20240722_184759

fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no [effect].

Below from Parkin GT LGE slidedeck

Potential Safety Issue	Probability of Occurrence	Impact	Rationale for Potential Issue	Proposed De-risking Strategy
Immunogenicity (Capsid)	High	Medium	Immune response to AAV therapy is common. Can effect efficacy and safety depending on the extent of the response. Development of excessive immune responses non-clinically can be program limiting. Cytotoxicity due to cell mediated destruction of transposed cells has been observed in some GT. In most cases toxicity is transient with a greater impact on long term transgene expression (efficacy) than as a safety liability There is a hypothesis by other group; formation of vector-antibody complexes (immunogenicity) could activate complement through the classic pathway or activate innate immunity through Fc-dependent uptake in antigen-presenting cell	Translatability of immune response observed in animals is not always clear. Excessive immune response resulting in toxicity should be evident in in vivo studies. The tropism of the capsid and the target tissue (CNS, muscle, liver etc.) should be taken into account when designing safety endpoints for in vivo studies.
Immunogenicity (Transgene)	Middle*	High	Immune response to transgene expression is common. Can effect efficacy and safety depending on the extent of the response. Cytotoxicity due to cell mediated destruction of transposed cells has been observed in some GT.
Cellular stress/ cytotoxicity	Low	High	Chronic activation of the unfolded protein response (UPR) due to endoplasmic reticulum (ER) stress can result in exacerbation of inflammation and programed cell death. In some cases activation of the UPR has been observed in gene therapy due to capsid processing and transgene expression. When transfected the dorsal root ganglia appears to be sensitive to these effects – the relevance of this risk to AAV GT is still theoretical AAV vectors activate the UPR pathways through three major UPR axes, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) during its infection (PLoS One. 2013;8(1):e53845) The IRE1 also activates the IKK leading to NF-κB upregulation. The activated NF-κB further activates the inflammatory genes thus inducing an inflammatory response (Proc Natl Acad Sci U S A. 2011 Mar 1; 108(9): 3743–3748)	If suspected as a potential source of toxicity the Activation of the UPR can be assessed via gene expression assays. Upregulation of the pathway signaling genes IRE-1, PERK, and ATF6 could be assessed in vitro or potentially in vivo via ddPCR or rtPCR
Genotoxicity	Very Low	High	Insertion of the transgene DNA into host chromosome has the possibility of being tumorigenic	AAV in general have very low insertion rate. Genotoxicity and carcinogenicity studies have not been historically required for GT programs – FDA is no longer concerned about insertional mutagenesis for AAV based GT

Study population

	Counterargument
HV	for most CGT trials, the benefit-risk profile is not acceptable for healthy volunteers.

Type of gene therapy

	Inherited genetic diseases	Cancer	Antisense oligonucleotide
	Glybera for Lipoprotein lipase deficiency	Kymriah (tisagenlecleucel) for ALL	Eteplirsen for DMD
Use of vector	o	O	X
	종양성 조직 내부에 직접적으로 사멸 유전자를 전달하거나, 항원을 발현시켜 면역세포가 신생물을 인식하고 공격하는 것을 촉진.

		Use of vector	Example
Inherited genetic diseases	Gene transfer/editing	O	Glybera for Lipoprotein lipase deficiency
			Luxturna for biallelic RPE65 mutation-associated retinal dystrophy
	Exon skipping (Antisense oligonucleotide)	X	Eteplirsen for DMD
Cancer	종양성 조직 내부에 직접적으로 사멸 유전자를 전달하거나, 항원을 발현시켜 면역세포가 신생물을 인식하고 공격하는 것을 촉진.	O	Kymriah (tisagenlecleucel) for ALL

Paper	condition	animal		Vector		administration	Efficiency	Efficiency measurement	Phenotype 개선
2017 Bengtsson	DMD	Mouse	CRISPR/Cas9	AAV	Non-tissue specific	IM	45% trasduction, 8-9% correction (muscle)	Deep sequencing	Not available
2017 Chan	WT	Mouse	Gene transfer	AAV	Non-tissue specific	IV	82% transduction (DRG neurons)	Immunostaining	Not available
2017 Kagiava	CMT1X	Mouse	Gene transfer	LentiV	Schwann cell-specific	Intrathecal	약 50% expression (Schwann cell)	Immunostaining	Improved
Toolgen	CMT1A	Mouse	CRISPR/Cas9	MFN	Nerve 부분선택적 (?)	Intraneural	11% correction (nerve)		?

Units (dose and transgene expression)

example		예	종	tissue	Quantification Method	transgene 구분 within individual?	transgene 구분: group level	Unit
Dose (=titer)		Prevail	NHP	Brain		필요없음.		vg/g brain
		Prevail	mice	Brain				vg
		Tak's biology	mice	Brain				vg/inj
		(Rocha et al. 2015, PMID)	Mice & rat	Brain				Genome copies/mL
	[Exposure, Biodistribution] vector genome copy levels,	Prevail NHP biodistribution study	NHP	Brain				- vector genome copy levels
거 Gene 이라 보면 됨. (전체, viral gene 임, target gene 아님)							= Vector copies/ug genome DNA = Vector copies/ ug DNA tissues Vector genome
제 viral mRNA		Tak's biology	mice	Brain	shire ID tag Q-PCR	구분함		미보고
		Prevail						미보고
		(Rocha et al., 2015)						미보고
		(Rockenstein et al., 2016)
		(Sardi et al. 2013, PMID)
		Tak's biology	mice	Brain	qRT-PCR	미구분
제 target mRNA					HA-tagging 시 가능하지 않나?
target Protein expression		(Rocha et al. 2015, PMID)	Mice & rat	Brain	IF	미구분	vehicle (ie AAV-GFP) treated control animal group 과 차이를 비교함으로써 transgene level 유추해냄.	% of vehicle treated control animal
				Brain	WB	미구분
		Prevail NHP :	NHP	Brain	an antibody-based automated assay, SimpleWestern	미구분
		(Rockenstein et al. 2016, PMID 27126635)	mice	brain	IF	미구분	No report	No report
		(Sardi et al. 2013, PMID)			IF	미구분	미보고	미보고
		Tak's biology	mice	Brain	ELISA	미구분이라함
		Ootake plan		CSF
	GBA GT NHP (snbr)	NHP	brain	HA-tagging → IHC	구분

one vector copy (vector genome) represent one ‘capsid’ (virus particle).

[PHARMACOLOGY]

Binding			examples	Binding site	Structure of the inhibitor	Conformational change of the [target]	Vmax	Km	Kd

Uncertain Spans

location	transcription	uncertainty
Top yellow-highlighted paragraph	(continuation describing DRG pathology meta-analysis across 33 nonclinical AAV studies in 256 NHP)	First sentence is mid-paragraph; the preceding sentences are on 20240722_184756.
Probability of Occurrence for Immunogenicity (Transgene)	Middle*	The literal token reads Middle* with an asterisk; preserved as printed.
Toolgen Vector cell	MFN	Three-letter code for the vector used by the Toolgen CMT1A study; legibility OK but the abbreviation is not expanded on this capture.
[PHARMACOLOGY] Binding header row	(header only)	Only the column header row is visible; the body of the Binding table continues on 20240722_184802.