Haploinsufficiency arises when one copy of a gene is functionally lost, often through nonsense or frameshift mutations or small chromosomal deletions. The resulting monoallelic expression is not sufficiently compensated for by the intact allele, ultimately leading to decreased expression of the gene product and resulting in pathologic phenotypes (1). What are the therapeutic options for diseases rooted in insufficient gene expression? One possible viable option is to restore normal gene expression levels by enhancing their transcription in a targeted fashion. On page 246 in this issue, Matharu et al.(2) report a CRISPR-based gene-activation approach that can increase the expression of normal endogenous genes in a tissue-specific manner, setting the stage for the development of new gene-regulating therapies for gene dosage–associated diseases.

Among the emerging applications of CRISPR-based gene editing are techniques that use a catalytically inactive Cas9 enzyme (dCas9) fused to a protein domain to modulate transcription (3). These fusion proteins can be recruited by way of guide RNAs (gRNAs) to specific genomic locations, including promoters and cis-regulatory elements such as enhancers, which regulate gene expression. If the recruitment site is transcriptionally competent, the result is activation (CRISPRa) or repression/interference (CRISPRi) of transcription. Although this strategy has been applied in human cell culture and animal models (45), the ultimate task of employing CRISPRa to therapeutically rescue pathologic gene expression has not been fully realized. Matharu et al. use CRISPRa to restore the expression of two haploinsufficient genes, single-minded 1 (Sim1) and melanocortin 4 receptor (Mc4r), to physiological amounts in mouse models of severe early-onset obesity. Haploinsufficiency of either gene causes severe obesity in humans, and previous work in mice established that SIM1 and MC4R control eating behavior through their expression in the hypothalamus (68); therefore, a relevant therapeutic intervention would target gene expression specifically in the hypothalamus.

Because Sim1 and Mc4r are expressed in multiple tissues, an important first step was to address whether it is feasible to modulate expression in a tissue-specific manner. The authors tested two approaches, focusing initially on Sim1: (i) Target CRISPRa to the promoter of the remaining functional Sim1gene to enhance expression wherever Sim1 was already active, and (ii) target CRISPRa to a 270-kb distal enhancer that controls Sim1 expression specifically in the hypothalamus (see the figure). Both approaches were employed in transgenic animals expressing the CRISPRa reagents (dCas9 fused to the transcriptional activator VP64), as well as recombinant adeno-associated virus (rAAV)–mediated delivery of CRISPRa directly into the hypothalamus. In all cases, hypothalamic Sim1 expression was restored to wild-type levels and the mice did not become obese, demonstrating robust prevention of a haploinsufficient phenotype by enhancing endogenous gene expression. Interestingly, the authors found that they could manipulate Sim1expression exclusively in the hypothalamus by targeting the hypothalamic enhancer instead of the Sim1 promoter, indicating that to obtain tissue-specific transcriptional modification, CRISPRa will likely need to be deployed to tissue-specific regulatory elements. Injection of rAAV-based CRISPRa into the hypothalamus of Mc4r haploinsufficient mice similarly prevented obesity, further demonstrating the strength of this approach.

This strategy illustrates what could emerge as an important new approach to treating gene expression disorders and raises the possibility of expanding the scope of CRISPRa and CRISPRi technology to treat diseases that involve pathogenic overexpression of a gene, particularly in cancer. For example, somatic mutations in a subset of pediatric T cell acute lymphoblastic leukemia (T-ALL) result in the formation of a highly active enhancer that drives oncogenic TAL1 gene overexpression (9). Moreover, MYC gene expression in human B cell acute myeloid leukemia (AML) was recently shown to be dependent on a 1.7-megabase distal enhancer element (10). Both studies demonstrated that disrupting these enhancer elements negatively affected cancer cell survival, providing a precedent for developing CRISPRi as a therapeutic approach to inactivate cancer-promoting enhancers. Although transcription factors such as TAL1 and MYC are among the most potent oncoproteins, targeting them with small-molecule inhibitors has proven challenging. The results presented by Matharu et al. suggest that it should be possible to circumvent protein-targeted therapies by quelling oncogene expression at its source—transcription.

Enhancing endogenous gene expression with CRISPRa

Most genes are biallelically expressed; however, heterozygous mutations can cause haploinsufficiency, resulting in 50% less functional protein. Recruitment of CRISPRa to the endogenous promoter or enhancer of the gene in mouse models causes up-regulation of the wild-type copy, leading to normal expression levels.



A key advancement in the study by Matharu et al. is their use of rAAV to deliver CRISPRa reagents in vivo. For a CRISPR-based therapeutic to be relevant for use in humans, it will likely need to be packaged within a virus and administered intravenously, because most targeted cell types will not be available for ex vivo manipulation and implantation. rAAV is nonpathogenic and displays a high delivery potential, making it a viable option for effectively introducing CRISPR reagents to human cells. CRISPRa and CRISPRi approaches have the added benefit of modulating gene expression without modifying the genome, thereby avoiding potential off-target mutations. Thus, pairing CRISPRa with rAAV to treat a gene expression disorder in vivo is an important step forward in the development of expression-based therapeutics.

Although Matharu et al. demonstrate that CRISPR-based up-regulation of a haploinsufficient gene can prevent obesity, this study also raises the important question of whether a disease phenotype can be reversed. Because the authors administered CRISPRa reagents to mice at 4 weeks of age—before the onset of obesity—they did not address the potential to rescue the phenotype later in life. Many haploinsufficient disorders in humans are likely to be therapeutically actionable only after the disease phenotypes are partially or fully established. Future experiments should test the therapeutic benefit of targeting gene expression with the goal of reversing a haploinsufficient phenotype. Additionally, it is important to recognize that many enhancers are dynamic, meaning that they may act at specific developmental stages and change their tissue specificity with time (11). Fortunately, the authors were able to capitalize on a developmentally stable tissue-specific enhancer, although it is unclear how often this will be the case for targeting enhancers of other haploinsufficient genes.

Naturally occurring and pathogenic gene regulatory DNA elements provide a tailored therapeutic route to targeting gene expression. The results presented by Matharu et al. underscore the importance of identifying and carefully characterizing the enhancers that control gene expression. Large-scale efforts have identified thousands of putative enhancers in hundreds of human cell types. However, cell types representing diverse disease states, particularly from human patients, remain understudied. Knowing the full repertoire of gene regulatory elements and their target genes (12) in these cell types is likely to provide critical insight that can be exploited for CRISPR-based therapeutic approaches to modify gene expression.



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