Parkinson's disease motor symptoms rescue by CRISPRa‐reprogramming astrocytes into GABAergic neurons

Abstract Direct reprogramming based on genetic factors resembles a promising strategy to replace lost cells in degenerative diseases such as Parkinson's disease. For this, we developed a knock‐in mouse line carrying a dual dCas9 transactivator system (dCAM) allowing the conditional in vivo activation of endogenous genes. To enable a translational application, we additionally established an AAV‐based strategy carrying intein‐split‐dCas9 in combination with activators (AAV‐dCAS). Both approaches were successful in reprogramming striatal astrocytes into induced GABAergic neurons confirmed by single‐cell transcriptome analysis of reprogrammed neurons in vivo. These GABAergic neurons functionally integrate into striatal circuits, alleviating voluntary motor behavior aspects in a 6‐OHDA Parkinson's disease model. Our results suggest a novel intervention strategy beyond the restoration of dopamine levels. Thus, the AAV‐dCAS approach might enable an alternative route for clinical therapies of Parkinson's disease.


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Appendix Figure S1: Design and evaluation of the dCAM line 2 Appendix Figure S2: Evaluation of dCAM x Gfap-Cre primary astrocytes 4 Appendix Figure S3: Evaluation of 6-ODHA induced lesion 5 Appendix Figure S4: Amount and regional distribution of GFP+ cells 6 Appendix Figure S5: In vivo reprogramming 5 wpi -dCAM 7 Appendix Figure S6: Activation of endogenous genes 8 Appendix Figure S7: Evaluation of AAV-dCAS system in vitro 9 Appendix Figure  Appendix Figure S18: dCAS reprogramming of primary astrocytes 22 Appendix Figure S19: AAV-dCAS injection into Gfap-Cre negative mice 23 Appendix Table S1: Sequences of gRNAs 24 Appendix  containing 6 sgRNAs and a reporter gene can be applied. E, If more than 6 sgRNAs shall be used for in vivo activation two AAVs containing 12 sgRNAs or 6 sgRNAs and a miRNA expression cassette can be applied with a split-reporter gene. AAVs contain sgRNAs, whose expression is driven by the different Pol III promoters (H1, hU6, mU6 and 7SK), and the marker gene FLEx-GFP, respectively split-FLEx-GFP, is expressed by the CBh promoter and also delivered by AAVs.
Appendix Figure S2: Evaluation of dCAM x Gfap-Cre primary astrocytes for the activation capacity.
Activation levels are depicted as fold change between cells transfected with and without sgRNAs.
All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.
Appendix Figure S4: Total amount and regional distribution of GFP + cells in vivo in dCAM x Gfap-Cre mice injected with FLEx-GFP reporter.
A, GFP + cells in the ipsilateral dorsal striatum of one slide after five weeks of injection Appendix Figure S7: Evaluation of AAV-dCAS system in vitro.
A, Western blot analysis evaluating the FLEx-N-dCas9 system in Neuro2A cells, using a C-Cas9 antibody. B, Western blot analysis evaluating the split-dCas9 system in Neuro2A cells, left blot -N-Cas9 antibody, right blot -C-Cas9 antibody. Correct fusion of the split-dCas9 parts at 175 kDa.  Pitx3 195 ± 19,FoxA2 32 ± 8) in primary astrocytic cultures. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin.
Error bars represent mean ± SD between technical replicates.  Appendix Figure S17: AAV combinations.

Appendix
A representation of the AAV combinations, which were used for the different approaches and experimental groups with detailed information to promoter and gRNA position and regulatory elements. A, Combinations used for the dCas9 activator mouse experiments. B, Combinations used for the adeno-associated virus (AAV)-based intein-split-dCas9 activator system (AAV-dCAS).
Reprogramming primary astrocytes by dCAS-mediated induction of Ascl1, Lmx1a and gRNA Gene Sequence 5´-3´ Table S1: Sequences of gRNAs used in this study