Publications by authors named "Bernd Gänsbacher"

41 Publications

A comparison of medical education in Germany and the United States: from applying to medical school to the beginnings of residency.

Ger Med Sci 2017 25;15:Doc15. Epub 2017 Sep 25.

Institute of Molecular Immunology & Experimental Oncology, Technical University Munich, Munich, Germany.

Both Germany and the United States of America have a long tradition of science and medical excellence reaching back as far as the nineteenth century. The same tribute must be paid to the medical educational system in both countries. Despite significant initial similarities and cross-inspiration, the paths from enrolling in a medical university to graduating as a medical doctor in Germany and the US seem to have become much different. To fill a void in literature, the authors' objective therefore is to delineate both structures of medical education in an up-to-date review and examine their current differences and similarities. Recent medical publications, legal guidelines of governmental or official organizations, articles in media, as well as the authors' personal experiences are used as sources of this report. Tuition loans of over $200,000 are not uncommon for students in the US after graduating from medical schools, which are often private institutions. In Germany, however, the vast majority of medical universities are tax-funded and, for this reason, free of tuition. Significant differences and surprisingly multiple similarities exist between these two systems, despite one depending on government and the other on private organizations. Germany currently employs an integrated medical curriculum that typically begins right after high school and consists of a 2-year long pre-clinical segment teaching basic sciences and a 4-year clinical segment leading medical students to the practical aspects of medicine. On the other hand, the US education is a two-stage process. After successful completion of a Bachelor's degree in college, an American student goes through a 4-year medical program encompassing 2 years of basic science and 2 years of clinical training. In this review, we will address some of these similarities and major differences.
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http://dx.doi.org/10.3205/000256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617919PMC
August 2018

Clinical Development and Commercialization of Advanced Therapy Medicinal Products in the European Union: How Are the Product Pipeline and Regulatory Framework Evolving?

Hum Gene Ther Clin Dev 2017 09 16;28(3):126-135. Epub 2017 May 16.

10 Finnish Medicines Agency, Helsinki, Finland .

The research and development of advanced therapy medicinal products (ATMPs) has been active in Europe and worldwide during recent years. Yet, the number of licensed products remains low. The main expected legal change in the near future in the European Union (EU) concerns the regulation on clinical trials (536/2014), which will come into force in 2018. With this new framework, a more harmonized and swift process for approval of clinical trials is anticipated, which is expected to support the entry of new innovations into the EU market. A survey on ATMPs in clinical trials during 2010-2015 in the EU was conducted in order to study the trends of ATMP development since the earlier survey published in 2012. According to the results, the number of clinical trials using ATMPs is slowly increasing in the EU. Yet, the focus is still in early development, and the projects are mainly carried out by small and medium-sized enterprises, academia, and hospitals. Oncology is the main area of clinical development. Yet, the balance between cell-based products and gene therapy medicinal products in this area may be changing in the future due to the new T-cell technologies. Many limitations and challenges are identified for ATMP development, requiring proportionate regulatory requirements. On the other hand, for such a novel field, the developers should be active in considering possible constraints and actively engage with authorities to look for solutions. This article provides up to-date information on forthcoming regulatory improvements and discusses the main challenges hampering the commercialization of ATMPs in the EU.
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http://dx.doi.org/10.1089/humc.2016.193DOI Listing
September 2017

Optimizing adenoviral transduction of endothelial cells under flow conditions.

Pharm Res 2012 May 30;29(5):1219-31. Epub 2011 Dec 30.

Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany.

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

Methods: Complexes of reporter gene expressing adenoviral vectors with positively charged magnetic nanoparticles were formed by electrostatic interaction in presence or absence of additional negatively charged poly(ethylene glycol)-based polymer. Transduction of HUVEC was analyzed in vitro under flow. Protection from inactivation by the immune system was analyzed by pre-incubation of AdV and complexes with neutralizing antibodies and subsequent reporter protein analysis of infected cells.

Results: Physical association of AdV with MNP and polymers was demonstrated by radioactive labelling of components and co-sedimentation in a magnetic field. Ad-MNP+/-polymer resulted in efficient transduction of HUVEC, depending on MOI and flow rate in presence of magnetic field, whereas no transduction was observed without complex formation with MNP or in absence of magnetic field. Association with MNP did result in protection from neutralizing antibodies, with slightly increased protection provided by the polymer.

Conclusions: Complex formation of AdV with MNP is a viable means for targeting of vectors to areas of magnetic field gradient. Additional coating with polymer might proof useful in protection from inactivation by the immune system.
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http://dx.doi.org/10.1007/s11095-011-0631-2DOI Listing
May 2012

YB-1 dependent virotherapy in combination with temozolomide as a multimodal therapy approach to eradicate malignant glioma.

Int J Cancer 2011 Sep 6;129(5):1265-76. Epub 2011 Jan 6.

Institut für Experimentelle Onkologie und Therapieforschung, Klinikum Rechts der Isar, Munich, Germany.

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.
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http://dx.doi.org/10.1002/ijc.25783DOI Listing
September 2011

Tetracycline-regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects.

Arthritis Rheum 2010 Jul;62(7):2037-46

Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1002/art.27461DOI Listing
July 2010

Tissue inhibitor of metalloproteinases-1-induced scattered liver metastasis is mediated by host-derived urokinase-type plasminogen activator.

J Cell Mol Med 2010 Dec;14(12):2760-70

Institut für Experimentelle Onkologie und Therapieforschung, Klinikum rechts der Isar der Technischen Universität, Munich, Germany.

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.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00951.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822726PMC
December 2010

Cancer gene therapy: present and future.

Hum Gene Ther 2009 Oct;20(10):1100

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http://dx.doi.org/10.1089/hum.2009.911DOI Listing
October 2009

Tissue engineering of the anterior cruciate ligament-sodium dodecyl sulfate-acellularized and revitalized tendons are inferior to native tendons.

Tissue Eng Part A 2010 Mar;16(3):1031-40

Department of Orthopaedic and Trauma Surgery, Technical University of Munich, Munich, Germany.

The acellularization of tendons using detergents (sodium dodecyl sulfate, Triton-X, tri-nitro-butyl-phosphate) is a new source of scaffolds for tissue engineering in anterior cruciate ligament (ACL) repair. In vitro testing demonstrated that acellular tendon scaffolds are biocompatible and show good biomechanical properties, but in vivo confirmation of these results is not yet available. Therefore, the aim of this study was to see in vivo if an acellular allogenic construct colonized with autologous fibroblasts improves the quality of ACL reconstruction. ACL replacement was performed in 31 New Zealand White rabbits using a standardized model. Fifteen animals received autologous semitendinosus tendon, whereas 16 animals were treated with a tissue-engineered construct. This construct was made by acellularization of allogenic semitendinosus tendons using sodium dodecyl sulfate and subsequent in vitro colonization with autologous fibroblasts. Eight weeks postoperatively, macroscopic, biomechanical (ultimate load to failure, elongation, stiffness; n = 8/9), and histological (n = 5) examinations were performed. Biomechanical testing showed decreasing strength of the constructs at 8 weeks after implantation compared with the direct postsurgical strength. However, tissue-engineered constructs (F = 19.7 +/- 20.3 N) were significantly weaker than autologous tendons (F = 61.2 +/- 31.2 N). Histologically, the autologous tendons showed signs of partial necrosis and tissue remodeling. The tissue-engineered constructs exhibited an inflammatory reaction and showed both repopulated and acellular regions. In conclusion, in vivo results were much more unfavorable than in vitro results had suggested. Further studies have to be performed to test if modifications of the acellularization process yield better results in vivo.
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http://dx.doi.org/10.1089/ten.TEA.2009.0043DOI Listing
March 2010

Therapeutic vaccination with an interleukin-2-interferon-gamma-secreting allogeneic tumor vaccine in patients with progressive castration-resistant prostate cancer: a phase I/II trial.

Hum Gene Ther 2009 Dec;20(12):1641-51

Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Klinikum rechts der Isar, 81675 Munich, Germany.

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.
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http://dx.doi.org/10.1089/hum.2009.101DOI Listing
December 2009

Combined reporter gene PET and iron oxide MRI for monitoring survival and localization of transplanted cells in the rat heart.

J Nucl Med 2009 Jul 12;50(7):1088-94. Epub 2009 Jun 12.

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Munich, Germany.

Unlabelled: There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation.

Methods: Human endothelial progenitor cells (HEPCs), derived from CD34+ mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by (124)I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs.

Results: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron- and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as (124)I accumulation. The (124)I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-5'-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation.

Conclusion: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.
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http://dx.doi.org/10.2967/jnumed.108.060665DOI Listing
July 2009

Physicobiochemical synergism through gene therapy and functional tissue engineering for in vitro chondrogenesis.

Tissue Eng Part A 2009 Sep;15(9):2513-24

Department of Orthopaedic and Trauma Surgery, University Medical Center, Albert-Ludwigs University Freiburg, Freiburg, Germany.

Mechanical and chemical stimulation have been shown to enhance in vitro chondrogenesis. The aim of this study was to analyze and compare combined physicobiochemical effects. Bovine articular chondrocytes were retrovirally transduced to express bone morphogenetic protein-2 (BMP-2) or left as naïve controls. Cells were seeded in three-dimensional polyurethane scaffolds and further cultured under static conditions or exposed to dynamic compression and shear in a joint-specific bioreactor. Four groups: control (A), load (B), BMP-2-infected (C), and BMP-2-infected plus load (D) were analyzed for DNA and glycosaminoglycan (GAG) content; collagen I, II, and X; aggrecan, (cartilage oligomeric protein (COMP), superficial zone protein, matrix metalloproteinase (MMP)-3; MMP-13 mRNA; histology; and immunohistochemistry at 7, 21, and 35 days post-seeding. Synergistic effects (D) were higher than the sum of the individual treatments (B and C) for GAG/DNA, collagen II, and COMP. Histology revealed a functional organization in D including an intense safranin O staining in C and D superior to that in A and B. Immunostaining for collagen II and aggrecan was detected in C and D and was strongest in D. The results show that both stimuli augment in vitro chondrogenesis better than in controls. Biochemical manipulation proved to be predominantly more effective than load, and synergistic effects were demonstrated.
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http://dx.doi.org/10.1089/ten.tea.2008.0479DOI Listing
September 2009

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.

Biomaterials 2009 Apr 31;30(12):2385-92. Epub 2009 Jan 31.

Department of Orthopaedics and Traumatology, TU Munchen, Ismaninger Str. 22, 81675 Munich, Germany.

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
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http://dx.doi.org/10.1016/j.biomaterials.2009.01.016DOI Listing
April 2009

Non-viral VEGF(165) gene therapy--magnetofection of acoustically active magnetic lipospheres ('magnetobubbles') increases tissue survival in an oversized skin flap model.

J Cell Mol Med 2010 Mar 14;14(3):587-99. Epub 2008 Nov 14.

Department of Plastic Surgery and Hand Surgery, Munich, Germany.

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.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00592.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823458PMC
March 2010

Searching for the right timing of surgical delay: angiogenesis, vascular endothelial growth factor and perfusion changes in a skin-flap model.

J Plast Reconstr Aesthet Surg 2009 Nov 23;62(11):1534-42. Epub 2008 Sep 23.

Department of Plastic and Reconstructive Surgery, Technische Universität München, Klinikum r. d. Isar, Munich, Germany.

Background: The angiogenic potential of vascular endothelial growth factor (VEGF) and its oxygen pressure-dependent regulation suggest a strong connection between this growth factor and the 'delay phenomenon'. In this study we focused on the chronological changes in VEGF concentration and flap perfusion in order to optimise the duration of surgical delay.

Methods: The VEGF concentration in skin and underlying muscle was measured in oversized, random-pattern flaps on 38 male Sprague-Dawley rats after 3, 5 or 7 days of surgical delay. Additionally, flaps were raised 5 or 7 days past preconditioning. The effect on flap perfusion was measured using indocyanine green fluoroscopy and the size of surviving and necrotic areas of the flaps were analysed. Microvessel density was assessed using a monoclonal CD31 antibody, and vessel diameter and morphometry were appraised by means of corrosion casting.

Results: VEGF expression in the distal half of the flaps was significantly increased 3 days after preconditioning and perfusion was significantly enhanced after day 5. An interval of 5 days between preconditioning and flap transposition resulted in a significantly reduced average necrosis rate. Microvessel density was significantly increased and vessel diameters were enlarged (P<0.05).

Conclusions: We illustrated the chronology of events from the ischaemic procedure to the rise in VEGF concentration and changes in flap perfusion, and demonstrated vasodilatation and the formation of new vessels. Most significantly, we were able to further specify the optimal length of surgical delay based on alterations on a molecular level as well as changes in vascularisation and perfusion.
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http://dx.doi.org/10.1016/j.bjps.2008.05.036DOI Listing
November 2009

Plasminogen activator inhibitor-2, but not cystatin C, inhibits the prometastatic activity of tissue inhibitor of metalloproteinases-1 in the liver.

Hum Gene Ther 2008 Oct;19(10):1039-49

Klinikum Rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, D-81675 Munich, Germany.

Formation of multiple and scattered metastases in target organs, leading to disruption of organ functional integrity, is the death-determining step for most lethal cancers. In the clinic, elevated expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is often associated with increased aggressiveness of cancer. We demonstrated that elevated host expression of TIMP-1 leads to the promotion of scattered liver metastases in mice, associated with increased activity of cysteine proteases (CPs). This study aimed for reduction of TIMP-1-promoted experimental liver metastases of lacZ-tagged human fibrosarcoma cells by overexpression of cystatin C, a natural inhibitor of CPs, in the murine host. Although CP inhibition reduced TIMP-induced proteolytic activity, the TIMP-1-induced increase in total tumor cell burden in livers was not significantly reduced. However, overexpression of cystatin C in livers with elevated TIMP-1 led to the formation of large multicellular metastatic foci in 42% of the mice. This formation was associated with increased expression of plasminogen activators (PAs). Additional overexpression of plasminogen activator inhibitor-2 prevented the formation of macrometastatic foci as well as the TIMP-1-induced increase in total tumor cell burden. This demonstrates that PAs are crucial for the prometastatic activity of TIMP-1 and led to the assumption that patients with elevated TIMP-1 expression may benefit from an antiproteolytic treatment directed against PAs.
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http://dx.doi.org/10.1089/hum.2008.078DOI Listing
October 2008

GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves.

Eur Urol 2008 Nov 15;54(5):1179-87. Epub 2008 Feb 15.

Department of Urology, Ludwig-Maximilians-Universität München, München, Germany.

Background: Schwann cell-seeded guidance tubes have been shown to promote cavernous nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regenerative capacity. The present study evaluates whether the transplantation of GDNF-overexpressing Schwann cells may enhance regeneration of bilaterally transected erectile nerves in rats.

Methods: Silicon tubes seeded with either GDNF-overexpressing or GFP-expressing Schwann cells were implanted into the gaps between transected cavernous nerve endings. Six (10 study nerves) or 12 wk (20 study nerves) postoperatively, erectile function was evaluated by relaparotomy, electrical nerve stimulation, and intracavernous pressure recording, followed by ultrastructural evaluation of reconstructed nerves employing bright-field and electron microscopy. Additional animals were either sham-operated (positive control; 20 study nerves) or received bilateral nerve transection without nerve reconstruction (negative control; 20 study nerves).

Results: The combination of GDNF delivery and Schwann cell application promoted an intact erectile response in 90% (9 of 10) of grafted nerves after 6 wk and in 95% (19 of 20) after 12 wk, versus 50% (5 of 10) and 80% (16 of 20) of GFP-expressing Schwann cell grafts (p=0.02). The functional recovery was paralleled by enhanced axonal regeneration in GDNF-overexpressing Schwann cell grafts, as indicated by larger cross-sectional areas and a significantly higher percentage of neural tissue compared with GFP-transduced controls.

Conclusions: These findings demonstrate that the time required to elicit functional recovery of erectile nerves can be reduced by local delivery of GDNF. In terms of clinical application, this enhanced nerve repair might be critical for timely reinnervation of the corpus cavernosum as a prerequisite for functional recovery in men.
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http://dx.doi.org/10.1016/j.eururo.2008.02.003DOI Listing
November 2008

Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector.

Biomaterials 2008 Mar;29(9):1242-9

Department of Orthopaedic and Trauma Surgery, Technical University Munich, Munich, Germany.

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.
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http://dx.doi.org/10.1016/j.biomaterials.2007.11.013DOI Listing
March 2008

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

Radiother Oncol 2008 Mar 29;86(3):419-27. Epub 2007 Oct 29.

Institute of Experimental Oncology, Technical University of Munich, Germany.

Background And Purpose: Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1.

Methods And Material: Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice.

Results: Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model.

Conclusions: Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.
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http://dx.doi.org/10.1016/j.radonc.2007.10.009DOI Listing
March 2008

In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects.

Biomaterials 2007 Oct 13;28(30):4480-7. Epub 2007 Jul 13.

Department of Trauma, Hand and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.

To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.
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http://dx.doi.org/10.1016/j.biomaterials.2007.06.027DOI Listing
October 2007

Inhibition of the multidrug-resistant phenotype by targeting YB-1 with a conditionally oncolytic adenovirus: implications for combinatorial treatment regimen with chemotherapeutic agents.

Cancer Res 2006 Jul;66(14):7195-202

Institute of Experimental Oncology and Department of Urology, Technical University of Munich, Klinikum rechts der Isar, Germany.

Bearing in mind the limited success of available treatment modalities for the therapy of multidrug-resistant tumor cells, alternative and complementary strategies need to be developed. It is known that the transcriptional activation of genes, such as MDR1 and MRP1, which play a major role in the development of a multidrug-resistant phenotype in tumor cells, involves the Y-box protein YB-1. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In addition, it has been reported previously that YB-1 is an important factor in adenoviral replication because it activates transcription from the adenoviral E2-late promoter. Here, we report that an oncolytic adenovirus, named Xvir03, expressing the viral proteins E1B55k and E4orf6, leads to nuclear translocation of YB-1 and in consequence to viral replication and cell lysis in vitro and in vivo. Moreover, we show that Xvir03 down-regulates the expression of MDR1 and MRP1, indicating that recruiting YB-1 to the adenoviral E2-late promoter for viral replication is responsible for this effect. Thus, nuclear translocation of YB-1 by Xvir03 leads to resensitization of tumor cells to cytotoxic drugs. These data reveal a link between chemotherapy and virotherapy based on the cellular transcription factor YB-1 and provide the basis for formulating a model for a novel combined therapy regimen named Mutually Synergistic Therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2339DOI Listing
July 2006

Characterization of the recombinant adenovirus vector AdYB-1: implications for oncolytic vector development.

J Virol 2006 Apr;80(8):3904-11

Institut fuer Experimentelle Onkologie und Therapieforschung, Technische Universitaet Muenchen, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 Munich, Germany.

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.
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http://dx.doi.org/10.1128/JVI.80.8.3904-3911.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440461PMC
April 2006

Novel three-pronged strategy to enhance cancer cell killing in glioblastoma cell lines: histone deacetylase inhibitor, chemotherapy, and oncolytic adenovirus dl520.

Hum Gene Ther 2006 Jan;17(1):55-70

Institute of Experimental Oncology, Technical University of Munich, Klinikum Rechts-der-Isar, 81675 Munich, Germany.

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.
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http://dx.doi.org/10.1089/hum.2006.17.55DOI Listing
January 2006

Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host.

Cancer Res 2005 Oct;65(19):8608-12

Klinikum rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung, Munich, Germany.

Tumor cell invasion and metastasis are associated with degradation of components of the extracellular matrix by different proteinases. Among those, papain-like cysteine proteases, such as cathepsin B, seem to play an important role, as they are associated with poor clinical outcome in different cancers. In this study, we tested whether cystatin C, a natural extracellular inhibitor of papain-like cysteine proteases, can inhibit metastasis when overexpressed at the tumor-host interface. Local overexpression of cystatin C in liver and lungs of CD1 nu/nu mice was achieved by gene transfer with a novel adenoviral construct, which also led to the presence of 60 ng/mL of cystatin C in the serum. Three days after gene transfer, these mice were challenged by i.v. inoculation of lacZ-tagged human fibrosarcoma cells (HT1080lacZ-K15), leading to the formation of experimental lung and liver metastases. In this model, formation of experimental metastatic foci correlated with expression of cathepsin B in lungs, whereas there was no correlation with metastasis to the liver. In mice overexpressing cystatin C, the number of lung metastases was significantly reduced by 92%, as compared with mice receiving control adenovirus. The efficacy of extravasation of HT1080lacZ-K15 cells into the liver was not affected, indicating the independence of this process from the activity of cysteine-cathepsins. The present report is the first evidence of successful reduction of metastasis by inhibition of cysteine-cathepsins by cystatin C overexpression in the host microenvironment. Furthermore, organ-specific protease expression during tumor-host cell interactions could affect the success of antiproteolytic intervention against metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-1572DOI Listing
October 2005

Non-invasive imaging of cardiac transgene expression with PET: comparison of the human sodium/iodide symporter gene and HSV1-tk as the reporter gene.

Eur J Nucl Med Mol Imaging 2005 Sep;32(9):1108-14

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675, München, Germany.

Purpose: Genes encoding for intracellular enzymes or transmembrane proteins are suitable as reporters, but may differ in terms of applicability for cardiac imaging. The aim of this study was to compare the human sodium iodide symporter gene (hNIS) with the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) as the reporter gene in non-invasive imaging of cardiac transgene expression with positron emission tomography (PET).

Methods: Equal doses of adenoviral vectors encoding for hNIS, wild-type HSV1-tk, mutant HSV1-sr39tk or LacZ as the control gene were directly injected into the myocardium of 34 animals. Two days later, dynamic PET was performed with a clinical scanner, using reporter probes specific for the respective reporter gene. Imaging with (13)N-ammonia was also performed to identify cardiac regions of interest.

Results: Kinetics differed significantly: (124)I as the probe for hNIS showed rapid early uptake, remaining stable over time. Maximal myocardial concentration was 3.61+/-1.15%. The nucleoside (18)F-FHBG, as the specific probe for HSV1-sr39tk, showed increasing uptake over time, but maximal accumulation was significantly lower (1.45+/-0.54%, P=0.0009). (124)I-FIAU, as the specific probe for wild-type HSV1-tk, showed early uptake with subsequent washout. Maximal accumulation was lowest (0.63+/-0.23%, P<0.0001). Post-mortem analysis by autoradiography and gamma counting confirmed the in vivo data.

Conclusion: Reporter genes encoding for transporter proteins such as hNIS are an attractive alternative to overexpression of intracellular enzymes for cardiac gene product imaging. hNIS yielded higher signal intensity and imaging contrast for PET than did HSV1-tk and HSV1-sr39tk. Therefore, this approach may be preferable for the future monitoring of cardiac gene- or cell-based therapy.
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http://dx.doi.org/10.1007/s00259-005-1854-4DOI Listing
September 2005

Optimization of radiation controlled gene expression by adenoviral vectors in vitro.

Cancer Gene Ther 2005 Jul;12(7):640-6

Institut für Experimentelle Onkologie & Therapieforschung, München, Germany.

The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.
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http://dx.doi.org/10.1038/sj.cgt.7700829DOI Listing
July 2005

Cardiac reporter gene imaging using the human sodium/iodide symporter gene.

Cardiovasc Res 2005 Jan;65(1):195-202

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 München, Germany.

Objective: Imaging of reporter gene expression holds promise for noninvasive monitoring of cardiovascular molecular therapy. We investigated the feasibility of myocardial gene expression imaging in living rats using the human sodium/iodide symporter gene (hNIS) and widely available scintigraphic techniques.

Methods: We injected adenovirus expressing hNIS under control of cytomegalovirus promoter (Ad(hNIS)) directly into left ventricular myocardium of Wistar rats. For detection of reporter gene expression, dynamic gamma-camera imaging was performed following intravenous injection of (123)Iodide or (99m)Technetium.

Results: For both radiotracers, focal cardiac accumulation was identified as early as 10 min, and remained detectable until 2 hrs after injection, while it was not present in animals injected with LacZ control virus. Intensity of tracer accumulation gradually decreased when decreasing titers of Ad(hNIS) were applied. Treatment with sodium perchlorate (a blocker of hNIS) abolished cardiac tracer uptake after Ad(hNIS)-infection. Serial imaging after cardiac gene transfer demonstrated a peak of tracer signal between days 1 and 3, and a subsequent decrease until day 12. Postmortem analysis of hearts yielded significant correlation between in vivo radiotracer accumulation and ex vivo gamma-counting. Autoradiography demonstrated specific regional radioactivity in Ad(hNIS)-infected myocardial areas.

Conclusions: hNIS offers a practical and reliable approach for myocardial gene expression imaging. Using suitable vectors, hNIS may be coexpressed with therapeutic genes or stably expressed in stem cells for future monitoring of cardiovascular molecular therapy.
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http://dx.doi.org/10.1016/j.cardiores.2004.10.001DOI Listing
January 2005

PET of cardiac transgene expression: comparison of 2 approaches based on herpesviral thymidine kinase reporter gene.

J Nucl Med 2004 Nov;45(11):1917-23

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, 81675 Munich, Germany.

Unlabelled: PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals.

Methods: Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest.

Results: In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography.

Conclusion: Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.
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November 2004

Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.

J Nucl Med 2004 Oct;45(10):1743-6

Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Munich, Germany.

Unlabelled: Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk).

Methods: Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro.

Results: In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003).

Conclusion: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.
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October 2004