Karwacz, K. (2012) Lentivector based gene transfer for immunotherapy – application of integration deficient vectors and PDL1 knockdown as tools to manipulate immune responses. Doctoral thesis, UCL (University College London).
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Lentiviral‐based vectors are effective and promising tools for the generation of cell mediated immunity. Multiple studies have demonstrated that subcutaneous injection of lentivectors encoding tumour antigens results in induction of strong CTL responses and often in tumour killing. However, integration of lentivectors into human genomic DNA poses a risk of insertional mutagenesis. Indeed, this possibility has been highlighted by gene therapy trials that resulted in the development of T cell leukaemia in several patients. For this reason, non‐integrating lentiviral vectors (NILVs) have been developed as a safer alternative for gene delivery. The first part of this thesis demonstrates that lentivectors carrying multiple mutations preventing integration are effective vaccines. Subcutaneous injection of these vectors resulted in induction of systemic dose‐dependant CD8+ T‐cell responses to the encoded antigen. The duration of the persistence of antigen presentation was measured using transfer of OT1 transgenic T cells into previously immunized mice. Measuring expansion of those cells revealed that the antigen was present and presented for at least 30 days. CD8+ T‐cell responses were further enhanced by addition of dendritic cell (DC) stimulators: p38 MAP kinase and NF‐κB stimulators. These activators led to a more rapid response peaking at day 7. Finally, NILVs expressing the antigen and DC activators were tested in a tumour therapy model and were found to be effective. The second part of this thesis focused on altering DC‐T cell interactions to enhance responses to immunization by lentivector‐mediated knockdown of PDL1 on DCs. The analysis of DCs infected with anti‐PDL1 shRNA showed that knocking down this molecule drives DCs towards a mature phenotype. The influence of PDL1 knockdown was assessed on co‐cultured T cells. The absence of PDL1 enhanced their proliferation and reduced antigenic stimulation induced TCR complex degradation. DCs transduced with lentivectors expressing PDL1 shRNA were also tested in vaccination and tumour therapy.
|Title:||Lentivector based gene transfer for immunotherapy – application of integration deficient vectors and PDL1 knockdown as tools to manipulate immune responses|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Infection and Immunity (Division of)|
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