1088 Toll Like Receptors Activation in the Innate and Adaptive Immune Response to Helper Dependent Adenoviral Vectors flow cytomctry analysis to study capsid and EGFP specific T cell responses Histolo[.]
Trang 1flow cytomctry analysis to study capsid and EGFP specific T cell
responses.Histologic analyses of EGFP expression,T cell
infiltra-tion, and pathology as well as molecular characterizations of vector
genomes in the livers were carried out Our data revealed that, at
day 8, efficient EGFP transduction was established in all livers
When compared to the results in mouse liver,ss vector transduced
macaque liver more efficiently but sc vector performed equivalently
in both species Virtually no clinical pathology, liver infiltration and
EGFP specific T cell responses were noticeable at day 8.However,
animals followed for 38 days demonstrated transient transaminase
elevations EGFP specific cytotoxic T cell response became evident
as early as week 2 in PBMCs, and secondary lymphoid and
non-lym-phoid tissues at the necropsy A complete loss of EGFP expression
and manifestation of CD8 infiltration were noticed in all 3 livers
harvested at day 38 Capsid T cell responses were undetectable in
all pre- and post- treatment PBMCs Analysis of vector gcnomes
in the livers suggested molecular pathways similar to what we
described in previous studies In summary, both ss and scAAV217
vector accomplished efficient but transient EGFP gene transfer in
macaque liver The EGFP expression in hepatocytes appeared to
be quickly terminated by the destructive T cell response directed
to the transgene, This contrasts with the results obtained in mice
where GFP expression is stable and transgene specific T cells arc
low to absent Our findings emphasize the possible negative impact
of transgene immunity on AAV vector mediated gene transfer to
primate liver
1087 Transient Blockade of Icas
Co-Stimulatory Pathways Induced Long-Term
Tolerance to Factor VIII Following Nonviral Gene
Transfer of Factor VIII into Hemophilia A Mice
Baowei Peng,' Peiqing Ye,' Bruce R Blazer.' Hans D Ochs,'
Carol H Miao.'
'Department ofPediatrics, ChildrensHospital and Regional
Medical Center and University ofWashington, Seattle, WA;
}Department ofPediatrics, University ofMinnesota, Minneapolis,
MN.
Approximately 2S-30% of hemophilia A patients produce
inhibi-tory antibodies in response to factor VIII (FVIII) protein replacement
therapy Potential gene therapy techniques used to treat hemophilia
A have also resulted in a significant humoral immune response to
FVIII in murine models.Nonviral gene transfer of a FVIII plasmid
into hemophilia mice induces strong humoral responses through
predominantly TH2 signals, representing a unique and useful model
for testing various immunomodulation strategies In this study, we
tested if transient blockade of the inducible costimulator
(ICOS)-ICOS ligand ((ICOS)-ICOSL) pathway can modulate the immune response
following gene therapy Two separate groups ofmice (n=S and n=11
per group,respectively) were subjected to administration of FVIII
plasmid via hydrodynamics-based tail-vein injection,and transient
immunosuppressive regimen using anti-ICOS monoclonal antibody
(mAb) (16 treatments in a 4 week period) None of the anti-ICOS
mAb treated mice developed inhibitory antibodies against FVIII
and all produced persistent, high-level FVIII (100-300nglml) for
at least ISO days (experimental period), whereas control FVIII
plasmid-treated mice all developed high-titer inhibitory antibodies
T cells isolated from spleen, blood,and lymph nodes of anti-I COS
mAb treated and untreated mice were further analyzed at different
time points The CD4+ T cells isolated from the spleen of
toler-ized mice did not proliferate in response to FVIII stimulation in
vitro, whereas cells from plasmid only-treated mice proliferated
significantly In the first couple of weeks post plasmid treatment,
induction of tolerance was associated with significantly increased
percentage (but not absolute numbers due to CD4+ T cell depletion)
ofCD4+CD2Sh i ghFOXP3+regulatory T cells in the peripheral blood,
Molecular Therapy Volume15 SupplementI ~ br 2007
C opyright © T he American So ci etyo r Gen eTherapy
spleen,and lymph nodes of tolerizcd mice compared to untreated and plasmid only-treated mice A decrease ofCD62L and CD4SRB expression and an increase of GITR expression distinguished the activated Treg cell subset observed in anti-ICOS mAb treated mice from Treg cells in mice receiving only FVIII plasmid Furthermore,
at 6 week time point and afterwards, the expansion of Treg cells was only observed in the lymph nodes of tolcrizcd mice but not in spleen and peripheral blood, suggesting that Treg cells prolifcr-ated selectively in the lymph nodes where FVIII antigen is being presented by APCs during the maintenance of tolerance Adoptive transfer ofTreg cells from tolerized mice can delay and/or reduce the production of inhibitors against FVIII in recipient hemophilia A mice treated with FVIII plasmid.Anti-ICOS mAb treatment has the potential for a novel immunomodulatory strategy to prevent the for-mation of inhibitory antibodies against FVIII following gene therapy Furthermore, we show that the CD4+CD2Shi g hFOXP3+regulatory
T cells play an important role in the induction and maintenance of long-term tolerance post anti-ICOS immunotherapy
1088 Toll-Like Receptors Activation in the Innate and Adaptive Immune Response to Helper Dependent Adenoviral Vectors
Vincenzo Cerullo,IMichael Seiler,'Daniel Wang; Viraj Mane,' Christian Clarke,IBrendan Lee.I,2
'Molecular and Human Genetics, Baylor College ofMedicine, Houston, TX; }Holl'ard Hughes Medical Institute, Baylor College ofMedicine, Houston , TX
Toll-like Receptors (TLRs) are innate receptors that sense mi-crobial products and trigger dendritic cell (DCs) maturation and cytokine production,thus bridging innate and adaptive immunity Cellular activation by most members of the TLR family involves
a signaling cascade that proceeds through myeloid differentiation primary response gene 88 (MyD88), interleukin-l (IL-I) recep-tor-activated kinase (IRAK) and tumour-necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), and culminates in the acti-vation of several transcription factors, including nuclear factor-kB (NF-kB) These transcription factors directly upregulate cytokine/ chemokine production Since the major obstacle to adenoviral vector gene delivery is the dose-dependent innate toxicity following the in-travenous administration, we sought to characterize it from a cellular base standpoint We first asked whetherTLRs are involved in the immune response following the intravenous injection of adenovirus vector To test that, we injected high dose (SxI012VP/Kg)of helper dependent adenoviral vector (HD-Ad) expressing a reporter gene into MyD88-/- mice and measured the eytokines and chemokines production 6 hours post-injection MyD88-/- mice showed a drastic reduction of proinflammatory molecules (IL6, RANTES,MCP-I, p<O.OO I) compared with wild type control mice, thus highlighting the important role that TLRs play in the innate toxicity to adenovi-ral vectors.We previously demonstrated that TLR9,an endosomal receptor, triggers part of the immune response to helper dependent vectors (I·[D-Ad).To test whether TLR 3 and 7 (two other intracel-lular TLRs) contribute to combinatorial signaling in response to HD-Ad, we injected Sx1O'2VP/Kg of HD-Ad in a mouse model carrying the Unc93bl mutation (known as "tripled" mutation, 3d) with impaired signaling of intracellular TLR3, 7 and 9; TLR9-/-mice were used as control No difference in cytokines production was found between the group of mice with 3d mutation and the TLR9-/- group suggesting that TLR9 is the primary intracellular TLR responsible for the innate immune response to I·ID-Ad.We then investigated the role of cell surface TLRs in similar fashion
We found that TLR2-/- mice exhibit a reduced proinflammatory cytokines production after intravenous administration of high dose ofHD-Ad (SxlO '2VP/Kg) While TLR9 recognizes DNA, surface receptors likely recognized portion of the Ad capsid To test this,
S41S
Trang 2we assayed response of primary macrophages from C57BLl6 mice
to Ad capsid components We found that I'ID-Ad DNA together
with penton proteins had the highest stimulatory effect In
conclu-sion, adenoviral vectors activate immunity through TLR-dependent
pathways leading to thc production of pro-inflammatory cytokines
and chemokines This is mediated cooperatively by cell surface and
endosomal receptors, TLR2 and TLR9,respectively Moreove,they
are dependent on a common TLR adaptorMyD88-1- By modulating
signaling via these receptors, we may be able to affect the innate
and adaptive immune responses to HDY in vivo
1089 An In Vitro Model of CD8+ T Cell-Mediated
Etiena Basner-Tschakarjan,' Daniel J Hui; Shangzhen Zhou;
Federico Mingozzi,' Katherine A High.P
I Hematology , The ChildrensHospital ofPhiladelphia,
Philadel-phia PA; ]Howard Hughes Medical Institute, PhiladelPhiladel-phia PA.
Adeno-associatedvirus (AAY) vectors have been successfully
used as gene therapy vectors, mainly to correct monogenic
disor-ders,where an advantage over other delivery systems is the mild
immune response evoked in animal models,allowing prolonged
expression of the transgene However,in a recent clinical trial of
AAY-mediated hepatic gene transfer for hemophilia B a CD8+ T
cell response to the vector capsid was documented We hypothesize
that this CD8+ T cell response eventually led to the destruction of
transduced hepatocytes with loss of transgene expression and an
accompanying reversible rise in serum transaminases In order to
further elucidate the underlying immune response as well as to try
to identify subjects prone to react to AAY capsid before treatment,
we established an in vitro system by which AAY capsid
epitope-expanded peripheral blood mononuclear cells (PBMC) can be tested
for their cytotoxic capacity towards virus-transduced target cells
We were able to identify several major T cell epitopes from normal
donors and from subjects enrolled in AAY gene therapy trialsusing
ELISpot assays and a peptide library derived from theAAY capsid
YP I protein sequence.Subsequently we used these peptide epitopes
to expand CD8+ T cells in vitro and also used them in a
cytotox-icity (CTL) assay with an HLA-matched hepatocyte cell line (an
immortalised primary human hepatocyte cell line kindly provided
by Arvind H Patel, University of Oxford, Oxford, UK) as target
cells Target cells were transduced with an AAY2 virus prior to the
assay and cytotoxicity could be shown as soon as four hours after
treatment (20% specific lysis at an E:T ratio of 10:I) with higher
levels after 48 hours (30% specific lysis) Peptide loading of the
hepatocytes with the relevant HLA-matched MHC class I epitope
led to constant high levels of cytotoxicity (30-40% specific lysis)
Our data suggest that human hepatocytes transduced in vitro with
AAY2 are able to process capsid antigen and present it in an MHC
I context to CD8+ T cells,recapitulating in vitro what we observed
in vivo in humans.Additional experiments with alternate serotypes
arc currently underway In conclusion we have established an in vitro
system for the expansion of human CD8+ T cells reactive to AAY2
capsid We have also established an in vitro cytotoxicity assay for
AAY2-transduccd human hepatocytes These results clearly
dem-onstrate that capsid-specific CD8+ T cells can lyse HLA-matched
AAY-transduced hepatocytes,providing strong supporting evidence
for our hypothesis that loss ofexpression and transaminitis observed
after hepatic artery infusion of AAY2-F.lX resulted from CD8+ T
cell-mediated lysis oftransdueed hepatoeytes The same system can
be used to test the performance of alternate serotypes This assay
system is particularly valuable since it has so far not been possible
to establish an animal model ofCD8+-mediated T cell destruction
ofAAY-transduced target cells
S416
GENE EXPRESSION IN ANIMAL MODEL SYSTEMS
1090 Prevention of Type I Diabetes Recurrence Following Islet Transplantation in Diabetic NOD Mice by Gene Therapy
Chaorui Tian,' Mohammed Javeed Ansari,'Jesus Paez-Cortez,' Jessamyn Bagley,' Mohamed Sayegh,' John Iacomini.'
'Transplantation Research Center; Brigham and Womens Hospi-tal and ChildrensHospital Boston, Boston, MA.
Introduction: We have recently shown that induction of mo-lecular chimerism by reconstitution of young NOD mice with autologous bone marrow cells transduced with retrovirus carrying genes encoding MHC class II~ chains from protective haplotypes can prevent occurrence of type I diabetes in NOD mice Here
we show that induction of molecular chimerism in mice that have spontaneously developed diabetes can be used to prevent recurrence
of type I diabetes following islet transplantation Methods: Bone marrow cells were harvested from 4-week old NOD mice treated with 5-fluorouracil (150mglkg) 7 days prior and then transduced with retrovirus carrying genes encoding either a protective MHC class II
~ chain fused to GFP (IAW-GFP) or GFP alone as a control Spon
-taneously diabetic NOD mice were lethally irradiated and recon-stituted the following day with eitherIA~d-G FP or GFP transduced bone marrow All mice were maintained normoglycemic with daily subcutaneous insulin injection Four weeks after reconstitution, all recipient mice were transplanted with 1000 NOD.scm islets under the kidney capsule Blood glucose levels were measured daily after islet transplantation Islet grafts were harvested upon hyperglyce-mia or 90 days after islet transplantation Results: All of the mice reconstituted with control GFPtransduccd bone marrow developed diabetes by 9 days after islet transplantation (Median onset time (MOT)=2 days) Immunohistochemical studies showed that there was massive CD3+T cell infiltration and no insulin storage in the islet grafts ofthese mice In contrast, 5 out of6 mice reconstituted with
protectiveIA~d-GFP transduced'bone remained normoglycemic for
90 days after islet transplantation (MOT= 90 days, n= 6, p<O.OOI) Histological studies showed that despite peri-islet infiltration of CD3+Tcells,insulin storage remained intact Interestingly,in these mice, we were able to detect significant numbers of Foxp.l" cells in the transplanted islets grafts Only lout of6 mice reconstituted with protective IAW-GFPtransduced bone marrow developed diabetes 42 days after islet transplantation.Conclusion: Induction of molecular chimerism can be used to prevent type 1 diabetes recurrence follow-ing islet transplantation in NOD miec with pre-existfollow-ing diabetes, Foxps" regulatory eclls may be involved in prevention ofdonor islet rejection by auto-reactive T cells in the recipients
1091 Induction of Molecular Chimerism in Rhesus Macaques Reconstituted with Autologous CD34+ Cells Transduced with the Gene Encoding the a(1-3)galactosyltranferase (aGT)
Lorenzo Benatuil,Robert Donahue, Aylin Bonifacino,Cynthia E Dunbar, John lacomini
'Transplantation ResearchCenter:Brigham and Womens Hos-pital and ChildrensHospital Boston , Harvard Medical School, Boston AlA; ]Hemat%gy Branch, NHBLI, NIH Bethesda, MD
Background: We have previously shown that genetic engineer-ing of autologous bone marrow stem cells to induce molecular chimerism can be used to establish B cell tolerance in mice In this study, we examined whether a similar approach could be used to induce B cell tolerance in Rhesus macaques,a preclinical animal model To this end,we investigated whether induction ofmolecular chimerism could be used to affect production of antibodies specific for the carbohydrate epitope Galal-3Gal~I-4GlcNAe-R (aGal)
Molecular Therapy Volume 15 Supplemen t I• \1 ,,)" 2007
C opyri ght © ' 111C A merican Societ y o f G ene Tllt'r.lP) '