Team Meetings / Update Meeting To Ceri Links, TM Assays Summary, NBB LLOQ Tables, Brain/CSF Comparison, pS65-Ub Reagents, Ubiquitin Linkage, IVC, And Start Of Parkin Activity Assay
| Team meetings |
- Mouse poc study 에서 BM? → DMPK 와 논의하자 - 내 생각: MC1 ARG IN MICE POC study or NHP BD study? [`https://mytakeda.sharepoint.com/sites/InterACT-RAD-Pipeline/Parkin%20Gene%20Therapy/Shared%20Documents/Forms/AllItems.aspx?RootFolder=%2Fsites%2FInterACT%2DRAD%2DPipeline...`] |
| Update meeting to Ceri | [`https://mytakeda.sharepoint.com/sites/InterACT-RAD-Pipeline/Parkin%20Gene%20Therapy/Shared%20Documents/Forms/AllItems.aspx?RootFolder=%2Fsites%2FInterACT-RAD-Pipeline%2FParkin%20Gene%20Therapy%2FShared%20Documents%2FMeeting%20Minutes%20and%20Materials&FolderCTID=0x0120007803EDB3EB43F94B88445EA2823B1719&View=%7B1B1D5A2D%2D5E8C%2D41AB%2D978F%2D618DC69212AC%7D`] |
Timeline
[Parkin GT Scenarios and Timelines.pptx]
Resources: my PGRN in this document
-
Parkin protein:
- Parkin KO mice
- Sensitivity, linearity, stability, selectivity
- Human pooled CSF → PD Patient CSF
- Check cross-reactivity to mouse parkin (although we will need to measure only human parkin)
- MPTP study에서는, mice parkin 이 많이 있으므로 구분해야 할 것.
- How?
- MPTP study에서는, mice parkin 이 많이 있으므로 구분해야 할 것.
-
Option 1. Mouse Brain homogenate에서 detectability 확인 (이거라 함)
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Option 2. 위 과정 없이 바로 CSF 측정
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Option 3. Mouse CSF 에서 detectability 확인 (right after we confirm detectability in human CSF)
- How long does it take?
-
Questions
- Who analysis brain pS65Ub?
- Can we freeze mouse CSF and measure when our assays are ready?
- May be feasible, caveat is that we don’t normaly test longterm stability
- What does it take for the assay to be ready for mouse studies?
- Parkin BM timeline 등 자세히 없으니 만들자
- Confidence 측면에서) asset generation timeline 과 맞춘다
- Resource prioritization 필요함.
TM Assays
| Assay | Buffer LLOQ | Concentration note | NBB result-CSF | CSF | Required volume |
|---|---|---|---|---|---|
| Parkin | 0.061 pg/mL | 10 times concentration from 1 mL |
n 7 Minimum 0.7 Maximum 2.500 Range 1.800 Mean 1.257 SD 0.5740 SEM 0.2170 |
NBB sample For 1ml, 1.3/0.061 pg/ml = 21x (this is window), For 500ul, LLOQ is 0.12 pg/ml, so then window is only 11x. For 100ul, LLOQ is 0.6 pg/ml, so then window is only 2.2x. | 1.5 mL 이라는데? |
| CSF Ps65-ub | 0.5 pg/mL | 10 times concentration from 0.5 mL |
n 7 Minimum 1.4 Maximum 10.2 Range 8.800 Mean 6.471 SD 3.705 SEM 1.4 | ||
| Parkin PET | |||||
| Mc1 |
| NBB results | mean (pg/ml) | sd | if we have x ul of CSF | LLOQ (pg/ml) | z score: (LLOQ-mean)/SD | probability (coverage, %) | window (mean/LLOQ) |
|---|---|---|---|---|---|---|---|
| CSF parkin in NHV | 1.257 | 0.574 | 1000 | 0.061 | -2.08 | 98.12 | 20.6 |
| CSF parkin in NHV | 1.257 | 0.574 | 750 | 0.081333333 | -2.05 | 97.98 | 15.5 |
| CSF parkin in NHV | 1.257 | 0.574 | 500 | 0.122 | -1.98 | 97.61 | 10.3 |
| CSF parkin in NHV | 1.257 | 0.574 | 250 | 0.244 | -1.76 | 96.08 | 5.2 |
| CSF parkin in NHV | 1.257 | 0.574 | 100 | 0.61 | -1.13 | 87.08 | 2.1 |
| CSF parkin in NHV | 1.257 | 0.574 | 50 | 1.22 | -0.06 | 52.39 | 1.03 |
| CSF parkin in NHV | 1.257 | 0.574 | 25 | 2.44 | -2.06 | 0.52 | |
| CSF parkin in NHV | 1.257 | 0.574 | 10 | 6.1 | -8.44 | 0.21 |
But it seems that, let’s say, 1ml (ie 500 ul for parkin and 500 ul for ps65ub) would still be able to generate a useful data for the assays?
20230322_pS65U_for Jaewon.pdf 에는 buffer-base LLOQ was 13 pg/mL라고 되어 있네?
Js compiled 에는 Functional LLoQ was 0.329 pg/mL in pS65Ub
| NBB results | mean (pg/ml) | sd | if we have x ul of CSF | LLOQ (pg/ml) | z score: (LLOQ-mean)/SD | probability (coverage, %) | window (mean/LLOQ) |
|---|---|---|---|---|---|---|---|
| CSF pS65Ub in NHV | 6.471 | 3.705 | 500 | 0.5 | -1.61 | 94.63 | 12.9 |
| CSF pS65Ub in NHV | 6.471 | 3.705 | 250 | 1 | -1.48 | 93.06 | 6.5 |
| CSF pS65Ub in NHV | 6.471 | 3.705 | 100 | 2.5 | -1.07 | 85.77 | 2.6 |
| CSF pS65Ub in NHV | 6.471 | 3.705 | 50 | 5 | -0.40 | 65.54 | 1.3 |
| Assay | Brain | CSF |
|---|---|---|
| TM SMCxPro | 0.41pg/mg | 0.06pg/Ml with 1 ml CSF |
| IPK LC-MS/MS | 32.5 pg/mg protein | |
| IPK SIMOA | 0.137-0.412 pg/mL (question: → 0.244 pg/ml 을 위해선 NSTM 은 250ul CSF 필요한데 어떻게 dmpk 는 50l 이면 되는지? → |
pS65-Ub assays
| Method/source | Detection Ab (for fluorescent label) | Capture Ab (for magnetic bead) | Recombinant Ps65Ub | |
|---|---|---|---|---|
| Takeda | SMCxPro | Anti-Ub antibody (detection): P4D1 (Cell Signaling Technology, Cat# 3936, mouse monoclonal antibody) | Anti-pS65Ub antibody: E2J6T (Cell Signaling Technology, Cat# 62802, rabbit monoclonal antibody) |
K48 Phospho-tetra-ubiquitin Chains (pS65-poly Ub, Boston Biochem, Cat#: UC-250) Phosphor-ubiquitin monomer (pS65-mono Ub, Boston Biochem, Cat#: U-102) K48 tetra-ubiquitin (poly-Ub, Cat#SBB-UP0070, South Bay Bio) |
| Cytokinin | SMCxPro | Ubiquitin: Ub P4D1 (many sources) | Phospho-ub: i) E2J6T clone (Cell Signaling) | K48 pS65-Ub (standard-Biotechne Catalog # UC-250), |
| Wolfdieter Springer |
[`https://www.michaeljfox.org/grant/ps65-ub-biomarker-parkinsons-disease`] (Watzlawik, 2020 #1760) | Ubiquitin (including P4D1) | Phospho-ub (including E2J6T) | |
| Malini Padmanabhan | [email protected] |
Ubiquitin linkage Ubiquitination occurs through a linkage between the C-terminus of ubiquitin and the ε-amino group of a lysine residue on the target substrate. Ubiquitin itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), ubiquitin to form polymers through various lysine
IVC
| Topic | Typical CSF volume per draw | Action |
|---|---|---|
| Rat | 100 uL |
Very low feasibility, mitigation? 5 rats pooled to make 500ul (ie minimum). For 100ul, LLOQ is 0.6 pg/ml, so then window is only 2.2x. |
| NHP | 1 mL |
Parkin activity assay?
Parkin activity assay (this should mean E3 ligase activity assay)
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Top SharePoint URLs | trailing path is wrapped and truncated by the photo edge | The first URL ends mid-path; preserve only the visible portion. |