Vetores lentivirais são ferramentas fundamentais para modificação celular. Sua utilização ganhou destaque devido à capacidade desses em integrar ao genoma de células que estão ou não em divisão. Grande parte dos vetores desenvolvidos são derivados do genoma do Vírus da Imunodeficiência Humana (HIV-1), portanto, modificações foram necessárias a fim de evitar a formação de Partículas Competentes em Replicação (RCLs) e garantir uma utilização segura. Com as modificações, foram produzidos os vetores lentivirais de terceira geração utilizados atualmente. Esses vetores podem ser usados para expressão constitutiva de genes, produção de proteínas recombinantes, produção de animais transgênicos e terapia gênica. Com isso, torna-se necessário o desenvolvimento de vetores lentivirais para aplicação em pesquisa básica e ensaios clínicos. Dessa forma, o presente estudo teve por objetivo a construção de vetores de expressão lentivirais aplicáveis à: 1- expressão constitutiva de genes de interesse e 2-vetores com promotores específicos para expressão de proteínas em megacariócitos. Esse trabalho descreve a construção desses vetores, sua importância e discute suas possíveis aplicações. As sequências selecionadas para produção dos vetores foram: os genes Runx1C e VkorC1 e os promotores proPF4 e proITGA2b. Todas as sequências encontram-se clonadas em vetor de clonagem e estoques de bactérias com esses vetores congeladas em glicerol foram confeccionados. Para a confecção dos vetores lentivirais, o gene Runx1C foi subclonado no vetor lentiviral base p1054-CIGWS sob controle do promotor forte CMV, enquanto o promotor proITGA2b foi subclonado no vetor base p1054-FVIII, em substituição ao promotor CMV, de forma a controlar a expressão de FVIII. Os dois vetores produzidos apresentam ainda o gene para proteína verde GFP precedida do sítio de ligação do ribossomo IRES, com expressão controlada pelo mesmo promotor interno do vetor. O trabalho possibilitou, portanto, a produção de dois vetores lentivirais bi-cistrônicos: p1054-Runx1C e pL-proITGA2b-FVIII. A construção p1054-Runx1C ainda não foi sequenciada, mas foi confirmada por restrição enzimática e apresenta potencial para aplicação em estudos de diferenciação hematopoética. Já a construção pL-proITGA2b-FVIII foi sequenciada, porém sem confirmação da região de ligação do proITGA2b ao vetor. Reações de PCR e de restrição enzimática confirmaram a ligação e sequenciamento mostrou 67% de similaridade entre a região sequenciada e o promotor ITGA2b depositado no banco de dados. Análise funcional foi realizada através da transfecção desse vetor em células HEK-293T. As células transfectadas apresentaram expressão positiva para GFP e secreção de FVIII no sobrenadante celular, evidenciando que o promotor proITGA2b clonado no vetor encontra-se ativo. Esse vetor apresenta potencial para aplicação em terapia gênica para hemofilias, pois apresenta expressão do fator de coagulação direcionado a megacariócitos e plaquetas, células que estão diretamente relacionadas ao processo de coagulação, representando grandes veículos para secreção desses fatores. Ainda, os dois vetores lentivirais gerados apresentam segurança e eficiência elevadas, pois são vetores de terceira geração auto-inativantes (SIN) e apresentam elementos regulatórios que melhoram o transporte e integração do DNA ao genoma hospedeiro. / Lentiviral vectors are fundamental tools for cell modification that gained prominence due to their ability to integrate the genome of non-dividing cells. Most of developed lentiviral vectors are derived from the genome of Human Immunodeficiency Virus (HIV-1), so modifications were necessary in order to avoid the formation of Competent Replication Particles (RCLs) and ensure safer operations. The modifications led to development of third generation lentiviral vectors currently used. These vectors can be used for constitutive gene expression, production of recombinant protein, production of transgenic animals and gene therapy. It\'s evident the need to develop lentiviral vectors for application in basic research and clinical trials. Thus this study aimed to construct lentiviral expression vectors applicable to: 1- constitutive expression of genes of interest and 2-vectors with specific promoters for expression of proteins in megakaryocytes and platelets. This paper describes the construction of these vectors, their importance and discuss their possible applications. Sequences were selected for production of the vectors: genes Runx1C and VkorC1 and proPF4 and proITGA2b promoters. All four sequences are cloned into cloning vectors and stocks of bacteria with these vectors frozen in glycerol were prepared. Lentiviral vectors were engineered from subcloning the sequence Runx1C into the basic lentiviral vector p1054- CIGWS under control of the strong CMV promoter, and from subcloning proITGA2b promoter into p1054-FVIII basic vector, replacing the CMV promoter in order to control the expression of FVIII. Both vectors exhibit the green fluorescence protein GFP gene preceded by a ribosome binding site IRES under control of vector\'s internal promoter. Therefore, this work resulted in the production of two bi-cistronic lentiviral vectors: p1054-Runx1C and pLproITGA2b-FVIII. The p1054-Runx1C construction has not yet been sequenced, but it was confirmed by digestion and has potential for use in hematopoietic differentiation studies. Though, pL-proITGA2b-FVIII construct was sequenced, but the technique didn\'t allow to confirm the binding region between proITGA2b and the vector. Although PCR reaction and digestion confirmed the construction. Sequence analysis showed 67% similarity between the sequenced region and ITGA2b promoter deposited in the database. Functional analysis was performed by transfection of this vector in HEK-293T cells. The transfected cells showed positive expression of GFP and FVIII secretion in cell supernatant, indicating that the proITGA2b promoter cloned into the vector is active. This vector has potential usage in gene therapy for hemophilia, since it can be used to express coagulation factors in megakaryocytes and platelets and these cells are directly related to the clotting process, representing great vehicles for secretion of these factors. Even more, the two lentiviral vectors generated have higher safety and efficiency, as they are self-inactivating (SIN) third-generation vectors and have regulatory elements that enhance transport and integration of DNA into the host genome.

La glycogénose de type Ia (GSDIa) est une maladie métabolique rare causée par un déficit en glucose-6- phosphatase (G6Pase), menant à l'absence de production endogène de glucose. Cette pathologie est caractérisée par des hypoglycémies sévères, une hépatomégalie et une stéatose hépatique ainsi qu'une néphromégalie. En absence de traitement curatif, la prise en charge de cette maladie repose actuellement sur des mesures diététiques très strictes. Cependant, des complications apparaissent avec l'âge comme le développement de tumeurs hépatiques et la progression de la néphropathie vers l'insuffisance rénale. Afin d'étudier l'évolution de cette pathologie à long terme, nous avons utilisé des modèles murins originaux présentant une invalidation du gène de la sous-unité catalytique de la G6Pase spécifiquement au niveau du foie ou des reins. Dans ce travail, nous avons démontré que la déficience en G6Pase uniquement au niveau des reins est suffisante pour entrainer le développement de la pathologie rénale de la GSDIa. Les souris déficientes en G6Pase hépatique nous ont permis de mettre en évidence les effets délétères d'une consommation modérée de fructose ou de galactose et d'une alimentation riche en lipides, de type « cafétéria », sur la pathologie hépatique de la GSDIa, en particulier sur le développement tumoral. Nous avons également démontré chez ces souris l'efficacité et l'innocuité d'un traitement par thérapie génique ciblant le foie. Le transfert de gène avec un vecteur lentiviral, permettant l'intégration du transgène au génome, semble plus efficace qu'avec un vecteur AAV pour prévenir le développement de la pathologie hépatique de la GSDIa et l'apparition des tumeurs / Glycogen storage disease type Ia (GSDIa) is a rare metabolic disease caused by glucose-6-phosphatase (G6Pase) deficiency, leading to the absence of endogenous glucose production. This pathology is characterized by severe hypoglycemia, hepatomegaly, hepatic steatosis and nephromegaly. In the absence of a curative therapy, the current treatments available consist in strict dietary management. However, various complications occur with aging, such as hepatic tumor development and progressive chronic renal disease leading to renal failure. In order to study the long term pathology development, we used original mouse models, presenting an invalidation of the gene encoding the G6Pase catalytic subunit, specifically in the liver or in the kidneys. In this work, we demonstrated that renal G6Pase deficiency alone is sufficient to induce the development of the GSDIa nephropathy. Mice with liver-specific G6Pase deficiency allowed us to highlight the deleterious effects of high-fat diet, such as « fast-food » diet, as well as moderate consumption of fructose or galactose on the hepatic GSDIa pathology, particularly on tumor development. Furthermore, we demonstrated the efficiency and innocuity of gene therapies targeting the liver in these mice. Gene transfer with a lentiviral vector, allowing transgene integration into the genome, seems to be more efficient than an AAV vector in preventing the development of hepatic GSDIa pathology and tumor formation

Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.

Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...