Abstract
Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo.
Highlights
The cerebellum functions as an important regulator of body movement and coordination, and, as such, disorders of the cerebellum typically result in ataxia, a clinical symptom characterized by lack of coordination and gait disturbances
Expression of fluorescent reporter proteins in lentiviral vectors appeared to be highly promoter dependent, since MND and murine stem cell virus (MSCV) promoters produced almost exclusively glial cell expression patterns, whereas the Ubiquitin C (UBC) promoter expressed well in granule neurons, and the phosphoglycerate kinase (PGK) promoter appeared to express in glial cells and Purkinje neurons
Because of the low Purkinje neuron transduction efficiency using lentiviral vectors, we switched to an associated virus (AAV) vector and fortuitously obtained excellent Purkinje neuron transduction with the first AAV vector tested, AAV serotype 1 (AAV1) with the CAG promoter
Summary
The cerebellum functions as an important regulator of body movement and coordination, and, as such, disorders of the cerebellum typically result in ataxia, a clinical symptom characterized by lack of coordination and gait disturbances. As the sole output of the cerebellar cortex, Purkinje neurons are critical for cerebellar information processing [1] and, may provide an ideal target for therapies designed to restore or improve cerebellar function. Cerebellar disease can be acquired (e.g. ethanol, drugs, stroke, or trauma) or inherited [2]. The inherited cerebellar ataxias, many of which have known genetic bases, may be amenable to treatments based on gene transfer. Studies of cerebellar function and pathophysiology would benefit from methods to genetically manipulate Purkinje neurons in adult model organisms. Viral vectors provide a promising gene delivery system in both basic research and gene therapy
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