New Adenoviral Vector Preparation And Its Tumor Immunotherapy And The Mechanism Of Chemokine Gene Vector

Successful gene therapy depends largely on vectors that can exciently deliver the therapeutic genes into the target tissues and cells. Recombinant adenovirus vectors (Adv) continue to be the preferred vectors for gene therapy because they can easily be grown to high titers and can exciently transfer genes into both dividing and nondividing cells. However, there are some limitations such as coxsackievirus-adenovirus receptor (CAR)-dependent gene transfer, immunologic side effects, lack of tissue specificity and lack of regulation of gene expression, etc. In this study, we inserted the biologically selected Arg-Gly-Asp (RGD) sequences into the HI loop of fiber knob to generated fiber mutated Adv (FM-Adv), and showed that the FM-Adv had enhanced gene transfer activity to cells, which lacked CAR expression but showed αv integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (αvβ3 & αvβ5)-dependent, CAR-independent cell entry pathway. Systemic administration of Adv leads to activation of innate and antigen-specific immunity. In an attempt to enhanced the physical stability of Adv and ablated humoral immune responses, free lysine groups of capsid proteins were PEGylated with activated monomethoxypolyethylene glycols (mPEGs). Athough the PEGylated Adv (PEG-Adv) exhibit antibody evasion activity and long plasma half-life, but the ability of PEG-Adv to bind CAR is diminished by the steric hindrance of conjugated PEG chains. So the transduction efficiency of PEG-Adv was reduced. Adv was PEGylated with RGD-mPEG which combined RGD peptides on the tip of PEG, to generated a RGD-PEG-Adv, which could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. Gene expression of RGD-PEG-Adv was almost equal to that of RGD-Adv, RGD-PEG-Adv maintained its activity against antibodies. In this study, we studied antitumor activity of chemokine ILC and fractalkine (FKN) in vivo. Tumor cells were infected with RGD-Ad-ILC and RGD-Ad-FKN, which encoding ILC and FKN, to express and release these chemokines. Tumor rejection experiments in vivo were carried out by inoculating OV-HM cells infected with RGD-Ad-ILC or RGD-Ad-FKN into immunocompetent mice. ILC significantly suppressed the tumor growth, whereas no such significant effect was observed by FKN. The antitumor activity induced by ILC was T cell dependent, involving both CD4~+ and CD8~+ T cells. Immunohistochemical analysis revealed accumulation of both CD3+ lymphocytes and NK cells in the tumor tissue transduced with ILC and FKN. However, there was a significant difference in the distribution of infiltrating cells. Furthermore, FKN appeared to have an angiogenic activity, which might have masked its tumor suppressiveactivity. Collectively, ILC may be a good candidate molecule for cancer gene therapy.