Studi pre-clinici per lo sviluppo di approcci terapeutici basati sui microRNA in un modello di Leucemia Linfatica Cronica

Despite its indolent nature, chronic lymphocytic leukemia (CLL) is an incurable disease. The involvement of microRNAs (miRNAs) in CLL pathogenesis has been established and it suggests that miRNAs are attractive candidates as therapeutic targets for CLL. Here, we employed the Eμ-TCL1 transgenic mouse model, which develops a chronic B-cell CD5+ leukemia similar to an aggressive form of human B-CLLs, to investigate miR-181b and miR-34a as a new therapeutic agents. In vitro, the enforced expression of miR-181b mimics induced a significant apoptotic effect in human B-cells (RAJI, DAUDI, 697, EHEB) as well as in mouse Eμ-TCL1 leukemic splenocytes. In addition molecular analyses revealed that miR-181b not only affected the expression of Tcl1, but also of Bcl2 and Mcl1 anti-apoptotic proteins, and reduced the phosphorylation levels of Akt and Erk1/2. Similarly, the restoration of miR-34a induced an increase of apoptosis in murine leukemic spleen cells, which could be related to the modulation of CDK6 and Bcl-2 targets. Finally, in vivo multiple treatments with miR-181b or miR-34a mimics induced a reduction/delay in the progression of leukemia and an increase of overall survival in treated mice. Our pre-clinical assessment of miRNA-based therapies in a CLL mouse model proves a sizable anti-leukemic activity of miR-181b and miRNA-34a as single agents, and suggests that the use of microRNAs could represent a novel potential therapeutic approach for the treatment of CLL

Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

none15siThe down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3' UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicityopenCallegari E; Elamin BK; D'Abundo L; Falzoni S; Donvito G; Moshiri F; Milazzo M; Altavilla G; Giacomelli L; Fornari F; Hemminki A; Di Virgilio F; Gramantieri L; Negrini M; Sabbioni SCallegari E; Elamin BK; D'Abundo L; Falzoni S; Donvito G; Moshiri F; Milazzo M; Altavilla G; Giacomelli L; Fornari F; Hemminki A; Di Virgilio F; Gramantieri L; Negrini M; Sabbioni

Description of the conditionally replication-competent oncolytic Adenovirus.

<p>(<b>A</b>) Schematic illustration of Adenovirus type 5 genome: the viral early and late genes and the Inverted Terminal Repeat (ITR) are indicated. (<b>B</b>) TheAd-Control viral genome encloses the E1A/E1B genes and the IRES-EGFP expression cassette. (<b>C</b>) The Ad-199T viral genome contains a specific sequence complementary to microRNA 199a-3p (miR-199 target) in the 3’ untranslated region of the E1A gene. (<b>D</b>) The miR-199 target sequence is made of 4 copies of a 22bp DNA segment complementary to the mature miRNA.</p

Ad-199T replicates in tumor cells <i>in vivo</i>.

<p>(<b>A</b>) 2x10⁶ HepLuc cells were intra-hepatically implanted in B6D2 wild type mice at 3 days of birth and examined at the In Vivo Imaging System (IVIS) two hours after cells implantation. After 24 hours, three experimental groups, consisting of six mice each, were defined: one was injected intra-hepatically with 1x10⁸ I.U. of the Ad-199T virus; one was infected with 1x10⁸ I.U. of the Ad-Control virus; one was not infected (no virus). Mice were monitored at the IVIS at 24h (<b>B</b>), 48h (<b>C</b>) and 72h (<b>D</b>) after virus inoculation. Reduction in pseudo-color images, representing bioluminescence intensity, indicates that the implanted tumor cells were eliminated, likely due to the active viral replication by either Ad-Control or Ad-199T viruses. (<b>E</b>) A quantitative photon analysis of the region-of-interest showed a highly significant decrease (p value <0.05) of luminescence in mice infected with Ad-Control and Ad-199T viruses versus uninfected animals.</p

miR-199 controls Ad-199T replication <i>in vitro</i>.

<p>(<b>A</b>-<b>B</b>) To asses viral replication in absence and in presence of miR-199, HepG2 and HepG2/199 cell lines were infected with Ad-199T or Ad-Control adenoviruses. A progressive accumulation of viral DNA, indicated as fold change copies of Adeno DNA referred the lower level of Adenovirus DNA copies, indicates that active viral replication is efficiently occurring in both cell lines infected with Ad-Control virus. A progressive accumulation of viral DNA in the HepG2 cells infected with Ad-199T indicates that active viral replication is efficiently occurring in this cell line; on the contrary, in HepG2/199 cells infected with Ad-199T the viral DNA did not increase over time, confirming that the virus replication was inhibited by the presence of miR-199. P-values of the comparisons are shown.</p

miR-199 controls Ad-199T replication <i>in vivo</i>.

<p>To assess replication properties of AD-199T <i>in </i><i>vivo</i>, 1x10⁸ I.U. of Ad-199T or 1x10⁸ I.U. of Ad-Control were intra-hepatically injected into 3 days old B6D2 mice. At 72 hours after infection, livers were collected and viral DNA quantified using analytical PCR (<b>A</b>) and quantitative PCR as fold change copies of Adeno DNA referred to the lower level of Adenovirus DNA copies (<b>B</b>) The replication of Ad-199T (mice 1375, 1376) was significantly suppressed in normal liver comparing with Ad-Control (mice 1370, 1372). This evidence confirms that miR-199 could control Ad-199T replication in normal liver cells <i>in </i><i>vivo</i>.</p

Differential replication of Ad-199T in normal liver versus tumor cells.

<p>2x10⁶ HepLuc cells were intra-hepatically implanted in six B6D2 wild type mice 3 days after birth. After 24 hours, the animals were injected intra-hepatically with 1x10⁸ I.U. of the Ad-199T virus. Mice were sacrificed at 24h and 48h after the treatment and livers as well as tumor cells masses were collected. Genomic DNA extracted both from normal liver and tumor masses was analyzed by analytical PCR (<b>A</b>) and quantitative PCR as fold change copies of Adeno DNA referred to the lower level of Adenovirus DNA copies (<b>B</b>). The results show a significant suppression of Ad-199T replication in normal liver (NL) compared to tumors (Tumor). P-values of the comparisons are shown.</p

Ad-199T therapeutic activity against DENA-induced tumors in HCC mouse model.

<p>A group of TG221 transgenic male mice was treated intra-peritoneum with the carcinogen DEN to boost the development of liver tumors. Three experimental groups, consisting of four mice each, were then defined: the first group was infected two times, at day 60 and 135 after DEN treatment, with 1x10⁸ I.U. of Ad-199T virus, through tail vein injection; the second group was infected with the Ad-Null-Control non-replicative adenovirus (Ad NR), at the same time points with the same I.U; the third group was the not infected control group. All the mice were sacrificed at five months of age and livers collected. (<b>A</b>-<b>B</b>) Macroscopically, tumors appeared to be less and smaller in mice treated with the Ad-199T virus in comparison with mice either untreated or treated with Ad NR. Quantitative parameters confirmed the qualitative observations. (<b>C</b>) Tumor burden was reduced in Ad-199T treated mice as shown by the significant reduction of liver weights. (<b>D</b>) The number of tumor nodules was also significantly lower in mice treated with Ad-199T in comparison with the control animals. (<b>E</b>) RNAs from normal livers and tumors were analyzed by Real-Time PCR to assess miR-199 levels: as expected tumors displayed a lower expression of miR-199. (<b>F</b>) The level of Ad-199T DNA was examined by quantitative Real-Time PCR in normal liver biopsies and tumor nodules of treated mice: a 2-3 fold increase levels in tumor tissues was detected.</p

E1A expression is miR-199 dependent.

<p>To assess the expression of E1A in presence or absence of miR-199, 7×10⁴ cells of HepG2 and HepG2/199 cell lines were seeded and infected with 1x10⁶ I.U. of Ad-199T or with 1x10⁶ I.U. of Ad-Control and harvested after 48 hours. E1A levels were evaluated by quantitative PCR analysis (<b>A</b>) and by Western Blot (<b>B</b>). In HepG2, E1A mRNA was expressed following infection by both viruses; conversely, in HepG2/199 the E1A mRNA levels were significantly lower in AD-199T infected cells compared to Ad-Control infected cells. P-values of the comparisons are shown. The same results were obtained at the protein level. This evidence confirms that miR-199, constitutively expressed in HepG2/199 cells, can control E1A expression by targeting the homologous sequences places downstream of the adenoviral E1A gene.</p

Ad-199T antitumor activity on HCC xenograft.

<p>CD1 nude mice (n=6) bearing Hep3B xenografts were treated intra-tumorally either with PBS or with Ad-199T (5x10⁸ I.U. each treatment, for a total of six). (<b>A</b>) A tumor growth curve was built by measuring the size of tumors every two days. The results shown a significant difference between the PBS-treated group and the Ad-199T-treated one (p=0.001), confirming the antitumor activity of Ad-199T virus. (<b>B</b>) Kaplan-Meier survival plot showed a median survival of 24 days for untreated animals and 45 days for Ad-199T treated animals, thus indicating a longer survival time in animals treated with the oncolytic adenovirus. This difference was highly significant according to the log-rank test (p <0.0001).</p