To target adenoviral vectors to cells of the vasculature and shielding vectors from inactivation by the immune system.

Experiments were performed with cold atmospheric plasma (CAP) to inactivate adenovirus, a non-enveloped double stranded DNA virus, in solution. The plasma source used was a surface micro-discharge technology operating in air. Various plasma diagnostic measurements and tests were performed in order to determine the efficacy of CAPs and to understand the inactivation mechanism(s). Different stages of the adenovirus ‘life cycle’ were investigated—infectivity and gene expression as well as viral replication and spread. Within 240 s of CAP treatment, inactivation of up to 6 decimal log levels can be achieved.

Human Gene TherapyVol. 21, No. 7 EditorialProstate Cancer Gene Therapy: Attempts to InnovateStefan Kochanek and Bernd GansbacherStefan KochanekDivision of Gene Therapy, University of Ulm, 89081 Ulm, Germany.Search for more papers by this author and Bernd GansbacherInstitute of Experimental Oncology and Therapy Research, Technical University of Munich, 81675 Munich, Germany.Search for more papers by this authorPublished Online:7 Jul 2010https://doi.org/10.1089/hum.2010.1216AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Prostate Cancer Gene Therapy: Attempts to Innovate." , 21(7), p. 791FiguresReferencesRelatedDetailsCited BySynthesis of Tat tagged and folate modified N-succinyl-chitosan self-assembly nanoparticles as a novel gene vectorInternational Journal of Biological Macromolecules, Vol. 72A universal gene carrier platform for treatment of human prostatic carcinoma by p53 transfectionBiomaterials, Vol. 35, No. 9Synthesis and preliminary cellular evaluation of phosphonium chitosan derivatives as novel non-viral vectorCarbohydrate Polymers, Vol. 97, No. 2Dendrimer Nanoscaffolds for Potential Theranostics of Prostate Cancer with a Focus on Radiochemistry24 January 2013 | Molecular Pharmaceutics, Vol. 10, No. 3Future Prospects in the Diagnosis and Management of Localized Prostate CancerThe Scientific World Journal, Vol. 2013Targeted expression of Escherichia coli purine nucleoside phosphorylase and Fludara® for prostate cancer therapy22 December 2011 | The Journal of Gene Medicine, Vol. 13, No. 12 Volume 21Issue 7Jul 2010 InformationCopyright 2010, Mary Ann Liebert, Inc.To cite this article:Stefan Kochanek and Bernd Gansbacher.Prostate Cancer Gene Therapy: Attempts to Innovate.Human Gene Therapy.Jul 2010.791-791.http://doi.org/10.1089/hum.2010.1216Published in Volume: 21 Issue 7: July 7, 2010PDF download

Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF 165 ‐cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane‐filled magnetic lipospheres (‘magnetobubbles’) from Tween60‐coated magnetic nanoparticles, Metafectene, soybean‐oil and cDNA and studied the effect in an oversized random‐pattern‐flap model in the rats ( n = 46). VEGF‐cDNA‐magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre‐operative. Flap survival and necrosis were measured 7 days post‐operatively. Flap perfusion, VEGF‐protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF‐cDNA‐magnetobubbles presented an increased flap survival of 50% and increased flap perfusion ( P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated ( P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile.

The human Y‐box binding protein 1 (YB‐1) is known to be a promising target for cancer therapy. We have demonstrated that YB‐1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2‐gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad‐Delo3‐RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB‐1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti‐glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad‐Delo3‐RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad‐Delo3‐RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor‐bearing nudemice treated with the YB‐1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad‐Delo3‐RGD has an anti‐angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB‐1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB‐1 based clinical setting.

OBJECTIVE:Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete.METHODS:Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system.RESULTS:Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions.CONCLUSION:The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.

We have earlier described the oncolytic adenovirus vector dl520 that was rendered cancer-specific by deletion of the transactivation domain CR3 of the adenoviral E1A13S protein; this deletion causes antitumor activity in drug-resistant cells displaying nuclear YB-1 expression. We hypothesized that the anticancer activity of dl520 could be further improved by introducing the RGD motif in the fiber knob and by deletion of the adenoviral E1B19K protein (Ad-Delo3-RGD). In this study, the in vitro and in vivo antitumor activity of Ad-Delo3-RGD was investigated focussing on two pancreatic cancer cell lines MiaPaCa-2 and BxPC3 alone and in combination with cytotoxic drugs. Furthermore, luciferin-based bioluminescence imaging was established to study the therapeutic response in vivo . In addition, to confirm the specificity of Ad-Delo3-RGD for YB-1 a tetracycline-inducible anti-YB-1 shRNA-expressing cell variant EPG85-257RDB/tetR/YB-1 was used. This TetON regulatable expression system allows us to measure adenoviral replication by real-time PCR in the absence of YB-1 expression. The results confirmed the YB-1 dependency of Ad-Delo3-RGD and showed that Ad-Delo3-RGD has potent activity against human pancreatic cancer cells in vitro and in vivo , which was augmented by the addition of paclitaxel. However, although high replication capacity was measured in vitro and in vivo , complete tumor regression was not achieved, indicating the need for further improvements to treat pancreatic cancer effectively.

The impact of the Vascular Endothelial Growth Factor (VEGF) on the angiogenic cascade is proven. Recently its neuroprotective effect after peripheral nerve injuries on α-motoneurons in the spinal cord was shown. Experiments on α-motoneurons demonstrated a decreased sensitivity to ischemia under VEGF-therapy. Aim of the study was to elucidate the effect of a localized VEGF-gene-therapy using an adenoviral vector construct in the model of a peripheral nerve defect in the rat treated with an autologous nerve transplant.

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases‐1 (TIMP‐1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP‐1‐induced modulation of a pro‐metastatic microenvironment in the liver. Indeed, in livers of uPA‐ablated mice elevated TIMP‐1 levels did not trigger HGF signalling and did not promote metastasis of a murine T‐lymphoma cell line. In contrast, lack of tumour cell‐derived uPA induced by gene silencing did not interfere with this pro‐metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP‐1 levels. This newly identified co‐operation between TIMP‐1 and host uPA suggests that therapies, simultaneously interfering with pro‐ and anti‐proteolytic pathways may be beneficial for patients with metastatic disease.

Immunotherapy with whole cell cancer vaccines has been tested in various tumor types. This study investigated the safety profile and antitumor activity of an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant human interleukin-2 and human interferon-gamma. Thirty HLA-A*0201-matched patients with progressive, castration-resistant prostate cancer received four intradermal injections on days 1, 15, 29, and 92, and then every 90 days, as long as no tumor progression occurred. Three patients received a dose level of 7.5 million cells, and 27 patients received 15 million cells per injection. The primary study criteria were safety and the difference in prostate-specific antigen doubling time (PSA-DT), determined in the pretreatment phase (before the start of vaccination) and in the trial treatment phase (during vaccination). No dose-limiting or autoimmune toxicity was seen. During vaccination there was a significant prolongation of the PSA-DT compared with the prevaccination period (prolongation from 63 to 114 days; p < 0.01; intention to treat). In addition, results showed a period of PSA stabilization of at least 12 weeks, together with stable bone scans in 12 of 30 patients, and 3 patients sustained a >50% decrease in PSA versus baseline. The median overall survival time from first vaccination was 32 months (mean value, 34 months). Immune monitoring revealed T cell stimulation in the majority of patients. This vaccine strategy was found to be safe and well tolerated and was accompanied by prolongation of PSA-DT. The results of this trial warrant clinical development of this vaccine.