Intraportal Injection Research Articles

Immunization with a tetramer derivative of an anti‐inflammatory pentapeptide produced by Entamoeba histolytica protects gerbils (Meriones unguiculatus) against experimental amoebic abscess of the liver

Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with several anti-inflammatory properties, including the inhibition of human monocyte locomotion (Monocyte Locomotion Inhibitory Factor (MLIF)). A construct displays the same effects as the native material. It remains to be seen if MLIF is used, or even produced in vivo by the tissue-invading parasite. If MLIF were to be relevant in invasive amoebiasis, immunizing against it could diminish this parasite advantage and prevent lesions. KLH-linked MLIF mixed with Freund's adjuvant was too aggressive an immunizing material to answer this question. However, immunization with a tetramer of MLIF (but not a scrambled version of MLIF) around a lysine core (MLIF-MAPS), that displays increased antigenicity, yet lacks excessive innate immunity activation, completely protects gerbils against amoebic abscess of the liver caused by the intraportal injection of virulent E. histolytica. Liver abscesses caused by Listeria monocytogenes were not prevented. Invasive E. histolytica may produce the parent protein of MLIF in vivo, and if appropriately cleaved, it may play a role in invasive amoebiasis. MLIF may join new vaccination strategies against amoebiasis.

SUCCESSFUL SOLITARY PANCREAS TRANSPLANTATION WITH PORTAL-ENTERIC DRAINAGE FOLLOWING UNSUCCESSFUL ISLET CELL TRANSPLANTATION

P424 Aims: Islet transplantation (ITx) and solitary pancreas transplantation (SPT) basically share the same indications. Although the results of ITx do not match those of SPT yet, a growing number of patients is attracted by the opportunity of achieving insulin-independence by simple percutaneous β-cell injection without the added morbidity entailed by a major operation such as SPT. Considering that, at least ideally, each patient who meets the indications for either ITx or SPT, and who undergoes immunosuppression, should eventually achieve insulin independence, it seems relevant to investigate the consequences of multiple islet injections in the portal vein on the outcome of subsequent SPT with portal venous drainage. We herein report our experience with SPT drained in the portal vein after failed ITx. Methods: Between October 16th, 2002 and December 30th, 2003, 3 SPT with portal venous drainage, including 2 PTA and 1 PAK, were performed in recipients of unsuccessful ITx. All recipients had undergone ITx due to frequent, acute, and severe metabolic complications and/or rapidly progressive diabetic complications at two different Italian transplant centers. Despite multiple intraportal islet injections (mean: 2.3 ijs.; range 2-3 ijs.) no patient actually achieved permanent insulin independence and initial endocrine function, measurable in each recipient, gradually disappeared between 2 and 4 months after the last injection. The three patients were subsequently evaluated for possible SPT. There were 2 females and 1 male aged 60, 49, 26 and years, respectively, and with a body max index of 19.5, 21.4 and 23.0 kg/m2, respectively. Daily insulin requirements were 30, 35 and 38 units, respectively. Pannel reactive antibody titer was 0% in all recipients. Pancreata were procured from brain dead cadaveric donors and allocated to each recipient according to AB0 compatibility and negative cross-match, without any specific degree of HLA matching. Results: Immunosuppression was based on a quadruple regimen including tolerance induction with either basiliximab (n= 2) or ATG (n= 1) and maintenance therapy with steroids, mycophenolate mofetil and tacrolimus. The final decision to drain each graft into the portal vein was taken during surgical exploration after verifying the absence of portal ipertension and following direct mesurement of portal pressure (13, 14 and 16 cm H20, respectively). Pancreas grafts were reperfused after a period of cold ischemia time of 835, 695 and 638 minutes, respectively. All grafts showed immediate endocrine function and keep their recipients insulin free at the longest follow-up (18, 16, and 3 months, respectively). One patient developed nonocclusive venous thrombus which resolved with intravenous heparin infusion. Conclusions: Our experience shows how unsuccessful ITx with multiple intraportal injections does necessarily precludes the possibility of subsequent successful SPT with portal venous drainage. Further experience is needed to define contraindications and possible complications of SPT with portal drainage following ITx.

A SIMPLE METHOD USING A POLYMETHYLPENTEN CHAMBER FOR ISOLATION OF HUMAN PANCREATIC ISLETS

P431 Aims: In order to easily reproduce the results of human islets isolations, we developed and evaluated a simplified digestion technique using a polymethylpenten chamber (PMPC) and compared this method with the automated islet isolation technique using a stainless steel chamber (SSC), described by Ricordi et al. (Diabetes, 1989) Methods: Twenty human pancreases obtained from cadaveric donors and preserved in UW solution were digested with either SSC (Group 1, n=5) or PMPC (Group 2, n=15). There was no significant difference between the two groups in terms of donors characteristics (age, CIT, WIT, BMI, amylase level). The pancreases digested with SSC were processed exactly following Ricordi’s method. Digestion with PMPC was performed with Liberase HI at 37°C without any circulation system. The digested tissue was collected after filtration through a 1000μm mesh screen and passed at 4°C on a column containing 6-mm glass beads and a 500μm mesh screen. Results: PMP chamber maintained a stable temperature at 37°C during the entire pancreas digestion. No significant difference was observed in term of pancreas digested tissue (>70%) between 2 groups. The islets equivalent yield by gram of digested tissue (IEQ/gram) was similar with both methods (5520 IEQ/gr for PMPC vs. 4679 IEQ/gr for SSC, p>0.05). A higher percentage of large islets (>100μm) was obtained with PMP chamber (p<0.05). The final volume obtained after pancreas digestion with PMPC (13±5 ml) was significantly reduced (4 fold) in comparison with the tissue volume obtained with SSC digestion (51±14ml, p<0.005).This small volume facilitated the purification step without Cobe 2991. Islet recovery (IEQ after digestion/IEQ after purification) with a simple discontinuous gradient (Group 2, n=15) was 63% and islets viability/purity reached 84±8% and 73±20%, respectively. The final cellular volume obtained with this technique, reached a mean of 880μl. Immunohistochemistry evidenced a similar (p>0.05) proportion of beta cells prior (56±10%)(n=6) and after (61±10%)(n=9) isolation. By transmission electron microscopy, the good preservation of the isolated islets containing many granulated cells was established. Islet function, assessed by in vitro systems (Glucose 1-15mM) revealed a stimulation index at 13.1±7.1. Three highly purified islets transplantation were carried out by intra-portal injection without any increase of portal pressure or changes of liver enzymes. In two cases, a significant decrease of daily insulin requirement (-40%) and no hypoglycemia (increase quality of life), after a single islets injection (<5,000 IEQ/kg) was observed at hospital discharge (follow-up: 12 months). Conclusions: Digestion technique of human pancreas with PMPC provided a simple method of pancreatic islets isolation, an equal yield of islets than previously described digestion methods as well as a preserved morphological integrity, viability and functionality. Last but not least, this technique might be carried out by only two persons and is significantly less expensive since all material are “one use” which is overwhelming for good medical practice.

Longterm recombinant adeno-associated, virus-mediated, liver-generated expression of an angiogenesis inhibitor improves survival in mice with disseminated neuroblastoma.

A gene therapy-mediated approach to the delivery of antiangiogenic agents using adeno-associated virus (AAV) vectors has a number of advantages including the potential for sustained expression. The purpose of this study was to attempt to restrict the growth of disseminated neuroblastoma through delivery of a truncated, soluble form of the vascular endothelial growth factor receptor-2 (VEGFR-2 fetal liver kinase [Flk]-1), a decoy receptor for VEGF. Mice underwent portal vein injection of vector, either AAV-CAGG-tsFlk-1 or control vector. Subsequent truncated soluble fetal liver kinase-1 (tsFlk-1) expression was confirmed and quantified by ELISA. After 8 weeks, mice were given human neuroblastoma cells through the tail vein to establish disseminated disease and were sacrificed after 40 days. The weight of liver metastasis was measured. Intrahepatic tumor microvessel density and levels of apoptosis were determined. Survival was assessed in a second cohort of mice. After intraportal injection of AAV-CAGG-tsFlk-1, high-level, stable transgene expression was generated. Sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug (BD Biosciences) neovascularization in vivo, suggesting that a systemic state of angiogenesis inhibition had been established. After neuroblastoma injection, mice expressing tsFlk-1 had a smaller tumor burden in the liver when sacrificed, as compared with control mice. The decrease in tumor weight in the liver correlated with lower intratumoral microvessel density and higher levels of tumor cell apoptosis in the tsFlk-1-treated tumors, supporting the hypothesis that decreased angiogenesis had occurred. In a second cohort of mice, survival of tsFlk-1-expressing mice after tumor cell challenge was longer than in control mice. Longterm in vivo expression of a functional VEGF inhibitor was established using an AAV-mediated gene therapy approach, and it demonstrated antiangiogenic and anticancer efficacy in a murine model of disseminated neuroblastoma.

863. Expression and Secretion of Human Glucocerebrosidase Mediated by Recombinant Lentivirus Vectors In Vitro and In Vivo

Gaucher disease is a lysosomal storage disorder resulting from a deficiency of glucocerebrosidase (GC). Most research on gene therapy for Gaucher disease has centered on ex vivo transduction of hematopoietic stem cells by retroviral vectors. In this study, we showed that vascular and hepatic delivery of a HIV-1 based recombinant lentivirus vector encoding human GC cDNA produced therapeutic levels of GC protein.Human cell lines and Gaucher fibroblasts were transduced at 10-50 multiplicity of infection (MOI) of lenti-EF-GC or lenti-CMV-GC. In vivo experiments were carried out in C57BL/6J mice by intravenous and intraportal injection with 1-5 107 transducing unit (TU) of lenti-EF-GC. Cells and tissues were assayed for GC activity. A high level of expression of GC was produced in cultured fibroblasts derived from patients with Gaucher disease by transducing the cells with lentivirus vectors. GC secreted by transduced fibroblasts was taken up by adjacent GC-deficient cells by endocytosis. Intraportal administration of recombinant lenti-EF-GC vector was resulted in efficient transduction of hepatic cells and expression of the GC. Vascular delivery of vector resulted in high levels of GC expression in liver, spleen, heart, lung, and bone marrow cells in mice that persisted in most organs over the four months. No significant abnormalities were found attributable to recombinant lentivirus vectors in any of the tissues examined. Recombinant lentivirus vector-mediated gene transfer appears to be effective and safe. This study represents an initial step toward gene transfer using recombinant lentivirus vectors for treatment of Gaucher disease.

161. Novel Pseudotyped AAV Vectors for Hemophilia B Gene Therapy

Novel AAV serotypes isolated from nonhuman primates and human tissues have shown promise as efficient gene transfer vectors, attributed to their high in vivo gene transfer efficiency and distinct immunology properties. In this study, we show that AAV2/7, 2/8, and 2/9 are more efficient and less immunogenic vectors for both liver and muscle-directed gene therapy for hemophilia B than the traditional serotypes. For a liver-directed approach, evaluation of the new serotypes was performed in mouse and dog hemophilic models. In hemophilia B mice (in C57BL/6 background), long-term superphysiological levels of canine FIX (41–70 μg/ml) and shortened aPTT time have been achieved following intraportal injection of 1 × 1011 GC of AAV2/7, 2/8, and 2/9-LSP-cFIX-WPRE vectors. A 10-fold lower dose (1 × 1010 GC) of AAV2/8 vector generated normal level of cFIX and aPTT time. In UNC hemophilia B dogs, we previously demonstrated that administration of an AAV2/8 vector into a dog previously treated with an AAV2 vector was successful; cFIX expression peaked at 10 μg/ml day 6 after the 2nd intraportal injection (dose = 5 × 1012 GC/kg), then gradually decreased and stabilized around 700 ng/ml (16% of the normal level) throughout the study (1 1/2 years). This level was about 3-fold higher than that from a hemophilia B dog that received a single injection of AAV2-cFIX at the similar dose. Recently, two naive hemophilia B dogs were injected with AAV2/8 vectors intraportally at the dose of 5.25 × 1012 GC/kg. cFIX levels in one dog (male) reached 30% of normal level (1.5 μg/ml) ten weeks after injection and has sustained at 1.3–1.5 μg/ml, while the second dog (female) maintained cFIX expression at about 10% of normal level. WBCT and aPTT were both shortened after the injection. Liver enzymes in both dogs remained in the normal range after surgery. We further assessed these new serotypes for muscle-targeted gene therapy of hemophilia B. AAV-CMV-cFIX-WPRE packaged with 6 different AAV serotypes were compared in hemophilia B mice (in C57BL/6 background) after intramuscular injection at the dose of 1 × 1011 GC. cFIX gene expression and antibody formation were monitored. Highest expression was detected in the plasma of the mice injected with AAV2/8 vectors (1460 ± 392 ng/ml at day 42), followed by AAV2/9 (773 ± 171 ng/ml) and AAV2/7 (500 ± 311 ng/ml). Levels were maintained for 5 months. To our surprise, cFIX expression by AAV2/1 ranged from 0–253 ng/ml (average: 66 ± 82 ng/ml). Anti-cFIX inhibitor (IgG) was detected in some of the AAV2/1-injected mice. Further screening of inhibitor formation was performed on day 28 samples for all serotypes. Hemophilia B mice showed highest inhibitor formation against AAV2/2, followed by AAV2/5, and AAV2/1. Only sporadic and low level inhibitors were detected in animals injected with AAV2/7, AAV2/8 and AAV2/9. Thus, we show the advantages of the new AAV serotypes 2/7, 2/8, and 2/9 for muscle-directed gene therapy for hemophilia B as more efficient and safe vectors without eliciting any significant anti-FIX antibody formation. These vectors could also be useful for gene therapy of other genetic diseases.

625. AAV Gene Therapy for Liver Metastatic Cancer

Metastatic liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of an adeno-associated-virus(AAV) vector led to long term and persistent expression of angiostatin and suppressed EL-4 tumors that metastasized to livers. In the present study, we seek strategies to improve the therapeutic efficacy of AAV mediated anti-angiogenic gene therapy in combating liver metastases. A recombinant adeno-associated-virus (AAV) vector encoding costimulator B7.1 was engineered. Incubation with AAV-B7.1 viruses resulted in rapid expression of B7.1 on the surface of 80% EL-4 cells. Transfected EL-4 cells vaccinated mice rejected the B7.1-engineered tumor cells, and resisted a secondary challenge with parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Combination of AAV-B7.1 vaccination and AAV-angiostatin therapy protected mice against the challenge of a heavy burden of EL-4 cells, and eradicated metastasized tumors that had disseminated to the liver. Furthermore, the combinational therapy promoted the survival rate of mice with advanced liver cancers. These encouraging results warrant future investigation of the use of AAV-mediated immunotherapy and anti-angiogenic gene therapy for targeting unresectable disseminated liver metastases.

Inhibition of acute rejection after skin or cardiac transplantation by administration of donor antigens in the rat.

We examined differences in host immunologic changes induced by the intravenous or intraportal administration of donor antigens at engrafting and evaluated their contribution to graft survival using a rat transplantation model. Lewis rat recipients were given either an intravenous or intraportal injection of donor splenocytes (1 x 10(8)) immediately after receiving skin grafts from Brown Norway donors. The immunologic responses were analyzed by mixed lymphocyte reaction (MLR) and profiles of interferon-Gamma, interleukin (IL)-2, and IL-10 in MLR supernatants. The effect on cardiac transplantation of perioperative administration of low-dose FK506 (0.1 mg/kg per day) was also examined. Mixed lymphocyte reactions using splenocytes and sera from recipients treated intraportally were greatly inhibited. Interferon-gamma, IL-2, and IL-10 levels were significantly higher after intraportal treatment compared with intravenous treatment (P < 0.05). When FK506 was injected from day 3, a significant enhancement of cardiac allograft survival was demonstrated by intraportal treatment (16.1 +/- 2.9 days) in comparison to the non-treatment (13.0 +/- 1.7 days, P < 0.05) and intravenous treatment rats (11.7 +/- 2.7 days, P < 0.05). The Th2 deviation induced with intraportal alloantigen administration immediately after engraftment was thus observed to produce a synergistic effect with immunosuppressant treatment to suppress acute rejection.

Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy.

Colorectal neoplasms remain a leading cause of cancer-related deaths. A recognized weakness of conventional 5-fluorouracil (5-FU) therapy relates to expression of the intracellular enzyme, thymidine phosphorylase (TP). Although TP promotes 5-FU cytotoxicity, TP-derived 2-deoxy-D-ribose (dRib) counterproductively stimulates tumor angiogenesis. Here, the newly discovered antiangiogenic drug rapamycin was combined with 5-FU to counteract the potential escape mechanism of dRib-induced angiogenesis. Orthotopic tumor growth was assessed in rapamycin and 5-FU-treated BALB/c mice with TP-expressing CT-26 colon adenocarcinoma cells. To examine liver metastasis, green-fluorescent protein-transfected CT-26 cells were visualized by fluorescence microscopy after intraportal injection. Cell counting and Ki67 staining were used to determine in vitro and in vivo cell expansion, respectively. In vitro angiogenic effects of dRib were assessed with endothelial cell migration and aortic ring assays. Western blotting detected dRib effects on p70/S6 kinase activation. Rapamycin treatment of mice bearing orthotopic tumors inhibited tumor growth more than did 5-FU, and mice treated with both drugs typically developed no tumors. In the liver metastasis assay, combination therapy blocked metastatic expansion of solitary tumor cells. Interestingly, complex drug activities were suggested by tumor-cell proliferation being more sensitive to 5-FU than to rapamycin in vitro, but more sensitive to rapamycin in vivo. With regard to angiogenesis, dRib-induced endothelial cell migration and aortic ring formation were completely abrogated by rapamycin, correlating with blockage of dRib-induced p70/S6 kinase activation in endothelial cells. Inhibition of dRib-induced angiogenesis with rapamycin counteracts a potential TP-based escape mechanism for colorectal cancer under 5-FU therapy, introducing a novel, clinically feasible, combination treatment option for this common neoplasm.

Induction of oral tolerance and the effect of interleukin-4 on murine skin allograft rejection.

We studied the effect of oral and portal vein administration of alloantigens on mouse skin allograft survival. Graft receptor BALB/c mice received spleen cells (30, 90, 150 or 375 x 10(6)) from donor C57BL/6 mice intragastrically on three successive days, starting seven days before the skin graft. Allograft survival was significantly increased with the feeding of 150 x 10(6) allogeneic spleen cells by one gavage (median survival of 12 vs 14 days, P< or =0.005) or when 300 x 10(6) cells were given in six gavage (12 vs 14 days, P<0.04). A similar effect was observed when 150 x 10(6) spleen cells were injected into the portal vein (12 vs 14 days, P< or =0.03). Furthermore, prolonged allograft survival was observed with subcutaneous (12 vs 16 days, P< or =0.002) or systemic (12 vs 15 days, P< or =0.016) application of murine interleukin-4 (IL-4), alone or in combination with spleen cell injection into the portal vein (12 vs 18 days, P< or =0.0018). Taken together, these results showed that tolerance induction with spleen cells expressing fully incompatible antigens by oral administration or intraportal injection partially down-modulates skin allograft rejection. Furthermore, these findings demonstrated for the first time the effect of subcutaneous or systemic IL-4 application on allograft skin survival suggesting its use as a beneficial support therapy in combination with a tolerance induction protocol.

Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype

AbstractDespite the popularity of adeno-associated virus 2 (AAV2) as a vehicle for gene transfer, its efficacy for liver-directed gene therapy in hemophilia A or B has been suboptimal. Here we evaluated AAV serotypes 2, 5, 7, and 8 in gene therapy of factor VIII (FVIII) deficiency in a hemophilia A mouse model and found that AAV8 was superior to the other 3 serotypes. We expressed canine B domain-deleted FVIII cDNA either in a single vector or in 2 separate AAV vectors containing the heavy- and light-chain cDNAs. We also evaluated AAV8 against AAV2 in intraportal and tail vein injections. AAV8 gave 100% correction of plasma FVIII activity irrespective of the vector type or route of administration.

Adult porcine islets produce MCP-1 and recruit human monocytes in vitro.

Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4(+) T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1beta and TNF-alpha. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of alpha- and beta-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1beta nor TNF-alpha were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1beta and TNF-alpha were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice.

Bovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 x 10(5) colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35000 units/kg of body weight) significantly (P < 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (P < 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (P < 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.

Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver.

Liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of adeno-associated-viral (AAV) particles that express angiostatin lead to long-term expression of angiostatin capable of suppressing the outgrowth of EL-4 tumors in the liver. Here we combine AAV-mediated angiostatin therapy with immunotherapy by employing an AAV vector encoding the T-cell costimulator B7.1. Incubation of EL-4 cells with AAV-B7.1 viruses resulted in the rapid expression of B7.1 on the surface of 80% of EL-4 cells. Mice that were vaccinated with B7.1-engineered tumor cells rejected the tumor cells and resisted a secondary challenge with unmodified parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Intraportal injection of AAV particles that express angiostatin into mice that had been vaccinated 1 month earlier with B7.1-engineered tumor cells protected mice against the challenge of a heavy burden of EL-4 cells and eradicated tumors that had disseminated to the liver. The combinational therapy increased the survival rate of mice with advanced liver cancer. These encouraging results warrant investigation of the employment of anti-angiogenic therapy subsequent to cancer immunotherapy for targeting unresectable disseminated liver metastases.

Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII

AbstractUsing separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats.

Adiponectin (Acrp30) is a physiologically active polypeptide hormone secreted by adipose tissue that shows insulin-sensitizing, antiinflammatory, and antiatherogenic properties. In humans, Acrp30 levels are inversely related to the degree of adiposity. In the current study, we tested the long-term weight-reducing and insulin-enhancing effects of Acrp30 cDNA delivered peripherally by a viral vector. To this end, we have generated a series of recombinant adeno-associated virus vectors of serotypes 1 and 5 encoding mouse Acrp30 cDNAs. The long-term expression of recombinant adeno-associated virus-Acrp30 vectors was tested after intramuscular or intraportal injection in female Sprague-Dawley rats with diet-induced obesity. We show that a single peripheral injection of 10(12) physical particles of Acrp30-encoding vectors resulted in sustained (up to 280 days) significant reduction in body weight, concomitant with the reduction in daily food intake. Acrp30 treatment resulted in higher peripheral insulin sensitivity measured by the i.p. glucose tolerance test in fasted animals. Ectopic expression of the Acrp30 transgene resulted in modulation of hepatic gluconeogenesis and lipogenesis, as demonstrated by the reduction of the expression of two key genes: PEPCK (phosphoenolpyruvate carboxykinase) and SREBP-1c (sterol regulatory element-binding protein 1c) in the liver. These data show successful peripheral therapy in a clinically relevant model for human obesity and insulin resistance.

Antitumor effect of a splenic injection of 5-fluorouracil on metastatic liver cancer in mice.

The regional administration of 5-fluorouracil (5-FU) has been the fundamental therapy against liver metastases for the improvement of patient prognosis; however, there have been few reports about the immunological effects of this agent. It is also unknown whether it affects the spleen, one of the major lymphoid organs. The objective of the present study was to determine the immunological effect of an intrasplenic injection of 5-FU against liver metastases. We investigated the effect of an intrasplenic injection of 5-FU on the formation of experimental liver metastasis resulting from an intraportal vein injection of colon 26 carcinoma cells in BALB/c mice and elucidated some of the underlying mechanisms involving the effects of this on cellular immunity. Liver metastases were significantly diminished by the splenic injection of 5-FU, particularly in comparison with the portal injection or systemic administration. This was followed by augmentation of the interleukin-12 (IL-12) level in the spleen and activation of hepatic mononuclear cells. In those cells, NK1.1+ (NKT) cells played a central role against metastases. A splenic injection of 5-FU is more effective on the involution of liver metastases than portal or systemic injection. This effect may be attributed to the augmentation of the IL-12 level in the spleen and of NKT cells in the liver rather than to the original effect of 5-FU, which is the so-called inhibition of DNA synthesis.

Survival benefit of KRN7000 immune therapy in combination with TNP470 in hamster liver metastasis model of pancreatic cancer

Pancreatic cancer is a solid malignancy with the poor prognosis largely due to frequent and lethal liver metastases. The combination of immunotherapy and anti-angiogenesis therapy might be a hopeful strategy for the treatment of distant metastases. The benefits of the combination therapy by an immune stimulator alpha-galactosylceramide (KRN7000) and an angiogenesis inhibitor AGM-1470 (TNP470) were evaluated on the hamster highly aggressive liver metastasis model using the syngeneic pancreatic cancer cell line HPD-NR. KRN7000 immediately activated hepatic mono-nuclear cells to produce IFN-gamma in vitro. Intraportal injection of KRN7000 exhibited a dense accumulation of CD4-CD8- natural killer T cells, around the liver metastases in vivo. KRN7000 treatment significantly inhibited the growth of liver metastases, and importantly, significant survival prolongation was confirmed when TNP470 treatment was added to it. Furthermore, cytotoxic T lymphocytes were induced at the sites of a few residual metastases in the liver of a long-term survivor. Thus, the combination of KRN7000 and TNP470 showed a high effectiveness for the treatment of liver metastases of pancreatic cancer.

Total vascular exclusion of the liver enhances the efficacy of retroviral-mediated associated thymidine kinase and interleukin-2 genes transfer against multiple hepatic tumors in rats

Backround. Recent advances in gene transfer technology render gene therapy an attractive treatment of disseminated liver metastases for which other treatments remain disappointing. In this setting, total vascular exclusion of the liver could improve gene transfer to cancer cells and prevent extrahepatic vector spreading during portal infusion of therapeutic genes. We evaluate the efficiency of combined herpes simplex virus type-1 thymidine kinase (HSV1-TK) and interleukin-2 retrovirus-mediated gene transfer through the portal vein, under total vascular exclusion of the liver, in a model of macroscopic multiple liver metastases in rats. Methods. Multifocal liver metastases were established in BDIX rats with intraportal injection of DHDK12 colon cancer cells. On randomization, rats received either vector-producing cells or saline solution under total vascular exclusion of the liver. Vector-producing cells released retroviral vectors encoding Lac-Z in marking studies or HSV1-TK or interleukin-2 in therapeutic studies. Rats were either killed for pathologic studies, or followed for survival. Results. Total vascular exclusion of the liver markedly improved gene transfer efficacy in marking studies. In therapeutic studies we observed a significant reduction in tumor volume of treated rats compared with untreated controls (2170 ± 310 mm3). Although singular HSV1-TK or interleukin-2 gene transfer showed significant efficacy, the greatest tumor volume regression was observed in rats treated with combined HSV1-TK + interleukin-2 gene therapy (145 ± 60 mm3; P =.0001 vs control). This translated into an increased median survival rate compared with either control rats (P =.006) or rats treated with single gene therapy. Conclusion. In a rat model, a significant antitumoral effect against macroscopic multifocal liver metastases can be observed after retrovirus-mediated HSV1-TK and interleukin-2 gene transfer through the portal vein under total vascular exclusion of the liver, followed by ganciclovir administration. We believe that this well-tolerated and efficient therapeutic approach deserves clinical evaluation in patients with disseminated colorectal liver metastases. (Surgery 2003;133:669-77)

Intra-portal injection of 400-mum microcapsules in a large-animal model

Abstract To date, encapsulated grafts have usually been implanted in the peritoneal cavity. This site is, however, not ideal, mainly because of its low blood supply. We have investigated the feasibility of intraportal injection of (400 μm) microcapsules in the pig. Ten-thousand microcapsules per kilogram body weight were injected into six Large White pigs. Portal pressure, various biological tests, portographies and liver histology were recorded before and at various time points after injection. As a result, portal pressure increased after injection (15 ± 2.3 vs 8.7 ± 1.7 mmHg) but remained within an acceptable range (<20 mmHg) and returned to normal values at 3 months (8.5 ± 3.7 mmHg). During the 3-month follow up, liver function and liver tests remained stable. Portographies showed a homogenous implantation of the capsule, with the portal flow always directed to the liver. At histological examination after 3 months the capsules demonstrated various degrees of fibrosis. We can thus conclude that these results demonstrate that intraportal injection of microcapsules is feasible in a large-animal model. Hemodynamic, biological and radiological results are similr to those observed in clinical free-islet transplantation.