The pharmacologic treatment of painful neuropathy continues to pose problems and challenges in clinical practice. This is largely due to a limited understanding of the underlying etiologies of such neuropathic pain and insufficient knowledge of the optimal effective doses that would cause only minimal systemic side effects. The use of molecular methods, such as gene deletion from knockout mice and the development of cellular mini-pumps for the delivery of biologic antinociceptive molecules have led to a better understanding of the underlying mechanisms involved in the induction of intractable neuropathic pain. It is now known that the initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, and the loss or down-regulation of the endogenous inhibitory spinal system and central sensitization, both of which cause such pain. Currently, there are novel approaches that use genetic therapy in the management of neuropathic pain. Two such approaches which have been determined to be safe are proposed to be investigated in this study using animal models of pain. The first approach involves cell-mediated delivery of antinociceptive molecules to the cerebrospinal fluid using cultivated spinal progenitor cells transplanted into the subarachnoid space. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. 1x106 spinal progenitor cells (SPCs) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days post surgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. Glutamic acid decarboxylase (GAD) immunoreactivity was revealed for 65% of the cultivated SPCs in our study. We also determined that transplanted cells could survive more than four weeks post intrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain. The second approach involves the use of a newly developed intrathecal electroporation probe in the delivery of antinociceptive peptides to reduce expression of endogenous nociceptive molecules in the spinal cord. To investigate the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection, pE-GFP C1 vector was used to achieve the goal. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of, followed by 5 electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and western blotting. At day 1, 3 or 7 following electroporation a clear green fluorescence protein (GFP) expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and western blotting. The expression of GFP protein was peaked between 3-7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery. Furthermore, the optimal pulse characteristics (voltage, pulse duration, number of shocks) were investigated for in vivo electroporation for gene transfer into the spinal cord. The expression of pre-opiomelanocortin (POMC) gene from electroporated plasmid DNA was then evaluated in this study using RT-PCR and western blot. We conclude that the optimal conditions for electroporation are a pulse voltage of 200 V, 75-ms duration, 925-ms interval, for five iterations. Also, electroporation treatment for neuropathic pain was attempted for CCI rats using plasmid DNA that expresses the POMC gene. Intrathecal administrations of the POMC plasmid elevated spinal beta-endorphin levels, as manifested in significantly elevated pain threshold for the CCI limbs. We also tested whether intrathecal electric stimulation would reduce the tolerance of chronic morphine usage and the severity of precipitated morphine withdrawal symptoms. Rats received intrathecal electrode catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/hr) or saline for seven days. Intrathecal electric stimulations (0, 20V, 200V) were performed once daily during the same period. Daily tail flick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal electric stimulation on antinociception and tolerance of morphine. Naloxone withdrawal (2mg/kg) was performed to assess morphine dependence, and changes in spinal neurotransmitters were monitored by microdialysis. The antinoceptive effect of intrathecal morphine was increased by 200V electric stimulation. The magnitude of tolerance was decreased in the rats receiving 2 nmol/hr infusion with daily intrathecal electric stimulation. The severity of naloxone-induced withdrawal symptom was lower in the rats receiving 200V stimulation. Intrathecal stimulation thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion. Increases in spinal glycine release may be the underlying mechanisms. The promise is that, both approaches attenuate or reverse persistent nociceptive states; they could be exploited for use in the development of gene therapy for the management of pain.

Die Gentherapie hat in den letzten Jahren wesentliche Entwicklungen im Vektordesign, der kontrollierte Expression sowie der Sicherheit ihrer Anwendung durchgemacht. Die Erkenntnis, dass die Tumorgentherapie allein nur in begrenztem Maße zum erhofften therapeutischen Benefit für den Patienten beitragen kann, führte zum Konzept der lokalen Gentherapie als Teil anderer, etablierter Tumortherapien. In diesem Zusammenhang wird die Gentherapie als eine moderne Option zur Steigerung der Effizienz von Chemotherapie, Strahlentherapie oder Hyperthermie verstanden. Zum Erreichen dieses Zieles ist die Etablierung Therapie-regulierbarer Vektorsysteme von besonderer Attraktivität. Im Rahmen der Strategie des lokalen Transfers therapeutischer Gene bietet inzwischen die Anwendung nicht-viraler Transfersysteme, wie z.B. in vivo-Elektrotransfer, Gene-Gun oder Jet-Injection eine klinisch applikable Technologie. Die Etablierung einer effizienten, auf der Jet-Injection basierenden nicht-viralen Transfertechnologie und die Analyse ihres Potentials für eine klinische Anwendung in einem multimodalen Therapiekonzept war ein wesentliches Ziel der Arbeit. Es wurde gezeigt, dass die Jet-Injection in tierexperimentellen Tumormodellen zur effizienten Expression der Transgene führt, dass sowohl Eindringtiefen, als auch Verteilung der Jet-Injection optimal für einen effizienten Gentransfer sind und die Höhe der Genexpression mit etablierten Gentransfer-Technologien, wie z.B. der in vivo-Lipofektion, vergleichbar ist. Basierend auf der Strategie des Einsatzes der Gentherapie in Kombination mit anderen Therapien, bestand ein weiteres Ziel der Arbeit in der Charakterisierung und Anwendung konditioneller Vektorsysteme, mit denen die Expression therapeutischer Gene durch Chemotherapie oder Hyperthermie kontrollierbar ist. Derartige Vektoren, in denen der humane Multidrug Resistenzgen 1- (mdr1) Promotor genutzt wurde, exprimierten vor allem Zytokingene, die die therapeutische Effizienz von Zytostatika oder der Hyperthermie verbessern. Die Zytostatika-und auch Hitze-Induzierbarkeit der mdr1-Promotor gesteuerten Genexpression konnte in verschiedenen Tumormodellen in vitro und in vivo erfolgreich demonstriert werden Diese Untersuchungen zeigten, dass eine Zytostatika-induzierte Gentherapie zu einer besseren Tumortherapie beiträgt. Die Kombinations-Experimente der konditionellen Gentherapie im Kontext einer Hyperthermie geben erste Hinweise, dass auch hier die therapeutische Effektivität in vitro und in vivo gesteigert werden kann. Im Rahmen des Konzepts der kombinierten Gen- und Chemotherapie von Tumoren ist in der Arbeit vor allem auf das chemosensitivierende Potential von Zytokinen gesetzt worden. Besonders für TNF-a, IL-2 sowie IFN-g konnte gezeigt werden, dass diese Zytokine zu einer Modulation der Expression MDR-assoziierter Gene, wie dem mdr1, MVP/LRP und auch MRP1 in der Lage sind und dadurch zur Chemosensitivierung in verschiedenen Tumormodellen führt. Diese Befunde bildeten eine wichtige Rationale für den Einsatz von Zytokingenen im Rahmen der Tumorgentherapie zur Überwindung der MDR. Gentransferexperimente mit TNF-a- und IL-2-exprimierenden Vektoren konnten analog zur Applikation rekombinanter Zytokine die Modulation der Gene mdr1 und MVP/LRP zeigen, die mit der Erhöhung der Sensitivität gegenüber Zytostatika wie Vincristin oder Adriamycin assoziiert ist. / Gene therapy has made great achievements in vector design, controlled gene expression and in safety. The fact, that gene therapy as single therapy has only limited potential for the benefit in the therapy for cancer patients, has led to the concept of local gene therapy as part of other, established therapies. In this context, gene therapy serves as a modern option to improve the efficiency of chemotherapy, radiotherapy or hyperthermia. To achieve this goal, the establishment of therapy-regulatable vectors is of particular attractiveness. For the concept of local transfer of therapeutic genes non-viral transfer systems, such as in vivo electrotransfer, gene gun or jet-injection represent clinically applicable transfer technologies. One major issue of this work was the establishment of an efficient, jet-injection based non-viral transfer technology and the analysis of its potential for clinical application in a concept of multimodal therapy. It has been shown in vivo, that efficient transgene expression can be achieved by jet-injection, that penetration and distribution of the transgene are optimal for an efficient gene transfer and that the level of gene expression is comparable to established gene transfer technologies, sch as in vivo lipofection. Based on the strategy of combination of gene therapy with other therapies, another goal of this work aimed at the characterization and utilization of conditional vector systems, by which expression of therapeutic genes is controllable by chemotherapy or hyperthermia. By such vectors, in which the human multidrug resistance gene 1 (mdr1) promoter was employed, cytokine genes were expressed, which are capable to improve the therapeutic efficacy of cytostatic drugs or of hyperthermia. The drug- and heat-inducibility of mdr1 promoter-driven gene expression has successfully been demonstrated in in vitro and n vivo tumor models. The studies have also shown, that drug-induced gene therapy leads to improved tumor treatment. Combination experiments of conditional gene therapy in the context with hyperthermia give first indication of an increased therapeutic efficiency in vitro and in vivo. For the concept of combined gene- and chemotherapy the chemosensitizing potential of cytokines was exploited. It has been shown, particularly for TNF-a, IL-2 and IFN-g, that these cytokines are capable to modulate the expression of MDR-associated genes, such as mdr1, MVP/LRP or MRP1 leading to chemosensitization in different tumor models. These observations represent an important rationale for the use of cytokine genes in gene therapy for MDR-overcoming. Gene transfer experiments with TNF- or IL-2 expressing vectors showed the modulation of mdr1 or MVP/LRP expression, associated with increased sensitivity towards cytostatic drugs, such as vincristine or adriamycin.

A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento. / The human growth hormone deficiency (GHD) is the most common deficiency related to pituitary hormones. The current therapy is based on daily injections of recombinant human growth hormone (r-hGH). This therapy, however, presents some disadvantages, as the need for frequent injections of r-hGH during a long life time, depending on the deficiency severity and the high cost of this hormone, due to the expensive purification processes. An alternative to the standard treatment should be to avoid these inconveniences via a sustainable hormone release, acting for a long time and providing normal and sustainable levels of insulin-like growth factor-I (IGF-I). A possible alternative is in vivo gene therapy, based on the administration of plasmid DNA in several organs/tissues, followed by electroporation. This methodology is considered very promising and has been the target of many different studies for several types of systemic deficiencies. In the present work several administrations of a plasmid containing the human growth hormone gene were carried out, in the exposed quadriceps or non-exposed tibialis cranialis muscle, followed by electroporation, using immunodeficient dwarf mice 40-80 days old. The goal was to obtain a phenotypic correction of dwarfism, through the evaluation of different growth parameters. The administration of this plasmid, in the tibialis cranialis muscle of 40 day old mice, was able to provide a normalization of mIGF-I levels, when compared to non GHD mice. Furthermore, catch-up increases of longitudinal growth parameters of 36-77% were obtained. Aiming a high efficiency on GH expression, parental plasmids were constructed and from these DNA minicircles were generated with CMV and Ubiquitin C promoter and hGH or mGH cDNA sequences. These DNA minicircles were transfected into HEK 293 cells and were even 2 times moren efficient than conventional plasmids with CMV promoter. This data are very promising and pave the way for more efficient assays utilizing this type of gene therapy protocol for GHD, aiming at a normalization of all growth parameters.

A deficiência de hormônio de crescimento (DGH) é a deficiência mais comum entre os hormônios pituitários. A terapia utilizada atualmente consiste de injeções diárias de hormônio de crescimento humano recombinante (r-hGH), entretanto esta terapia apresenta alguns inconvenientes, como a necessidade de frequentes injeções de r-hGH durante um longo período de vida, dependendo da severidade da deficiência, e o alto custo do hormônio, em razão dos dispendiosos processos de purificação. Uma alternativa ao tratamento padrão seria aquele no qual fossem evitados estes tipos de inconvenientes e o processo de liberação da proteína fosse sustentável, por um longo período e promovesse níveis normais e sustentáveis do fator de crescimento semelhante à insulina I (IGF-I), o principal mediador dos efeitos do GH. Uma alternativa é a terapia gênica in vivo, baseada na administração de DNA plasmidial em diversos órgãos/tecidos, seguida de eletroporação. É considerada uma metodologia bastante promissora e que tem sido alvo de vários estudos para diversos tipos de deficiências sistêmicas. Neste trabalho foram realizadas diversas administrações de um plasmídeo contendo o gene do hormônio de crescimento humano, nos músculos quadríceps exposto ou tibial anterior sem exposição, seguidas de eletroporação, em camundongos anões e imunodeficientes (lit/scid) com 40-80 dias de idade, na tentativa de obter uma correção fenotípica do nanismo, mediante a avaliação de parâmetros de crescimento. A administração deste plasmídeo no músculo tibial anterior, em camundongos com a idade inicial de 40 dias, foi capaz de proporcionar uma normalização dos níveis de mIGF-I, quando comparados aos dos camundongos não-deficientes de GH. Além disso, foram obtidos valores de catch-up dos parâmetros de crescimento longitudinal de 36-77%. Visando uma maior eficiência na expressão de GH, foram construídos plasmídeos parentais, e a partir destes, foram produzidos minicírculos de DNA com os promotores do CMV e Ubiquitina C e com os cDNAs de hGH e mGH. Estes minicírculos de DNA foram transfectados em células HEK 293 e foram até 2 vezes mais eficientes em relação aos plasmídeos convencionais com o promotor do CMV. Estes dados são bastantes promissores e abrem caminho para ensaios mais eficientes, utilizando este tipo de protocolo de terapia gênica para a DGH, visando uma normalização de todos os parâmetros de crescimento. / The human growth hormone deficiency (GHD) is the most common deficiency related to pituitary hormones. The current therapy is based on daily injections of recombinant human growth hormone (r-hGH). This therapy, however, presents some disadvantages, as the need for frequent injections of r-hGH during a long life time, depending on the deficiency severity and the high cost of this hormone, due to the expensive purification processes. An alternative to the standard treatment should be to avoid these inconveniences via a sustainable hormone release, acting for a long time and providing normal and sustainable levels of insulin-like growth factor-I (IGF-I). A possible alternative is in vivo gene therapy, based on the administration of plasmid DNA in several organs/tissues, followed by electroporation. This methodology is considered very promising and has been the target of many different studies for several types of systemic deficiencies. In the present work several administrations of a plasmid containing the human growth hormone gene were carried out, in the exposed quadriceps or non-exposed tibialis cranialis muscle, followed by electroporation, using immunodeficient dwarf mice 40-80 days old. The goal was to obtain a phenotypic correction of dwarfism, through the evaluation of different growth parameters. The administration of this plasmid, in the tibialis cranialis muscle of 40 day old mice, was able to provide a normalization of mIGF-I levels, when compared to non GHD mice. Furthermore, catch-up increases of longitudinal growth parameters of 36-77% were obtained. Aiming a high efficiency on GH expression, parental plasmids were constructed and from these DNA minicircles were generated with CMV and Ubiquitin C promoter and hGH or mGH cDNA sequences. These DNA minicircles were transfected into HEK 293 cells and were even 2 times moren efficient than conventional plasmids with CMV promoter. This data are very promising and pave the way for more efficient assays utilizing this type of gene therapy protocol for GHD, aiming at a normalization of all growth parameters.