256 the comparison of various promoters for wiskott aldrich syndrome gene therapy using insulated lentiviral vectors

256 The Comparison of Various Promoters for Wiskott Aldrich Syndrome Gene Therapy Using Insulated Lentiviral Vectors Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Societ[.]

Molecular Therapy Volume 19, Supplement 1, May 2011

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I

Immunodefi ciency by Lentiviral Gene Therapy

Requires Strong Expression of RAG2

Niek P van Til,1 Helen de Boer,1 Roya Sarwari,1 Trudi P Visser,1

Barbara Cassani,2 Anna Villa,3 Gerard Wagemaker.1

1 Department of Hematology, Erasmus University Medical Center,

Rotterdam, Netherlands; 2 Fondazione Humanitas per la Ricerca,

Rozzano, Milan, Italy; 3 CNR-ITB; Telethon Institute for Gene

Therapy-HSR, Milan, Italy.

Recombination activating gene 2 (RAG2) defi ciency results in

severe combined immunodefi ciency (SCID) due to a lack of T and B

lymphocytes, caused by a block in differentiation Patients succumb

due to recurrent infections within the fi rst year of life, unless treated

with an allogeneic bone marrow (BM) transplant Unfortunately, the

paucity of compatible donors affects the effi cacy of treatment and

the survival of treated patients Gene therapy using gammaretroviral

vectors for X-linked SCID gene therapy and adenosine deaminase

(ADA)-SCID has been shown to be highly effective, but RAG2 SCID

may require more tightly controlled expression as its expression is

heavily regulated during normal T and B cell development Previously,

it has been shown that gammaretroviral vectors can effectively restore

immune function in Rag2 -/- mice To improve effi cacy and reduce

the risk of genotoxicity we developed and tested self-inactivating

lentiviral vectors constituting a codon-optimised RAG2 (RAG2co)

driven by the spleen focus forming virus promoter (SF) This vector

was then compared to its native RAG2 counterpart as well as to weaker

cell-restricted promoters based on minimum elements of the RAG2

and γc promoter 5x105 Rag2 -/- transduced lineage negative BM cells

were transplanted in sublethally irradiated Rag2 -/- mice, monitored

for six months Codon-optimization of RAG2 increased detectable

RAG2 protein 3-fold, determined in HeLa cells The SF-RAG2co

vector resulted in a signifi cant reduction in the developmental block

during T and B cell differentiation in thymus and BM, respectively,

and in robust reconstitution of CD4 and CD8 T cell subsets, as

well as mature IgM+IgD+ B cells in peripheral blood and spleen

Reconstitution of T and B cells following transplantation of cells

transduced with the vector carrying the native RAG2 was signifi cantly

slower, and most peripheral T cells expressed a single dominant T cell

receptor subtype In contrast, the RAG2 and γc promoter reconstituted

T cells effectively, but hardly any B cells Vector copy number per

cell determined in bone marrow was considerable higher in the

SF-RAG2co (2.3±2.0, n=17) and SF-RAG2 treated mice (3.0±0.9, n=7)

than in those treated with the RAG2 and γc promoters (0.5±0.5,

n=9 and 0.5±0.4, n=11 respectively), and proportional to the levels

of reconstitution In the SF-RAG2co treated mice, T cell responses

to mitogens, plasma immunoglobulin levels and T cell dependent

(tetanus toxoid) and independent (pneumococcal polysaccharide

vaccine) antibody responses were fully restored, but incomplete

in the other treatment groups We conclude that complete immune

restoration can be achieved in Rag2 -/- mice by use of the strong SF

promoter driving constitutive RAG2co expression and that further

development towards safe promoter cassettes is required for clinical

application

Beauty-Mediated Gene Transfer into Hematopoietic Cells

Kendra A Hyland,1 Erik R Olson,1 Nicola Philpott,2 Xianzheng

Zhou,3 R Scott McIvor.1

1 Discovery Genomics, Inc., Minneapolis, MN; 2 Medicine,

Rheumatic and Autoimmune Diseases, University of Minnesota,

Minneapolis, MN; 3 Pediatrics, University of Minnesota, Medical

School, Minneapolis, MN.

Primary immune defi ciencies comprise a group of inherited genetic

disorders caused by interruption of normal lymphoid development

X-linked severe combined immunodefi ciency (X-SCID) is one of the most common primary immunodefi ciencies, and is caused by

a mutation in the gene encoding the common gamma chain (γc)

of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 In the absence of γc protein, cytokine signaling via the JAK-STAT pathway

is interrupted, thereby blocking normal lymphoid development

Clinical trials employing retroviral vectors for ex vivo treatment of

X-SCID have resulted in complete restoration of patient immunity However, serious adverse events have emerged, highlighting the need for development of alternate and less genotoxic approaches

for therapeutic gene transfer The Sleeping Beauty (SB) transposon

system mediates stable integration of sequences into mammalian chromosomes, supporting long term expression of both reporter and therapeutic genes Advantages of the SB system include ease

of manufacture, reduced immunogenicity in comparison with viral vectors, and a more random and potentially less genotoxic integrant profi le in comparison with retroviral and lentiviral vectors We fi rst demonstrated successful transposon-mediated expression of γc protein and restoration of JAK-STAT signaling in a T-cell line, lacking the

γc gene Introduction of plasmid DNA by electroporation into mouse hematopoietic stem cells (HSC) is not currently feasible, so we will deliver the SB system to mouse HSC using a non-integrating lentiviral vector system After transplantation of transduced HSC in γc-defi cient mice, we will assay for correction of lymphoid development by measuring the presence of B, T, and NK cells as well as restoration

of normal immunity Transposition will be verifi ed by recovery and molecular characterization of transposon integrants and fl anking chromosomal sequence Results from these studies will provide a platform for establishment of SB-mediated transposition of HSC in the treatment of human X-linked SCID

Wiskott Aldrich Syndrome Gene Therapy Using Insulated Lentiviral Vectors

Rachel M Koldej,1 Gael Carney,1 Matthew Wielgosz,1 Satyanarayana Pondugula,1 Arthur W Nienhuis.1

1 Hematology, St Jude Children’s Research Hospital, Memphis, TN.

Wiskott Aldrich Syndrome (WAS) is a severe disease which affects virtually all lineages of the hematopoietic system Thrombocytopenia, eczyma and recurrent or persistent infections are common disease manifestations Patients with a matched sibling donor can be cured

by bone marrow transplantation but transplantation from alternative donors leads to a poorer outcome Development of effective lentiviral vector mediated gene therapy for WAS would provide alternative curative therapy Our efforts to develop a safe and effective self-inactivating lentiviral vector for gene therapy of WAS have focused

on the evaluation of vectors with an insulator element in the integrated long terminal repeats (LTRs) and various internal promoters driving the WAS protein (WASp) cDNA The ideal promoter for WAS gene therapy would achieve physiological expression levels at the majority of integration sites in all relevant blood lineages, particularly T-cells, B-cells, monocytes and megakaryocytes Several promoters including a γ-retroviral LTR (MND), a short fragment (240 bp) from the elongation factor 1-α (EF1α) promoter and various length fragments of the proximal WAS gene promoter are being evaluated These promoters were cloned into non-insulated, 400bp or 650bp chicken HS4 insulated lentiviral vectors and examined in mouse bone marrow transplant studies using GFP as a surrogate marker

for hWASp expression Lineage negative B6.SJL-Ptprc a Pepc b/ BoyJ mouse bone marrow was prestimulated for 28 hours prior to transduction on retronectin coated plates for 21 hours with virus at

an MOI of 20 after which the transduced cells were administered

via tail vein to irradiated recipient C57BL/6J mice The mice were

monitored with periodic eyebleeds and sacrifi ced 22 weeks post transplant In peripheral blood all mice exhibited equal levels of

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy

S100

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I

engraftment (>80%) with percent GFP positive ranging from

40-100% in CD45.1 positive white blood cells The presence of the

400bp insulator increased relative GFP expression in lymphoid

lineages when combined with the short (500bp) WAS promoter

fragment but not the full length (1.6kb) WAS promoter or the EF1α

promoter Platelet counts stabilized at 600 to 1,200 x 103/ul and GFP

expression, when normalized to bone marrow copy number, showed

that the presence of the 400bp insulator increased expression in the

platelets independent of the promoter utilized In bone marrow, the

400bp insulator decreased GFP expression from the EF1α promoter

in every lineage examined In contrast, expression from either the

short or full length WAS promoter was not affected Secondary

transplants are ongoing In vitro studies in WAS patient peripheral

blood mononuclear cells using insulated hWASp expression vectors

have demonstrated that while all the promoters examined will express

to wild-type levels in transduced cells, the MND promoter gives a

higher proportion of cells expressing at this level

Expression after Hematopoietic Stem Cell Gene

Transfer in a Large Animal Model

Aravind Ramakrishnan,1,2 Beverly Torok-Stord,1 Mortimer Poncz,3

Nathaniel P Williams,1 Christina Ironside,1 Hans-Peter Kiem,1,2

Brian C Beard.1

1 Clinical Research Division, Fred Hutchinson Cancer Research

Center, Seattle, WA; 2 Department of Medicine, University of

Washington, Seattle, WA; 3 Pediatric Hematology, The Children’s

Hospital of Philadelphia, Philadelphia, PA.

Efficacy of gene-modified hematopoietic stem cell (HCS)

transplantation for the treatment of patients with single allele genetic

disease has unequivocally been demonstrated for adenosine deaminase

deficiency severe combined immunodeficiency (ADA-SCID),

X-linked severe combined immunodefi ciency (SCID-X1), chronic

granulomatous disease (CGD), adrenoleukodystrophy, (ALD), and

Wiskott-Aldrich Syndrome (WAS) For a subset of these patients

proto-oncogene activation as a result of insertional mutagenesis have

resulted in both clonal dominance and frank leukemia The strong

promoter enhancer elements in the long terminal repeats (LTR) were

likely responsible for the adverse events seen in these patients In

order to maintain the effi cacy of these pioneering clinical trials while

improving the safety for future patients a promising strategy is to

eliminate the LTR promoter and enhancers and instead use inducible

or lineage-directed promoters In mouse and human cells, the platelet

factor 4 (PF4) promoter is largely megakaryocyte lineage-specifi c

and has been used to study megakaryopoiesis We hypothesized

that the PF4 promoter could potentially be used as a tool to target

transgene expression specifi cally to the megakaryocytic lineage In

theory, this could be used to correct underlying genetic defects that

affect platelets or utilize platelets as delivery vehicles to deposit

specifi c gene products at sites of thrombosis/injury To effectively

test hematopoietic lineage expression of the PF4 promoter in a large

animal, we used the clinically relevant dog model Specifi cally,

autologous CD34-selected bone marrow cells were transduced with

a VSVG-pseudotyped HIV-derived self-inactivating (SIN) lentivirus

vector with PF4 expressing green fl uorescent protein (GFP) using

a 24-hour protocol at a multiplicity of infection of ∼10 Following

ablative conditioning ∼1.0E+06 cells/kg were infused and the dog

displayed normal gene-modifi ed hematopoietic engraftment kinetics

At the most recent follow-up, 77 days after transplantation, the dog

has stable in vivo gene marking of ∼5.4% in granulocytes, ∼2.4% in

red blood cells, and ∼19.0% in platelets Importantly, compared to

historical controls at similar time points for dogs that received cells

gene-modifi ed with HIV-derived SIN lentivirus vectors expressing

GFP from a constitutive promoter (phosphoglycerate kinase-PGK)

the fold difference in mean fl uorescence intensity of GFP+ relative

to GFP- gene marked granulocytes was 34.9 (PGK) compared to 12.2 (PF4) and gene marked platelets was 12.9 (PGK) compared to 72.1 (PF4) These data shows that the PF4 promoter is preferentially active in platelet lineages and far less active in myeloid lineages and have important implications for improving the safety and effi cacy of gene therapy strategies by limiting transgene expression primarily to affected subsets or specifi cally for this application, sites of thrombosis/ injury

Busulfex 8 mg/kg or 200 cGy TBI Is Equivalent

in Gene Therapy of Canine Leukocyte Adhesion Defi ciency Using Foamy Viral Vectors

Thomas R Bauer, Jr.,1 Erik M Olson,2 Laura M Tuschong,1 Tanya

H Burkholder,3 David W Russell,2 Dennis D Hickstein.1

1 Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD;

2 Department of Hematology, University of Washington, Seattle, WA; 3 Division of Veterinary Service, Offi ce of Research Services, National Institutes of Health, Bethesda, MD.

The optimal non-myeloablative regimen for hematopoietic stem cell gene therapy for treatment of phagocytic diseases remains unclear

In particular, the use of low doses of ionizing radiation, although widely use in reduced intensity allogeneic hematopoietic conditioning regimens, has not been used in human gene therapy clinical trials To address this issue, we compared the results with non-myeloablative conditioning with Busulfex 8 mg/kg to conditioning with 200 cGy total body irradiation (TBI) in the engraftment and chimerism of foamy viral (FV) vector transduced CD34+ cells in animals with canine leukocyte adhesion defi ciency (CLAD) Similar to children with LAD-1, dogs with CLAD suffer from recurrent bacterial infections and early death within the fi rst six months of life due to infections resulting from the absence of the CD18 leukocyte integrin

on the neutrophil surface and the resultant lack of adherence and migration of neutrophils lacking CD18 Initially, four CLAD pups were treated using a non-myeloablative conditioning regimen of 200-cGy total body irradiation (TBI) administered the day before receiving genetically-corrected cells The four dogs survived for four years,

at which time the study was completed Of note, CD18+ neutrophil levels ranged from 2.9 to 3.9% in the peripheral blood, and CD18+ T-cell levels ranged from 10.7 to 22.9% at one-year post-transplant

In the companion study four CLAD pups received a reduced intensity conditioning regimen with Busulfex 8 mg/kg given IV two days prior

to infusion of genetically-corrected cells All four dogs survived more than 1 year, free of severe CLAD disease CD18+ neutrophil levels ranged from 1.8 to 4.5% and CD18+ T-cell levels ranged from 14.1 to 21.6% at one-year post-transplant Thus, comparison of the chimerism levels in the dogs in each group revealed little difference between the conditioning regimens at 1 year post-transplant The slight variation

in chimerism could be attributed to the corrected cell doses infused

No animal with either regimen became neutropenic nor required platelet infusions due to conditioning These results demonstrate the effi cacy of both conditioning regimens for achieving suffi cient levels of CD18+ cells to reverse the CLAD disease phenotype, and further show that Busulfex 8 mg/kg appears to represent an effective substitute for 200 cGy TBI as a conditioning regimen for the treatment

of myeloid disease such as LAD by hematopoietic stem cell gene therapy