Bioanalytical Method To Differentiate Human And Monkey Parkin

Test-1 and Test-2: Different promoter (ubiquitous vs neuron-specific)

For IPa

Table 1: Study Design: One control group and four test article groups

Group	Test and Control Articles	Dose Level (vg/body)	Dose Volume (mL/body)	Concentration (vg/mL)	Number of Animals (Animal No.)*
1	Vehicle	-	TBD	-	2
2	Test-1	Low	TBD	2.0 × 1013	3
3	Test-1	High	TBD	2.0 × 1013	3
					8#

Do we need sero-test in the case of IPa injection?

	HA-tag GBA	HA-tag PRKN
AAV1, 5, 9, DJ
CSF collection		Acclimation day 3 → day 1 (dosing day, 57, 90 (kill)
MBM	Transgene expression: >20% neurons IHC	Transgene expression: >20% neurons by IHC/ISH in SN

No safety concerns, Manufacturability

Takeshi is asking whether parkin protein level can be measured in the brain by NSTM’s method. (JS: He will assess HA in the brain by IHC so I guess he wants to know whether Parkin protein can be measured in frozen tissues as an additional option.) → Ootake-san says we will get the data by end of May.

Parkin protein : it’s difficult to explain as we don’t know how much increase after GT treatment to NHP. But I think we need the same volume with human (1.0mL). If we can only get 500uL, we will try. For cross reactivity, our current result shows we do not detect parkin from NHP CSF, so there are low cross reactivity between human and NHP.

• CSF Biomarkers
  • CSF Parkin protein
    • Previous communications on SMCxPRO LLOQ of CSF Parkin protein was that 1.22 pg/mL in 100 μL (neat CSF), and 0.12 pg/mL in 1 mL CSF (by 10x concentration)..
    • We will have only 1 ml from each animal, and we need to share it with DMPK function. So, will it be feasible that we measure parkin protein in the NHP study?
  • Will we include to measure CSF Ps65ub and other biomarkers (eg TOM20)?

Ps65Ub analysis from NHP CSF samples, I realized that it may not work because we use normal monkeys (not parkin mut monkey) in this study. Damaged mt 가 (substrate로서) 있어야 작동할 테니, 이거 측정은 무의밀 할 듯.

1. Do we need collection into Protein LoBind (Eppendorf) tubes? → yes, we usually use the Eppendorf Protein LoBind® tubes for CSF studies (Cat# 0030108116 (1.5mL) or 0030108132 (2mL)).

2. Should the sampled CSF be centrifuged (4° C, 12000 rpm, 1 minute) to remove the blood component? → Yes, if possible, could you tell them to process 4C, 2000g × 5min. centrifugation and dispense the supernatant to a new tube? → Regarding centrifuging condition, the original condition you mentioned (4° C, 12000 rpm, 1 minute) is OK for us. I realized that this condition is the same with that in the GBA study

3. Do we need aliquoting? → We would like to obtain as much volume of the CSF as possible (possibly >1.0mL). so it’s not needed to aliquot.

4. Where should the samples be shipped to? → If they can send them to TSHO directly, please send to the following address: Neuroscience Drug Discovery Unit Takeda Pharmaceutical Company Limited 26-1, Muraoka-Higashi 2-chome, Fujisawa-shi, Kanagawa, 251-8555, Japan Recipient: Yasuko Matsuki Phone#: +81-466-32-1619

Parkin: HA-tagged parkin only? WHO? Cross-reactivity? : NHP brain homogenate will be used? P23(Can you measure Parkin protein level in the bain by the system established at NSTM?) Takeshi: We should perform double staining using anti-HA antibody for HA-Parkin expression and anti-TH antibody for visualization of DA neurons in the SN, putamen, and caudate. Ps65-ub also? Others? Blood (prevail measured GBA activity, GlcSph in blood)?

LLOQ	ELISA	MSD	SMCxPRO (pg/mL)
Buffer	125 (pg/mL)	24.4 (pg/mL)	0.61
CSF			-1.22 pg/mL in neat CSF (100 μL) -0.12 pg/mL in 10x concentrated CSF (1mL),

Bioanalytical method to differentiate human and monkey Parkin,

Homology between human and NHP is large (97%)

HA Tag	In situ hybridization Three major candidate methods		LCMS (dmpk)	SMCxPro (NSTM)	IHC (of HA-PRKN)	Apomorph?
Gabi: need to establish RNA-protein expression	Sarah Jacobo		6 aa 가지고는 differentiation 힘들걸		Parkin expression 이 제일 많은 곳을 고르자 (jaewo 에 질문)	SZ suggested
	This may replace HA-taggd NHP BD study? iPS 에서조차 expression was low WANT to avoid d/t RA (toxicity assessment)?		If we don't use HA-tag in the upcoming 1M bioD study, we should have one of the above assay developed by Jan2023, and build the rational to justify why it's the best method by considering the future IND application.	Test NHP brain ((nontreatment NHP used in GBA-PD PJ) in Aug 2022		cost
	In situ hybridization is a technique that is used for localization and detection of specific DNA and RNA sequences in cells, preserved tissue sections, or entire tissue (whole mount in situ hybridization, Fig. 1) by hybridizing the complementary strand of a nucleotide probe to a particular sequence, [RNAScope] within TBOS, Elizabeth's Galbreath group and Rare DDU histopharmacology group has protocols already for RNAScope (including tissue pre-treatment) so as not to detect vector genome with the antisense probe. If so desired, the DNAScope for AAV vector genome and BaseScope for mRNA can be performed simultaneously on the same specimen. These two groups would be a good resource: • For RNA preservation in FFPE specimen • For the Takeda account manager from ACDBio - TBOS is a big customer of ACDBio, and this is useful when ordering probes. In my old labs, I'd been able to get as much as 40% discount • Both of these groups in TBOS makes use of Halo analyses software (Indica Labs). ACDBio has an "RNAScope/Basescope" script that can be downloaded for use in Halo AI [20220901]• I had submitted the "PRKN codopt 1" or "CpG5_2" sequence to the ACD Bio's in silico probe analyzer. The scientists there have confirmed that it is possible to design an RNAScope probe to distinguish this sequence from the wt human/cyno/ms PRKN CDS. • There are outstanding action items:   o Does the team vote to pursue ISH by RNAScope? This consensus will trigger custom probe synthesis (2 weeks to delivery)   o If yes, what is the desired chemistry by DMPK/DSRE? Is it colorimetric or fluorimetric? Is it manual or automated staining?   o If yes, will the probes be directly shipped to Japan from ACD Bio? [Hironobu Yasuno] From the view point of pathology, colorimetric detection would be my preference to classify the cell type, and its semipermanent Aspect of stainability would be benefit for our future biodistribution study in monkeys. Axcelead has capability for RNAscope using the Leica Bond RX system according to the automated ISH protocol for RNAscope 2.5 LS Reagent Kit-Brown (ACDBio) (We, pathologist at TSHO, have an experience to order RNAscope to Axcelead).	Mitsuhiro to discuss with Rare-DDU DMPK(?) for the method and lead the assay development for Parkin (Will take several months?) 3 pairs of species specific peptides were identified in in house database for LC/MS measurement of parkin proteins. → DMPK will develop the LC/MS method using the 3 pairs of peptides. 20220801: We have already ordered the necessary signature peptides. After obtaining them, Nao-san will initiate to develop the method 3 pairs of species-specific peptides were found in in-house database (brain)

Uncertain Spans

location	transcription	uncertainty
Ps65Ub follow-up	이거 측정은 무의밀 할 듯	The character 밀 reads as written in the source; it is likely an authoring typo for 무의미, but the visible glyph and both OCR engines agree on 무의밀.