Samady, Laila; (2001) Development of herpes virus vectors for gene delivery to dendritic cells and the potential immunotherapy of cancer. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The greatest challenge in cancer immunotherapy is to overcome the fact that the immune system does not elicit an effective immune response to the tumor. This is partly due to the fact that tumors are deficient in presenting peptide antigens to T cells. One approach has been to use dendritic cells which are professional antigen presenting cells, to initiate an immune response. Dendritic cells may, therefore, provide the basis for generating a more effective anti-tumor response if a safe and efficient way is found to deliver genes encoding tumor antigens to them and to activate their T-cell stimulatory capacity. The development of a gene delivery system for dendritic cells is described in this thesis using herpes simplex virus (HSV-1) vectors for potential application in cancer immunotherapy. Minimally disabled HSV-1 was found to infect dendritic cells efficiently even though dendritic cells were found not to be permissive for viral growth. This suggests that HSV-1 may naturally infect these cells However, slight viral growth was observed indicating that minimally disabled vectors may be toxic to the cells and affect their function. Thus, a panel of more disabled HSV-1 vectors were tested in dendritic cells. Two vectors were found to give optimal results in terms of balance of gene delivery and toxicity to cells. A partially disabled vector with deletions in the non-essential genes ICP34.5 and UL43, and inactivations in VP16 and vhs gave very high gene delivery (81% GFP expression at multiplicity of infection MOI=1) and was not toxic to cells. Furthermore, no viral growth was detected and only very low levels of immediate early (IE) proteins ICPO and ICP22, but no ICP47 were expressed at MOI=1. A further disabled replication incompetent vector with deletions in essential genes ICP27 and ICP4, and deletions and/or inactivations in the non-essential genes ICP34.5, vmw65 and vhs, was also found to give high level transgene expression. Selected HSV-1 based vectors were then used to study their effect on dendritic cell function. The inhibition of proteasomes in transduced cells showed that actual gene delivery was at higher efficiency than recorded by apparent protein expression levels. This suggested that cells infected with HSV process the antigen delivered to them via the vector. The effect of HSV-1 was then assessed on several surface markers that are important for T-cell co-stimulation and activation. Minimally disabled HSV-1 prevents the up-regulation of these molecules in response to infection and this may be a possible route of escape of the virus from the immune system. However, a replication incompetent vector with vhs inactivated specifically up-regulated e.g. CD86 expression compared to an equivalent vector with vhs intact. This implicates the vhs protein of HSV-1 in immune escape. A replication incompetent vhs inactivated HSV-1 vector encoding hepatitis B surface antigen (HBsAg) was constructed in order to test the ability of transduced dendritic cells to specifically present a delivered antigen to T cells and thus elicit an immune response. Dendritic cells transduced with this vector stimulate a CD4+ T cell response specific to HBsAg in vitro. Finally a vector expressing the tumor-associated MUC-1 antigen was constructed which may be useful in the treatment of MUC-1 expressing cancers including breast and ovarian cancers. Based on these results, replication incompetent vhs deleted HSV-1 vectors may provide an optimal means of delivering tumor antigens to dendritic cells for use in immunotherapy.

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