In vitro GBA-PD POC, GBA-GT PK-PD strategy, Immunogenicity assays, NHP studies / SNBR

In vitro GBA-PD model (continued)

GT continued

현재 Human GBA-PD cell model 은 Oxford 것 밖에 없는 듯 (N370S PD)

• However, phospho-Syn has not been observed in iPS-DA (20200731 Tak’s slide)
• → 그래서, PFF 쳐보겠다.
• 아래 그림처럼 해서 (Tx 반응까지 봐서) in vitro POC 를 치리겠다.
• PhReT 에 있는지?
• Phenotype 은? aSyn, lysosome?
• Tak 이 할 건가? If no, 우리 자체적으로 해야 할 것.

In vitro POC workflow figure labels:

1. PFF injection into iPS-DA
2. Find iPS-DA in which P-Syn can be detected with PFF
3. P-Syn accumulation rescued with CBE treatment and/or suppression with rescue
4. Rescue phenotype with GBA activator
5. In vitro POC

Right-side bullets: GBA mutation, A53T iPS, α-syn tri. iPS, Oxford iPS, PhReT, Phenotype, Tak.

GBA-GT [PK-PD strategy]

	PK	PD
20210120 Sato	Based on previous discussion with Mitsuhiro, Misawa and Hidenori in the Parkin GT meeting, GBA protein level should be dealt as PK (transgene product) rather than mRNA, so please prioritize assay development for GBA protein.	PD part in PKPD strategy will not be changed so much compared with the previously shared one in terms of GlcSph (GlcCer). If newly found soluble biomarker in CSF is available as drug responder, we will replace it from GlcSph (GlcCer) or add it on GlcSph in the PKPD strategy.
Prc1	Viral genome and mRNA quantification procedures will be developed for the PK/PD and biodistribution studies in preclinical species by using ddPCR with the first priority and qPCR as a backup for ddPCR. To understand the tissue exposure translatability of human GBA1 (js: protein 의미?) from rodents to NHP, two (or maximum of three) dose levels will be evaluated for serum, CSF, peripheral and CNS target tissues. In addition to human GBA1 levels, the mRNA and vector genome levels will be measured.

a mechanistic PKPD model will be developed to account for the distribution of the capsid to both peripheral and CNS tissues, the uptake of the capsid into the target cells, cellular and intracellular distribution of the viral genome, uptake of the viral genome into the nucleus and finally transgene transduction, transcription and translation rate of GBA1. To develop the mechanistic PK/PD model, vector genome/mRNA/GBA1 expression/GBA1 activity/GlcSph relationship and their species difference will be clarified in a predictive GBA-PD or GD rodent/NHP model(s).

GBA GT [Immunogenicity assays] prc1

(To the selected candidates that will be found as of PE),

• pro-inflammatory cytokine in CSF,
• anti-viral capsid total ADA assay.
• anti-viral capsid Neutralizing antibody assay
• anti-transgene product total ADA assay) will be performed in collaborative discussion with DSRE.

NHP studies

ATUKA, USD ($) 660,496, 35w (ie 8m)

Treatment Group	Treatment (Day 1, ICM)	Dose (vg/brain)	Necropsy	N
1	buffer control	0	Day 85	1
2	AAV1-PD018-HA	2E13	Day 85	3
3	AAV5-PD018-HA	2E13	Day 85	3
4	AAV9-PD018-HA	2E13	Day 85	3
5	AAV9-PD019-HA	2E13	Day 85	3

Brain readout	CSF readout
[Histology]: - H&E staining, - HA (Transgene label): Following immunolabeling techniques, HA will be revealed if present in addition to a nuclear counterstain.	-

SNBR ((Shin Nippon Biomedical Laboratories)

Biodistribution Study of AAV1, 5, and 9 After Single Intra-Cisterna Magna Injection in Cynomolgus Monkeys

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 (1 and 2)
2	AAV1	2.0 × 1013	PD018	1	2.0 × 1013	3 (3 to 5)
3	AAV5	2.0 × 1013	PD018	1	2.0 × 1013	3 (6 to 8)
4	AAV9	2.0 × 1013	PD018	1	2.0 × 1013	3 (9 to 11)
5	AAV9COMP (this should be Prevail's PR001)	2.0 × 1013	PD016	1	2.0 × 1013	3 (12 to 14)

Table 1: Study Design: One control group and two 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	-	1	-	1 (1)
2	AAV9	2.0 × 1013	1	2.0 × 1013	2 (2 and 3)
3	AAV-DJ	2.0 × 1013	1	2.0 × 1013	2 (4 and 5)

CSF

• Collection: acclimation day 3, day 1 (dosing day), day 57, day 90 (final), 1 mL each

Sacral DRG / HA staining results

	Method	Results
Sacral DRG toxicity (Neurodegeneration) on AAV5	HE staining and IHC	Strong (IHC) HA reactivity in DRG → Neurodegeneration In sacral DRG, positive reaction of neuron were most prominent in all AAVs.
HA staining (IHC)	IHC (anti HA antibody 이겠지). Criteria for grading (in case, neuron): 1 (+): <5% of neurons were positive 2 (+): 6-30% of neurons were positive 3 (++): 31-80% of neurons were positive	- Brain: 대부분 1 이하 The distribution/stainability of AAV 1 and 9 is almost comparable, and AAV 5 showed weaker distribution/stainability. No positive reaction was noted in SN in any animals at all groups. No IHC positive cells were noted in substantia nigra or striatum in any animals dosed with AAV9 or AAV-DJ. AAV9 (만 시험): Grade 2 or more positive reaction was noted in temporal, frontal and parietal cortex and cerebellar medulla at 1 month, while all brain regions were less than Grade 1.5 at 3 months. No concern was confirmed in IHC for HA in the other peripheral organs.
VG / mRNA in CSF	Both VG and mRNA in CSF on Day 57 and 90 was under LLOQ. Earlier time points should be selected in the future assessments. - LLOQ for VG: 20 copies/μg gDNA which is less than 50 copies/μg gDNA required for FDA guidance CSF GCase protein level (almost specific to human) and its enzymatic activity were increased by AAVs treatments at Day 57 and Day 90. - CSF GCase level by AAV1 and AAV5 showed decrease after 57 days of AAVs injection, while that by AAV9 showed delayed expression and continued increase. Possibly, the increased CSF GBA level/activity might reflect AAV transduction in other cell types in the brain than neurons? + It might come from other region outside brain (from vascular endothelial cells + peripheral?)
GCase in plasma	GCase (almost specific to human) in plasma increased after 10 days of AAVs injection. - Plasma GCase expression was higher in AAV9-treated groups (vs that in AAV1 and 5 groups.) - AAVs treatment did not have significant effect on GCase enzymatic activity in plasma
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.