estimated in cultures of 7-8 days after transfection

In conclusion, activation of human gamma-globin by episomal gene

transfer of a synthetic activator, in three different hemopoietic cells,

is documented, including the CD43+ cells, that are the target cells for

gene therapy of the Hemoglobinopathies This is the  rst time that an

S/MAR based episomal vector is used for gene transactivation in a

cell line and progenitor cells, aiming at speci c gene therapy

341 Engineered Human Regulatory T Cells

Expressing Lentiviral PDL1 under Cell Fate Control

Prevent Lethal Xenogeneic GVHD

Shoba Amarnath,1 James Wang,2 Courtney Mangus,1 James Riley,3

Bruce Levine,3 Carl June,3 Jeffrey Medin,2,4 Daniel Fowler.1

1 ETIB, CCR, NCI, Bethesda, MD; 2 IMS, Toronto, ON, Canada;

3 UPENN, Philadelphia, PA; 4 UHN, Toronto, ON, Canada.

Programmed death ligand-1 (PD-L1) represents a regulatory T

cell (Treg) mechanism that controls immunity mediated by effector

memory T cells expressing PD1 We hypothesized that genetically-engineered human T cells forced to express PD-L1 would manifest a Treg phenotype, and thus be capable of inhibiting human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) Advantages of this proposed therapy include a capacity to: (1) enforce long-term, stable expression of PD-L1; (2) utilize cellular delivery vehicles composed

of functional T cells that persist in vivo; and (3) incorporate an enhanced ‘cell fate control’ cDNA to permit in vivo control of therapy

We thus developed a recombinant lentiviral vector (LV) that encodes cDNA for a fusion protein consisting of human CD19 and mutated TMPK that activates the prodrug AZT, followed by full-length human PD-L1 We previously found that ex vivo T cell expansion in rapamycin induces an anti-apoptotic phenotype that permits in vivo T cell persistence in murine models and xenogeneic transplant models

We thus manufactured human CD4+ T cells using co-stimulation (anti-CD3/28), Th1 polarization (IFN-α), and rapamycin After 6 days of culture and subsequent  ow sorting, >90% of transduced T cells expressed PD-L1 Next, we utilized a x-GVHD model to assess

in vivo persistence of the modi ed T cells and transgene expression

Two separate experiments demonstrated that gene-modi ed T cell recipients had increased numbers of human T cells in the spleen that co-expressed CD19 and PD-L1 relative to non-transduced T cell recipients (p=0.02) Harvested T cells were secondarily co-stimulated

ex vivo and propagated for 5 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells (p=0.0001) To test the ability of PD-L1-transduced T cells to prevent x-GVHD, a 3rd experiment involved cohorts that received: human Th1 cells alone (C#1) or in combination with PD-L1 LV-transduced T cells (C#2); puri ed human Treg cells (C#3); or control LV-transduced T cells (C#4) On day 5, mice were challenged with LPS to induce cytokine-mediated x-GVHD Cohorts did not differ with respect to human T cell engraftment Relative to C#1, both C#2 and C#3 had reduced numbers of human T cells that expressed IFN-γ (each comparison, p<0.03) and also had reduced serum human TNF-α (each comparison, p<0.04) Recipients of either Treg cells or PD-L1-transduced T cells had reduced lethality from x-GVHD (C#2< C#1, p=0.002; C#3< C#1, p=0.0001) In conclusion, CD19/TMPK.PD-L1 LV results in stable and functional transgene expression in human T cells in vitro and in vivo, thereby opening

an avenue to assess PD-L1 mediated immuno-gene therapy under cell fate control

342 Correction of Murine Hemophilia A Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Express

a Bioimproved Human FVIII Using a Safety-Augmented, B-Lymphoid Lineage-Restricted Lentiviral Vector

Ali Ramezani, Sara Karandish, Lynnsey A Zweier-Renn, Teresa S

Hawley, Robert G Hawley

The George Washington University Medical Center, Washington, DC.

Hemophilia A is an X-linked recessive genetic bleeding disorder caused by a de ciency or functional defect in coagulation factor VIII (FVIII) The clinical success of hemophilia A gene therapy requires overcoming a number of obstacles, necessitating development of a strategy that will ideally achieve therapeutic FVIII expression and induce immune tolerance to the FVIII neoantigen with minimal risk of insertional mutagenesis We recently reported correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells (HSCs) engineered to express an enhanced human FVIII variant (eFVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/

R489A/P492A) for reduced immunogenicity—using a safety-augmented retroviral vector The current study evaluates the potential

use of insulated SIV-derived lentiviral vectors as a safer alternative to gammaretroviral vectors, and the transcriptional targeting of eFVIII expression to B cells A B-lymphoid lineage-speci c transcriptional regulatory element was constructed by combining an intronic locus control region from the Igµ heavy chain gene (LCRµ) with a minimal

Igµ variable region promoter (Pµ) The LCRµ consists of the Ig heavy chain enhancer flanked by matrix attachment regions required for transcriptional activation of the Igµ gene during normal B cell development The speci city of the LCRµPµ transcriptional regulatory element to direct GFP reporter gene expression was evaluated in a series of hematopoietic cell lines While all cell lines tested expressed high levels of GFP from a control SIV-MSCV-GFP vector, only the B cell line (Sp2/0) expressed signi cant levels of GFP when transduced at equivalent MOIs with the SIV-LCRµPµ-GFP vector

To evaluate in vivo ef cacy, BALB/c hemophilia A (BAL-FVIIIKO)

mice were conditioned with a busulfan-based nonmyeloablative preparative regimen and transplanted with Sca-1+ HSCs transduced with the FB.SIV-LCRµPµ-eFVIII vector, which is a safety-augmented derivative harboring a novel 77-bp enhancer-blocking element in its deleted 3’ U3 region (designated FB for FII/BEAD-A) In parallel, a second cohort of similarly conditioned BAL-FVIIIKO mice received Sca-1+ HSCs transduced with the FB.SIV-MSCV-eFVIII vector as

a positive control (in addition to eFVIII, both vectors also express the GFP reporter) At 16 weeks posttransplant, 4.7 ± 2.6% and 5.3 ± 2.7% GFP+ peripheral blood cells were detected in FB.SIV-MSCV-eFVIII- and FB.SIV-LCRµPµ-eFVIII-treated mice, respectively (n =

9 mice per cohort) The plasma eFVIII levels in the FB.SIV-MSCV-eFVIII-treated animals were 39 ± 19 ng/ml (20% of normal) More exciting, therapeutic eFVIII levels were also detected in the plasma

of the FB.SIV-LCRµPµ-eFVIII-treated mice (49 ± 26 ng/ml; 25%

of normal) Characterization of the cell types expressing eFVIII, and investigation of the mechanism and extent of eFVIII immune hyporesponsiveness are ongoing and the  ndings obtained will be presented

343 Lentiviral Vectors Containing an Endogenous Btk Promoter Restore B and Myeloid Lineage Expression and Function in a Murine Model of X-Linked Agammaglobulinemia

Hannah M Kerns,1 Byoung Y Ryu,1 Brigid V Stirling,1 Blythe D

Sather,1 Alexander Astrakhan,2 Stephanie Humblet-Baron,4 Denny Liggitt,5 David J Rawlings.1,2

1 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 2 Department of Immunology, University of Washington School of Medicine, Seattle, WA; 3 Center for Cellular and Molecular Therapy, University of Liège, Liège, Belgium; 4 Department of Comparative Medicine, University of Washington, Seattle, WA.

The immunode ciency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton’s tyrosine kinase (Btk) Btk is required for pre-B cell clonal expansion and B cell antigen receptor (BCR) signaling XLA patients lack mature B cells and immunoglobulin, and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy

We have recently demonstrated successful ex vivo gene therapy

using a B lineage-speci c, lentiviral vector (LV) containing the immunoglobulin heavy chain enhancer (Eµ) and Igβ (B29) minimal promoter to drive human Btk expression in Btk/Tec-/- mice, a strain

that reproduces the features of human XLA (Blood, 2010 in press)

Notably, in addition to its critical role in B cells, Btk is also expressed

in all myeloid-derived lineages; and recent data indicate that Btk de ciency impacts innate immune function in both human and murine cells For example, Btk de cient macrophages and Btk/Tec-/- bone marrow-derived macrophages exhibit altered TLR-driven cytokine production and reduced survival in response to M-CSF, respectively

Thus, an optimal Btk gene therapy LV should ef ciently restore Btk function in both B and myeloid cells To address this issue, we developed a series of LV containing the endogenous Btk promoter (Btkp; 790bp containing all of the proximal DNA binding elements as well as the two recently described distal NFκB binding sites), alone or

in association with either the immunoglobulin enhancer (Eµ); or the ubiquitously acting chromatin opening element (UCOE) in order to potentially minimize positional variegation While the Btkp-Btk LV failed to correct the B cell defects in Btk/Tec-/- recipient mice, UCOE-Btkp-Btk LV showed partial, and EµUCOE-Btkp-Btk LV exhibited complete developmental and functional reconstitution (equivalent to or greater than EµB29-Btk) Speci cally, EµBtkp-Btk LV treated mice showed rescue of mature B cells in the bone marrow, peripheral blood, spleen and peritoneal cavity, and improved responses to both T-independent and T-dependent antigens LV-treated B cells manifested enhanced BCR signaling and an in vivo selective advantage in the peripheral

vs central B cell compartment Secondary transplantation showed sustained Btk expression, viral integration and functional responses, consistent with long-term stem cell marking; without evidence for cellular or systemic toxicity Most notably, EµBtkp-Btk-, in contrast

to EµB29-Btk-, LV-treated mice exhibited sustained, WT-levels of Btk expression and improved activity in myeloid lineages As this vector closely mimics the endogenous expression pattern of human Btk, it appears to represent an optimal, clinically-relevant LV platform for gene therapy in XLA

344 Early Gestational Gene Transfer of IL-10 by Lentiviral Vector Can Prevent Arthritis in a Murine Model

Jessica L Roybal,1 Masayuki Endo,1 Antoneta Radu,1 Carlyn Todorow,1 Philip W Zoltick,1 Alan W Flake.1

1 Department of Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA.

Rheumatoid arthritis is an autoimmune disease thought to be caused by the imbalance of pro- and anti-in ammatory cytokines Anti-in ammatory IL-10 has shown promise in preventing arthritis in mouse models, but prolonged joint expression has been problematic Our laboratory has found that early gestational intravascular injection

of lentiviral vector leads to ef cient transduction and sustained expression of transgene in a number of tissues including articular cartilage and synovium We hypothesized that early gestational gene transfer of IL-10 could prevent or decrease pathology in a murine model of rheumatoid arthritis

Time-dated B10.Q fetal mice at embryonic day 9 underwent ultrasound-guided intracardiac injection of 107 infectious particles

of an HIV-1-based vector encoding murine IL-10 and a GFP reporter driven by the CMV promoter Serum IL-10 levels were measured

at 4, 7 and 10 weeks of age by ELISA Naive mice and injected mice with undetectable serum IL-10 were used as controls An arthritis model was generated by the immunization of mice against type II collagen at 6 weeks with a booster at 8 weeks Each paw was scored subjectively (0-4/paw, 4 for maximal in ammation) for arthritis by 3 blinded observers Upon sacri ce (12 weeks after immunization), serum antibodies to type II collagen were measured

by ELISA Forelimbs and hindlimbs were collected for histology and immunohistochemistry Histological sections were scored subjectively for in ammation by 2 blinded observers (0-3/limb, 3 for maximal in ammation) Expression of IL-6, IL-1B, and TNF-a in the knee joint homogenate was assessed by qRT-PCR and corresponding cytokine sera levels were obtained by ELISA

Thirty percent of fetuses injected with the CMV-IL-10-GFP lentiviral vector were born compared to 38% with the CMV-GFP reporter vector Serum IL-10 was undetectable in the control group, but ranged from 121-3728pg/ml in the experimental mice (n=16) Comparing mice with initial IL-10 levels greater than 700pg/ml

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