PARKN GT PRC1 Dataset, FXN Benchmark, In-Vivo Models, KOL Notes

Parkin KO Mice Biomarker Matrix Continuation

The top table continues the biomarker expected-change matrix from 20240722_182021.

Biomarker	Expected change in the model (basal)	Expected Tx response in the model	Mechanistic relevance with Parkin-PD patients
CSF Parkin protein	none	당연히 ↑	May help PK-PD
CSF Parkin mRNA	None	당연히 ↑	May help PK-PD
CSF pS65-Ub	Very low (from postmortem papers)	↑	May help PK-PD
Brain MC1 proteins	↓ (from Palacino)	↑	May help PK-PD

PRC1 In-Vivo Disease-Relevant / Function Model Dataset

Title:

Candidates Of In Vivo Disease-relevant/Function Model For PRC1 Dataset (Draft)

Model Establishment In Parkin KO Mice Compared To WT Mice

Subheader:

Model establishment in Parkin KO mice compared to WT mice (-Oct 10, 2021)

Model	Minimum data	Preferable data	Best data
6-OHDA model or Ethanol model or Spontaneous model (young adult)	Reduction of Parkin activity (e.g. phospho-poly ubiquitination)	Reduction in mitophagy - Increase in mitochondrial protein (Tom 20) - Decrease in autophagy levels (LC3-II)	Reduction in number of DA neurons (e.g. TH cell or TH fiber)

Efficacy Study Of rAAV9-Parkin In Parkin KO Mice

Subheader:

Efficacy study of rAAV9-Parkin in Parkin KO mice (-Oct 29, 2021)

Model	Minimum data	Best data	Nice to have
One model among above 3 models	Significantly increase in Parkin activity	Significantly reverse mitophagy deficits	Reverse reduction of TH

Externalization: Publication And IP

Heading:

Externalization

Item	Publication	IP
PRKN protein	Publication planned for brain parkin assay (collaboration with Juntendo-U, publication timing TBD). IP는 안 할거지만 publication 은 해서 so that we can protect the assay originality from competitors	Takeda does not care about IP for assay method because it's not our core business
pS65Ub	Will; IP는 안 할거지만 publication 은 해서 so that we can protect the assay originality from competitors	X (since we've got help from Mitokinin, it's not good to get IP by ourselves.)

FXN GT Benchmark Executive Summary

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FXN GT (Benchmark) Executive summary

Benchmark rows:

Topic	Stable text
NHP biodistribution study	Normal NHP, hemagglutinin (HA)-tagged lead expression construct, -> after 4w -> analysis: viral genome copy number, FXN mRNA & protein, histological (safety)
ROA? (after capsid selection)	untagged FXN expression cassette in NHPs (up to 3 months) with standard toxicology endpoints, as well as analysis of FXN expression levels

FY22 Goals

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FY22 Goals

Text:

1. Set a (preliminary) target CSF parkin protein level with transgene expression
 
My understanding is that this will be achieved by the following two approaches:
Direct approach: by NBB CSF samples (already measured) and antemortem CSF samples (we will plan this, right?).
Indirect approach: correlation of Parkin level between brain and CSF, in NBB samples and from NHP biodistribution study
 
Increase confidence of Parkin protein and pS65-Ub as PS, TE and PD marker for clinical use:
  Obtain CSF-brain correlation with NBB samples (parkin, pS65-Ub)
  Confirm transgene protein expression in monkey CSF (parkin)

In Vivo: MPTP / Prkn Knockout Mouse

Top treatment table:

Treatment group	AAV treatment (Day 1, Neurosurgery)	Dose (vg/brain)	MPTP treatment (D1, 2, 3, 4, and 5, i.p.)	Necropsy	Number of animals (Animal number)
1	Buffer control	0	Saline control	Day 19	16 (101-116)
2	Buffer control	0	MPTP (25 mg/Kg)	Day 19	16 (201-216)
3	AAV9-PK044	1x10^8	MPTP (25 mg/Kg)	Day 19	16 (301-316)
4	AAV9-PK041	1x10^8	MPTP (25 mg/Kg)	Day 19	16 (401-416)

Results table:

Model	Type	Construct	Dose (VG/brain)	Readout	Results/Current status	Data delivery
6-OHDA	blank	hSYN1-coPRKN (PK041)	1x10^8, 10^9, 10^10	TH in SN and striatum (STR) (IHC); Dopamine in STR (LC/MS-MS); Mitophagy (pUb)	~20% DA neuron recovery in SN and STR (IHC); No effect on DA contents in STR	Completed
6-OHDA	Re-test	hSYN1-coPRKN (PK041)	1x10^8	TH in STR (WES); TH in SN and STR (IHC)	~30% DA neuron recovery in STR (WES)	Mid-Feb 2023
MPTP	blank	hPGK1-coPRKN (PK044); hSYN1-coPRKN (PK041)	1x10^3	TH in SN and STR (IHC); Dopamine in STR (LC/MS-MS); Mitophagy (pUb)	Necropsy is finished (Jan 5); IHC and biochemical analysis is ongoing.; DA analysis is ongoing	End Feb 2023 (flash); End Mar 2023 (final)
α-Syn PFF	1M	hSYN1-coPRKN (PK041)	0.3x10^8, 1x10^8	TH in SN (IHC)	~23% DA neuron recovery in STR (IHC)	End of Jan 2023
α-Syn PFF	3M	hSYN1-coPRKN (PK041)	0.3x10^8, 1x10^8	TH in SN (IHC)	Necropsy is finished	Mid-March 2023
LPS	blank	TBD	TBD	Iba1 (WES)	Trend of increase in Iba1	End of Mar 2023
Naïve KOM	Nysno's	TBD	TBD	Brain IL-6	Under measuring (ELISA)	Early Feb 2023
Naïve R275W	M	TBD	TBD	TH in SN (IHC); Dopamine in STR (LC/MS-MS)	STR DA is not altered at 25M; IHC is on going	End of Mar 2023

IP Filing

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IP filing

Link / note:

provisional IP filing.

The full left margin of this section is partially cut; use the full-body evidence for exact placement.

KOLs: Hattori

Text under KOLs / Hattori:

- 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

Astrocyte table fragments:

State	Fragments
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?

Hattori Collaboration Questions

Text:

- 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.

Attachment/link:

Questions for Prof. Hattori.xlsx

KOLs: Michael Schlossmacher / Redox Biomarker Notes

Text under Michael Schlossmacher:

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.

Uncertain Spans

• Michael Schlossmacher/redox notes continue into the lower adjacent scroll position; use the next photo before treating this as a complete KOL subsection.