Combining gene-editing with brain imaging: from genes to molecules to networks

Abstract

"Receptors, transporters and ion channels are important targets for therapy development in neurological diseases, [...] but their mechanistic role in pathogenesis is often poorly understood. Gene-editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on whole-brain level. Here, we combine CRISPR/Cas9 gene-editing with in vivo PET and fMRI to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the VMAT2, involved in the storage and release of DA, makes it an excellent target for studying the gene networks relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the SNc of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR/Cas9-mediated VMAT2 KD. Simultaneous PET/fMRI was performed to inspect the molecular and functional brain adaptations, beyond the predicted dopaminergic changes. We found a regional increase in postsynaptic DA receptor availability, preceded by a reorganization of brain networks that adapt to reduced DA transmission states by becoming functionally connected and organized. We observed that FC adaptations are stage-specific and follow the selective impairment of presynaptic DA storage and release. Further, the observed hyperconnectivity within and between brain networks spreads from the contralateral thalamus and prefrontal cortical regions to the striata and hippocampi. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a novel tool to investigate the impact of specific genes on brain molecular and functional dynamics which will help to develop tailored therapies for normalizing brain function. The method can easily be transferred to higher- order species allowing for a direct comparison of the molecular imaging findings" [1]. 86 Future studies could benefit from in vivo reporter gene PET probes to quantitatively assess and monitor the Cas9 and sgRNA brain expression over time [38, 220]. Indeed, in vivo reporter gene imaging is a powerful tool to monitor gene therapy and image exogenous gene expression in the brain of preclinical models of neurological diseases. Despite several reporter genes have been developed in the last years, these show major limitations. Indeed, most available reporter gene systems are based on endogenously expressed genes, resulting in high background binding or low brain uptake. Here, we characterized the pharmacokinetics and metabolism of [11C]TMP, a novel PET reporter probe which binds to EcDHFR-engineered cells and shows potential for imaging ectopic gene expression in xenografted tumor models in vitro and in vivo [47]. We found that [11C]TMP presents several unfavorable characteristics; dependency on PgP efflux transport at the BBB, hindering its brain uptake in the rat species, and in vivo metabolism, hampering the PET data quantification. Our study shows that [11C]TMP is not a suitable PET reporter gene probe to image exogeneous gene expression in the rat brain, presenting low brain uptake and fast metabolism. Future studies should focus on the investigation of different targets and the development of [11C]TMP analogs with more favorable pharmacokinetics and detectability, which are neither PgP substrate nor rapidely metabolized.

[1].Marciano et al., PNAS, 2022