In vivo gene delivery by embryonic-stem-cell-derived astrocytes for malignant gliomas.

Currently, there is no optimal treatment for malignant gliomas, the most common malignant brain tumors in adults. Patients with these tumors have a dismal prognosis, with a median survival of less than 1 year.1 Although chemotherapy has been incorporated into the treatment of various intracranial neoplasms, the resistance and invasive nature of these tumors call for new treatment strategies. These include targeting brain tumor cells while sparing normal tissue and the use of gene therapy approaches. For brain tumors, the success of gene therapy depends on the ability to deliver genes to the infiltrating tumor cells interspersed within the normal cells. Viral vectors have been considered the most effective in vivo gene delivery reagents. Retrovirus, adenovirus (Adv), and herpes simplex virus-1 (HSV-1) are the best-studied viral brain tumor therapy vectors.2 Replication-competent retroviruses have infection rates of 97%, with tumor specificity and lack of spread to nontumor tissue. Potential disadvantages, however, include insertional mutagenesis.3 Adv and HSV-1, used in both replicating and replication-deficient forms, offer numerous advantages, including high transgenic capacity (Adv) and persistent gene expression (HSV-1).4,5 Potential disadvantages, however, include short-term transgene expression and potential toxicity for Adv, and neuro-virulence and recombination with wild type for HSV-1. Oncolytic viruses have also been used successfully for clinical trials,6 because they replicate selectively within tumor cells, leading to increased intratumoral viral titers. To date, however, their clinical effectiveness for brain tumors remains to be substantiated.7,8 Recent evidence suggests that stem cells can be used as delivery vehicles for brain tumor therapy.9,10 In vitro migration assays confirm the ability of isolated stem cells to migrate toward factors produced by glioma cells.11 There is, however, concern that these undifferentiated stem cells can interfere with baseline functions.12 Thus, ideally, differentiated cells such as astrocytes should be considered as vectors. Astrocytes are native to the CNS, which should maximize their survival and function after transplantation. Astrocytes are highly secretory cells and can generate large amounts of proteins.13 Therefore, transplanted astrocytes should be protective within the host brain. Finally, normal astrocytes can migrate along white matter tracts after transplantation into the brain.14,15 This migratory capacity may be useful for delivery of gene therapy to infiltrative tumors such as malignant gliomas. Autologous astrocytes can be derived from the pluripotent neural precursor cells in the periventricular area or from fetal tissue.16,17 These approaches are hazardous, with limited capability of generating large numbers of cells. Our recent work shows that we can generate a pure population of astrocytes from mouse embryonic stem cells (ESCs).18 Thus, it is our hypothesis that ESC-derived astrocytes conditionally expressing transgenes can be used as an alternative source for gene delivery in malignant glioma therapy. Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), a member of the TNF family, has been shown to induce apoptosis in a variety of transformed cell lines but to spare most normal cells.19,20 Systemic administration of recombinant TRAIL protein was associated with minimal toxicity in recent clinical trials with cancer patients.21 Recombinant TRAIL, however, does not cross the blood–brain barrier and has a short half-life.22 To obviate these limitations, convection-enhanced delivery (CED) of TRAIL has been used in a malignant glioma xenograft model with good results.23 Although CED is a promising technique to bypass the blood–brain barrier, side effects in the clinical setting include invasive implant of multiple catheters and difficult calculation of drug delivery.24 To circumvent these side effects, alternative delivery methods have been sought. Cellular gene delivery methods are gaining importance as stem cells have shown to have migratory and homing capacity.25 Our recent work shows that we can generate a pure population of transgenic ESC-derived astrocytes with a highly regulated, robust conditional expression of TRAIL under the control of a tetracycline (tet)-inducible promoter.26 Furthermore, we have shown that transgenic delivery of TRAIL by ESC-derived astrocytes induced apoptosis in malignant glioma cells in vitro.26 Based on our recent work, we hypothesize that ESC-derived astrocytes conditionally expressing TRAIL may represent an effective and novel approach for treatment of human malignant gliomas in vivo. Using a heterotopic xenograft model in the mouse, we have found that ESC-derived astrocytes conditionally expressing TRAIL can be used as a vector for gene therapy in malignant gliomas in vivo.