Table 19.1.

Summary of all major gene therapy studies for treatment of RHO-adRP, grouped by therapeutic strategy

Allele specificity (target)	Silencing and/or replacement reagents	Delivery vector	Animal model	Salient results	References
Treatment strategy: knockdown
Mutation dependent (mouse P23H)	Ribozyme Hp11	AAV2-BOPS-Hp11	P23H-3 rat	15% KD of mutant RNA compared to control eye. 12% ONL loss at P60–90 vs. 40% in control eyes. Scotopic ERG b-wave 30% >control eye	(Lewin et al. 1998)
	Ribozyme Hh13	AAV2-BOPS-Hh13		11% KD of mutant RNA compared to control eye. 20% ONL loss at P60–90 vs. 40% in control eyes. Scotopic ERG b-wave 45% >control eye
Mutation dependent (mouse P23H)	Ribozyme Hp11 Ribozyme Hh13	AAV2-BOPS-Hp11; AAV2-BOPS-Hh13	P23H-3 rat	Long-term (8 months) ONL and ERG rescue PI at P15 (before RD onset) 0.3-month ONL and ERG rescue PI at P60–P90 (40% PR loss)	(LaVail et al. 2000)
Mutation dependent (mouse P23H)	siRNA0, shRNA0 (shP23H)	AAV2/5-U1-shP23H	P23H-3 rat	68% KD (at 3–4 months) 61% KD (at 4–7 months) of mouse P23H RNA; ERG decline and no ONL rescue	(Tessitore et al. 2006)
Mutation dependent (mouse P23H; mouse RHO)	Antisense oligonucleotide: ASO2, ASO3	Intravitreal ASO injection	WT RHO+/+mouse	50% (ASO3) −70% (ASO2) KD of mouse RHO	(Murray et al. 2015)
			P23H-1 rat	30% KD of mouse P23H RHO (ASO3); limited ERG rescue; ONL and OS rescue
Mutation independent (mouse, dog RHO)	Ribozyme Rz397	AAV2-mOP-Rz397	RHO+/+ mouse	50% KD of RHO protein (compared to control eye); reduced ERG b-wave amplitude but no ONL or OS loss	(Gorbatyuk et al. 2005)
			RHO+/− mouse	80% KD of RHO protein (compared to control eye); reduced ERG b-wave amplitude and 30% ONL loss
Mutation independent (mouse, dog, human RHO)	Ribozyme: Rz525	AAV2/5-mOP-Rz525	P23H-3 rat	46% KD of mouse P23H RNA; no change in protein levels; ONL rescue; ERG rescue but decline over time	(Gorbatyuk et al. 2007a)
Mutation independent (mouse, dog, human RHO)	shRNA: shRNA301	AAV2/5-H1-shRNA301	RHO+/+ mouse	49% KD of mouse RHO RNA	(Gorbatyuk et al. 2007b)
			RHO+/− mouse	30% KD of mouse RHO RNA; 60% KD of RHO protein; reduced ERG amplitudes and ONL loss
Mutation independent (human RHO)	shRNA: shBB	AAV2/5-H1-shBB	NHR+/− RHO−/− mouse	90% KD of human RHO RNA in FACS sorted PRs	(O’Reilly et al. 2007)
Mutation independent (human RHO)	shRNA: shQ1	AAV2/5-H1-shQ1	NHR+/− Rho−/− mouse	95% KD of human RHO RNA in FACs sorted PRs; reduced ERG and loss of rod OS and RHO immunostaining	(Chadderton et al. 2009)
			hP347S+/− RHO+/−	Improved ONL thickness and ERG up to 10 weeks PI but not stable: loss of ONL thickness between 5 and 10 weeks PI
Mutation independent (human RHO CRE)	Zinc finger artificial transcription factors: ZF-R2; ZF-R6	AAV2/8-RKp-ZF-R6	hP347S+/− Rho+/+ mouse	26% KD of hP347S RHO RNA in Tx area; partial ERG and ONL rescue	(Mussolino et al. 2011)
Mutation independent (human and pig RHO CRE)	Zinc finger DNA-binding domain: ZF6-DB	AAV2/8-CMV-ZF6	RHO+/+ pig	45% KD of WT pig RHO at 15 days PI, collapse of OS	(Botta et al. 2016)
			hP347S+/− Rho+/+ mouse	ERG rescue at P30 (injection at P14)
Mutation independent (dog, human RHO)	shRNA: shRNA820	scAAV2/5-H1-shRNA820	RHO+/+ dog	8 weeks PI: RHO RNA 0–3%, RHO protein 15% of control at highest safe viral dose. Shortening of OS, loss of immunolabeling	(Cideciyan et al. 2018)
			Light sensitive RHOT4R/+ dog	6–8 weeks PI: RHO RNA and protein levels, structural changes, similar to seen in treated RHO+/+. ONL preservation in treated area after 8–10 weeks PI, 2 weeks after light exposure
Treatment strategy: replacement
Mutation independent	RHO-M (resistant human RHO)	Tg RHO-M mouse	RHO-M+/− RHO−/− mouse	Rescue of rod ONL and ERG loss	(O’Reilly et al. 2007)
				Single copy of resistant human RHO transgene rescues ONL, OS, and ERG loss; leads to RHO RNA expression (∼ 75% of RHO+/+) and expression of RHO in OS	(O’Reilly et al. 2008)
Mutation independent	Various RHO-BB (resistant human RHO)	AAV-mOP RHO-BB24	Rho−/− mouse	ONL rescue + OS formation; rescue of rod ERG but decline from 6 to 12 weeks of age	(Palfi et al. 2010)
Treatment strategy: augmentation
Mutation independent	RHO301 (mouse RHO resistant to shRNA301)	AAV2/5-mOP-RHO301	hP23H+/− RHO+/+ mouse	Twofold increase in total RHO RNA and 58% increase in RHO monomer protein; ERG and ONL rescue up to 6-month PI (at Pl5)	(Mao et al. 2011)
Mutation independent	RHO-BB (human RHO resistant to shBB)	AV2/8–1.7 RHOp-RHO-BB; AAV2/rh10–1.7 RHOp-RHO-BB	RHO−/− mouse	75% of RHO RNA levels as in NHR+/− Rho−/−; ONL rescue; rod expression in OS, formation of OS, ERG rescue, visual acuity rescue	(Palfi et al. 2015)
Treatment strategy: knockdown and replacement
Mutation independent (mouse RHO)	shRNA: shMR3 siRNA: siMR3 Resistant RHO: MR7	shMR3 and resistant RHO MR7 (as plasmids)	WT mouse (liver)	shMR3 + mouse RHO, 90% KD (in liver); shMR3 + MR7, 0% KD	(Kiang et al. 2005)
Mutation independent (human RHO)	shRNA: shBB Resistant RHO: rBB	AAV2/5-H1-shBB-mOP-rBB	hP23H+/− Rho+/− mouse	ONL: 33% thicker than control eye at P10	(O’Reilly et al. 2007)
	shRNA: shQ1 Resistant RHO: rQ1	AAV2/5-H1-shQ1-mOP-rQ1	WT mouse (liver)	ONL: 33% thicker than control eye at P10
Mutation independent (mouse, dog, human RHO)	shRNA: shRNA301 Resistant mouse RHO: RHO301	AAV2/5-H1-shRNA301-mOP-RHO301	hP23H+/− RHO+/− mouse	74% KD of endogenous (human P23H and mouse RHO) RNA; 2X increase in total RHO RNA (compared to control eye); 2X increase in RHO protein (compared to control eye); long-term (9 months) ERG, and ONL and OS rescue	(Mao et al. 2012)
Mutation independent	ZF6 and hRHO	AAV2/8-RHOΔ-ZF6-GNAT1-hRHO-WPRE	RHO+/+ pig	38% KD of pig RHO; replacement with hRHO protein; OS structure better preserved than with ZF6 alone	(Botta et al. 2016)
Mutation independent (dog, human RHO)	shRNA: shRNA820 Resistant human RHO: human RHO820	scAAV2/5-hOP-RHO820-H1-shRNA820	Light-sensitive RHOT4R/+ dog; complete ONL degeneration in 2 weeks post light exposure.	9 weeks PI: Dog RHO RNA 15% of untreated control eye; human RHO RNA 5–9% of canine RHO in untreated control eyes. Total RHO protein: 18% compared to untreated area 13 weeks PI: Dog RHO RNA 1–2% of untreated control eye; human RHO RNA 118–132% of canine RHO in untreated control eyes. 32% compared to untreated area Preservation of ONL, OS, and ERG in the treated area even after repeated light exposure (light exposure at 11, 15, 25, and 37 weeks PI; retinal assessment at 13, 17, 27, and 37 weeks)	(Cideciyan et al. 2018)
Treatment strategy: CRISPR-Cas9 gene editing
Mutation dependent (mouse RHO, S334 locus)	spCas9/sgRNA	sgRNA-spCAs9 plasmid	S334ter-3 rat	Cleavage efficiency: 33–36%; ONL rescue (8 rows vs 1 in Ctrl); OS formation, improved optokinetic response, no ERG rescue	(Bakondi et al. 2016)
Mutation independent (human RHO)	hSpCas9/sgRNA1, sgRNA3, or 2 sgRNAs	CRISPR-Cas9-2sgRNA plasmid	hP23H+/− RHO−/− (very fast RD) mouse	Editing efficiency, 4–33% in transfected rods; KD of RHO protein, 56–77% in transfected rods; no structural or functional rescue shown	(Latella et al. 2016)
Mutation dependent (human P23H)	saCas9/sgH23	AAV2/5-sgH23–2-saCas9	hP23H Tg pig	NHEJ editing in 2 out of 5 pigs but low efficiency (3.4–4.4% alleles showed NHEJ)	(Burnight et al. 2017)

KD knockdown, PR photoreceptors, Tg transgenic, ONL outer nuclear layer, OS outer segment, PI postinjection, CRE cis-regulatory element, WPRE woodchuck hepatitis virus posttranscriptional regulatory element. Promoters listed: BOPS bovine opsin promoter, mOP mouse proximal opsin promoter, U1 human U1 small nuclear RNA promoter, H1 human H1 RNA polymerase III promoter, GNAT1 human guanine nucleotide-binding protein 1 promoter, CMV cyto-megalovirus promoter, RKp human rhodopsin kinase promoter, 1.7 RHOp 1.7 kb mouse rhodopsin promoter