GT PK Comparison, Dose Scaling, And Promoter Section

GT PK comparison (continuation)

	Affected by	Capsid	Distribution → cell uptake (ie transduction) → intracellular trafficking → degradation of vector in the cell - Secretion (so shedding marker) - Can be affected by immune responses - Durability of VS (ie loss of episomal DNA)		Transcription rate from DNA to mRNA (특히 이부분이 종간차 커서, NHP은 mouse 대비 수십배까지 적음, 더구나 human potentially has 3x lower transduction & expression efficiency than NHP)(1 vg당 1 mRNA copy니 오시 않고 보통 수십개 나옴. - mRNA stability - secretion (글 수 있음)		- Translation rate from mRNA to protein (낭연히 1 vg낭 1 protein 나오는 것이 아님! - Secretion (글 수 있음) - Can be affected by immune responses
20210210 Translational science forum	Capsid concentration assay may build an exception as established AAVx ELISA may not be feasible for novel (engineered) capsid.	이건 PK 에 불포함	-		-		이것도 pk에 포함 (TG product 임)	이것도 pk 에 포함 (TG product 임)	-	-	-	-
Hunter,, TAK-073	여러 dose 의 rAAV-GT vg/kg (Mouse) 두 dose 의 rAAV-GT vg/kg (NHP)		안 보네? 안 보네?				I2S (Serum & tissue) I2S (Serum )	Not shown, (correlate) I2S activity	- Not shown	↓ GAG -	↓ LAMP1 (IHC, for lyso vol) Normal NHP 라 무의미	- -
Fabry	rAAV-GT vg/kg (Mice) rAAV-GT vg/kg (Human)						αGal (serum & tissue) αGal (serum & tissues)		Gb3 & Lyso-Gb3 (tissue) Gb3 & Lyso-Gb3 (tissue)
hemophilia	rAAV-GT cp/kg (Human) cp is capsid particle						FVIII	FVIII activity
	DMPK: no need to measure (Parkin GT meeting)		The correlation between vector and DNA may not be linear (eg. DNA production may be saturated) Parra-Guillén2010
assay	AAVx ELISA may not be feasible for novel (engineered) capsid.		qPCR		RT-PCR	ELISA
Discriminate transgene?	NA	- shire ID tag qPCR (GBA GT preclinical) - GBA GT (Pharm Sci): the ID-Tag (target region for the dose measurement assay) is incorporated into all potential transgene candidates - Frataxin gt: NHP biodistribution studies for StrideBio's lead capsids will be performed with a HA-tagged version of the lead expression construct.   • For the lead cassette epitope tagged versions are being developed which are necessary for biodistribution studies in NHP. Human influenza (HA) tag in 3 different versions and Myc-tag are considered.			- Sarah Melissa Jacobo: anti-human Parkin (or anti-HA antibody) 가 혹시 있으면 endogenous parkin과 구분할 수 있을지도 (mouse와 구분에 국한될 듯) - While the antibodies against frataxin protein can discriminate between mouse and human, they do not discriminate between NHP and human (어쨌든 Clinical study에서는 무상관)		-	-	-	-	-	-
		- 구분 필요 없을 듯: Because   이차피 Transgene-specific PD 안 존재 - 구분 필요 할 듯, because   • Endogenous protein 의 functional viability 모르므로 (반론: gba환자에서 대개 gba amount 와 activity가 비례함)					-	-	-	-	-	-

The average life span of adult human hepatocytes is around 500 days (20210207 Estimation of Human Efficacious Dose TSF GT modeling)

Dose Scaling for GT

Dose_Human = Dose_Animal · (Scaling factor) · (Activity factor)

• Dose_Animal
  • Pharmacologically active dose (PAD; usually in vg or vg/kg) in animal models
  • No-observed-adverse-effect level (NOAEL) dose in toxicological species
• (Scaling factor)
  • Scaling factor based on:
    • Body weight, or
    • Target organ volume
• (Activity factor)
  • “Relative activity factor” based on perceived species difference in transduction
  • Examples:
    • Internal hemophilia GT programs cited a mouse-to-human factor of 30
    • Mouse-to-human factor of 20 to 100 from external programs
    • NHP-to-human factor: Key issue #1

Species difference (Activity factor)

아래 표 의미: mouse 에서 NHP 거쳐 human 으로 가면서, vg (biodistribution)도 줄고, mRNA 도 줄고, protein 생산도 줄어, 종국에는 human 이 120 배 준다. (from 20221005 Tomoki_modeling strategy for NS GTs for PRKN GT.ppt)

Analytes	Mouse	NHPs	humans
Vg	0.7-3.8x		-
mRNA	57-134x		-
Transgene proteins	-		2.9x
Overall	~120x (previous benchmark)

참고: gene efficiency factor (GEF) ({Tang, 2021 #2153})

Scenarios of Objectives of dose exploration in P1 (

• MTD
• Range of optimal effective doses
  • achieving a specified target range of exposure that was derived by significant practical limits on the dose of the product that can be produced or delivered

stopping rule, treatment plan (staggering administration ) 등 관해 fda guidance (Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products) 봐라!

Promoter

(P)1

Relatively small size (1,179 bp), which is good

enhancer 와 promoter 와 intron 는 붙어다니는구나.

• (CAG?)I: CMV early enhancer fused to modified chicken β-actin promoter
• (CAG?)G: CMV early enhancer fused to modified chicken β-actin promoter
• V: Human cytomegalovirus immediate early enhancer/promoter
• K: phosphoglycerate kinase 1 promoter
• C: Human ubiquitin C promoter
• A: Human eukaryotic translation elongation factor 1 α1 promoter

(EF1A short form): Human eukaryotic translation elongation factor 1 α1 short form

Table 1

Comparison of Selected Ubiquitous and Cell-specific Promoters.

Promoter	Specificity	Relative Strength	Size (bps)	Reference(s)
CMV	Ubiquitous	+++	750–800	Xu et al., 2001; Gray et al., 2011
CBA (including derivatives: CAG, CBh, etc.)	Ubiquitous	+++	248–1,600	Klein et al., 2002; Ohlfest et al., 2005; Gray et al., 2011
EF-1α	Ubiquitous	++	2,500	Gill et al., 2001; Xu et al., 2001; Ikeda et al.,
UBC	Ubiquitous	+	403	Gill et al., 2001; Qin et al., 2010
GUSB (hGBp)	Ubiquitous	+	378	Husain et al., 2009

Uncertain Spans

location	transcription	uncertainty
GT PK comparison header (NHP transduction note)	(1 vg당 1 mRNA copy니 오시 않고 보통 수십개 나옴.	The Korean note is partly wrapped onto a thin column; tokens 오시 않고 may be a transcription of 오지 않고 (“does not come”), but the visible glyphs remain as written.
GT PK Hunter,, TAK-073 row	안 보네? (twice)	The same text fills two adjacent merged columns; the source likely intends one statement spanning both columns rather than a duplicate, but the rendering here preserves both visible cells.
GT PK assay row	qPCR / RT-PCR / ELISA row alignment	Three sparse cells are spread across many narrow columns in the source; column-cell alignment beyond the visible labels is not fully reconstructed.
Promoter abbreviation list	(CAG?)I and (CAG?)G	Two leftmost abbreviations are partly obscured by the Promoter heading and a leading paren glyph; both appear to point to CAG-style enhancer/promoter combinations but the exact key letter is not legible.