Direct conversion of astrocytes to neurons utilizing VPR and SAM

Using the SAM split-dCas9-VPR system astrocytes were successfully converted to neurons in chapter 5.2.5. These results are very promising as direct conversion of somatic cells had not been achieved with SAM or VPR systems before. Furthermore, astrocytes have not been directly converted with any CRISPR/Cas9 based system up to now. Therefore, these results act as proof-of-principle for the SAM split-dCas9-VPR system. It was not surprising that direct overexpression of Ascl1 resulted in a higher reprogramming efficiency then the CRISPR/Cas9-based approach. When inducing just a single gene the CRISPR/Cas9 based approach has some downsides including the limiting effect of the required co-transduction by two viruses (SAM-N-dCas9 and C-dCas9-VPR vs Tet-O-Ascl1). This means that only a fraction of FlagTag⁺ (N-dCas9⁺) cells were also co-transduced by a C-dCas9-VPR encoding lentivirus similar to the findings for Ascl1 and Nurr1 at the beginning of this thesis in chapter 5.1.1. Therefore, the percentage of MAP2⁺ cells per FlagTag⁺ cells underestimates the reprogramming potential of SAM split-dCas9-VPR.

Furthermore, N-dCas9 and C-dCas9-VPR have to assemble in order to become a functional dCas9-VPR protein. Finally, protein separation at P2A, which was used to separate SAM and N-dCas9 is also a limiting factor. However, these calculated downsides of the SAM split-dCas9-VPR system should only be prominent when a single gene is induced. The underlying idea to generate this new system was based on the simultaneous induction of several genes where this new technology may show its full potential. In this case, direct overexpression of multiple transcription factors would require co-transduction of several viruses whereas gRNA multiplexing can still be applied to express all required gRNAs from the two established vectors SAM-N-dCas9 and C-dCas9-VPR due to the small size of gRNA expression cassettes.

Indeed, in a recent article Black et al., report a twofold increase in reprogrammed neurons when inducing the expression of endogenous genes Brn2, Ascl1 and Myt1l in MEFs by VP64-dCas9-VP64 compared to direct overexpression of the three transcription factors [107].

So far, this is the only report of directly converted somatic cells to neurons by dCas9 mediated gene induction. Naturally, the performance of this system was compared to the SAM-VPR system. RT-qPCR analysis revealed no significant activation of Ascl1 expression when using VP64-dCas9-VP64 with gRNAs mA1 and mA2 (appendix, Figure 29). This reflects earlier findings of this thesis which suggested that a single kind of transcriptional activator (VP16) and the relatively weak hUBC promoter are not suited for a strong transcriptional induction (chapters 5.2.2.4 and 5.2.4). It could be argued that this is mainly based on the different performance of promoters used (weak hUBC for VP64-dCas9-VP64 versus strong Tet-O for SAM split-dCas9-VPR). However, Chavez et al., 2016 came to a

6 | DISCUSSION

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similar solution comparing Tet-O-VP64-dCas9-BFP-VP64 with Ef1a-SAM or CMV-VPR systems [110]. The latter three promoters reach comparable levels of expression [120]

confirming actual differences in the activation potential of the tested systems. Here, gene induction by VPR and VP64-dCas9-BFP-VP64 were comparable for human ASCL1 and NEUROD1 but SAM performed approximately ten-fold stronger in both cases [110].

Along these lines, when testing the hUBC-VP64-dCas9-VP64 system with gRNAs mA1 and mA2 for reprogramming of astrocytes to neurons no significant increase in reprogrammed cells was observed (transfer by lipofection, appendix Figure 30). Besides the weak hUBC promoter the combination of specific gRNAs and the transcriptional activator system also seems to play a role. This was already suggested by the results regarding the synergistic effect of SAM and VPR systems for Ascl1 induction and the findings of Chavez et al. 2016, [110] who reported differential gene induction properties of dCas9 fusion proteins depending on gene and cell type. This might also explain the missing synergistic effect in the reprogramming potentials of SAM and VPR when used alone or in combination in lipofection based reprogramming experiments. However, the underlying mechanisms in astrocytes have to be analyzed in more detail before a final conclusion can be drawn.

Taken together, the successful conversion of astrocytes to neurons by CRISPR/Cas9 based gene induction of Ascl1 demonstrates the suitability of this system for the direct reprogramming of cells. It is therefore a promising starting point for further tests including simultaneous induction of multiple genes and in vivo experiments to analyze the functionality of reprogrammed cells. In vivo, CRISPR/Cas9-based gene induction may have further advantages by modifying the epigenetic landscape of targeted promoters thus resembling natural gene expression [107]. This was found to result in sustained levels of gene expression even when using transient expression systems [107]. This system may therefore be helpful to overcome the limitations of exogenous transcription factor expression as described in this thesis and by Theodorou and Rauser et al., [118].

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CONCLUSION

AND FUTURE PERSPECTIVES

7 | CONCLUSION AND FUTURE PERSPECTIVES

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7 Conclusion and future perspectives

In this thesis limitations of exogenous gene expression for direct reprogramming were shown due to low efficiencies of co-transduction and the generation of fusion proteins. Promisingly, forskolin was found to support the direct conversion of Ascl1, Lmx1a and Nurr1 transduced MEFs to dopaminergic neurons and enabled the generation of TH⁺/PITX3⁺ midbrain dopaminergic neurons. These findings are highly promising as overexpression of Ascl1, Lmx1a and Nurr1 alone was not sufficient to generate PITX3⁺ mdDA neurons which therefore do not resemble the DA neuron subtype affected in PD. As next steps, the functionality of these neurons should be further characterized by electrophysiology and finally transplantation in lesioned mouse models to investigate the therapeutic benefits.

As an alternative to exogenous gene expression a combination of SAM and dCas9-VPR was found to strongly induce endogenous Ascl1 levels in murine Neuro 2a cells. Interestingly, the newly observed synergistic effect of these two systems seemed to depend on the chosen set of gRNAs and was specific for Ascl1 and the murine cell system used. For the first time SAM and VPR systems were utilized in this thesis to directly convert astrocytes to neurons. These promising data are a basis for further improvements in order to further increase the reprogramming efficiency and also guide reprogrammed cells towards a specific neuronal subtype such as mdDA neurons.

Currently, optimized versions are tested such as a SAM split-dCas9-VPR system driven by a GFAP promoter which restricts expression to astrocytes. This should help simplifying the quantitative analysis where newly converted neurons must be distinguished from neurons already present at the beginning of the primary cortical cell population. One central task for the future will be the simultaneous induction of multiple genes in order to generate specific neuronal subtypes. For this, multiplexing is required which allows expression of several gRNAs from a single vector.

Finally, the developed system will be tested in vivo by injecting the SAM dCas9-VPR system into the murine cortex to analyze the reprogramming and rescue potential in disease models such as 6-OHDA treated mice.

Taken together, in this thesis successful proof-of-principle experiments show the highly promising potential of the CRISPR/Cas9 technology for the direct conversion of somatic cells which will now be a basis for further developments.

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MATERIAL AND METHODS

8 | MATERIAL AND METHODS

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8 Material and methods