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Gene therapy for chronic granulomatous disease
Başlık:
Gene therapy for chronic granulomatous disease
Yazar:
Bellantuono, Ilaria, author.
ISBN:
9780438083295
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (254 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Chronic Granulomatous Disease is a rare inherited disorder of phagocytic cells due to a lack of NADPH-oxidase, the enzyme responsible for the killing of infectious pathogens in the phagosome. This complex enzyme consists of various membrane and cytosolic subunits whose lack of any of which result in serious and often life-threatening repetitive infections. The most common forms of CGD, Gp91-phox and p47-phox deficiencies, are in principle amenable to gene therapy through the transfer and expression of the cDNA encoding the missing subunit of the enzyme into haemopoietic stem cells and mature progeny, possibly with a long term correction of the defect. A MoMuLV based retroviral vector containing either the cDNA of gp91-phox or p47-phox was used and the appropriate amphotropic packaging cell lines, L47 and L91 respectively, were derived. The L47 packaging cell line showed a moderate titre which was useful to infect p47-phox-defective EBV-transformed B-cell line and demonstrated the presence of recombinant p47-phox protein of about 10% of the normal B-cell line in the transduced cells. This showed that the L47 virus could infect haemopoietic cells. Given that gp91-phox is missing in 60% of the patients and that this category of patients showed the highest morbidity and mortality the attention was focused mainly on the transfer of the gp91-phox cDNA and this proved to be more difficult than expected. The missing gp91-phox protein is expressed in a tissue specific manner only in late stage of differentiation of myeloid cells. The present study showed that expression of the recombinant protein in the producer cells, which do not normally express this protein, interferes with the viral production, probably due to a detrimental effect of the protein on the cells. This resulted in difficulties in deriving a packaging cell line with a high titer. To investigate the potential of retroviral gene transfer to correct gp91-phox deficiency, the cell line X-CGD PLB-985 was transduced with L91 virus. A superoxide activity of 56-89% of normal levels was achieved in bulk cultures. Gp91-phox protein was detected at significantly lower levels in clones of transduced cells than in wild type cells, however this was sufficient to generate enzyme activity of between 66-100% of wild type in gp91-phox positive clones. The transduction efficiency was measured 3 weeks after infection, the time necessary for clonal expansion, and showed 30-35% positive clones as determined by PCR and NBT. Studies of clones derived from single cell sorting after transduction showed loss of respiratory burst activity in up to a third of the clones possibly due to rearrangement, mutation or silencing of the LTR promoter. This finding is in agreement with the data on the fibroblast producer lines, indicating that expression of gp91-phox protein may be detrimental in cells, including primitive haemopoietic cells, which do not normally express it, and strict regulation of its expression is required. These results point to a need for alternative strategies for gene therapy of CGD, perhaps including redesigns of vectors to achieve strict tissue-regulated expression of gp91-phox.
Notlar:
School code: 1543
Konu Başlığı:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(686871.1) | 686871-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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