Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: A novel perspective for seizure control

Abstract

Cell therapies based on focal delivery of the inhibitory neuromodulator adenosine were previously shown to provide potent seizure suppression in animal models of epilepsy. However, hitherto used therapeutic cells were derived from rodents and thus not suitable for clinical applications. Autologous patient-derived adenosine-releasing cell implants would constitute a major therapeutic advance to avoid both xenotransplantation and immunosuppression. Here we describe a novel approach based on lentiviral RNAi mediated downregulation of adenosine kinase (ADK), the major adenosineremoving enzyme, in human mesenchymal stem cells (hMSCs), which would be compatible with autologous cell grafting in patients. Following lentiviral transduction of hMSCs with anti-ADK miRNA expression cassettes we demonstrate up to 80% downregulation of ADK and a concentration of 8.5 ng adenosine per ml of medium after incubating 10 5 cells for 8 hours. hMSCs with a knockdown of ADK or cells expressing a scrambled control sequence were transplanted into hippocampi of mice 1 week prior to the intraamygdaloid injection of kainic acid (KA). While mice with control implants expressing a scrambled miRNA sequence or sham treated control animals were characterized by KAinduced status epilepticus and subsequent CA3 neuronal cell loss, animals with therapeutic ADK knockdown implants displayed a 35% reduction in seizure duration and 65% reduction in CA3 neuronal cell loss, when analyzed 24h after KA-injection. We conclude that lentiviral expression of anti-ADK miRNA constitutes a versatile tool to generate therapeutically effective adenosine releasing hMSCs, thus representing a model system to generate patient identical autologous adult stem cell grafts.

The adenosine kinase hypothesis of epileptogenesis

“…Human mesenchymal stem cell grafts with a knockdown of ADK significantly reduced acute brain injury and duration of SE in a mouse model of KAinduced epileptogenesis (Ren et al, 2007). Since epileptogenesis in patients is not a completed pathophysiological state, but rather a dynamic process leading to progressively increasing frequency and severity of seizures, to pharmacoresistance and eventually to cognitive deficits (Blumcke et al, 2002;Elger et al, 2004;Engel, 2002), the time course of disease progression might provide a sufficient time window for therapeutic augmentation of the adenosine system to prevent the progression of the disease.…”

Adenosine dysfunction in astrogliosis: cause for seizure generation?

“…Due to the existence of an active substrate cycle between adenosine and AMP involving ADK and 5′-nucleotidase, small changes in ADK activity are sufficient to translate into rapid and major changes in ambient adenosine (Arch and Newsholme, 1978;Bontemps et al, 1983). Thus, genetic disruption or RNAi mediated downregulation of ADK is an efficient means to induce cellular adenosine release (Fedele et al, 2004;Ren et al, 2007) and rapid downregulation of ADK following stroke has been proposed to be an endogenous neuroprotective mechanism of the brain (Pignataro et al, 2008).…”

Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity

“…Cells deficient in the metabolizing enzyme ADK have proved to be an attractive therapeutic tool for the local delivery of adenosine. Initial experiments applied non‐neural cells, which resulted in strong but temporally limited antiepileptic activity 18, 19, 20. Using mouse ADK‐deficient embryonic stem cell‐derived neural cell populations, we demonstrated (a) strongly enhanced levels of secreted adenosine compared to cells derived from wild‐type controls 33, (b) short‐term protection of murine brains against experimental epileptogenesis upon grafting encapsulated ADK‐deficient cells into the ventricles of rats subjected to kindling 34, and (c) efficient suppression of kindling‐induced epileptogenesis and seizure severity upon direct transplantation of ADK −/− cells into the hippocampus of rats 21.…”

“…ADK deficient myoblasts or fibroblasts transplanted in encapsulated polymer membranes were able to temporarily protect animals from seizures in a rat kindling model 18, 19. In the human system, small interfering RNA(siRNA)‐mediated knockdown of ADK in mesenchymal stem cells resulted in an 80% decrease in enzymatic activity, leading to decreased seizure events in the mouse kainate acid model by 35% 20. Non‐neural cells, which have to be encapsulated, elicit their effects via a passive paracrine and non‐activity dependent mode of action and show only limited survival times.…”