{"id":2547,"date":"2019-01-19T21:39:47","date_gmt":"2019-01-19T12:39:47","guid":{"rendered":"http:\/\/163.180.4.222\/lab\/?p=2547"},"modified":"2019-01-19T21:39:47","modified_gmt":"2019-01-19T12:39:47","slug":"gene-therapy-for-pathologic-gene-expression","status":"publish","type":"post","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=2547","title":{"rendered":"Gene therapy for pathologic gene expression"},"content":{"rendered":"

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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<\/em><\/a>). 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\u00a0et al.<\/em>(2<\/em><\/a>) 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\u2013associated diseases.<\/p>\n

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<\/em><\/a>). 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 (4<\/em><\/a>,\u00a05<\/em><\/a>), the ultimate task of employing CRISPRa to therapeutically rescue pathologic gene expression has not been fully realized. Matharu\u00a0et al.<\/em>\u00a0use CRISPRa to restore the expression of two haploinsufficient genes, single-minded 1 (Sim1<\/em>) and melanocortin 4 receptor (Mc4r<\/em>), 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 (6<\/em><\/a>\u20138<\/em><\/a>); therefore, a relevant therapeutic intervention would target gene expression specifically in the hypothalamus.<\/p>\n

Because\u00a0Sim1<\/em>\u00a0and\u00a0Mc4r<\/em>\u00a0are 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\u00a0Sim1<\/em>: (i) Target CRISPRa to the promoter of the remaining functional\u00a0Sim1<\/em>gene to enhance expression wherever\u00a0Sim1<\/em>\u00a0was already active, and (ii) target CRISPRa to a 270-kb distal enhancer that controls\u00a0Sim1<\/em>\u00a0expression 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)\u2013mediated delivery of CRISPRa directly into the hypothalamus. In all cases, hypothalamic\u00a0Sim1<\/em>\u00a0expression 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\u00a0Sim1<\/em>expression exclusively in the hypothalamus by targeting the hypothalamic enhancer instead of the\u00a0Sim1<\/em>\u00a0promoter, 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\u00a0Mc4r<\/em>\u00a0haploinsufficient mice similarly prevented obesity, further demonstrating the strength of this approach.<\/p>\n

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\u00a0TAL1<\/em>\u00a0gene overexpression (9<\/em><\/a>). Moreover,\u00a0MYC<\/em>\u00a0gene expression in human B cell acute myeloid leukemia (AML) was recently shown to be dependent on a 1.7-megabase distal enhancer element (10<\/em><\/a>). 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\u00a0et al.<\/em>\u00a0suggest that it should be possible to circumvent protein-targeted therapies by quelling oncogene expression at its source\u2014transcription.<\/p>\n

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