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Characteristics of inherited retinal dystrophies include deficiencies in light perception and nervous conduction within the retina, leading to reduced vision or even blindness. In this context, the loss of function of photoreceptor-specific genes causes a variety of clinically and aetiologically distinct syndromes - each of them belonging to the group of rare diseases. With a prevalence of 1 in 2500, however, inherited retinal diseases are clinically significant and important - especially since these diseases lead to restrictions of a patient's fitness for work and overall quality of life. More than 250 genetic mutations causing the various types of inherited retinal dystrophies have been identified by now (https://sph.uth.tmc.edu/Retnet). In recent years, preclinical research on suitable animal models has yielded important progress in the understanding of the mutations underlying the pathological and molecular biological processes of these diseases. These findings have led to the development of novel and innovative therapeutic strategies for the treatment of inherited retinal dysfunctions, which are still incurable. Meanwhile, many of the successful preclinical studies have led to translational research projects aiming to find treatment options for human patients. However, some preliminary results of these human translational studies indicate the need to optimise and refine the underlying therapeutic concepts.

Poxviruses as expression vectors are widely used in medical research for the development of recombinant vaccines and molecular therapies. Here we review recent accomplishments in vaccine research using recombinant modified vaccinia virus ankara (MVA). MVA is a highly attenuated vaccinia virus strain that originated from serial tissue culture passage in chicken embryo fibroblasts more than 40 years ago. Growth adaptation to avian host cells caused deletions and mutations in the viral genome affecting about 15% of the original genetic information. In consequence, MVA is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. Because of its safety for the general environment MVA can be handled under conditions of biosafety level one. Non-replicating MVA can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. Therefore, recombinant MVA have been established as an extremely safe and efficient vector system for vaccine development in medical research. By now, various recombinant MVA vaccines have been found safe and immunogenic when used for phase I/II clinical testing in humans, and suitable for industrial scale production following good practice of manufacturing. Thus, there is an obvious usefulness of recombinant MVA vaccines for novel prophylactic and therapeutic approaches also in veterinary medicine. Results from first studies in companion and farm animals are highly promising.

Gene therapy for inherited retinal diseases (IRDs) is currently being validated in several clinical trials and is becoming a promising therapeutic option for these previously incurable diseases.The aim of this review is to give an overview of the concept, the application and the challenges associated with gene therapy. In particular, the pertinence of gene therapy for IRDs will be highlighted along with ongoing clinical trials in the field.A systematic review of relevant entries on gene therapy and on gene therapy for IRDs, in particular in PubMed and ClinicalTrials.gov.Gene therapy is emerging not only as a therapy for monogenetic retinal diseases but also for complex genetic diseases, such as neovascular age-related macular degeneration. The discovery of adeno-associated viral vectors (AAVs) has marked a great improvement for IRD gene therapy. All clinical studies since 2006 demonstrated the safety and initial efficacy; however, not all expectations based on very successful preclinical studies were met.In future we can expect gene therapy to continue to become more clinically relevant. More than ever, it is now essential to generate precise characterizations of the natural disease progression of IRDs through observational or retrospective studies in order to guarantee a most effective study design.

Corneal endothelium is an interesting target for in vitro gene transfer strategies as it is readily accessible thanks to its anatomic structure as a monolayer and its direct contact to culture medium. Whereas the use of adenoviruses as viral vectors (carriers) to endothelial cells (EC) has been described as problematic as to its immunogenicity, lentiviruses and adeno-associated viruses (AAV) are potent vectors for the transfer of genetic DNA into EC. Lentiviral vectors, developed on the basis of HI-viruses, can integrate the transferred gene into the host DNA and thus lead to a permanent protein expression. Evaluating apathogen alternatives to lentiviral vectors for humans, we herein compared non-integrating AAV to lentiviral gene transfer.A comparison was made of the kinetics of expression of a green fluorescent protein after transduction using a lentiviral vector and AAV 2 / 2 in a murine EC line, human EC line and human primary cells (flow cytometry). A proof of principle experiment was conducted to demonstrate the function after lentiviral gene transfer of the anti-apoptotic gene Bcl-xL.The kinetics of protein expression after transduction of EC using a lentiviral or an AAV vector show fundamental differences. Contrary to gene transfer using AAV, a high expression of the reporter protein was readily detectable only hours after transduction using the lentiral vector. In addition, we could demonstrate distinct differences in protein expression characteristics between human and murine EC as well as human EC line and primary human EC. Function could be demonstrated by showing a significant reduction in apoptosis in both murine and human EC.AAV vectors are an alternative to lentiviral vectors for gene transfer to corneal EC. Given a cultivation time of donor corneas of up to 4 weeks before transplantation, translation to eye banking, e. g., to decrease apoptosis in corneal allografts, is conceivable.

Endothelial keratoplasty is a promising surgical procedure which may replace penetrating keratoplasty in cases of endothelial cell diseases of the cornea. This method may thereby help to prevent postoperative astigmatism and transplant rejection.A survey of publications reporting about results after endothelial keratoplasty shows that the main problem of this transplantation technique is a postoperative endothelial cell loss which is comparable to or even higher than that observed in penetrating keratoplasty. Improving surgical techniques led to a reduction of the endothelial cell loss, however, cell-based strategies to prevent postoperative cell loss or to enhance the cell densities of donor corneas or endothelial lamellae are rare.This review presents an overview of clinical results after endothelial keratoplasty. Current strategies in the field of cell biology and tissue cultivation of corneal endothelial cells, genetic manipulation of the corneal endothelium and tissue engineering strategies aiming at the production of transplantable endothelial cell sheets are described.The limited availability of donor corneas makes it mandatory to develop methods in the field of tissue engineering in order to improve corneal endothelial cell survival or to increase corneal endothelial cell density, using interdisciplinary approaches.

Gene therapy holds great potential for the treatment of various acquired and inherited pulmonary diseases. Among various viral vectors, adeno-associated viral (AAV) vectors have been most frequently used in different clinical trials of pulmonary gene therapy. In the present study, we examined the kinetics and duration of transgene expression, vector biodistribution and development of neutralizing antibodies (NAB) in mice after pulmonary application of AAV2/9 vector. The pulmonary route of application did not affect any of the measured parameters. Transgene expression and biodistribution analysis at day 450 post-application confirmed the systemic spread of the vector after pulmonary delivery. Using SPB(-/-) mice, the study shows that AAV2/9-mediated gene expression is influenced by animal gender but not mouse genotype and is insensitive to the presence of lung inflammation. Lower expression levels were observed in male compared with female mice, and transient immunosuppression with dexamethasone significantly reduced the development of NAB in both genders of mice. The study thus advances this serotype for further development and use as a therapeutic vector.

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease of unknown aetiology that is characterized by the progressive destruction of articular structures. The accumulation of inflammatory cells in the joint and the transformation of the healthy synovial membrane into a hyperplasic and aggressive pannus tissue constitute important steps in the pathology of RA. The synthesis and secretion of inflammatory factors such as cytokines and chemokines as well as the release of matrix degrading enzymes play a decisive role and have, therefore, become of interest for novel therapeutic strategies, among the gene therapy. This article summarizes the principles and current developments of gene therapy in RA and gives an overview about available vector systems and target genes.

Diseases of the inner ear such as presbycusis, tinnitus, sudden hearing loss, and vertigo affect many patients, but so far there are no specific therapy options. Gene therapy might become a potential modality of treatment. Viral vectors are standard in animal models to date. Future considerations, however, call for a further evaluation of non-viral transfection methods.The non-viral transfection agents Metafectene, Superfect, Effectene, and Mirus TransIT were incubated with a plasmid coding for GFP. In vivo, the plasmid-agent mix was injected via the membrane of the round window, and 48 h later the inner ear was perfused, harvested, decalcified, and histologically evaluated for GFP expression.Cationic lipids (Metafectene) and dendrimers (Superfect) were able to transfect cells in the area of the organ of Corti and lead to GFP expression. The polyamine (Mirus TransIT) did show expression of GFP in the area of Rosenthal's canal and in the area of the inner hair cell. The combination of a non-liposomal lipid with a DNA condensing component (Effectene) did not show transfection of the organ of Corti. In the area of the spiral ganglia cells, GFP expression was seen with all the transfection agents.Non-viral transfection agents are able to introduce a reporter gene in cells of the inner ear in vitro and in vivo. There are, however, differences in the efficiency of the transfection. They might be an alternative in gene therapy of the inner ear. Further investigations to elucidate their potential are needed.

Ein neuer gentherapeutischer Ansatz adressiert nicht den Erhalt dopaminerger Neurone, sondern setzt auf die Unterstützung des inhibitorischen GABAergen Systems.

Current advances in CRC treatment have led to significant but slight improvements in patient survival with curative outcomes only seen in earlier stage cancers. Consequently, much effort is being put into developing completely novel therapies that fulfil a number of criteria including greater efficacy and fewer side effects. Many of these conditions are met by the wide range of gene therapy strategies currently in pre-clinical or clinical trial phases. Gene therapy approaches may be broadly broken down into three main areas: Following a few tragic events in the context of clinical gene therapy studies, safety is currently the prime concern. Further progress in the field is expected from the combination of the described approaches with conventional treatment modalities.

Objective Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. Methods We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. Results Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. Conclusion These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.

To help in the control of fowl plague caused by highly pathogenic avian influenza A viruses of hemagglutinin (HA) subtypes H5 and H7 several vaccines have been developed. A prophylactic immunization of poultry with inactivated influenza viruses in non-endemic situations is questionable, however, due to the impairment of serological identification of field virus-infected animals which hinders elimination of the infectious agent from the population. This problem might be overcome by the use of genetically engineered marker vaccines which contain only the protective influenza virus hemagglutinin. Infected animals could then be unambiguously identified by their serum antibodies against other influenza virus proteins, e.g. neuraminidase or nucleoprotein. For such a use, purified HA or HA-expressing DNA vaccines are conceivable. Economically advantageous and easier to apply are modified live virus vaccines in use against other poultry diseases, which have been modified to express influenza virus HA. So far, recombinant HA-expressing fowlpox virus (FPV) as well as infectious laryngotracheitis and Newcastle disease viruses have been asssessed in animal experiments. An H5-expressing FPV recombinant is already in use in Central America and Southeast Asia but without accompanying marker diagnostics. Advantages and disadvantages of the different viral vectors are discussed.

In der hier vorliegenden Arbeit werden unter Verwendung eines adenoviralen Vektors der dritten Generation, eines sogenannten gutless Vektors, zunächst die Transduktionseigenschaften sowie die Transduktionseffizienz verschiedener Stammzellpopulationen untersucht. Zu diesen Zellen gehören sowohl glial restringierte Vorläuferzellen der Ratte und des Menschen sowie embryonale Stammzellen der Maus. Stellvertretend für eine adulte Stammzelle wird die Möglichkeit einer Transduktion der autolog verfügbaren mesenchymalen Stammzellen aus dem humanen Knochenmarkstroma untersucht. Für die Transduktion werden Vektoren mit verschiedenen Reportersequenzen eingesetzt, sodass nach der Infektion der Transduktionserfolg anhand der Transgenexpression ermittelt werden kann. Zu den Reportergenen gehört einerseits das grün fluoreszierende Protein (GFP), das LacZ (ß- Galaktosidase) sowie als Markerenzym zur Darstellung der Syntheserate der transduzierten Zellen, die sezernierte alkalische Phosphatase des Menschen (SEAP). Unter Verwendung steigender infektiöser Partikel (10, 50 und 100 MOI) kann in den untersuchten Zellpopulationen eine dosisabhängige Transduktionsrate ermittelt werden. Dabei werden die höchsten Transduktionsraten in den glial restringierten neuralen Vorläuferzellen erzielt. Nach Infektion der neural differenzierten embryonalen Stammzellen zeigt sich ebenfalls die Tendenz einer bevorzugten Transduktion von glial differenzierten Zellen. Die Verwendung von adulten Stammzellen resultiert ebenfalls in Transduktionsraten von bis zu 85%. In einem weiteren Versuchsabschnitt werden glial restringierte neurale Vorläuferzellen nach Infektion mit dem adenoviralen Vektor in das Striatum adulter Ratten injiziert. Dieses Vorgehen soll die Möglichkeit eines ex-vivo gentherapeutischen Einsatzes in Verbindung mit astrozytären Zellen als mögliche Trägerzellen näher beleuchten. Die Befunde dieses Teilabschnittes zeigen, dass sich die transduzierten Zellen im Empfängergewebe integrieren und innerhalb des Beobachtungszeitraumes von 6 Wochen nur in begrenztem Maße in das umliegende Gewebe migrieren. Dabei zeigen die Zellen die normalen Differenzierungscharakteristika entlang der glialen Zelllinie, wie das für Astrozyten in-vivo beschrieben wurde. Der zweite Teil der Arbeit beschäftigt sich mit der Möglichkeit der direkten Vektorinjektion in das zentrale Nervensystem. Dabei wird der adenovirale Drittgenerationsvektor in verschiedene Hirnareale injiziert. Mittels immunhistochemischen Färbungen für NeuN und GFAP kann gezeigt werden, dass nach Injektion in das Striatum analog zur Situation in-vitro eine präferentielle Transduktion von Astrozyten erzielt wird. Innerhalb dieses Hirnkompartimentes kann eine Diffusion des Vektors bis zu einer Distanz von 1000 µm von der Injektionsstelle ermittelt werden. Nach Injektion der Vektorpartikel in das Corpus callosum kommt es zu einer Verteilung des Vektors innerhalb des gesamten Fasertraktes der weißen Substanz, einschließlich des kontralateralen Abschnittes. Mit diesen Befunden wird deutlich, dass die Verteilung des Vektors abhängig von der Struktur und Beschaffenheit des Empfängergewebes ist. Wird der Vektor in den Liquorraum des Seitenventrikels injiziert, kann anschließend eine Transgenexpression (GFP oder LacZ) in den Ependymzellen des gesamten Ventrikels dargestellt werden. Diese Befunde deuten darauf hin, dass sich der Vektor innerhalb des Liquor cerebrospinalis verteilt und als Folge eine generalisierte Transduktion der Ventrikelependymzellen erfolgt. Nach Anlegen einer permanenten Sonde in den Seitenventrikel kann kontinuierlich Liquor zur Analyse des Markerenzyms SEAP entnommen werden. Mittels des luminometrischen Nachweisverfahrens kann nach Vektorinjektion über einen Zeitraum von bis zu 42 Tagen das Enzym SEAP im Liquor cerebrospinalis nachgewiesen werden. Insgesamt zeigen die Befunde, dass die Verwendung des adenoviralen Vektors zumindest unter experimentellen Bedingungen eine zukunftsträchtige Therapieoption für Erkrankungen des zentralen Nervensystems darstellt., In this study the characteristics of transduction as well as the efficacy of transduction of different stem cell populations are being investigated by using a 3rd generation adenoviral vector, a so called gutless adenoviral vector. These cells include glial restricted precursor cells of rat and human origin as well as murine embryonic stem cells. As representatives for an adult stem cell population the possibility of a transduction of the autologous available mesenchymal stem cells deriving from the human bone marrow stroma are examined. For cell transduction, vectors with different reporter sequences are applied, resulting in the possibility of measuring the transduction success after infection by the transgene expression. The reporter genes include the green fluorescent protein (GFP), the LacZ (ß-galactosidase) as well as the human secreted placenta alkaline phosphatase (SEAP). Applying increasing numbers of infectious particles (10, 50 and 100 MOI) a concentration-dependent transduction rate can be ascertained in the different investigated cell populations. The highest transduction rates are achieved in association with the glial restricted neural precursor cells. Following infection of the neural differentiated embryonic stem cells similarly a tendency towards a preferred transduction of glial differentiated cells is observed. The use of adult stem cells also results in transduction rates up to 85%. In further investigations glial restricted neural precursor cells, after having been infected with the adenoviral vector are injected into the striatum of adult rats. This procedure was performed in order to test the possibility of an ex-vivo gene therapeutical use in association with glial cells as possible carrier cells. The results show that the transduced cells are integrated into the receiving tissue and during the investigation period of six weeks only migrate into the surrounding tissue to a restricted extent. At a closer look the injected cells show normal characteristics of differentiation along the glial cell linage as it has been described for astrocytes in-vivo. The second part of this study focuses on the direct vector injection into the central nervous system. Therefore the 3rd generation adenoviral vector is injected into different brain areas. Using immunohistochemical investigations using antibodies against NeuN and GFAP it can be shown that after injection into the striatum analogous to the situation in-vitro a preferential transduction of astrocytes is achieved. Within this brain compartment a diffusion of the vector up to a distance of 1000 µm from the injection site can be determined. Following injection of vector particles into the corpus callosum, a vector distribution within the entire fibre tract of the white substance including the contralateral hemisphere is observed. With these results it can be shown that the allocation of the vector is dependent on the structure and features of the receiving tissue. Injecting the adenoviral vector into the cerebrospinal fluid results in transgene expression (GFP, LacZ and SEAP) in the ependymal cells surrounding the entire ventricle. These results suggest that the vector distributes within the cerebrospinal fluid and therefore would result in a generalized transduction of ependymal cells. After installing a permanent catheter in the lateral ventricle cerebrospinal fluid may be removed in a continuous manner in order to analyse/evaluate the marker enzyme SEAP. Using a luminometric screening procedure the enzyme SEAP can be demonstrated up to a period of 42 days following vector injection into the cerebrospinal fluid. Summarizing the results, the use of an adenoviral vector at least under experimental circumstances might be a pioneer therapeutical option for diseases of the central nervous system.

Resistance can be overcome by modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. We constructed an adenoviral vector with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter (designated Ad/TRAIL-F/RGD), and evaluated its antitumor activity in vitro and in vivo.The induction of apoptosis by the new vector Ad/TRAIL-F/RGD was evaluated in human carcinoma cells derived from hepatocellular carcinoma (Hep G2, Hep 3b), pancreatic carcinoma (Panc-1, Capan-1), and colon carcinoma (LOVO, SW 620) . Cell viability was measured by the XTT assay and GFP expression and apoptosis induction by fluorescence-activated cell sorting (FACS) and Western blot. In vivo experiments were performed in an orthotopic pancreas tumor model in nu/nu nude mice.Treatment with Ad/TRAIL-F/RGD and Ad/gTRAIL resulted in significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in all examined human carcinoma cell lines. In addition, mice treated with Ad/TRAIL-F/RGD showed a significantly decreased tumor growth than both control groups.Our results suggest that Ad/TRAIL-F/RGD may become a potent therapeutic agent for the treatment of different human solid carcinomas.

Surface coating with mitogenic or morphogenic proteins can improve the healing of bone adjacent to implants and increase the bone-implant interface. Clinical surveys have shown liposome-mediated gene transfer to be a promising and safe new therapeutic method. The aim of our study was to evaluate an experimental model of new approaches for topical treatment of the implant surface and of periimplant defects by using DNA liposomes encoding for BMP-2 (bone morphogenetic protein).A total of 27 implants (3.5 x 14 mm) were placed in critically sized defects of the frontal skull bone of adult pigs (n=3). The bottom of the implant was placed in the base of the defect which guaranteed primary stability, whereas the superior part of the implant (10 mm) represented an implant in a defect area. Liposomes containing DNA encoding for BMP-2 and GFP (green fluorescence protein) were used. In a first trial GFP-DNA liposomes on a collagen matrix were directly applied to the periimplant defect. In a second stage, the surface of the implants was encoded with BMP-2 DNA liposomes. Subsequently, these implants were inserted in the manner described. The resulting bone samples were prepared for immunohistochemical staining. Staining for GFP was performed in the area of the defect and for BMP-2 on the bone-implant interface.Immunohistochemical staining on day 3 postoperatively revealed an increased GFP expression in the periimplant defect. Therefore, the effectiveness of the liposomal vector was verified for the chosen animal model. On the surface of the implants encoded with BMP-2 DNA liposomes an increased BMP-2 expression was found. Thus, the liposomal vector system was validated also for BMP-2 DNA transfer in the chosen animal model. Further, the established system allows a sustainable and delayed release of BMP-2 in the area of the bone-implant interface.As a result of the study we were able to collect data concerning the influence of implant surface conditioning on the bone-implant interface and on therapeutically relevant options for the treatment of periimplant defects. These approaches are currently being evaluated in a long-term study.

A specific antigenic ligand and a costimulatory signal are required for optimal T cell activation. We constructed and tested recombinant Vaccinia viruses (recVV) expressing hB7-1 or hB7-2 as a gene expression vector system for delivery of costimulatory function in vitro. Upon infection with replication incompetent and non-cytopathic recVV B7, all human tumour cell lines tested expressed the recombinant molecules. Cell lines expressing recombinant B7 molecules provided effective costimulation for proliferation of resting CD4+ T helper cells in the presence of suboptimal PMA concentrations. The costimulatory effect could be blocked with soluble CTLA-4 proteins. RecVV B7-1 infected EBV transformed B-lymphocytes overexpressed the costimulatory molecules resulting in enhanced costimulation. The capacity of these cells to stimulate autologous CD4+ memory cells of VV immunocompetent donors was not impared by the recVV infection indicating an intact capacity for processing and presenting antigenic proteins in the context with MHC Class II molecules remained. RecVV encoding human B7 molecules therefore appear to be promising experimental and clinical tools to enhance immune responses.

Gastroenterological oncology requires new strategies with new mechanisms of action and without cross-resistance to currently available treatment regimes. Virotherapy which is based on the employment of replication-competent viral vectors exhibiting strong oncolytic properties is such an approach currently under preclinical/clinical investigation. Techniques of molecular virology are required for further improvement of current vectors, particularly with respect to oncolytic activity, tumour selectivity, tumour spread capacity, and safety.

Gene therapy is based on the transfer and the expression of therapeutic genes in specific target cells. For the treatment of genetic diseases gene therapeutic approaches aim at replacement of the deficient gene or at the correction of the genetic defect. Malignant diseases and an increasing number of other acquired diseases are additional targets for gene therapeutic strategies. For gene therapy to become a potential future treatment option, safe and therapeutically efficient gene transfer into specific target cells is a central requirement. A variety of nonviral and viral vector systems have been developed. Nonviral vectors transfer genes are far less efficient than viral vectors, but they have advantages due to their low immunogenicity and their large size capacity for therapeutic DNA. To improve the function of nonviral vectors, the addition of viral functions such as receptor mediated uptake, enhanced endosomal release and nuclear translocation of DNA may finally lead to the development of an artificial virus. In contrast, natural viruses are already highly developed structures for the transfer of nucleic acids. Recombinant viruses can be used for efficient gene transfer. Retroviruses, adeno-associated viruses and lentiviruses are suitable for gene therapeutic approaches which are based on the permanent expression of therapeutic genes such as the correction of enzyme deficiencies or the manipulation of hematopoetic stem cells, as they are able to integrate nucleic acids into the cellular genome. In contrast, adenoviral vectors result in highly efficient, but transient gene expression and are therefore especially useful for the treatment of malignant tumors. Novel developments of viral vectors mainly aim at the reduction of immunogenicity, increase of capacity for therapeutic genes and at improved vector production. Viruses which replicate selectively in tumor cells leading to tumor cell lysis represent a novel generation of viral vectors, which can further be improved by the addition of therapeutic genes resulting in enhanced tumor toxicity.

Gene therapy has gained increasing attention and a number of ongoing clinical trials have been initiated. This article provides current perspectives and limitations on gene therapy in ophthalmology. Since a number of comprehensive studies on gene therapy for retinal diseases already exist, we focus attention to the treatment of anterior segment disorders of the eye.We undertook a reference search (DIMDI, PubMed) of articles published between (1989-2000) using the key words cornea, conjunctiva, eye, gene therapy, and keratoplasty. The search was restricted to publications in English, French and German. In addition, we incorporated some results of our recent experiments on cytokine gene transfer to the cornea.Attention to gene therapy in ophthalmology is currently focused on retina and choroidea (40 articles) however, an increasing number of publications includes the cornea (12 articles). The majority of these contributions deals with improvements in the design of gene therapy vectors in particular for targeted application.Gene therapy to the cornea may offer interesting new venues. Currently, insufficient gene transfer technologies and safety concerns prevent the broad application in humans. However, a broad spectrum of applications can be supposed.

A variety of reasons can afflict wound healing. Current research is focussed on the acceleration of wound healing by stimulating molecular processes. Gene therapy may offer completely new ways to treat chronic wounds. Possible advantages of gene therapeutic modulation of wound healing might be a long term efficiency, systemic or local regulation of gene expression and low side-effects. Current goals comprise the improvement of transfection efficiency and specificity. In vivo applications are therefore focussed on optimized inducible or even cell-type specific promotors, as well as on improved local application techniques. Studies from our laboratory demonstrate the possibility to combine modern cell culture techniques with different types of gene transfer. This enables the simultaneous grafting of manipulated cells to the wound with the continuous delivery of specific proteins of interest. Experimentally, this lead to accelerated closure of partial and full thickness animal wounds. Clinically, gene therapy for the treatment of chronic wounds seems to be a realistic goal within the next years and might be applicable for a variety of novel indications.

Background: Research into the molecular and genetic basis of disease is continually expanding. How does the increasing knowledge about the genetic basis of eye diseases contribute to the development of new therapeutic strategies? Materials and methods: Gene therapy, here defined as the introduction of genetic material into human cells, offers great opportunities. Gene transfer strategies can be used for gene replacement in recessive disease, gene inactivation in dominant disease, expression of “rescue factors” and apoptosis modulators in degenerative disease, “suicide genes” for example in proliferative diseases and expression of immunmodulatory factors in immunological disorders. Viral vector systems have been developed to introduce the gene of interest into the target cell. Results: Most of the published strategies include the use of vectors for gene transfer. Adenovirus (AV), adenoassociated virus (AAV), encapsulated adenovirus mini-chromosomes (EAMs), herpes simplex virus (HSV) and lentiviruses are the most frequently used viral vector systems to date. Their advantages and disadvantages, the in vivo models used for gene transfer in retinal degeneration, and the results obtained to date by different research groups in the field will be reviewed. Conclusions: Gene transfer into ocular tissues has been demonstrated with growing functional success and may develop into a new therapeutic tool for clinical ophthalmology.

The involvement of cytokines in degeneration and inflammation of human tissue is well established. Interleukin-1 (IL-1) is a major agent in the pathophysiology of periarticular bone resorption in rheumatoid arthritis and in osteoporosis. Because the use of recombinant cytokines and growth factors is limited due to their short half lives, techniques are needed to get a permanent release of these therapeutic proteins. The rational of this study was to show that retroviral transduction of human osteoblastic cells is possible in vitro using the marker gene LacZ and the potentially therapeutic gene encoding for human interleukin-1 receptor antagonist protein (IL-1Ra). Different transduction techniques were combined to improve the rate of transduction in vitro.Osteoblastic cells were isolated from human spongious bone and cultured in vitro. The beta-galactosidase (LacZ) gene and the cDNA of IL-1Ra were introduced into the isolated cells by retrovirus mediated gene transfer. LacZ activity was determined by Xgal staining, IL-1Ra was measured quantitatively by ELISA.The transfer of retroviral IL-1Ra led to IL-1Ra expression of 8614 to 10,089 pg IRAP/50,000 cells/48 h. By combining different techniques to improve transduction, the X-gal staining established a rate of transduction of 60%.Our results demonstrate that retroviral transduction of human osteobalstic cells is possible in vitro, and leads to high levels of the synthesized transgene product. The rate of retroviral transduction can be accelerated in vitro.

Based upon our increasing knowledge of mechanisms underlying tissue destruction in RA patients, new therapeutic principles have been developed, aimed at blocking proinflammatory cytokines or using antiinflammatory cytokines. Both principles, however, have proven to be very effective. In addition, the availability of the methodology to transduce cells with genes has initiated first experiments in animal models to test whether gene therapy for arthritis is suitable, followed by a first, very carefully formulated protocol for human RA. Gene therapy for RA has to still be considered as an experimental form of therapy in a very early stage, not allowing even now any serious treatment offer to RA patients. Several problems, such as the question of a suitable vector system have not yet been solved. With more experiments this therapeutic principle might become available and might prove effective even in a disease with a systemic character like RA in the coming years.

Growth factors have the potential to enhance native repair responses in ligamentous and meniscal lesions. However, methods for applying these cytokines to sites of injury for extended periods are lacking. We suggest that local transfer of genes which encode the relevant healing factors merits investigation as a potential solution to this problem. In the present study different viral vectors and liposomes were evaluated for their ability to deliver genes to cells of ligamentous and meniscal origin. The anterior (ACL) and posterior (PCL) cruciate ligaments and the medial collateral ligament (MCL), semitendinosus tendon, patellar tendon, and menisci were removed from New Zealand white rabbits Cells grown from these tissues were then investigated for their susceptibility to genetic alteration by these vectors. Based upon the ability of these vectors to convert cells in culture to a lacZ(+) phenotype, adenovirus was the most effective vector in short-term experiments. However, expression was transient. Although retrovirus gave lower initial transduction efficiencies, the percentage of transduced cells was increased by the use of the selectable marker gene neo(r). Cells infected with adeno-associated virus containing the neo(r)-gene were also selected in this way. Liposomes showed low efficiency of gene transfer and expression. In an in vivo marker study we injected adenovirus into the rabbit patellar tendon. Transduced cells were observed mainly in the subsynovial layer at a declining frequency over a 6-week period. The allogeneic transplantation of retrovirally-transduced fibroblasts into the patellar tendon resulted in a greater number of transduced cells. Although the number of lacZ(+) cells declined with time, positive cells were still present 6 weeks after transplantation. Furthermore, the transplanted cells, unlike cells transduced in situ with adenovirus, migrated from the injection site and integrated into the crimp of the tendon.

Growth factors have the potential to enhance native repair responses in ligamentous and meniscal lesions. However, methods for applying these cytokines to sites of injury for extended periods are lacking. We suggest that local transfer of genes that encode the relevant healing factors merits investigation as a potential solution to this problem. In the present study, different viral vectors and liposomes are evaluated for their ability to deliver genes to cells of ligamentous and meniscal origin. The ACL, PCL, MCL, semitendinosus tendon, patellar tendon, and menisci were harvested from New Zealand white rabbits. Cells grown from these tissues were then investigated for their susceptibility to genetic alteration by these vectors. Based upon the ability of these vectors to convert cells in culture to a lacZ(+) phenotype, adenovirus was the most effective vector in short-term experiments. However, expression was transient. Although retrovirus gave lower initial transduction efficiencies, the percentage of transduced cells could be increased by the use of the selectable marker gene neo(r). Cells infected with adeno-associated virus containing the neor-gene could also be selected in this way. Liposomes showed low efficiency of gene transfer and expression. In an in vivo marker study, we injected adenovirus into the rabbit patellar tendon. Transduced cells could be observed preferentially in the subsynovial layer at a declining frequency over a 6-week period. The allogeneous transplantation of retrovirally transduced fibroblasts into the patellar tendon resulted in a greater number of transduced cells. Although the number of lacZ(+) cells declined with time, positive cells were still present 6 weeks after transplantation. Furthermore, the transplanted cells, unlike cells transduced in situ with adenovirus, migrated from the injection site and integrated into the crimp of the tendon.

The many different oncological concepts of gene therapy can be classified into three major groups. These include immunopotentiation, compensation of oncogene and tumor suppressor gene alterations, and suicide gene strategies. Gene marker studies and the transfer of drug resistance genes to normal cells have also shown to be of some importance in oncology. Although there is no clear demonstration of the efficacy of gene therapy so far, it will most likely become part of the therapeutic armamentarium of oncologists within the next two to three decades.