I I U M A N G E N E T H E R A P Y 6:1029-1037 (August 1995) Mary Ann Liebert, Inc.

A M o d i f i e d U r o k i n a s e P l a s m i n o g e n Activator I n d u c e s L i v e r

R e g e n e r a t i o n W i t h o u t B l e e d i n g

ANDRE LIEBER,! MARIE-JEANNE T.F.D. VRANCKEN PEETERS,'^ ALLEN GOWN,^ JAMES PERKINS,2 and MARK A. KAYi*

ABSTRACT

Direct retrovirus-mediated hepatic gene transfer results in permanent gene expression; however, gene trans-

fer requires surgical hepatectomy (to stimulate cell division) and has been inefTicient. W e recently used re-

combinant adenovirus vectors that transiently expressed urokinase from mouse hepatocytes to induce hepa-tocellular regeneration in place of a partial hepatectomy. The adenovirus method allowed for five-fold more

efficient retrovirus transduction in vivo compared to the conventional partial hepatectomy approach. The m a -

jor problem with the urokinase-mediated hepatic regeneration was the transient secretion of urokinase into the bloodstream that led to hypocoagulation. T o circumvent this side-effect, the urokinase protein was mo d -

ified by adding amino-terminal and carboxy-terminal endoplasmic reticulum retention signals. The recombi-

nant urokinase molecules expressed from adenoviral vectors remained in hepatocytes, were enzymatically ac-

tive, and resulted in similar rates of hepatic regeneration as found with the secreted urokinase. Modified urokinase-mediated liver regeneration was equally capable of allowing retrovirus-mediated gene transfer in

vivo. Thus, the method of direct retrovirus transduction of hepatocytes becomes clinically relevant as the tech-nology becomes safer.

OVERVIEW SUMMARY

A cDNA encoding a modified uroldnase plasminogen acti-vator was constructed and shown to induce liver regenera-tion without transient secretion of urokinase into the blood-stream when introduced into mouse hepatocytes in vivo with a recombinant adenovirus vector. The advantage ofthe non-secreted urokinase was that it did not cause hypocoagula-tion and spontaneous bleeding. This study demonstrates that retroviral vectors can transduce the dividing hepato-cytes in the absence of a surgical partial hepatectomy. Thus, transient gene expression from recombinant adenovirus vectors can be used to induce liver regeneration and allow for relatively efiicient permanent retroviral-mediated gene transfer.

E S fTRODUCTION

THE LIVER REPRESENTS AN IMPORTANT TARGET TISSUE for he-patic gene fransfer because of the large number of genetic

and acquired disorders that are potentially freatable. Refrovirus-

based gene transfer remains an important prospect to achieve such goals because of the persistent nature of gene expression after gene transfer. The major drawbacks to this gene fransfer system are the relatively low rate of hepatocyte fransduction (0.25-2%) in in vivo/ex vivo gene therapy trials in animals and humans (Kay et al, 1992, 1993; Grossman et al, 1994), and the requirement for cell division for gene fransfer (Miller et al, 1992; Moscioni et al, 1993; Branchereau et al, 1994). As a re-sult, direct in vivo Uver gene transfer requires a 2/3 surgical he-patectomy for stable rettovirus-mediated gene expression. The general sfrategy for in vivo gene fransfer is to perform partial hepatectomy and then infuse refrovirus into the portal cttcula-tion (Kay et al, 1992, 1993; Branchereau et al, 1994). Recently, we have described a new method for inducing liver regeneration using an adenovirus vector system (Lieber et al, 1995 a). Adenoviral vectors can fransduce virtually all the he-patocytes witiiout partial hepatectomy and result in fransient gene expression. A recombinant adenovirus was used to express urokinase (uPA). Urokinase is involved in diverse physiologi-cal and pathological processes inclucUng fibrinolysis, ovulation, inflammation, and tumor cell invasion (Sakelsa and Rifkin, 1988). The ectopic expression of uPA in hepatocytes after ade-

'Division of Medical Genetics RG-25, Department of Medicine, Markey Molecular Medicine Center, ̂Division of Transplant Surgery, Department of Surgery RF-25, ̂ Department of Pathology SM-30, and ''Department of Pediatrics, University of Washington, Seattle, W A 98195.

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1030 L I E B E R E T AL.

novttus-mediated gene transfer resulted in the slow tumover of the majority of hepatocytes and stimulated asynchronous liver regeneration over a period of 8 days.

Urokinase is produced as a single chain (sc-uPA) (reviewed in Saksela, 1988). In hepatocytes fransduced by adenovirus vec-tors that express uPA, ttace amounts of plasminogen are con-verted to plasmin, which is capable of converting the sc-uPA to the more active two-chain uPA (tc-uPA). The tc-uPA can generate more plasmin. The protease activity of plasmin is most likely dttected against a number of different cellular proteins that resuhs in hepatocyte death. During this period of hepato-cyte regeneration, it was possible to ttansduce hepatocytes di-rectly in vivo with transduction efficiencies five- to nine-fold greater to that achieved with partial hepatectomy (Lieber et al, 1995a).

The major limitation to this system was the ttansient hypoco-agulable condition created from the presence of secreted uroki-nase in the plasma that reached lOOX the normal concentra-tion. Generally, secreted proteins are first ttanslocated across the endoplasmic reticulum (ER) membrane, and are further ttansported in vesicles through the Golgi apparatus to the plasma membranes. In the case of uPA, the ttanslocation across the E R membranes is triggered by a characteristic signal se-quence at the amino terminus of the precursor protein (Nagai et al, 1985; van Hinsbergh, 1988). This signal peptide is cleaved off by signal peptidase during the ttansfer of the polypeptide across the membrane (for review, see Nothwehr and Gordon, 1990). To inhibit the secretion of uPA into the sys-temic cttculation, the protein had to be modified in a way that the export of the protein from the ER was prevented. Additionally, because the hepatic toxicity of uPA is primarily based on the inttacellular activation of plasminogen, following proteolytic self-digestion, a catalytically active intracellular uPA should function in the same way for stimulating hepato-cyte regeneration. Thus, we genetically modified the uPA c D N A with different E R retention signals and found that the modified enzyme activity was locaUzed inside ttansduced cells in culmre and mouse hepatocytes in vivo. Furthermore, the mod-ified uPA was found to induce hepatic regeneration as previ-ously shown for secreted uPA and could be used for rettoviral fransduction of hepatocytes in vivo.

MATERIALS AND METHODS

Modification of urokinase plasminogen activator

The human uPA cDNA (1,326 bp) (Nagai et a/., 1985) cloned as a Xba I/Asp-718 fragment in p G E M 3 (Promega) was kindly provided by B. Hinzmann ( M D C BerUn). For the modification AC-uPA, the 3' end of uPA c D N A was extended by a sequence coding for the K D E L signal with additional upsfream residues as described in Fig. 1. The additional amino acids were included because some of the acidic residues upstream of the tetrapep-tide K D E L may be important for the conformation of this do-main or for the interaction with the K D E L receptor (Pelham, 1988).

The sequence for the modified carboxyl terminus (75 nu-cleotides) was cloned as synthetic double-stranded oUgonu-cleotide instead of the Bam HI (position 1,257)/Asp-718 (Fig. 1) fragment of uPA. The 75-nucleotide-long double-stranded

oligonucleotide was generated by annealing the following phos-phorylated single-sfranded oUgonucleotides (I-V):

I 5' -G ATCCCGC AGTC AC ACC A AGG AAGAG AATGGC-CTGGCCCTC

II 5' -G A AG A AG AT ACCTCTG A A A A AG ATG AGCTCT-GAGG

III 5'-TCTTCCTTGGTGTGACTGCG IV 5' -CAGAGGTATCTTCGAGGGCCAGGCCATTC V 5'-GTACCCTCAGAGCTCATCTmT

For the AN-uPA modification, the 25 amino-terminal amino acids, including the pre-uPA signal peptide, were substituted by the amino-terminal RR-retention signal (Strubin et al, 1986; Schutze et al, 1994) together with the fransmembrane anchor (TM) separated by a spacer peptide (31 amino acids) from the membrane II protein Iip33 (Stinbin etal, 1986) (Fig. IC). The Iip33 fragment was obtained by P C R from the Iip33 cDNA, kindly provided by P. Petersen and M. Jackson (R.W. Johnson Pharmaceutical Research Institute, San Diego). The forward primer was designed for the Iip33 sequence coding for the Met -l-l and following amino acids with a 5' extention containing a Xba 1 site and a Kozak sequence upsfream of the Iip33-ATG: 5 ' - G G C T C T A G A T C G C C A C C A T G C A C A G G A G G A G A A G C A G G A G C . The reverse primer was specific against the Iip33 sequence coding for amino acids 68-74 with a 5' exten-sion for the in-frame fusion with uPA by a Taq 1 site at posi-tion 108 of the uPA cDNA: 5'-GTT C G A T G G A A C C T G CTG CTG CTA C A G G A A GTA GGC.

The PCR was carried out with 50 ng of Iip33 D N A by Pfu-D N A polymerase (2.5 units) (Sfratagene) with 2 % DMSO, 300 m M dNTPs, 20 p M of each primer, and the Pfu reaction buffer #2 (20 m M Tris-HCl pH 8.2, 10 m M KCl, 6 m M (NH4)2S04, 1.5 m M MgClz, 0.1% Triton X-100) in 40 cycles (45 sec at 95°C, 45 sec at 65°C, 90 sec 72°C) followed by 10 min of 72°C incubation. The amplification product (251 bp) was separated in a 3 % NuSieve agarose, purified, and digested with Taq 1 and Xbal.

A deletion of the wild-type uPA 5' end containing the sig-nal peptide (Xba 1/Taq I, position 108) was not possible due to multiple Taq I sites within the uPA cDNA. Thus, a 526-bp Xba 1-Bal 1 uPA fragment was first isolated and then recut with Taq I (Fig. IA). The remaining 421-bp Taq 1-Bal I fragment was then ligated with the Xba VTaq I P C R product and the Bai I-truncated uPA gene/vector.

The correctness of the structure of all modifications was con-firmed by sequencing from the T7 promoter (AC-uPA) and Sp6 promoter (AN-uPA) in pGEM.

To create the ANAC-uPA D N A gene, the amino terminus of AC-uPA was modified in the same way like described for uPA. The resulting modified uPA genes: AC-uPA, AN-uPA, and ANAC-uPA were cloned as a HinA llll Asp-11% fragment into the HinA III/Asp-718 site of pAdL.l/RSV and pAdL.l/PGK (Fang et al, 1994; Kay et al, 1995). The resulting adenoviral plasmids were called:

pAd.RSV-AC-uPA pAd.RSV-AN-uPA pAd.RSV-ANAC-uPA pAd.PGK-AN-uPA pAd.PGK-ANAC-uPA

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MODIFIED UROKINASE INDUCES LIVER REGENERATION

A

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Xbal TaqI HIII 1 108

Ball 530

BamHI Asp7l8 1257 1326

Sp6 prom uPAcDNA

TV prom

B

C-terminal KDEL-signal(synthetic)

uPA

AC-uPA

...WIRSHTKEENGLALcT 'C"̂ *""'

.WIRSHTKEENGLAL EEDTSEKDELci C-Tem)

.TGG ATC CGC . GAG CTC TGA GGGTACC Asp718

c

N-terminal Ilp33-signal ( P C R cloning)

.' signal peptide I pre-uPA (N-Term M R A L L A R L L L C V L V V S D S K G S N E L H Q V P S N . ,

AN-uPA N-Terifll

GGCTCTAGATCGCCACCL^TGICAC Xbal Kozak

Iyqqqvpsn.

..CCA TCG AAC TaqI

FIG. 1. Endoplasmic reticulum retention of uPA. A. The u P A c D N A . B. Carboxy-terminal modification. The amino acids that differ from the original sequence are in bold. The (—) represents the negatively charged glutamine residue. C. Amino-terminal modification. The arrow represents the signal peptidase cleavage site. The 5' primer contained a Kozak sequence. The details of the modifications and cloning are described in Materials and Methods.

Production of recombinant adenoviral vectors

The construction of the Ad.RSV-uPA vector has been pre-viously described (Lieber et al, 1995a). For generating E 1 A ~ recombinant adenoviruses, these plasmids were cofransfected with p J M 1 7 into 293 cells by calcium phosphate coprecipita-tion and plaques were isolated as described (Lieber et al, 1995b). The supematant and cell lysate from 10 plaques of each adenovirus were analyzed for u P A in supematant and cell lysate by ELISA. For generating the cell lysate for E L I S A analysis, an equal number of cells from 12 well plates was pelleted, re-suspended in 10 m M Tris-Cl p H 7.5, incubated in ice for 10 min, and homogenized in a glass Dounce homogenizer (20 sfrokes). NaCl was added to a final concenfration of 137 m M .

Vu u s from positive plaques was expanded, purified by CsCl ulttacentrifugation, and titered by O D measuring and plaque as-say. Transduction of Chinese hamster ovary cells was per-formed as previously described (Lieber et al, 1995b).

Immunohistochemistry for uPA analysis

Deparaffinized Uver sections were incubated with a 1:200

dilution of a 1:1 mixture of two monoclonal antibodies against different epitopes on the u P A (provided by Dr. H. WiU, M D C -Berlin). Immunohistochemical studies were performed using a modified avidin biotin immunoperoxidase technique on a Biotek Techmate automated immunostainer with Biotek sec-ondary antibody reagents.

uPA ELISA

The E L I S A is based on monoclonal antibodies against the catalytic and receptor-binding domain of uPA. The E L I S A has a linear range from 1 ng/ml to 50 ng/ml. The methods were as described in Lieber (1995 a).

Fibrin plaque assay

The procedures were modified as described by Jespersen and Astmp (1983). Fifteen milUgrams of fibrinogen were dissolved in 50 m M Tris-HCl, 140 m M NaCl pH 7.5 (TBS) and mixed with 2 % Agar Noble in TBS, 25 m M CaCh, 12.5 m M M g C b at 58-60°C. Five micrograms of plasminogen and 60 units of throm-bin were quickly added to the agar-fibrinogen mix and plated

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1032 LIEBER ET AL.

out (thickness 5 m m ) . Fibrin as the product of fibrinogen acti-vation forms polymers that are visible in the agar as cloudy back-round. Twenty-microUter samples or uPA standard dilutions (Sigma) were added in wells with 4-inm diameter and incubated for 4 hr in a humidified chamber. After the diffusion in agar, the uPA forms clear plaques around the cavities. The plaque diam-eter was measured and the fibrinolytic activity of samples in Plough units/ml was estimated based on the standard curve.

For enzymatic analysis of cell lysates, the cell pellets were resuspended in 0.14 M NaCl, 1 m M U g C h , 10 m M Tris-HCl p H 8.5, 0.5% NP-40, and incubated on ice for 20 min.

Animal studies

All animal stadies were performed on female C57B1/6 mice (Jackson Labs) in accordance with the University of Wash-ington's animal guidelines. All experimental methods have been previously described (Lieber, 1995a). A U vims infusions were performed via the portal vein.

Biochemical assays

The methods for analysing the semm SGPT and PT have been described (Lieber et al, 1995a). The method of Paulsen and Reicheh was used for the determination of hepatic [^HJthymidine uptake. One microcurie of [e-^H]thymidine (5 Ci/mmol, Amersham) in 0.9% saline per gram body weight was injected infraperitonealy three times at 24 hr, 12 hr, and 45 min prior to sacrificing the animals. The D N A content in the Uver exfract was detemiined by O D reading and by the dipheny-lamine method (Bucher, 1963). Specific activity was expressed as cpm/jLig of D N A . Autoradiography was performed as de-scribed (Lieber et al, 1995a).

Retrovirus-mediated gene transfer

The methods for propagation and infusion of rettovttus into mice were identical to that as previously described by Lieber (1995a).

RSV-aCuPA RSV^NuPA RSV-ACANuPA PCK-ANuPA PGK-aCaiNuPA

supernatant H cell lysate

B

RSV-uPA RSV-4NuPA PCK-aNuPA RSV-hAAT RSV-iCuPA RSV-AC4^uPA PGK-aCiNuPA

supernatant ffl cell lysate

FIG. 2. Expression of uPA and modified uPAs in tissue cultare cells. Chinese hamster ovary cells were transduced with 1,000 pfu/cell of the respective adenovims vectors. The supematants and cell lysates were obtained 48 hr later and analyzed for: A. Antigen levels by ELISA (ng antigen/per total supernatant or ceU lysate from a tissue culture dish containing 1 X 10* cells). B. Enzymatic activity expressed as Plough units/1 X 10* cells for supemants or lysates. Each experiment was performed in dupli-cate.

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MODIFIED UROKINASE INDUCES LIVER REGENERATION 1033

RESULTS

Modification ofthe urokinase cDNA

The methods used to modify the uPA cDNA for creating dif-ferent E R retention signals are summarized in Fig. 1. A car-boxy-terminal E R K D E L signal (for review, see Pelham, 1989) was cloned onto the 3' end as described in Materials and Methods and referred to as AC-uPA (Fig. IB). The AN-uPA modification was produced by substitating the 25 amino-ter-minal amino acids with an RR-retention signal together with tiie fransmembrane anchor (TM) separated by a 31-amino-acid spacer from the membrane n protein lip 33 (Fig. IC), thereby deleting the signal peptide. A thttd constmct contained a com-bination of the amino- and carboxy-terminal modifications (ANAC-uPA). Recombinant adenovims vectors that express the unmodified uPA (Lieber, 1995a) or the modified constiucts were prepared from expression cassettes under the franscrip-tional confrol of the Rous sarcoma vims long terminal repeat (RSV-LTR) or P G K promoters.

Cellular localization of uPA arui modified uPAs in tissue culture cells

To determine how much of the modified uPA protein was localized in the cell or secreted into media, Chinese hamster ovary cells were exposed to enough adenovuus to fransduce most ofthe ceUs (Lieber et al, 1995b). T w o days later, the su-pematants and cell lysates were analyzed for uPA antigen by

ELISA (Fig. 2A) and enzyme activity by fibrin plaque (Fig. 2B) assays. The results showed that as predicted, the majority ofthe unmodified urokinase antigen and enzymatic activity was found to be secreted into the media while all three of the modified uPAs were predominantiy found in cells. Based on the amount of uPA protein produced from each adenovttus vector, the spe-cific enzymatic activity of the modified uPAs was relatively similar to the wild-type protein. A small proportion of the AC-uPA and AN-uPA was found in the media; however, there was virtually no detectable uPA protein in the media of double ANAC-uPA modified protein. Thus, the double-modified con-stmct was used for all subsequent studies. The total amount of uPA production was slightly greater when the RSV-LTR pro-moter was used compared with the P G K promoter (Fig. 2).

Ad.PGKAN AC-uPA delivery to mouse hepatocytes in vivo

To determine whether ANAC-uPA was retained in mouse hepatocytes in vivo, Ad.PGKANAC-uPA vims was infused into the portal vein of mice and at different times the semm and Uver tissue were analyzed for uPA (Fig. 3). The results showed that no uPA was detected in the semm of recipients over an 11-day period after gene ttansfer, whereas the protein was de-tectable at relatively high concentrations in Uver tissue on days 3-6 after gene ttansfer. In conttast, in a previous stady, mice infused with a recombinant adenovims that expressed wild-type uPA had ttansient semm concenttations of up to 350 ng/ml

norm. 10 11 day

uPA-serum (ng/ml)

S G P T (u/ml)

uPA - liver (ng/100>ig)

PT (sec)

FIG. 3. Biochemical stadies after adenovttus adminisfration. Mice were given 5 X 10' pfu of Ad.PGKANAC-uPA adenovims by portal vein infusion. At different times, samples were taken for measurements of semm SGPT, prothrombin times (PT), uPA antigen in Uver (per 100 ̂ g tissue) and semm. The vertical bars represent the standard deviation. At least three independent sam-ples were analyzed for each point.

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FIG. 4. Immunohistochemical staining for uPA. Mice were infused with 5 X 10' pfu of Ad.RSV-uPA or Ad.PKG-ANACuPA adenovims. Four or 6 days later, the liver sections were stained for uPA. A. Normal liver. B. Ad.RSVuPA 6 days later. C. Ad.PKG-ANACuPA 4 days later. D. Ad.PKG-ANACuPA 6 days later. Magnification, lOOX.

(Lieber et al, 1995a). In that study, the elevation in the semm urokinase caused a marked increase in the prothrombin time, an indication of hypocoagulability. In this stady, the PTs were found to be in the normal range in animals infused with Ad.PGK-ANAC-uPA (Fig. 3). Furthermore, unlike wild-type uPA, modified uPA expression in hepatocytes did not cause he-morrhage in any of the animals studied. To monitor for hepa-tocellular injury, semm SGPTs were detemiined. An elevation in semm SGPT in animals transduced with Ad.PGK-ANAC-uPA was observed between days 3 and 8 after gene transfer (Fig. 3) and indicated that these animals had hepatocellular in-jury similar to what was previously described with wild-type uPA (Lieber et al, 1995a). Taken together, the modified uPA caused hepatocellular damage without secretion of the protein into the bloodstream or detectable alterations in hemostasis.

As additional evidence that the ANAC-uPA was locaUzed in the cell, animals were transduced with Ad.RSV-uPA or Ad.PGK-ANAC-uPA and liver sections were immunohisto-chemically stained for uPA (Fig. 4). Although infusion of ei-ther vector resulted in the characteristic histologic appearance of degenerating hepatocytes (Lieber et al, 1995a), the uPA staining pattems showed marked differences. Secreted uPA was rarely detectable immunohistochemically while a large number of the Ad.PGKANAC-uPA transduced cells were stained with a pattem suggestive of a membrane localization (Fig. 4). Six days after Ad.PGKANACuPA adenovims administration, uPA stained hepatocytes had the characteristic degenerative changes, while the patches of hepatocytes with normal histologic ap-

pearance did not stain. This suggested that the normal hepato-cytes represent nontransduced regenerating cells.

Ad.PGK-ANACuPA induces liver regeneration

To determine whether intracellular production of ANACuPA caused liver regeneration, animals infused with either 5 X 1 0 ' pfu of Ad.RSV-uPA or Ad.PGKANAC-uPA were analyzed for incorporation of [̂ H]thymidine into hepatic DNA. Both vectors led to similar rates of radioactive incorporation occurring from days 3-11 after gene fransfer (Fig. 5). Autoradiography was performed on the liver sections of 2 animals that received Ad.PGK-ANACuPA on days 3, 5, and 11. The mean propor-tion of labeled hepatocytes was 4 0 % (range 38, 42), 5 0 % (range 48, 52), and 3 4 % (range 30, 38) on days 3, 5, and 11, respec-tively. These resuUs were identical to what was observed with Ad.RSV-uPA in a previous study (Lieber et al, 1995a). The mean proportion of labeled hepatocytes during the same peri-ods following infusion of an irrelevant control adenovims var-ied between 4 % and 11% (Lieber et al, 1995a). Thus, the ANACuPA protein produced in hepatocytes caused asynchro-nous liver regeneration in a manner similar to wild-type uPA.

Ad.PGKANACuPA-treated hepatocytes can be transduced with recombinant retroviral vectors

The application of urokinase-induced liver regeneration was developed as a means to ttansduce hepatocytes with recombi-nant retrovims vectors with five-fold greater retrovims-medi-

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MODIFIED UROKINASE INDUCES LIVER REGENERATION 1035

<

a

u

160

140H

120

100

80H

60

40H

20

0

9 10 11 12 13 14 day

2/3-hepatectomy Ad.RSV-uPA Ad.PGK-muPA

FIG. 5. Hepatic [̂ HJtiiymidine uptake. Mice were infused with 5 X 10' pfu of Ad.PGKANACuPA (Ad.PGK-muPA) aden-ovirus and at various times the animals were infused with [^H]thymidine at 24 hr, 12 hr, and 45 min prior to sacrifice as de-scribed (Lieber et al, 1995a). The specific activity was expressed as cpmlpg of D N A . The vertical Unes represent the standard deviation. At least 3 animals were analyzed per time point. Animals receiving partial hepatectomy or Ad.RSV-uPA were included for comparison (Lieber et al, 1995a).

E

c

1-< < E 3 O (0

1000 •

800-

600-

400-

200-

0 4

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•

1000

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20 40 60 Days post injection

BO 10 20 30 Days post Injection

FIG. 6. Rettovttus-mediated gene ttansfer. Mice were infused with 5 X 10' pfu of Ad.RSVuPA (A) or Ad.PGKANACuPA (B) and 1 ml of LNAlbhAAT (2 X 10* cfu) rettovttus on days 3 and 5 after adenovttus infusion. S e m m was periodically quantitated for h A A T in at least dupUcate. Each Une represents an individual animal.

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1036 LIEBER E T AL.

ated gene ttansfer efficiencies compared to the surgical hepa-tectomy approach (Lieber et al, 1995a). To establish that the Ad.PGKANACuPA and Ad.RSV-uPA adenovttus-freated ani-mals were equally susceptible to retrovims-mediated gene transfer in hepatocytes in vivo, 3 and 5 days after adenovims infusion 2 X 10* cfu of LNAlbhAAT refrovims was infused into the portal vein (Fig. 6). This rettovims expresses human ai-antitrypsin from transduced cells (Kay et al, 1992; Lieber et al, 1995a). The semm h A A T concenttations were similar in both tteatment groups, establishing that the new urokinase ade-novims vectors were equally able to allow for refrovims-medi-ated hepatic gene ttansfer.

D I S C U S S I O N

Recently, we reported on the use of the urokinase recombi-nant adenovims to induce liver regeneration and allow for retto-vttal-mediated gene fransfer without a partial hepatectomy. This type of approach may be useful for ex vivo gene therapy as well because urokinase-induced regeneration prior to cellular trans-plantation may allow for reconstitation with a much greater pro-portion of genetically altered hepatocytes due to proliferation of ttansplanted hepatocytes. To avoid the risk of hemorthage secondary to uPA secretion, the uPA protein was retained within the cell by the addition of ER retention signals. ER retention signals have been identified for soluble ER-luminal proteins (KDEL-signal) (Pelham, 1989) for type I ttansmembrane pro-teins (KK-motif) (Jackson et al, 1990) and for type II trans-membrane proteins (RR-motif) (Schutze et al, 1994). The ER retention is based on the interaction of these signals with spe-cific membrane receptors. The prevention of secretion of post-ttanslationally modified proteins is probably not due to perma-nent ER retention but due to a continious retrieval from a post-ER compartment (e.g., Golgi) back to the ER (Pelham, 1988; Jackson ef a/., 1993). The uPA carboxyl-terminus (ACuPA) was modified with the

highly conserved K D E L motif (Munroe and Pelham, 1987; Pelham, 1988), which is a characteristic feature of soluble pro-teins residing in the ER lumen, Uke BiP (Ig heavy chain bind-ing protein) (Munroe and Pelham, 1986). Because the prote-olytic activity of uPA is located in the carboxy-terminal protein domain (van Hinsberg, 1988; Lu et al, 1992), it was not clear a priori whether the modified ACuPA would remain catalyti-cally active. Furthermore, the signal peptide on the uPA amino terminus was substitated by the R R retention signal and ttans-membrane region, which was shown to be present in type II transmembrane proteins Uke invariant chain protein lip 33 (Schutze et al, 1994) or T R A M (Goriich et al, 1992). The R R motif is based on surtounding arginine residues M H R R R S R lo-calized close to a membrane anchor in Iip33. Generally, the membrane orientation of the first membrane anchor depends on the distribution of nearby charged amino acids, with the more positive charged regions facing the cytosol (Hartmann et al, 1989). According to this mle, the carboxyl terminus of the AN-uPA protein would be directed into the ER lumen. In the com-bination of both uPA modifications (ANAC-uPA), the K D E L signal could interact with a receptor in the way like other lu-minal ER resident proteins. W e anticipated that an additive ef-fect might occur when both signals were combined. Two ER

retention signals were found for other proteins. The ER resi-dent protein T R A M (GorUch et al, 1992) has in its amino ter-minus characteristics of a R R motif, whereas the carboxy-ter-minal sequences contains a K K ER targeting motif

The study here demonsttated that both the amino- and car-boxy-terminal modifications of uPA prevent the secretion of uPA, whereas the K D E L signal seemed to work better in ER retention as the amino-terminal modification. The most effec-tive retention of uPA (no detectable secretion) occurted when both termini were modified as with ACANuPA. Although there was no direct proof that the protein is exclusively localized in the ER, the pattem of uPA antibody staining in Ad.PGK-ANAC-uPA-ttansduced hepatocytes was suggestive. The mod-ified uPA caused similar histologic changes found in a uPA-fransgenic mouse model (Sandgren et al, 1991). Ulfrastmctural examination of the Uvers showed a characteristic cytoplasmic vacuolization. The vesicles were lined with membrane that con-tained polyribosomes, suggesting an origin from the rough ER. The modified uPA used in this study was present on similar stmctares. The presence of modified uPA protein on these vac-uolized stmctures was further evidence that the protein is as-sociated with the ER membrane. The majority of unmodified uPA is secreted yet still induced high rates of hepatocellular de-generation. Thus, it was unclear whether inttacellularly retained uPA would be more potent at inducing Uver regeneration. Therefore, we elected to use Ad.PGK-ANACuPA rather than the Ad.RSV-ANACuPA vector because the P G K promoter has been shown to be about 10-fold less active than the RSV-LTR promoter in hepatocytes in vivo (Kay et al, 1994, 1995).

The hepatotoxic effect of uPA is based on the hypothesis that uPA activates plasminogen to plasmin within the ER. One hy-pothesis is that tc-uPA and plasmin could be involved in en-hanced protein degradation within the ER and cause intermp-tions in normal protein export from the ER. Additional stadies are needed for dttect proof Nonetheless, the infracellular uPA led to similar rates of hepatocellular degradation and regener-ation as the unmodified uPA without the side-effects related to tendency to bleed.

The ability to use recombinant adenovims vectors as a means to fransduce refrovkus into hepatocytes is an atfractive model for precUnical studies because of the permanence of refrovims-mediated gene expression. Resolution of the fransient hypoco-agulable state created by secreted urokinase is one important hurdle that has been resolved prior to considering this approach for clinical gene therapy.

ACKNOWLEDGMENTS

We thank Marilyn Skelly and Leonard Meuse for tiiett tech-nical assistance. This work was supported by the Lucille P. Markey Charitable Tmst and National Institutes of Health grant DK47754. A.L. is a recipient of a Deutscher Akademischer Austauschdienst fellowship.

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Address reprint requests to: D r Mark A. Kay

Division of Medical Genetics RG-25 University of Washington

Seattle, W A 98195

Received for publication March 15, 1995; accepted after revi-sion April 20, 1995.

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