Chinese scientists have shown for the first time that the down-regulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), locally converted glial cells to neurons and showed promise for treating the symptoms of neurodegenerative diseases in mice. Reported in the April 8, 2020, online edition of Cell, the study also established the in vivo efficiency of RNA editing using the recently developed CRISPR-CasRx system, which may be useful both for cell replacement and for treating diseases requiring down-regulation of specific gene products.

Neurodegenerative diseases are associated with progressive potentially devastating neuronal loss throughout the nervous system. For example, degeneration of retinal ganglion cells (RGCs) is the leading cause of permanent blindness among retinal diseases.

“We believe that if RGCs could be regenerated efficiently, then this approach could be applied to the treatment of glaucoma and other retinal degenerative diseases,” said study co-leader Hui Yang, a postdoctoral researcher in the Institute of Neuroscience at the Chinese Academy of Sciences Center for Excellence in Brain Science and Intelligence Technology in Shanghai.

One such approach, the differentiation of Müller glia (MG) cells into RGCs, has been proposed as a means of restoring visual function, but MG-to-RGC conversion has yet to be achieved in mature mammalian retinas.

Recent in vitro studies have shown that down-regulating expression of a single gene, Ptbp1, which encodes for the Ptbp1 RNA binding protein, could convert cultured mouse fibroblasts and stem cells into functional neurons. But this conversion remains unexplored in vivo.

A recently identified RNA-guided and RNA-targeting CRISPR protein family, Cas13, potentially offers an efficient approach for the manipulation of RNA transcripts.

Among the different Cas13 proteins, a CRISPR-Cas13d ortholog, known as CasRx, is the smallest and shows a particularly high RNA-targeting specificity and efficiency, potentially making it ideal for in vivo therapeutic application.

In the new Cell study, researchers led by Yang and his postdoctoral colleague, Haibo Zhou, demonstrated that MG cells could be converted into RGCs by injecting adeno-associated viruses (AAVs) expressing CasRx and two guide RNAs (gRNAs) targeting Ptbp1 messenger RNA in both intact and in damaged mature retinas.

“The AAVs serve as vectors to deliver gene editing tools into MG cells, while the gRNAs with CasRx enable the specific and efficient knockdown of Ptbp1,” Yang told BioWorld.

Notably, the converted RGCs were shown to establish central projections to the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC), partially restoring visual function in a mouse model with drug-induced retinal injury.

This is a significant finding, as “it means that induced RGCs could establish functional and corrective connections in the brain and this could be observed by fluorescent axon and visually evoked potentials (VEP), which could be recorded in the primary visual cortex,” said Yang.

“We demonstrated improved sight using the dark/light preference test and found that treatment group had a marked increase in the duration spent in the dark compartment, to a level close to that seen in uninjured control mice,” he said.

The researchers then investigated the possibility of RNA editing using the CRISPR-CasRx system to convert glial to neuronal cells being used in Parkinson’s disease, which is associated with loss of dopaminergic neurons.

Parkinson’s disease

Using a similar approach to that used in MG-to-RGC conversion, the researchers showed that knockdown of Ptbp1 also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson’s disease mouse model.

“The induction of striatal neurons having dopaminergic features was demonstrated by the presence of dopaminergic markers, and by functional spike and dopamine release experiments,” said Yang.

“We analyzed the injected mice using apomorphine- and amphetamine-induced net rotation and found that the rescued mice showed marked reductions in net rotation to a level comparable to that found in non-lesioned wide-type mice.”

The researchers also analyzed the effects of treatment in the mouse models using forelimb-use asymmetry and motor coordination, as assessed using cylinder and rotarod tests, respectively.

“We found that the rescued animals also showed significantly lower percentages of ipsilateral touches of the cylinder and longer duration on the rotarod, as compared to control mice,” he said.

Collectively, these findings suggest that CasRx-mediated Ptbp1 knockdown could achieve efficient glia-to-neuron conversion that replenishes desired neuronal types, representing a promising new in vivo genetic approach for treating a variety of neurodegenerative disorders.

Said Yang, “We believe this represents a promising approach to treat many types of neurodegenerative diseases in humans, but the next and most important step will be to establish the safety of these gene editing tools and demonstrate that safety in non-human primate models.”

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