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.
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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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