669 involvement of myd88 and TLR9 in the innate immune response elicited by replication incompetent adenovirus vectors

669 Involvement of MyD88 and TLR9 in the Innate Immune Response Elicited by Replication Incompetent Adenovirus Vectors anti adenoviral immunity may not be an obstacle to the application of adenoviral[.]

anti-adenoviral immunity may not be an obstacle to the application

of adenoviral gene transfer in islet transplantation

668 Effect of Pre-Existing Anti-Adenovirus

Immunity and Anti-Tumor Immunity on

the Efficacy of Adenovirus Vectors in

Immunocompetent Syrian Hamster Model

Debanjan Dhar,'JacquelineF.Spencer; Karoly Toth,'William S

M.Wold '

'Molecular Microb iology andImmunology,St Louis University,

St Louis , MG.

The field of Oncolytic adenovirus (Ad) vectors needs a proper

animal model to study these vectors Due to the species specificity

of Ads, such vectors are commonly evaluated in immunodeficient

mice bearing human tumor xenografts Because these mice are

immunodeficient and nonpermissive for human Ads, this model

cannot adequately address the effect of the host immune system on

the vector-infected tumor or the toxicity and biodistribution of the

667 Periocular Triamcinolone Enhances

Intraocular Gene Expression Following Delivery

by Adenovirus

Marisol D Cano,IChoul Y Park.l-'Roy S Chuck,IMargaret

Yew,'Vict Nguyen,'Jack Parker,I Kcisuke Mori,' PeterL

Gchl-bach.'

'Ophthalmology; Johns Hopkins UniversitySchool ofMedicine

Baltimore MD; lOphthalmolog)\ Dong-Guk UniversitySchool of

Medicine , llsan, Korea; 'Biomedical Engineering Johns Hopkins

University Baltimore, MD ; "Ophthalmology; Saitama Medical

University Iruma, Saitama, Japan

Immunogenicity isa recognizeddrawbackofadenovirusmediated

gene transfer in vivo While relative immune toleranceis present

in the eye, immune responses may damage delicate ocular tissues

and alter transgene expression.A variety of vector modifications

and treatment strategies have been employed to diminish vector

induced immune response,ineluding deletionof nonessential viral

genes; selective retention ofgenes for evasion of the host immune

response and the use of more efficient vectors in smaller doses

Immune modulation is standard component of the treatment of

a variety of ocular diseases However, the extent to which local

immune modulationaffects viral transgene expression is not well

understood In this study we monitored expression of the firefly

luciferase reporter gene in mouse eyes following intravitreous

delivery of adenovirus vcetor Gene expression was assessed by

serial measurement of ocular bioluminescence with an IVIS' 200

ImagingSystem(Xenogen).Patternsofluciferase expressionin mice

receiving periocular injection of the corticosteroid, Triamcinolone

acetonide (TA), were compared to those in mice with no local

im-mune modulation.The resultsindicatethat local imim-munemodulatory

treatmentsmaysignificantlyprolongthe durationofpeakadenovirus

mediated gene expression and the total period of gene expression

in previously naive eyes The study also indicates that non-invasive

imaging is a useful approach to longitudinal assessment of in vivo

ocular gene expression in mice

vector in normal tissues Wehave recently developeda novel Syrian hamster model for the study of oncolytic Ads (Thomas M.Aet al.

Cancer Res 66:1270-1276) We have been exploring the benefits

of direct intratumoral injection of our oncolytic Ad5-based vector VRX-007 intosubcutaneousSyrianhamstertumors.VRX-007 lacks most E3 genes and overexpresses the Adenovirus Death Protein which mediates efficient infected cell lysis and vector spread.This vector is very effective in suppressing the growth of tumors.A key question regarding the use of Ad5-based oncolytic Ad vectors in humans is whether pre-existing immunity to Ad5 will limit the use

of such vectorsfor cancergene therapy.To address this question,we compared the effectiveness ofVRX-007 innaiveand Ad5-immune hamsters, We found that, following intratumoral injcction,

VRX-007 suppressed the growth of tumors equally well in both groups Thus, pre-existing immunity to Ad5 does not affect vector efficacy following intratumoral injection However, pre-existing immunity greatly prevented the dissemination of vector from the tumor to lung and liver Wealso examined the role of pre-existing immunity

to Ad in vector replication and toxicity in the liver following i.v, injection of VRX-007 with and without immunosuppression with cyclophosphamide (CP) The CP prevented an immune response against the vector and allowed us to evaluate thc duration of the original immunization All the non-immunized hamsters that were immunosuppressed and injected with VRX-007 were moribund by day) and had extremely high levels of serum ALT/AST The livers from non-immunized group were infected to a much greater extent than their immunized counterparts These results indicate that pre-existing antibody to Ad5 does not affcet the efficacy ofVRX-007

in suppressing tumor growth following intratumoral injection but it does reduce the toxicity ofthe vector Wealso investigated whether

an immune response develops against the tumor and if the vector acts as an adjuvant Hamsters were immunizedagainst the tumor by establishingsubcutaneousHaK(hamsterkidneycarcinoma)tumors Tumors were either injected with VRX-007 or vehicle After two weeks,tumorswcre surgicallyremovedfrom bothgroups HaKcells were injected into the other hind flank of both the groups and also into the hind flank of naive hamsters The growth of tumors in both VRX-007 and vehicle injected groups was suppressed to similar extent whereas in naiveanimals the tumorgrowth was not restricted Thus, an immune responsedevelops against the tumor.No evidence for an adjuvant effect of the vector was obtained; however further research on this important question is needed,

669 Involvement of MyD88 and TLR9 in the Innate Immune Response Elicited by Replication-Incompetent Adenovirus Vectors

Tomoko Yamaguchi,Kenji Kawabata,' Naoya Koizumi.t-' Fu-minori Sakurai,IKazuko Nakashima,' Tomomi Sasaki,INaoki Okada,' Hiroyuki Mizuguchi.P

JLaborator yofGene Transferand Regulation, N ational lnsti-tute ofBiomedical Innovation , Ibaraki, Japan; "Departmentof PharmaceuticalSciences, Showa Pharmaceutical University, Machida , Japan; 'Graduate School ofPharmaceuticalSciences, Osaka University Suita , Japan

A replication-incompetent adenovirus (Ad) vector is arousing growing interest for both gene therapy and immunotherapy A major limitation of the use of Ad vectors is the innate immune response, whieh causes inflammatory cytokine production and tissue damage; however,the precise mechanism of it remains to

be clarified Previous studies demonstrated that antigen-presenting cells express varioustypes of pattern-recognizingreceptors (PRRs), such as toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I),and nucleotide binding oligomerization domain (NOD)-I, and that innate immune responses in antigen-presenting cells were augmented by recognition of pathogens by PRRs TLRs recognize

· 00 ·· - ., - 5)

-· ··· ·· ·~· · - - ~

Mm utes I fl.« gl.lcO se Il ~ on I p&02 15

'"~300

g

§2 00 :2

iii

_ _ 1 liIn p~ 00

A G lucose tol et'~ 25 dsys posl lr am plent

O + r- ,. - ""T"""- ,o 30 60 90 120

Mi"lul ~ net g "'CO ~ 1l;c.d1 on r;- · 0 00 8

.00

microbial components and trigger the signal cascade that activates

innate immune responses Following the recognition of microbial

components by TLRs,they,except for TLR3,transduce

intracel-lular signaling through the adaptor protein,myeloid differentiating

factor 88 (MyD88), which initiates a signaling cascade leading to

NF-kB activation Among 13 members of TLRs,TLR3,TLR4,

TLR7,TLR8, and TLR9 have been demonstrated to be involved in

the recognition ofviral components.Here,we show that Ad vectors

elicit innate immune responses through a MyD88 /TLR9-dependent

and/or -independent manner according to cell types Following

stimulation with Ad vectors, the production of interleukin (IL) -6

was significantly decreased in MyD88- orTLR9-deficient dendritic

cells (DCs), compared with wild-type DCs In addition,the surface

expression of maturation marker protein, such as CD86,was lower

in Ad-infected MyD88-dcficicnt granulocyte-macrophage

colony-stimulating factor (GM-CSF) induced DCs than in wild-type DCs

On the other hand, MyD88- or TLR9-deficient peritoneal

macro-phages produced the same level of IL-6 as wild-type macromacro-phages

after infection with Ad vectors We did not findany differences in

the mRNA expression levels of the molecules involved in innate

immunity,such as MyD88, TLR3,TLR 7,and TLR9,between DCs

and macrophages The intravenous injection of

luciferase-exprcss-ingAd vectors into wild-type,MyD88-deficient,orTLR9-deficient

mice resulted in almost comparable levels of IL-6 production and

luciferase expression These results suggest that Ad vectors can

activate the innate immunity via MyD88/TLR9-dependent and

-independent mechanisms

Nondepleting CD4 Antibody and Cyclosporine Is

Efficient at Attenuating Humoral but Not Cellular

Immunity after Adenovirus Serotype 5 Mediated

Gene Transfer

Jenny H Mclntosh,' Melanie Cochrane,' Andrew M.Davidoff.'

AmitC Nathwani.P

I Department ofHaematology, University College London,

Lon-don, United Kingdom; 2Haematology , National Blood Service,

London United Kingdom; J Department ofSurgery, St Jude

ChildrensResearch Hospital, Memphis, TN.

Gene therapy is being explored for chronic disorders such as

haemophilia B The development of neutralising antibodies to the

FIX transgcne would be a potentially serious complication

of'hacmo-philia B gene therapy.We have previously shown that a combination

ofcyclosporine and non-depleting CD4 antibody (NDCD4), when

co-administered with recombinant adeno-associated virus (rAAV)

vectors prevented a humoral response to capsid proteins, enabling

successful gene transfer upon re-administration ofvectors based on

the same serotype Here we investigate whether the same

immune-modulating strategy can be used to prevent the development of a

humoral immune response to the FIX transgene using a challenging

experimental design that involves intramuscular administration of

an early generation (E IA-E3 deleted) adenovirus serotype-5 vector

encoding human FIX (hFIX) gene under the control ofthe CMV

pro-moter (Ad5-CMV-hFIX) Adenoviral vectors were chosen in

prefer-ence to AAV because they are more efficient at mediating humoral

and cellular immune responses to the adenoviral and transgenic

proteins Balb/c mice were treated on days -1,0 +1, +3, +6, and+8

with NDCD4 (0.5mglmouse) and cyclosporine (CyA) 25mglkg On

day 0,Ix I01"vector particles ofAd5-CMV-hFIX were administered

into the hindlimb muscles Control mice received the same vector

dose without immunosuppression hFIX at therapeutic levels was

detectable in the plasma ofboth the control and immunosuppression

cohorts immediately after vector administration but rapidly declined

to undetectable levels within two weeks of vector administration

In the control animals, this decline in expression coincided with

C opyright © T he American So ci etyo r Gen eTherapy

the development ofncutralizing anti-hFIX antibodies (inhibitors) Inhibitors were, however, not detected in animals treated with im-munosuppression Instead these animals had developed a robust T cell response to the transgene,which most likely eliminated Ad5-CMV-FIX transduced myocytes, Thus, the combination ofNDCD4 antibody and CyA is cffieient at attenuating humoral immunity to the transgenic hFIX proteins cven after administration ofadcnoviral vectors However,our studies suggest that further optimization of the immunosuppressive regimen is required to moderate cellular responses to the transgene following Ad5-CMV-hFIX mediated gene transfer

GENE EXPRESSION IN ANIMAL MODEL SYSTEMS

Fusion Protein into the Regulated Secretory Pathway in Murine and Rat Salivary Gland

Yuval Samuni,IAna P Cotrim,IChangyu Zheng,' Gabor Z Racz,I

Niamh X.Cawley,"Peng Y Loh.? Bruce J Baum.'

'Gene Therapy and Therapeutics Branch, NIDCR, NIH, DHHS, Bethesda, MD; 2 LaboratOlY ofDevelopmental Neurobiology, NICHD, NIH, DHHS, Bethesda, MD

Salivary glands are considered classical exocrine glands that sort and secrete proteins in both regulated (saliva) and constitutive (bloodstream) directions The mechanisms involved in this differ-ential sorting arc believed to be based on structural determinants composed ofamino acid sequences providing a sorting signal or "zip

secreted via the constitutive secretory pathway into the regulated secretory pathway (RSP) and thus into saliva, we investigated whether we could redirect a constitutively secreted protein into the RSP by fusing it with a RSP protein.Human growth hormone (hGH), an endocrine protein normally secreted into the bloodstream via the RSP in somatotrophs of the anterior pituitary, is secreted from salivary glands into saliva by the RSP (Baum et al,Hum Gene Ther, 1999) Conversely,human erythropoietin (hEpo),normally secreted via the constitutive secretory pathway by kidney cells, is secreted constitutively to the bloodstream when expressed in sali-val)' cells (Voutetakis et al, PNAS, 2004).We therefore constructed

a hEpo-hGH fusion protein expression construct and showed that the chimeric product was correctly expressed in HEK 293 cells us-ing ELiSAs and Western blottus-ing specific for both hEpo and hGH Experiments in AtT20 cells,a model endocrine cell line containing both constitutive and regulated secretory pathways,demonstrated significant sorting of the hEpo-hGH fusion protein into the RSP compared to hEpo alone The chimeric eDNA was then packaged

in a serotype 5 adenoviral (Ad5CMVEpoGH) vector and delivered

to murine and rat submandibular cells in vivo viaretroductal can-nulation.Over multiple experiments with different cohorts of mice (n=48) and rats (n=20) we showed that the hEpo-hGH fusion protein was sorted more frequently into saliva,versus thc bloodstream, than the native hEpo protein,demonstrating a redirection of hEpo from the constitutive to the regulated secretory pathway in salivary cells The serum/saliva ratios of hEpo and hEpo-hGH were shown to be statistically different (p<O.OOI).Currently, we are trying to define the specific hGI-I sequence that is sufficient to re-direct hEpo into the RSP Overall,these studies show that a normally constitutively secreted proteincan be dirceted into the RSP and thus into the saliva from salivary gland cells in vivo This may be beneficial for develop-ing novel treatments for multiple upper GI tract disorders

S257