Gene delivery of apoptin-derived peptide using an adeno-associated virus vector inhibits glioma and prolongs animal survival

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Highlights

  • An AAV vector for intracranial delivery of the secreted HSP70-targeted peptide APOPTIN derived from Apoptin to GBM tumors was designed.

  • In U87MG and U251MG cells, conditioned medium from AAV2-apoptin-derived peptide (ADP)-expressing cells induced 83% and 78% cell death.

  • In mice bearing intracranial U87MG tumors treated with AAV2-ADP, treatment resulted in a significant decrease in tumor growth and longer survival in mice bearing orthotopic invasive GBM brain tumors.

Abstract

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of the secreted HSP70-targeted peptide APOPTIN derived from Apoptin to GBM tumors. We applied this therapy to GBM models using human U87MG glioma cells and GBM xenograft models in mice. In U87MG and U251MG cells, conditioned medium from AAV2-apoptin-derived peptide (ADP)-expressing cells induced 83% and 78% cell death. In mice bearing intracranial U87MG tumors treated with AAV2-ADP, treatment resulted in a significant decrease in tumor growth and longer survival in mice bearing orthotopic invasive GBM brain tumors. These data indicate that ssAAV2-ADP injection in the left hemisphere effectively prevented ipsilateral tumor growth but was insufficient to prevent distal tumor growth in the contralateral hemisphere. However, the systemic route is the most effective approach for treating widely dispersed tumors. In summary, systemic delivery of AAV2-ADP is an attractive approach for invasive GBM treatment.

Introduction

Glioblastoma multiforme (GBM) is classified as WHO grade 4 astrocytic glioma and is the most common and aggressive type of primary brain tumor [1], [2], [3]. Although chemotherapy, radiotherapy and surgery are used clinically to treat GBM, the median survival time of patients with gliomas is only 13–16 months [4]. Thus, there is a strong unmet medical need for the development of novel therapies with improved clinical efficiency and enabling longer survival times in GBM patients.

Gene therapy, defined as the introduction of nucleic acid polymers into a host cell as a source of new drugs, is mainly used for the treatment of serious diseases and is very promising for the treatment of tumors. More than 2000 clinical trials employing gene transfer have been performed and generally established that a number of vehicles or vectors are safe [24], [25]. To increase the success of cancer gene therapies, however, a major hurdle must be overcome: the development of gene delivery vectors that can efficiently, safely and specifically deliver genetic material to a patient's cells. In particular, AAV vectors are increasingly successful due to their gene delivery efficacy, lack of pathogenicity, and strong safety profile and are among the most commonly used viral vectors for gene therapy. Based on these properties, AAV vectors have enabled clinical successes in a number of recent clinical trials that have established the promise of gene therapy in general, including for the treatment of diseases such as Leber's congenital amaurosis (LCA) [10], [11], [12]. AAV vectors with excellent gene delivery properties have been harnessed for cancer studies in vitro, cancer preclinical models in vivo, and more recently, cancer clinical trials under development, such as vectors based on adeno-associated virus serotype 2 (AAV2). These vectors have been used as effective vectors for delivering therapeutic genes to the tumor and effectively suppressing the growth of malignant gliomas in the brains of mice [13]. We previously demonstrated that an AAV vector mediated the co-expression of IL-24 and apoptin and significantly suppressed hepatocellular carcinoma cell growth both in vitro and in vivo [14].

Apoptin, a 14-kDa viral protein (chicken anemia virus protein-3,VP3), selectively induces apoptosis in cancer cells [5], [6]. Apoptin-mediated cell toxicity depends on its cellular localization; nuclear localization promotes cell death, whereas cytoplasmic localization does not [7], [8]. The 70-kDa heat shock protein (HSP70) facilitates the folding, assembly, transport and degradation of biological macromolecules. HSP70 inhibits the release of cytochrome C, thus preventing induction of apoptosis by procaspase-9, which is highly expressed in tumor tissue and poorly expressed in normal tissues. Thus, HSP70 is considered a molecular target of tumors. Apoptin directly binds the promoter of HSP70 and inhibits HSP70 transcription [26]. We aimed to promote the apoptosis-inducing capacity of apoptin in tumor cells using a polypeptide sequence of reduced length. Thus, we created an apoptin-derived-peptide (ADP) to target the promoter region of HSP70. The HSE-targeting ADP that we designed comprised four parts: a TAT tag, the core sequence of NLS1, the LRS, and NLS2. With this design, the LRS creates a flexible connection between the two NLSs to effectively maintain the necessary three-dimensional structure. We demonstrated that compared to apoptin, ADP more strongly promotes tumor cell apoptosis, reduces tumor growth, improves animal survival and inhibits glioma invasion and migration in vivo.

To assess the possibility of glioma gene therapy using an AAV vector expressing ADP, we performed both in vivo and in vitro experiments by inhibiting human glioma growth in the brains of nude mice and analyzing cultured U87MG glioblastoma cells. Our results demonstrated that ADP gene transfer effectively suppressed U87MG brain glioma growth in mice and that systemic delivery of an AAV2-ADP vector may be effective for GBM treatment.

Section snippets

Cell culture

The human GBM cells lines U87MG and U251MG and human embryonic kidney 293 cells were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were routinely cultured at 37 °C in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum.

Plasmids

To construct an AAV expression vector, Apoptin-Derived-Peptide cDNA was amplified from I.M.A.G.E. by PCR using the primers 5′-TTTCAATTGACCTATGGCCGTAAAAAACGTCGT-3′ and 5′-TTTCAATTGACGACGACGCTGACGACG-3′, which contain MunI

The construction of ssAAV-apoptin-derived peptide and expression levels in glioma cells

We constructed the ssAAV-apoptin-derived peptide and assessed the expression level in glioma cells. We constructed a recombinant AAV that expressed apoptin alone or ADP alone (Fig. 1A). To evaluate apoptin-derived peptide (ADP) expression in U87MG glioma growth and cell proliferation, we established U8MG cells that stably expressed ssAAV2-ADP or ssAAV2-Apoptin and a EGFP control. For the ADP and apoptin sequences, we designed constructs that contained a TAT sequence. We were therefore able to

Discussion

GBM accounts for the majority of all primary brain tumors. The standard therapy to increase survival in GBM patients consists of maximal surgery followed by radiation and various chemotherapy agents. However, these tumors are remarkably resistant to both radiation and chemotherapy and typically recur within months of treatment. Although studies of angiogenic growth and proliferation pathways of primary tumors have uncovered GBM-sensitive targets, systemic delivery of immunotherapies to block

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Grant No: 81502676) and the Heilongjiang Province Postdoctoral Science Foundation (Grant No: LBH-Z14157) to Dr. Wu Liande well as by Foundation (WLD-QN110).

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    1

    Xiuli Zhong and Hengyu Zhao contributed equally to this study.

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