Enhancement of AAV gene therapy by transient expression of adenoviral early gene products

HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2 0 0 1

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UROKINASE-TYPE PLASMINOGEN ACTIVATOR GENE THERAPY I N LIVER C I R R H O S I S IS M E D I A T E D BY A C T I V A T I O N O F METALLOPROTEINASES AND D O W N - R E G U L A T I O N O F C O L L A G E N S G E N E EXPRESS I O N . J u a n A r m e n d a r i z - B o r u n d a , U n i v e r s i t y of Guadalajara, Guadalajara, Jal. Mexico; M i r i a m Bueno, Silvia Salgado, Vidal Delgado, U n i v e r s i t y of Guadalajara, Guadalajara Mexico; Estnardo Aguilar-Cordova, H a r v a r d G e n e T h e r a p y Initiative, Boston, MA

E F F I C I E N T E X P R E S S I O N O F T R U N S D U C E D G E N E S BY A N O V E L HYBRID RETROVIRUS VECTOR IN ItEPATOCYTES ARE REGULATED BY T W O E L E M E N T S I N THE UPSTREAM CONTROL REGION OF THE SFFVP LTR. N a o k i Ohnishi, Yoshito Itoh, Kanji Yamaguchi, Takeshi Okanone, T h i r d Dept of Internal Medicine, Kyoto Prefectural U n i v of Medicine, K y o t o J a p a n ; K a t s u h i k o I t o h , J u n Fujita, Dept of Clin Molecular Biology, Fac of Medicine, Kyoto Uuiv, Kyoto J a p a n

Viral infections (Hepatitis B and C) and toxic damage (mostly by alcohol) represent an insult to the liver with high worldwide prevalence leading to liver fibrosis which is a major health care burden. During chronic damage to the liver, there is an over-accumulation of matrix proteins (mainly collagens) which reflects alterations in their synthesis and/or degradation. Consequently, conventional therapies for the treatment of liver fibrosis has focused on withdrawal of the causal agent a n d / o r use of drugs to decrease collagens synthesis, though, no ultimate remedy is reachable so far. Recently, we showed (Salgado et al., MOLECULAR THERAPY 2(6):545-551, (2000), that one single injection of an adenoviral vector bearing a modified cDNA coding for a nonsecreted form of human uPA (AdAhuPA) efficiently reverts liver cirrhosis induced in rats by chronic intoxication with CCl4 mimicking human alcoholic cirrhosis. Here, we injected via iliac vein 6x10 11 vp/kg of either Ad-AhuPA, AdGFP or saline to cirrhotic animals to gain further insight on the molecular mechanisms controlling degradation of exacerbated liver fibrosis. To this end we obtained RNA from liver biopsies and semiquantitative RT-PCR was performed to search for gene expression of collagens I,IIl and IV; collagen-degrading enzymes (MMP-2, MMP-9 and MMP-13), and key molecules involved in regulation of MMPs activity like Tissue Inhibitor Metalloproteinases-i (TIMP-1) and Plasminogen Activator Inhibitor-1 (PAI-]). Thus, liver samples were obtained from animals in each experimental group over a period of 0, 2, 4, 6, 8 and 10 days after Ad infusion. Gene expression for Collagen I decreased notably 58% by day 4 to 70% at day 10, while Collagen III mRNA transcripts expression dropped from 60-70% at the end of the treatment. Collagen IV gene expression was the most affected with a 80% reduction as compared with saline- and AdGFP-treated animals. These findings strongly correlated with a dramatic reversal in liver fibrosis by day 10 as measured with computerized image analyesis of liver sections stained with Masson s and Sirius red dyes. Gene expression for the three MMPs shared similarpatterns where their corresponding mRNAs transcripts were higher in the first six days of treatment dropping thereafter, though, still greater than normal and saline-injected cirrhotic animals. TIMP-1 and PAI-I gene expression decreased as the Ad-AhuPA-treatment advanced suggesting a decreased action of the regulatory brake for MMPs activity. This was not found in AdGFPinjected animals. We then measure MMP-2 protein activity by ELISA and we found it significantly elevated in liver of Ad-AhuPA-treated animals. Since it is known that in liver fibrosis TIMP-1 inhibits the active forms of all MMPs by a noncovalent binding forming complexes MMP/TIMP-1, we reasoned that huPA might be inducing the separation of these complexes, rendering free TIMP-I and active MMP species. Therefore, we measured free TIMP-1 species by Western-blotting using an anti-TIMP-i specific antibody. An specific band of 29 Kda was revealed by chemiluminescence in liver extracts from Ad-AhuPAtreated animals which by densitome~ry showed 800% higher intensity as compared with AdGFP- and saline-injected animals. These findings suggest that uPA is activating in vivo MMPs in order to trigger a cascade of fibrinolytic mechanisms. This work was supported by a grant from CONACyT # 28832-M to Juan Armendariz-Borunda.

AIM: A novel hybrid retrovirus vector (FMEV-type) was developed, which conmined the spleen focus-forming virus (SFFVp) LTR and the leader sequence from the murine ES cell virus (MESV). The aim of this study is 1) to examine whether or not this FMEV-type retrovirus vector show efficient expression of transduced genes in hepatocytes and, if so, 2) to clarify the mechanisms of efficient gene expression. METHODS: Experiment 1. Promoter activities of LTRs of SFFVp or Moloney murine leukemia virus (MoMLV) were evaluated by transient expression of a reporter gene, CAT, in h u m a n hepatocenuler carcinoma (HCC) cell lines, PLC/PRL/5, Huh-7, HLE, or HepG2, and cholangiocellular carcinoma (CCC) cell lines, KMCH-1, or KMC- 1. These cells were also transduced by FMEV-type or MoMLV-based retroviral vectors and the expression of marker genes, mCD8 or MDR-1, were compared among these vectors. Experiment 2. After partial hepatectomy, FMEV-type or MoMLV-based vectors were infused via the tail vein in C57BL/6 mice. Both vectors carried a CAT gene as a marker and the expression of CAT was compared among these vectors. The transduced livers were homogenized and the concentrations of CAT protein in the transduced livers were measured by EL1SA and the proportions of retroviral integration were evaluated by real-time PCR. Experiment 3. To provide additional insights into the regulatory mechanism in retroviral gene expression, isolation and functional characterization of regulatory elements in the LTR of SFFVp was attempted. RESULTS: 1) In transient expression assays, the SFFVp LTR provided superior gene expression to the MoMLV LTR in well-differentiated HCC cell lines such as PLC/PRL/5 or Huh-7, but not in undifferentiated HCC cell lines or CC C cell lines. 2) These results were further confirmed by the retroviral transduction experiments using FMEV-type and MoMLV-based vectors. 3) Although the proportions of genomic integration by these two vectors were almost the same, the expression of a marker gene by the FMEV-type vector was higher than that by the MoMLV vector in mouse liver in vivo. 4) Strong enhancer activity was observed in the upstream control region (UCR) of the SFFVp LTR. Two elements were defined within the UCR, which cooperatively regulated the reporter gene expression. These findings were confirmed by the retroviral trunsduction experiments using the LTRs, in which these elements were deleted. CONCLUSION: 1) The FMEV-type retrovirus vector was superior to MoMLV-based vector in the transgene expression in welldifferentiated HCC cell lines in vitro and in murine hepatocytes in vivo. 2) Two elements within the UCR of the SFFVp LTR were defined, which, at least in part, regulated the high expression of a reporter gene in a wen-differemiated HCC cell lines.

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GENE THERAPY BY TRANSFORMING GROWTH FACTOR-BETA (TGF-18) RECEPTOR-IGG FC CHIMERA, SOLUBLE TGF-/8 RECEPTOR, SUPPRESSES DIMETI-IYLNITROSAMINE-INDUCED HEPATIC FIBROSIS IN RATS. Makoto N a k a m u t a , D e p a r t m e n t of Medicine a n d Bioregulatory Science, K y u s h u University, F u k u o k a Japan; Shusuke Morizono, D e p a r t m e n t of Medicine, F u k u o k a City Hospital, F u k u o k a Japan; M u n e c h i k a Enjoji, Satorn T s u r u t a , Marie F u k u t o m i , M a s a m i Kuniyoshi, Takashi Irie, D e p a r t m e n t of Medicine a n d Bioregulatory Science, K y u s h u University, F u k u o k a Japan; Yuichi Tanabe, D e p a r t m e n t of Medicine, F u k u o k a City Hospital, F u k u o k a Japan; H a j i m e N a w a t a , D e p a r t m e n t of Medicine a n d Bioregulatory Science, K y u s h u University, F u k u o k a J a p a n

EI-IANCEMENT O F AAV G E N E T H E R A P Y BY T R A N S I E N T E X P R E S S I O N O F A D E N O V I R A L EARLY G E N E P R O D U C T S . O g n j e u Petras, UCSF Liver Center, San Francisco, CA; G r e g M Enns, Stanford U n i v Div of Medical Genetics, Stanford, CA; T i m o t h y J D a v e r n II, UCSF Liver Center, San Francisco, CA

Background: Several lines evidence indicates that t r a n s f o r m i n g g r o w t h factor-~ ( T G F - ~ ) is a key m e d i a t o r in the pathogenesis of fibrotic disease including hepatic fibrosis. In this study, w e e v a l u a t e d a strategy for a n t i - T G F - ~ g e n e t h e r a p y against hepatic fibrosis b y transfecting a TGF-~8 r e c e p t o r - I g G Fc chim e r a , soluble T G F - ~ receptor, into skeletal muscle. Methods: E x p e r i m e n t a l hepatic fibrosis was i n d u c e d b y d i m e t h y l n i t r o s a m i n e ( D M N ) in rats. T h e expression v e c t o r c o n t a i n e d a n extracellular d o m a i n of the TGF-~8 type II receptor fused to the lgG-Fc d o m a i n . I n t r a m u s c u l a r injection of the p l a s m i d D N A after b u p i v a c a i n e p r e t r e a t m e n t was p e r f o r m e d at day 1 (right femoral m u s c l e ) and day 14 (left femoral m u s c l e ) after the start of D M N treatment. S e r u m levels of soluble TGF-j8 r e c e p t o r w e r e d e t e r m i n e d by a n ELISA m e t h o d . Hepatic fibrosis e v a l u a t e d by i m a g e analysis, m e a s u r e m e n t s of h y d r o x y p r o f i n e contents. Tissue levels of T y p e I collagen was also e v a l u a t e d by semi-quantitative RT-PCR. E x p r e s s i o n of a l p h a - s m o o t h m u s c l e actin was evaluated b y i m m u n o histological analysis. Finally, hepatic tissue levels of IL-12 a n d IL-10 w e r e m e a s u r e d b y ELISA. Results: S e r u m levels of soluble T G F - ~ r e c e p t o r at day 7 a n d d a y 28 after the first D N A injection w e r e a r o u n d i 0 0 n g / m l a n d 50 ng/ml, respectively. This gene t h e r a p y significantly decreased the o c c u r r e n c e of D M N - i n d u c e d hepatic fibrosis e v a l u a t e d b y i m a g e analysis, a n d also r e d u c e d h y d r o x y p r o l i n e c o n t e n t s in the liver. Soluble TGF-]3 r e c e p t o r s u p p r e s s e d the expression of a l p h a - s m o o t h m u s c l e expression in the liver. Semiquantitative RT-PCR revealed that soluble TGF-/3 r e c e p t o r significantly decreased in type I collagen m R N A level. Interestingly, hepatic tissue levels of IL-12 w e r e decreased, a n d those of IL-10 w e r e increased in the soluble TGF-/3 receptortreated rats. Conclusion: O u r data indicated that b l o c k a d e of TGF-]3 after i n t r a m u s c u l a r transfer of soluble TGF-]3 r e c e p t o r gene s u p p r e s s e d hepatic fibrosis, a n d also suggested that this strategy m a y be a useful a n d feasible gene t h e r a p y against hepatic fibrosis.

BACKGROUND:Adeno-associatedvirus (AAV)vectorshave severalattractiveattributes for hepatic gene therapy including the ability to stably integrate and express transgenes in non-dividing cells such as hepatocytes in viva. In addition, AAVvectorsare nontoxic and appear to preferentiallytransduce the liver when administeredintravenously.However,the efficiencyof AAVtransduction is very low and this has severelylimited clinicalapplicationof this otherwisepromisingvector system. Previouspublished work suggeststhat expression of the earlyadenovira]gene products, E40RF6 and (to a lesser extent) EIB 55K, markedlyenhancesAAVtransducdonby increasingsecond (leading)strand vectorDNAsynthesis. The alto of this study was to define whether transient expression of these two adenoviral early gene products enhance AAVvector mediatedgene expressionboth in cultured cell lines arid in vivo. METHODS:AAV vectors expressingeither the gene for LacZ (AAV-CMV-LacZ)or green fluorescentprotein (AAV-CMVGFP) were produced by an adenovirus-free,triple transfection method and titered as transducing units (TU) on 293 cells in the presenceof adenovirus.Cell lines, including293 (which constitutivelyexpresses E1B 55K), Hela and Hepa 1-6 (a mouse hepatoma cell line), were transfectedby a liposoma]method with plasmids expressingeither E40RF6 a one, E1B 55K alone, both, or neither (control). Expression of the adenoviralgeneproductswas confirmedby immunofluorescenceusingfluorescentmonoclonalantibodies. Cells were transducedwith AAV-CMV-LacZ(MO1= t0) twelvehours after transfection,and then maintained in culture for 48-72 hours, fixed, assayedand scored for beta-galactosidaseactivity. For in vivo experimenis, mice(20-30g) wereadministered60 ug ofplasmidand 7.7 x 106TU ofAAV-CMV-GFPin 2-3 ml of normal saline by rapid tail vein injection in order to achievehydrodynamictransfectionof hepatocytes. Twelve hours after transfeetion, hepatocyteswere isolated by collagenase perfusion, elntsiated, assessedforviabilityby nypan blue exclusion,and allowedto adhere to coflagen-coatedplates.At 72 hours, hepatocyteswere scoredfor green fluorescenceby fluorescencemicroscopy.RESULTS:Transienttransfection of E40RF6 and EIB 55K in cultured cells subsequentlytransducedwith AAV-CMV-LacZresulted in the unexpectedfindingssummarizedin the Table.While transient transfecaonof E40RF6 aloneincreased beta-galactosidaseexpression in 293 (EIB 55K+) cells by over 150-fold, E40RF6 expression only increased expression in Hela or Hepa 1-6 (both E1B 55K-) cells by approximately30 fold. Transienttransfection of EIB 55Kaloneincreasedthe number of LacZpositiveHelaand Hepa 1-6 ceilsby severalhundred fold, and co-transfectionof E40RF6 augmentedthis further. In vivo co-administrationof AAV-CMV-GFP with both EIB 55K and E40RF6 plasmidsby hydrodynamictransfeetionresulted in a 500-foldincreasein GFP expressing hepatoeytes compared with administration of control plasmids with the same vector. SUMMARY:Contraryto prior published work, we havedemonstratedthat transient expressionof EIB 55K aloneis necessaryand sufficientto achievea dramatic (500-fold)enhancementof AAVvectorexpressionin non-293 cell lines. In addition, in preliminary experimentswe have shown for the first time that AAV transduction is enhanced by transient expressionof adenoviral early genes in vivo. These findingshave important implicationsfor the use of AAVvectors for hepatic gene therapy.

Fold increase in Beta.Galactosidase Positive Cells Following Transient Transfection of Adenoviral Eady Genes Cell Type E1B 55K E40RF6 EIB 55K + E40RF6 293 Hela Hepa 1-6

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