H U M A N G E N E T H E R A P Y 6:1039-1044 (August 1995) Mary Ann Liebert, Inc.

G e n e T h e r a p y for H e m o p h i l i a B : H o s t I m m u n o s u p p r e s s i o n

P r o l o n g s the Therapeutic Effect o f A d e n o v i r u s - M e d i a t e d

Factor I X E x p r e s s i o n

B. FANG,i R.C. EISENSMITH,! H. WANG,^ M.A. KAY,^ R.E. CROSS,^ CN. LANDEN,^ G. GORDON,^ D.A. BELLINGER,4-6 M.S. READ,̂ '̂ P.C. HU,^ K.M. BRINKHOUS,4'6 and S.L.C. WOO^^

ABSTRACT

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correc­

tion of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1 - 2 % of nor­

mal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of pro­ducing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration

ofthe cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings sup­

port a recent report (Yang et al, 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocom­

petent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with

CsA, the prolonged expression of the transgene indicates that immunosuppression m a y be applicable in at­taining long-term treatment of clinically relevant disorders.

OVERVIEW SUMMARY

Adenovirus-mediated gene transfer in vivo results in effi­cient but transient transgene expression. Recent studies have suggested that this loss of expression is caused by the destruction of adenovirally transduced cells by the host im­mune system. If true, suppression of the inunune system should significantly prolong the effects of adenoviral treat­ment This hypothesis was examined by combining adeno­virus-mediated transfer of the canine factor IX cDNA with continuous immunosuppression in a dog model for hemo­philia B. Immunosuppression significantly increased the persistence of transgene expression following adenovirus-mediated transfer in vivo. This result suggests that further modifications of adenoviral vectors that reduce their im­munogenicity may significantly increase their persistence in

INTRODUCTION

HEMOPHILIA B IS CAUSED BY A HEREDrtARY DEFECT in blOOd clotting factor IX (FIX). The incidence of this disease is

about 1 in 30,000 males, representing about 2 0 % of all hemo­philiacs (Roberts and Lozier, 1991). Affected individuals re­quire regular administration of FIX concentrates prepared from human plasma to prevent or stop bleeding episodes. Although the complications resulting from contamination with blood-bome pathogens, such as human immunodeficiency and hep­atitis B vimses, can be reduced through the use of recombinant FIX, severely affected patients are still at risk for life-threaten­ing hemorrhage and other complications from repeated bleed­ing episodes due to the relatively short half-Ufe of FIX in the circulation. Continuous supply of this coagulating factor is therefore preferable to intermittent administration of concen-tiates. Curtent research efforts are focusing on the development

'Department of Cell Biology, ̂ Howard Hughes Medical Institute, Baylor College of Medicine, Houston TX 77030. 'Department of Medicine, Markey Molecular Medicine Center, University of Washington, Seattle, W A 98195. Departments of ''Pathology and 'Pediatrics and ̂Center for Thrombosis and Hemostasis, University of North Carolina, Chapel Hill, NC 27599.

1039

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.

1040 FANG ET AL.

of novel therapies for hemophilia B based on the deUvery of tiie FIX gene or c D N A into appropriate target cells, so that suf­ficient amounts of FIX will be constantly produced to aUeviate or cure the disease (Dai et al, 1992; Kay et al, 1993, 1994; Smith et al, 1993; Lozier and Brinkhous, 1994; Yao et al, 1994).

Previous studies have shown that infusion of a retroviral vec­tor containing the cFIX c D N A into the portal vasculature could directly tiansduce hepatocytes of the hemophiUa B dog in vivo, resulting in constitutive expression of low levels of cFTX for more than 11 months (Kay et al, 1993; Lozier and Brinkhous, 1994). Reductions of whole blood clotting and partial throm­boplastin times were also observed in the treated animals. However, retrovims-mediated in vivo gene deUvery is ex­tremely invasive, necessitating the removal of 7 0 % of the Uver from the individuals. Levels of transgene expression are also too low to have a therapeutic effect in many cases. L o w levels of expression were also observed with ex vivo gene transfer ap­proaches (Dai et al, 1992; Yao et al, 1994). In contrast, in vivo gene delivery using recombinant adenoviral vectors has been shown to be highly efficient (Smith et al, 1993; Kay et al, 1994). At relatively safe adenoviral doses, supernormal levels of plasma FIX could be achieved in hemophilia B dogs 1-3 days after infusion of a recombinant adenoviral vector con­taining the cFIX cDNA, resulting in a complete phenotypic cor­rection ofthe hemophilic state (Kay et al, 1994). Because ade­noviral vectors can transduce quiescent cells, partial hepa­tectomy is not required in adenoviras-mediated gene therapy. Unfortunately, initial studies of adenovims-mediated gene ther­apy showed that high levels of transgene expression were only transient. FIX levels in treated hemophilia B dogs declined by about 3 log units during the first 3 weeks of treatment, and slowly decreased to undetectable levels by 6 months after tieat­ment (Kay et al, 1994). More recent studies have suggested that this loss of transgene expression is largely due to a host immune response directed against the cells transduced by the recombinant adenovims (Yang etal, 1994). Because long-term expression of a tiansgene at therapeutic levels is necessary for the treatment of most genetic defects, including hemophilia B, we have examined whether the coadministration of the cFIX-expressing adenoviral vector and the immunosuppressive agent cyclosporin A (CsA) could significantiy prolong the expression of therapeutic levels of FIX in hemophilia B dogs.

MATERIALS AND METHODS

Construction of an adenoviral vector expressing canine factor IX

The constmction of the adenoviral vector containing the cFIX c D N A under the transcriptional control of the Rous sar­coma vims (RSV) long terminal repeat (LTR) promoter has been described (Kay et al, 1994). The recombinant adenovims was purified by two cycles of CsCl banding and dialyzed ex­tensively with 10 m M Tris HCl p H 7.4, 1 m M M g C h , and 1 0 % glycerol. The purified viras was stored at — 80°C. The vims titer, as determined by OD26o> ranged from I to 3 X 10" pfu/ml. Titers measured by plaque assay (Graham and Prevec, 1991) were usually within one order of magnitude of the OD260 titers.

All viral preparations were proven to be free of endotoxin by assays with a third-generation pyrogen testing kit from Bio­Whittaker (Catalog No. 50-648U).

Animal studies

Hemophilia B dogs completely deficient in FIX were main­tained at the Francis O w e n Blood Research Laboratory, University of North CaroUna at Chapel Hill. All animals were cared for according to the Guide for the Care and Use of Laboratory Animals (National Institiites of Health publication no. 85-23). Experiments for non-cyclosporin A-treated dogs have been reported (Kay et al, 1994). Adenoviral infusion into cyclosporin A-treated dogs was performed via a catheter to the cephalic vein. Cyclosporin A was administrated to the dogs orally at a dosage of 300 mg/dog-day. This is equivalent to an average dose of 19.5 mg/kg. The optimal dose of cyclosporin in the dog has been reported as 18 mg/kg (Hasegawa et al, 1992). The first dose of cyclosporin was given prior to or im­mediately after vector infusion (Schrieber and Crabtree, 1992).

Hemostatic analysis

Whole blood clotting times (WBCTs) and FIX levels were determined as previously described (Langdell et al, 1953; Brinkhous et al, 1989, 1993). The ELISA immunoassay for quantitation of FIX antigen was perfonned using a polyclonal rabbit anti-dog FIX antibody as previously described (Kay et al, 1994).

Cyclosporin assay

Whole blood CsA values were determined as described (Wang era/., 1990).

Plaque neutralization assay

The plaque neutralization assay was performed according to the method described by Pacini et al (1984).

RESULTS

FIX levels in hemophilia B dogs treated with the adenoviral vector Ad.RSV-cFIX and CsA

The adenoviral vector used in the present study, Ad.RSV-cFIX, is the same as that reported previously (Kay et al, 1994). Before infusion into dogs, all viral preparations were tested for function and toxicity in vivo by infusion of 10"^ purified viral particles into the tail vein of B A L B / C mice. S e m m cFIX anti­gen levels determined by ELISA were similar to those observed in previous studies (data not shown). Endotoxin assays were negative.

As reported previously (Kay et al, 1994), infusion of 1.6 X 10" pfu/kg of Ad.RSV-cFIX into the splenic veins of two he­mophilia B dogs resulted in 250-300% of normal plasma FIX levels at 1-2 days post-treatment (Fig. 1). Plasma FIX levels in these 2 animals rapidly decreased to about 1 % of normal lev­els at 3 weeks after infusion, and then slowly decreased to less than 0.1% of normal levels by 2 months. In the present exper­iment, 2 hemophilic animals were infused via the cephalic vein

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.

GENE THERAPY FOR HEMOPHILIA B

1000001

1041

10000

c C, 1000

>< li. o ra E (A ra Q.

100

10-

Normal range

30 60 90 120 150 180 210 240 270 300

Days Post-treatment

FIG. 1. Plasma cFIX levels in 4 hemophiUa B dogs after treatment with Ad.RSV-cFIX in the presence or absence of cyclosporin A. Dogs 1 (•) and 2 (D) each received 1.6 X 10" pfu of Ad.RSV-cHX/kg. Dog 3 (•) received 2.2 X 10" pfu of Ad.RSV-cFIX/kg plus 300 mg/day of cyclosporin A, while dog 4 (O) received 1 X 10" pfu of Ad.RSV-cFIX plus 300 mg/day of cy­closporin A. Dog 4 also received a second infusion of 1.34 X 10" pfu of Ad.RSV-cFIX/kg at the point indicated by the solid arrow. Statistical analysis of the differences between conttol and cyclosporin A-treated animals was performed using Student's f-test by comparing semm FIX levels expressed as a percentage of peak values. These differences were statistically significant (p < 0.05) at all time points greater than 2 weeks after infusion.

with the cFIX-expressing adenoviral vector Ad.RSV-cFIX and the immunosuppressant CsA. One dog, which received 2.2 X 10" pfu/kg, attained a peak level of plasma FIX of 4 8 0 % of normal at day 2; FIX levels were maintained at 108% of nor­mal through day 21 (Fig. 1). Plasma FIX levels were about 1 0 % of normal at 2 months and about 2 % of normal at 6 months af­ter ti-eatment. Thus, in this 1 animal, cyclosporin treatment largely prevented the rapid decrease in factor IX expression ob­served during the first 3-week period in the original experiment. To confirm this resuh, a second dog was treated with CsA but received a lower viral dose (1 X 10" pfu/kg). Although the peak level was lower than in the previous animal, reflecting the lower viral dose administered, a similar pattem of persistence was observed (Fig. 1).

Whole blood clotting time in hemophilia B dogs treated with Ad.RSV-cFIX and CsA

Before administration of Ad.RSV-cFIX, whole blood clot­ting times (WBCTs) in all ofthe hemophilia B dogs were greater than 50 min. Within 1-8 days after infusion of 1.6 X 10" pfu/kg of Ad.RSV-cFIX alone, W B C T s became normal or near normal but increased slowly to over 20 min at the end of 3 months (Fig. 2). These changes in W B C T were consistent with changes in plasma FIX levels. Although the differences in W B C T between cyclosporin A-treated and non-cyclosporin A-treated animals were not statistically significant due to high variability in control animals and low number of animals ex­

amined, administiation of cyclosporin A maintained near nor­mal W B C T s for up to 10 montiis (Fig. 2).

CsA trough levels in treated animals

Trough levels of CsA in whole blood of dogs receiving im­munosuppression were monitored periodically by fluorescence polarization assays. The cyclosporin A ttough levels were usu­ally in the range of 300-600 ng/ml, but were always between 100 ng/ml and 1100 ng/ml (data not shown).

Anti-adenovirus antibodies in cyclosporin A-treated dogs

Sera from CsA-tteated dogs were periodicaUy monitored for anti-adenovims antibodies by plaque neuttaUzation assay. High levels of anti-adenovims antibodies were detected in both dogs 1 week after viral infusion (Fig. 3). After achieving peak lev­els of 1:1,280 at 3 weeks, seram anti-adenovims antibodies were maintained at 1:480 to 1:160 for at least 6 months.

Repeated administration of Ad.RSV-cFIX in CsA-treated hemophilia B dogs

To determine whether the gradual decrease in semm levels of neuttalizing antibodies would permit additional cFIX- ex­pression from the recombinant adenovims, 1 dog was given a second dose of Ad.RSV-cFIX, and plasma FIX levels and W B C T s were monitored before and after this second infusion.

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.

1042 FANG ET AL.

The viral dose for the second infusion was 1.34 X 10" pfu/kg, for a total of 2.1 X 10'^ pfu. Changes in plasma FIX levels af­ter the second infusion were minimal (Fig. 1). A slight increase in plasma FIX from 1.8% to 3.1-3.4% of normal was observed at 1-2 days after infusion. Changes in W B C T s were also min­imal (Fig. 2). This minimal change in W B C T was anticipated, because W B C T in this dog was still in near normal range be­fore the second administration. Antibody levels were increased dramatically by the reinfusion of the adenoviral vector (Fig. 3).

D I S C U S S I O N

The roles of host cellular immune response to human ade­novims 5 have been evaluated in mice by Miillbacher et aL (1989). The cytotoxic T lymphocyte response to AdS was class I major histocompatibiUty complex (MHC)-restricted and mapped to the k end of M H C in C B A (H-2'') mice. Experiments with different viral mutants suggested that the El and E2 re­gions of the Ad5 genome are important for target cell sensitiv­ity to lysis by Ad5-inmiune T cells. Target cell lysability was much reduced, but still detectable in cells infected with ElA-or ElB-deleted Ad5 vims (Miillbacher et al, 1989). More re­cently, cellular immunity to an El-deleted recombinant aden­oviral vector has also been reported (Engelhardt et al, 1994; Yang et al, 1994). Following instillation of a recombinant ade­novims encoding lacZ into immunocompetent (CBA) and ge­netically athymic (nu/nu) mouse sttains, ttansgene expression diminished from > 8 0 % of hepatocytes at day 2 to undetectable levels by day 21 in C B A mice, but was maintained essentially unchanged for 60 days in nu/nu mice. Progressive loss of viral

D N A was detected in C B A mice but not in nu/nu mice (Yang et al, 1994). Following treatment of immunocompetent mice with CsA, lacZ transgene expression was extended to 21 days as compared with 14 days in untreated animals (Engelhardt et al, 1994). In the present study, the therapeutic effects of ade­novims-mediated gene therapy could be extended for nearly 6 months in adult hemophilia B dogs by the administtation of CsA. Thus, a combined therapy of adenovims-mediated gene ttansfer and immunosuppression might be applicable in some cUnical cases, especially where tteatment of a life-threatening disorders require long-term high-level ttansgene expression.

Cytotoxic T lymphocytes are evoked by class I M H C re­stricted antigens, which are synthesized within cells and pre­sented together with class I M H C on the cell surface. Because El-deleted adenovirases can still replicate without El comple­mentation in trans in cells infected at high multiplicity (Shenk et al, 1980), viral protein expression from the El-deleted ade­noviral genome in transduced cells is not unexpected. In fact, Ad5 DNA-binding protein and hexon protein were detected in the livers of the mice infused with El-deleted adenoviral vec­tor (Yang et al, 1994). Provoked cytotoxic T lymphocytes will desttoy ttansduced cells, leading to the eUmination of ttansgene and thus limiting the therapeutic effect of adenoviral-mediated gene therapy to a short period of time. By blocking host cellu­lar immune response with an immunosuppressive agent, the ge­netically modified cells can elude host immune surveillance, re­sulting in prolonged expression of the ttansgene and the therapeutic effect of adenovims-mediated gene ttansfer. This hypothesis was tested and supported by the present studies in hemophiUa B dogs. Cyclosporin A, a fungal metabolite, ex­hibits sttong immunosuppressive properties and is widely used

70

60 o Deficient range

30 60 90 120 150 180 210 240 270 300

D a y s Post-treatment

FIG. 2. Whole blood clotting times (WBCTs) in hemophilia B dogs after treatment with Ad.RSV-cHX in the presence or ab­sence of cyclosporin A. The experimental design is identical to that described in Fig. 1.

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.

GENE THERAPY FOR HEMOPHILIA B

10000 1

1043

> •D O n c < o> c N 3 0)

1000

100

300

Days post-treatment

FIG. 3. Anti-adenovims neuttaUzing antibodies in the 2 hemophiUa B dogs tteated with Ad.RSV-cFIX and cyclosporin A. The soUd artow indicates the point at which 1 dog was reinfused with 1.34 X 1 0 " pfu of Ad.RSV-cFIX/kg.

as an immunomodulator for combating allograft rejection due to its low myelotoxicity and specificity for T lymphocytes (Borel et al, 1977; Shevach, 1985). The results from the pres­ent stady showed that C s A can be used to extend the effects of adenovims-mediated gene therapy. Although the mechanisms of CsA-mediated immunosuppression are not fully understood, it is known that C s A can inhibit early events in T cell activa­tion, such as activation of interleukin-2 gene expression (Borel and Gunn, 1986; Heitman et al, 1993). CsA is also reportedly able to inhibit ttanscription from the adenovkus major later pro­moter (Mahajan and Thompson, 1993). It is unclear if this func­tion of CsA is also involved in its effects on adenovims-mediated gene therapy in the hemophilia B dogs. Nevertheless, in the present stadies, host humoral immune response was not suppressed, since a high level of adenovims neuttalization antibodies was detected in the CsA-tteated dogs and repeated administtation of adenoviral vector in 1 dog 6 months after the first infusion showed only minimal effects on plasma FIX levels.

A slower decline of plasma FIX levels over time was ob­served in CsA-tteated dogs. It is unclear if this decUne was due to unsuppressed host immune function from residual cytotoxic T lymphocytes and/or antibody-dependent cellular cytotoxicity (ADCC), or due to "nataral" loss of the nonintegrated aden­oviral gene in ttansduced cells. This gradual decUne of plasma FIX levels occurted even though the C s A dose was optimal, and no antibodies against canine factor IX were detected in these animals (data not shown). Not surprisingly, rejection of allografts has previously been reported under such optimal dos­ing (Hasegawa et al, 1992; Fujisawa etal, 1993). Because the adenoviral genome is not integrated into the host cellular genome, degradation of the adenoviral genome within ttans­duced cells or loss of the ttansduced cell through the natural

process of cell tamover may also have resulted in reduction of ttansgene expression in the target tissues or organs. A better understanding of the mechanisms underlying this slow decUne of ttansgene expression will be important for the future design of adenoviral vectors aimed at long-term gene therapy.

ACKNOWLEDGMENTS

We thank the animal support staff at the Francis Owen Blood Research Laboratory at the University of North CaroUna at Chapel Hill. This work was supported in part by National Institates of Health Grants D K 44080 (Baylor College of Medicine) and H L 01648-46 and H L 26309-12 (University of North Carolina).

REFERENCES

BOREL, I.K., and GUNN, H.C. (1986). Cyclosporine as a new ap­proach to therapy of autoimmune diseases. Ann. N.Y. Acad. Sci. 475, 307-318.

BOREL, I.F., FEURER, C, M A G N E E , C.A., and STAHELIN, H. (1977). Effects of the new anti-lymphocytic peptide cyclosporine A in animals. Immunology 32, 1017-1025.

BRINKHOUS, K.M., HEDNER, U., GARRIS, I.B., DINESS, V., and READ, M.S. (1989). Effect of recombinant factor VHa on the he­mostatic defect in dogs with hemophiUa A, hemophiUa B and Willebrand disease. Proc. Nati. Acad. Sci. U S A 86, 1382-1386.

BRINKHOUS, K.M., LANDEN, C.N., and READ, M.S. (1993). Evaluation of sensitivity of whole blood clotting time (WBCT), par­tial thromboplastin time (PTT), and F.K one-stage bioassay tests with low plasma F.IX levels observed with transfusion or gene ther­apy in canine hemophilia B. Blood 82, Suppl. 1, 595.

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.

1044 FANG ET AL.

DAI, Y., R O M A N , M., NAVIAUX, R.K., and VERMA, I.M. (1992). Gene therapy via primary myoblasts: Long-term expression of fac­tor IX protein following transplantation in vivo. Proc. Natl. Acad. Sci. USA 89, 10892-10895.

ENGELHARDT, J.F., YE, X., DORANZ, B. and WILSON, J.M. (1994). Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA. 91, 6196-6200.

FUnSAWA, T., SAITOH, Y., URABE, N., TAKEDA, T., SERINE, Y., BABA, M., and YAMAGUCHI, Y. (1993). Dose study of the immunosuppression of FK 506 in canine lung allotransplantation. Surgery Today 23, 338-343.

G R A H A M , F.L., and PREVEC, L. (1991). Manipulation of adenovirus vector. In Methods in Molecular Biology: Gene Transfer and Expression Protocols, vol. 7. E.J. Murray, ed. (The Humana Press, Clifton, NJ) pp. 109-128.

HASEGAWA, S., YOKOMISE, H., HIRAI, T., FUKUSE, T., MURO, K., TAKAHASHI, Y., INUI, K., AOKI, M., HITOMI, S., and W A D A , H. (1992). Combination use of suboptimal dose of FK 506 and cyclosporine in canine lung transplantation. J. Thorac. Cardiovasc. Surg. 104, 1340-1348.

HEITMAN, J., KOLLER, A., KUNZ, J., HENRIGUEZ, R., SCHMIDT, A., M O W A, N.R., and HALL, M.N. (1993). The immunonosup-pressant FK506 inhibits amino acid import in Sacciiaromyces cere­visiae. Mol. Cell. Biol. 13, 5010-5019.

KAY, M.A., ROTHENBERG, S., LANDEN, C.N., BELLINGER, D.A., LELAND, F., TOMAN, C, FINEGOLD, M., THOMPSON, A.R., READ, M.S., BRINKHOUS, K.M. and W O O , S.L.C. (1993). In vivo gene therapy of hemophilia B: Sustained partial correction of factor DC-deficient dogs. Science 262, 117-119.

KAY, M.A., LANDEN, C.N., ROTHENBERG, S.R., TAYLOR, L.A., LELAND, F., WIEHLE, S., FANG, B., BELLINGER, D., FEME-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. Natl. Acad. Sci. USA 91, 2353-2357.

LANGDELL, R.D., WAGNER, R.H., and BRINKHOUS, K.M. (1953). Effect of antihemophilic factor on one-stage clotting tests. J. Lab. Clin. Med. 41, 637-647.

LOZIER, J.N., and BRINKHOUS, K.M. (1994). Gene therapy and the hemophilias. J. Am. Med. Asso. 271, 47-51.

PACINI, D.L., BUBOVI, E.J., and CLYDE, W.A., Jr. (1984). A new animal model for human respiratory tract disease due to adenovirus. J. Infect. Dis. 150, 92-97.

MAHAJAN, P.B., and THOMPSON, E.A., Jr. (1993). Cyclosporine A

inhibits the activity of a TATA box-binding protein that is required for transcription from the adenovirus major late promoter. J. Biol. Chem. 268, 16693-16698.

MfJLLBACHER, A., BELLETT, A.J.D., and HLA, R.T. (1989). The murine cellular immune response to adenovirus type 5. Immunol. Cell Biol. 67, 31-39.

ROBERTS, H.R., and LOZIER, J.N. (1991). Clinical aspects and ther­apy for hemophilia B. In Hematology: Basic Principles and Practice. R. Hoffman, E. Bens, Jr., S. Shattil, B. Furie, and H. Cohen, eds. (Churchill Livingston, New York) pp 1325-1331.

SHENK, T., JONES, N., COLBY, W., and FOWLKES, D. (1980). Functional analysis of adenovirus-5 host-range deletion mutants de­fective for transformation of rat embryo cells. Cold Spring Harbor Symp. Quant. Biol. 44, 367-375.

SCHREIBER, S.L., and CRABTREE, G.R. (1992). The mechanism of action of cyclosporin A and FK506. Immunol. Today 13, 136-141.

SHEVACH, E.M. (1985). The effects of cyclosporine A on the immune system. Annu. Rev. Immunol. 3, 397-423.

SMITH, T.A.G., MHAFFEY, M.G., KAYDA, D.B., SAUNDER, J.M., YEI, S., TRAPNELL, B.C., McCLELLAND, A., and KALEKO, M. (1993). Adenovirus mediated expression of therapeutic plasma lev­els of human factor IX in mice. Nature Genet. 5, 397^02.

W A N G , P., MEUCCI, V., SIMPSON, E., MORRISON, M., LUNETTA, S., ZAJAC, M., and BOECKX R. (1990). A monoclonal antibody fluorescent polarization immunoassay for cyclosporine. Transplant. Proc. 22, 1186-1188.

YANG, Y., NUNES, F.A., BERENCSI, K., FURTH, E.E., GONCZOL, E., and WILSON, J.M. (1994). Cellular immunity to viral antigens limits El-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91, 4407^1411.

YAO, S.-N., SMITH, K.J., and KURACHI, K. (1994). Primary my­oblast-mediated gene transfer: Persistent expression of human factor IX in mice. Gene Ther. 1, 99-107.

Address reprint requests to: Dr. Savio L. C. Woo

Department of Cell Biology Room T721

Baylor College of Medicine One Baylor Plaza

Houston, TX 77030

Received for publication December 12, 1994; accepted after revision April 21, 1995.

Dow

nloa

ded

by S

tanf

ord

Uni

vers

ity M

edic

al C

ente

r fr

om w

ww

.lieb

ertp

ub.c

om a

t 10/

09/1

8. F

or p

erso

nal u

se o

nly.