1062 functional correction of t cells from wiskott aldrich syndrome patients by retroviral and lentiviral vector mediated gene transfer

1062 Functional Correction of T Cells from Wiskott Aldrich Syndrome Patients by Retroviral and Lentiviral Vector Mediated Gene Transfer Molecular Therapy �������� ��� ���� ���������������� �������� ��[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

STEM CELL GENE THERAPY FOR GENETIC DISEASES

1060 Functional Analysis of an Oncoretrovirus

Vector for Human Gamma-Globin in Mouse

Models of Beta Thalassemia

Tamon Nishino,1 Julie Tubb,1 Yumiko Nishino,1 George

Stamatoyannopoulos,1 David W Emery.1

1 Medicine / Medical Genetics, University of Washington, Seattle,

WA.

Many advances have been made over the past few years in the

development of recombinant virus vectors for human globin genes

designed to treat patients with beta-thalassemia and sickle cell disease

However, many of these advances have relied on the use of lentivirus

vectors We have recently described the development of an

oncoretrovirus vector for human gamma-globin that is resistant to

silencing position effects and expresses gamma-globin at 6 ±4% of

total endogenous alpha-globin in normal mice transplanted

long-term with transduced marrow [Blood 100:2012, 2002] In order to

determine whether this is a potentially therapeutic level of expression,

we performed studies in a mouse model for beta-thalassemia

intermedia Transduction and transplantation of bone marrow cells

from heterozygous Hbbth-3/+ donors into myeloablated syngeneic

recipients resulted in a modest improvement in both the RBC count

(3.8±0.1 x106/ul vs 2.6±1.1 for mock and 6.0±1.8 for wild type)

and total hemoglobin (6.7±0.2 g/dl vs 4.7±1.9 for mock and 12.2±3.3

for wild type) However, this effect was only seen early after

engraftment and in mice with greater than 25% gamma-expressing

RBC Likewise, studies with this vector in a mouse model for severe

beta-thalassemia major also showed only a partial therapeutic effect

In this case, transduction and transplantation of fetal liver cells from

Hbbth-3/Hbbth-3 homozygotes into congenic C57BL6 recipients

increased survival to 34±20 days (n=13), compared to only 16±7

days for the mock-transduced controls (p=0.006) However, the

level of gamma-globin provided by the optimized vector was not

sufficient to support long-term survival in this model In order to

determine the amount of gamma-globin necessary for a full

therapeutic correction in these mouse models of beta-thalassemia,

we crossed Hbbth-3/+ heterozygotes with a transgenic line expressing

gamma-globin at a level 2-3 fold higher (10-15% of total endogenous

alpha-globin) than that of the optimized oncoretrovirus vector

Compound heterozygotes exhibited a significant improvement in

both RBC counts (11.4±0.3 x106/ul vs 10.5 ±0.5 for Hbbth-3/+ and

11.6±0.7 for w.t.) and total hemoglobin (15.0±0.4 g/dl vs 13.1 ±0.6

for Hbbth-3/+ and 17.9±1.5 for w.t.), suggesting a nearly full correction

However, the level of gamma-globin produced by the transgene was

insufficient to rescue the Hbbth-3/Hbbth-3 embryonic lethality

phenotype These studies are currently being repeated with a

transgenic line expressing gamma-globin at twice the level of the

original line Taken together these studies demonstrate that an

optimized oncoretrovirus vector for human gamma-globin is capable

of functionally improving the phenotypes of two mouse models of

beta-thalassemia, but that full correction of these phenotypes will

require further improvement in the level of vector expression

1061 Reconstitution of the IL-12 Signaling Pathway in IL-12 Receptor beta-1 (IL-12R βββββ1) KO

Mice upon Transplantation of Retrovirally Transduced Hematopoietic Stem Cells

Marita Bosticardo,1 Francesco Novelli,2 Jean-Laurent Casanova,3 Fabio Candotti.1

1 Genetics and Molecular Biology Branch, NHGRI/NIH, Bethesda,

MD, United States; 2 Centro Oncologico Ematologico Subalpino, Centro Ricerche Medicina Sperimentale, Ospedale San Giovanni Battista, Torino, Italy; 3 Unité Clinique d’Immunologie et d’Hématologie Pédiatriques, Hơpital Necker-Enfants Malades, Paris, France.

The functionality of the IL-12-mediated signaling pathway is critical for the elimination of intracellular pathogens Patients carrying genetic defects in the 12 signaling pathway (12 p40 or IL-12Rβ1) indeed show increased susceptibility to weakly pathogenic strains of mycobacteria and salmonella Infections can be treated by administration of IFN-γ and antibiotics; however, reversion of patients’ susceptibility by corrective gene transfer could be beneficial We were able to restore the IL-12-mediated signaling pathway in PHA-activated T cells blasts of IL-12Rβ1 deficient patients through retroviral-mediated gene correction In addition, to test the feasibility and safety of retroviral-mediated gene correction

in vivo, we established a murine model of gene therapy in IL-12R β1-deficient mice Lineage-negative cells isolated from bone marrow of IL-12Rβ1-deficient mice were cultured for 5 days and transduced using a retroviral vector carrying the murine IL-12Rβ1 cDNA This resulted in a high percentage of IL-12Rβ1+ cells (50-100%) Gene-corrected bone marrow cells were transplanted into lethally irradiated recipient mice, which were then analyzed at 8-16 weeks after the transplant Treated mice did not show adverse effects upon gene

lymphocytes, spleen and thymus from the majority of treated mice Moreover, splenocytes isolated from mice transplanted with gene-corrected bone marrow cells acquired the ability to respond to

IL-12, as demonstrated by the production of IFN-γ, which is completely impaired in IL-12Rβ1 KO mice However, membrane expression of the IL-12Rβ1 chain as well as the production of IFN-γ in response

to IL-12 where significantly lower than those detected in wild type control mice In vivo experiments of IFN-γ production and challenge with intracellular pathogens are planned to establish if the degree of gene correction reached in our experimental model is sufficient to overcome their immunodeficiency

1062 Functional Correction of T Cells from Wiskott-Aldrich Syndrome Patients by Retroviral and Lentiviral Vector-Mediated Gene Transfer

Lọc Dupré,1 Sara Trifari,1 Francesco Marangoni,1 Antonia Follenzi,2 Antonio Bernad,3 Silvana Martino,4 Shigeru Tsuchiya,5 Luigi Naldini,1,2 Claudio Bordignon,1 Alessandro Aiuti,1 Maria-Grazia Roncarolo.1

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2 IRCC, University of Turin, Turin, Italy; 3 National Center for Biotechnology, Autonomous University of Madrid, Madrid, Spain; 4 Department of Pediatrics, University of Turin, Turin, Italy; 5 Department of Pediatric Oncology, Tohoku University, Sendai, Japan.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by infections, severe hemorrhage, and lymphomas resulting in a median survival below the age of 20 WAS is caused by mutations in the gene encoding the WAS protein (WASP) which is expressed in hematopoietic cells and is crucial for actin cytoskeleton organization Since limited success has been reported with bone marrow transplantation, gene therapy could

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright ®The American Society of Gene Therapy

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STEM CELL GENE THERAPY FOR GENETIC DISEASES

represent an alternative treatment for this disease Defective T

lymphocytes play a central role in the pathogenesis of WAS and we

demonstrated that WASP is required for the assembly of the

immunological synapse and for optimal T-cell activation In this

study, we compared the efficiency of different MMLV-based

oncoretroviral and HIV-based lentiviral vectors in transferring the

WASP gene into T lymphocytes to restore normal immune functions

Stable packaging cell lines designed to produce MMLV-based

oncoretroviral vectors were associated with low virus titers and

infectivity, which correlated with high WASP expression These

low titers could be overcome by transient transfection and

production, which resulted in higher titers and higher transduction

efficiency in patients T cells The use of transiently produced

HIV-based lentiviral vectors led to higher transduction rates, compared

to oncoretroviral vectors All vectors tested, including oncoretroviral

vectors containing the WASP cDNA either under the LTR or the

internal SV-40 promoter and lentiviral vectors containing the WASP

cDNA either under the PGK promoter or the autologous WASP

promoter, led to normal levels of WASP expression in the transduced

T cells Correction of the functional defects, including proliferation

and IL-2 production, was achieved in transduced patients T cells

Functional correction of transduced T cells was further proven by

normal clustering of the lipid raft marker GM1 upon TCR activation,

thus indicating normal immunological synapse assembly These

results show that WASP-encoding retroviral and lentiviral vectors

can lead to functional restoration of WAS T cells, providing the

basis for the development of further gene therapy approaches for

WAS patients using either T cells or hematopoietic stem cells as

target cells

1063 HIV Mediated Expression of Bruton´s

Tyrosine Kinase in Hematopoietic Stem Cells

Promotes B Cells Development but Not Restore

Immunoglobulin Production in X-Linked

Immunodeficient Mice

Hiroko Tanabe,1,2 Koichi Miyake,1 Takashi Shimada.1

1 Deaprtment of Biochemistry and Molecular Biology, Nippon

Medical School, Tokyo, Japan; 2 Department of Internal Medicine

1, Nippon Medical School, Tokyo, Japan.

X-linked agammaglobulinemia (XLA) is characterized by profound

hypogammaglobulinemia affecting all isotypes, an absence of

antigen-specific antibodies, and less than 1% of the normal number of B

cells Mutations of the Bruton´s tyrosine kinase (Btk) gene have

been implicated in the pathogenesis of human XLA A specific

mutation was also found in the mouse Btk (mBtk) gene in a

spontaneously occurring mouse model of immunodeficiency, murine

X-linked immunodeficiency (Xid) Although the clinical picture of

the mouse model is mild compared to that of XLA patients, Xid

mice should act as a useful model to evaluate new strategies for

treatment of patients with XLA As a step toward gene therapy for

patients with XLA, we constructed a VSV-pseudotyped HIV vector

containig the human Btk (hBtk) gene under the control of the internal

murine stem cell virus (MSCV) promoter Freshly isolated and

magnetically selected hematopoietic stem cells (HSCs) from Xid

mice (5-10 weeks) were transduced with the HIV vector without

cytokine stimulation The vector sequence was detected in more

than 90 % of colony-forming units in vitro by PCR Transduced

HSCs (1 - 3 x 105) were injected into irradiated adult Xid mice (4

weeks old) through the tail vein All mice were sacrificed 30 weeks

post-transplantation for examination of immunological function The

HIV vector sequence and hBtk mRNA were detected in bone marrow,

spleen and peripheral blood The number of differentiated B cells

(IgMlowIgDhigh) was increased in treated animals However, there

was little or no restoration of serum immunoglobulin concentrations

and antibody response to NP-Ficoll challenge (T-cell-independent

type II antigen) We also Similarly, the immunological reconstitution was not achieved even if the Xid mice was treated by stem cell gene therapy within 48 hours after birth To elucidate the reason why gene transfer of the Btk gene into HSCs was inadequate, we generated chimeric mice that received a mixture of 10% normal HSCs from CBA/J mice and 90% mutant-HSCs from Xid mice after lethal irradiation Normalization of IgM and IgG3 concentrations was confirmed 7 weeks after transplantation, indicating that partial reconstitution with normal bone marrow cells is sufficient for phenotypic correction of Xid mice The ratio of human Btk+B cells

in total B cells in Xid mice treated by HIV mediated gene therapy was 60 % in bone marrow and peripheral blood, but markedly decreased to 5 % in spleen, suggesting that bone marrow cells expressing Btk may have selective growth disadvantage during B cell development In contrast, the ratio in mice treated by partial bone marrow transplantation was 25 % in bone marrow, but increased

to 60 % in spleen These results indicate that HIV mediated expression

of Btk in bone marrow stem cells promotes B cell development, but not sufficient to restore immunoglobulin production in Xid mice Other factors in addition to Btk may be required for normal B lymphopoiesis

1064 Investigation into the Possible Use of HoxB4 in Order To Enhance Reconstitution of Gene Modified Haemopoietic Stem Cells Following Bone Marrow Transplantation

Michael D Milsom,1 Lorna B Woolford,1 Dorothy Gagen,1 Rachel Duxbury,1 Claire M Heyworth,2 Geoff Margison,3 Leslie

J Fairbairn.1

1 Gene Therapy Group, Paterson Institute for Cancer Research, Manchester, United Kingdom; 2 Experimental Haematology Group, Paterson Institute for Cancer Research, Manchester, United Kingdom; 3 Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester, United Kingdom.

We have previously described the construction of a novel tricistronic retroviral vector, which facilitates the high level co-expression of HoxB4 and the O6-alkylguanine-DNA alkyltransferase P140K mutant (ATaseP140K), along with the selective marker gene enhanced green fluorescent protein (eGFP) In order to assess the relative selective advantage conferred upon haemopoietic stem cells

(HSCs) transduced with this vector, we have devised a novel in vivo

competitive repopulation assay that enables a direct comparison of the performance of two retroviral vectors within the same mouse Using this assay, we find that the tricistronic vector described above

is able to confer a profound post-engraftment selective advantage over bone marrow cells transduced with a vector containing only ATaseP140K and eGFP Furthermore, following treatment with O 6-benzylguanine followed by temozolomide, HSCs containing this tricistronic vector expanded to provide virtually all of the peripheral blood cells in the recipient mice Clearly this not only demonstrates

the potential use of HoxB4 to facilitate the in vivo selection of

HSCs containing a second therapeutic gene, but also reveals the possibility of using HoxB4/ATaseP140K co-expression to enable a further enhanced expansion of gene modified bone marrow cells

In an effort to evaluate the safety implications of using HoxB4 in any future gene therapy protocol, we utilised the FDCP-mix haemopoietic progenitor cell line as a model system in an attempt to elucidate the mechanism of action of HoxB4 We found that retroviral mediated constitutive expression of HoxB4 lead to a block/delay in myeloid differentiation as measured by analysis of both cellular morphology and colony forming ability following a differentiation stimulus We are currently investigating the nature of this perturbation of differentiation in order to determine if this biological phenomenon significantly contributes to the observed improvement

in haemopoietic reconstitution described above