689 AAV5 Mediated CD39/CD73 Expression Effects of Adenosine and Regulation of Its Receptors in Primary Cells From Healthy Donors and Rheumatoid Arthritis Patients Molecular Therapy Volume 22, Suppleme[.]
Trang 1Molecular Therapy Volume 22, Supplement 1, May 2014
Copyright © The American Society of Gene & Cell Therapy S266
HEMATOLOGIC & IMMUNOLOGIC DISEASES II
growth of erythroid colonies at low erythropoietin concentration
Erythropoietin dependence was maintained since colonies did not
grow in the presence of anti-erythropoietin blocking antibodies The
assessments of the effi cacy and the safety of the new LentiGlobin
BB305-tEpoR vector in mice and cynomolgus monkey transplant
models after reduced conditioning are ongoing
Normal and Leukemic Stem Cells
Chen Glait-Santar,1 Xingmin Feng,1 Jichun Chen,1 Ronan
Desmond,1 Benjamin Mizukawa,2 James C Mulloy,2 Neal S
Young,1 Cynthia E Dunbar.1
1 Hematology Branch, National Heart, Lung, and Blood
Institute,National Institutes of Health, Bethesda, MD; 2 Cancer and
Blood Disease Institute, Cincinnati Children’s Hospital Medical
Center, Cincinnati, OH.
Hematopoietic stem cells (HSCs) reside in a specialized niche that
regulates their proliferative capacity and their fate The functions of
HSCs are regulated by both intrinsic signals from the cells themselves
and from surrounding bone microenvironment These signals enable
tight regulation of stem cell pool size together with steady-state
and injury-responsive hematopoiesis There is increasing evidence
for similar roles of marrow niches in impacting on the behavior of
leukemic stem cells, for instance protecting LSCs from cytotoxic
drugs, however whether normal HSCs and leukemic cells reside in
or could compete for the same marrow niche is unknown We used
a murine competitive repopulation model and MLL-AF9 myeloid
leukemia to investigate whether normal and leukemic stem cells
compete for the same niches Mice were transplanted with progenitor
cells transduced with an MLL-AF9 vector expressing GFP in a murine
syngeneic model
We co-transplanted a fi xed number of MLL-AF9 cells generated
by transduction of primary murine progenitors with MLL-AF9/GFP
vector along with increasing numbers of normal whole bone marrow
(WBM) cells, derived from syngeneic or dsRed transgenic mice, into
irradiated recipient mice WBM cell doses were above the threshold
permitting full hematopoietic recovery from irradiation Control
groups received MLL-AF9 and normal WBM cells only Time to a
moribund state, spleen weight and marrow cellularity at sacrifi ce as
well as leukemic infi ltration were assessed Interestingly, survival was
increased and leukemic progression delayed proportional to increasing
doses of normal WBM cells, in three independent experiments
Confocal microscopy confi rmed co-localization of engrafting
WBM and MLL-AF9 progenitors in proximity with marrow
microenvironment MLL-AF9 cells isolated from transplanted
recipient marrows were cycling more actively in the presence of
increasing doses of WBM, suggesting competitive exclusion from the
protective marrow niche Osteopontin (OPN) is a cytokine produced
by niche components and demonstrated to promote anchoring and quiescence of normal and acute lymphoblastic leukemia stem cells Quantifi cation of OPN expression in BM supernatants collected from femurs pointed to higher OPN levels correlated with lower cycling of recovered ALL-MF9 cells measured at the time mice were moribund The competitive model suggests that normal and myeloid leukemic stem cells compete for the same functional niche, thus niche manipulation could impact on response to anti-leukemic therapies, and the extent of normal hematopoiesis could impact on outcomes, informing approaches to cell dose in the context of stem cell transplantation
Effects of Adenosine and Regulation of Its Receptors in Primary Cells From Healthy Donors and Rheumatoid Arthritis Patients
Susanne A Snoek,1,2 Ciska Braam,1,2 Ronne Brunekreef,1,2 Lisa van Baarsen,2 Niels Broekstra,1,2 Jan van Ittersum,1,2 Paul P Tak,1,2 Margriet J Vervoordeldonk,1,2 Jonathan D Finn.1,2
1 Arthrogen B.V., Amsterdam, Netherlands; 2 Academic Medical Center, Amsterdam, Netherlands.
The conversion of extracellular ATP to adenosine is an important mechanism of immune suppression by Tregs, and this is done by expression of ENTPD1 (CD39) and 5NTE1 (CD73) CD39 is a membrane bound ATPase that converts extracellular ATP and ADP
to AMP, whereas CD73 is a membrane bound ecto-nucleotidase that converts AMP to adenosine We have found evidence suggesting that the balance between pro-infl ammatory ATP and anti-infl ammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis (RA) patients Therefore, we aim to ameliorate this balance by AAV mediated expression of CD39 and CD73 Here we present data investigating the effects of adenosine and expression of its 4 receptors (ADORA-1, -2A, -2B, -3) in two of the major cell types involved in the pathogenesis of RA, monocytes and fi broblast-like synoviocytes (FLS) Also, we show that AAV-mediated expression of CD39 and CD73 is effi cacious in primary cell based in vitro infl ammation assays Primary monocytes were isolated from healthy donor blood and FLS from synovial biopsies from RA patients These cell types showed a distinct ADORA expression profi le, with ADORA1/2B predominant on FLS, and ADORA3/2B predominant on monocytes Interestingly, both cell types showed a striking upregulation of ADORA2A, but not of ADORA-1, -2B or -3, following stimulation with LPS (monocytes: 102-fold) or TNF/IL-1β (FLS: 432-fold) When monocytes were stimulated with LPS, adenosine signifi cantly reduced pro-infl ammatory cytokine production, resulting in a >70% decrease
in CCL2 and TNF In similar studies using FLS, adenosine was found
to have a much more modest effect, showing only a <20% decrease in CCL2 and no change in IL-6 production in TNF/IL1β stimulated cells
As ADORA2A signaling activates anti-infl ammatory pathways, this was surprising given the 432-fold increase in ADORA2A expression seen after TNF/IL-1β stimulation
As we wanted to investigate the use of CD39 and CD73 as a novel therapeutic strategy for the treatment of RA, we designed
an in vitro infl ammation assay that mimics the predicted in vivo scenario Primary FLS cells (n=6) were transduced with an AAV5 vector expressing CD39 and CD73 After a 5 day incubation, LPS activated THP1 cells (monocyte cell line) were added in the presence
of ATP After overnight incubation, pro-infl ammatory cytokines were measured in the supernatant We observed a signifi cant inhibition of CCL2 (68% ± 4.5%), TNF (88% ± 3%), and IL-1β (74% ± 6.4%) (p<0.00099)
Together these data show that the primary cells involved in the pathogenesis of RA dynamically change their ADORA expression following infl ammatory stimulus, and that this change is refl ected in the cells response to adenosine Further, we show that AAV mediated
Trang 2Molecular Therapy Volume 22, Supplement 1, May 2014
HEMATOLOGIC & IMMUNOLOGIC DISEASES II
expression of CD39 and CD73 from FLS is effective in reducing
pro-infl ammatory cytokine production in a THP1 based in vitro
infl ammation assay Thus, the modulation of ATP:adenosine levels
by CD39 and CD73 may be an effective approach for the treatment
of infl ammatory disease such as RA
CD34+ Cells in Hypoxic Conditions Does Not
Improve Lentiviral Transduction Effi ciency in
Long-Term Repopulating Hematopoietic Stem
Cells
Ayla Cash,1 Zanetta Chang,1 Brian Ichwan,1 Jean-Yves Metais,1
Cynthia E Dunbar,1 Andre Larochelle.1
1 NHLBI, Hematology Branch, National Institutes of Health,
Bethesda, MD.
Mobilized CD34+ cells from 4 rhesus macaques were divided
in two equal fractions for transduction with SIN-lentiviral vectors
expressing GFP or TdTomato In 3 animals, cells were transduced
with GFP vectors in hypoxia and TdTomato vectors in normoxia
These conditions were reversed in one animal Cells from both
fractions were combined and injected intravenously in lethally
irradiated rhesus macaques The numbers of total live cells, CD34+
cells and CD34+CD38- cells after 2 days of transduction were not
signifi cantly different compared to baseline for both hypoxic and
normoxic conditions After 2 days of transduction, the total number
of colony-forming cells increased 1.4-fold under both hypoxic and
normoxic conditions (p=0.69) compared to freshly isolated CD34+
cells At baseline, the majority (50.6 ± 4.4%) of CD34+ cells were
in the G0 phase of the cell cycle After 2 days of transduction
under hypoxic or normoxic conditions, the percentages of cells
in G0 were 19.9 ± 8.8% and 18.6 ± 5.9%, respectively (p=0.82)
The pO2 also had no impact on cell death (18.2 ± 5.3% in hypoxia
and 16.8 ± 4.8% in normoxia, p=0.69) or on the percentages of
apoptotic cells (11.1 ± 6.7% in hypoxia and 8.4 ± 2.3% in normoxia,
p=0.47) When considering the bulk of CD34+ cells, transduction
effi ciencies were similar in normoxia (70.7 ± 11.7%) and hypoxia
(68.0 ± 16.3%) (p=0.80) The contribution to long-term in vivo
gene marking measured by fl ow cytometry and quantitative PCR in
peripheral blood granulocytes up to 21 months after transplantation
was comparable between cells transduced in 21% pO2 (9.0 ± 4.5%)
and 5% pO2 conditions (8.4 ± 4.9%) (p=0.87) Consistent with
these data, GFP and TdTomato-labeled hematopoietic cells were
equally visualized, using a confocal/2-photon hybrid microscopy
approach, in BM biopsy specimens collected at various time points
after transplantation Given recent evidence suggesting that lower
oxygen concentrations (£1.5%) may be required to stabilize
HIF-1α, a key sensor of hypoxic conditions, transduction was performed
under extreme hypoxic conditions (1% O2) in one animal While the
overall viability of the bulk CD34+ cells after 2 days of transduction
under extreme hypoxia was comparable to cells cultured in normoxia,
their contribution to long-term in vivo marking was negligible (0.3%)
compared to cells transduced under normoxic conditions (2.4%),
suggesting toxicity of very low oxygen levels on HSCs Overall,
when current methodologies used for the genetic manipulation of
HSCs for gene therapy applications were performed under hypoxic
conditions, susceptibility to lentiviral transduction of CD34+ cells was
not ameliorated compared to cells cultured in 21% O2 conditions in
the rhesus macaque transplantation model The short culture times (2
days) used in recent lentivirus-based gene therapy clinical trials and
replicated in this study may not be suffi cient to impact the phenotype
of long-term repopulating HSCs and do not warrant incorporation of
hypoxia in current gene therapy protocols
Products to Treat Hereditary Pulmonary Alveolar Proteinosis
Kevin A Link,1 Takuji Suzuki,1 Brenna Carey,1 Paritha Arumugam,1 Punam Malik,1 Bruce Trapnell,1 Carolyn Lutzko.1
1 Cincinnati Childrens Hospital Medical Center, Cincinnati, OH.
Pulmonary Alveolar Proteinosis (PAP) is a group of rare lung disorders characterized by the inability of alveolar macrophages to breakdown and remove surfactant from the lungs Greater than 90%
of PAP results from impaired GM-CSF signaling hereditary PAP (hPAP) develops as a result of mutations in the genes that encode the GM-CSF receptor, CSF2RA and CSF2RB Using several mouse models, our group has established the foundation for both cell and gene therapy approaches as viable treatment options for hPAP (Suzuki
et al., in preparation) For example, direct installation of normal
or gene corrected macrophages into the lungs of mice with hPAP resulting from GM-CSFRB deletion have a sustained physiological correction of disease The current study is focused on the development
of conditions for the GMP manufacture of macrophages for clinical application Our approaches use: 1) expanded/differentiated CD34+ cells for pulmonary macrophage transplant (PMT) (allogeneic) and 2) expanded/differentiated autologous CD34+ cells that have been genetically modifi ed to express the normal gene In this study, CD34+ cells from human bone marrow or mobilized peripheral blood were analyzed for in vitro expansion and differentiation towards myeloid precursors and macrophages We compared 3 different GMP grade media formulations: X-VIVO10, Stemline, and SCGM Stemline outperformed the other medias with >5-fold expansion over X-VIVO10 and >2-fold over SCGM over 2 weeks Cytokine concentrations and timing were optimized to maximize expansion and differentiation ability All cells were expanded in KIT ligand, Flt3 ligand, GM-CSF, and IL-6 (K/F/GM/6) In protocol 1, M-CSF (M) was added after 2 weeks of expansion In protocol 2, cells were moved after one week of expansion into M-CSF and IL-1b (M/1b) containing media for differentiation Differentiation was identifi ed
by flow cytometry for CD14 and CD11b Differentiation was consistently higher at two weeks in M/1b media compared to K/F/ GM/6/M media Cell morphology evaluated on cytospins confi rmed enhanced differentiation in the M/1b medium Four BM samples of ~ 20ml were also obtained from hPAP patients with CSF2RA mutations and CD34+ cells were isolated A mean of 10x106 CD34+ cells were isolated with a >95% CD34+ purity and viability CD34+ cells were transduced with CSF2RA-GFP SIN lentiviral vector at 1x10^7 iu/ml with 25% GFP transduction effi ciency Transduced cells had increased expansion compared to mock with 16.6 fold and 3.3-fold expansion, respectively Expanded transduced cells were able to differentiate as evidenced by expression of CD11b (68%) and CD14 (63%) The cells had an increased phagocytosis of E coli beads (44-65%) compared
to control cells (22%) and restored GM-CSF signaling through Stat5 Together, these data demonstrate the feasibility of expanding CD34+ cells from normal or gene corrected hPAP patients, and highlight the potential of this approach as a treatment for hPAP Future studies will evaluate the in vivo potential of these cells to functionally correct hPAP in an immune defi cient mouse model of hPAP