Ruben Hernandez-Alcoceba

New Oncolytic Adenoviruses with Hypoxia- and Estrogen Receptor-Regulated Replication

Oncolytic adenoviruses with restricted replication can be produced if the expression of crucial transcription units of the virus is controlled by tissue- or tumor-specific promoters. Here we describe a method for the rapid incorporation of exogenous promoters into the E1A and E4 regions of the human adenovirus type 5 genome. Using this system, we have generated AdEHT2 and AdEHE2F, two conditionally replicative adenoviruses for the treatment of breast cancer. The expression of the E1A gene in both viruses is controlled by a minimal dual-specificity promoter that responds to estrogens and hypoxia. The tight regulation of E1A expression correlated with the ability of these viruses to replicate and kill human cancer cells that express estrogen receptors, or are maintained under hypoxic conditions. The telomerase reverse transcriptase (TERT) promoter and the E2F-1 promoter are preferentially activated in cancer cells. They were introduced into the E4 region of AdEHT2 and AdEHE2F, respectively. The telomerase core promoter failed to block the replication of the virus in telomerase-negative cells. In contrast, AdEHE2F was attenuated in nontransformed quiescent cells growing under normoxic conditions, suggesting that an intact pRB pathway with low levels of E2F transcription factors acts as a negative modulator for the virus. These data indicate that the simultaneous regulation of E1A and E4 viral transcription units by the appropriate combination of promoters can increase the tumor selectivity of oncolytic adenoviruses.

A Novel, Conditionally Replicative Adenovirus for the Treatment of Breast Cancer That Allows Controlled Replication of E1a-Deleted Adenoviral Vectors

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER(+) human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER(-) cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1a-deleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.

Intrapulmonary expression of macrophage inflammatory protein 1α (CCL3) induces neutrophil and NK cell accumulation and stimulates innate immunity in murine bacterial pneumonia

ABSTRACT Macrophage inflammatory protein 1α (MIP-1α) (CCL3) is an important mediator of leukocyte recruitment and activation in a variety of inflammatory states, including infection. A recombinant human type 5 adenovirus containing the murine MIP-1α cDNA (AdMIP-1α) was constructed to determine the effect of transient intrapulmonary expression of MIP-1α on leukocyte recruitment, activation, and bacterial clearance in a murine model of Klebsiella pneumoniae pneumonia. The intratracheal administration of AdMIP-1α resulted in both time- and dose-dependent expression of MIP-1α mRNA and protein within the lung. Importantly, the intrapulmonary overexpression of MIP-1α resulted in a maximal 35- and 100-fold reduction in lung and blood bacterial burden, respectively, in animals cochallenged with K. pneumoniae, which was associated with a significant increase in neutrophil and activated NK cell accumulation. Furthermore, the transgenic expression of MIP-1α during bacterial pneumonia resulted in enhanced expression of gamma interferon mRNA, compared to that observed in Klebsiella-challenged animals pretreated with control vector. These findings indicate an important role for MIP-1α in the recruitment and activation of selected leukocyte populations in vivo and identify this cytokine as a potential immunoadjuvant to be employed in the setting of localized bacterial infection.

Evaluation of a new dual-specificity promoter for selective induction of apoptosis in breast cancer cells.

The conditional expression of lethal genes in tumor cells is a promising gene therapy approach for the treatment of cancer. The identification of promoters that are preferentially active in cancer cells is the starting point for this strategy. The combination of tissue-specific and tumor-specific elements offers the possibility to artificially develop such promoters. We describe the construction and characterization of a hybrid promoter for transcriptional targeting of breast cancer. In many cases, breast cancer cells retain the expression of estrogen receptors, and most solid tumors suffer from hypoxia as a consequence of their aberrant vascularization. Estrogen response elements and hypoxia-responsive elements were combined to activate transcription in cells that present at least one of these characteristics. When a promoter containing these elements is used to control the expression of the pro-apoptotic gene harakiri, the induction of cell death can be activated by estrogens and hypoxia, and inhibited by antiestrogens such as tamoxifen. Finally, we show evidence that these properties are maintained in the context of an adenoviral vector (AdEHhrk). Therefore, infection with this virus preferentially kills estrogen receptor–positive breast cancer cells, or cells growing under hypoxic conditions. We propose the use of this promoter for transcriptional targeting of breast cancer. Cancer Gene Therapy (2001) 8, 298–307

Deletion of the E3-6.7K/gp19K region reduces the persistence of wild-type adenovirus in a permissive tumor model in Syrian hamsters.

A partial deletion of the adenovirus E3 region, comprising the overlapping 6.7K/gp19K genes, has been described for the incorporation of therapeutic genes in ‘armed’ oncolytic adenoviruses. This deletion allows the insertion of up to 2.5 kb genetic material into the virus and ensures strong expression of transgenes without reducing the replication and cytolytic potency of viruses in vitro. E3-gp19K and 6.7K proteins are involved in avoiding recognition and elimination of infected cells by the host immune system. Therefore, we have studied the effect of this deletion on the replication and transgene expression of the virus in immunocompetent models based on Syrian hamsters. Tumors were established by intrahepatic injection of pancreatic cancer cells with moderate (HaP-T1, HP-1) or low (H2T) permissivity for adenovirus replication. The wild-type human adenovirus 5 (Ad5) or a modified version containing the luciferase gene in the E3-6.7K/gp19K locus (Ad-WTLuc) were injected intratumorally. We found that elimination of Ad-WTLuc was faster than Ad5 in HaP-T1 and HP-1 tumors. In contrast, no differences were observed when the same tumor was established in severely immunocompromised NOD-scid IL2Rγnull mice. In addition, virus-mediated luciferase expression was more stable in these animals. These results suggest that the lack of E3-6.7K/gp19K genes may accelerate the clearance of oncolytic adenoviruses in some immunocompetent tumor models.

Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal+) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal+ infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca2+-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal+ infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK½, ERK½) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal+ in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

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.

Brain copper storage after genetic long-term correction in a mouse model of Wilson disease

Wilson disease is a rare autosomal recessive condition caused by mutations in the copper-transporting ATPase ATP7B gene (OMIM: 606882) provoking loss of function and resulting in variable hepatic and neurologic symptoms. Currently, the treatment of Wilson disease focuses on achieving a negative copper balance either with chelators (e.g., d-penicillamine, trientine, and tetrathiomolybdate) or zinc, which reduces copper absorption, or a combination thereof.1 However, these lifelong treatment regimens often cause side effects and do not restore normal copper metabolism.

Inhibition of adenovirus infection by mifepristone

&NA; 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. Graphical abstract Figure. No caption available. HighlightsMifepristone, a synthetic steroid, has been evaluated for its antiviral activity.Treatment of mice with mifepristone interferes with the transgene expression of HAdV‐based vectors.Mifepristone shows a dose‐dependent anti‐HAdV activity at low micromolar concentration.Mifepristone targets HAdV DNA transport to the nucleus after endosomolysis.Mifepristone represents a potential candidate for the development of an antiviral drug to treat HAdV infections.

164. Improvement of Gene Therapy for Wilson Disease

Wilsons disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs, which may cause side effects and do not restore normal copper metabolism. We have recently demonstrated that the administration of an AAV vector expressing ATP7B under the control of a liver specific promoter induces full restoration of copper homeostasis in a mouse model of Wilsons disease. However, the size of the vector genome surpasses the optimal size for AAV packaging, limiting the use of large promoters or additional regulatory sequences such as introns or bigger poly A sequences. In the present work we have designed two truncated versions of ATP7B protein (T1 and T2), and have analyzed their functionality and therapeutic efficacy. ATP7B contains 6 metal binding sites (MBS) in the N-terminal region. T1 presents a deletion of 1 MBS and a fragment of the adjacent one, whereas T2 lacks the first 4 MBS. Both proteins were active since they were able eliminate intracellular excess of copper in an in vitro system. The administration of recombinant AAV vectors carrying the truncated proteins under the control of a liver specific promoter was able to reduce liver damage and urinary copper excretion in the murine model, but T1 was less efficient than the WT ATP7B gene. In contrast, T2 showed full capacity to normalize these parameters. Furthermore, T2 was more efficient than WT in restoring ceruloplasmin oxidase activity in serum. In conclusion, we have identified a fully functional truncated version of ATP7B protein that will reduce the size of the therapeutic AAV vector genome and allow the introduction of additional regulatory elements.