DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV) genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt) AAV integration and recombinant (r) AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy.

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of Receptor Activator of Nuclear Factor Kappa B Ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remains the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to tumor necrosis factor related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Towards uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage.

Key Points• The study developed a mouse model of bone disseminated myeloma disease as in humans.• The study established therapeutic potential of OPG variants to revert myeloma bone damage in vivo.

Current treatments for osteolytic cancers include a combination of radiation, chemotherapy and cytotoxic products, but toxic side effects are still of major concern. Studies have shown that osteoclast activity is increased in patients with osteolytic cancers such as Multiple Myeloma (MM), through increased expression of Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) leading to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Moreover, Osteoprotegerin (OPG) is drastically decreased in these patients who presents with bone lesion. Thus, the use of OPG as a therapeutic molecule would greatly decrease osteolytic damage and reduce morbidity. However, despite the of OPG potential in inhibiting the activation of bone resorbing osteoclast, OPG also binds to tumor related apoptosis-inducing ligand (TRAIL) making tumor cells resistant to apoptosis. TRAIL binds to the Death Receptor 4 and 5 (DR4, DR5) and initiate cell death of Transformed cells such as MM. The present study was designed to develop a novel therapeutic approach to the treatment of osteolytic bone damage by use of genetically altered OPG retaining RANKL binding but abolished of TRAIL binding. In order to eliminate TRAIL binding while maintaining RANKL binding, we created mutant OPG-Fc constructs by site directed mutagenesis based on interactive domain identification and by superimposing structural models of TRAIL, OPG and DR5. The mutant OPGs were produced in HEK 293 cells for characterization of potential mutants and their TRAIL binding ability. We conclude that using site-directed mutagenesis at the N-terminal of OPG effectively retains RANKL binding property but abolishes TRAIL binding property as determined by osteoclast and TRAIL assays respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4943. doi:1538-7445.AM2012-4943

Multiple Myeloma (MM) remains the second most common hematologic malignancy occurring in adults, which affects primarily the skeletal system. Current therapies, which include chemotherapy, radiotherapy, autologouos stem cell transplantation and in some cases surgery, have the extended median survival between 3 and 10 years. However, MM is still incurable and therefore improving current therapies or developing novel ones to extend survival rate will be highly beneficial for patient management. The long term goal of this study is to develop a novel therapeutic approach to the treatment of MM using genetically-engineered mesenchymal stem cells (MSC) for inhibiting osteoclast activity by stable expression of osteoprotegin (OPG). Studies have shown that osteoclast activity is increased in myeloma patients through increased expression of Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) leading to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Osteoprotegerin is a soluble decoy receptor for RANKL, and is decreased in expression in myeloma patients possibly because of a marked decrease in osteoblast and or MSC, which produce OPG. Thus, we hypothesize that therapy with MSC, overexpressing OPG, will greatly decrease osteolytic damage and reduce morbidity. Despite the potential of OPG in inhibiting osteoclast activation, OPG also binds to tumor related apoptosis-inducing ligand (TRAIL) making MM cells resistant to apoptosis. In order to eliminate TRAIL binding while still possessing RANKL binding, we have created mutant OPG constructs by site-directed mutagenesis based on interactive domain identification, and superimposing structural models of TRAIL, OPG and DR5, the death receptor that binds to TRAIL. The mutant OPGs were produced in HEK 293 cells for characterization of the five potential mutants and their TRAIL binding ability. One of the mutants abolished TRAIL binding while possessing RANKL binding as determined by TRAIL assay and osteoclast assay respectively. Finally, to achieve biphasic effects of MSC-OPG therapy with anti-tumor activity, the regenerative MSC therapy will be tested in combination with chemotherapy and anti-angiogenic therapy. These studies are performed in preclinical mouse models of MM. Currently, other OPG mutants are being characterized for their RANKL and TRAIL binding properties which will be confirmed via the assays mentioned and immunoprecipitation. Thus far, we conclude that using site-directed mutagenesis targeted at the N-terminal of OPG effectively retains RANKL binding and abolishes TRAIL binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3391. doi:10.1158/1538-7445.AM2011-3391