comparison of adenoviral and adeno-associated viral...

HUMAN GENE THERAPY 15405ndash413 (April 2004)copy Mary Ann Liebert Inc

Brief Report

Comparison of Adenoviral and Adeno-Associated ViralVectors for Pancreatic Gene Delivery In Vivo

ALFRED Y WANG12 PETER D PENG2 ANJA EHRHARDT2

THERESA A STORM2 and MARK A KAY2

ABSTRACT

Although effective gene therapy vectors have been developed for organ systems such as the liver an effectivedelivery vector to the pancreas in vivo has remained elusive Of the currently available viral vectors adeno-virus and adeno-associated virus (AAV) are two of the most efficient at transducing nondividing cells Wehave constructed recombinant adenovirus (AdVLacZ) adeno-associated virus serotype 2 (AAV2LacZ) andpseudotyped adeno-associated virus serotype 5 and 8 (AAV5LacZ AAV8LacZ) carrying the LacZ reporterand compared the transduction efficiency of these four vectors in the pancreas of mice in vivo We showedthat adenovirus AAV2 and AAV8 are capable of transducing the pancreas in vivo but with different ex-pression kinetics efficiencies of transduction and persistence AdVLacZ-transduced pancreas exhibited max-imum LacZ expression at 1 week postdelivery with greater than 90 of expression lost at 4 weeks AAV2LacZ-transduced pancreas displayed peak LacZ levels at 4 weeks postdelivery with no significant decrease inexpression for up to 8 weeks AAV8LacZ was at least 10-fold more efficient than AAV2LacZ in transducingthe pancreas in vivo with significant levels of expression detectable at 1 week whereas AAV5LacZ did notresult in any detectable transgene expression at all tested time points All three vectors primarily transducedpancreatic acinar cell types with limited transduction of pancreatic endocrine cells AdVLacZ elicited a sig-nificant leukocyte infiltration early after delivery into the pancreas whereas none of the AAV vectors eliciteda significant leukocyte response None of the tested vectors caused significant changes in serum amylase orblood glucose levels suggesting that they do not significantly alter pancreatic function These vectors will beuseful for studying novel gene delivery based treatments in animal models for diabetes and other pancreaticdisorders

405

INTRODUCTION

THE PANCREAS is a complex organ that serves many criticalendocrine and exocrine functions crucial for survival (Ed-

lund 2002) Common maladies of the pancreas include diabetesmellitus pancreatitis and pancreatic cancer No effective long-term therapies are available for any of these Viral-mediatedgene delivery is currently the most efficient gene transfermethod and of these viral vectors recombinant adenovirus(AdV) is probably the most efficient and has been shown totransduce a variety of organ systems including the liver skele-tal muscle heart brain and lung (Breyer et al 2001) In mouse

and rat pancreas adenovirus has been shown to efficiently trans-duce pancreatic cells in vivo but expression of the transgene istransient and the viral proteins elicit a potent immune response(Raper and Dematteo 1996 McClane et al 1997a) In addi-tion administration of AdV to the mouse pancreas in vivo re-sults in significantly decreased intracellular amylase levels andassociated histologic damage suggesting that the vector is quitetoxic to the organ (McClane et al 1997b)

Adeno-associated virus (AAV) is a single-stranded DNAvirus which unlike adenoviral vectors does not elicit a signif-icant inflammatory response making it a particularly promis-ing gene delivery vector (Snyder 1999 Monahan and Samul-

Departments of 1Molecular Pharmacology 2Pediatrics and Genetics School of Medicine Stanford University Stanford CA 94305

ski 2000 Zaiss et al 2002 Stilwell and Samulski 2003) AAVhas been shown to efficiently transduce a variety of cell typesin vivo including liver muscle and lung resulting in long-termgene expression from these tissues (Carter and Samulski 2000)Ex vivo AAV has been shown to transduce pancreatic endo-crine cells (Flotte et al 2001) but successful transduction ofthe pancreas by AAV has not been demonstrated in vivo It hasbeen demonstrated that ex vivo viral transduction efficienciesdo not always correlate with in vivo efficiencies (Kay et al1992 Snyder et al 1997 Park et al 2000) so we wanted todetermine whether AAV could also efficiently and safely trans-duce pancreatic cells in vivo Several novel serotypes of AAVhave been identified each which exhibit different structural andfunctional characteristics (Rutledge et al 1998 Gao et al2002 Grimm and Kay 2003a) Recent studies have shown thatthe different serotypes of AAV have different cellular speci-ficities and in particular AAV serotype 5 was shown to trans-duce human pancreatic islets ex vivo with a much higher effi-ciency than AAV serotype 2 (Flotte et al 2001) We alsowanted to determine if different serotypes of AAV could resultin greater efficiency transduction of the pancreas in vivo

MATERIALS AND METHODS

Recombinant viruses

The LacZ expression cassette (consisting of a Rous Sarcomavirus [RSV] promoter driving the expression of an Escherichiacoli b-galactosidase gene with a nuclear localization signalflanked by a bovine growth hormone polyadenylation site) wasexcised from plasmid pRSVnlsLacZ using BglII and cloned intothe BglII site of the pDE1 adenovirus shuttle plasmid or blunt-ended and cloned into the NotI site of the pAAV shuttle plas-mid AdVLacZ was amplified in 293 cells and subsequently pu-rified in a cesium chloride (CsCl) density gradient as previouslydescribed (Kay et al 1994) AAV2LacZ (AAV serotype 2) wasproduced by the triple-transfection method in 293 cells and pu-rified using a high-performance liquid chromatography (HPLC)method as described earlier (Nakai et al 2000) PseudotypedAAV5LacZ and AAV8 LacZ were produced by cross-packag-ing the AAV2 vector genome (containing the ITRsLacZ ex-pression cassette) into AAV5 or AAV8 capsids by cotransfec-tion of the pAAV plasmid with DF5 (containing AAV2 RepAAV5 capsid and adenovirus helper genes) or triple transfec-tion of pAAV with R2C8 (containing AAV2 Rep and AAV8capsid genes) and plAd5 (containing adenovirus helper genes)plasmids (Gao et al 2002 Grimm et al 2003b) The viruseswere purified using CsCl density centrifugation AdVLacZ was titered by a plaque-forming assay whereas AAV2LacZAAV5LacZ and AAV8LacZ were titered by a quantitative den-sitometric dot-blot assay To confirm the expression of the LacZtransgene from the two recombinant viruses 293 cells weretransduced with 1 3 108 AdVLacZ and 1 3 108 AAV2LacZand stained with X-gal 2 days posttransduction Positive LacZexpression was observed in AdVLacZ- AAV2LacZ- AAV5-LacZ- and AAV8LacZ-transduced 293 cells

Surgical procedures

All animal procedures were done according to the guidelinesfor animal care at Stanford University (Stanford CA) Eight-

to 12-week-old male C57Bl6 mice were anesthetized using asingle intraperitoneal injection of a mixture of ketamine ace-promazine and xylazine suspended in phosphate-bufferedsaline (PBS) Mice were subjected to laparatomy and injectedwith 100 ml of purified virus solution suspended in PBS usinga 26-gauge needle into a single location in the splenic lobe ofthe pancreas Laparotomies were subsequently closed with atwo-layer suture Mice were then sacrificed at various dayspostinjection and pancreatic tissue was harvested for X-galstaining and immunohistochemistry

Tissue embedding and X-gal staining

Pancreatic tissue was washed in PBS and placed in optimalcutting temperature (OCT) compound and immediately frozenon dry ice Ten-micrometer sections were then cut on a cryo-stat and placed on VWR Superfrost plus microscope slides (ErieScientific Company Portsmouth NH) Tissue sections werefixed in 125 glutaraldehyde in PBS for 10 min and rinsed 3times in PBS prior to staining Slides were then placed in X-Gal solution (1 mgml X-gal 5 mM K3Fe(CN)6 5 mMK4Fe(CN)6 and 1 mM MgCl2 in PBS) and incubated overnightat 37degC Slides were then washed three times in PBS and thencounterstained with hematoxylin

Immunohistochemistry

Rat anti-mouse CD45 antibodies were obtained from BDPharmingen (San Diego CA) Slides were processed using theVectastain ABC kit (Vector Labs Burlingame CA) Pancreassections were blocked with 10 normal goat serum and avidinfor 30 min and then incubated with 1250 dilution of primaryrat anti-mouse CD45 antibody in PBS1 bovine serum albu-min (BSA)10 goat serum and biotin for 2 hr Subsequentlythe slides were then washed 3 times in PBS and biotinylatedmouse anti-IgG2 secondary antibody diluted 1500 in PBS1BSA10 goat serum was applied for 30 min Slides werewashed 3 times in PBS and then incubated with the ABCreagent for 30 min The sections were thoroughly washed andincubated with 006 339-diaminobenzidine and 001 H2O2

for 5 min and counterstained with hematoxylin

Monitoring changes in pancreatic function

Whole blood and sera from mice injected with saline AdVLacZ AAV2LacZ AAV5LacZ and AAV8LacZ were col-lected at various days postinjection by retro-orbital eye bleedsGlucose levels were measured using a Glucometer Elite glu-cometer (Bayer Healthcare Pittsburgh PA) and Ascensia Eliteblood glucose test strips (Bayer Healthcare) Amylase levelswere measured from the collected sera samples by the StanfordUniversity Department of Comparative Medicine DiagnosticLaboratory

RESULTS AND DISCUSSION

Transduction of mouse pancreas by AdV and AAV

To compare the transduction efficiencies of AdV and AAVin the pancreas we delivered 1 3 108 plaque-forming units(pfu) AdVLacZ and 1 3 1011 particles AAV2LacZ AAV5LacZor AAV8LacZ by direct injection into the splenic lobe of the

WANG ET AL406

pancreas of 8- to 12-week-old C57Bl6 mice Successful ad-ministration was confirmed by a uniform swelling of the injectedarea The majority of the mice survived this operation with noobserved complications and histologic examination of mousepancreas injected in this manner showed no significant inflam-matory or mechanical lesions Mice were sacrificed at 1 2 4and 8 weeks postinjection (n 5 3 or 4 per group) and their pan-creas harvested for X-gal staining and immunohistochemistry

Significant LacZ expression was detected in the pancreas ofmice injected with AdVLacZ starting at 1 week postinjectionwith expression decreasing over 99 by 4 weeks (Figs 1 and2) In contrast immunodeficient C57Bl6 SCID mice injectedintrapancreatically with 1 3 108 pfu of AdVLacZ displayed in-creased persistence of gene expression with significant LacZexpression detectable for at least 4 weeks (Fig 2) in agreementwith previously published results (McClane et al 1997a) In

COMPARISON OF Ad AND AAV VECTORS 407

FIG 1 Pancreas sections from C57Bl6 mice injected with 1 3 108 plaque-forming units (pfu) AdVLacZ 1 3 1011 particlesAAV2LacZ or 1 3 1011 particles AAV8LacZ stained with X-gal at various days postinjection AdVLacZ-injected mouse pan-creas at 7 days (A) 14 days (D) 28 days (G) or 56 days (J) postinjection AAV2LacZ-injected mouse pancreas at 7 days (B)14 days (E) 28 days (H) or 56 days (K) postinjection AAV8LacZ-injected mouse pancreas at 7 days (C) 14 days (F) 28 days(I) or 56 days (L) postinjection Magnification 310

addition LacZ expression was primarily concentrated in a smallarea near the area of injection with much of the pancreas leftuntransduced Transduction of other tissues (liver spleen) wasnot observed

LacZ expression was detectable in the pancreas at 7 dayspostdelivery in mice injected with AAV2LacZ but expressiondid not peak until 4 weeks postdelivery The total number of

cells transduced by AdVLacZ was at least 10-fold higher thanthe number of cells transduced by AAV2LacZ at the time pointsof maximum expression AAV8LacZ transduced pancreas ex-hibited at least 10-fold higher levels of expression compared toAAV2LacZ and significant levels of expression were de-tectable beginning at 1 week postinjection The more rapid on-set of expression from AAV8 is likely caused by the more rapid

WANG ET AL408

FIG 3 Dose response of 5 different titers of AAV8LacZ (33 3 109 1 3 1010 33 3 1010 1 3 1011 33 3 1011 particles) ad-ministered intrapancreatically to C57Bl6 mice at 28 days postinjection

FIG 2 Quantitation of the number of LacZ-positive cellsslide obtained by AdVLacZ AAV2LacZ or AAV8LacZ transduc-tion of the pancreas in C57Bl6 mice or by AdVLacZ in C57Bl6 SCID mice The number of LacZ-positive cells were deter-mined by counting the total number of LacZ expressing cells per section (n 5 4mouse) and averaging the number of cells countedbetween the different mice (n 5 3 or 4 mice per time point)

uncoating of the AAV8 capsid in the cell compared to AAV2(Thomas et al 2004) Expression from AAV8LacZ-transducedpancreas peaked at 4 weeks postdelivery but interestingly ex-pression declined dramatically (90) by 8 weeks This de-crease in gene expression may be the result of an immune re-sponse elicited to the LacZ transgene or a result of pancreaticcell turnover The half-lives of murine pancreatic acinar cellsand islet cells are approximately 70 days and 47 days respec-tively (Magami et al 2002) so it is not unlikely that many of

the transduced cells were lost because of cell cycling In con-trast mice injected intrapancreatically with AAV5LacZ did notresult in any detectable LacZ expression in the pancreas in vivoat any of the tested time points (data not shown) Expressionof the LacZ transgene from both AAV2LacZ and AAV8LacZwas present through the entire pancreas and transduction ofother organs was not observed The ability of the adeno-asso-ciated viral vectors to transduce a larger area of the pancreaswas likely caused by the smaller particle size of AAV com-


FIG 4 Sections from C57Bl6 mouse pancreas injected with AdVLacZ AAV2LacZ or AAV8LacZ at 7 days (AdVLacZ) or4 weeks (AAV2LacZ AAV8LacZ) postinjection showing the different transduced cell types Pancreatic acinar cells transducedby AdVLacZ (A) AAV2LacZ (B) or AAV8LacZ (C) 203 magnification Pancreatic islets of Langerhans transduced by AdVLacZ (D) AAV2LacZ (E) or AAV8LacZ (F) and stained with anti-insulin antibody Magnification 340

TABLE 1 QUANTITATIO N OF THE CELL TYPES TRANSDUCED BY AdvLacZAAV2LacZ AAV5LacZ AND AAV8LacZ

1 week 2 weeks 4 weeks 8 weeks

AdvLacZAcinar cells 292 177 08 08Beta cells 027 01 01 01

AAV2LacZAcinar cells 02 07 05 12Beta cells 01 01 01 01



Numbers represent the percentage of the total cells in a 103 field of view The maxi-mum number of LacZ-positive cells in a 103 field of view for each cell type was deter-mined and averaged between the different mouse pancreas samples (n 5 3 or 4 per group)

WANG ET AL410

FIG 5 Sections of AdVLacZ- AAV2LacZ- and AAV8LacZ-transduced C57Bl6 mice pancreata at various days postinjectionstained with rat anti-mouse CD45 antibody AdVLacZ-injected mouse pancreas at 7 days (A) 14 days (D) 28 days (G) or 56days (J) post-injection AAV2LacZ injected mouse pancreas at 7 days (B) 14 days (E) 28 days (H) or 56 days (K) postinjec-tion AAV8LacZ-injected mouse pancreas at 7 days (C) 14 days (F) 28 days (I) or 56 days (L) postinjection Magnification 310

pared to adenovirus allowing it to diffuse more readily throughthe injected tissue

Dose response of AAV8

In order to test the maximum levels of transduction achiev-able with AAV8LacZ we performed a dose-response experi-ment to determine if higher doses of AAV8LacZ would resultin greater transduction levels Five doses of AAV8LacZ (33 3

109 1 3 1010 33 3 1010 1 3 1011 33 3 1011 particles) weredelivered intrapancreatically to 8- to 12-week-old C57Bl6mice and the mice were sacrificed at 4 weeks and their pan-creas harvested for X-gal staining Increasing doses ofAAV8LacZ resulted in increased of levels of LacZ expressionin the pancreas of mice with comparable levels of expressionachieved by the highest two doses (Fig 3) Higher doses ofAAV8LacZ could not be tested because of the relatively lowtiter of our AAV8LacZ viral preparation (29 3 1012 particles

per milliliter) and the risk of inducing pancreas damage by us-ing larger injection volumes It is likely that higher doses ofAAV8 (33 3 1011 particles) may result in even greater lev-els of transduction of the pancreas in vivo

Cell types transduced by AdV and AAV

AdVLacZ- AAV2LacZ- and AAV8LacZ-injected pancreatictissue stained with X-gal and an antibody for insulin revealedsimilar cellular distributions for the three viruses Pancreas trans-

duced with AdVLacZ showed expression primarily in the pan-creatic acinar cells with some pancreatic endocrine cells ex-pressing LacZ at the periphery of the insulin-positive beta cellclusters (Fig 4) The location of the transduced cells were pri-marily clustered around the site of injection Pancreas transducedby AAV2LacZ and AAV8LacZ showed positive LacZ expres-sion primarily in the acinar cells with punctate LacZ positivestaining throughout the entire pancreas Few insulin-positive cellswere transduced by AAV2LacZ whereas AAV8LacZ clearly re-sulted in transduction of several insulin-positive cells


FIG 6 Nonfasting blood glucose (A) and serum amylase (B) measurements of C57Bl6mice injected intrapancreatically withsaline (r ) 1 3 108 plaque-forming units (pfu) AdVLacZ (j ) 1 3 1011 particles AAV2LacZ (m ) 1 3 1011 particles AAV5LacZ(3) and 1 3 1011 particles AAV8LacZ (d ) at various days postinjection (n 5 3ndash5 mice per group)

A

B

Ex vivo the pancreatic islets of Langerhans have been suc-cessfully transduced by AAV (Prasad et al 2000) but therehas been no demonstration thus far of successful islet trans-duction by AAV in vivo AdVLacZ was able to transduce bothpancreatic acinar and endocrine cell types but transduction ofthe endocrine islet cells was primarily limited to the peripheryof the cell clusters This may be because of the limited accessto the islets which do not feed into the vast network of pan-creatic ducts that connect the acinar cells AAV2LacZ wasshown primarily to transduce pancreatic acinar cells with lim-ited transduction of pancreatic endocrine cells This may be-cause of the lower efficiency at which AAV2 transduces pan-creatic cells AAV8LacZ transduced both acinar and endocrinecells with similar efficiency (Table 1) Delivery of immuno-protective molecules to the islets in vivo using AAV8 may bea promising method of inhibiting the onset of type 1 diabetes(Prudhomme 2000 Kapturczak et al 2001 Bottino et al 2003)

Histopathology in treated animals

To begin to unravel the cause of the loss of transgene ex-pression from the AdV transduced cells we stained the trans-duced pancreas sections for CD45 (leukocyte common anti-gen) a transmembrane glycoprotein that is found on all cellsof hematopoetic origin except erythrocytes (Ledbetter andHerzenberg 1979) As expected significant leukocytic in-filtration was observed in AdVLacZ injected pancreas start-ing at day 7 (Fig 5) decreasing to negligible levels by day28 which corresponds to the decrease in LacZ expressionlevels AAV2LacZ and AAV8LacZ injected pancreata hadsignificantly less leukocytic infiltration at day 7 and littleif any CD45 positive cells were observed at day 28 eventhough there was no decrease in LacZ expression with thesevectors

Pancreatic function parameters in treated animals

In order to determine if the two delivery vectors induced anychanges in pancreatic function in vivo we monitored serumamylase levels a key pancreatic enzyme useful for the diagno-sis of pancreatitis (Moridani et al 2003) In addition we mon-itored the nonfasting blood glucose levels of these mice to de-termine if there were any changes in glucose homeostasis inresponse to AdV or AAV delivery Intrapancreatic delivery ofthe AdV and AAV vectors to the pancreas of mice resulted ina slight (2-fold) elevation of serum amylase levels comparedto saline at 1-day postinjection but amylase levels returned tonormal at 7 days postinjection and remained normal for the re-mainder of the measurement period (Fig 6B) This suggeststhat neither AdV nor AAV delivery to the pancreas inducespancreatitis in mice in vivo Intrapancreatic delivery of AdVand AAV also resulted in no significant changes in nonfastingblood glucose levels with the mice in all groups remaining nor-moglycemic (250 mgdl) at all measured time points (Fig6A) These data suggest that AdV and AAV do not cause anysignificant alteration in insulin secretion or islet function in thepancreas of mice in vivo

Our studies suggest that AAV8 is a highly useful vector forsafe efficient and long-term gene delivery to the pancreas Al-though our direct injection delivery method would not be suit-

able for human applications because of the high risk of pan-creatitis safer methods for vector delivery such as endoscopicretrograde cholangiopancreatography (ERCP) exist and are cur-rently in wide use in humans and could be adapted for deliv-ery of gene transfer vectors This in combination with the im-proved safety of helper dependent adenoviral vectors (Ehrhardtand Kay 2002 Yant et al 2002) and the relative safety recordof AAV suggest that these approaches may be useful for de-veloping new therapies for diabetes

REFERENCES

BOTTINO R LEMARCHAND P TRUCCO M and GIAN-NOUKAKIS N (2003) Gene- and cell-based therapeutics for typeI diabetes mellitus Gene Ther 10 875ndash889

BREYER B JIANG W CHENG H ZHOU L PAUL R FENGT and HE TC (2001) Adenoviral vector-mediated gene transferfor human gene therapy Curr Gene Ther 1 149ndash162

CARTER PJ and SAMULSKI RJ (2000) Adeno-associated viralvectors as gene delivery vehicles Int J Mol Med 6 17ndash27

EDLUND H (2002) Pancreatic organogenesis Developmental mech-anisms and implications for therapy Nat Rev Genet 3 524ndash532

EHRHARDT A and KAY MA (2002) A new adenoviral helper-dependent vector results in long-term therapeutic levels of humancoagulation factor IX at low doses in vivo Blood 99 3923ndash3930

FLOTTE T AGARWAL A WANG J SONG S FENJVES ESINVERARDI L CHESNUT K AFIONE S LOILER SWASSERFALL C KAPTURCZAK M ELLIS T NICK H andATKINSON M (2001) Efficient ex vivo transduction of pancreaticislet cells with recombinant adeno-associated virus vectors Diabetes50 515ndash552

GAO G ALVIRA MR WANG L CALCEDO R JOHNSTONJ and WILSON JM (2002) Novel adeno-associated viruses fromrhesus monkeys as vectors for human gene therapy Proc Natl AcadSci USA 99 11854ndash11859

GRIMM D and KAY MA (2003a) From virus evolution to vectorrevolution Use of naturally occurring serotypes of adeno-associatedvirus (AAV) as novel vectors for human gene therapy Curr GeneTher 3 281ndash304

GRIMM D KAY MA and KLEINSCHMIDT J (2003b) Helpervirus-free optically controllable and twondashplasmid-based productionof adeno-associated virus vectors of serotypes 1 to 6 Mol Ther 7839ndash850

KAPTURCZAK MH FLOTTE T and ATKINSON MA (2001)Adeno-associated virus (AAV) as a vehicle for therapeutic gene de-livery Improvements in vector design and viral production enhancepotential to prolong graft survival in pancreatic islet transplantationfor the reversal of type 1 diabetes Curr Mol Med 1 245ndash258

KAY MA LI QT LIU TJ LELAND F FINEGOLD M andWOO SLC (1992) Direct hepatic gene delivery in mice results inpersistent expression of human alpha-1-antitrypsin in vivo HumGene Ther 3 641ndash647

KAY MA LANDEN CN ROTHENBERG SR TAYLOR LALELAND F WIEHLE S FANG B BELLINGER D FINE-GOLD M THOMPSON AR READ M BRINKHOUS KMand WOO SLC (1994) In vivo hepatic gene therapy Complete al-beit transient correction of factor IX deficiency in hemophilia B dogsProc Natl Acad Sci USA 91 2353ndash2357

LEDBETTER JA and HERZENBERG LA (1979) Xenogeneicmonoclonal antibodies to mouse lymphoid differentiation antigensImmunol Rev 47 63ndash90

MAGAMI Y AZUMA T INOKUCHI H MORIYASU FKAWAI K and HATTORI T (2002) Heterogeneous cell renewal

WANG ET AL412

of pancreas in mice [3H]-Thymidine autoradiographic investigationPancreas 24 153ndash160

MCCLANE SJ HAMILTON TE DEMATTEO RP BURKE Cand RAPER SE (1997a) Effect of adenoviral early genes and thehost immune response system on in vivo pancreatic gene transfer inthe mouse Pancreas 15 236ndash245

MCCLANE SJ HAMILTON TE BURKE CV and RAPER SE(1997b) Functional consequences of adenovirus-mediated murinepancreas gene transfer Hum Gene Ther 8 739ndash746

MONAHAN PE and SAMULSKI RJ (2000) AAV vectors Is clin-ical success on the horizon Gene Ther 7 24ndash30

MORIDANI MY and BROMBERG IL (2003) Lipase and pan-creatic amylase versus total amylase as biomarkers of pancreatitisAn analytical investigation Clin Biochem 36 31ndash33

NAKAI H STORM TA and KAY MA (2000) Increasing the sizeof rAAV-mediated expression cassettes in vivo by intermolecularjoining of two complementary vectors Nat Biotechnol 18 527ndash532

PARK F OHASHI K CHIU W and KAY MA (2000) Efficientlentiviral transduction of liver requires cell cycling in vivo NatGenet 24 49ndash52

PRASAD KM YANG Z BLEICH D and NADLER JL (2000)Adeno-associated virus vector mediated gene transfer to pancreaticbeta cells Gene Ther 7 1553ndash1561

PRUDHOMME GJ (2000) Gene therapy of autoimmune diseaseswith vectors encoding regulatory cytokines or inflammatory cytokineinhibitors J Gene Med 2 222ndash232

RAPER SE and DEMATTEO RP (1996) Adenovirus-mediated invivo gene transfer and expression in normal rat pancreas Pancreas12 401ndash410

RUTLEDGE EA HALBERT CL and RUSSELL DW (1998) In-fectious clones and vectors derived from adeno-associated virus(AAV) serotypes other than AAV type 2 J Virol 72 309ndash319

SNYDER RO (1999) Adeno-associated virus-mediated gene deliv-ery J Gene Med 1 166ndash175

SNYDER RO MIAO CH PATIJN GJ PRATT SK DANOS

O GOWN AM WINTHER B MEUSE L COHEN LKTHOMPSON AR and KAY MA (1997) Persistent and thera-peutic concentrations of human factor IX in mice after hepatic genetransfer of recombinant AAV vectors Nat Genet 16 270ndash276

STILWELL JL and SAMULSKI RJ (2003) Adeno-associatedvirus vectors for therapeutic gene transfer Biotechniques 34 148ndash150

THOMAS CE STORM TA HUANG Z and KAY MA (2004)Rapid uncoating of vector genomes is the key to efficient liver trans-duction with AAV pseudotyped vectors J Virol (in press)

YANT SR EHRHARDT A MIKKELSEN JG MEUSE LPHAM T KAY MA (2002) Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo NatBiotechnol 20 999ndash1005

ZAISS AK LIU Q BOWEN GP WONG NC BARTLETTJS and MURUVE DA (2002) Differential activation of innateimmune responses by adenovirus and adeno-associated virus vectorsJ Virol 76 4580ndash4590

Address reprint requests toMark A Kay MD PhD

Departments of Pediatrics and GeneticsStanford University School of Medicine

300 Pasteur DriveGrant Building Room G305

Stanford CA 94305

E-mail markaystanfordedu

Received for publication March 8 2003 accepted after revi-sion February 23 2004

Published online March 17 2004


ski 2000 Zaiss et al 2002 Stilwell and Samulski 2003) AAVhas been shown to efficiently transduce a variety of cell typesin vivo including liver muscle and lung resulting in long-termgene expression from these tissues (Carter and Samulski 2000)Ex vivo AAV has been shown to transduce pancreatic endo-crine cells (Flotte et al 2001) but successful transduction ofthe pancreas by AAV has not been demonstrated in vivo It hasbeen demonstrated that ex vivo viral transduction efficienciesdo not always correlate with in vivo efficiencies (Kay et al1992 Snyder et al 1997 Park et al 2000) so we wanted todetermine whether AAV could also efficiently and safely trans-duce pancreatic cells in vivo Several novel serotypes of AAVhave been identified each which exhibit different structural andfunctional characteristics (Rutledge et al 1998 Gao et al2002 Grimm and Kay 2003a) Recent studies have shown thatthe different serotypes of AAV have different cellular speci-ficities and in particular AAV serotype 5 was shown to trans-duce human pancreatic islets ex vivo with a much higher effi-ciency than AAV serotype 2 (Flotte et al 2001) We alsowanted to determine if different serotypes of AAV could resultin greater efficiency transduction of the pancreas in vivo

MATERIALS AND METHODS

Recombinant viruses

The LacZ expression cassette (consisting of a Rous Sarcomavirus [RSV] promoter driving the expression of an Escherichiacoli b-galactosidase gene with a nuclear localization signalflanked by a bovine growth hormone polyadenylation site) wasexcised from plasmid pRSVnlsLacZ using BglII and cloned intothe BglII site of the pDE1 adenovirus shuttle plasmid or blunt-ended and cloned into the NotI site of the pAAV shuttle plas-mid AdVLacZ was amplified in 293 cells and subsequently pu-rified in a cesium chloride (CsCl) density gradient as previouslydescribed (Kay et al 1994) AAV2LacZ (AAV serotype 2) wasproduced by the triple-transfection method in 293 cells and pu-rified using a high-performance liquid chromatography (HPLC)method as described earlier (Nakai et al 2000) PseudotypedAAV5LacZ and AAV8 LacZ were produced by cross-packag-ing the AAV2 vector genome (containing the ITRsLacZ ex-pression cassette) into AAV5 or AAV8 capsids by cotransfec-tion of the pAAV plasmid with DF5 (containing AAV2 RepAAV5 capsid and adenovirus helper genes) or triple transfec-tion of pAAV with R2C8 (containing AAV2 Rep and AAV8capsid genes) and plAd5 (containing adenovirus helper genes)plasmids (Gao et al 2002 Grimm et al 2003b) The viruseswere purified using CsCl density centrifugation AdVLacZ was titered by a plaque-forming assay whereas AAV2LacZAAV5LacZ and AAV8LacZ were titered by a quantitative den-sitometric dot-blot assay To confirm the expression of the LacZtransgene from the two recombinant viruses 293 cells weretransduced with 1 3 108 AdVLacZ and 1 3 108 AAV2LacZand stained with X-gal 2 days posttransduction Positive LacZexpression was observed in AdVLacZ- AAV2LacZ- AAV5-LacZ- and AAV8LacZ-transduced 293 cells

Surgical procedures

All animal procedures were done according to the guidelinesfor animal care at Stanford University (Stanford CA) Eight-

to 12-week-old male C57Bl6 mice were anesthetized using asingle intraperitoneal injection of a mixture of ketamine ace-promazine and xylazine suspended in phosphate-bufferedsaline (PBS) Mice were subjected to laparatomy and injectedwith 100 ml of purified virus solution suspended in PBS usinga 26-gauge needle into a single location in the splenic lobe ofthe pancreas Laparotomies were subsequently closed with atwo-layer suture Mice were then sacrificed at various dayspostinjection and pancreatic tissue was harvested for X-galstaining and immunohistochemistry

Tissue embedding and X-gal staining

Pancreatic tissue was washed in PBS and placed in optimalcutting temperature (OCT) compound and immediately frozenon dry ice Ten-micrometer sections were then cut on a cryo-stat and placed on VWR Superfrost plus microscope slides (ErieScientific Company Portsmouth NH) Tissue sections werefixed in 125 glutaraldehyde in PBS for 10 min and rinsed 3times in PBS prior to staining Slides were then placed in X-Gal solution (1 mgml X-gal 5 mM K3Fe(CN)6 5 mMK4Fe(CN)6 and 1 mM MgCl2 in PBS) and incubated overnightat 37degC Slides were then washed three times in PBS and thencounterstained with hematoxylin

Immunohistochemistry

Rat anti-mouse CD45 antibodies were obtained from BDPharmingen (San Diego CA) Slides were processed using theVectastain ABC kit (Vector Labs Burlingame CA) Pancreassections were blocked with 10 normal goat serum and avidinfor 30 min and then incubated with 1250 dilution of primaryrat anti-mouse CD45 antibody in PBS1 bovine serum albu-min (BSA)10 goat serum and biotin for 2 hr Subsequentlythe slides were then washed 3 times in PBS and biotinylatedmouse anti-IgG2 secondary antibody diluted 1500 in PBS1BSA10 goat serum was applied for 30 min Slides werewashed 3 times in PBS and then incubated with the ABCreagent for 30 min The sections were thoroughly washed andincubated with 006 339-diaminobenzidine and 001 H2O2

for 5 min and counterstained with hematoxylin

Monitoring changes in pancreatic function

Whole blood and sera from mice injected with saline AdVLacZ AAV2LacZ AAV5LacZ and AAV8LacZ were col-lected at various days postinjection by retro-orbital eye bleedsGlucose levels were measured using a Glucometer Elite glu-cometer (Bayer Healthcare Pittsburgh PA) and Ascensia Eliteblood glucose test strips (Bayer Healthcare) Amylase levelswere measured from the collected sera samples by the StanfordUniversity Department of Comparative Medicine DiagnosticLaboratory

RESULTS AND DISCUSSION

Transduction of mouse pancreas by AdV and AAV

To compare the transduction efficiencies of AdV and AAVin the pancreas we delivered 1 3 108 plaque-forming units(pfu) AdVLacZ and 1 3 1011 particles AAV2LacZ AAV5LacZor AAV8LacZ by direct injection into the splenic lobe of the

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A

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Stanford CA 94305












WANG ET AL408














WANG ET AL410












A

B








REFERENCES















WANG ET AL412






















Stanford CA 94305
















WANG ET AL410












A

B








REFERENCES















WANG ET AL412






















Stanford CA 94305











A

B








REFERENCES















WANG ET AL412






















Stanford CA 94305












REFERENCES















WANG ET AL412






















Stanford CA 94305


























Stanford CA 94305





comparison of adenoviral and adeno-associated viral...

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