204 IL 28b enhances cellular immunity by reduction of t regulatory cell populations during DNA vaccination

204 IL 28B Enhances Cellular Immunity by Reduction of T Regulatory Cell Populations during DNA Vaccination Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene[.]

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

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resistant to stretch, or more stiff, compared to BL10 mice In summary,

the absence of dystrophin increases stiffness (decreases compliance)

at the MTJ The decreased mechanical compliance of the MTJ may

explain the reduced mobility seen in DMD patients Gene therapy for

DMD should consider both the muscle and the MTJ

Observation Period after bFGF Gene Therapy To

Promote Healing of Injured Flexor Tendons

Jin Bo Tang,1,2 Yi Cao,1 Chuan Hao Chen,1 Ya Fang Wu,1 Xiao Tian

Wang,2 Paul Y Liu.2

1 Hand Surgery Research Center, Department of Hand Surgery,

Affi liated Hospital of Nantong University, Nantong, Jingsu, China;

2 Gene Therapy, Department of Surgery, Roger Williams Medical

Center, Boston University School of Medicine, Providence, RI.

Introduction Previously, we demonstrated that AAV2-bFGF gene

therapy signifi cantly increases the strength of fl exor tendons but does

not increase adhesions An ideal gene therapy for wound healing

should produce an increase in transgene expression during the healing

process, but “shut off” of this expression when healing is achieved

For this reason, we investigated expression of the transgene—bFGF

gene—over a prolonged period (16 weeks) in injured fl exor tendons

Methods 50 long toes of 25 chickens were used The fl exor digitorum

profundus (FDP) tendons of 20 toes were transected completely, and

were repaired surgically with modifi ed Kessler repair method The

tendons were subsequently injected with AAV2-bFGF at 2´109 viral

particles/tendon, into 4 sites in the proximal and distal tendon stumps

The AAV2-bFGF harbors bFGF gene of rat origin 20 FDP tendons

in 20 long toes of 10 chickens received no injection 10 tendons from

10 long toes of the remaining 5 chickens were not injured, serving as

normal controls At postoperative weeks 2, 4, 8, 12, and 16, tendons

were harvested and stained immunohistochemically to determine

the presence of bFGF Real-time PCR analysis was performed to

determine levels of overall bFGF gene expression Results Compared

with the controls, compound expression of bFGF and expression of

delivered bFGF genes was drastically increased at 2 to 8 weeks in the

tendons that had undergone bFGF gene therapy From weeks 8 to 16,

the amount of bFGF in the AAV2-bFGF treated tendon declined At 16

weeks, the amount of bFGF in the gene therapy tendons was identical

to that in the controls Expression of the bFGF gene decreased

from week 12 to 16 At week 16, compound expression level of the

endogenous and delivered bFGF gene returned to the level identical

to that in the uninjured tendon The expression of delivered rat bFGF

was not detectable in real-time PCR analysis Discussion: In a clinical

relevant animal model involving complete cut and surgical repairs of

the tendon, our investigation showed that the compound expression

of bFGF gene in the AAV2-bFGF treated tendon peaks in the most

critical period of the tendon healing The transgene expression peaks

in that healing period However, supernormal expression of the bFGF

transgene, and excessive production of bFGF, cease when tendon

healing is completed This information is important in that it shows

that bFGF gene therapy does not result in problems of bFGF

over-production after tendon wounds have healed

203 An Investigation of Effi ciency of Gene Delivery Methods and Time-Course of Transgene Expression in Injured Tendons and Tissue Reactions Caused by Different Vectors

Jin Bo Tang,1 Chuan Hua Chen,1 Ya Fang Wu,1 Bella Avanessian,2

Xiao Tian Wang,2 Paul Y Liu.2

1 Department of Hand Surgery, Affi liated Hospital of Nantong University, Nantong, Jiangsu, China; 2 Gene Therapy, Department

of Surgery, Roger Williams Medical Center, Providence, RI.

Introduction Comparisons of effi ciency of gene delivery—through

different vector systems—for in vivo tendon healing have not been

reported previously We investigated effi ciency of gene delivery to injured tendons and tissue reactions caused by different vectors

Methods Plasmid, adeno-associated viral (AAV), and adenoviral

vectors were used to transfect the injured tendon using 72 digital

fl exor tendons of bilateral toes of 18 white leghorn chickens After transverse tendon cuts, pCMV-EGFP, pCAG-EGFP, rAAV2-EGFP, and Ad5-EGFP were injected into the tendons At 3, 7, 14, and 21 days, the tendons were subjected to examination under a fl uorescence microscope for GFP expression to determine the efficiency of transgene delivery by different vectors These sections were also stained with DAPI, and the percent of GFP-positive cells in the sections were recorded The tendon sections were also stained with hematoxylin and eosin to examine the infl ammation caused by these vectors Infl ammatory cells were counted under a microscope and were compared statistically The results of cell counts were analyzed

statistically using ANOVA followed by post hoc Tukey test Results,

The GFP expression, compared with normal tendons, was observed

in experimental tendons at 3, 7, 14 and 21 days post-injection; for all vectors, it was highest at 7 days At 14 days, a marked decrease in GFP expression was observed The GFP expression in the tendons injected with either rAAV2-EGFP or Ad5-EGFP was higher than in those injected with pCMV-EGFP or pCAG-EGFP vectors At days 3, 7, and 14, the percentage of GFP-positive cells in AAV2-GFP and Ad5-GFP treated tendons was signifi cantly higher than that in the plasmid vector treated tendons (p < 0.05 or p < 0.001) At days 7 and 14, the AAV2-GFP treated tendons had the highest percentage of the cells showing positive GFP (p < 0.05 or p < 0.01) Tissue reactions of the tendons induced by the liposome-plasmid vectors (including pCMV-EGFP and pCAG-pCMV-EGFP) were the most prominent Infl ammatory reactions were least severe in tendons injected with AAV2 vector

Conclusions Among the 4 vectors tested, effi ciency of gene delivery

was highest using AAV2 and Ad5 vectors The AAV2 vector caused the mildest tissue reaction This study suggests that the AAV2 vector

is a promising gene delivery vector for tendon healing gene therapy Its time-course of transgene expression the expression starts very early in the tendon healing process and its expression drops as the healing proceeds can be favorable to the tendon healing, and avoids potentially detrimental effects of prolonged gene expression

Infectious Diseases and Vaccines I

Reduction of T Regulatory Cell Populations during DNA Vaccination

Matthew P Morrow, Panyupanok Pankhong, Dominick J Laddy, Kimberly A Kraynyak, Jian Yan, Neil Cisper, David B Weiner

Pathology and Laboratory Medicine, The University of Pennsylvania, Philadelphia, PA.

Improving the potency of immune response is paramount among issues concerning vaccines to deadly pathogens, such as HIV IL-28B belongs to the newly described Interferon Lambda (IFNλ) family of cytokines, and has not yet been assessed for its potential ability to act as a vaccine adjuvant We tested the ability of plasmid encoded

Molecular Therapy Volume 17, Supplement 1, May 2009

IL-28B to boost immune responses to a multi-clade consensus HIV

Gag plasmid during DNA vaccination We show here for the fi rst

time that IL-28B robustly enhances immune responses during in an

adjuvant setting Moreover, we describe for the fi rst time how

IL-28B reduces Regulatory T cell populations during DNA vaccination

while increasing the percentage of splenic CD8+ T cells in vaccinated

animals These cells are more granular and have higher

antigen-specifi c cytolytic degranulation when compared to cells taken from

animals that received antigen alone Lastly, we are the fi rst to report

that plasmid encoded IL-28B can induce 100% protection from

mortality after a lethal infl uenza challenge These data suggest

that IL-28B is a strong candidate for further studies of vaccine or

immunotherapy protocols

Therapy Effi cacy in HBV Transgenic Mice:

Constitutive Versus Inducible Expression

Lucia Vanrell,1 Cristina Olague,1 Africa Vales,1 Lilian

Tenembaum,2 Gloria Gonzalez-Aseguinolaza.1

1 Division of Gene Therapy, School of Medicine, Center for Applied

Medical Research, University of Navarra, Pamplona, Navarra,

Spain; 2 Laboratory for Experimental Neurosurgery, Université

Libre de Bruxelles, Brussels, Belgium.

Background: Hepatitis B virus (HBV) and hepatitis C virus

(HCV) constitute the main etiologic factors for the chronic viral

hepatitis affecting more than 550 million people worldwide Interferon

alpha (IFNα) has demonstrated therapeutic effi cacy in both chronic

hepatitis B and C However, the monotherapy response rate

(25%-35%) can be increased, and side effects limited, by improving the

pharmacokinetic of IFNalpha therapy with stabilising ligands Gene

therapy allows a continuous in vivo expression of the transgene at

the desired site AAV vectors lack pathogenicity in humans and

have demonstrated prolonged expression of numerous transgenes,

in several tissues and immunocompetent animal models Aims:

Therapeutic effi cacy evaluation of a recombinant AAV expressing

murine IFN alpha (AAVIFN) under the control of a constitutive

or an inducible promoter in HBV transgenic mice Methods: We

have produced AAV vectors expressing luciferase or murine

IFN-alpha1 into two different expression cassettes (AAVIFN) The fi rst

one contains the promoter for the human Elongation Factor-1 alpha

(hEF1alpfa), the transgene, and the SV40 polyadenilation signal

sequence The second one contains a CMV-TetON promoter, the

transgene, and the SV40 polyadenilation signal AAV serotype 8

virus production was performed by cotransfection into 293 T cells

of the plasmid containing the expression cassette fl anked by the

AAV ITRs, and the plasmids containing the necessary AAV and

adenoviral proteins, using linear polyethylenimine (25 kDa) Viruses

were purifi ed by iodixanol centrifugation gradient and viral titers

were determined by real time PCR IFN-alpha concentration was

determined by the cytopathic effect (CPE) reduction assay Serum

viral load was determined by qPCR after viral DNA purifi cation from

serum samples Results AAV constitutively expressing murine

IFN-alpha was able to inhibit HBV viral replication to undetectable levels

in HBV transgenic mice, while the administration of AAV expressing

luciferase had no effect over viral replication However, prolonged

IFN-alpha expression resulted in severe side effects as shown by a

profound pancytopenia that led to animal’s death Experiments are

now being performed to determine the antiviral effi cacy and safety

of the AAV vector expressing IFN-alpha in an inducible manner

Conclusion This report highlights the promises and the limitations

of IFNα-gene therapy in viral hepatitis

Adenovirus-Based Ebola Virus Vaccine

Jason S Richardson,1 Michel K Yao,1 Kaylie N Tran,1,2 Maria

A Croyle,5,6 James E Strong,1,3,4 Heinz Feldmann,1,3 Gary P Kobinger.1,3

1 Special Pathogens, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, MB, Canada; 2 Department

of Microbiology, University of Manitoba, Winnipeg, MB, Canada;

3 Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 4 Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; 5 Division

of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, TX; 6 Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX.

Background: Zaire ebolavirus (ZEBOV) causes hemorrhagic fever

with an associated death rate estimated as high as 90% in infected humans Vaccine based on virus-like particles, vesicular stomatitis virus, human parainfl uenza virus type 3 or adenovirus containing

or expressing the ZEBOV envelope glycoprotein (ZGP) have successfully protected nonhuman primates from an otherwise lethal challenge of Ebola virus Currently Phase I testing in humans using

a recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZGP sequence from a human cytomegalovirus immediate early gene (CMV) promoter (Ad-CMVZGP) Ebola vaccine

is underway Objective: 1) Increase the expression of the ZGP antigen

from an adenovirus serotype 5 vector Improvements of the expression cassette included codon optimization for translation in mammalian cells, inclusion of a consensus Kozak sequence and engineering of a hybrid CAG promoter which combines the human cytomegalovirus immediate early gene enhancer and a modifi ed chicken beta-actin promoter (Ad-CAGoptZGP) 2) Evaluate protection effi cacy and T and

B cell immune response elicited by Ad-CAGoptZGP compared to the standard Ad-CMVZGP vector on a dose-to-dose basis against lethal

ZEBOV challenge in mice Signifi cant fi ndings: In vitro, evaluation

of the Ad-CAGoptZGP demonstrated a 3 to 7-fold increase in the expression of the ZGP antigen when compared to the Ad-CMVZGP vaccine The Ad-CAGoptZGP vaccine fully protected mice against

a lethal challenge with mouse-adapted ZEBOV at a dose 100 times lower than the minimal dose required to attain full protection with the Ad-CMV/ZGP vaccine Mice administered the Ad-CAGoptZGP vaccine resulted in improved immune responses even at a dose 10 times lower for the T cell response and 100 times lower for the B cell response than with the Ad-CMVZGP vaccine Unexpectedly, the improved Ad-CAGoptZGP vaccine afforded 100% protection for mice when administered 30 minutes post-exposure with mouse-adapted ZEBOV, although weight loss was observed Performance of the improved Ad-CAGoptZGP vaccine in the presence of pre-exisitng immunity to the vaccine carrier is currently being evaluated in guinea pigs and results will be presented Understanding and improving the molecular components of adenovirus-based vaccines may be useful for vaccination and post-exposure therapy