Browsing by Subject "Lentiviral gene therapy"

Gene therapy trials are becoming more common place with the first approved products having arrived onto markets within the last 5 years. Gene therapy trials have focused on diseases that are monogenic and curable through the reintroduction of autologous, gene-corrected hematopoietic stem cells with promising results. A popular method for gene-correction is through the integration of the wild-type gene into the patient’s genome with a class of retroviruses called lentivirus. APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) is a rare, monogenic autoimmune disease that is caused by a recessive mutation within the AIRE gene coding region. APECED has a high prevalence within the Finnish population (1/25,000) and is theoretically curable through the transplantation of autologous gene-corrected hematopoietic stem cells. Therefore, creating and studying a lentivector carrying the endogenous AIRE promoter and coding region could provide insight and a preliminary foundation on how to begin to develop a viable gene therapy for APECED. The aim of this thesis was to generate and characterise the first lentivector containing the wildtype AIRE gene, specifically the proximal AIRE promoter and the AIRE open reading frame (ORF). The lentivector was constructed and then transfected into a human cell-line, HEK293T, from which qPCR and immunohistochemistry were used to detect for AIRE mRNA and protein, respectively. The presence of significant amounts of AIRE mRNA and protein indicated that the constructed plasmid was transcriptionally functional. It is the first plasmid to have Aire transcription regulated by its endogenous promoter and can provide future insight into gene regulation. As a lentiviral plasmid, it demonstrates the integrating of the AIRE gene into an existing lentiviral system and serves as a first step and a technical proof-of-concept in developing a gene therapeutic cure for APECED.