Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome.

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

Application of a split-Cre system for high-capacity adenoviral vector amplification.

BACKGROUND AND AIMS: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. METHODS AND RESULTS: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. CONCLUSIONS: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.

Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations.

Dravet syndrome is a genetic encephalopathy characterized by severe epilepsy combined with motor, cognitive, and behavioral abnormalities. Current antiepileptic drugs achieve only partial control of seizures and provide little benefit on the patient's neurological development. In >80% of cases, the disease is caused by haploinsufficiency of the SCN1A gene, which encodes the alpha subunit of the Nav1.1 voltage-gated sodium channel. Novel therapies aim to restore SCN1A expression in order to address all disease manifestations. We provide evidence that a high-capacity adenoviral vector harboring the 6-kb SCN1A cDNA is feasible and able to express functional Nav1.1 in neurons. In vivo, the best biodistribution was observed after intracerebral injection in basal ganglia, cerebellum, and prefrontal cortex. SCN1A A1783V knockin mice received the vector at 5 weeks of age, when most neurological alterations were present. Animals were protected from sudden death, and the epileptic phenotype was attenuated. Improvement of motor performance and interaction with the environment was observed. In contrast, hyperactivity persisted, and the impact on cognitive tests was variable (success in novel object recognition and failure in Morris water maze tests). These results provide proof of concept for gene supplementation in Dravet syndrome and indicate new directions for improvement.

Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 1012 viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1 -/- mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients.

Adenovirus-Mediated Inducible Expression of a PD-L1 Blocking Antibody in Combination with Macrophage Depletion Improves Survival in a Mouse Model of Peritoneal Carcinomatosis.

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.

Local administration of IL-12 with an HC vector results in local and metastatic tumor control in pediatric osteosarcoma.

Osteosarcoma is the most frequent and aggressive bone tumor in children and adolescents, with a long-term survival rate of 30%. Interleukin-12 (IL-12) is a potent cytokine that bridges innate and adaptive immunity, triggers antiangiogenic responses, and achieves potent antitumor effects. In this work, we evaluated the antisarcoma effect of a high-capacity adenoviral vector encoding mouse IL-12. This vector harbored a mifepristone-inducible system for controlled expression of IL-12 (High-Capacity adenoviral vector enconding the EF1α promoter [HCA-EFZP]-IL-12). We found that local administration of the vector resulted in a reduction in the tumor burden, extended overall survival, and tumor eradication. Moreover, long-term survivors exhibited immunological memory when rechallenged with the same tumor cells. Treatment with HCA-EFZP-IL-12 also resulted in a significant decrease in lung metastasis. Immunohistochemical analyses showed profound remodeling of the osteosarcoma microenvironment with decreases in angiogenesis and macrophage and myeloid cell numbers. In summary, our data underscore the potential therapeutic value of IL-12 in the context of a drug-inducible system that allows controlled expression of this cytokine, which can trigger a potent antitumor immune response in primary and metastatic pediatric osteosarcoma.

Epilepsy and neuropsychiatric comorbidities in mice carrying a recurrent Dravet syndrome SCN1A missense mutation.

Dravet Syndrome (DS) is an encephalopathy with epilepsy associated with multiple neuropsychiatric comorbidities. In up to 90% of cases, it is caused by functional happloinsufficiency of the SCN1A gene, which encodes the alpha subunit of a voltage-dependent sodium channel (Nav1.1). Preclinical development of new targeted therapies requires accessible animal models which recapitulate the disease at the genetic and clinical levels. Here we describe that a C57BL/6 J knock-in mouse strain carrying a heterozygous, clinically relevant SCN1A mutation (A1783V) presents a full spectrum of DS manifestations. This includes 70% mortality rate during the first 8 weeks of age, reduced threshold for heat-induced seizures (4.7 °C lower compared with control littermates), cognitive impairment, motor disturbances, anxiety, hyperactive behavior and defects in the interaction with the environment. In contrast, sociability was relatively preserved. Electrophysiological studies showed spontaneous interictal epileptiform discharges, which increased in a temperature-dependent manner. Seizures were multifocal, with different origins within and across individuals. They showed intra/inter-hemispheric propagation and often resulted in generalized tonic-clonic seizures. 18F-labelled flourodeoxyglucose positron emission tomography (FDG-PET) revealed a global increase in glucose uptake in the brain of Scn1aWT/A1783V mice. We conclude that the Scn1aWT/A1783V model is a robust research platform for the evaluation of new therapies against DS.

Short-Term Local Expression of a PD-L1 Blocking Antibody from a Self-Replicating RNA Vector Induces Potent Antitumor Responses.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.

Inhibition of adenovirus infection by mifepristone.

The repurposing of drugs approved by the regulatory agencies for other indications is emerging as a valuable alternative for the development of new antimicrobial therapies, involving lower risks and costs than the de novo development of novel antimicrobial drugs. Adenovirus infections have showed a steady increment in recent years, with a high clinical impact in both immunosuppressed and immunocompetent patients. In this context, the lack of a specific drug to treat these infections supports the search for new therapeutic alternatives. In this study, we examined the anti-HAdV properties of mifepristone, a commercially available synthetic steroid drug. Mifepristone showed significant in vitro anti-HAdV activity at low micromolar concentrations with little cytotoxicity. Our mechanistic assays suggest that this drug could affect the microtubule transport, interfering with the entry of the virus into the nucleus and therefore inhibiting HAdV infection.

Adaptation of vectors and drug-inducible systems for controlled expression of transgenes in the tumor microenvironment.

Biological therapies based on recombinant proteins such as antibodies or cytokines are continuously improving the repertoire of treatments against cancer. However, safety and efficacy of this approach is often limited by inappropriate biodistribution and pharmacokinetics of the proteins when they are administered systemically. Local administration of gene therapy vectors encoding these proteins would be a feasible alternative if they could mediate long-term and controlled expression of the transgene after a single intratumoral administration. We describe a new vector platform specially designed for this purpose. Different combinations of transactivators and promoters were evaluated to obtain a fully humanized inducible system responsive to the well-characterized drug mifepristone. The optimal transactivator conformation was based on DNA binding domains from the chimeric protein ZFHD1 fused to the progesterone receptor ligand binding domain and the NFkb p65 activation domain. The expression of this hybrid transactivator under the control of the elongation factor 1α (EF1α) or the chimeric CAG promoters ensured functionality of the system in a variety of cancer types. Expression cassettes with luciferase as a reporter gene were incorporated into High-Capacity adenoviral vectors (HC-Ad) for in vivo evaluation. Systemic administration of the vectors into C57BL/6 mice revealed that the vector based on the EF1α promoter (HCA-EF-ZP) allows tight control of transgene expression and remains stable for at least two months, whereas the CAG promoter suffers a progressive inactivation. Using an orthotopic pancreatic cancer model in syngeneic C57BL/6 mice we show that the local administration of HCA-EF-ZP achieves better tumor/liver ratio of luciferase production than the intravenous route. However, regional spread of the vector led to substantial transgene expression in peritoneal organs. We reduced this leakage through genetic modification of the vector capsid to display RGD and poly-lysine motifs in the fiber knob. Safety and antitumor effect of this gene therapy platform was demonstrated using interleukin-12 as a therapeutic gene. In conclusion, we have developed a new tool that allows local, sustained and controlled production of therapeutic proteins in tumors.

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling.

Development of reporter systems for in vivo examination of IFN-ß induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-ß induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.

Enhanced therapeutic effect using sequential administration of antigenically distinct oncolytic viruses expressing oncostatin M in a Syrian hamster orthotopic pancreatic cancer model.

BACKGROUND: The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. METHODS: We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs. RESULTS: NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed. CONCLUSION: Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.

Transient depletion of specific immune cell populations to improve adenovirus-mediated transgene expression in the liver.

BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.

Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer.

Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs.

Efficient gene delivery by EGF-lipoplexes in vitro and in vivo.

AIMS: In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. MATERIALS & METHODS: We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. RESULTS: The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. CONCLUSION: These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.

Treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin-12 in Syrian hamsters.

Pancreatic cancer is an aggressive malignancy resistant to most conventional and experimental therapies, including conditionally replicative adenoviruses (CRAds). The incorporation of immunostimulatory genes such as interleukin-12 (IL-12) in these viruses may overcome some of their limitations, but evaluation of such vectors requires suitable preclinical models. We describe a CRAd in which replication is dependent on hypoxia-inducible factor (HIF) activity and alterations of the pRB pathway in cancer cells. Transgenes (luciferase or IL-12) were incorporated into E3 region of the virus using a selective 6.7K/gp19K deletion. A novel permissive model of pancreatic cancer developed in immunocompetent Syrian hamsters was used for in vivo analysis. We show that, in contrast with nonreplicating adenoviruses (NR-Ad), active viral production and enhanced transgene expression took place in vivo. A single intratumor inoculation of the CRAd expressing IL-12 (Ad-DHscIL12) achieved a potent antitumor effect, whereas higher doses of replication-competent adenoviruses carrying luciferase did not. Compared to a standard NR-Ad expressing IL-12, Ad-DHscIL12 was less toxic in hamsters, with more selective tumor expression and shorter systemic exposure to the cytokine. We conclude that the expression of IL-12 in the context of a hypoxia-inducible oncolytic adenovirus is effective against pancreatic cancer in a relevant animal model.

Characterization of cisplatin cytotoxicity delivered from PLGA-systems.

Biodegradable lactic acid-glycolic acid copolymer (PLGA) formulations incorporating cisplatin have been developed to evaluate the cytotoxicity of this agent in cultured cells. Two different W/O/W protocols were used to formulate micro- (MP) and nanoparticles (NP) under the solvent evaporation method. Although the amount of cisplatin encapsulated was higher in the MP, the efficiency of encapsulation was similar: 10.33% vs. 11.23%, for both MP and NP, respectively. The "in-vitro" release profiles displayed a significant difference in the initial burst effect, which had a significant impact in the antiproliferative effect of cisplatin. In addition, a duality in the cell cycle distribution was found for both formulations and low doses of free cisplatin (2.5, 10 microM) in comparison with the high doses of free cisplatin. The 50 microM caused a rapid inhibition of cells growth followed by a significant loss of cells in phases G0/G1 and G2/M which correlated with an increase in the number of cells in sub-G1. However, cisplatin released from controlled formulations induced an accumulation of cells in the phase G2/M. These results led to enhance the caspase-3 activity for MP and NP. These findings indicate that controlled release formulations of cisplatin are able to induce a more effective apoptosis than free cisplatin.

Human adenovirus replicates in immunocompetent models of pancreatic cancer in Syrian hamsters.

The preclinical evaluation of toxicity and antitumor effect of conditionally replicative (oncolytic) adenoviruses is hampered by the inability of human adenoviruses to replicate efficiently in murine cells. The Syrian golden hamster (Mesocricetus auratus) has been suggested as a permissive animal for adenoviral replication, and cancer cell lines derived from various hamster tumors are available. We provide evidence that wild-type adenovirus type 5 is able to infect and replicate in the pancreatic cancer cell lines HaP-T1 and H2T both in vitro and in vivo. Determination of cytopathic effect, viral spread, progeny production, and the expression of late viral proteins indicates that the complete viral cycle of adenovirus takes place, albeit less efficiently than in highly permissive human cancer cell lines A549 and HuH7. Intrahepatic inoculation of HaP-T1 and H2T cells gave rise to tumors in the liver of hamsters that resemble metastases of pancreatic cancer. The growth of HaP-T1-induced nodules was faster compared with those derived from H2T, but both caused progressive liver infiltration and peritoneal dissemination. When adenovirus was inoculated in these lesions, productive replication took place and newly formed infective virions could be recovered 4 days after administration. In conclusion, the Syrian hamster models described here offer the opportunity to evaluate the effect of oncolytic adenoviruses in an immunocompetent animal and may be a valuable tool in the preclinical evaluation of these agents.

Serum-resistant lipopolyplexes for gene delivery to liver tumour cells.

In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.