Flt3-ligand in combination with CD40-ligand gene transfer following radiation-incudible HSV-TK gene therapy improves survival in mice with diffuse hepatocellular carcinoma

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AASLD ABSTRACTS

HEPATOLOGYO c t o b e r 2001

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TUMOR NECROSIS FACTOR-a IS REQUIRED FOR CHOLESTASIS-INDUCED LIVER FIBROSIS IN THE MOUSE. Blair U Bradford, Erwin Gabele, Matthias Froh, Gavin E Arteel, Takehiko Uesugi, University of North Carolina, Chapel Hill, NC; Michael Marino, Sloan-Kettering Ludwig Institute for Cancer Research, New York, NY; Ronald G Thurman, University of North Carolina, Chapel Hill, NC

SINGLE-STRANDED OLIGONUCLEOTIDES CAN MEDIATE SITE-SPECIFIC NUCLEOTIDE CONVERSION IN CULTURED CELLS AND I N U T E R O . . Betsy T Kren, Rod Felsheim, Angela Panoskaltsis-Mortari, Rorl T McE1murry, Bruce R Blazar, Clifford J Steer, University of Minnesota Medical School, Minneapolis, MN

Tumor necrosis factor-a is elevated in acute and chronic liver diseases in patients and is a mediator of hepatotoxicity in several animal models. Furthermore, TNFo~ plays an important role in hepatic stellate cell activation in culture. In addition, TNFc~ receptor knockout mice were protected from the development of fibroproliferative lesions in a model of pulmonary" fibrosis. It was also shown that bile duct ligation leads to activation of the transcription factor NF-KB and production of TNFa. Accordingly, this study was designed to investigate if hepatic injury and fibrosis due to bile duct ligation would be reduced in TNFoe knockout mice. TNFa knockout (TNFc~-/-) and wild-type mice (SV129 backcrossed five times to C57B1/6) underwent bile duct ligation or sham operation and were sacrificed after 3 weeks. Briefly, under i. p. pentobarbital anaesthesia (50 mg/dl), the abdominal cavity was opened and the common bile duct was double ligated and cut between the ligatures. Sham operations were performed by gently touching the common bile duct with a forceps three times. Alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin were analyzed by standard procedures and livers were harvested for histology. Sections were stained with sirius red and fast green and collagen content was quantitated by image analysis, a-Smooth muscle actin, a indicator of stellate cell activation, was detected by imunhistochemistry. RNAse protection assays were performed using the RiboQuant muhiprobe assay system (Pharmingen) or individual probes. Bile duct ligation decreased body weight to about 70% in wildtype mice, but not in T N F a knockout mice. Liver to body weight ratios were elevated from around 4% in sham operated mice to about 7% in wild-type mice, changes not observed in TNFa knockout mice. Three weeks after bile duct ligation, survival in wild-type mice was 60%; however, survival was increased significantly to 90% in bile duct ligated TNFa knockout mice. Serum alanine transaminases (ALT) levels were about 60 U/l_ in sham-operated mice. As expected, bile duct ligation elevated ALT to about 270 U/L; however, in TNFo~knockout mice, transaminases were 60 % lower. Serum alkaline phosphatase (ALP) and total bilirubin were increased from about 35 U/L and 0.1 mg/dl to about 350 U/L and 11 mg/dl in wild-type mice after bile duct ligation; however, in TNFa knockout mice values were actually higher. TGF-~31 mRNA levels were not different in all groups. Focal necrosis and periportal fibrosis were observed 3 weeks after bile duct ligation in wild-type mice, pathological changes which were minimized in T N F a knockout mice. Bile duct ligation increased the percentage of sirius red stained areas (i. e., collagen) from about 1% to about 4%. This effect was blocked in TNFa knockout mice. Furthermore, a-smooth muscle actin positive cells observed in wild-type mice were barely detectable in TNFa knockout mice. In conclusion, hepatic fibrosis due to bile duct ligation was reduced significantly in TNFa knockout mice. One possibility is that prevention of cell death reduced activation of steflate cells. Alternatively, TNFa, together with TGF/3, could directly activate hepatic stellate ceils (supported, in part, by grants from NIAAA).

The ideal gene therapy is one that only repairs the precise genetic defect so that the regulation of gene expression remains under control of the endogenous promoter. A novel nonviral strategy has emerged for site-directed single-nucleotide (nt) alteration of genomic DNA. The RNA/DNAoligonucleotide (RDO), was originally synthesized as a chimeric 68 mer with 2'-O-methylated RNA residues flanking a stretch of DNA, poly-T hairpin loops a 3' GC clamp and a complementary aI1-DNAstrand. The RDOs are designed to be compl'ementary to the target gene sequence, except for a single nt mismatch, which activates endogenous DNA repair pathways and genomic conversion. The process is independent of both transcription, and cell replication and does not involve strand transfer. It can replace or exchange, and insert or delete genomic DNA bases in cultured cells as well as in vivo. The aim of these studies was to determine whether the all-DNA strand of the RDO could reproduce the activity of geue conversion. Thus, short single-stranded oligonucleotides (SSOs) were designed to target alteration of single base pairs in a cell-free assay cultured cells and in utero. We previously described the RDO correction of single-point mu'tations in plasmid antibiotic resistance genes in vitro using protein extracts from isolated rat liver mitochondria, RDOs and a bacterial read-out system. We used a similar in vitro approach for the SSOs, targeting a mutated copy of the lacZ gene on a plasmid conferring ampicillin resistance. Our results indicated that both nuclear and mitochondrial extracts from rat liver contained the required enzymatic activity to mediate targeted nt repair. Interestingly the results suggested that the repair process occurs one strand at a time. We investigated the ability of SSOs to repair the single-point mutation in the factor 1X gene in the Chapel Hill strain of hemophilia B dog fibroblasts. We observed correction using 45-mer SSOs that were modified at the 5' end with 2 phosphothioate nucleotides, a single phosphothioate at both the 5' and 3' end as well as unmodified. Greater conversion (--8%) was observed using the 45-mers blocked at both the 5' and 3' ends or entirely unblocked. The conversion observed 1 week post-transfection by RFLP analysis of PCR amplified genomic DNA indicated similar conversion frequency with targeting to either geuomic strand. We then examined whether this technology for site-directed genomic DNA modification would work if the molecules were delivered in utero. Thus, we used 45-mer wild-type unmodified SSOs to target the tyrosinase point mutation in albino mice. Correction of this nt defect results in the loss of a DdeI endonuclease site, permitting RFLP screening for site-directed repair of the mutation. On day 15/16 of gestation, 50/zg of SSOs were injected into the livers of fetuses. When the animals were 4 weeks old, liver DNA was isolated, and PCR amplification of the genomic DNA spanning the target site performed. RFLPanalysis of the amplicons indicated conversion of the targeted nt in utero. Similar conversion was observed irrespective of the genomic strand targeted by the SSOs. Finally, we have recently shown that the SSOs also function in vivo to correct the UDP-glucuronosyltransferase-1 gene defect in the Gunn rat model of Crigler-Najjar syndrome type 1. In conclusion, SSOs are capable of mediating site-directed conversion in cell-free extracts, primary cultured cells, in utero and in vivo. The repair frequency in utero was independent of the targeted genomic strand and significant repair occurred using unmodified DNA SSOs. Moreover, unlike the RDOs, the 45-met SSOs are easy and inexpensive to manufacture. The ability of these molecules to promote targeted genomic modification of single-base pairs in primary cultured cells and in utero suggests the potential for gene therapy using this approach in the coming decade.

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FLT3-LIGAND IN COMBINATIOn W I T H CD40-L1GAND GENE TRANSFER FOLLOWING RADIATION-INDUCIBLE HSV-TK GENE THERAPY IMPROVES SURVIVAL IN MICE W I T H DIFFUSE HEPATOCELLULAR CARCINOMA. Y Kawashita, Nj Deb, Mk Garg, Z Fan, Aa Alfieri, Sj Shah, Pk Chakravarty, Department of Radiation Oncology, Albert einstein college of medicine, Bronx, NY; N Roy-Chowdhury, Department of Medicine, Albert einstein college of medicine, Bronx, NY; B Vikram, Department of Radiation Oncology, Albert einstein college of medicine, Bronx, NY; J Roy-Chowdhury, Department of Medicine, Albert einstein college of medicine, Bronx, NY; C Guha, Department of Radiation Oncology, Albert einstein college of medicine, Bronx, NY

SEQUENCE-SPECIFIC CLEAVAGE OF HCV RNA BY THE MODIFIED RIBONUCLEASE H CONJUGATED TO ItCV ANTISENSE OLIGODEOXYNUCLEOTIDES. Toshiko Fukuma, Cherie M Walton, University of Connecticut School of Medicine, Farmington, CT; Catherine H Wu, Universty of Connecticut School of Medicine, Farmington, CT; George Y Wu, University of Connecticut School of Medicine, Farmington, CT

Purpose: Multi-focal hepatocellular carcinomas (HCC) are often unresectable and incurable at the time of presentation. Efficacyof regimens consisting of combinations of chemoand radiation therapy (RT) is limited by radiation-induced liver injury. Although, suicide gene therapy with herpes simplex viral thymidine kinase (HSV-TK) could enhance the tumor control after RT, local and systemic recurrence occur almost invariably. We hypothesized that HCC cells treated by a combination of HSV-TKgene transfer and RT should serve as a source of tumor antigen and provide the necessary "danger signals", thereby activating dendritic cells (DC), in vivo. A sequential therapy with systemic Fh3-1igand (Fh3L) and CD40-Ligand (CD40L) should induce DC proliferation and maturation, respectively. Materials and Methods: Murine hepatoma cells (BNL1ME)were stably transfected with pEgrTK plasmids, which expresses HSV-TK,driven by a radio-inducible early growth response-1 (Egr-1) promoter. Multifocal HCC were induced by intra-portal inoculation of BALWcJmice with 5 x I04 pEgrTK-transfected HCC cells. The mice were treated with RT (25Gy/whole liver), 7 days after tumor inoculation, followed by ganciclovir (GCV) (100mg/kg, x 14 days). Recombinant adenoviruses expressing human FIt3L (AdEh3L), CD40L (AdCD40L), or LacZ (AdLacZ) were injected intravenously, singly or in combination, 1 and 8 days post-RT. Treatment cohorts, non-treated (n=11), RT alone (n=9), GCV+RT (+AdLacZ) (n~26), GCV+RT+ AdCD40L (n=8), GCV+RT+AdFh3L (n=24), and GCV+RT+AdFlt3L÷AdCD40L (n=8) were compared for survival (log rank test), lymphocyte activation and cytotoxic T lymphocyte (CTL) activity. Resuhs: RT alone did not impact overall survival (median, 28 days) over untreated controls (median, 23 days). In the GCV+RT-treated group, stimulated splenocytes exhibited modest CTL activation (--15% lysis of target GCV/RT-treated HCC cells by 51Cr release assay) and the median survival was prolonged to 44 days (p=0.002) and all mice died by 51 days. In contrast, splenocytes from GCV+ RT+AdFh3L-treated mice exhibited markedly enhanced CTL activation (40-50% lysis) and increased IL-12 secretion. In this group, the median survival was prolonged to 63 days (p=0.00003 vs. GCV+RT group) and 37% of the mice survived beyond 70 days. Parallel studies performed in nude mice did not show prolongation of survival (median, 37 days) following TK+ RT+AdFh3L therapy, indicating that the improvement of survival was related to anti-tumor immune response. Addition of AdCD40L further improved the survival of GCV +RT +AdFh3L-treated mice (median 70 days, p=0.03 vs. GCV+RT+AdFh3L group) with 85% of the mice surviving >70 days. Conclusion: A novel three-pronged immuuostimulatory regimen consisting of hepatic irradiation, radio-inducible HSV-TK gene therapy and DC-stimulating eytokines (FIt3L+ CD40L) results in cure in a high proportion of muhifocal HCC.

BACKGROUND: Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of RNA-DNA hybrids. Although RNase H catalytic activity lacks useful sequence-specific recognition properties, a genetically modified RNase H, coupled to an antisense oligodeoxynucleotide complementary to a specific RNA sequence, has been reported to produce a RNase H-oligonucleotide conjugate that can cleave a specific RNA sequence. AIM: To apply the RNase H-oligonucleotide conjugates to Hepatitis C Virus (HCV), a single-stranded RNA virus, to inhibit the viral gene expression and subsequently the viral replication. METHODS: Recombinant E.coli. RNase H was expressed and purified from a bacterial expression system. Two HCV RNA antisense oligodeoxynucleotides complementary to two different HCV RNA sequences were designed (#1: antisense 14-mer oligonucleotide targeting HCV 5'nontranslated region [5'-NTR], #2: antisense 13-mer oligonucleotide targeting HCV core region). RNase H was conjugated to 5'-amino modified oligonucleotides using a water soluble crosslinker. The conjugates were purified by anion exchange chromatography. Purified conjugates were analyzed by SDS-PAGE and 3'-OH end labeling of oligonucleotides. The conjugates were tested by the cleavage of a 1.4 Kb HCV RNA transcript including 5'-NTR and core region. Cleavage assay was performed by incubation of 5pM transcript and RNase H conjugates at various concentrations for 1 hr at 37°C. Cleaved RNA samples were analyzed on formaldehyde gels. RESULTS: RNase H-oligonncleotide conjugates were prepared, and RNase H and oligonucleotide were found to be in one to one molar ratios. Chemical coupling of the conjugates was confirmed by SDS-PAGE analysis of the purified protein, showing a band vdth approx. 22 KDa molecular size compared with 18 KDa molecular size of control RNase H alone. The presence of free RNase H or oligonucleotide in the purified protein was also excluded by the 3'-OH end labeling of oligonucleotides. Both RNase H conjugates targeting HCV 5'-NTR and core region showed HCV sequence-specific cleavage by the presence of shorter fragments of RNA on the formaldehyde gel. Both RNase H conjugates cleaved target transcript in a dose-dependent manner at conjugate concentrations between 60 nM and 600 nM. Cleavage was not seen by RNase alone, or ofigonucleotides alone. A control 1.2 Kb HBV mRNA transcript was not cleaved by either RNase H-oligonucfeotide conjugate. The cleavage reaction was inhibited by EDTA, a Mg2+chelator. CONCLUSION: Modified RNase H conjugates containing HCV RNA antisense oligodeoxynucleotides were successfully synthesized and purified. The conjugates cleaved HCV transcript in a sequence-specific manner. The RNase H-oligonucleotide conjugates directed towards HCV genome may be useful to inhibit the HCV gene expression, thus contribute as a new antiviral therapy for hepatitis C virus infection. (This work was supported by grants from the NIDDK: DK-42182 IGYW], and the Herman Lopata Chair in Hepatitis Research [GYW].)