RDDU’s PE I Imaging GBA Plan (continued), PK/PD Strategy, 20201007 / 2021005 DMPK Meetings, AAV/PK Rationale, PRC1 Parkin Team Considerations, And [Benchmark] FA GT PRC1 PE Narrative

Dose-dependent change of SB)

PK (→ rapid clearance, metabolite, stability of labeling position)

Blocking (pretreatment, occupancy) 필요없겠지? 대신 parkin KO mice 에서 brain transgene protein level data 로 충분할 듯

Imaging (hot): Reginal distribution, Blocking (불필요?)

Drug candidate; rodent; DMPK (겠지); Drug candidate; NHP; QTS

아래처럼 하면 되지 않을까?

Date	Plan
-2022 01 (PE 직후)	Identify selective compound series
2022 01 - 2022 11 (CN)	BMax assessment
2022 11 (CN) - 2023 12 (CS)	Optimization of physical properties and non-imaging triaging of potential candidates (blocking 불필요하니 ttimeline 줄일까? (- 202307?)
2023 12 (CS) - IND?	In vivo PET studies in relevant preclinical species (e.g. NHP) – specific binding, dosimetry

PK/PD Strategy

Model	Plan	Timing
WT Rat	See DMPK's plan
PRKN KO Rat		After CN (Mar 2023) RATIONALE: i) The results will be available by Mar 2023. ii) Promoter will be selected by Mar 2023 (MPTP, etc.). iii) We don't need SN biodistribution data.
Kimio Tohyama	ELISA in brain tissues (animal only?)

• 20201007 meeting with DMPK
  • Mice CSF
    • Necessary?, not necessary but helpful, nhp is costly,
    • Who’s responsible for CSF Parkin assay of mice ? & NHP?
    • Conclusion
      • Immunoassay needs minimum 25 uL, but mice usually can produce 10uL, so we may need rat (RAT 은 100 UL제공가능)
      • Plan
• DMPK’s pk/pd modelling plan:
  • Brain protein -brain VG → brain – CSF (to be establish before PE)
• NSTM
  • We won’t change current timeline of parkin protein assay
  • But, Mice expression study시작시점인 2021 Jan 전까지 우리 method의 mouse CSF 에서의 detectibility를 확인함!
• Hioki상에게 확인: Mice expression study 에서 CSF채출 여부, 채출양은?

2021005 meeting with DMPK WHOLE PURPOSE: i) To predict human efficacious exposure ii) clear understanding of PK-PD-DR BM in an animal model (cf: WT animal 에서 하면 2등급)

Dose	PK	PD	DR BM
dose	Brain vg		: DA
	Brain Parkin mRNA
	Brain Parkin protein
	CSF vg
	CSF Parkin mRNA
	CSF Parkin protein

What’s the role of Parkin mRNA in PK/PD relationship? Dose → Tx effect 로 바로 가기에는, 종간 차이 등에 의해 중간자인 pk 가 달라서 결과가 다를 수 있으므로, 중간자인 pk → Tx effect 를 하자. PD 는 역할이 별로 없을 것 같긴 함.

Nishimura: CSF VG assay: almost complete Nishimura: mRNA assay: complete (in mouse), translation to NHP should be easy, but they haven’t tried CSF!

Finally DMPK will try to do the following items

1. To measure mRNA in mouse CSF, because the minimum CSF volume to measure mRNA is 5 uL in the current assay system, which suggest the possibility to detect Parkin mRNA in CSF, although the sensitivity is unknown. I have already asked Hioki-san to prepare the CSF samples in the Parkin KO or normal mouse administered PGK-1. Hioki-san, please discuss the detail later.
2. To measure endogenous Parkin protein in rat CSF by SMCxpro assay We don’t know whether this challenge works well, but it is worth to try. If the measurement success, we will try to the mouse CSF next. Kamiguchi-san, please share your human CSF assay method and protocol.

Anyway, we confirm whether Parkin expression in CSF can work as a PK parameter in PK/PD analysis prior to NHP study.

Rationale: Select AAV which can deliver sufficient VG (at least above 1x10^4 VG per ug genome DNA shown in AAV9 of Prevail study) in NHP brain. Estimate the dose to recover the healthy Parkin level in model mice, after confirming the preliminary concept that Parkin level in brain correlates with protein or mRNA in CSF of NHP or model mouse.

VG data alone is not sufficient – we will need to generate exposure data (RNA or protein) – I do not think that there is any data showing that AAV0 can achieve delivery to SN/striatum by ICM – as measured by expression? CSF is may not be representative for SN/substanita nigra WHY limit to AAV9?

We would like to confirm that the AAV selected by GBA exploratory study can deliver sufficient VG in next NHP study with HA-tag. In addition, we will try to confirm the relationship between mRNA in CSF and protein in brain by Parkin KO mice prior to the NHP study. I understand that CSF may not be representative for SN, but I believe that Parkin level in CSF is the most promising PK parameter at this point. Due to the available CSF limitation in mice, this study is challenging, but we need to confirm the PK/PD relationship in model mice. Thus, I seek the possibility to obtain the data by mice after PE.

Hioki: 2-5 uL CSF/mouse can be sampled.

PRC1

efficacious dose(s) observed in the Parkin KO mouse disease model, biodistribution in NHP

PK/PD) correlations dose-exposure correlations

Parkin team consideration	Note / limitation	Cf) Methods that GBA team uses	Parkin Team's request
AAV capsid			No need to measure
Vg (viral DNA) in CSF or plasma	only detects DNA from virus	shire ID tag qPCR.	May I ask about the feasibility of measurement and your thought?
mRNA	No distinguish	RT-PCR	Hioki'san: A separate slide
mRNA NDE	No distinguish
Protein CSF	No distinguish, Parkin Protein Sarah Melissa Jacobo: anti-human Parkin (or anti-HA antibody) 가 혹시 있으면 endogenous parkin 과 구분할 수 있을지도	ELISA
Protein NDE	No distinguish

Parkin team consideration	Cf) Methods that GBA team uses	Parkin Team's request	When we need this by
AAV capsid		DMPK: No need to measure
Vg (viral DNA) in CSF or plasma	shire ID tag qPCR.	DMPK: May I ask about the feasibility of measurement and your thought?	By ? to support NHP biodistribution study?
mRNA CSF (/EV)	RT-PCR	Hioki'san: A separate slide is requested for LGE proposal
mRNA NDE
Protein CSF (/EV)	ELISA
Protein NDE

[Benchmark] Molecular biomarker development of FA GT PRC1 PE Narrative

7: Biomarker Probability of Technical Success (PTS)

Type	Biomarkers	PTS	Priority	Needed by
Target Engagement (TE)	Frataxin mRNA in exosomes	Low	Low	By CS to support biodistribution study
Pharmacodynamic (PD)	Frataxin protein in CSF	Medium	High	IND filing
Pathway Modulation (PM)	Plasma NfL	High	N/A	Assays developed for clinical use
	Plasma GFAP	High	Low
	Plasma UCHL1	High	Low
Disease Related (DR)	Brain atrophy (vMRI)	High (brain)	High	IND filing
Disease Related (DR)	Mitochondrial function (MC1 PET)	High (brain) Low (heart)	High; if MRI QSM or SWI not available	IND filing
Disease Related (DR)	Iron accumulation (MRI – QSM or SWI)	Medium (brain)	High; if MC1 PET not available	IND filing

Topic | Action

Uncertain Spans

location	transcription	uncertainty
20201007 / DMPK plan	to be establish before PE, detectibility, CSF채출	reads as written; establish/detectibility likely authoring spelling issues; 채출 is unusual Korean and may be a typo for 채취.
AAV / PK boxed right	AAV0, substanita nigra, CSF is may not be representative	reads as written; AAV0 may be AAV9, substanita is likely a typo for substantia, and the doubled is may not is likely an authoring slip.
PRC1 Parkin team consideration	shire ID tag qPCR., Hioki'san	reads as written; shire may be a vendor name and Hioki'san uses a non-standard apostrophe between name and san.
FA GT benchmark	leftmost type-label cells	The type column on the left is partially cut by the photo edge; values reconstructed from visible endings.