SNBR/NHP tail, Otake-san’s update, RD-DDU GBA GT, TM GT GBA-PD planning matrix, GBA PET / mRNA / pathway

SNBR / NHP results (continued)

GCase

• Plasma
• AAVs

relationship of VG with mRNA level

There was a large gap in the relationship of VG with mRNA level between mouse and NHP. For example, let’s assume that 10⁵ copies are enough for mRNA expression to be detected in mouse. On the other hand, 10⁸ copies are required for mRNA expression to be detected in NHP. This suggests the promoter used in the current cassette does not work well in NHP. It would make PK/PD prediction among species very complicated. We need to consider whether the promoter works in NHP using i.e. monkey fibroblast.

Otake-san’s update

	project	TM
20220202	[NHP biodistribution study] ongoing Completed by 2022/03~04, ICM, AAV 1/5 vs 9 (Prevail) Readouts: VG, Mrna, HA-tag (staining), in SN	Assays are in place (GBA Activity, GlcSph, gba protein, cath B/D) establishment of dynamic range between PD vs HC is important, but MJF refused to grant (antemortem) PD csf samples, requiring QC data, consider re-request after NHP biodistribution study result
	[In vivo efficacy test] aSyn A53 TG mice + CBE, Readout (Following Tx): ↑ GB activity, (inconsistent & not clear) HMW aSyn, (maybe different regions can be the reason)	[GBA PET] Candidate compounds optimization, (we have 2 compounds but they bind to GBA only in acidic ph)

RD-DDU GBA GT

GBA GT RD DDU	iv leveraging on transferrin receptor

TM GT GBA-PD

	Patient Selection	PK		Proximal PD (Target function)			Pathway Modulation	Disease (Pathophysiologic process)	Modifying / system	Clinical endpoint
		PK	GBA protein Expression (이걸 pk에 포함?)	GBA activity	GlcCer	GlcSph
Description	PD with GBA heterozygous mutation (		GBA protein Expression (이걸 pk에 포함?)	GBA activity	GlcCer	GlcSph		a-syn, (regional consideration: nigrostriatal 아니면 DA/motor 아님.	Dopamine system	Motor function
Clinical	[GBA] genotyping, Patients with reduced GBA protein using PET (?) [PD] Early PD (H and Y) stage ≤2, aSyn PET (in vivo Braak staging)		-GBA PET ligand (preTx: regional expression pattern of GBA) (postTx: temporal and anatomical profile of increased GBA expression, CNS penetration), pharmacologically active concentration range in the CNS, Exposure-response relationship, proof of adequate exposure at the site of action,	GBA activity in CSF	GlcCer in CSF	GlcCer in plasma	GldSph in CSF, 이게 중요하긴 하나 most pateints 에서 below the LLOQ 임.	Autophagy?, calcium?, ER stress?	? (CSF a-syn no correlation)	DAT scan
Nonclinical	PET (in vivo Braak staging)	-	-GBA protein level in CSF	GBA protein level in CSF and brain	GBA activity in CSF and brain	GlcCer in CSF and brain	GldSph in CSF and brain		a-syn aggregation in brain	TH neurons, DAT
	human GBA-L444P heterozygous KI x human SNCA-A53T Tg mice							human GBA-L444P heterozygous KI x PFF injection (or AAV9-SCNA)
In vitro	iPSC-derived DA neurons from patients with GBA-PD

결국 KEY 는 GBA activity CSF · GlcCer CSF · GlcCer plasma · a-syn brain · DAT 일 것 같으므로 이걸 NHP 에서 drug study 해야 하지 않나? 그러려면 결국 Atuka NHP a-syn spread model 에서 해야 하네.

내 summary 보면 다수에서 normal control vs GBA+PD 이 측면에서 이미 differentiation 되고 있는 것 같음. So, actually depleted subgroup (or individual level 에서) 찾으려 노력할 필요. 없을듯.

PD+GBA 군에서 대개 normal control 대비 GBA activity ↓25% 인데, PD-GBA 군 대비도 이 정도 감소되어 있나? 이 정도로 PD phenotype acceleration 시키는 것 같음 (ie vs GBA-PD).

N=325 per arm would be needed to detect 3.4 (7.7) vs 2.0 (6.6) (per year). This difference (1.4=3.4-2.0) corresponds to 41.2% slowing of 3.4.

1. 이것은 Davis 2016 자료인데 PPMI 자료 필요함.
2. 325가 너무 많으므로 더 subgroup 필요할 듯.

Further stratification rows

Further stratification	GBA pathway	biomarker	note
	GBA activity	(2019 Moors) GBA activity postmortem brain: Good separation from normal control,	Normal control 의 define 이 어려움 Cf) HER2: Normal 은 zero니까 define됨. Cf) PD-L1: 개체내에서 tumor tissue 에서 50%미만은 정의될 수 있음 (normal cell은 zero니까).
	GlcCer
	GlcSph		Feasible? PD patients with detectible GlcSph are rare! (Most PD patients had lyso-GL-1 levels below the LLOQ at baseline and Week 4, Venglustat P2)
	Cathepsin B
	Cathepsin D	동반 ↓ (2019 Moors)
	Lysosomal function?
	Drug	In vitro screening like Ambroxol (Narita)

Patient selection / BM discovery matrix

Patient selection		rationale	Support	Natural Hx study
dementia				Not feasible, 임상에서 define 안 되므로 (normal 군과 늘 비교해야 하므로), 그리고 nc 대비 대개 differentiated
reduced GBA activity
altered lysosome		BM discovery (BMP)	Disease relevance to be checked: post mortem? Tx relevance to be checked (in response to Tx) BM feasibility 행여 가능?	Csf (or fibroblast) 에서 ALP 망가진 군 define되나? (LC3 제시한 논문 참조하자) 이게 결국 위의 ALP 보는 것 아닌가?
cathepsin
increased GlcSph				Not feasible because in CSF: venglu p2 후로 GlcSph > lower detection limit 인 환자 전혀 없다고 함!
confirmed GBA response (fibroblast)				AAV-GBA 주면 GBA activity, GlcSph 변화 당연하니 무의미? BMP 가 1. 위에서 Validation되고 2. fibroblast 에서도 변화가 validation 된다면 가능성 있지 않나?

Rebecca

• Modifier
  • PDD
    • V MRI
• Patient selection
  • Pathogenic vs non-pathogenic
  • In vitro screening
  • Reduced GBA vs normal level GBA
• Pff NHP
  • Atuka model
  • CBE+Atuka
• GBA PET

v MRI

method

• prospective
  • Assess cross-sectional and Develop sensitive imaging biomarkers from longitudinal data (patient segmentation and/or disease progression)
• Retrospective
  • Identify a region of interest with high sensitivity to disease progression

GBA PET

• Rationale
  • PET measures (uptake of the ligand) of gene expression is proportional to histological measures of gene transduction.
• Goal
  • determining the extent of gene delivery and expression.
• Timing
  • NHP biodistribution study에서 correlation of regional distribution of AAV-GBA between PET-histology (pk, =‘brain exposure’= vector genome level, = gene expression = GBA immunoreactivity, the quantity (number and density) of AAV-infected cells, GBA activity) - biochemical consequence (GlcSph & GlcSph) - aSyn

GBA protein & mRNA

• Assay
  • Protein
  • M RNA
  • Hirozane-san: need to improve performance
• Example
  • FA GT:
    • FXN expression in CSF or plasma
    • FXN mRNA in exosomes in plasma
  • GlcCer & GlcSph
    • PK/PD (GlcCer & GlcSph CSF) analysis by DMPK performed

GBA pathway

• Objective
  • Delivery of AAV-GBA
  • GBA expression (mRNA → protein)
  • Clinic 에서 measure 하려고, Assay development 만 하면 되나? 약 줘야 되나?
  • 어차피 response 로서의 correlation 보려는 경우, 약 줘야겠네. (AAV-GBA로)