Hypoxia-inducible transgene expression in differentiated human NT2N neurons – a cell culture model for gene therapy of postischemic neuronal loss

Abstract

Expression of anti-apoptotic or neurotrophic transgene proteins in hypoxic neurons may provide a novel therapeutic strategy for neuroprotection and alleviation of damage to ischemic brain areas. NT2, a human neoplastic cell line which terminally differentiates into postmitotic neuronsIschemia of the brain due to major vessel thrombosis causes hypoxia in the respective vascular territory and subsequently induces neuronal death which is at least in part caused by apoptosis, especially in the penumbra of the lesion. 1,2 Hypoxia-inducible expression of anti-apoptotic or neurotrophic transgenes in hypoxic neurons may provide a novel therapeutic strategy for neuroprotection and alleviation of damage to ischemic brain areas.Hypoxia has been known as a potent inducer of a large number of genes including those encoding erythropoietin, vascular endothelial growth factor (VEGF), and some glycolytic enzymes, eg phosphoglycerate kinase. These genes have specific regulatory sequences in their promoter regions, known as hypoxia responsive elements (HRE). 3 Under hypoxic conditions, the upregulated hypoxia-inducible transcription factor 1 (HIF-1) binds to the HRE and initiates transcription of 3Ј sequences. This physiological mechanism has been utilized for develop- ment of hypoxia-responsive gene transfer vectors with HRE-based promoters. Hypoxia-inducible expression of transgenes has been reported in tumor cells and in cardiac myocytes. [4][5][6][7][8][9] Recently, new hypoxia-responsive vectors for tumor-specific gene therapy were developed. 10

Liposome-mediated transfer of the bcl-2 gene results in neuroprotection after in vivo transient focal cerebral ischemia in an animal model

“…Previous studies demonstrated efficient VEGF promotermediated induction of transgene expression in hypoxic human neurons in culture. 6 The present study employed two different types of bcl-2 expression cassettes in two different plasmid vectors: one driven by the strong constitutive cytomegalovirus promoter (pCMV) and another based on the hypoxia-inducible VEGF promoter (pHRE). 6 The expression plasmids pCMV-bcl2 and pHRE-bcl2 could be efficiently transfected into rat brain in vivo.…”

“…6 The present study employed two different types of bcl-2 expression cassettes in two different plasmid vectors: one driven by the strong constitutive cytomegalovirus promoter (pCMV) and another based on the hypoxia-inducible VEGF promoter (pHRE). 6 The expression plasmids pCMV-bcl2 and pHRE-bcl2 could be efficiently transfected into rat brain in vivo. Lipoplexes were injected intrathecally immediately after occlusion of the middle cerebral artery (MCA).…”

“…13 We have recently shown that postmitotic human neurons may be efficiently transfected by lipoplexes. 6 Hypoxia-inducible transgene expression was also demonstrated in cultured hypoxic neurons. Furthermore, transfection with liposomes carrying the human bcl-2 gene protected cultured neurons against hypoxic cell death.…”

“…3 Bcl-2 also protects human neurons in cell culture against apoptosis induced by withdrawal of nerve growth factor (NGF) and by amyloid ␤-peptide, as well as against oxidative and hypoxic insults. [4][5][6] Delivery of the bcl-2 gene and expression of transgenic bcl-2 in ischemic brain tissue results in neuroprotection and may inhibit hypoxic cell death in vivo. [7][8][9][10] Most of the previous studies on neuroprotective gene transfer used genetically engineered virus vectors, such as herpes-simplex type I virus (HSV), adenovirus (AV), or adeno-associated virus (AAV).…”

“…12,14,15 It has been shown that DOTAP is more efficient than LipofectAmine (Gibco BRL, Berlin, Germany) for gene transfer in lung and in cultured neurons. 6,12 In the present study, the therapeutic transgene bcl-2 complexed with DOTAP liposomes was delivered to the brain by means of a single intrathecal injection.…”