Highly Efficient Gene Delivery for Bladder Cancers by Intravesically Administered Replication-Competent Retroviral Vectors

Departments of Urology and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA
Clinical Cancer Research (Impact Factor: 8.72). 09/2007; 13(15 Pt 1):4511-8. DOI: 10.1158/1078-0432.CCR-07-0151
Source: PubMed


In an attempt to improve viral delivery of potentially therapeutic genes via an intravesical route, we have recently developed murine leukemia virus-based replication-competent retrovirus (RCR) vectors.
We evaluated the transduction efficiency of intravesically administered RCR vectors to bladder tumor using orthotopic animal models to determine their potential as delivery vectors for bladder cancer.
The RCR vector containing green fluorescent protein (GFP) marker gene achieved efficient in vitro transmission of the GFP transgene. Murine bladder tumor-2 mouse bladder tumors exposed to intravesically administered RCR vectors exhibited 0%, 9.2 +/- 2.9%, and 30.0 +/- 6.2% of GFP expression at 9, 18, and 27 days after exposure in the orthotopic model, respectively. Orthotopic KU-19-19 human bladder tumors exposed to intravesically administered RCR vectors exhibited 3%, 85 +/- 1.0%, and 100% of GFP expression at 7, 21, and 35 days after exposure, respectively. GFP staining was observed only in the tumor cells in the bladder. No detectable PCR products of GFP gene could be observed in distant organs. Treatment with RCR vectors containing yeast cytosine deaminase (CD) gene plus 5-fluorocytosine (5-FC) dramatically inhibited the growth of preestablished murine bladder tumor-2 tumors. A single course of 5-FC treatment resulted in a 50% animal survival in mice exposed to RCR-CD compared with a 0% survival in all controls over a 70-day follow-up period.
Intravesically administered RCR vectors can efficiently deliver genes to orthotopic bladder tumor without viral spread in distant organs. RCR-CD/5-FC suicide gene therapy promises to be a novel and potentially therapeutic modality for bladder cancer.

14 Reads
  • Source
    • "To enhance the transgene, histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA) and n-butyrate, have been used. Actually, these agents reactivated the virally transduced genes and amplified the expression of transgenes encoded by the recombinant adenovirus or retrovirus vector (Chen et al., 1997; Dion et al., 1997; Khalighinejad et al., 2008; Kikuchi, et al., 2007). They regulated the histone acetylation levels in chromatin structure by competing with histone deacetylase (HDAC), and thus affected the level of transcription. "
    [Show abstract] [Hide abstract]
    ABSTRACT: One major problem of gene therapy for the treatment of tumors is poor transgene expression. To seek enhancing substances of the transgene, we screened Actinomycetes bioproducts with a luciferase reporter assay using the herpes simplex virus thymidine kinase (HSV-tk) promoter. Methanol extracts from the Actinomycetes MK616-mF5 strain enhanced the HSV-tk promoter activities significantly. The bioactive product was identified as Trichostatin A (TSA), a histone deacetylase inhibitor. TSA also enhanced the activities of the cytomegalovirus (CMV) promoter, and other promoters by 4~28-fold, although the HSV-tk promoter showed the highest response (100-fold), and the CMV promoter was the strongest. We determined the most relevant region responsive to TSA in the HSV-tk promoter, and made chimeric promoters combining the TSA-responsible region with the strongest promoter, CMV. The chimeric promoters amplified the downstream luciferase activities by 200-fold in response to TSA, and the strength was comparable to that of the CMV promoter. Unexpectedly, we also found that the long 3’-untranslated region from SV40 further enhanced the gene expression in response to TSA, by nearly 400-fold. These findings may be beneficial for the cancer gene therapy, by lowering the adverse effects on normal cells and enhancing the killing effects on cancer cells.
    International Journal of Integrative Biology 01/2009; 5(2).
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities. Adenovirus geeniterapia on yksi kehitteillä olevista syövän hoitomuodoista. Adenoviruksia voidaan käyttää joko kuljettimina, joiden avulla kohdekudokseen viedään hoitogeenejä tai syöpäsoluja voidaan tuhota hyödyntämällä adenoviruksien jakautumissykliä, minkä seurauksena infektoitu solu tuhoutuu. Vaikka adenoviruksilla on monia etuja verrattuna muihin viruksiin, niiden kyky infektoida syöpäsoluja on rajallinen. Tässä työssä olemme tutkineet eritavoin muokattujen adenoviruksien kykyä infektoida ei pieni soluisen keuhkosyövän solulinjoja ja lisääntyä niissä. Lisäksi olemme tutkineet mahdollisuutta käyttää mesenkymaalisia kantasoluja kuljettimina, jotta virus saataisiin paremmin kohdennettua tuumorikudokseen. Lisätäksemme adenoviruksien spesifisyyttä, olemme selvittäneet aktivoituuko paljon käytetty tuumorispesifinen promoottori, Cox-2 normaalisoluissa adenovirus infektion seurauksena ja voidaanko promoottorin tuumorispesifisyys palauttaa erillisellä 3'UTR säätelytekijällä. Lopuksi tutkimme, voidaanko adenoviruksen geeninsiirtotehokkuus säilyttää myös adenovirukselle spesifisen immuunivasteen vallitessa vaihtamalla toistamiseen injektoivan viruksen fiber erilaiseen kuin ensimmäisellä hoitokerralla annetulla viruksella. Saadut tulokset osoittavat, että muokatut adenovirukset, jotka on kohdennettu esim. integriineihin tai hepariinisulfaattiproteoglykaaneihin viruksen primäärireseptorin asemesta infektoivat tehokkaammin ei pienisoluisen keuhkosyövän soluja ja lisääntyvät niissä. Cox-2 promoottorin todettiin aktivoituvan adenovirus infektiosta, mutta spesifisyys saatiin palautettua 3'UTR säätelytekijän avulla. Lisäksi adenovirukset saatiin mesenkymaalisien kantasolujen avulla kuljetettua tehokkaammin tuumorikudokseen. Osoitimme myös, että adenoviruksen fiberin vaihtaminen injektiokertojen välillä auttaa virusta välttämään ensimmäisellä kerralla nousseen immuunivasteen vaikutuksen. Yhteenvetona voidaan todeta, että adenoviruksien geeninsiirto tehokuutta ja spesifisyyttä voidaan lisätä käyttämällä muokattuja adenoviruksia ja mesenkymaalisia kantasoluja kuljettimina sekä samanaikaisesti vähentää vasta-aineiden vaikutusta adenoviruksien infektointitehokkuuteen.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Various animal models of bladder tumor have been developed for the preclinical evaluation of therapeutic modalities for the treatment of bladder cancers. The ideal model for the investigation of therapeutic effects of proposed novel intravesical treatments requires the mass of the implanted tumor to be confined to the urothelium of the bladder at least for the initial phase. However, previously reported bladder tumor models are not suitable for the evaluation of intravesical therapies for the treatment of superficial bladder cancer, since the muscle invasive tumors have developed from the beginnings of the experiments. These models are too aggressive to study local treatment effects. In the current study, we demonstrated that careful instillation of MBT-2 mouse bladder cancer cells into the bladder of a syngenic C3H/HeJ mouse could establish a superficial bladder tumor with an incidence of 100%. The procedure and technique for handling animals are simple for standard animal investigators. Maintenance of the in vitro conditions of MBT-2 cells without contamination of Mycoplasma and careful selection of the substrain of C3H mouse seem to be essential for stable tumor establishment. This bladder tumor model appeared to be easy to reproduce among several investigators in different institutions. The orthotopic bladder tumor model, which was confined to urothelium, lets us evaluate various intravesical treatment strategies.
    Human Cell 09/2008; 21(3):57-63. DOI:10.1111/j.1749-0774.2008.00055.x · 1.41 Impact Factor
Show more