254 AAV In Vivo Gene Transfer Suppresses Adaptive Immune Responses Against α 1 Antitrypsin evaluate possible inflammatory and cytotoxic side effects caused by overexpression of transactivators We hav[.]
Trang 1evaluate possible inflammatory and cytotoxic side effects caused by
overexpression of transactivators We have previously developed a
novel HC-Ad vector, HC-Ad-mTetON-~-Gal, encoding the
tetra-cycline dependent transactivator (rtTA2S-M2)and silencer (tTSk;d)
under the control of a constitutive mCMV promoter We observed
robust, inducible ~gal transgene expression in vitro and in vivo
following administration of Doxycyline (Dox) Here, we
charac-terized the effect of immune responses against the transactivator
(constitutive expression) and the transgene (inducible expression)
on HC-Ad-mTetON-~-Gal mediated expression after its delivery
into the brain ofC57BL/6mice We immunized C57BLl6 mice
with either pCI-TetON or pCI-~Gal plasm ids by intra-muscular
injection.HC-Ad-mTetON-~-Gal vector was injected three weeks
later into the striatum (Ix107blue forming units) We administered
Dox(in chow) for 3 or 7 weeks beginning twenty-four hours prior
to intracranial HC-Ad injection We demonstrate that intra-muscular
expression of the rtTA2S-M2and Bgalusing TetON and
pCI-~Gal plasmids induced a cellular immune response directed against
the rtTA2S-M2transactivator and~gal protein We observed higher
numbers of IFN-ysecreting specific T cells by ELISPOT in the~gal
and TetON immunized animals (p<O.05) Quantification of F4/80
and CD3 immunoreactive cells in the brains ofimmunized animals
indicated increased numbers of immune cells in thepCI-~gal
im-munized group than in the pCI-TetON imim-munized group Levels
of~gal activity and quantification ofBgalexpressing cells in the
striatum was reduced significantly inpCI-~gal immunized group
(p<O.05) while the pCI-TetON immunized group did not exhibit any
significant difference when compared to non-immunized animals
(p>O.05) In conclusion, the TetON protein is less immunogenic and
when delivered into the periphery it does not affect long term HC-Ad
mediated transgcne expression in the brain.Funded by NINDS IRO I
NS 42893.01, U54 NS045309-01, IR21 NS047298-01; and Bram
& Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS
1R01 NS44556.01, 1R21 NS054143-01 and1R03 TW006273-01
to MGC; The Linda Tallen&David Paul Kane Foundation and the
Board ofGovernors at CSMS.
Responses in a Plasmid DNA Treated Hemophilia
A Mouse Model by Transgenic FOXP3+CD4+
Regulatory T Cells
Benjamin R.Harmeling,IStevenZiegler;' Troy Torgerson.P
Lip-ing Chen,IBaoweiPeng,' Hans D Ochs.l-' David 1 Rawlings,1.3
Carol H.Miao.P
/Pediatrics, ChildrensHospital and Regional Medical Center;
Seattle, IfA; l1mmunology, Benaorya Research Institute, Seattle,
WA; 'Department a/Pediatrics, University a/Washington,
Se-attle, IE4
The suppressive capabilities of FOXPYCD4+CD25+
regula-toryT(T", ) cells have made them a compelling tool for potential
therapeutic treatments that target a wide variety of autoimmune
and alloimmune responses Treatment of a factor VIII (FVIII)
deficient (I-IemA) mouse model with a non-viral plasmid
(pBS-HCRHPI-FVIIIA) via hydrodynamics-based gene delivery results in
supra-physiologic expression ofhuman FVIII Succesful expression
triggers a robust humoral immune response, generating high-titer
FVIII-specific antibodies This model mirrors the response seen in
the 25-30% of hemophilia A patients that produce FVIII-specific
inhibitory antibodies following protein replacement therapy We
hypothesize that regulatory T cells could modulate the formation
of inhibitory antibodies against FVIII, and allow for prolonged
expression of FVIII in plasmid gene therapy recipients In order
to evaluate whether FOXP3 expressing T", cells can suppress the
antigen-specific immune response in the ifemA mouse model, we
have developed a transgenic mouse of a HemA phenotype with
Molecular Therapy Volume1S t Supplement 1 ~tl)· 2007
Copyright © The American Society of Gene Therapy
overexpressed FOXP3 (HemAlFOXP3-Tg) FACS analysis dem-onstrated that approximately 60-80% ofCD4+Tcells from HemAl FOXP3-Tg mice expressed FOXP3, and treatment of transgenic mice with FVIII plasmid did not induce any detectable humoral response against FVIII Furthermore, T cells isolated from FVIII plasmid treated HemA mice were shown to rapidly proliferate and secrete significant amounts oflL-1 0 cytokine when stimulated with human FVIII in vitro, whereas T cells from FVIII plasmid treated
HemAlFOXP3-Tg did not display either response Finally, HemAl FOXP3-Tg CD4+ T cells were clearly shown to be of suppressive nature by significantly downregulating proliferation of anti-CD3 stimulated wild-type CD4+ effectorT cells By adoptively transfer-ring splenic T", cells from FVIII exposed HemAlFOXP3-Tg mice,
we havedemo~strated transient control of FVIII-specific immune responses in recipient HemA mice following treatment with FVIII plasmid In approximately halfof recipient HemA mice, FVIII lev-els have been maintained at higher levlev-els and have persisted over
a longer duration than in mice not receiving Treg cells Inhibitory antibody titer levels, as measured by Bethesda Assay, have also been fully or partially suppressed In ongoing experiments, we are optimizing the suppression ofinhibitory antibodies in hemophilia A mice in combination with anin vitro suppressive assay to delineate
the mechanisms and conditions required to specifically control these immune responses
Antitrypsin
EkaterinaBreous,' Suryanarayan Somanthan,'Luk1-1 Vandenber-ghe.' James M Wilson.'
'Gene Therapy Program Department0/Pathology and Labora-tory Medicine, University ofPennsylvania, Philadelphia, PA.
A cell-mediated destructive immune response to the transgene expressed from adeno-associated virus (AAV)-based vectors is seldom seen, resulting in long-term transgene expression We characterized the mechanisms responsible for this apparent lack
of immune response to humana-I antitrypsin (AIAT) encoded by AAVscrotype-S in murine models AAV encoding A IAT is currently being pursued in clinical trials for therapeutic treatment of AIAT deficiency Immunocompetent C57BLl6 and BALB/c mice injected intravenously (IV) with AAV-AIAT displayed sustained Al Al' expression and no A IAT-specific antibodies for up to 3 months
No CD8+ T cell response was detected against mapped dominant epitopes of AIAT We next asked whether AAV-immunized mice will continue to express thetransgene after the administration of adenoviral vector (Ad), which is a strong activator of cytotoxic T lymphocytes (CTL) As anticipated, C57BLl6 mice that received
an IV injection ofAd-AIAT only had transientAIAT expression in conjunction with high frequencies ofA IAf-specific CD8+ T cells Anti-A IAT Ig levels were sometimes detected in these mice but not found to be a factor in the elimination of detectable transgene expression A IAT expression remained stable following administra-tion ofAd-A IAT in animals treated with AAV-AIAT simultaneously
or prior to(i.e., 2 or6weeks before) the Ad vector Most striking, however, was the fact that we could not detect any AIAT-specific CD8+ T cell response following the Ad vector in AAV pretreated animals, which strongly suggests an active AAV-specific suppres-sion effect of A IAr-antigen-specific CTLs Similarly, BALB/c mice administered Ad-A IAT only expressed A IAT transiently and had high numbers of A IAT-specific CD8+ T cells However these mice also generated high titers of anti-AI AT Ig Using the above-described modcl of Ad challenge, we were able to demonstrate thatAIATexpression was sustained in mice pre-administered with AAV for 2 to 6 weeks even though it was significantly reduced More importantly, these mice showed the complete absence of
S97
Trang 2antibodies to AIAT On the other hand, an AlAT-specific CDS+
therefore seems likely to be responsible for the observed reduction
to suppress COST and B cell responses is relevant for the use of
AAV in therapeutic gene transfer as well as viral immune evasion
The mechanisms of active suppression including the involvement
of regulatory'I'cells are discussed
255 Cross Presentation of AAV2 Capsids
Activates Cytotoxic T Cells and Hinders
Muscle-Directed Gene Transfer but Does Not Render
Hepatocytes Effective Cytolytic Targets
James M Wilson}
[Gene Therapy Program Department ofPathology and
Labora-tory Medicine University ofPennsylvania Philadelphia, PA.
Livertoxicityobserved in a recentclinicaltrial ofAAV2 delivered
systemically to patients with hemophilia was ascribed to killing of
evaluated the biology ofT eell activation to AAV capsids in murine
models COST cell epitopes were mapped to capsids from AAV2,
7 and S A tetramer was generated to a dominant capsid epitope in
Balb/C mice shared between these AAVserotypes, Administration
evidenced by binding to tetramer and production ofcapsid induced
IFN g and TNF a expression; this was not observed with AAV7or
AAVSvectors.COST cells toAAV2capsids demonstratefunctional
AAV capsid (Ad.Cap) were able to decrease the muscle-directed
gene transfer efficiencyofAAV2.cFIXby 2-fold compared to mice
pre-injected with an empty adenovirus However, the performance
ofliver directed AAVmediated gene transfer was not diminished in
that cross presentation of AAVcapsids does result in activation of
CTL in a serotype specific manner and the pre-existing
capsid-spe-cific'l-cells arc capable to hinder the muscle-directedgene transfer,
however there is no evidence that vector transduced hepatocytesare
targets for CTL effector activity
256 Ultrasound Guided Gene Transfer to the
Fetal Thymus Results in Thymic Epithelial Cell
Transduction and Deletion of Transgene Specific
T Cells
Masayuki Endo, William H Peranteau, Phillip W Zoltick,
Anto-neta Radu, Nidal Muvarak, Alan W Flake
[The Children's Center for Fetal Research, The Children's
Hospi-tal ofPhiladelphia Philadelphia PA.
Fetal immune ontogeny provides a unique opportunity for
ma-nipulationof tolerancefor prenataland anticipatedpostnatalgenetic
and cellular therapies Postnatal intra-thymic cell or gene transfer
has been shown to result in antigen specifictolerance in rodents, but
these results have not been successfully translated to large animals
Whereas in rodents the thymus plays a key role in determination of
T-cellrepertoireand generationofT-regulatorypopulationswell into
adult life, in higher mammals this role is confinedto early gestation
Our aim in this study was to develop a murine model for thymic
antigen presentation, which would be analogous to the anticipated
timing required for effective thymic presentation of antigen, in a
large animal system We hypothesized that thymic presentation
of antigen early in gestation would result in deletion of antigen
differ-S9S
ent ultrasound guided approaches for achieving intrathyrnic gene transfer in fetal mice First, we injected lentiviral vector carrying the green fluorescent protein (OFP) reporter gene into the murine amniotic cavity from post coital day 8 (E8) to Ell, when the thymic primordiumconsists of pharyngeal pouchepitheliumand is in direct contact with the amniotic fluid Second, we injected vector directly into the fetal thymus at E17 We analyzed the injected mice under fluorescence stereornicroscopyat sequential time points after birth and identifiedOFP expressingcell types by immunohistochemistry Furthermore, we assessed deletion of specific TCR bearing 'l-cells after early intra-amniotic gene transfer (lAGT) with
Balb/c (MHC II I-E+, mtv7-) fetal mice underwent IAOTat E9 with either Lenti-mtv7(experimentalgroup), Lenti-mtv9(irrelevant mtv vector) or no vector.The peripheral blood (PB), thymus and spleen
of experimental and control mice were analyzed at intervals after
are normally deleted in mtv?",1-£+ strains of mice Sustained OFP
and after direct intra-thymic injection at E17.Histological analysis confirmed the expression of OFP in cortical and medullar thymic epithelial cells Expressionofmtv7 in the thymus resulted in partial deletion of relevant VJ36 TCR bearing T-cells in the PB and spleen Experimentalanimals maintaineda significantreduction in the level oftransgene specific'I'cells for the duration ofthe study, i.e beyond
6 months of life, The current study supports the ability of in utero thymic gene transfer to I) transduce thymic cortical and medullary antigen presenting cells and 2) inducedeletion oftransgene specific
T cells This model system should be valuable for testing strategies for specific tolerance induction that may translate to large animal systems
257 Characteristic of Responsive CD8+ T Cells
to AAV 2/8 and Ad C7 HIV Gag Vector Vaccines
'Gene Therapy Program Department ofPathology and Labora-tory Medicine, University ofPennsylvania, Philadelphia, PA.
Adeno-associated viruses (AAV) and adenoviruses (Ad) have been developed as vectors for gene therapy and genetic vaccines
In this study, we characterized the peak and long term COST cell
AdC7 vectors expressing HIV-I gag in CB6Fl hybrid mice The gag specific COST cell responses were evaluated by monitoring the frequency, phenotype and functional properties of gag specific
tetramer (H2-Kd AMQMLKETI) equal to 24 and 60% of all CDS
were found to persist as long as 140days post immunization.A
of tetramer positive COST cells (6 and 2S% in spleen and liver, respectively).These tetramer positive'I'cells were further classified
as effector, effector memory and central memory subsets based on the expression of CD62L and CDI27 cell surface markers The majority of the gag tetramer specific COST cells (93%) following
AAV2/Sadministrationwere effectorcells which is in contrastto the
effec-tor memory gag specific cells were elicited at the peak of response and a small percentage of central memory cells were observed as long as day 127 post immunization In addition to phenotyping for
induced an equal proportion of CDS T cells secreting IFN-g alone and IFN-glTNF-a in all tissues studied In the case of AdC7gag,
Molecular Therapy Volume 15.Supplement I• \br W07
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