AAV cassette structure, clinical trial, dose selection, and glossary

AAV / capsid table continuation

visible row/item	visible content
AAV (U. Heidelberg)	CNS; ICM, IV; 2021 TBD
New opportunities (Under discussion)	CNS; IV, (ICM); TBD
PHP.eB)	`↑ brain delivery in rodents via the LY6A receptor on brain endothelial cells.`
AAV9	`AAV9 has been shown to have the highest distribution through the CNS, following either direct stereotactic injection or IV injection (25). AAV9 is one of the few serotype suitable for IV injection as it is capable of freely crossing the BBB (26).`
AAV9 note	`AAV9 has been shown to give widespread transduction of neurons and glial cells, as well as transduction of astrocytes.`

Capsids are the main determinants of AAV tropism and transduction characteristics.
The amino acid similarity in the capsid proteins of various AAV clades is about 45%, with the most divergent serotypes being AAV2/4 and AAV2/5 [9].
Different AAV capsid serotypes utilize specific cellular receptors and co-receptors for attachment and internalization into the host cell.
AAV2 and 3 bind primarily to heparin sulfate proteoglycan.
AAV1, 4, 5 and 6 bind to sialylated glycoproteins.
AAV9 binds to galactose.
AAV8 has no known primary receptor [8].

row	5’ ITR	Enhancer-promoter-intron	transgene	Regulatory elements	pA	3’ ITR
label	ITR (Inverted terminal repeats)	Enhancer-promoter-intron	transgene	Regulatory elements	pA (polyadenylation signal)	ITR (Inverted terminal repeats)
function	ITRs base pair to allow for synthesis of the complementary DNA strand.	Promoter carries clusters of binding sites (BSs) for transcription factors (TFs) and stimulates transcription initiation at the transcription start site, mediated by binding of RNA polymerase II in the core promoter. The recruitment of widely expressed, spatially restricted and/or cell-type-specific TFs is noted.		to achieve fine regulation and boost the efficiency of transgene expression	pA marks the end of the transcriptional cassette	ITRs base pair to allow for synthesis of the complementary DNA strand.
eg	`Marianthi: necessary for packaging the genome into the AAV capsid.`	EF1	hGBA1	WPRE (woodchuck hepatitis virus post-transcriptional regulatory element)		`Marianthi: necessary for packaging the genome into the AAV capsid.`
size / limit	Maximum 4.7 kb	1179 bp		592

Capsid: base case appears as AAV9 (Prevail); alternatives include AAV5 and improved CNS tropism, with an example mentioning Stridebio.
Enhancer-Promoter: ubiquitous expression; high CBh, EF1a variations; mid PGK; low UBC.
AAV Genome Conformation: base case Single stranded; alternative Self-complementary; no fit.
Route of administration: primary intra-cisterna magna (ICM) injection; alternatives intracerebroventricular (ICV), intrastriatal injection, or IV.
Transgene: base case Native hGBA coding sequence; alternatives Codon-optimized hGBA and protein-engineered transgene.
3’ Regulatory Elements: base case None; alternative WPREdel6mutATG.

section	visible content
Early	Due to the possibility of permanent unintended effects, along with adverse effects due to invasive procedures that may be necessary for product administration, it is generally not acceptable to enroll normal, healthy volunteers into GT studies.
Early	Well-controlled, randomized, placebo, needed.
Early	It is dangerous to conduct large-scale first-in-human trials. To reduce the potential risk, FDA suggests sponsors stagger administration to consecutively enrolled subjects, followed by staggering between dose cohorts [3,8].
Late	`[adaptive design]`
Late	`(Zang 2021) To address Sample size problem, Huang and Chow proposed an innovative method based on a probability monitoring procedure to calculate the sample size in 2019 [11].`

open label, dose-escalation protection study in a total of 12 patients, 3 doses
js: P2 was not done.
Phase 3 study was an open-label, randomized, controlled, cross-over trial
2:1 ratio
primary efficacy at 1y
After one year of study, patients in the control group were crossed over to Luxturna

item	visible content
Traditional allometric scaling	Species specificity and immunogenicity make extrapolation from a CGT product dose administered in animals to a clinical dose less reliable than customary allometric scaling typically used for small-molecule pharmaceuticals.
Starting dose FIH	Eg. TAK-754, between the lowest efficacious dose in mice and the highest dose tested in the model.
Dose escalation	Clinical development of CGT products has often included dose escalation in half-log (approximately three-fold) increments.
Max dose	Eg. TAK-754 around NOAEL.

CRISPR/Cas9
Efficacy
maintenance
Electroporosis

전기자극으로 세포막을 일시적으로 열어 plasmid 들 들어가게 함.
Plasmid 는 -극이라 원래는 못 들어감 (세포막도 -)

term / visible label	visible definition or note
expression cassette	`a distinct component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell`
expression cassette	`composed of one or more genes and the sequences controlling their expression. [3] An expression cassette comprises three components: a promoter sequence, an open reading frame, and a 3' untranslated region`
transfection	`(js: this should be restricted to non-viral systems!) the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred:`
transduction	`the process by which foreign DNA is introduced into a cell by a virus or viral vector.`
capsid	`the protein shell of a virus`
null vector
MOI	`the number of infected viral particles per cell, Plaque forming units (pfu) of virus used for infection / number of cells.`
transient transfection	Constructed plasmids are introduced into mammalian cells, but foreign genes are not integrated into the genome; `the new gene will not be replicated`; foreign genes are gradually and totally lost after several days. Notes: `Only plasmid administered`; `No AAV, No MOI, No`.
stable transfection	`AAV used, MOI used, 9일후에 분석가능`

Selection of the expression cassette is the next step of this project.
...: Raav9

The page starts and ends mid-content; adjacent photos should be checked for table continuation and clearer boundaries.
Dense rows in the AAV capsid table, cassette structure table, Luxturna Phase 3 text, plot labels, and glossary entries need manual verification before being used as exact values.
Korean notes under cassette structure, electroporosis, null vector, and stable transfection are OCR-assisted and should be manually checked.
Citations such as (25), (26), [8], [9], [3,8], and [11] are visible/OCR-supported but should be verified against the image before final KB use.
Product/program names and IDs such as TAK-754, WPREdel6mutATG, hGBA1, AAV9, AAV5, CBh, EF1a, PGK, UBC, ICM, ICV, and Raav9 are high-risk exact strings.