334 efficient gene transfer in cultured aortic smooth muscle and endothelial cells by magnetically responsive nanoparticles impregnated with adenovirus

334 Efficient Gene Transfer in Cultured Aortic Smooth Muscle and Endothelial Cells by Magnetically Responsive Nanoparticles Impregnated with Adenovirus and display high transduction efficiencies in a[.]

and display high transduction efficiencies in a wide range of cell

types,both in cell culture and in animals However,data obtained

from different studies indicate that gene transfer by Ad vectors is

confronted with differenthurdles,Onc ofthcsc hurdles is poor

trans-duction efficiency in target cells lacking expression of the primary

Ad receptor, CAR (Coxsackie and Adenovirus Receptor) Besides,

following in vivo systemic delivery,different tissues are transduced,

and particularly the liver, with a high risk of toxicity We first have

shown that RGD inelusion in thc HVR5 loop of the hexon monomer

(AdHRGD) providesAd with anew integrin-mediated entry pathway

in cells in vitro (Vignc ct aI.,J.Virol, 1999) While performing

bio-distribution studies in mice, we observed that transgene expression

afforded byAdHRGD was dramatically decreased at48h p.i in liver

compared with that obtained with unmodified control Ad (Adwt)

[n order to analyze whether this decrease was due to RGD motif or

to [-[VR5modification itself, we constructed different Ad

contain-ing either Gly-Ala repetitions of different lengths (AdH(GA)8 and

AdH(GA)24) or HVR5 sequences from other Ad serotypcs (Ad2,

Ad 19 and Ad30) into hexon protein, Following systemic

adminis-tration in different strains of mice (C57BLl6,BALB/c,C3H),we

similarlyobserveda dramatic decrease(>95%),in

transgeneexpres-sion in liver Iysates, at 48h p.i., whatever the hexon modification

was This severe decrease in transgene expression was correlated

with an important reduction (84 to 97%) ofAd DNA content in liver

as documented by Real-Time Q-PCR.However, measurement of

liver viral genome content at an earlier time point (30 min) showed

a similar accumulation of vector genome in total liver extract for

AdH(GA)24 and the control Adwt Thus, vectors containing HVR5

modifications are cleared more rapidly from the liver than Adwt

Studies are on their way to examine whether this elimination of viral

DNA is linked to a reduction ofhepatocytes transduction and/or to

a higher uptake by non-parenchymal cells Besides,we observed

that AdH(GA)24 was as efficient as Adwt for transducing tumor

cells confirming that Ad infectiousness was not impaired in vivo

by hexon HVR5 modification.Altogether,our results suggest that

enhanced tumor gene transfer could be achieved following

inser-tion of targeting peptides into hexon protein while avoiding a risk

of transgene expression into hepatocytes

333 Converging Evidence for a Critical Role

of the Diameter of Sinusoidal Fenestrae in

Hepatocyte Transduction after Adenoviral Gene

Transfer

Frank Jacobs,' Eddie Wisse, Yingmei Feng,'Jan Snoeys,' Joke

Lievens,' Changchun Ling; Hans Duirnel,' Desire Collen,' Peter

Frederik,' Bart De Geest.'

'Center/or Molecular and Vascular Biology University0/

Leuven, Leuven, Vlaams Brabant Belgium; 2EM Unit Pathology,

University Maastricht Maastricht Limburg Netherlands

Background: Anatomical barriers constitute a major obstacle

for gene transfer The presence of fenestrae in the sinusoidal

endo-thelium is critical for efficient transduction of parenchymal liver

cells (PC) Previously,we have demonstrated that straindifferences

of transgeneDNA uptake in PC after adenoviral gene transfer in

rabbits correlate with the diameter of sinusoidal fenestrae In

addi-tion, interventions that increase the diameter of fenestrae in New

Zealand White (NZW) rabbits lead to increased transgenc DNA

uptake in PC Objective: The objective of the current study was to

provide further evidence for the role of the diameter of fenestrae

in hepatocyte transduction Therefore,filtration experiments using

filters containing 80 nm, 100nm and 200 nm pores were performed

Second, hepatocyte transduction and the diameter of'fenestrae were

compared in NZW rabbits,C57BLl6 mice and Sprague Dawley

(SD) rats Third,NZW rabbits were comparedwith a newly

gcncr-atcd substrain ofNZW rabbits with significantly higher transgcnc

SI26

expression Results: Using Vitrobot" technologyand cryo-electron microscopy, the diameter of human adenoviral serotype 5 vectors was shown to be 93 nm with protruding fibers of30 nm Filtration experiments demonstrated that the recovery of adenoviral vectors

in the filtrate determined by real-time PCR analysis was 0.43±

0.091 %,51 ± 7.3 % and 91± 1.9 % for a 80 nm, 100 nm and

200 nm filter, respectively Scanning electron microscopy of the filters was in agreement with these data.The average diameter of fenestrae determined by transmission electron microscopy in NZW rabbits (103±1.3 nm; n""IO) was lA-fold (p<O.OOOI) lower than in C57BLl6 micc (14[±5.4 nm; n""4) and 1.6-fold lower than in SD rats.This was associatedwith 8.8-fold (FO.O[) and 12-fold(p<O.O[) lower transgene DNA levels in PC at day 3 after adenoviral transfer

in NZW rabbits than in C57BLl6 mice and SO rats,respectively

In contrast, the copy number in the non-parenchymal liver cells (NPC) at day 3 after transfer in NZW rabbits was 5.I-fold(p<O.05) and 4.6·fold (p<0.05) higher than the copy number in the NPC of C57BLl6 mice and SD rats,respectively,Thus, species differences

of intrahepatic transgcnc DNA distribution arc consistent with an anatomical barrier for hepatocyte transduction in NZW rabbits but not in C57BLl6 mice and SO rats Compared to C57BLl6 mice, human apo A-[ transgeneexpression was 63-fold lower inNZW rab-bits and not significantly different in SD rats.In the course of these studies, one NZW rabbit and his F2 offspring (n""4) showed a more than 50-fold higher transgene expression compared to a series of 16 New Zealand White controls The diameter of fenestrae in these 5 rabbits was significantly larger than in control NZW rabbits (113±

1.5 nm versus 103± 1.3nm;p==O.0005) Conclusion: The current study provides further converging evidence for a critical role of the diameter ofsinusoidal fenestrae in hepatocyte transduction

334 Efficient Gene Transfer in Cultured Aortic Smooth Muscle and Endothelial Cells

by Magnetically Responsive Nanoparticles Impregnated with Adenovirus

Michael Cherny,'Robert J Levy.'

'Cardiology Research The ChildrensHospital0/Philadelphia Philadelphia , I'll.

Replication deficient adenoviruscs (Ad) arc a promising vector for gene therapeutic applications due to their high gene transfer efficiency in both quiescent and dividing cells and high transgene capacity.However their clinical use is limited by side effects due

to distribution to non-target tissues, inflammatory response and reduced efficiency upon readministration Entrapment of Ad in bioresorbable magnetically responsive nanoparticles (MNP) may enable magnetically targeted delivery and minimize the inflam-matory response, thereby improving the efficiency and safety of these vectors We hypothesized that magnetically driven delivery

of Ad formulated in bioresorbable MNP would result in efficient gene transfer in vascular cells in culture.Ad entrapment in iron oxide laden MNP (290± 20 nm) was achieved by controlled ag-gregation of ferrofluid in the presence of calcium with or without poIY-L-arginine (pA) The efficiency and kinetics of gene transfer usingMNP-GF~d was studied in rat aortic smooth muscle (A10) and bovine aortic endothelial cells (BAEC) as a function of MNP composition and amount, and magnetic exposure The cells were incubatedwithMNP-GF~d for 15 min with or without a magnetic field and the transgene expression was assayed fluorimetrically in cells I - 21 days post treatment usingAJ)"omof 485 nm/535 nm The gene expression in both cell types was strongly dependent on the MNP dose and the formulation amount of pA, and peaked at

72 hr equaling 194 and 94 RFU in BAEC and A10 cells, respec-tively,treated with AMNP-GF~d in the presence of magnetic field Cell treatment with"MNPwithout a magnctie field exposure or pA modificationresulted in 42-fold and 140-foldlower gene expression

Molecul ar Therapy V olume 15 S upplement I•.\by 2<)07

C opyright © The Am eric m Society o f Gene Therapy

rates, respectively,at this timcpoint, reflecting the difference in the

nanopartiele cellular uptake under these conditions.Interestingly,

the gene expression following ANP-GF~d treatment was protracted

in both cultured A 10 and BAlk cells, equaling 15% of the peak

value after 21 days in the latter It is concluded that entrapment

and magnetically driven delivery of Ad using bioresorbable MNP

results in high levels ofgene expression in cultured smooth muscle

and endothelial cells

335 Spliceform Specific

Coxsackie-Adenovirus Receptor Interactions with MAGI-1b

Nicholas D Ganserner,'Joseph Zabner,' Katherine J D A

Excoffon.'

'Internal Medicine , University ofIowa , Iowa City , IA.

The Coxsaekicvirus and Adenovirus Receptor (CAR) is a

trans-membrane viral receptor that also functions in homotypic cell

adhesion The gene encoding human CAR,Cxadr,was previously

described as consisting of 7 alternatively spliced exons Mouse

Cxadr consists of8alternatively spliced exons with 2 possible

c-termini: one that is similar to the previously described human

Cxadr terminating in exon 7 (CARE'7), the other terminating in

exon8(CARE,S) Both splice forms contain a carboxy-terminal PDZ

binding domain and differ only in thc last 26 (Exon 7,-GSIV) or 13

(Exon8,-ITVV) amino acids We hypothesized that a human CARE,s

isoform exists and will have similar virus receptor characteristics to

that ofCARE'7,but will differ in biological aspects making it

evo-lutionarily relevant Human CARE,S was successfully cloned from

primary human airway epithelia and the cDNA was inserted in the

pcDNA3 I expression vector Adenoviral receptor activity, protein

expression,localization,and PDZ-interactions were subsequently

determined in vitro As expected,both CARE'7and CARE'"showed

similar adenovirus infection Expression levels,as determined by

western blot,and localization,as determined by immunostaining,

were also similar However, a difference was seen when

co-ex-pressed with the PDZ-domain containing and CAR-interacting

protein MAGI-I b CARE,7 alters the localization ofMAGI-I b from

diffusely cytoplasmic to sites of cell junctions In contrast, when

CARE'"was co-expressed with MAGI-Ib, CARE'S was lost from the

ceil CARE'Sexpression was maintained when co-expressed with

MAGI-Ib in the presence of proteasomaI inhibitors This suggests

that the CARE,S isoform undergoes proteosomal degradation in the

presences of MAGI-I b To determine if'the 4 amino acid PDZ

bind-ing domain was responsible for this,the CARE,7 motif(GSIV) was

swapped with the CARE,Smotif(lTVV).Interestingly,localization

of both CARE'7ITVV and CARE,SGSIVwere unaltered and both were

able to translocate MAGI- Ib from the cytoplasm to the junctions

To determine whether the extracellular and transmembrane domain

were required for this isofonn specific degradation, the c-tenninus

of each isoforms was fused to RFP (RFpE'7, RFpExS) and expressed

in the presence of MAGI-I b When expressed on its own, both

RFpE,7 and RFpE,s showed mainly ER or nuclear localization In

the presence ofMAGI-lb, RFpEx7 co-localized with MAGI-Ib and

was diffusely cytoplasmic RFpE,8 remained only in the nucleus,

isolated from MAGI-lb In summary, whereas CARE'7 regulates

the localization of MAGI-Ib and other PDZ domain containing

proteins,MAGI-Ib is able to regulate CARE'"

Molecular Therapy Volume15 Supplement 1 ~ br 2007

336 Protein IX-Modified Adenovirus Gene Transfer Vectors Can Efficiently Be Targeted to LRP by the Receptor-Associated Protein RAP

Stephanie Corjon,Tatjana Engler,Stefan Kochanek,Florian Kreppcl

'Division ofGene Therapy ; University ofUlm , VIm , Germany.

Successful usc of adenovirus type 5 based vectors for gene de-livery requires retargeting to the organs or cell types of interest by modification ofAd capsid proteins Multiple strategies to generate re-targeted Ad vector particles have been described Recently,the minor capsid protein piX has gained a high level ofattention since it allows for genetic incorporation of relatively large protein moieties which may serve as ligands for specific receptor targeting However,up to date, no full-length protein ligand has been demonstrated to mediate successful and robust targeting to its eorrcsponding receptor when fused to pIX In this study,we present (i) a geneti-chemical targeting technology for specific modification of the piX protein, (ii) the first example of a full-length protein ligand that is capable of efficient receptor targeting when coupled to pIX, and (iii) a detailed compari-son of the targeting efficiencies of different ligands when coupled

to fiber or pIX capsid proteins To allow for specific and efficient coupling ofligands to the C-tenninus of pIX in a similar manner as described for fiber (Kreppel et al.,Mol Ther.2005 Jul; 12:107-117),

we genetically added a cysteine residue to the C-tenninus ofpIX via

a 75-Angstrom spacer sequence This vector could be produced to high titers and did not show vector particle aggregation As a proof

of concept, we successfully coupled rnaleimide-activated biotin specifically and efficiently to the genetically engineered cysteine on pIX as demonstrated by Western transfer experiments To analyze receptor targeting by ligands coupled to pIX,we chose to couple the Receptor-Associated Protein RAP to piX, which is known to strongly bind to the low-density lipoprotein receptor-related protein (LRP) and might, therefore, be used as a ligand for hepatocyte targeting FACS experiments, including competition experiments with excess free RAP and performed with LRP-positive (CI-IO

K I) and l.Rl negative cells (CHO LRP-) showed a strong recep-tor-specific targeting when RAP was coupled to pIX In the same manner,we tested targeting to CD71 by coupling of transferrin to piX Surprisingly, no targeting but slight detargeting occurred with the appropriate cell line (K562) Finally, when linked to fiber,both RAP and Transferrin-modified vectors were strongly targeted to LRP and CD71, respectively This data suggest that the intracellular fate

of the ligand can playa crucial role for the targeting efficiency of plX.modified vectors.This assumption is supported by the fact that transferrin and its receptor arc known to bc recycled after uptake and might carry the modified pIX vectors back to the cell surface

In contrast., RAP is known to detach from its receptor LRP,which might allow the vectors to escape from the early endosomes and to reach the nucleus In this study, we describe the first example of an adenovirus vector that is strongly targeted to LRP by carrying the full-length protein ligand RAP covalently linked pIX Our finding that the intracellular fate of ligands together with their position on the capsid (pIX versus fiber) can strongly influence the success of targeting is important for all targeting strategies used today

SI27