H U M A N GENE THERAPY 7:1693-1699 (September 10, 1996) Mary Ann Liebert, Inc.

A d e n o v i r u s - M e d i a t e d H e p a t i c G e n e Transfer in M i c e :

C o m p a r i s o n o f Intravascular a n d Biliary A d m i n i s t r a t i o n

MARIE-JEANNE T.F.D. VRANCKEN PEETERS,'' GUSBERT A. PATIJN, ANDRE LIEBER,^ LEONARD MEUSE,! nd MARK A. KAY^^

ABSTRACT

Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of

their ability to transduce m a n y cell types with high efficiencies in vivo. After intravenous infusion, the m a ­

jority of the vector is found in hepatocytes, but vector D N A is found to varying degrees in other tissues. In

an attempt to restrict adenovirus-mediated gene transfer to the liver, w e developed a microsurgical method

that allowed for vector administration directly into the biliary tract of a mouse. W e demonstrate that gene

transfer w a s 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular in­

fusion. Intravascular infusion of recombinant adenovirus elicits a powerful i m m u n e response that limits gene

expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser i m m u n e re­

sponse as determined by the lower neutralizing antibody titers directed against the vector compared with an­

imals treated by intravascular infusion. There was no difference in the persistence of gene expression, sug­

gesting a similar cell-mediated i m m u n e response against the vector containing cells in animals administered

vector by either method. A s future-generation adenovirus vectors that are safer and less immunogenic be­

c o m e available, the m o r e liver specific gene transfer via the biliary tract m a y offer advantages over intra­

venous infusion for hepatic gene therapy.

O V E R V I E W S U M M A R Y al, 1993). Numerous studies have demonsfrated that systemic adminisfration of recombinant adenoviral vectors leads to ther-

A microsurgical method for cannulating the biliary tract in apeutic levels of gene expression in the liver, resulting ki com-a mouse, allowing for local adenovirus-mediated gene trans- plete amelioration of the clinical phenotype in a number of an-fer into the liver is described. This method results in less imal models for an inherited metabolic disease (Ishibashi era/., adenovirus D N A in nonhepatic tissues compared with in- 1993; Fang era/., 1994; Kay era/., 1994; Kozarsky era/., 1994). travascular infusion. Because biliary cannulation can be Unfortunately, two major problems arise when using aden-performed in a noninvasive manner in humans, it repre- ovkal vectors for in vivo hepatic gene therapy. The fkst is re­sents a potential method for liver-directed gene transfer in lated to the host knmune response that limits the persistence of a clinical setting. gene expression and the ability to readminister the vector (Barr

et al, 1995; Yang et al, 1995). Transient gene expression re­sults from cellular immunity dkected agamst cells tiansduced

I N T R O D U C T I O N with vector whereas neufralizing antibodies, directed against the vims, prevent secondary gene fransfer. A second potential prob-

INCE THE ISOLATION OF ADENOVIRUSES ovcr three decades lem is that after inttavenous admmisfration, the adenovims.

s ago the recombmant El-deficient adenovkal vectors have while highly hepatotrophic, does fransduce most otiier tissues emerged as a promising technology for in vivo gene tiierapy be- to varying degrees (Vrancken Peeters et al, 1996). For kistance, cause of their ability to ttansduce many tissues in vivo with rel- the spleen and lung contained about one-tenth tiie amount of atively high efficiency (SttaOFord Perricaudet et al, 1992; Li et vkal D N A contained in the liver, whereas other tissues con-

'Markev Molecular Medicine Center, Division of Medical Genetics, Box 357720, Department of Medicine, ^Departments of Biochemistry, Patiiology and Pediatrics, University of Washington, Seattle W A 98195.

Current Address: Department of Surgery, University of Leiden, The Netheriands.

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1694 VRANCKEN PEETERS ET AL.

tained variable but lesser amounts of D N A . Interestmgly, there was no difference in D N A distribution when tiie vector was ad­ministered via the portal or peripheral vein. The promiscuous nature of adenovims is an unatttactive feature for several rea­sons. Gene products made in other tissues may be harmful, and the uptake of adenovuus by reticuloendothelial tissues such as the spleen may exacerbate the immunological response.

A previous report (Yang et al, 1993) showed that when the appropriate amount of adenovuus is administered into the bil­iary ttact of a rat, by means of rettograde mjection into the com­m o n bile duct, 8 0 % of the hepatocytes were ttansduced. However, this type of perfusion will more than likely allow for antegrade outflow of vims, leading to the fransduction of other tissues. W e hypothesized that biliary infusion of adenovirus could lead to a more liver-specific fransduction when the infu­sion is performed in such a way as to limit the outflow of the vims. Therefore, w e developed a new method that allows for refrograde biliary infusions in a mouse model by placing a can­nula in the cystic duct of the biliary fract. During adminisfra­tion, the common bile duct was clamped off, preventing ante­grade outflow ofthe vector. Subsequently, this method was used to study adenovkal-mediated gene fransfer. Recombinant adenovims D N A was quantitated within different tissues after either bile duct infusion or infravenous injection. The im­munological response was determined by comparing the per­sistence of gene expression and neufralizing antibody titers when the vector was administered via these two routes.

MATERIAL AND METHODS

Animals

C3H/HeJ mice (Jackson Laboratory, Bar Harbor, M E ) , ages 5-6 weeks, were used in the described experiments. All stud­ies were performed in accordance with the institutional guide­lines at the University of Washington.

Adenovirus production

T w o recombinant adenoviral vectors were used, Ad.RSV/3-Gal (Sfratford Perricaudet er a/., 1992) and A d / R S V h A A T (Kay er al, 1995a). The vectors produce nuclear Escherichia coli p-galactosidase (;S-Gal) and human al-antitrypsin, respectively. The vectors were prepared, purified, and tested for the absence of helper vector as previously described (Barr et al, 1995). Purified vims was stored in 10 m M Tris HCl p H 8.0, 1 m M MgCl2, 1 0 % glycerol ki aliquots at -80°C and freshly diluted in semm-ffee Dulbecco's modified Eagle media ( D M E M ) (Life Technologies, Gaithersburg, M D ) prior to infusion.

Adenovirus administration into the biliary tract through a bile duct cannula

To allow infusion of adenoviras via the bUiary fract, a can­nula was placed into the bile duct. Mice were anesthetized with an inttaperitoneal adminisfration of 0.5 ml of 20 mg/ml Avertin (2,2,2-tribromoethanol, Aldrich Chemicals, Milwaukee, WI). Bile duct cannulation was performed under the operating mi­croscope (with 4-6.4X magnification). A midline abdominal incision was made and the intestinal duct displaced to allow for exposure of the liver. The falciform ligamentum anterior was

separated and the median liver lobe was displaced to expose the gallbladder, cystic duct, hepatic ducts, and c o m m o n bile duct (Fig. IA). Exposure was maintained with small rettactors and one clamp witii which the xyphoid was grasped and re­ttacted. Subsequentiy, die falciform ligamentum posterior was separated, and a 6.0 silk sumre (David and Geek, Inc., American Cyanamid, Manati, PR) was placed loosely around tiie proxi­mal site of the gallbladder. A lO-mm-long P E 10 (0.011"I.D., 0.024"O.D., Clay Adams, Parsippany, NJ) polyethylene tube, connected to a silicone tube (0.02"I.D., 0.037"O.D., S/P Medical Grade Silicone Tubing, Baxter, IL) (Vrancken Peeters et al, 1996) was inserted into the distal site of die gallbladder, and moved up to the origin of the cystic duct (Fig. IB). Hereafter the suture was tied off around the caimula. Prior to infusion of the adenovkus, the common bile duct was flushed with saline and clamped off where k opens into the duodenum, to avoid antegrade outflow ofthe vkus. Different amounts of adenovims, diluted in 100 p\ of semm-free D M E M , were slowly mfused into the caimula with an approximate flow rate of 10 /il/min. After infusion, the distal end of the polyethylene m b e was co­agulated, all retractors and the xyphoid clamp relieved, and the mtestinal duct placed back in its original position. One hour af­ter adenoviras administtation, the antegrade flow from tbe bile duct to the duodenum was restored by removing the clamp from the common bile duct. Finally, the abdomen was closed in two layers (continuous suture, 4.0 silk).

Intravascular administration of the adenovirus

Different amounts of adenovkus, diluted in 100 yal of semm-free D M E M medium were injected into either the taU vein or the portal vein through a previously placed permanent portal vein catmula, as described earlier (Vrancken Peeters et al, 1996).

X-Gal staining

Mice tiiat were administered with the Ad.RSV/SGal were sac­rificed 3 days after injection to study the efficiency of adeno­viras infection. A partial hepatectomy was performed to obtain liver tissue that was embedded in O C T compound (Miles Inc., Elkhart, IN), frozen in metiiyl butane cooled in liquid nittogen, and stored at -80°C. To detect )3-Gal activity, 10-/tm-tiiick frozen sections were cut, fixed witii 1.25% glutaraldehyde in phosphate-buffered saline (PBS) for 10 min and tiien stained for 4 hr with 5-bromo-4-chloro-3-indolyl-/3-D-galactosidase (X-Gal) (Sigma Chemical, St. Louis, M O ) as described (Vrancken Peeters et al, 1996).

DNA blot analysis

Anunals tiiat received tiie Ad.RSV^SGal, eitiier by means of systemic administtation or by mjection mto tiie biliary tract, were sacrificed 4 days after injection. Liver, spleen, lung, brain, and mtestme of each individual mouse were excised and stored at -80°C until whole-tissue D N A preparation was perfonned as previously described (Vrancken Peeters et al, 1996). A 10-/Ag amount of D N A was digested witii Hind m and subjected to Soutiiem analysis usmg rapid hybridization buffer (Amersham, hidianapohs, IN) according to tfie manufactiirer's protocol.

The probe was prepared by random D N A priming of a 3 4-kb -Gal D N A fragment. The blot was washed in IX sodium sahne cittate (SSC) at room temperature for 20 min, followed

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INFUSION OF RECOMBINANT ADENOVIRUS VECTORS 1695

FIG. 1. Placement and function of biliary caimula. A. Schematic drawing of the hepatobiliary anatomy. The site of catheter in­sertion is shown. B. Insertion of the catheter mto the gallbladder, clamping of the common bile duct and ligature around the gall­bladder are shown.

by a 30-mki wash in O.IX SSC at 65°C. It was then exposed to X-ray film, followed by analysis by a Model 400S Phosphorimager (Molecular Dynamics, Sunnyvale, CA). The blot was subsequently washed in O.IX SSC at 100°C for 30 m m and reprobed with die 2-kb Hind JU-Eco RI mouse metiial-lothionem I gene fragment from plasmid m M M T - 1 (Searle et al, 1984) to adjust for small variations in D N A loading and

ttansfer between lanes.

Biochemical analysis

H u m a n al-antitrypsin was quantified with an enzyme-linked immunosorbent assay (ELISA) using a human-specific anti­body as previously described (Kay er al, 1995a). Neuttalizing

antibodies dkected against the adenoviras were analyzed in du­plicate as described previously (Kay et al, 1995b). The titer of inactivating antibodies for each seram sample is reported as the dilution of seram for which there is 7 5 % kihibition of gene ttansfer in cultured cells.

RESULTS

Development of permanent access into the biliary tract

A new technique was developed that allowed for injection dkectiy into the biliary ttact (Fig. 1), without allowing ante­grade flow out of the liver for 60 min. This was accomplished

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1696 VRANCKEN PEETERS ET AL.

•TV

B

FIG. 2. /3-Gal staining of liver after adenoviras-mediated gene transfer. A total of 5 X 10' pfu of Ad.RSV /3 gal vector was infused into the biliary tract or administered by tail vein injection. Three days later, liver sections were obtained for X-gal staining. A. Tail vein injection. B. Biliary tract infusion. C. Biliary fract infusion. The arrows show stained biliary epithe­lial cells. Original magnifications: A and B, lOOX; C, 200X.

by clampkig off the common bile duct at the origin of the duo­denum. Adminisfration of 100 pl of methylene blue into this caimula resulted in an equal staining of the whole liver, demon­strating efficient patency and flow through the biliary fract, whereas mfusion without clamping of the common bile duct led to flow of methylene blue into the duodenum (not shown). By placing the proximal end of the catheter in a subcutaneous pocket (Vrancken Peeters et al, 1996), secondary infusion into the cannula was possible for at least a week after its initial place­

ment. Moreover, m more recent smdies, tiie catiieter could be removed at the end of the procedure. The mortality rate during this procedure was less than 3%. O n rare occasions, tiie liver would bleed extensively when the falcifonn ligaments were sep­arated, hi all cases, the cannula remained in the mice without causing any detectable complications.

Efficiency of adenoviral-mediated gene expression

In vivo gene fransfer to the liver was first compared by the infroduction of 5 X 10^ pfu of Ad.RSV/3Gal into tiie biliary tract or the portal vein. Livers that were recovered 3 days after infusion showed expression of the exogenous gene m hepato­cytes, as seen by the nuclear blue staming, characteristic of -Gal activity (Fig. 2A,B). Although there was variability be­tween tissue sections from an individual liver, both methods led to sunilar pattems of parenchymal cell staining. The only dif­ference between the biliary and intravascular infusion was that the former resulted in gene transfer into the biliary epithelial cells as well as into hepatocytes (Fig. 2C). The two routes of infusion of Ad/RSVhAAT led to equal secretion of the seram human al-antitrypsin into the ckculation (Fig. 3). The rate of fall-off of gene expression was the same between the two groups and similar to that previously reported (Barr et al, 1995).

The inability to perform multiple adenovkal gene ttansduc­tion is related to a humoral immune response (Smith et al, 1993; Barr et al, 1995; Vrancken Peeters et al, 1996). W e de­termined whether or not the block to secondary gene ttansfer by production of neuttalizing antibodies could be overcome by means of biliary infusion. Mice received 5 X 10' pfu of Ad/RSVhAAT into either the portal vein or the biliary ttact. All animals developed neuttalizing antibodies dkected against the vkal vector as shown in Table 1. Interestingly, statistically significant lower titers were obtained after biliary infusion in comparison with systemic infusion. Animals given the same amount of Ad/RSVhAAT by inttavascular infusion developed high anti-adenoviral neuttalizing antibodies, were infused with 5 X 10' pfu of Ad.RSV)3gal via the bUiary ttact 3 months later. N o X-Gal staining was observed ki tiiese anknals, whereas naive control animals had blue livers (not shown). This sug­gests that the biliary fract contained neuttalizing antibodies or that there were sufficient sinusoidal or inti cellular antibodies to block secondary gene ttansfer.

Table 1. NEtrrRALiziNG Antibody Titers

Infusion method Week 4^ Week 6" Week 10^

Bile 64(16-256) 64(32-64) 16 (<16-64) duct

Portal 256(256-1024) 256(256-512) 256(256-1024) vein

Animals (n = 5 per group) infused with 5 X 10' pfu Ad/RSVhAAT were analyzed at different periods for neuttal­izing antibodies against adenoviras. The titers are expressed as the median reciprocal titers. The range of the reciprocal titer is given in parentheses.

> values were <0.005.

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INFUSION OF RECOMBINANT ADENOVIRUS VECTORS 1697

10000

<

E

1000

100 •;

20 30 40 50 Da y s post infusion

FIG. 3. Seram hAAT concenttations after Ad/RSVhAAT ad­ministtation. A total of 5 X 10' pfu of A d / R S V h A A T aden­ovirus was infused via the portal vein {n = 5; open circles) or biliary ttact (n = 5; closed ckcles). Periodic seram samples were obtained and seram h A A T concenttations determined.

Tissue distribution of recombinant adenovirus

The tissue distiibution pattem of adenoviras after biliary ttact infusion versus systemic administtation was determined by in­jecting 1 X 10'" plaque forming units (pfu) of Ad.RSVjSgal into the bile duct cannula (n = 4) or into the tail vein (n = 4) of mice. Four days after the adenoviras administtation, mice were sacrificed and D N A was prepared from liver, spleen, lung, and heart, digested with Hind in, and subjected to Southem analy­sis. As shown in Fig. 4A, the relative quantity of adenoviral D N A in the liver was the same in animals receiving intravas­cular versus biliary infusion. In conttast, all the other organs showed greater amounts of vector D N A after tail vein injection as compared to biliary infusion.

To quantitate adenovkal D N A in various tissues more accu­rately, after the blot was reprobed with the mouse metallo­thionein exon I D N A probe to adjust for small variations ki D N A loading and ttansfer between lanes, the relative conected intensities were determined on a phosphorimager (Fig. 4B). The number of adenovkus copies per diploid genome was calcu­lated by comparing the relative )3-Gal hybridization signal m each lane with a /3-Gal D N A standard. N o significant differ­ence in the amount of adenovkal D N A copies per diploid genome was found in liver between systemic and biliary infu­sion. The range of adenovkal D N A varied between 12 and 20 adenoviral D N A copies per hepatocyte (Fig. 4B). Consistent with previous studies (Vrancken Peeters er al, 1996), in­ttavascular administtation resulted m about 10 tunes less vec­tor D N A kl tissues other than liver, including the spleen (3 copies/cell), lung (6.5 copies/cell), and heart (2 copies/cell) whereas there was only 0.6 genome copy per cell in spleen and heart, and 0.7 copy per cell in die lung of anknals receivmg bil­iary infusion. Thus, biliary infusion decreased the amount of vector D N A found in other tissues by about 4- to 10-fold com­pared with inttavascular mfusion. This was a statistically sig­

nificant difference.

DISCUSSION

The liver has been an important target for gene transfer stud­ies (for review, see K a y and W o o , 1994). Whereas dkect in­ttavascular infusion results in efficient parenchymal cell gene transfer, a large amount of vector ttansduces a wide variety of other organs. W e n o w describe a surgical technique that m a k e s the biliary ttact mo re accessible for hepatic gene transfer in the m o u s e by placement of an indwelling cannula in the cystic duct. This method allows for retrograde injections into the biliary ttact without losing the injected vector through antegrade out­flow into the d u o d e n u m at the time of infusion. It is not possi­ble to exclude that s o m e of the vector is lost kito the duode­n u m at the time the duct is u n d a m p e d 1 hr later. This would depend on whether or not significant amounts of infectious vec­tor particles are still present in the liver 1 hr after infusion. After assessing the advantages biliary infusion might have over in­ttavenous infusion, this study was designed to deliver aden­oviras m or e selectively to the liver in a preclinical model that could be adapted to humans.

The immunological response against the vector and ttansduced cells m a y be exacerbated by adenovkus uptake by reticuloen­dothelial cells and antigen-presenting cells present outside the liver. Because the spleen is also a target for adenovkus after in­ttavascular infusion, it was hoped that by decreaskig gene ttans­fer to nonhepatic tissues the i m m u n e response would be reduced. Previous smdies have targeted the liver by biliary infusion of viras (Yang et al, 1993), but no mechanism to limit antegrade flow into nonliver tissue was used and the presence of vector in other tissues was not evaluated. In the cunent study, a micro­surgical technique allowed this to be performed. Although gene fransfer to nonhepatic tissues was substantially reduced compared with inttavascular infusion, gene ttansfer to other tissues did oc­cur to varying degrees. There w a s a reduced humoral i m m u n e response in biliary duct ttansduced animals; however, it is un­likely that this is of clinical importance. Furthermore, there w a s no change in the persistence of gene expression, suggesting no differences in ceU-mediated immunity.

T h e ttansfer of adenovkus D N A into nonhepatic organs m a y have been the result of adenoviras ttansversing the biliary spaces, possibly due to the disraption of tight junctions between hepatocytes and leakage into the hepatic vasculamre. If this is conect, it m a y be possible to reduce vascular spread by infus­ing vector at a slower rate and reduced pressure. This vascular leak m a y have been sufficient to elicit an i m m u n e response. Altematively, antigen production/presentation from ttansduced liver cells m a y have been sufficient to elicit the i m m u n e re­sponse. Until the leakage of adenoviras to other tissues can be conttolled, it will be difficult to differentiate between these two possibilities.

O u r present s m d y shows that the same levels of gene ttans­fer, reflected by the same levels of recombinant D N A per he­patocyte, can be achieved w h e n the adenovirus is administered via the biliary ttact. However, biliary ttact infusion allowed for m o r e liver-specific ttansduction. In comparison with intta­venous administration, four to ten times less adenoviras D N A was detected in the spleen, lung, and heart, which is an impor­tant safety consideration w h e n adenoviras vectors are consid­ered for clinical protocols.

It is of general interest that biliary infusion also results in

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1698 VRANCKEN PEETERS ET AL.

T ^ ^ T3 T4 B1 Ba B3 B4 T t 2 T3 T4 B1 B2 B3_B4

Spleen "f^^* „ bi b2 b3 b4 11 T2 T3 T4 B1 B2 B3 B4 Tl T2 T3

o a 60

Ol

_4> 'S, O o < Q

Spleen

p<0,01

Lung

p<0,05

Heart

p<0,05

ili.i - - i l l l l - -- l . , - i - _ _

B

HHhH gpammco g > > > > > > > >

cS c c c c 5 U V 4> 4> ' _« _2J Ji .2 Q4 o. o. D. «3 C/5 (/5 t/5

c e U 4> ... .V JS JS iJ .2 Q. O. Q. 0. c/3 C/5 C/5 V5

c - _ _ 3 3 S 3 3 3 3 3

CQ CQ 03 C3 ^ CO w TO

FIG. 4. Recombinant adenovkal D N A detection in mice ttansduced with Ad.RSV^gal. A. Three days after infusion of 1 X 10'° pfu of Ad.RSV/Sgal kito the taU vein or biUary ttact, total D N A s were analyzed by Soutiiem blot. Ten micrograms of Hind ni-digested D N A was loaded hi each lane. The blot was hybridized sequentiaUy with a ^P-labeled /3-Gal gene or mouse metal­lothionein gene (not shown) fragment. Each lane represents D N A from an individual organ. T, Tail vein; B, bUiary ttact infu­sion. Each number represents an mdividual animal. B. The number of genome copies per diploid genome was calciUated in the following manner. The /3-Gal gene hybridization signal was quantitated on a phosphorimager and adjusted by using tihe signal obtained from the metaUothionein probe. The adjusted signal was compared with linearized plasmid pCMV-/3gal concenttation markers (not shown). Then, 5, 30, and 120 pg of plasmid was mixed with 10 /xg of Hind Hl-digested normal mouse D N A . The 30-pg amount represents one genome equivalent. Statistical p values were calculated using Student's r-test.

gene transfer mto a proportion of biliary epithelial ceUs, whereas inttavascular uifusion, although equally efficient at parenchymal cell gene fransfer, did not result in bUiary epithe­lial gene fransfer. Following both methods of infusion, the rel­ative amounts of D N A per diploid genome of the liver were equivalent suggesting that the number of genome copies per he­patocyte would be slightly lower after bUiary infusion. The abil­ity to fransduce biliary epithelium wUl offer littie advantage for

the freatment of most metabolic diseases and genetic disorders resulting in loss of plasma proteins such as hemophdia. Nevertheless, gene transfer into the bUiary epithelium may bave specific uses for the freatment of specific liver diseases such as cystic fibrosis, primary biliary ckrhosis, and cholangiocarci-noma.

This study and otiiers estimate that between 10 and 50 copies of adenoviras D N A molecules are present per Uver ceU in a

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INFUSION OF RECOMBINANT ADENOVIRUS VECTORS 1699

mouse (Smitii et al, 1993; Vrancken Peeters et al, 1996) after adenoviras-mediated gene ttansfer. The variation in D N A copy number reported between studies is most lUcely the result of dif­ferences between vkal preparations, titering, age of animals, and dosage of vector administered. Assuming that only 6 0 % of cells are hepatocytes, then the acmal number of genomes per nucleus would be two-fold greater titan tiiat determined for D N A copy number per haploid genome. Moreover, because a relatively large proportion of mouse hepatocytes are more than diploid in D N A content, it is conceivable that some nuclei wUl have close to 100 adenoviras genomes.

This study is important because depending on safety issues related to improved, less immimogenic vectors, the smaller the amount of adenovims dissemination into nonhepatic tissues, the better. In humans k should be feasible to deliver recombmant viruses by a noninvasive approach, endoscopic rettograde cholangiopancreaticography (ERCP). This common procedure, mainly used in clinical practice to visualize the biliary ttact by deUvering radio-opaque conttast agents through an endoscopi­cally placed bUiary cannula, should be effective for the infu­sion of recombinant virases. It allows for rettograde infusion of the adenovirus while antegrade outflow can be limited by balloon-catheterization ofthe distal common bile duct. The abil­ity to infuse via the biliary duct in mice allows the preclinical development of a number of different vectors as well as adenovkus as they are developed.

ACKNOWLEDGMENTS

We thank Brian Winther and H. Deutman for their technical assistance. This work was supported by National Institutes of Healtii grant DK49022. M.J.T.F.D.V.P. and G.P. were sup­ported m part by the Dutch N W O 901-01-096 fellowship. A.L. was supported by a D F G fellowship.

REFERENCES

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FANG, B., EISENSMTTH, R.C, LI, X.H.C., FINEGOLD, M.I., SHEDLOVSKY, A., DOVE, W., and W O O , S.L.C. (1994). Gene therapy for phenylketonuria: Phenotypic correction in a genetically deficient mouse model by adenovirus-mediated hepatic gene ttans­fer. Gene Ther. 1, 247-254.

ISHIBASHI, S., B R O W N , M.S., GOLDSTEIN, J.L., GERARD, R.D., H A M M E R , R.E., and HERZ, I. (1993). Hypercholesterolemia in low density lipoprotein receptor knockout mice and hs reversal by adeno­virus-mediated gene delivery [see comments]. J. Clin. Invest. 92,

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KAY, M.A., LANDEN, C.N., ROTHENBERG, S.R., TAYLOR, L.A., LELAND, F., WIEHLE, S., FANG, B., BELLINGER, D., FINE-GOLD, M., THOMPSON, A.R., READ, M., BRINKHOUS, K.M., and W O O , S.L.C. (1994). In vivo hepatic gene therapy: complete al­beit transient correction of factor IX deficiency in hemophilia B dogs. Proc. Natt. Acad. Sci. USA 91, 2353-2357.

KAY, M.A., GRAHAM, F., LELAND, F., and W O O , S.L. (1995a). Therapeutic serum concentrations of human alpha-1-antitrypsin af­ter adenoviral-mediated gene transfer into mouse hepatocytes. Hepatology 21, 815-819.

KAY, M.A., HOLTERMAN, A.-X., MEUSE, L., GOWN, A., OCHS, H., LINSLEY, P.S., and WILSON, C.B. (1995b). Long-term hepatic adenovirus mediated gene expression in mice following CTLA4Ig administration. Nature Genet. 11, 191-197.

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STRATFORD PERRICAUDET, L.D., MAKEH, I., PERRICAUDET, M., and BRIAND, P. (1992). Widespread long-term gene transfer to mouse skeletal muscles and heart. J. Clin. Invest. 90, 626-630.

VRANCKEN PEETERS, M.J.T.F.D., LIEBER, A., PERKINS, J., and KAY, M.A. (1996). A method for multiple portal vein infusions in mice; quantitation of adenovirus-mediated hepatic gene ttansfer. BioTechniques 20, 278-285.

YANG, Y., RAPER, S.E., COHN, J.A., ENGELHARDT, J.F., and WILSON, J.M. (1993). An approach for treating tiie hepatobiliary disease of cystic fibrosis by somatic gene transfer. Proc. Nafl. Acad. Sci. USA 90, 4601^605.

YANG, Y., LI, Q., ERTL, H.C, and WILSON, J.M. (1995). Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69, 2004-2015.

Address reprint requests to: Dr. Mark A. Kay

Markey Molecular Medicine Center Division of Medical Genetics

Department of Medicine Box 357720

University of Washington Seattle, W A 98195

Received for publication April 25, 1996; accepted after revi­sion June 17, 1996.

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