PARKIN GT Biomarker Plan, KOL Notes, And Natural History Meeting Notes

Prior Table Continuation And IP Filing

Top-row continuation fragments:

Fragment	Text
Model / mutation	Naive R275W; M
Status cells	TBD; TBD
Readouts	TH in SN (IHC); Dopamine in STR (LC/MS-MS)
Result / status	STR DA is not altered at 25M; IHC is on going
Timing	End of Mar 2023

IP note:

IP filing
Provisional IP filing.

KOL Notes: Hattori, Michael Schlossmacher, Christein Klein

Hattori

Stable fragments:

- In general, patients are reluctant to undergo neurosurgery by DBS treatment, but he recommends it for 100% of his PD patients
- Regarding GT in PRKN-PD, local administration makes sense since the disease is confined to the basal ganglia. An option could be to put the virus together when inserting DBS electrodes.
 
- In postmortem brain analysis in PRKN-PD, GFAP level in astrocytes is reduced, suggesting an immaturity (prkn-PD 환자들이 20세 되서야 발병하는 것과 일맥상통) of astrocytes and astrocyte dysfunction for pathogenesis.
His team observed a metabolic change (reduction of lactate consumption) in the culture cell system (PRKN-PD iPS-astrocyte? or KO astrocyte?) --> GABA neuron 이상 -> ↓ motor function

Hattori table fragments:

Row	Stable text
Normal	mitophagy is required on astrocytes to efficiently release lactate and maintain; Mitophagy and maintain mito copy number
In PD	immaturity (prkn-PD 환자들이 20세 되서야 발병하는 것과 일맥상통) of astrocytes; reduction of lactate consumption
Js questions	Lactate as a BM?

Follow-up notes:

- Prof. Hattori is open to the draft collaboration plan about the astrocytic role of Parkin using mono and co cultured iPSC derived astrocytes.
 
He further agreed that Takeda researchers visit his lab to receive data and experimental protocols related to astrocytic Parkin studies.
 
He is open to all options of mouse models we have including human Parkin R275W knock in mouse for in vivo study.
As he did not know the details about the various mouse models, an additional discussion (possible with his lab members) is needed to understand the scope of a research collaboration
 
- Dyskinesia in PRKN-PD is dominant in the lower limb, so-called dancing-feet dyskinesia.
His team is trying to elucidate its mechanism by electrophysiological approach by using Prkn KO rat.
Flash result suggests D1 signal is associated with dyskinesia.

Linked attachment fragment:

Questions for Prof. Hattori.xlsx

Michael Schlossmacher

Stable fragments:

Canada: Dr. Michael Schlossmacher at Ottawa Hospital, who is at the redox mechanism for Parkin, and also at biomarkers. (Tokarew, 2021 #2156)
 
Normally: parkin has an anti-oxidant function (unrelated to E3 ligase activity!).
In Pakrin-PD, ↑ Oxidative stress: ↑ H2O2 in i) post-mortem human cortex (fig 5i) and ii) dopamine-treated human M17 cells expression prkn point mutation (C431F, G321E)(fig5j),
 
(El Kodsi, 2023 #2215) prkn-/-//Sod2± adult animals did not develop dopamine cell loss in the S. nigra, but, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins.
 
We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling.
 
Js question: how can we distinguish from mitochondrial dysfunction-induced ↑ oxidative stress?

Continuation row:

There are multiple intracellular sources of ROS eg. Endoplasmic reticulum, mitochondria etc.
If we'd use GSH as a biomarker in a clinical trial that can monitor the treatment effect of a drug targeting parkin, if GSH was modulated in patient biofluid, how can we know which pathway (cellular organelle) we have modulated?
 
Intracellular Sources of ROS, ER (Konno, 2021 #2212)

Christein Klein

Stable fragment:

BMs: 31P MRS (but not specific?), aSyn seeding assay (eg RT-Quic), Neuromelanin imaging

High-Level Plan Of Key Biomarkers

Heading:

GE (20201023 SDIB)
high level plan of key biomarkers

Hypothesis / target validation:

AAV-mediated delivery of Parkin gene will result in increased expression of Parkin in the CNS and modify disease progression in early onset Parkinson's disease patients with Park2 mutations.

Biomarker Table

BM Category	Type	Description	Stable plan / rationale
Target Engagement (TE)	MBM	Parkin protein in CSF	Transgene expression; Some literatures: Parkin is measured in CSF; We: CSF, EV
Target Engagement (TE)	MBM	Parkin PET	Direct evidence of target engagement in the targeted brain regions; most Parkin mutation catalytic site is preserved; possible active-site binder may bind both endogenous and exogenous protein; compare pre-Tx and post-Tx; Plan: Paul McQuade & Makoto Fusimi
Pharmacodynamic (PD)	MBM	pS65-Ub	Parkin plays a key role in mitophagy; feedforward process to form phosphorylated polyubiquitinated chain on mitochondrial outer membrane; PINK1 phosphorylates ubiquitin at s65, Parkin is recruited and activated, then adds ubiquitin repeatedly; hypothesis that pS65-Ub represents degree of mitophagy and functional Parkin
Pharmacodynamic (PD)	MBM	pS65-Ub	In PD brain with PINK1 or PARK2 mutation, pS65-Ub signaling is very low or absent; plan to examine correlation of pS65-Ub chain level with mitophagy (WT CCCP, KO CCCP); human evidence: association of pS65-Ub chain level in human CSF with PARK2 mutation; assess whether pS65-Ub chain level increases by treating with AAV-PARK2 in KO model (in vitro and/or vivo)
IMG	MC-1 (Mitochondria Complex I) imaging	MC1 PET	Develop mitochondrial complex 1 (MC1) PET as a translatable in vivo brain imaging biomarker of mitochondrial integrity in Parkin-PD; MC1 is the largest part of the mitochondrial respiratory chain and critical for ATP production; MC1 level is considered to represent viable mitochondria
IMG	MC-1 (Mitochondria Complex I) imaging	MC1 PET	Parkin-PD patients show reduction of MC1 activity in dermal fibroblasts by 45% by spectrophotometric assessment; plausible considerable reduction in MC1 density, but not specifically assessed in these patients; [18F]BCPP-EF binds selectively to MC1 and has been used to quantify MC1 levels in non-human primates, healthy human subjects, and individuals with neurological conditions
IMG	MC-1 (Mitochondria Complex I) imaging	de-risking / calibration	Demonstrate reduced [18F]BCPP-EF PET signal in brains of Parkin-PD individuals; first determine sufficient reduction of MC1 density in Parkin-PD dermal fibroblasts using in vitro immunoassay of MC1 subunit, e.g. NDUFB8; if MC1 deficit meets predefined criteria, proceed to in vivo brain MC1 PET imaging; calibrate MC1 PET signal preclinically to other mitochondrial dysfunction/loss markers
Disease Related	IMG	DaTScan	a reduced presynaptic dopamine transporter density; putamen predominantly affected, consistent with Parkinson disease of other etiologies; DATScan can be used in clinic to show modulation in disease progression
Disease Related	unclear	aSyn-related biomarker note	Because a-Syn pathology is rarely observed in the target population, not planning on aSyn-related biomarkers for this project

Additional name fragment:

Mitsuhiro Nishihara

Biomarker Development Pathway Notes

Fragments:

[MC1 imaging:]
Survey of available external options and/or partnerships & (Nonclinical enabler study) --> BFA
Vendor/collaborator/technology assessments (PO STUDY) --> BTV (Laura ... validation, if it measures what it is intended to measure)
Clinical characteristics of marker in relevant population (face validity) Animal study (Preclinical calibration, Tx response) --> BCS
Assessment in relevant population for drug development use case (construct validity) --> BFP (ready for use in TAK study)

Team Meeting And Natural History Study Notes

Team Meeting

Date	Stable note
20201016	Vector production: Xiuxia Sun, GeneWiz make plasmids and send to Umass ??? Tsho 언제?

Natural Hx Study

Leading notes:

Cross sectional first? -> 대개 불필요, datscan 은 원래 이거 불필요, mc1, ps65-ub 등은 미리 이거 얻어올 것.
 
Longitudinal natural history study in Parkin PD - 10-07-2021 - Simen AA.docx

Meeting 20220521 notes:

Patient recruitment: GP2 is not active cohort but more of sample contribution, PDGen is more active,
Working with individual PI is needed, and MJF can catalyse, (ie site-based model), they can inform 'who is PI at x site'?
 
'Centogene model': ROPAD Study
Cost: Jamie Eberling will review, they recommend 6 month VMAT (AV133) as repeat -> ↓ variability,
Funding: overal with PPMI
Assay: circle back to samantha, perhaps around assays, there could be some interest in cost-sharing.

Contact fragments in the left column include:

Xiao <[email protected]>;
[email protected];
Jaya Padmanabhan <...@takeda...>;
Mara Blumenstein <...@michaeljfox...>;
[email protected];
Kopil <[email protected]>

Meeting 20220625 notes:

MC1 을 ppmi cohort 에 계획 (n=150), (Rationale: mind-map study에선 patients were not well charactersied. And there were noise)
Possibility: mc1 in prkn-pd as a satellite.

Contact fragment:

Jaya Padmanabhan <...@takeda...>

Uncertain Spans

• Development pathway: the Korean/English Laura parenthetical is partially abbreviated in the transcription and should be checked before KB promotion.
• Contact emails are only partially transcribed and should not be promoted to structured KB fields.