Correction: The state of the art of adeno-associated virus-based vectors in gene therapy

It has come to our attention that, on writing our manuscript (dos Santos Coura and Nardi, 2007), we unintentionally included unquoted passages from the work by Wu et al. (2006), collected during the period when we were doing extensive readings on the subject, and not adequately referenced. We regret this error and offer our sincere apologies

The state of the art of adeno-associated virus-based vectors in gene therapy

The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches

Uso de SIG na avaliação da contaminação por chumbo e cobre no Canal do Cunha e no oeste da Baía de Guanabara , Rio de Janeiro, RJ.

8 p. il.As well as other subbasins of Guanabara Bay, the Canal do Cunha's sub - basin, located in the city of Rio de Janeiro, suffered a severe environmental degradation as from the 1950s due to the accelerated urban - industrial development. Having a fluvial dynamics changed - by deforestation, dredging, occupying valleys and corrections - the channel is responsible for the flow of effluents and sediments that contribute to environmental degradation and has direct influence on water quality aggravation indices in the western portion of the Guanabara Bay. This study intends to evaluate, through GIS techniques, the spatial distribution of the levels of lead (Pb) and copper (Cu) launched by domestic sewage and industrial effluents using data provided by six points monitoring State Environmental Institute (INEA) . The representation of distributions allowed us to observe the influence of seasonality and the concentration of suspended sediment concentrations of metals. This work integrates the Canal do Cunha's characterization project, developed by the Laboratory of Environmental Impacts and Sustainability (LIMA) Nuclear Engineering Institute (IEN)

Guidelines for the management of neuroendocrine tumours by the Brazilian gastrointestinal tumour group

Neuroendocrine tumours are a heterogeneous group of diseases with a significant variety of diagnostic tests and treatment modalities. Guidelines were developed by North American and European groups to recommend their best management. However, local particularities and relativisms found worldwide led us to create Brazilian guidelines. Our consensus considered the best feasible strategies in an environment involving more limited resources. We believe that our recommendations may be extended to other countries with similar economic standards.Univ Sao Paulo, Inst Canc Estado Sao Paulo, BR-01246000 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Radiol & Oncol, BR-01246903 Sao Paulo, BrazilHosp Sirio Libanes, BR-01308050 Sao Paulo, BrazilHosp Moinhos de Vento Porto Alegre, BR-90035000 Porto Alegre, RS, BrazilOncoctr, BR-30360680 Belo Horizonte, MG, BrazilUniv Fed Rio Grande do Sul, Dept Cirurgia, BR-90040060 Porto Alegre, RS, BrazilHosp Clin Porto Alegre, BR-90035903 Porto Alegre, RS, BrazilUniv Fed Ceara, Fac Med, Dept Fisiol & Farmacol, BR-60020180 Fortaleza, Ceara, BrazilHosp Univ Walter Cantidio, BR-60430370 Fortaleza, Ceara, BrazilInst Nacl Canc, BR-20230240 Rio De Janeiro, BrazilUniv Sao Paulo, Fac Med, Disciplina Endocrinol & Metabol, BR-01246903 Sao Paulo, BrazilAC Camargo Canc Ctr, Dept Surg, BR-01509010 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Gastroenterol, Sao Paulo, BrazilUniv Fed Ciencias Saude Porto Alegre, BR-90050170 Porto Alegre, RS, BrazilHosp Albert Einstein, BR-05652900 Sao Paulo, BrazilHosp Base, Fac Med Sao Jose do Rio Preto, BR-15090000 Sao Paulo, BrazilSanta Casa Sao Jose do Rio Preto, BR-15025500 Sao Jose Do Rio Preto, BrazilPontificia Univ Catolica Parana, Hosp Erasto Gaertner, BR-81520060 Curitiba, Parana, BrazilUniv Fed Rio Grande do Norte, BR-59300000 Natal, RN, BrazilUniv Sao Paulo, Inst Coracao, BR-05403900 Sao Paulo, BrazilAC Camargo Canc Ctr, Med Oncol, BR-01509010 Sao Paulo, BrazilUniv Fed Sao Paulo, Disciplina Gastroenterol, BR-04021001 Sao Paulo, BrazilHosp Sao Rafael, BR-41253190 Salvador, BA, BrazilHosp Canc Barretos, Dept Cirurgia Aparelho Digest Alto & Hepatobiliop, BR-14784400 Sao Paulo, BrazilUniv Sao Paulo, Fac Med, Dept Patol, BR-01246903 Sao Paulo, BrazilClin AMO, BR-1950640 Salvador, BA, BrazilHosp Sao Jose, BR-01323001 Sao Paulo, BrazilUniv Nove de Julho, BR-02111030 Sao Paulo, BrazilUniv Fed Sao Paulo, Disciplina Gastroenterol, BR-04021001 Sao Paulo, BrazilWeb of Scienc

Estabelecimento de um protocolo pré-clínico de terapia gênica, baseada em vetores adeno-associados, direcionado para o tratamento das manifestações do sistema nervoso central em um modelo murino de mucopolissacaridose tipo I

As mucopolissacaridoses (MPS) constituem um subgrupo de doenças lisossomais de depósito, causadas pela deficiência em uma enzima lisossomal envolvida no catabolismo de glicosaminoglicanos. A ausência de degradação destas moléculas leva ao seu acúmulo nos lisossomas, conduzindo a um processo de dano ou degeneração celular. A maior parte das MPS apresenta desordens neurológicas. As formas serveras de MPS I [déficite em α-L-iduronidase (IDUA)] caracterizam-se por manifestações neurológicas severas. Atualmente, as abordagens terapêuticas para MPSI resumem-se ao transplante de medula óssea (TMO) e à terapia de reposição enzimática (TRE). O TMO haplo-idêntico ou geno-idêntico pode desacelerar ou mesmo parar o processo patológico, inclusive a neurodegeneração. As taxas de mortalidade e morbidade, entretanto, são elevadas. A TRE não é eficaz contra o comprometimento do sistema nervoso central (SNC), já que a enzima não pode atravessar a barreira hemato-encefálica. As lesões de depósito lisossomal são difusas por todo o SNC. Assim, o objetivo é fornecer a enzima para todo ou quase todo o encéfalo. Isto pode ser alcançado através de terapia gênica in situ, injetando vetores virais diretamente no parênquima cerebral, através de método estereotáxico, criando assim uma fonte intracerebral da enzima. Os vetores derivados de vírus adeno-associados (AAV) são capazes de transduzir os neurônios e as células gliais do SNC. Neste trabalho, nós apresentamos um protocolo pré-clínico de terapia gênica com vetor AAV2.5-IDUA injetado via intra-estriatal, bilateralmente, em camundongos com 6 semanas de vida. Nossos resultados indicam que a transferência gênica induz uma expressão persistente de IDUA nas células e leva à reversão das lesões de depósito lisossomal na quase-totalidade do SNC. Assim, nós demonstramos a eficácia e a viabilidade desta abordagem terapêutica na correção e/ou prevenção das manifestações do SNC em um modelo murino de MPSI. Assim, sugerimos que a associação deste protocolo de terapia gênica com uma terapia sistêmica (ERT ou TMO), em idade precoce, poderia ser uma abordagem terapêutica adequada e eficaz para o tratamento global da MPSI.Mucopolysaccharidosis (MPS) is a type of lysosomal storage disease, caused by a deficiency in lysosomal enzymes involved in glycosaminoglycan catabolism. The absence of degradation of these molecules leads to cellular damage or degeneration process. The majority of MPSs shows neurological disorders. Severe forms of MPSI (defficiency in α-L-iduronidase (IDUA)) are characterized by severe neurological manifestations. Currently, therapeutic approaches for MPSI are limited to bone marrow transplantation (BMT) and enzyme replacement therapy (ERT). Haplo-identical or geno-identical BMT is able to slow or stop the pathological process, including neurodegeneration. However, mortality and morbitiy rates are high. The ERT is not efficient to correct or prevent central nervous system (CNS) damage, since the enzyme is not able to cross the blood-brain barrier. Storage lesions could be diffusely found in all CNS. Thus, the aim is the widespread delivery of the enzyme for the whole brain. This can be reached through in situ gene therapy, by injecting viral vectors directly in brain parenchyma, through stereotaxical methods, creating a brain source of the enzyme. AAV vectors are able to transduce neurons and glial cells of the CNS. In the present work, we have established a gene-therapy pre-clinical protocol with AAV2.5-IDUA vector which have been administered bilaterally in the striatum of 6-weeks old mice. Our results indicate that gene transfer leads to persistent IDUA expression in the cells and to the reversion of storage lesions in almost all the CNS. We have demonstrated the effectiveness and viability of this therapeutic approach in the correction or prevention of storage lesions in the MPSI mice brain. Thus, we suggest that the association of this gene therapy protocol with systemic therapy (ERT or BMT), in early age, could be an adequate and efective therapeutic approach to the global treatment of MPSI.Les mucopolysaccharidoses (MPS), un sous-groupe des maladies de surcharge lysosomale, sont causées par le défice d'une enzyme lysosomale intervenant dans le catabolisme des glycosaminoglycanes. L'absence de dégradation de ces molécules entraîne leur accumulation dans les lysosomes conduisant à une souffrance cellulaire. La plupart des MPS s'accompagnent de désordres neurologiques. Les formes sévères de MPS I (défice en α-L-iduronidase (IDUA)) se caractérisent par des manifestations neurologiques sévères. Actuellement, les approches thérapeutiques pour les MPSI s'agitent de la greffe de moelle osseuse (GMO) ou de l'injection d'enzyme purifiée (TRE). La greffe de moelle osseuse haplo-identique et géno-identique peut stopperle processus de la maladie y compris la neurodégénérescence mais les taux de mortalité et de morbidité sont élevés. L'enzymothérapie substitutive n'est pas efficace contre les atteintes du système nerveux central (SNC) car l'enzyme ne traverse pas la barrière hématoencéphalique. Les lésions de surcharge lysosomale étant diffuses dans tout le SNC, l'objectif est d'apporter l'enzyme dans la totalité de l'encéphale. Ceci peut se concevoir grâce à la thérapie génique in situ en injectant des vecteurs viraux directement dans le parenchyme cérébral par une méthode stéréotaxique, créant ainsi une source intracérébrale d'enzyme. Les vecteurs AAV sont capables de transduire les neurones et les cellules gliales du SNC. Dans ce travail nous présentons un protocol prè-clinique de thérapie génétique avec des vecteurs AAV2.5-IDUA injectés dans le striatum des souris agées de 6-semaines. L'injection a été bilaterale. Nos résultats indiquent que le transfert de gène induit une expression persistante de l'IDUA dans les cellules et entraîne une disparition des lésions de surcharge. Nous avons démontré l'efficacité et faisabilité de cette approche therapeutique pour traiter les lesions de surchage dans le cerveau des souris MPSI. Ainsi, nous proposons l'association de ce protocol de thérapie génique avec une thérapie systemique (TRE ou GMO), dans un age precoce, comme une approche thérapeutique adéquate et efficace pour le traitement global du MPS I

Prevalência e valor prognóstico de marcadores moleculares em tumores colorretais esporádicos

O câncer colorretal (CCR) é o terceiro tumor em freqüência e o segundo em mortalidade nos países desenvolvidos. No Brasil, está entre as seis neoplasias malignas mais encontradas e é a terceira em mortalidade. Dentre todos os casos de CCR, cerca de 85% são esporádicos. A avaliação de prognóstico se baseia atualmente apenas em parâmetros clínico-patológicos e morfológicos, uma vez que o valor de marcadores moleculares para o prognóstico ainda precisa ser melhor esclarecido. Mutações em genes de reparo de malpareamento de DNA (MMR) estão associadas principalmente com CCR hereditário, em especial na Sìndrome do Câncer Colorretal Hereditário Não-Polipomatoso (HNPCC), podendo também ser observadas em alguns casos de CCR esporádico. Essas mutações em genes MMR (hMSH2 e hMLH1, principalmente) resultam em falha na correção dos erros de replicação, que como conseqüência geram instabilidade de microssatélites (IMS). A freqüência de IMS é maior que 90% no HNPCC e cerca de 15% em CCRs esporádicos. Tumores com IMS tendem a apresentar melhor prognóstico. Mutações no oncogene K-ras estão presentes em alta freqüência em CCRs e desempenham um papel importante na patogênese da doença, tendo também sido associadas ao prognóstico. Este trabalho avalia a prevalência e o valor prognóstico de um painel de cinco marcadores para análise de instabilidade de microssatélites e de mutações no códon 12 do gene K-ras em tumores de reto. Além disso, também faz uma análise populacional descritiva do perfil dos microssatélites BAT-25 e BAT-26 em duas amostras do Rio Grande do Sul: uma de indivíduos com ascendência africana e outra de doadores de sangue do Hospital de Clínicas de Porto Alegre (HCPA). A freqüência de IMS-H nos tumores de reto foi de 7.3%, e a de IMS-L foi de 51.2%. A freqüência de mutação no códon 12 do gene K-ras foi de 26.8%. Estes resultados estão de acordo com o descrito na literatura para tumores colorretais esporádicos. Não houve correlação significativa entre os achados moleculares, e os dados clínicos e histopatológicos. Mutações no códon 12 do gene K-ras apresentaram associação com o aspecto da cromatina nuclear e com a circularidade do núcleo. Pacientes com tumores com alto grau de instabilidade (IMS-H) mostraram uma melhor sobrevida. Mutações no códon 12 do gene K-ras não apresentaram correlação significativa com o prognóstico. Os locos BAT-25 e BAT-26 são polimórficos na população do Rio Grande do Sul, o que torna necessária a análise comparativa entre tecido normal e tecido tumoral para detecção de IMS. Nossos resultados descrevem o perfil destes dois marcadores moleculares em carcinomas de reto na população do Rio Grande do Sul. Além de sugerirem tendências importantes do seu possível valor prognóstico.Colorectal cancer is the third tumor in frequency and the second in mortality in developed countries. In Brazil, it is one of the six most common cancers and the third in mortality. Prognostic evaluation is currently based only on clinicopathological and morphological parameters, since the prognostic value of molecular markers requires further elucidation. Microsatellite instability (MSI), which is caused by mutations in mismatch repair genes, are found in more than 90% of tumors of HNPCC and in about 15% of sporadic CRC. Mutations in the oncogene K-ras are present in a high frequency in CRCs and play an important role in pathogenesis of the disease. In this study we evaluate the prevalence and prognostic value of mutations in codon 12 of K-ras gene and of a panel of five markers for MSI analysis in sporadic colorectal tumors. Besides we made a descriptive populational analysis of the BAT-25 and BAT-26 microsatellites profile in two samples from Rio Grande do Sul. We found a high-level instability (MSI-H) frequency of 7.3%. Low-level instability and stability were found in 51.2% and 41.5% of patients, respectively. MSI-H showed significant association with better overall survival. We found a frequency of K-ras codon 12 mutations of 26.8%. There was not significant association of K-ras mutation with clinicopathological variables. K-ras mutation showed significant association with two morphological features: aspect and roundness. BAT-25 and BAT-26 showed polymorphism in the studied samples, what requires the comparative analysis between normal and tumoral tissue to assess IMS status. Our results have described the profile of these two molecular markers in rectal carcinomas in our population. Furthermore, they suggest important tendencies of their possible prognostic value that need to be well elucidated in a larger sample

Estabelecimento de um protocolo pré-clínico de terapia gênica, baseada em vetores adeno-associados, direcionado para o tratamento das manifestações do sistema nervoso central em um modelo murino de mucopolissacaridose tipo I

As mucopolissacaridoses (MPS) constituem um subgrupo de doenças lisossomais de depósito, causadas pela deficiência em uma enzima lisossomal envolvida no catabolismo de glicosaminoglicanos. A ausência de degradação destas moléculas leva ao seu acúmulo nos lisossomas, conduzindo a um processo de dano ou degeneração celular. A maior parte das MPS apresenta desordens neurológicas. As formas serveras de MPS I [déficite em α-L-iduronidase (IDUA)] caracterizam-se por manifestações neurológicas severas. Atualmente, as abordagens terapêuticas para MPSI resumem-se ao transplante de medula óssea (TMO) e à terapia de reposição enzimática (TRE). O TMO haplo-idêntico ou geno-idêntico pode desacelerar ou mesmo parar o processo patológico, inclusive a neurodegeneração. As taxas de mortalidade e morbidade, entretanto, são elevadas. A TRE não é eficaz contra o comprometimento do sistema nervoso central (SNC), já que a enzima não pode atravessar a barreira hemato-encefálica. As lesões de depósito lisossomal são difusas por todo o SNC. Assim, o objetivo é fornecer a enzima para todo ou quase todo o encéfalo. Isto pode ser alcançado através de terapia gênica in situ, injetando vetores virais diretamente no parênquima cerebral, através de método estereotáxico, criando assim uma fonte intracerebral da enzima. Os vetores derivados de vírus adeno-associados (AAV) são capazes de transduzir os neurônios e as células gliais do SNC. Neste trabalho, nós apresentamos um protocolo pré-clínico de terapia gênica com vetor AAV2.5-IDUA injetado via intra-estriatal, bilateralmente, em camundongos com 6 semanas de vida. Nossos resultados indicam que a transferência gênica induz uma expressão persistente de IDUA nas células e leva à reversão das lesões de depósito lisossomal na quase-totalidade do SNC. Assim, nós demonstramos a eficácia e a viabilidade desta abordagem terapêutica na correção e/ou prevenção das manifestações do SNC em um modelo murino de MPSI. Assim, sugerimos que a associação deste protocolo de terapia gênica com uma terapia sistêmica (ERT ou TMO), em idade precoce, poderia ser uma abordagem terapêutica adequada e eficaz para o tratamento global da MPSI.Mucopolysaccharidosis (MPS) is a type of lysosomal storage disease, caused by a deficiency in lysosomal enzymes involved in glycosaminoglycan catabolism. The absence of degradation of these molecules leads to cellular damage or degeneration process. The majority of MPSs shows neurological disorders. Severe forms of MPSI (defficiency in α-L-iduronidase (IDUA)) are characterized by severe neurological manifestations. Currently, therapeutic approaches for MPSI are limited to bone marrow transplantation (BMT) and enzyme replacement therapy (ERT). Haplo-identical or geno-identical BMT is able to slow or stop the pathological process, including neurodegeneration. However, mortality and morbitiy rates are high. The ERT is not efficient to correct or prevent central nervous system (CNS) damage, since the enzyme is not able to cross the blood-brain barrier. Storage lesions could be diffusely found in all CNS. Thus, the aim is the widespread delivery of the enzyme for the whole brain. This can be reached through in situ gene therapy, by injecting viral vectors directly in brain parenchyma, through stereotaxical methods, creating a brain source of the enzyme. AAV vectors are able to transduce neurons and glial cells of the CNS. In the present work, we have established a gene-therapy pre-clinical protocol with AAV2.5-IDUA vector which have been administered bilaterally in the striatum of 6-weeks old mice. Our results indicate that gene transfer leads to persistent IDUA expression in the cells and to the reversion of storage lesions in almost all the CNS. We have demonstrated the effectiveness and viability of this therapeutic approach in the correction or prevention of storage lesions in the MPSI mice brain. Thus, we suggest that the association of this gene therapy protocol with systemic therapy (ERT or BMT), in early age, could be an adequate and efective therapeutic approach to the global treatment of MPSI.Les mucopolysaccharidoses (MPS), un sous-groupe des maladies de surcharge lysosomale, sont causées par le défice d'une enzyme lysosomale intervenant dans le catabolisme des glycosaminoglycanes. L'absence de dégradation de ces molécules entraîne leur accumulation dans les lysosomes conduisant à une souffrance cellulaire. La plupart des MPS s'accompagnent de désordres neurologiques. Les formes sévères de MPS I (défice en α-L-iduronidase (IDUA)) se caractérisent par des manifestations neurologiques sévères. Actuellement, les approches thérapeutiques pour les MPSI s'agitent de la greffe de moelle osseuse (GMO) ou de l'injection d'enzyme purifiée (TRE). La greffe de moelle osseuse haplo-identique et géno-identique peut stopperle processus de la maladie y compris la neurodégénérescence mais les taux de mortalité et de morbidité sont élevés. L'enzymothérapie substitutive n'est pas efficace contre les atteintes du système nerveux central (SNC) car l'enzyme ne traverse pas la barrière hématoencéphalique. Les lésions de surcharge lysosomale étant diffuses dans tout le SNC, l'objectif est d'apporter l'enzyme dans la totalité de l'encéphale. Ceci peut se concevoir grâce à la thérapie génique in situ en injectant des vecteurs viraux directement dans le parenchyme cérébral par une méthode stéréotaxique, créant ainsi une source intracérébrale d'enzyme. Les vecteurs AAV sont capables de transduire les neurones et les cellules gliales du SNC. Dans ce travail nous présentons un protocol prè-clinique de thérapie génétique avec des vecteurs AAV2.5-IDUA injectés dans le striatum des souris agées de 6-semaines. L'injection a été bilaterale. Nos résultats indiquent que le transfert de gène induit une expression persistante de l'IDUA dans les cellules et entraîne une disparition des lésions de surcharge. Nous avons démontré l'efficacité et faisabilité de cette approche therapeutique pour traiter les lesions de surchage dans le cerveau des souris MPSI. Ainsi, nous proposons l'association de ce protocol de thérapie génique avec une thérapie systemique (TRE ou GMO), dans un age precoce, comme une approche thérapeutique adéquate et efficace pour le traitement global du MPS I

A role for adeno-associated viral vectors in gene therapy

Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV) are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors