Spell Checking Nature: Development of a CRISPR- Mediated Gene Editing Approach for the Treatment of Pathogenic Duplications

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to progressive muscle deterioration, loss of ambulation, and respiratory complications. It is caused by genetic mutations that result in the absence of dystrophin protein expression needed for muscle function. Despite significant advances in our understanding of the pathogenesis of DMD, no curative treatment has been identified to date and the disorder has a life-limiting disease trajectory. Recently, we have pioneered an approach to successfully remove large duplications in patient cells. We first tested this approach in vitro by removing a multi-exon (18-30) duplication of 139 kb in the DMD gene using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated Nuclease (Cas9) with a single guide. To test our treatment approach in vivo, I first generated a mouse model harboring a multiexon duplication of 136.8 kb in Dmd using CRISPR/Cas9. This first multiexon duplication model of DMD specifically mimics a patient duplication of Exons 18-30. Molecular and functional characterization of this model reveals dystrophin deficiency with characteristic markers of dystrophic muscle. Furthermore, using our previously described CRISPR/Cas9 single guide strategy, we have for the first time treated a large genomic duplication in vivo and shown successful removal of the duplication fragment leading to restoration of full-length dystrophin in skeletal and cardiac muscles. Additionally, histopathological analysis shows that treated mice have less indications of dystrophy including fewer centrally localized nuclei as well as significantly improved muscle function. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders caused by duplications.Ph.D.2021-06-22 00:00:0

The POZ-ZF Transcription Factor Kaiso (ZBTB33) Induces Inflammation and Progenitor Cell Differentiation in the Murine Intestine

<div><p>Since its discovery, several studies have implicated the POZ-ZF protein Kaiso in both developmental and tumorigenic processes. However, most of the information regarding Kaiso’s function to date has been gleaned from studies in <i>Xenopus laevis</i> embryos and mammalian cultured cells. To examine Kaiso’s role in a relevant, mammalian organ-specific context, we generated and characterized a Kaiso transgenic mouse expressing a murine Kaiso transgene under the control of the intestine-specific <i>villin</i> promoter. Kaiso transgenic mice were viable and fertile but pathological examination of the small intestine revealed distinct morphological changes. Kaiso transgenics (<i>Kaiso^Tg/+</i>) exhibited a crypt expansion phenotype that was accompanied by increased differentiation of epithelial progenitor cells into secretory cell lineages; this was evidenced by increased cell populations expressing Goblet, Paneth and enteroendocrine markers. Paradoxically however, enhanced differentiation in <i>Kaiso^Tg/+</i> was accompanied by reduced proliferation, a phenotype reminiscent of Notch inhibition. Indeed, expression of the Notch signalling target HES-1 was decreased in <i>Kaiso^Tg/+</i> animals. Finally, our Kaiso transgenics exhibited several hallmarks of inflammation, including increased neutrophil infiltration and activation, villi fusion and crypt hyperplasia. Interestingly, the Kaiso binding partner and emerging anti-inflammatory mediator p120^ctn is recruited to the nucleus in <i>Kaiso^Tg/+</i> mice intestinal cells suggesting that Kaiso may elicit inflammation by antagonizing p120^ctn function.</p></div

<i>Kaiso^Tg</i>^/+ mice display decreased HES-1 expression in the small intestine.

<p>Both Non-Tg and <i>Kaiso^Tg</i>^/+ tissues displayed nuclear HES-1 expression in the crypts of the small intestine, however <i>Kaiso^Tg</i>^/+ tissue displays significantly decreased HES-1 expression in the villi. Quantitative RT-PCR showed a significant decrease in HES-1 expression in <i>Kaiso^Tg</i>^/+ mice. Values were first normalized to the GAPDH housekeeping gene, followed by normalizing to non-Tg HES-1 expression (** represents p<0.05).</p

Generation of transgenic mouse lines ectopically expressing <i>villin</i>-Kaiso.

<p>(<b>A</b>) Myc-tagged murine <i>Kaiso</i> cDNA was cloned downstream of the 9 kb v<i>illin</i> promoter sequence. (<b>B</b>) The transgene copy number in each transgenic line was evaluated via PCR. Line A transgenic animals have the greatest copy number. (<b>C</b>) RT-PCR confirmed expression of the Kaiso transgene in <i>villin</i>-expressing tissues of transgenic mice, <i>i.e.</i> the small intestine, large intestine, and kidneys. (<b>D</b>) Immunoblot analysis shows increased Kaiso expression in both small and large intestines in Kaiso transgenic (<i>Kaiso^Tg</i>^/+) Line A mice compared to non-transgenic (Non-Tg) siblings.</p

Kaiso transgenic mice exhibit inflammation of the intestinal mucosa.

<p>(<b>A</b>) <b><u>H</u></b>ematoxylin and <b><u>e</u></b>osin (H&E) stained sections were used to measure villi length (red bracket; ∼80 villi/mouse) and crypt depth (black bracket; ∼800 open crypts/mouse). <i>Kaiso^Tg</i>^/+ display increased crypt depth compared to their Non-Tg siblings, p = 0.001. (<b>B</b>) <i>Kaiso^Tg</i>^/+ mice exhibit increased immune cell infiltration of the lamina propria (yellow demarcated area) accompanied by increased MPO activity compared to their Non-Tg siblings, p = 0.014. (<b>C</b>) Line B mice do not exhibit immune cell infiltration or enhanced MPO activity compared to Non-Tg siblings. ** represents significance.</p

Subcellular localization and expression of ectopic Kaiso in Line A <i>Kaiso^Tg</i>^/+ small intestines.

<p><i>Kaiso^Tg</i>^/+ mice display strong nuclear Kaiso in the villi and crypt cells, compared to non-transgenic mice (Non-Tg), which mainly display weak Kaiso staining in the cytoplasm. Additionally, <i>Kaiso^Tg/+</i> mice display strong nuclear c-Myc staining corresponding to ectopic myc-tagged Kaiso expression, while Non-Tg mice display cytoplasmic c-Myc expression.</p

Secretory cell lineages are expanded in the intestines of <i>Kaiso^Tg</i>^/+ mice.

<p>(<b>A</b>) PAS stain for Goblet cells (black arrowheads) revealed increased numbers of Goblet cells in both the villi and crypts of <i>Kaiso^Tg</i>^/+ intestines, p = 0.011 & 0.002. (<b>B</b>) Lysozyme staining revealed increased Paneth cell numbers in <i>Kaiso^Tg</i>^/+ mice, p = 0.017. (<b>C</b>) Synaptophysin positive enteroendocrine cells (arrowheads) are increased in <i>Kaiso^Tg</i>^/+ mice, p = 0.031. n = 3 mice/genotype; measurements performed by two independent blind observers; T-test used for p-value. ** represents significance.</p

Targeted genome editing in vivo corrects a Dmd duplication restoring wild‐type dystrophin expression

Abstract Tandem duplication mutations are increasingly found to be the direct cause of many rare heritable diseases, accounting for up to 10% of cases. Unfortunately, animal models recapitulating such mutations are scarce, limiting our ability to study them and develop genome editing therapies. Here, we describe the generation of a novel duplication mouse model, harboring a multi‐exonic tandem duplication in the Dmd gene which recapitulates a human mutation. Duplication correction of this mouse was achieved by implementing a single‐guide RNA (sgRNA) CRISPR/Cas9 approach. This strategy precisely removed a duplication mutation in vivo, restored full‐length dystrophin expression, and was accompanied by improvements in both histopathological and clinical phenotypes. We conclude that CRISPR/Cas9 represents a powerful tool to accurately model and treat tandem duplication mutations. Our findings will open new avenues of research for exploring the study and therapeutics of duplication disorders