SUPT4H1-edited stem cell therapy rescues neuronal dysfunction in a mouse model for Huntington's disease

Abstract

Huntington's disease (HD) is a severe inherited neurological disorder caused by a CAG repeat expansion in the huntingtin gene (HTT), leading to the accumulation of mutant huntingtin with polyglutamine repeats. Despite its severity, there is no cure for this debilitating disease. HTT lowering strategies, including antisense oligonucleotides (ASO) showed promising results very recently. Attempts to develop stem cell-based therapeutics have shown efficacy in preclinical HD models. Using an HD patient's autologous cells, which have genetic defects, may hamper therapeutic efficacy due to mutant HTT. Pretreating these cells to reduce mutant HTT expression and transcription may improve the transplanted cells' therapeutic efficacy. To investigate this, we targeted the SUPT4H1 gene that selectively supports the transcription of long trinucleotide repeats. Transplanting SUPT4H1-edited HD-induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) into the YAC128 HD transgenic mouse model improved motor function compared to unedited HD iPSC-NPCs. Immunohistochemical analysis revealed reduced mutant HTT expression without compensating wild-type HTT expression. Further, SUPT4H1 editing increased neuronal and decreased reactive astrocyte differentiation in HD iPSC-NPCs compared to the unedited HD iPSC-NPCs. This suggests that ex vivo editing of SUPT4H1 can reduce mutant HTT expression and provide a therapeutic gene editing strategy for autologous stem cell transplantation in HD.

Fig. 1. SUPT4H1 gene editing increased neural differentiation and reduced mHTT protein in Q57 HD iPSC-NPCs.

a In vitro experimental scheme. The Q57 HD iPSC line was maintained for approximately 7 d (D-7). The dissociated cells were cultured in SFEBq media for 8 d (D-8). Embryoid bodies were dissociated in NPC media, then attempted Neon electroporation. The NPCs were maintained for several days and showed neuronal differentiation after 42 d (after iPSC culture, D-number; after gene editing, number day; blue color). b NPC immunostaining (positive marker: NESTIN, SOX2) following gene editing. c mHTT protein immunostaining (EM48) demonstrated a decreased HD phenotype in SUPT4H14-edited Q57 HD iPSC-NPCs (SUPT4H1 gRNA). d Western blot analysis of EM48 expression (n = 3, *p < 0.05, **p < 0.01). e MAP2 immunostaining for morphology exhibited neuronal dendrite maturation in SUPT4H1-edited Q57 HD iPSC-derived neurons. f. Western blot analysis of HMW and LMW MAP2 expression (n = 3, *p < 0.05, **p < 0.01). g The number of neurite and mean neurite length were increased in MAP2-positive cells of the SUPT4H1 gRNA group compared to the AAVS1-edited Q57 HD iPSC-derived neuron (AAVS1 gRNA) (each group: 100 cells, scale bar: 20 μm). h GFAP immunostaining. i Western blot analysis for GFAP and Kir4.1 revealed normal astrocytic function in the SUPT4H14 gRNA group. (n = 3, *p < 0.05, **p < 0.01). j Real-time PCR analysis for astrocytic development in AAVS1-edited Q57 HD iPSC-derived NPCs. The CTL (control) and SUPT4H1-edited groups showed significant increases in CDH2, a radial glial cell marker, and S100β, a maturate astrocyte marker, mRNA compared to the AAVS1-edited group (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001). Data were analyzed using two-way ANOVAs followed by Tukey’s post hoc tests with SPSS software or GraphPad Prism. The error bars on the bar charts represent the standard deviation.

Fig. 2. Transplanted SUPT4H1-edited Q57 HD iPSC-NPCs promote neuronal regeneration and functional recovery in 9 m-old YAC128 mice.

a, b Functional motor recovery was monitored every month using the accelerating rotarod and grip strength tests. YAC128 mice transplanted with SUPT4H1-edited Q57 HD iPSC-NPCs showed significantly accelerated motor recovery compared to the Q57 HD iPSC-NPCs transplants. All transplant mice significantly recovered at 1 m post-transplantation and this recovery was maintained until 3 m post-transplantation in the SUPT4H1-edited Q57 HD iPSC-NPCs group (WT = 9, Media = 8, Q57 = 8, and Q57/SUPT4H1 = 8; Media vs. Q57 or Q57/SUPT4H1: *p < 0.05 and **p < 0.01; Q57 vs. Q57/SUPT4H1: ⁺p < 0.05). c Immunostaining revealed an increased NeuN-positive area density in the striatum of YAC128 mice transplanted with SUPT4H1-edited Q57 HD iPSC-NPCs (scale bar: 100 μm). d Image J analysis showing a significant increase in the striatal density of mice that received SUPT4H1-edited Q57 HD iPSC-NPCs transplants (n = 3, *p < 0.05, **p < 0.01). Data were analyzed using two-way ANOVAs followed by Tukey’s post hoc tests with SPSS software. The error bars on the bar charts represent the standard deviation.

Fig. 3. SUPT4H1-edited Q57 HD iPSC-NPC transplantation reduced SUPT4H1 expression and increased cell survival in 9-m-old YAC128 mice.

a Double staining for transplanted cell markers (hNESTIN) and Spt4 protein revealed that transplanted unedited or SUPT4H1-edited Q57 HD iPSC-NPCs decreased Spt4 expression (scale bar: 20 μm). b The number of pixels for Spt4 expression quantified using ZEN black software analysis were significantly decreased in transplanted SUPT4H1-edited Q57 HD iPSC-NPCs (n = 3, **p < 0.01). c Immunostaining for hNu 12 week post-transplantation showed human cell survival in the striatum of YAC128 mice transplanted with unedited or SUPT4H1-edited Q57 HD iPSC-NPCs (scale bar: 20 μm). d Quantification of hNu-positive cell numbers in the 6–7 sections included the striatal lesion site (n = 5). Data were analyzed using two-way ANOVAs followed by Tukey’s post hoc tests with SPSS software. The error bars on the bar charts represent standard deviation.

Fig. 4. Transplanted unedited or SUPT4H1-edited Q57 HD iPSC-NPCs have different neuronal differentiation potential in YAC128 mice.

a Double staining for the transplanted cell marker (hNu) and MSN marker (DARPP-32) revealed that transplanted unedited or SUPT4H1-edited Q57 HD iPSC-NPCs differentiated into MSNs (scale bar: 20 μm). b Double staining for the human-specific MSN marker (hDARPP-32) and HD phenotype marker (EM48) in the striatum demonstrated decreased EM48 expression in transplanted SUPT4H1-edited Q57 HD iPSC-derived MSNs (scale bar: 20 μm). c Immunostaining to identify hMAP2-positive cells exhibited neuronal dendrite degeneration in the transplanted unedited Q57 HD iPSC-NPCs-derived neurons (scale bar: 20 μm). d Quantification of neurite numbers and max neurite length indicated more neuronal maturation in transplanted SUPT4H1-edited Q57 HD iPSC-NPCs-derived MAP2-positive cells than the unedited cells. (Photograph: same color is one cell for analysis, scale bar: 100 μm, for two regions in 6–7 brain sections of each group, ***p < 0.001). Data were analyzed using two-way ANOVAs followed by Tukey’s post hoc tests or Student’s t tests with GraphPad Prism. The error bars on the bar charts represent the standard deviation.

Fig. 5. Transplanted unedited or SUPT4H1-edited Q57 HD iPSC-NPCs have different astrocytic differentiation potential in YAC128 mice.

a Double staining for the transplanted cell marker (hNu) and astrocyte marker (GFAP) revealed that transplanted unedited or SUPT4H1-edited Q57 HD iPSC-NPCs differentiated into astrocytes (scale bar: 20 μm). b Double staining for the human-specific astrocyte marker (hGFAP) and HD phenotype marker (EM48) in the striatum revealed decreased EM48 expression in transplanted SUPT4H1-edited Q57 HD iPSC-derived astrocytes (scale bar: 20 μm). c Immunostaining to identify C3 expression revealed reactive astrocytes in transplanted unedited Q57 HD iPSC-NPCs-derived astrocytes (scale bar: 20 μm). d Quantification of the GFAP-positive area and intensity indicated more activation in the transplanted SUPT4H1-edited Q57 HD iPSC-NPCs-derived astrocytes than the unedited cells. (Photograph: same color is one cell for analysis, scale bar: 100 μm, for two regions in 6–7 brain sections of each group, ***p < 0.001). Data were analyzed using two-way ANOVAs followed by Tukey’s post hoc tests or Student’s t tests using GraphPad Prism. The error bars on the bar charts represent standard deviation.