257 Characteristic of Responsive CD8+ T Cells to AAV 2/8 and Ad C7 HIV Gag Vector Vaccines antibodies to AIAT On the other hand, an AlAT specific CDS+ ''''I'''' cell response was observed This partial CTL s[.]
Trang 1antibodies to AIAT On the other hand, an AlAT-specific CDS+
'I'cell response was observed This partial CTL suppression effect
therefore seems likely to be responsible for the observed reduction
in AIATexpression,This strain-specific dominant effect ofAAVS
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
Lili Wang,1Joanita Figueredo; Roberto Caicedo; Jianping Lin;
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
vector transduced hepatocytes by capsid specific'I'cells This study
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
ofAAV2vectors resulted in the activation ofcapsid CD8'I'cells as
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
cytolyticactivityin vivoto peptideloadedtargetcells.The frequency
ofcapsid'I'cells was much higher in liver than blood or spleen The
pre-existing capsid'I'cells induced by an adenovirus expressing the
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
animals with high levelsofpre-existing capsid'I'cells Weconclude
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
specific 'l-cells, To test this hypothesis, we developed two
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 Icntiviralvec-tor encoding an exogenous mammary tumor virus oncogene(rntv), 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 birth by flow cytometry forTscells expressing VJ36 TCR which are normally deleted in mtv?",1-£+ strains of mice Sustained OFP expression was observed in the thymus after lAO'I'from E8 to ElO 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
Jianping Lin,' Joanita M Figueredo,' Yan Zhi,' Roberto Caicedo,1 Lili Wang,1Zhe Zhang,' James M Wilson.'
'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 responses elicited by recombinantAAV2/S and simian derived 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 'I'cells in mouse spleen and liver Intramuscular administration
of lEI I OC ofAAV2/S gag resulted in peak frequencies of gag tetramer (H2-Kd AMQMLKETI) equal to 24 and 60% of all CDS 'I'cells in spleen and liver, respectively.About 50% of these cells were found to persist as long as 140days post immunization.A 10-fold reduction in dose ofAAV2/Svector still produced high levels
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 response achieved with AdC7 vector where botheffectorand 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 memory markers,'I'cell responses were characterized with respect
to antigen induced cytokine secretion At the peak,AAV2/S gag 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
Copyright © "1111; Arncricm $Ol;(f,:ty of Gene Therapy
Trang 2the majority of the gag specific CD8 T cells secreted both IFN-g
and TNF-a in multiple organs at the peak of response Long term
evaluation revealed 20% of gag specific CD8 T cells secreting
IFN-g, TNP-a and IL-2 in AdC7 immunized mice Moreover, both
T cells expressing granzyrne B In vivo CTL assays demonstrated
that both AAV and Ad immunizations can elicit the production of
CTLs with the capability of lysing in vivo antigen loaded target
cells In conclusion,AAV2/8gag immunization induces a robust
and relatively long-lasting antigen specific effector T cell response,
while AdC7 gag immunization resulted in the generation ofeffector
memory and central memory T cells
258 Lentiviral-Mediated Gene Transfer to Lung
Activates Transgene and Vector Specific T Cells
MariaL.Limberis,IChristie Bell,IRoberto Caicedo,I Luk1-1.
Vandenberghe; Deirdre Mclvlenamin,' Regina Munden; Di WU,1
Julie C Johnston,IPeter Bell,IJames M Wilson.'
'Gene Therapy Program, Department ofPathology and
Labora-tory Medicine, University ofPennsylvania, Philadelphia, PA.
Gene transfer vectors based on HIV-I (Ientiviral vectors) are
attractive gene transfer agents for airway genetic diseases such as
cystic fibrosis due to their inherent ability to transduce quiescent
cells in a stable manner Since less than I% of the airway
epithe-lium is actively dividing at any given time, the use of these vectors
is warranted To improve targeting efficiency of lentiviral vectors
in airway epithelium much effort has been placed on modifying
envelopes from viruses with known affinity for airway One of
these viruses is the Ebola Zaire virus, the glycoprotein of which
has been mutated (NTDL6) to successfully pseudotype a Ientivirus
vector [Medina et aI., 2003, Mol Ther;8 (5) 777-89] and
signifi-cantly improve its transduction efficiency in rodent and non-human
primate airway (Kobinger, unpublished) To further characterize the
potential of a lentivirus vector for lung-directed gene transfer we
studied the likelihood ofT and B cell activation to the vector and
encoded transgene product following delivery to the lung which
may compromise the therapeutic potential ofthis approachin vivo.
For dosing, vectors wcrc delivered in 50 ml (4E+8 transducing
units) in either C57B1I6 or BalbC mice(n=41group) For delivery
to lung, mice were subjected to an intratracheal (IT) instillation of
NTDL6-pseudotyped lentivirus vector expressing GPP under the
control of'the CMV promoter As negative control for gene transfer,
a VSVG-pseudotyped lentivirus vector expressing GFP was used To
activate T cells, groups of mice were also injected intramuscularly
(1M) with the same vectors (i.e., NTDL6- or VSVG- pscudotyped
HIV-GFP vector) Spleens were harvested from each mouse (treated
either IT or 1M) as well as the draining superficial cervical lymph
nodes from mice treated IT Isolated lymphocytes were assayed by
IFN-g ELISPOT following stimulation with the entire GFP library
(to assay transgene-specific responses) or the p24/p 17 library (to
assay vector-specific responses) NTDL6-pseudotyped H1V-GFP
vector resulted in significant transduction of the alveolar and less
so of the conducting airway epithelium VSV-G pseudotyped HIV
vector resulted in no GFP transduction in lung Immunologic studies
were performed in all animals dosed with pseudotyped HIV
vec-tors Following 1M injection, there was significant T cell activation
to GPr, and B cell responses to both GPP and p24 in C57BI/6 and
BalbC mice, with responses being more pronounced in the latter
POl' [T delivery, responses were lower than those achieved by 1M
injection but appeared to be sustained up to 6 wks post injection
There was a trend in the decrease of GFP-cxpressing cells in lung
with time possibly reflecting immuno-toxicity We arc currently
investigating the impact of the OFP-specific T cells to decipher
Molecular Therapy Volume15.Suppkmen.l ~br 2007
Copyright © The American So(,.;C!;}" of Gene
Therapj-whether the decrease in gene expression in lung is due to the normal cell turnover of the epithelium or due to the cytotoxic nature of the OPP-specific CD8 T cells
259 Characterization of AAV Capsid Responses in Humans Using Normal Donor Splenocytes
Daniel J Hui,' Federico Mingozzi,' EtienaBasner-Tschkaraian,' Shyrie Edmonson,IKatherine A.High.l-'
PA; lHemat%gy, Howard Hughes Medica/Institute, Philadel-phia, PA.
Although adeno-associated viral (AAV) vectors remain a promising gene therapy approach for the treatment of genetic disorders, concerns over potential immune responses are a major obstaele facing successful implementation in a clinical setting It has recently been reported that liver-directed gene transfer of fac-tor IX using AAV serotype 2 to correct hemophilia B activated a
T cell response against AAV capsid, thereby causing destruction
of transduced hepatocytes leading to a transient transaminitis (Manno et al Nat Med 2006) In order to further elucidate T cell responses against AAV capsid and to help predict potential im-mune responses in future subjects undergoingAAV-mediated gene transfer, a novel approach using normal donor human splenocytcs
as a source for T cells was developed Splenocytes offer two major advantages over a conventional peripheral blood mononuclear cell (PBMC)-based approach First,cells are harvested directly from the spleen, a lymphatic compartment, as opposed to the peripheral circulation; and second, splenocytes are harvested in much higher cell numbers than PBMCs, which allows multiple experiments to
be performed using cells from a single donor To date, a total of 43 donor splenocyte samples have been collected from normaladults
or children undergoing splenectomy Once harvested, splenocytes are utilized forT cell functional assays such as intracellular cytokine staining (ICCS) or Enzyme-Linked Immunospot (ELI Spot) assays
To improve detection of low-frequency T cell responses, an expan-sion strategy was used in which splenocytes were incubated with candidate cpitopes in culture prior to testing Ofthe 19 samples that wcre expandcd, 12 had detectable T cell responses by ELiSpot assay representing a much higher percentage (63%) than previous PBMC data (Blood 2006; 108( [ I): [38a) In conjunction with bioinformatics prediction algorithms, 8 new epitopes are reported here for 7 MI-IC Class I HLA haplotypes, including 6 of the most frequently occur-ring haplotypes in the normal population Although most subjects recognized a single AAV epitope, 2 of these subjects recognized
2 or more cpitopes Expanded splenocytes wcrc also successfully used as effector cells in cytotoxicity (CTL) assays against HLA-matched AAV-transduced human hepatocytes, further verifying functionality of T cell epitopes There was no correlation between the magnitude ofthe T cell response and anti-AAV IgO levels from ELISA assays performed on matched serum samples for [2 of the donors screened, although all donors did have detectable levels of anti-AAV antibodies While responses were seen in donors as young
as 5 years ofage, more robust responses were detected in donors >20 years old, which has implications for selecting candidates for future clinical trials Current studies are focused on utilizing splenocytes
to examine cross-reactive T cell responses among AAV serotypes and phenotyping memory responses In conclusion, the cumulative results from these splenocyte studies validate this approach as being more informative for characterizing T cell responses than current PBMC-based methods
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