542 the innate immune response to intravenously injected adenoviral vectors: role of endosomal escape in MAP kinase activation and the cytokinechemokine response

542 The Innate Immune Response to Intravenously Injected Adenoviral Vectors Role of Endosomal Escape in MAP Kinase Activation and the Cytokine/Chemokine Response Molecular Therapy Volume 18, Supplemen[.]

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IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY

poorly de ned The genome of Ad encodes two approximately

160-nucleotide-long non-translated RNA polymerase III transcripts:

the virus-associated (VA) RNAs, VA RNA I and VA RNA II To

determine whether RNA transcribed by RNA polymerase III plays a

role in IFN induction by Ad vector, we treated MEFs with ML-60218

(RNA polymerase III inhibitor) and then transduced the cells with

Ad vectors IFN-β production by Ad vector was markedly reduced

by ML-60218 treatment These results demonstrated that RNA

polymerase III is indeed involved in the innate immune response

by Ad vectors We prepared VA-RNA-deleted Ad, which can help

our understandings of the role of VA-RNA in the Ad-induced innate

immune response Ad elicited signi cant amounts of IFN-β in MEFs,

whereas its production level in response to VA-RNA-deleted Ad was

largely decreased To investigate the role of VA-RNA in Ad-induced

in ammatory cytokines and type I IFN in other types of cells,

GM-CSF-DCs were transduced with wild-type or VA-RNA-deleted Ad

There was no difference in IL-6 production between wild-type and

VA-RNA-deleted Ad In contrast, Ad elicited signi cant amounts of

type I IFN, whereas VA-RNA-deleted Ad showed largely decreased

type I IFN secretion These results indicate that VA-RNA can activate

the signaling pathways to induce type I IFN This study provided

important insights into mechanisms of Ad vector-triggered innate

immune responses

538 Immunological Effects of Oral and

Intravenous Cyclophosphamide in Cancer Patients

Treated with Oncolytic Adenoviruses

Vincenzo Cerullo,1 Sophie Escutenaire,1 Sari Pesonen,1 Iulia

Diaconu,1 Liisa Sirrka,1 Anna Kanerva,1 Lotta Kangasniemi,4 Elina

Haavisto,4 Minna Oksanen,1 Tiimo Joensuu,3 A Karioja-Kallio,

Petteri Arstila,2 Akseli Hemminki.1

1 Molecular Cancer Biology, University of Helsinki, Helsinki,

Finland; 2 Haartman Institute, Helsinki, Finland; 3 Cancer Center

Docrates, Helsinki, Finland; 4 Oncos Therapeutics Inc., Helsinki,

Finland.

Accumulating evidence indicates that a dynamic cross-talk

between tumors and the immune system can regulate tumor

growth and metastasis In this respect, use of oncolytic viruses

to break immunological tolerance to tumors is an exciting new

area of research Control of Regulatory T cells and reduction of

the immunosuppressiveness of the tumor microenvironment may

be useful for enhancing the efficacy of T cell mediated tumor

clearance It has been suggested in preclinical and clinical studies that

administration of cyclophosphamide results in selective reduction of

circulating regulatory T cells, associated with a suppression of their

inhibitory functions on conventional T cells and NK cell, which can

lead to a restoration of peripheral T cell proliferation and innate killing

activities However, the effect of cyclophosphamide in cancer patients

treated with oncolytic adenovirus has not been reported thus far

Patients with advanced metastatic solid tumors progressing after

conventional chemotherapy were treated either with metronomic

daily oral cyclophosphamide at 50mg/day starting one week prior

to oncolytic adenovirus injection and lasting weeks or months after

virus treatment, or with one single intravenous administration of 1000

mg one hour before the virus application Also, the combination of

oral and intravenous cyclophosphamide was studied Immunological,

virological and clinical aspects were compared with a retrospective

case-control approach Oncolytic adenovirus was given intratumorally

on a single day Safety was assessed over time and laboratory values

were collected Pro-in ammatory cytokines were also measured

(IL-6, TNF-alpha, IL-8) Safety in general was good and there were no

signi cant differences in side effects between the four groups

The use of cyclophosphamide did not seem to interfere with the

generation of anti-tumor and anti-Adenovirus speci c cytotoxic T

cells as shown by IFN-gamma ELISPOT performed on PBMCs from the treated patients

CD4+CD127-Foxp3high regulatory T cells were also analyzed

by  ow cytometry Interestingly, although still preliminary in all the patients analyzed at the time of the submission we observed a decrease

of the total number of T-Reg

Taken together our data suggests that the use of cyclophosphamide has immunological activity in advanced cancer patients treated with oncolytic adenoviruses Speci cally, it may be able to reduce circulating T-Regs Further, our preliminary non-randomized data suggests that cyclophosphamide is safe and may increase the biological and immunological activity of oncolytic adenoviruses

Complete data will be presented at the meeting

539 The Role of TGF-ȕ and IL-10 in the Development of Immune Tolerance Following

Hepatic Gene Therapy

Brad E Hoffman,1 Ashley T Martino,1 Brandon K Sack,1 Roland

W Herzog.1

1 Pediatrics, University of Florida, Gainesville, FL.

Hepatic gene transfer using an adeno-associated virus vector has been shown to ef ciently induce tolerance to a variety of proteins

in several strains of mice It is also become widely accepted that the microenvironment of the liver favors immune tolerance, by a combination of tolerogenic antigen-presenting cells and cytokines

Previously, we have shown that immune tolerance following liver directed gene transfer is mediated by transgene product-speci c CD4+CD25+FoxP3+ regulatory T cells (Tregs), which suppress humoral and cellular immune responses against the transgene product Additional  ndings by others suggest that various molecules, secreted or expressed on the cell surface of Tregs, such as IL-10 and transforming growth factor beta (TGF-β), may be directly

or indirectly contributing to their suppressive functions In this current study we use two different strains of mice that have been shown to differ in their ability to be tolerized to examine the role of TGF-β and IL-10 in the development of immune tolerance following AAV induced hepatic expression of the human coagulation protein, Factor IX (hF.IX) The C57BL/6 (B6) strain is widely accepted as conferring the highest degree of tolerance, while the C3H/HeJ strain

of mice is considered to have the lowest degree of tolerance In different cohorts, groups of mice were either administered 25mg/kg anti-TGF-β (clone 1D11) or PBS intraperitoneal (i.p.) 1 hour prior to delivery of 1e11vg of AAV2-apoE/hAAT-hFix vector via the splenic capsule Subsequently, the mice received repeated i.p injections of the neutralizing antibody or PBS, every third day At two weeks post gene transfer, the B6 mice showed no statistical difference in their serum levels of hFIX and were negative for antibodies In contrast, the less tolerogenic C3H mice that received the anti-TGF-β had a 2.5 to 3.5-fold decrease in serum hFIX protein and formed low level inhibitors as determined by ELISA Interestingly, when the B6 mice were subsequently challenged subcutaneously with hFIX in CFA, only mice from the group that continued to receive anti-TGF-β developed antibodies against the transgene Additionally, when dnTGFBRII mice (manifest impaired TGF-β signaling, B6 background) were subjected

to the identical vector and CFA/hFIX challenge, they continued to express the transgene without any inhibitor formation These results suggest alternative pathways, perhaps utilizing alternative receptors

To determine if the suppressive effects of Tregs depended upon

IL-10, the previous conditions were used with IL-10 de cient mice (B6 background) The results for serum protein levels and inhibitors were similar to the results of the WT-B6 mice that received anti-TGF-β

Interestingly, suppression of anti-hFIX formation was successfully transferred using tolerized IL-10 de cient CD4+CD25+ Tregs but not using Treg from anti-TGF-β treated mice Taken together, our data suggest a crucial role for TGF-β in immune regulation and

suppression of antibody formation upon hepatic gene transfer, while IL-10, although a likely contributor is not absolutely required

540 Murine Hepatocytes Transduced with Y-F Mutant Capsid Are More Resistant to Killing by Capsid Speci c CD8+ T Cells

Ashley Martino,1 David M Markusic,1 Arun Srivastava,1 Roland

W Herzog.1

1 Pediatrics, University of Florida, Gainesville, FL.

In clinical trial, liver gene transfer of an AAV2 vector expressing factor IX resulted in transient correction of hemophilia B due to a CD8+ T cell response against the viral capsid Recently, we found that mutations of surface exposed tyrosine residues to phenylalanine (Y-F) on the AAV2 capsid improved gene transfer ef ciency and reduced ubiquitination of the capsid protein Poly-ubiquitinated proteins are degraded in the proteasome, and subsequently displayed

as peptides on the surface of MHC I molecules We hypothesized that Y-F capsid mutants would be less ef ciently presented on MHC I molecules because of a reduction in viral dose required for therapeutic expression and in poly-uqibuitination as compared to wtAAV2 To evaluate MHC I presentation of wtAAV2 and AAV2-Y444+500+730F (AAV2TRP) capsids, we established an in vitro CTL

assay using murine hepatocytes Preliminary experiments showed that an AAV2-Y-F capsid mutant vector induced a similar frequency

of IFN-γ + cells as compared to wtAAV2 upon in vitro re-stimulation

with VPQYGYLTL peptide, the dominant, Ld-restricted CD8+ T cell epitope for this strain and also a HLA-B*0702 restricted epitope

in humans, suggesting comparable T cell activation Subsequently, capsid-speci c CD8+ T cells were generated in BALB/c (H-2d) mice using a prime-boost protocol based on IM administration of AAV-2 vector followed by boost with VPQYGYLTL peptide Capsid-speci c CD8+ T cells were expanded in vitro and co-cultured at different

effector:target (E:T) ratios with BALB/c H2.35 adult hepatocytes, transduced 24 hrs earlier with wtAAV2 or AAV2TRP vectors expressing hF.IX Prior to co-culture, hepatocytes were labeled with a  uorescent dye (DiOC18), which labels 100% of cells After 24hrs co-culture, cells were stained with 7-AAD, which speci cally stains dead cells

Flow cytometry was used to determine percent 7- AAD+ hepatocytes

of total DiOC18+ hepatocytes Parallel co-cultures were analyzed 48hrs after gene transfer using immunostaining and  ow cytometry to determine hF.IX+ DiOC18+ cells In two independent experiments we observed speci c killing of AAV transduced hepatocytes, con rming that murine hepatocytes present AAV capsid to CD8+ T cells In order

to achieve comparable transduction ef ciency, an approximately 10-fold higher MOI of wtAAV2 was required Although the extent of hepatocyte killing was MOI-dependent, a reduction in the MOI from

105 to 103 vg/cell resulted in only a 23% decrease in killing at the highest E:T ratio tested for wtAAV2 capsid In contrast, a reduction

in the MOI from 105 to 103 of AAV2TRP transduced cells resulted

in a 60% decrease in killing At equal MOIs, killing of AAV2TRP transduced hepatocytes was reduced 56% and, when adjusted for equal transduction ef ciency, was reduced by 67% These results strongly suggest that AAV2TRP transduced hepatocytes are more resistant to killing by capsid speci c CTL, presumably because of reduced peptide presentation Studies are ongoing to exclude the possibility of shifts in epitope usage

541 Abstract Withdrawn

542 The Innate Immune Response to Intravenously Injected Adenoviral Vectors: Role of Endosomal Escape in MAP Kinase Activation and the Cytokine/Chemokine Response

Jeffrey S Smith,1 Zhili Xu,1 Jie Tian,1 Donna J Palmer,2 Philip

Ng,2 Andrew P Byrnes.1

1 Division of Cellular and Gene Therapies, Food and Drug Administration, Bethesda, MD; 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.

Innate immune responses, characterized by a cytokine/chemokine burst, are a signi cant hindrance to the systemic use of adenoviral vectors for treatment of disease A better understanding of innate signaling pathways may suggest targets for improving the safety of adenoviral gene therapy In order to gain insight into which features

of the adenovirus virion trigger the innate immune response in vivo,

we have investigated the response to a mutant Ad2 virus (ts1) that

is defective for endosomal escape The innate response triggered by ts1 was compared to a helper-dependent Ad2 (HDAd2) vector whose genome contains only non-coding mammalian DNA sequences, without a transgene We found that ts1 was unable to activate early kinase pathways in the liver and spleen and that ts1 was defective for

induction of some, but not all, cytokines and chemokines Results:

We  rst investigated the mitogen-activated protein kinase (MAPK)

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response in the liver and spleen at an early timepoint (30 min)

MAPKs comprise a group of highly conserved intracellular signaling

kinases that are involved in many cellular processes, including

cytokine synthesis Of interest, MAPKs are known to be activated by

toll-like receptors and other pro-in ammatory pathways We injected

mice i.v with 5 x 1012 vp/kg of ts1 or HDAd2, and livers and spleens

were collected 30 min later to examine phosphorylation of MAPKs

by Western blot We found that HDAd2 increased phosphorylation of

p38 and ERK in mice In contrast, ts1 did not cause any signi cant

changes in the phosphorylation of these MAPKs as compared to buffer

controls We also investigated the serum cytokine response to Ad at

6 h Ts1 failed to induce a number of cytokines and chemokines that

were robustly induced by HDAd2, including KC, IL-12p70, IL-6,

TNF-alpha, IL-1beta and G-CSF Interestingly, however, ts1 was able

to induce IP-10, RANTES and IL-10 to similar levels as did HDAd2

Ts1 was also able to partially induce MCP-1 and IFN-gamma

Conclusions: Because we found that ts1-induced kinase and cytokine

responses were severely impaired, we conclude that most of the innate

immune response to adenovirus in vivo is triggered during or after

endosomal escape of the virion However, a subset of cytokines and

chemokines appear to be induced even without endosomal escape,

suggesting a direct response to the viral capsid

543 AAV8 Vectors Evade CTL Mediated

Destruction by Failing To Activate a Potent

In ammatory Response

Suryanarayan Somanathan, Ekaterina Breous, Peter L Bell, James

M Wilson

Gene Therapy Program, Department of Pathology & Laboratory

Medicine, University of Pennsylvania, Philadelphia, PA.

We found previously that liver-directed gene transfer in mice using

AAV serotype-8 vectors resulted in stable long-term expression of

vector encoded transgenes Persistence correlated with absence of

cytotoxic T lymphocyte (CTL) response to the transgene Stable

expression from AAV8 vectors could not be abolished even when mice

were challenged with Ad5; an adenovirus known to induce potent

cytolytic T cell responses Most notably, there was an absence of IFN-γ

secreting CTLs in mice systemically administered with AAV8 when

compared to animals that only received an adenoviral vector Thus,

it remains unclear whether AAV8 transduced cells can be cleared

upon manifestation of a potent CTL response To study the effect

of CTLs on AAV8 transduced hepatocytes, we adoptive transferred

Ad5 activated CD8 T cells from donor syngeneic mice (C57B/6) into

recipient mice (Rag-/-) stably expressing AAV8 encoded transgene

The use of Rag -/- mice allowed us to monitor the effect of donor

CTLs without interference from recipient T or B cells Although Ad5

induced CTLs could clear HAdV5 nlacZ expression in Rag-/- mice,

they failed to eliminate AAV8 nlacZ expression The absence of

CTL killing could be due to insuf cient “danger signals” that cause

ignorance of AAV8 transduced hepatocytes by circulating CTLs To

test our hypothesis we co-administered TLR ligands LPS and CpG

at the time of adoptive transfer LPS and CpG activate TLR 4 and

9 respectively, and are potent inducers of a systemic in ammatory

response Our results show that co-administration of LPS or CpG

with the CTLs led to the elimination of AAV8 transduced hepatocytes

with a concomitant decrease in AAV8 vector genome in the liver

In control experiments we established that CTLs to an irrelevant

transgene (eGFP) even when co administered with TLR ligands failed

to eliminate transgene expression Livers of C57B/6 mice injected

with LPS and CpG showed upregulation of MHC class I and cell

adhesion molecules such as ICAM-1 and VCAM-I Similar results

were also observed in mice that received adenovirus but not AAV8

Our results demonstrate that the propensity for in ammation arising

from genetic or environmental factors may negatively in uence AAV

gene transfer to the liver

544 Robust and Sustained Factor IX Expression by IV Administration of AAV8-Based Vectors in Macaques

Hiroaki Mizukami,1 Jun Mimuro,2 Akira Ishiwata,2 Hiroya Yagi,1 Tsukasa Ohmori,2 Seiji Madoiwa,2 Tomonori Tsukahara,1 Masashi Urabe,1 Akihiro Kume,1 Yoichi Sakata,2 Keiya Ozawa.1

1 Div of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan; 2 Div of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

Adeno-associated virus (AAV) vectors are promising for gene therapy approaches especially for hemophilia Among the increasing number of serotypes and strains in AAV, serotype 8-based vectors remain particularly suitable for liver-directed trials In order

to evaluate therapeutic efficacy of AAV vectors in hemophilia gene therapy, we have been working on preclinical studies using cynomolgus macaques Earlier results indicate that the AAV8-based vectors are capable of transducing macaque liver ef ciently by intravenous injection On the other hand, inhibitory actions of pre-existing neutralizing antibody (NAb) against vector capsid, even at marginal levels, are also demonstrated, suggesting the necessity of more sensitive assay for NAb detection To solve this issue, we have improved NAb detection system against AAV8 capsid with higher sensitivity Five sero-negative male cynomolgus macaques were selected AAV8-based vector encoding mutant macaque factor IX driven by liver-speci c promoter was injected intravenously at 2 ×

1012 vg/kg (2 animals) or 5 × 1012 vg/kg (3 animals), which are relevant vector dose in human trials All of the animals exhibited robust and sustained factor IX expression at the therapeutic window (3.0 – 20.2%

of normal) No differences were found in factor IX concentration

by the vector dose High copy numbers of vector genome (12.9 ± 1.3 vg/dge) were detected in the liver biopsy specimen Our results indicate a prospect of hemophilia gene therapy using AAV8 as well as the suf cient sensitivity of our improved NAb assay, which would be useful in human clinical trials using AAV8-based vectors

This study was performed in collaboration with Tsukuba Primate Research Center, National Institute for Biomedical Innovation, and The Corporation for Production and Research of Laboratory Primates, Japan

545 Induction of Transgene Immunological Tolerance by a Single Injection of miR-142-Regulated Integrase Defective Lentiviral Vector

Andrea Annoni,1 Alessio Cantore,1 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2 Vita-Salute San Raffaele University, Milan, Italy.

We have developed a microRNA-regulated lentiviral vector (LV) platform, which can maintain long-term sustained transgene expression in the liver of immune competent mice and induce tolerance to the transgene product

In order to reduce the risk of insertional mutagenesis and to broaden its applications, we evaluated the use of integrase-defective lentiviral vectors (IDLV) for liver gene transfer IDLV can drive low-level transgene expression from the non-integrated proviral forms that transiently accumulate in transduced cells Because this episomal DNA is progressively lost in actively dividing cells, transgene expression is only transient in proliferating cells while IDLVs have been reported to drive prolonged transgene expression

in non-dividing target cells

We administered intravenously to mice matched doses of integrase-competent lentiviral vectors (LV) or IDLV encoding GFP under the control of the hepato-speci c promoters and carrying target sequences for miR-142 (ET.142T) At 1 week post-injection we found detectable

GFP expression in the liver of IDLV-treated mice but to a much lesser extent than that observed in the liver of mice treated with the integrating LV counterpart At 6 weeks post-injection, GFP expression was substantially, but not completely, reduced in IDLV-treated mice while it remained stable in those injected with LV Similar  ndings were obtained measuring vector DNA content in the liver

We then investigated whether IDLV are suitable to generate tolerance to a speci c antigen for immunomodulatory purposes

Six weeks after IDLV treatment mice were re-challenged with the antigen to evaluate the induction of secondary anti-transgene immune response The absence of response to transgene indicated that IDLV.142T treatment generate a state of transgene-speci c immunological tolerance in recipient mice On the contrary, secondary expansion of transgene-speci c CD8+ T cells were detectable in mice injected with IDLV encoding GFP under the control of an ubiquitously expressed promoter, indicating that those mice maintained GFP responsiveness In addition, residual levels of vector DNA were detected only in mice receiving the tolerogenic IDLV.ET.142T, while transduced hepatocytes in those treated with the control vector were completely cleared Mechanism of tolerance induction and role of regulatory T cells are currently under investigation

Overall, these data indicate that a single administration of IDLV

ET.142T can tolerize treated mice towards the encoded protein minimizing the risks of insertional mutagenesis This important and unexpected feature candidates hepatocytes-targeted IDLV to the development of novel immunomodulatory strategies that may

be useful to tolerize individuals to protein replacement therapies

or to prevent the development of autoimmune disease in at-risk individuals

546 T Cell Hyperactivity in Cmah Null Mice: A

Pre-Clinical rAAV Immunotoxicity Model?

George Buchlis,1,2 Federico Mingozzi,2 Daniel J Hui,2 Harre Downey,2 Virginia Haurigot,2,4 Shangzhen Zhou,2 Ajit Varki,3 Katherine A High.1,2,4

1 University of Pennsylvania School of Medicine, Philadelphia, PA; 2 Pediatrics-Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 3 Medicine, University of California at San Diego, La Jolla, CA; 4 Howard Hughes Medical Institute, Philadelphia, PA.

Gene transfer with AAV treats a range of diseases in animal models, but shows more limited success in humans In a clinical trial involving liver-directed AAV2 transfer of factor IX(F.IX) for the treatment of Hemophilia B, two subjects developed transient liver enzyme elevation 4-6 weeks after vector administration, accompanied by a decline in circulating levels of factor IX[Nat Med 2006] This occurred in spite of long-term transgene expression and absence of vector immunity in canine hemophilia models and

in non-human primates We showed that a resident pool of AAV2 capsid-speci c CD8+ T-cells expanded upon vector administration and may have destroyed transduced hepatocytes, resulting in loss

of transgene expression and concomitant transaminitis[Nat Med 2007] Thus animal models of rAAV-mediated gene transfer have not been immunologically predictive of the human outcome Of note, glycosylation pro les of human T-cells differ substantially from even those of the closest primate relatives: an inactivating mutation of the sialic acid(Sia) converting enzyme CMAH caused the elimination of expression of Sia Neu5Gc from human cells and alterations in expression and binding of inhibitory receptors(Siglecs) that bind these Sias A correlative study showed that human TCR’s were hyperactivatable when compared to primates and that responses could be lowered upon Siglec expression[PNAS 2006] In an attempt

to model human immunity, we harvested Cmah-/- and C57BL/6

mouse splenocytes, labeled them with CFSE, and plated them with anti-CD3/anti-CD28 beads We assayed cells after 3 and 5 days for

T cell activation markers CD62L and CD44, with CFSE dilution as

a measure of the degree of proliferation At day 3, CD62L levels

were signi cantly lower in Cmah-/- T cells compared to C57BL/6

controls, as this lymph node localizing lectin is shed upon activation This was true for both CD4(3% v 24% low) and CD8(5% v 18% low)

T cells Analysis at day 5 showed signi cantly lower Cmah-/- CD4

CD62L levels (71% v 85% low) and CD8 CD62L levels(68% v 79% low), albeit not as great of a difference as that seen at day 3 CD44 expression, a positive marker for activation, was signi cantly higher

in Cmah-/- CD8 T cells at day 3(78% v 49% high) and Cmah-/- CD4

T cells at day 5(47% v 31% high) In addition to higher activation

levels, a signi cantly higher proportion of Cmah-/- CD8 T cells(67%

v 44% low) and CD4 T cells(23% v 9% low) proliferated by day 5 vs C57BL/6 T cells as measured by low CFSE expression These results detail a novel phenotype in a mouse model of a human mutation Since humans exhibit a signi cant T cell response to hepatic administration

of rAAV, mice with the human inactivation of CMAH that show greater T cell activation may serve as a better pre-clinical model for rAAV-mediated liver gene transfer, and experiments are ongoing to determine whether or not this is the case

547 Complement-Mediated Clearance of AAV by Tissue Resident Macrophages Limits Transduction

of Target Cells

Chelsea M Bolyard,1 Jeffrey S Bartlett.1

1 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH.

We have previously shown that AAV particles are opsonized

by components of the complement system, yet are still capable of ef cient gene transfer following systemic administration Postulating that increased circulatory retention might improve bioavailability and enhance gene transfer, we were interested in characterizing the role

of circulating opsonins in the clearance of AAV We hypothesized that limiting “dead-end” interactions with phagocytic cells might enhance extravascular transport and transduction of target tissues The fact that AAV elicits very little in ammatory response suggested that the mechanism of AAV clearance was different than that of other microorganisms, since most phagocytic cells require in ammation

to become “activated” and clear foreign particles Recently, tissue resident macrophages have been shown to clear C3-opsinized particles via a newly identi ed complement receptor of the immunoglobin super-family, CRIg This receptor preferentially binds iC3b-opsinized particles in the absence of in ammation and activation Having previously established a threshold response for hepatic in ammation following AAV delivery, we hypothesized that lower doses of AAV might be cleared via CRIg expressed on Kupffer cells residing in the lumen of the liver, but when very high doses of AAV were delivered, these cells became activated by engagement of a second receptor (CR3), causing perpetuation of a systemic response, and recruitment and activation of other phagocytes

To characterize the interaction of complement-opsinized AAV with CRIg, we utilized CHO cells expressing murine CRIg (mCRIg) iC3-opsonized AAV bound readily to these cells Binding was dependent

on mCRIg, since CHO cells that did not express the receptor bound very little vector; and was also dependent on the presence of iC3b

on the vector surface, since only vector treated with complement-containing mouse serum, or with iC3b generated from C3 in the presence of serum factor H (fH) and factor I (fI) was able to bind mCRIg-CHO cells In order to model a situation wherein tissue-resident macrophages expressing CRIg might limit AAV-mediated transduction of parenchymal cells by sequestering complement-opsonized AAV, we developed a dual cell culture system in which both target cells (HeLa C12) and cells expressing CRIg (mCRIg-CHO) at approximately physiological ratios were treated with AAV vectors that had been exposed to serum complement components

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