Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.
BACKGROUND
After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2).


RESULTS
We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents.


CONCLUSIONS
RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.
Ehlert
Erich M
EM
Department of Neuroregeneration, Netherlands Institute for Neuroscience, an institute of the Royal Academy of Arts and Sciences, Amsterdam, the Netherlands.
Eggers
Ruben
R
Niclou
Simone P
SP
Verhaagen
Joost
J
D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D005727:Ganglia, Spinal / Q000502:physiology; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009410:Nerve Degeneration / Q000209:etiology*; D009474:Neurons / Q000502:physiology*; D039942:Neuropilin-1 / Q000235:genetics / Q000378:metabolism; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012012:Red Nucleus / Q000502:physiology; D039961:Semaphorins / Q000378:metabolism
D039943:Neuropilin-2; D034741:RNA, Small Interfering; D039961:Semaphorins; D039942:Neuropilin-1