2019 Lukina (re-shown), Animal models of GD (continued), hGBA-L444P-tg8 mouse, GCB Mouse Model Substrate Comparison
2019 Lukina Eliglustat (re-shown)
| Biomarker | Normal Range | Baseline | Year 8 | Percent Reduction |
|---|---|---|---|---|
| Glucosylceramide (GL-1) (μg/mL) (n=18) | <2.0 to 6.6 | 12.15 | 2.70 | -80% |
| Plasma glucosylsphingosine (Lyso-GL-1) (ng/mL) (n=16) | <5 | 624 | 47.6 | -92% |
| Chitotriosidase (nmol/hr/mL) (n=17) | <15 to 181 | 8084 | 902 | -91% |
| CCL18 (ng/mL) (n=18) | 17 to 246 | 3560 | 442 | -87% |
Lukina chart x-axis: Years on Eliglustat (0 to 8) with sample sizes (n=24-25), (n=20-21), (n=15-19), (n=17-19), (n=17-18), (n=16-18), (n=17-18), (n=16-18), (n=16-18). y-axis: Median Percent Change from Baseline (0% to -100%). Legend: GL-1, CCL18, Chitotriosidase*, Lyso-GL-1.
Animal models of GD (continued)
| Generation method | GBA activity | Phenotype | |||
|---|---|---|---|---|---|
| KO | GBA KO the GBA-/- | mouse | insertion of a neo-cassete in exons 9 and 10 in the GBA gene | <4% | glucosylceramide accumulation in the lung, liver, and brain. However, the GBA-/- mouse exhibits neonatal death as a result of a marked transepidermal water loss due to altered skin permeability. (This is likely due to the lesser hydrolytic efficiency of the N370S enzyme toward the longer chain fatty acid acyl moieties on GC in murine skin vs. human13,14,15) |
| Point mutation | RecNcil (L444P/A456P) point mutant mice | A single insertion mutagenesis procedure (SIMP) | <10% | died soon after birth due to compromised skin barrier function | |
| L444P/L444P point mutant mice | <20% | died soon after birth due to compromised skin barrier function, ↑ a-syn in striatum | |||
| N370 |
| ||||
| GBAL444P/L444P;Ugcg+/+ | crossing L444P+/- mice with a mouse bearing a knockout in glucosylceramide synthase (Ugcg), | 15-20% | ↑ improved life span (50% of the mice survive for up to a year), However, these mice lack the typical features of GD such as: ↑ glucosylceramide or Gaucher cells | ||
| human GBA-L444P heterozygous KI mice (Gba-wt/L444P) human GBA-L444P x 1 copy + mouse Gba-wt x 1 copy (그냥 GBA heteroz carrier 모델이네) | (generated at Shonan) |
[shonan findings]
| |||
| Point mutation | D409H, D409V, or V394L | 4-10% | similar life span and also lack gross phenotypic abnormalities and glucosylceramide accumulation | ||
| The hGBA-L444P-tg8 mouse: 8 copies of human GBA ((no mouse Gba gene) (this mice is 'hemizygous' and still called 'transgenic') 그럼 이게 homoz 결과인 셈이고 GD model 이라고 분명히 GBA activator PRC1 에서 말함.. |
GBA activator project uses this! this strain expresses human GBA L444P mutant protein only, (Sanders, 2013 #909) Cerebrum: gba activity is 32% control (table2) So, =GlcCer, ↑(x2, ~7 vs ~12.5 at 45w, at 25w: 6 vs 8) GlcSph cerebellum: So, =GlcCer, ↑(x2) GlcSph | ||||
hGBA-L444P-tg8 figures and notes
Panels A-D show Glucosylsphingosine (y-axis label Glucosylsphingosine [pmol/mg prot]) per Transgenic strain (Gba+/-, N370S, L444P) across SPLEEN, LIVER, CEREBRUM tissues. Panel B y-axis range visible: 0.0 - 16.0.
- Ambroxol response (100mpk 14 days): js no response
Chaperone-therapy panel legend:
- Vehicle
- Ambroxol: 100 mg/kg/day
- Isofagomine: 20 mg/kg/day
Caption (under tissue GCase activity panel):
Tissue GCase activity after 14-day chaperone therapy: Data points … orally dosed mice (A) hN370S-tg4/Gba-/-; (B) hL444P-tg8/Gba-/-
- RD DDU (20200916): ↑ GlcCer, ↑ GlcSph
- 20201030 Natalia) . Liver histology data from the L444P model natural history study indicates that there might be mosaic transgene silencing. I am waiting for the rest of the histology. If we see the same silencing in other tissues, the model will not be usable for testing small molecules. I have attached paper on the L444P model that Gene Therapy team is using. Below is a liver image stained for transgene. Transgene is expressed under ubiquitous promoter and all liver cells are expected to stain. Instead, we observe patchy pattern (js: this may explain not very high GlcCer & GlcSph accumulation). This means that even a very potent small molecule can clear substrate from a small fraction of the cells that actually express mutant GBA that can be corrected. Efficacy will not be evident from tissue homogenate or in any functional tests because most of the cells will still have the same level of substrate as in untreated animals. Therefore, we need to see what is going on with transgene expression in other organs before we can decide if this model can be used
- 20201117: male에서 GBA staining↑, variable substrate, natalie: 왜 hepatocyte 만에서만 gba보이나 (not Kupper cell), ubiquitous promoter인데.
- ‘RD_NS joint team meeting December 2020_V2’: very little Brain GlcSph accumulation, and (only) little brain GlcSph reduction by GT
GCB Mouse Model Substrate Comparison
GCB Mouse Model Substrate Comparison Lyso-GL1 chart (grouped bars):
- Series:
L444P tg (14-15 wks),N370S tg (14-19 wks),D409V (8-14 wks) - Tissues:
Spleen,Bone Marrow,Liver,Brain,Serum
GCB Mouse Model Substrate Comparison Total-GL1 chart (grouped bars):
- Series:
L444P tg (10 wks),N370S tg (15-20 wks),D409V (8-14 wks) - Same tissue groups
Lyso-GL1 Comparison in Mice Treated with rhGCB (L444P CIT20-09(A), D409V GCB.06.19):
- Legend:
Vehicle,rhGCB - Tissues:
Spleen,Bone Marrow,Liver,Brain,Serum
Note: below 20200930_L444P,D409V mic_BL (by DMPK)
Cortex GlcSph plot — y-axis Concentration; lower portion clipped at the photo edge.
D409V hetero (start)
- below 20200930_L444P,D409V mic_BL (by DMPK): ↑ GlcSph
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| GCB Mouse Model figures / x-axis units | Concentration y-axis units | the y-axis unit text is too small to read on this capture and is omitted. |