Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid. Madrid 28049, Spain.

Laboratory of Cellular and Molecular Neuroendocrinology, Cajal Institute, CSIC, Madrid 28002, Spain.

The development of gene therapies for human cerebellar disorders is currently hampered by the lack of a safe and efficient method to transfer genes into cerebellar neurons in vivo. We have explored the transduction of cerebellar neurons in vivo with Herpes Virus Simplex 1 (HSV-1) amplicon carrying the lacZ gene after injection of the vector in the cerebellar cortex, ventricles and inferior olive of adult rats.

Injection into the cerebellar cortex resulted in transduction of Purkinje cells which were restricted to the vicinity of the needle tract. Injection into the ventricles yielded no transduced neurons, although ependymal and meningeal cells were transduced. In contrast, high transduction efficiency of cerebellar Purkinje cells was achieved by vector injection into the inferior olive, resulting in one out of three positive Purkinje cells all over the ipsilateral and contralateral cerebellar hemispheres. As neurons in the deep cerebellar nuclei are also transduced, we suggest that the vector is delivered from the inferior olive to the cerebellar nuclei and then to Purkinje cells by retrograde axonal transport. Expression of the lacZ gene within Purkinje cells was surprisingly persistent and was maintained at the same level for at least 40 days.

Importantly, no signs of either toxicity or inflammation were observed in the cerebellum after vector injection, except for the borders of the needle tract where some reactive astrocytes were detected. Indeed, motor coordination of treated animals was entirely normal, as assessed by the rota rod test.

These results demonstrate that HSV-1 amplicon vectors can effect safe and stable transgene expression in Purkinje cells in vivo. Thus, this system will allow specific gene transfer into Purkinje neurons in vivo for physiological studies in rodents and raises the possibility of using these vectors for long-term gene therapy of human cerebellar disorders.