Genome Editing for CNS Disorders

Abstract

Central nervous system (CNS) disorders have a social and economic burden on modern societies, and the development of effective therapies is urgently required. Gene editing may prevent or cure a disease by inducing genetic changes at endogenous loci. Genome editing includes not only the insertion, deletion or replacement of nucleotides, but also the modulation of gene expression and epigenetic editing. Emerging technologies based on ZFs, TALEs, and CRISPR/Cas systems have extended the boundaries of genome manipulation and promoted genome editing approaches to the level of promising strategies for counteracting genetic diseases. The parallel development of efficient delivery systems has also increased our access to the CNS. In this review, we describe the various tools available for genome editing and summarize in vivo preclinical studies of CNS genome editing, whilst considering current limitations and alternative approaches to overcome some bottlenecks.

Keywords: CNS; CRISPR/Cas; TALEs; ZFs; genome editing.

FIGURE 1

Gene editing tools and therapeutic approaches. (A) Gene editing tools are based on TALEs, ZFs and CRISPR/Cas platforms. Site-specific TALENs and ZFNs consist of two modules of TALEs and ZFs fused to the FokI nuclease. Both modules recognize adjacent sequences in opposite strands to promote the dimerization of FokI and sequence cleavage in a staggered fashion. In contrast, CRISPR/Cas systems hold intrinsic nuclease activity. Cas nucleases or Cas nickases are explored to produce either DSBs or SSBs in the targeted sequence, respectively. Alternatively, paired nickases targeting adjacent sequences in opposite strands generate staggered DSBs. (B) Gene editing therapeutic approaches rely on the intrinsic DNA repair mechanisms NHEJ and HDR after generation of DSBs. Gene disruption by NHEJ involves the introduction of indels after generation of DSBs at the coding region of a pathogenic gene, resulting in the formation of a premature stop codon. Gene correction by NHEJ implicates the targeting of the non-coding region of a pathogenic gene. It includes the removal of deleterious exons by the simultaneous cleavage in both upstream and downstream intronic regions and/or disruption of splicing regulation sites. Both gene repair and gene insertion by HDR involve the use of donor templates containing intended sequences flanked by homology arms. In the first case, the template is targeted to the pathogenic gene and contains the corrected sequence allowing gene restoration. In contrast, gene insertion by HDR targets safe harbor locations in the genome to introduce therapeutic transgene expression cassettes.

FIGURE 2

Base editing tools and therapeutic approaches. (A) Base editors consist of Cas nickases fused to cytosine (CBEs) or adenine ssDNA deaminases (ABEs). CBEs are fused to either AID or APOBEC1 (pink), which convert C into U, whereas ABEs are fused to an evolved TadA (TadA*) followed by a wild-type TadA fusion (brown), which convert A into I. The consequent G:U and T:I mismatches are then corrected by the cellular DNA repair mechanisms. To favor the correction of the non-edited nucleotides by the DNA mismatch repair machinery, the nickase introduces a “nick” in the unedited strand. The correction of the non-edited strand results in a final conversion of C:G into T:A base pairs and A:T into G:C base pairs by CBEs and ABEs, respectively. CBEs are usually fused to the UGI to prevent the rapid removal of uracil by BER (blue). (B) Base editing therapeutic approaches include the repair of pathogenic genes by correcting point mutations or the inactivation of toxic genes by generating a premature stop codon.

FIGURE 3

Transcriptional regulators, epigenetic modifiers, and therapeutic approaches. (A) Gene expression regulation tools are generated by fusing TALEs, ZFs or dCas proteins to scaffold transcriptional modulators or to epigenetic modifiers (B) Therapeutic approaches by transcriptional regulation. Transcriptional activation or repression is explored to upregulate therapeutic genes or to downregulate deleterious genes, respectively. Transcriptional activators are targeted at the promoter region whereas transcriptional repressors are usually targeted downstream to the transcription starting site to further block the RNA polymerase activity. (C) Therapeutic approaches through histone modification. Histone (de)acetylases and (de)methylases are the most common employed enzymes to modify histone marks and the epigenetic activation or inhibition effect of such modifications is frequently context-specific. (D) Therapeutic approaches by editing the DNA methylation state. Epigenetic editors based on DNA demethylases are used to activate gene expression whereas the ones based on DNA methylases result in gene expression inhibition.