PARKIN GT Expression Timeline And Parkin Delivery Evidence

Parkin Gene/Protein Delivery In Vivo Continuation

Model / row label	Study / citation	Intervention / timing	Result notes
MPTP mice	(Paterna et al. 2007, PMID 17299411)	mice treated transiently with MPTP; preventive, Parkin overexpression; AAV2-Parkin into unilateral SN; after 21-26 days; MPTP Injection (ip, 4 consecutive); 7 days after MPTP, killing -> analysis	prevented DA cell loss; But no effect on striatal DA, DOPAC, HVA (fig 2b,c,d); possible explanation that surviving TH neurons may be compromised in ability to synthesize dopamine
MPTP mice	(Yasuda et al. 2011, PMID 21760537)	chronic MPTP-Minipump mouse; preventive, Parkin overexpression; AAV-Parkin into unilateral SN; after 14 days; MPTP pump implantation; At 7 or 28 days after implantation of the minipumps, killing -> analysis	prevented motor deficits & DA cell loss; But no effect on striatal DA, DOPAC, HVA (fig 4a,b,c); discrepancy may be related to enhanced dopamine release of surviving DA neurons that overexpress parkin
6OHDA Rat	(Vercammen et al. 2006, PMID 16914382)	Lentiviral vectors encoding human WT parkin were stereotactically injected into the SN 2 weeks prior to a striatal 6-OHDA lesion; preventive	no report on DA, DOPAC, HVA
6OHDA Mice	(Chung et al. 2020, PMID 32494688) Cellivery	iCP-Parkin (iCP=intracellular delivery of CPP-Parkin protein); (iv delivery) in 6OHDA mice/Rat or AAV-aSyn mice model; pff 용어는 미사용

Chung / Cellivery note:

Takeshi) To overcome solubility issue (otherwise precipitate) -> they added solubilization domain
to produce iCP-Parkin. Even though they adopt this strategy, they couldn't produce soluble
protein in E.coli. Although they refold insoluble iCP-Parkin from the inclusion body, iCP-Parkin
has not a correct structure, resulting in constitutive active protein (?).

iCP-Parkin Model Notes

Parkin-KO Mouse 아니니 parkin level 안 봄!
 
1. Mouse: (fig5A), 6OHDA unilateral lesioning -> 4일후 ICP-PARKIN (for 3 days)
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 (FIG5E, DA loss 도 봤고)
Symptomatic 이네 (이 시점에 ↓ DA일것) therapeutic!
-> 6ohda lesioning으로부터 12일 후 kill -> TH level을 아예 No Tx group의 90%수준으로 만들었네,
(figS6d) restored COX4 & VDAC1, pole test에서 slowing progression 아니라
치료첫날부터 호전시켰고, 시간이 지나면서 vehicle 군은 거의 불변인데, 치료군은 BL보다 improved. (fig5c)
 
2. Rat: (fig5G), 6OHDA unilateral lesioning -> 2w후 ICP-PARKIN (for 4 w)
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 (FIG5j,k, DA loss 도 봤고)
Symptomatic 이네 therapeutic! (이 시점에 ↓ DA일것)
-> 7주후 kill -> TH level을 No Tx group의 80-90%수준으로 만들었네
normalized DA loss, rotarod 에서 slowing progression 아니라 치료첫날부터 호전시켰고,
시간이 지나면서 vehicle 군은 거의 불변인데 치료군은 BL보다 improved. (fig5h)
 
3. Mouse: (fig s7) MPTP lesioning -> 4d후 ICP-PARKIN (for 4d) -> 8d 후 kill
-> ↑ rotarod 에서 치료첫날부터 호전시켰음., TH not quantified
 
4. Mouse: (fig6a), AAV-(human WT) aSyn administration -> 8주후 ICP-PARKIN (30 mg/kg)
was intravenously injected three times per week for 4 weeks
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 (FIG5j,k, DA loss 도 봤고)
Symptomatic 이네 therapeutic! -> normalized preventive아니라 therapeutic!
-> 12주후 kill -> restored DA loss (fig 6c, no quantification), ↓ aSy (fig 6d,e),
rotarod에서 vehicle군은 시간에 따른 변화 무, 치료군은 BL보다 호전
 
5. Mouse: (fig6f), AAV-aSyn administration -> 8주후 ICP-PARKIN (10 mg/kg)
was intravenously injected three times per week for 4 weeks
(icp-parkin 직전에) apomorphine rotation test로 확인했으니 preventive아니라 therapeutic!
-> TH level을 No Tx group의 90%수준으로 만들었네 (fig 6h),
↓ aSy (fig 6h,j), Pole test에서 vehicle군은 악화되고 치료군은 BL 대비 불면

Summary:

종평: 대체로 Sx reversal 을 보였고, TH level 도 (vehicle treated 군의 80-90%를 달성했으니)
reversal 을 보인 듯 함, author's argument on mechanism: TH level are neuronal loss are separate process.
Parkin -> TH expression (measured by IHC) -> DA

Bian MPTP Mouse And p62/SQSTM1 Notes

Bian row:

Model / row label	Study / citation	Intervention / model	Result notes
MPTP mice	(Bian et al. 2012, PMID 22792139)	Parkin (preventive Parkin overexpressing) TG mouse model in which expression of WT parkin was driven by neuron specific enolase (NSE) promoter.	↑ parkin mRNA by 2x (fig 1c), ↑ parkin protein by 1.5x (fig 1d), ↑ TH-positive cells (fig2), ↓ damaged mt (fig 3, only ~20%), ↓ aSyn (fig6), no report on DA, DOPAC, HVA

p62/SQSTM1 note:

p62/SQSTM1
- a multi-signaling adaptor molecule
- can directly interact with ubiquitin and LC3, promoting autophagic degradation of ubiquitinated aggregated protein

Symptomatic row:

Section	Study / row	Model	Result
Symptomatic	(Manfredsson, 2007 #1763)	6OHDA rat + AAV Parkin	미저장으로 인해, 추후 작성) =TH+ neurons, =striatal TH, ↑NIGral TH, ↑(DOPAC, HVA 등), ↓Amphetamine-induced rotation

PARKIN GT Executive Summary And Expression Timeline

Milestone headers and fragments:

Column / milestone	Text
PARKIN GT	Expression cassette test (plasmid): 12 tested -> 9 identified & nominated as candidates of lead cassette for LGE
LGE	2020 Oct; To select Expression cassette (여전히 cassette임) in AAV tools
PE	2021 /12
CN	2023 10 (on manufacturing of xx)
CS	2024/06 ->; EDE: ? ->; IND 2025/86 ->; P1 start ?

In Vitro Expression

Text:

iPSC-DA neurons (WT & Parkin KO), Cell lines (HEK293 at GGTR,
Glial cell type: U87 > 1-fold expression compared to endogenous, WT Parkin
-> 9 cassettes were selected (rAAV9-PK031, rAAV9-PK022, rAAV9-PK030,
rAAV9-PK012, rAAV9-PK013, rAAV9-PK023, rAAV9-PK024, rAAV9-PK025, rAAV9-PK026)

Continuation in LGE/expression column:

iPSC-DA neurons (WT & Parkin KO), Cell lines:
9 cassettes (근데 cassette대신에 계속 vector prioritization이라 칭하네?)
to be tested in two AAV tools (AAV5 & 9) -> ~5 exp cassette will be selected
for in vivo expression test (Parkin KO mice, ICM).
 
20210202 & 20210315:
AAV9-Parkin ->
1) ↑ (매우많이) transgene expression
2) ↑ PHOSPHO-UBIQUITIN
3) ↑ mitophagy (reversed 증가되었던 TOM20)

CS column in-vitro note:

iPSC-DA neurons (KO, R275W): -> phospho-poly Ubiquitin,
>50% neuroprotection 이 실험이 decision tree에는 x

In Vivo Mouse Expression

Text:

In vivo expression:
(Parkin KO mouse, 4 m old (젊은쥐)
20210617) 6 exp cassette with AAV9 were tested
): ICM rAAV9 administration of (1x10**) vg/10 uL injection, single injection,
(n=7 per cassette)
) -> dissection 1.5 m post-injection -> analysis (ELISA, IHC):
> 1-fold expression compared to endogenous WT Parkin,

Later POC columns:

[in vivo POC: 6ohda + Parkin KO]
Necropsy was finished on June 1st. Flash report will be available in early July.
 
in vivo POC: MPTP + Parkin KO
-> dose finding study (final product), May-Nov2023

Uncertain Spans

• nav_path: lower half clearly starts PARKIN GT (Takeshi Hioki, PFR-4249-100), but the upper half is a continuation of Parkin PD / Parkin GT in-vivo evidence from 20240722_182007.
• Manfredsson row was apparently added later after an unsaved state (미저장으로 인해, 추후 작성); treat the entire result row as provisional.
• The vector dose in the in-vivo expression row appears as (1x10**) vg/10 uL; the exponent is not safely readable.
• 2025/86 may be 2025/8?, 2025/6?, or another date-like notation; leave unresolved.