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Doctoral Thesis
DOI
10.11606/T.87.2012.tde-21092012-102415
Document
Author
Full name
Daniela Bertolini Zanatta
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2012
Supervisor
Committee
Strauss, Bryan Eric (President)
Galante, Pedro Alexandre Favoretto
Janini, Luiz Mario Ramos
Martins, Elizabeth Angelica Leme
Pierulivo, Enrique Mario Boccardo
Title in Portuguese
Sequenciamento de um código de barras como ferramenta para quantificação de alterações na dinâmica de populações celulares transduzidas com vetores lentivirais.
Keywords in Portuguese
Lentivirus
Mutagênese
Populações celulares
Sequenciamento genético
Transferência de genes
Vetores lentivirais
Vetores retrovirais
Abstract in Portuguese
Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional.
Title in English
Sequencing of a barcode as a tool for the quantification of changes in the dynamics of cell populations transduced with lentiviral vectors.
Keywords in English
Genetic sequencing
Lentiviral vectors
Lentivirus
Mobile populations
Mutagenesis
Retroviral vectors
Transfer of genes
Abstract in English
Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called "barcodes". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.
 
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Publishing Date
2012-10-17
 
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