NHP Studies Current Plan, CSF Sampling, And Parkin Correlation Evidence

NHP Studies: Current Plan

Study Columns / Timeline

Study	Stable text
ROA study	ROA study = neurosurgery (a pilot study to determine the feasibility of intraparenchyal (ipa) delivery into snpc and putamen in cynomolgus monkeys
ROA study timeline	(after PE) 2022 Oct - 2023 Mar (CN)
1M NHP biodistribution study	targeted a Feb'23 start of study -> 4mths to have data -> CN, expecting to have it around June '23.
1M NHP biodistribution study slot	NBR has held a slot in their schedule for pre-surgery MRI scans (~14 NHP) in Jan '23 and a surgery slot in Feb '23. The NBR reference for that study will be 047-052.
1M NHP biodistribution study milestones	Injection: Feb/Mar, 2023; Necropsy: Mar/Apr, 2023; Report (IHC/ISH): Jun, 2023
Toxin induced NHP study	(after CN); 2023/02-2023/12; (before CS); (1-3 mon in-life, 3 mon analysis); POC

AAV / Route / Criteria Notes

AAV
ubiquitous promoter
Hattori 말들으면 astrocyte 도 중요하니까 ubiquitous promoter 사용가능성
 
AAV9 PK044; PGK1 as priority
*
AVV9 PK041; Syn1Dose: just highest dose possible?

20221004 takeshi: @Fergus: Is there a possibility to select one ROA from two (SN and SN/putamen) by the pilot study?
This may suppress the cost of the BD study.
 
Based on the results of the ICM-NHP study by the GBA-PD GT project, ICM isn't in our scope now
 
AAV capsid / ROA evaluation
PK/PD (?) assessment and immunogenicity assessment (?)
 
Infusion coverage: >30%? Of SN with acceptable AE
Transgene expression: >30% neurons by IHC/ISH in SN (근데 HA-tagging 안 한다는 거지?)
No safety concerns

Pilot / Device Notes

1. Buffer in SNpc, N=2
2. Buffer in SNpc+Putamen, N=3
3. Buffer in SNpc+Putamen, N=3 ("Clear Point").
 
Cf) Current our default is a "Neurochase" device.CP)

Route / Dose Table

Group	Route	Number of Animals	Test Article	Dose Volume per Hemisphere (uL)	Necropsy Study Day
1	Substantia Nigra	3	AAV	25 - 50	29 +/- 4
2	Substantia Nigra + Putamen	4	AAV	25-50; 150 - 250	29 +/- 4
3	Substantia Nigra + Putamen	2	Control	25 - 50; 150 - 250	29 +/- 4
4	Putamen	3	AAV	150 - 200	29 +/- 4

Cost / Schedule / Results

The Northern Biomedical Research (NBR) costs we estimated at $750k. We would add device related costs which will be in the range $125k - $200k depending on final study design.
 
~550,000 USD (~300K in FY2022 + ~250K in FY2023)
for in-live portion
 
Prescreen -> D1: Dosing -> D29: necropsy

We propose neurosurgery with AAV9 and/or AAV5, testing direct delivery to SN and putamen, and combination of both. Interim
 
biodistribution data are expected to available in June 2023 (in live completes March 21)

TM Consideration / CSF And Blood Collection

TM consideration text:

CSF/blood/plasma collection (CSF collection before, during and end of study) 하기는 하지만, WT NHP 이므로 endogenous CSF parkin 있는데, transgene expression-(human) parkin 과 구분할 방법이 없음. -> but gabi 는 하자네. Protein - SMC assay for CSF (and time points) SMC assay for CSF control (vehicle-treated)와 비교하자는 거겠지.
 
Volume, timepoints, csf blood, serum, brain? it should be collected (e.g. in RNAlater), stored and shipped.

CSF Sampling / Aliquoting / Shipment

CSF sampling block:

[CSF Sampling]
- Volume (mL): 1.0 mL or as much as possible, (Cisterna Magna?),
- Schedule: section 13.9, Day -15, 15, 29
- centrifuged (4° C, 2000g, 5 minutes)

Aliquoting block:

[Aliquoting:]
- A 200 uL aliquot is collected in 2 mL DNA LoBind tube pre-filled with 600 uL of DNA/RNA shield.
- The remaining aliquot is collected in 2 mL Protein LoBind tube (Eppendorf, cat# 0030108132) (at least 500 uL, and as much as possible). -> After aliquoting, samples will be snap frozen on dry ice immediately, and transferred to an ultra-low freezer (-80 C) until shipment

Shipment block:

[shipment]: section 15. Naomi Kamiguchi (Tohyama: Will need to specify which samples will shipped to where and who is the recipient.)

Blood / Analytics Needed

Blood / analytics-needed block:

Want to confirm if we need to measure VG and RNA. Otherwise, this may be collected for further use.
• VG DNA tag
• RNA method
• ISH need samples Dec/January if first probes work
• Protein? LC MS? started to assess
Protein SMC assay for CSF (and time points
• Histology
• IHC? To be tested could be ok test do set up IHC on NHP brains
• CSF and blood
• Necropsy additional peripheral tissues similar to GBA study
• Liver, DRG, heart? For VG

Lower-row hematology fragments:

Hematology, Serum Chemistry: Prior to surgery, day 14 and prior to necropsy
Total Cell Count and Chemistry: Total Cell Count and Chemistry: Day-14, -7, Day 14, and prior to necropsy

NBB / PrecisionMed And NHP BD Correlation Plan

Row content:

No.	Plan text	Source / evidence column	Date
1	Normal level in human CSF (의 실측치의 평균) (이미 1 pg/mL로 나옴) (this is the target!)	HC CSF (NBB & PrecisionMed)	2022 Aug
2	Normal level in human brain (의 실측치의 평균)	HC brain (NBB)	2022 Aug
3	Correlation between Brain tissue (SN only) and CSF (regression, normal level 결정 자체에는 안 사용) ... 임상에서 SN의 Parkin level 을 예측하는 데에 이 correlation은 직접적인 증거는 안 될 듯.	Brain tissue and CSF from PD and HC (NBB & PrecisionMed)
4	Normal level in NHP CSF (의 실측치의 평균) (혹시 volume 문제로 이것 못 얻으면, 3에서 extrapolation 하면 됨)	NHP CSF (NHP BD study 의 vehicle group)	2023 Mar?
5	Normal level in NHP brain (의 실측치의 평균)	NHP brain (NHP BD study 의 vehicle group)	2023 Mar?
6	Correlation between Brain tissue and CSF (regression, normal level 결정 자체에는 안 사용) ... 임상에서 (csf을 이용하여) SN의 Parkin level 을 예측하는 데에 이 correlation이 직접적인 증거가 될 듯.	NHP BD study 의 vehicle group & AAV group	2023 Mar?

Purpose note:

*correlation 의 목적: 1. 임상에서 (csf을 이용하여) SN의 Parkin level 을 예측하는 데에 2. NHP (& rodent)에서 CSF 못 얻을때를 대비하여 (즉 이때는 SN으로부터 CSF를 예측).

Scenario table:

Scenario	cortex	SN	CSF
Normal human	3	3	3
ICM 후 1m 후	3	1.5	3
Intraparencymal 1m후	1.5	3	1.5

After CN note:

결국 하고자 하는 것: Prevail 처럼, 임상에서 몇 몇이 normal level 획득. 임상에선 endogenous 와 HA 합쳐서 NORMAL level 인지 볼 것이므로, 우리 assay 가 둘 구분없이 total 재는 것 정당함.

Toxin-induced NHP study note:

Efficacy (DA neuroprotection)과 연결시킬 수 있음. Eg. 75%이상의 parkin protein expression 이면 efficacious 하다.
 
(KO NHP 는 아니지만) 여기서도 다음을 또 얻음. : Correlation between Brain tissue and CSF (regression, normal level 결정 자체에는 안 사용) (NHP BD study 는 임상에서의 ROA 와 동일하므로, 임상에서 (csf 로부터) SN 의 Parkin level 을 예측하는 데에 이 correlation 이 직접적인 증거가 될 듯.

Previous Plan / ICM ROA Capsid Profiling

This bottom table is partly cut by the crop.

Heading and top rows:

Previous plan?
~Jul 2022
2022 Oct - 2023 Mar (CN)
ICM ROA Capsid Profiling (NHP)
Normal Cyno, 8 males
(Vehicle; N=2, Test-1; N=3, Test-2; N=3)
Necropsy at day 85
For ICM
Table 1: Study Design: One control group and four test article groups

Study-design rows:

Group	Test and Control Articles	Dose Level (vg/body)	Dose Volume (mL/body)	Concentration (vg/mL)	Number of Animals (Animal No.)
1	Vehicle	-	1	-	2
2	Test-1	2.0 x 10^13	1	2.0 x 10^13	3
3	Test-2	2.0 x 10^13	1	2.0 x 10^13	3
					8+a#

Footnote:

# Additional animals are required for sero-test