تحویل موضعی پایدار آدنوویروس آنکولیتیک آنکولیتیک جهت درمان سرطان با ویروس با ژل تزریقی آلژینات

ABSTRACT Local sustained delivery of oncolytic adenovirus with injectable alginate gel for cancer virotherapy

The efficacy of adenovirus (Ad) vectors in cancer gene therapy has been heavily investigated. Several characteristics make Ad attractive gene therapy candidates, including high gene-transfer efficiency in both dividing and non-dividing cells, easy production of high-titer Ad stocks, low risk of insertional mutagenesis and induction of oncolysis by viral replication. Among the 1600 clinical trials studying gene therapy since 1990, investigators have chosen Ad as a delivery vector in over 390 clinical trials because of these characteristics http:// www . wiley . co . uk / genmed / clinical /. A significant advance in the second generation of oncolytic Ad is the capability of replicating selectively in cancer cells.2, 3, 4, 5, 6, 7, 8, 9, 10 After the lysis of the infected cancer cells, progenies of oncolytic Ad disseminate locally and infect neighboring cancer cells. This sequential replication and lysis of cancer cells makes oncolytic Ad a promising candidate for cancer gene therapy, overcoming the modest efficacy of non-replicating virus-mediated cancer gene therapy.11, 12, 13, 14, 15, 16, 17 Cancer cell-specific replication of oncolytic Ad also reduces toxicity and maximizes safety by not replicating in normal healthy cells18, 19, 20 Moreover, the combination of therapeutic gene expression and lytic ability of oncolytic Ad further increase the likely impact of Ad-mediated cancer gene therapy. Combining virotherapy and expression of genes with anti-cancer roles, such as decorin, relaxin, cytokines and vascular endothelial growth factor- or interleukin-8-specific short hairpin RNA, have greatly increased the efficacy of cancer gene therapy.11, 13, 14, 21, 22, 23, 24, 25, 26, 27

Despite these therapeutically advantageous characteristics, the use of oncolytic Ads has thus far had low efficacy in clinical settings because of induction of immune responses against Ad and the short duration of therapeutic gene expression.28 ONYX-015 oncolytic Ad administered intratumorally into patients with head and neck cancer demonstrated limited local viral replication as a result of robust innate immune responses.29, 30 The limited efficacy of this oncolytic Ad is a result of viruses being engulfed by Kupffer cells and hepatocytes.31 Effective treatment of large tumors requires maintenance of the therapeutic dose of the virus at targeted disease site. To increase the effectiveness of Ad virotherapy, experiments have been conducted involving high Ad titer and repeated injections, but efficacy was hampered by fast clearance of the virus from tumor tissue to the blood microvasculature and normal tissues.32, 33, 34

Efficacy is further diminished by the fact that Ad is slow to diffuse through the extracellular matrix of a very large tumor. The limited viral infection of neighboring cells far from the initial injection site greatly limits the ability of virus-mediated cancer gene therapy to be a successful form of cancer gene therapy. In a phase I clinical trial using p53-expressing Ad to treat recurrent glioma, the zone of transduced cells did not extend beyond 5 mm from the injection site.35 Moreover, tumor biopsies taken from subjects in a phase II clinical trial studying head and neck cancer demonstrated that ONYX-015 oncolytic Ad are present in tumor tissues, but the pattern of viral dissemination was very focal in nature, indicating that the virus did not distribute evenly throughout the tumor tissue.30 With the aim of improving oncolytic viral spread and tumor penetration to enhance cancer gene therapy, we recently demonstrated the importance and utility of the peptide hormone relaxin. Relaxin has been shown to be a potent inhibitor of collagen expression when collagen is overexpressed, but does not markedly alter basal levels of collagen expression, in contrast to other collagen-modulatory cytokines, such as interferon-γ36 and transforming growth factor-β.37 DWP418 is an oncolytic Ad that expresses relaxin and demonstrates markedly improved viral distribution in large tumors, resulting in higher transduction efficiency and enhanced antitumor efficacy.2, 22

The development of optimized Ad and an effective Ad delivery system would further advance Ad vector therapy by maximizing safety, efficacy and duration of transgene expression. Recently, a locally injectable virus delivery system has been used for bone morphogenic protein-2-expressing Ad gene therapy of osteogenesis using a collagen carrier,38 antibody complexation with Ad in a collagen carrier,39 lentivirus entrapment in hyalulonic acid/collagen matrix40 and mixture of silk elastin-like protein polymers/Ad for breast and head and neck cancer xenograft tumor models.34 The use of a matrix-based Ad transduction system assisted widespread and uniform virus transduction.34, 41 In addition, the local injection of a matrix-based Ad delivery system prevented rapid viral dissemination and infusion from tumor to normal organ during intratumoral injection, which results in low normal cell transduction efficiency.42, 43 However, no previous studies have used localized and sustained oncolytic Ad delivery to clearly demonstrate the therapeutic effects of this Ad in vivo. Moreover, it is as yet unknown whether spatial Ad delivery using a matrix-based sustained delivery retains sufficient Ad biological activity to facilitate long-lasting oncolytic Ad replication and cancer cell killing.

In this study, we tested a locally injectable Ad in a matrix-based depot system involving a biodegradable alginate gel, a natural polymer that is frequently used in biomedical applications.44 Alginate has been previously used as an injectable gel carrier for protein45, 46 and cells47 because of its biocompatibility, low toxicity, relatively low cost and mild gelation behavior with divalent cations. Here we report the in vitro characterization of Ad entrapped in alginate gel, prolonged gene expression as a result of the sustained Ad release and protective microenvironment within the gel, and enhanced oncolytic Ad-mediated cancer cell killing. We also demonstrate that local injection of oncolytic Ad-loaded gel resulted in sustained release of Ad with prolonged activity in vivo, leading to enhanced antitumor therapeutic effects. Enhanced local preferential replication of oncolytic Ad mediated by sustained release in the protective gel was determined by immunohistochemical analysis and quantitative PCR (Q–PCR).