METHODS AND COMPOSITIONS FOR TREATING PAX6-DEFICIENCY RELATED DISEASE

Abstract

The present invention relates to an agent for increasing PAX6 gene and/or protein expression in a subject in need thereof. The insertion of a nonsense mutation found in patients allowed inventors to identify altered gene expression of PAX6 target genes, enhanced cell adhesion and reduced cell migration and proliferation. Remarkably, these parameters were rescued by recombinant PAX6 protein. Using this unique cellular model in which they inserted multiple copies of PAX6 responsive elements and tdTomato, they screened a FDA-approved library and identified in a first hit, 3 small compounds that enhance production of PAX6 protein while two of them did not modulate PAX6 gene expression. More particularly, they observed that the small compounds efficiently rescue in vitro migration of mutated limbal cells. Moreover, in the art, it is known that a decrease of PAX6 leads to an induction of diabetes. Small compounds identified by the inventors are suitable to treat PAX6-related deficiencies diseases such as aniridia and diabetes.

Claims

1-7. (canceled)

8. A pharmaceutical composition comprising an agent which increases PAX6 gene and/or protein expression.

9. The pharmaceutical composition according to claim 8, wherein said agent is duloxetine, ritanserin, topotecan and/or a derivative thereof.

10. (canceled)

11. A method for treating a PAX6-related deficiency diseases in a subject need thereof comprising a step of administering to said subject a therapeutically effective amount of an agent which increases PAX6 gene and/or protein expression.

12. The method of claim 11, wherein the PAX6-related deficiency disease is aniridia and/or diabetes.

13. The method of claim 11, wherein the agent is an aptamer, a small molecule, an antibody, a peptide, a polypeptide peptidomimetic or glycomimetic.

14. The method of claim 11, wherein the agent is duloxetine, ritanserin, topotecan and/or a derivative thereof.

15. The method of claim 14, wherein the duloxetine, the ritanserin, the topotecan and/or the derivative is/are formulated in a combined preparation.

16. The method of claim 11, wherein said agent is formulated for oral, cutaneous, topical or ocular use.

Description

FIGURES

[0104] FIG. 1: Topotecan can rescue PAX6 haploinsufficiency in vitro. Mutated (mut-LT) imbal stem cells were treated (dark blue) with 0.5 mM topotecan or DMSO (light blue) for 10 h and compared to normal limbal cells treated with DMSO (LT, white) by either qRT-PCR for PAX6-target gene expression (A) or western blot analysis for PAX6 protein production (B). The data illustrate several independent experiments (n=5 for A and n=3 for B). Oneway ANOVA followed by Dunnett's test was performed *p<0.05, **p<0.01.

[0105] FIG. 2: Topotecan can rescue migration of mutated limbal cells. Normal LT (blue) and mut-LT (orange and red) were tested for migration rate on IBIDI chambers. Mut-LT were treated (red) or not (orange) with 0.5 mM topotecan 2 h before migration start. DMSO was used as control. Numbers represent the percentage of remaining open area. n=2

[0106] FIG. 3: Topotecan activates the WT allele transcription in mutated cells but not in normal LSCs. qRT-PCR analysis for PAX6 expression was done on WT and mut-LSC+/− topotecan. Amplicons of mut-LSCs+Topotecan was sequenced and did not identified any amplification of the mutated allele. It suggests that topotecan does not act as forced read through the stop codon (like ataluren). Of interest, PAX6 in treated WT-LSCs is not enhanced, suggesting a fine regulation like in vivo.

[0107] FIG. 4: Ritanserine rescued PAX6 protein production. (A). Mutated LT (W) were treated (grey) or not (orange) with Ritanserine (1 mM) for 16 h and cell proliferation was measured and compared to untreated WT limbal cells (LT; blue). PAX6-target gene expression was analyzed by qRT-PCR. (B). Representative (over 3) western blot analyses on LT and W cells untreated and treated with MetOH buffer (buf) or Ritanserine (Rit) at 0.5, 1 or 5 mM.

[0108] FIG. 5: Duloxetine rescued PAX6 protein production. (A) qRT-PCR analysis of PAX6-target gene expression on WT control limbal cells (blue), or W mutant limbal cells (grery and orange) following treatment with buffer (grey) or 1 mM of Duloxetine (orange). (B) The data illustrate several independent experiments (n=5 for A and n=3 for B). One way ANOVA followed by Duneett's test was performed *p<0.05, **p<0.01. (C). Normal LSC (blue) and mut-LSC (orange and grey) were used as control for migration rate on IBIDI chambers. Mut-LSC were treated (orange) or not (grey) with 0.5 μM duloxetine 2 h before migration start. DMSO was used as control. Numbers represent the percentage of remaining open area; n=2.

EXAMPLE

[0109] Material & Methods

[0110] Cells and culture conditions: Human mutated limbal epithelial stem cells (mut-LSCs) were obtained by genome editing (Crispr/cas9) of primary limbal stem cells, as described in Roux et al. (Roux, 2018 Stem cells). mut-LSCs and WT controls were cultured, as described earlier (Roux, 2018 Stem cells), in Keratinocyte serum-free medium (K-sfm) (Gibco™, Life Technologies), supplemented with 25 μg/mL Bovine Pituitary Extract (BPE; Gibco™, Life Technologies), 0.2 ng/mL Epidermal Growth Factor (EGF, Peprotech), 0.4 mM CaCl.sub.2, 2 mM Glutamine (Gibco™, Life Technologies) and 100 U/mL Penicillin/Streptomicin (Gibco™, Life Technologies). Routine subcultures were obtained by detaching cells with StemPro™ Accutase™ Cell Dissociation Reagent (Gibco™, Life Technologies) and replating at 2,000 cells/cm.sup.2 (T-LSCs) or 3,000 cells/cm.sup.2 (PAX6.sup.+/− T-LSCs). All cells were negative for mycoplasma contamination (monthly tested).

[0111] Rescue experiments: Mut-LSCs were treated with each compound (Duloxetine, Ritanserine and Topotecan) directly added into the culture medium from 0.5 μg/mL to 4 μg/mL. No cell toxicity has been observed up to 10 μg/mL. The optimal concentration for each experiment (qRT-PCR, cell migration and cell adhesion) was 0.5 μg/mL. As positive control, 2 μg/mL recombinant PAX6 protein coupled with an 11R-tag (recPAX6, LD Biopharma Inc) was added, as described elsewhere (Roux et al. 2018 Stem cells).

[0112] qRT-PCR analysis: Cells were treated for 10 h with either compound or recPAX6 for rescue experiments, and harvested as a dry pellet. RNA was then extracted using RNEasy Mini kit (Qiagen) and cDNA were synthetized from 1 μg RNA using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR were performed in triplicate using 2λ SYBR Green PCR Master Mix (Absource Biotools). Expression of each gene was calculated using the 2.sup.−ΔΔCt method. Results are presented as fold change normalized to B2M house-keeping gene and relative to control (treated with protein buffer or untreated) T-LSCs. Specific primers sequences used are listed in Suppl. Table I.

[0113] Western blot analysis: Cells were harvested as a dry pellet, after 16 h or 24 h of treatment with each compound or 2 μg/mL recPAX6 protein or its buffer for rescue experiments, lysed in RIPA buffer supplemented with Protease and Phosphatase Inhibitor Cocktail EDTA-Free (Roche) for 15 min on ice and centrifuged for 15 min at 4° C. at 15,000×g. Protein concentration was measured using Pierce BCA Protein Assay kit (Thermo Fisher Scientific) following manufacturer instructions. Thirty micrograms of total protein or 10 ng of recPAX6 protein were loaded on a SDS-PAGE gel (10%), transferred to nitrocellulose membranes using semi-dry method. Membranes were pre-stripped using Re-Blot Plus Strong solution (EMD Millipore) for 15 min, blocked twice for 5 min in 5% milk solution and incubated in primary antibody (PAX6, ab2237, Millipore, 1/1,000) overnight at 4° C. After 3 washing of 10 min in TBS with 0.2% Tween (TBS-T), membranes were incubated for 1 h with secondary antibody (Goat anti-rabbit HRP, BD Pharmingen) diluted in TBS-T 5% milk solution at 1/1,000. Proteins were visualized using chemiluminescence detection (Clarity™ Western ECL Substrate, Bio-Rad) on a gel imaging system (ImageQuant LAS 4000). Membranes were stripped again, incubated 90 min at room temperature with ACTIN (sc1615, 1/500, Santa Cruz) antibody diluted in TBS-T 5% milk, washed 3×10 min in TBS-T and incubated for 1 h in secondary antibody (Rabbit anti-goat HRP, 1/20,000, Jackson ImmunoResearch) at room temperature before revelation. Signal quantifications were made using ImageJ 1.49 software (NIH). Results are presented after ACTIN normalization.

[0114] In vitro migration test: WT and mut-LSCs were seeded into culture-inserts 2 well at 25,000 cells per well in 6-well plates (IBIDI®). After adhesion, cells were treated with each compound at 0.5 μg/mL or recPAX6 as positive control for rescue experiments. Sixteen hours after plating, the inserts were removed creating a gap of 500 μm between cells. Patches were overlaid with culture medium and closure of the gap was monitored for 10 h, taking pictures at regular time intervals. Gap width was measured using ImageJ 1.49 software (NIH). Results are presented as percent of gap width normalized at 100% at t=0 h.

[0115] Statistical analysis: Data are expressed as means +/−SEM except for Gene Ontology charts as indicated and analyzed by Prism v7.04 (GraphPad software, Inc). Normality was first evaluated using Shapiro-Wilk test. Then, unpaired t-test (WT-LSCs vs mut-LSCs experiments), one-way ANOVA followed by multiple comparison Dunnett's test (rescue experiments, proliferation and WB figures) or two-way ANOVA followed by Bonferroni's test (migration assays) were performed, as indicated in legends, to calculate p-values. Differences are considered to be statistically significant from a p-value below 0.05.

[0116] Results:

[0117] As illustrated on FIGS. 1 to 5, each of the three compounds: Topotecan (FIGS. 1 to 3), Ritanserine (FIG. 4) or Duloxetine (FIG. 5) is able to rescue the production of endogenous PAX6 protein, PAX6-target gene expression and cell migration of mutated limbal stem cells, comparable to the rescue effect of recombinant PAX6 protein treatment, as described in Roux et al. (Roux 2018 Stem cells). Topotecan is able to rescue PAX6 gene expression at the RNA level. It suggests that duloxetine or ritanserine stabilizes PAX6 protein while Topotecan enhances its transcription.

REFERENCES

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