Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p < 0.05) when compared to control transfections while not affecting expression of the nondisease allele. This study demonstrates the potential of allele-specific gene modification using TALE proteins, and provides a foundation for targeted treatment for individuals suffering from Huntington's or other genetically linked diseases.

Figure 1.

Transcription activator-like effector (TALE) design. The repeat variable diresidue (RVD) sequence can be seen by the colored lettering. The target sequence is in black text.For single-nucleotide polymorphism (SNP) targeting, the allele mismatch can be seen in the outlined box. The effector domain for each plasmid is shown in the right column.

Figure 2.

TALE binding assay. (A) TALE binding was assessed using a luciferase-binding assay in which the TALE was cloned with an activator effector domain (VP64) and cotransfected into HEK293 cells with a luciferase reporter plasmid containing the TALE target region. All TALE constructs demonstrated luciferase expression when compared to control transfections (n = 4). (B) Toxicity was measured 48 h posttransfection with Lipofectamine 3000 by flow cytometry with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI). Cells were considered viable if they were negative for annexin V/PI. No differences were observed in toxicity between the phosphoglycerate kinase (PGK) empty vector and the TALE targeting SNP sites or CAG regions (n = 3). (C) Protein aggregation was quantified with a Western blot for anti-ubiquitin. No overall between-group differences were observed in the expression of ubiquitin protein aggregation [F(8, 26) = 0.512, p = 0.832] (n = 5). (D) Allele expression was quantified using a Life Technologies SNP genotyping assay. One-way ANOVA revealed no significant between-group differences in expression of the healthy allele [F(4, 18) = 0.994, p = 0.443], suggesting that the TALE does not target the healthy allele. A significant between-group difference was observed for expression of the mutant allele [F(4, 18) = 3.021, p = 0.05]. LSD post hoc analysis revealed a significant difference in mutant allele expression between control (pPGK empty) and CAG-FR (p = 0.007), T7 (p = 0.009), T3γ (p = 0.021), and T2 (p = 0.010). LSD post hoc analysis revealed a significant difference between mutant and healthy allele expression in CAG-FR (p = 0.001), T3γ (p = 0.05), T2 (p = 0.022), and a trend in T7 (p = 0.061) (n = 6). *Significant from Control.

Figure 3.

Transfection efficiency was measured using fluorescent microscopy. (A) All TALE transfections resulted in approximately 15–23% efficiency. (B) Fluorescent activated cell sorting to purify only the cells receiving TALE demonstrated approximately 68% reduction of the mutate allele 24 h after sorting (n = 3). GFP, green fluorescent protein.

Figure 4.

Application to the patient population. In theory, allele-specific targeting using TALE could potentially cover 100% of the patient population. The use of multiple TALEs may be capable of covering 100% of the patient population, allowing flexibility for either allelic silencing, pending SNP allele analysis, or CAG collapse in regards to potential gene therapies. HTT, Huntingtin gene. Adapted from Kay et al. (22).