High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a ‘double-unit’ AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact ‘excisional-expression’ unit consisting of a target cDNA ‘upstream’ of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the ‘double-unit’ AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity

Breast imaging technology: Recent advances in imaging endogenous or transferred gene expression utilizing radionuclide technologies in living subjects - applications to breast cancer

A variety of imaging technologies is being investigated as tools for studying gene expression in living subjects. Two technologies that use radiolabeled isotopes are single photon emission computed tomography (SPECT) and positron emission tomography (PET). A relatively high sensitivity, a full quantitative tomographic capability, and the ability to extend small animal imaging assays directly into human applications characterize radionuclide approaches. Various radiolabeled probes (tracers) can be synthesized to target specific molecules present in breast cancer cells. These include antibodies or ligands to target cell surface receptors, substrates for intracellular enzymes, antisense oligodeoxynucleotide probes for targeting mRNA, probes for targeting intracellular receptors, and probes for genes transferred into the cell. We briefly discuss each of these imaging approaches and focus in detail on imaging reporter genes. In a PET reporter gene system for in vivo reporter gene imaging, the protein products of the reporter genes sequester positron emitting reporter probes. PET subsequently measures the PET reporter gene dependent sequestration of the PET reporter probe in living animals. We describe and review reporter gene approaches using the herpes simplex type 1 virus thymidine kinase and the dopamine type 2 receptor genes. Application of the reporter gene approach to animal models for breast cancer is discussed. Prospects for future applications of the transgene imaging technology in human gene therapy are also discussed. Both SPECT and PET provide unique opportunities to study animal models of breast cancer with direct application to human imaging. Continued development of new technology, probes and assays should help in the better understanding of basic breast cancer biology and in the improved management of breast cancer patients

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

Perinatal Gene Transfer to the Liver

The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.Vaikka syöpähoitojen viimeaikainen kehitys on ollut nopeaa, etäpesäkkeitä lähettäneet syövät ovat harvoin parannettavissa. Onnistuneen hoidon jälkeenkin resistentit solukloonit aiheuttavat syövän uusiutumisen, jolloin syöpä kaikkiaan on tullut hoidoille yhä vastustuskykyisemmäksi. Resistenssin aiheuttavat tekijät ovat yhteisiä eri hoitomuotoilla, jolloin hoitovaste tyypillisesti huononee nopeasti resistenssin ilmenemisen jälkeen. Uusien eri vaikutusmekanismeihin perustuvien hoitojen tarve onkin ilmeinen. Onkolyyttiset adenovirukset on geneettisesti muokattu jakautumaan syöpäsoluissa ja tappamaan niitä. Nämä virukset edustavat lupaavaa täsmälääkettä syövän hoitoon, ja erityisesti muiden hoitomuotojen lisänä. Tässä työssä avaamme uusia mahdollisuuksia onkolyyttisten adenovirusten käyttöön mahasyövän, haimasyövän ja rintasyövän hoidossa. Kapsidimuokkauksilla ohjataan virus sitoutumaan syöpäsolujen pinnassa ilmentyviin reseptoreihin. Kapsidimuokattujen adenovirusten transduktiotehokkuutta mitattiin ensin maha- ja haimasyöpäsoluissa sekä potilasnäytteissä, ja sitten hiirimalleissa. Varmistimme vastaavalla tavalla kapsidimuokatuilla onkolyyttisillä viruksilla lisääntyneen transduktiotehokkuuden vaikutuksen myötävaikuttavan virusten onkolyyttiseen tehoon. Onkolyyttiset virukset myös pidensivät ortotooppisten haima- ja mahasyöpää mallintavien hiirien elinaikaa. Onkolyyttinen adenovirusgeeniterapia saattaa osoittautua lupaavaksi hoitomuodoksi haima- ja mahasyövän hoidossa, ja hoidon tarkkuuteen, tehoon ja turvallisuuteen voidaan vaikuttaa kapsidimuokkauksen avulla. Kuvaamme myös uuden virusten annostelumenetelmän vatsaonteloon, joka kestollisesti parantaa geeninsiirtoa vatsaontelon haima- ja mahasyöpäkasvaimiin. Silikageeli-implanteilla saatiin ohjattua virusta vapautumaan kasvaimen välittömään läheisyyteen tasaisesti ja pitkittyneesti, joka paransi ortotooppisten syöpämallien selviytymistä in vivo. Silikageeliannostelu madalsi proinflammatorista sytokiinivastetta sekä kokonais- ja neutraloivien vasta-aineiden (NAb) tuottoa. Toisaalta, silika suojasi virusta jo olemassa olevilta NAbeilta, joka johti suotuisampaan biodistribuutioon immunisoiduissa hiirissä. Silika saattaa olla käyttökelpoinen vatsaonteloon levinneen syövän virushoidoissa. Syövän kantasolut ovat resistenttejä yleisille syöpähoidoille, ja ne ovat oleellisia syövän uusimisessa ja etäpesäkkeiden lähettämisessä. Tutkimme, voidaanko näitä soluja tappaa jakaantumiseltaan kohdennetuilla onkolyyttisillä adenoviruksilla. Virukset tappoivat täysin CD44+CD24-/low solupopulaatiot, joissa syövän kantasolut ovat, ja ne olivat tehokkaita myös in vivo CD44+CD24-/low –soluista kasvatetuissa kasvaimissa. Geneettisesti muokatuilla onkolyyttisillä adenoviruksilla on mahdollista tappaa syövän kantasoluja, mikä voi osoittautua kliinisesti merkittäväksi tiedoksi

Regulation of adenovirus replication by miR-199 confers a selective oncolytic activity in hepatocellular carcinoma

Oncolytic virotherapy represents a growing field of experimental cancer therapy. For safe and effective virotherapy, restricted tissue expression and replication of the virus is desirable. Various methods have been developed to achieve such restricted expression. They included the engineering of viral genomes through the insertion of tissue-specific promoters or genes encoding for tissue specific binding proteins. Here, we employed a new approach based on the use of microRNAs (miRNAs) to achieve tumor-specific viral expression and replication. miRNAs are approximately 22-nucleotide (nt)- long non-coding RNAs that are able to bind the 3’ untranslated regions (UTRs) of homologous target mRNAs and causing either their degradation or translation inhibition. Since miRNA are differentially expressed in cancer versus normal cells, it is theoretically possible to make virus expression restricted to cancer cells in a miRNA-dependent manner. Several studies have shown that miR-199 is significantly down-regulated in primary hepatocellular carcinoma (HCC) tissue and HCC cell lines. With this notion in mind, we developed a conditionally replication-competent oncolytic adenovirus, Ad-199T, by introducing four copies of miR-199 target sites within the 3′ UTR of the E1A gene, which is essential for adenovirus replication. In vitro studies of the properties of Ad-199T virus revealed that E1A expression was indeed tightly regulated both at RNA and protein levels depending upon the expression of miR-199. Consequently, Ad-199T could replicate in the HCC derived cells HepG2, negative for miR-199 expression, while its replication was strictly controlled in HepG2-199 cells, which were engineered to express high level of miR-199. A replication-competent miRNA independent Ad-Control was also generated,. Thus, these in vitro studies proved that cytotoxicity of Ad-199T was effective in HCC derived cells, which lacks expression of miR-199, and could be successfully controlled in cells that express miR-199 at high level. To assess in vivo properties of Ad-199T, we tested an orthotopic tumor model. HepG2 cells were implanted in the liver of newborn B6D2 mice. The cells could survive at least one week in this environment, enough for testing in vivo properties of Ad-199T. These studies revealed that intrahepatic delivery of Ad-199T led to virus replication in HepG2 derived xenograft tumors and a faster removal of cancer cells. Conversely, Ad-199T replication was not detected in normal, miR-199 positive, liver parenchyma. These results demonstrate that Ad-199T is a conditionally replicative adenovirus (CRAd) miR-199 dependent, with antitumor activity in vivo. This system allows replication of the oncolytic virus in HCC cells and, at the same time, tightly control replication in normal liver tissues, thus avoiding or reducing hepatotoxicity