Table 2.

Overview of gene editing in large animal models

Disease	Animal/disease model	Strategy	In vivo or ex vivo	Target	Gene editing system	Delivery	Conditioning	Results	Reference
HIV	NHP (pigtail macaque)	inactivate CCR5 in HSCs to enable HIV-resistant hematopoiesis	ex vivo	CCR5 in HSPCs	ZFN	electroporation of ZFN mRNA	myeloablative (TBI)	64% CCR5 editing in infusion product, 3%–5% long-term engraftment	52
HIV	SHIV+ NHP (pigtail macaque)	inactivate CCR5 in HSCs to enable HIV-resistant hematopoiesis	ex vivo	CCR5 in HSCs	ZFN	electroporation of ZFN mRNA	myeloablative (TBI)	∼50% CCR5 editing in infusion product, 3%–4% long-term engraftment, trafficking to secondary lymphoid tissue, trends toward delayed viral rebound after ART removal	53
HIV	SIV+ NHP (rhesus macaque)	inactivate CCR5 in HSCs to enable HIV-resistant hematopoiesis	ex vivo	CCR5 in HSPCs	CRISPR (SpCas9)	SIV-based LV	non-myeloablative (busulfan)	<16% CCR5 editing in infusion product, ∼1% long-term engraftment, all but one animal rebounded after ART removal	54
HIV	SHIV+ NHP (rhesus macaque)	inactivate CCR5 in anti-HIV CAR T cells to confer HIV resistance and enable virus-specific effector function	ex vivo	CCR5 in anti-HIV CAR T cells	CRISPR (SpCas9)	electroporation of CRISPR RNPs	none	<36% CCR5 editing in infusion product	55
HIV	SIV+ NHP (rhesus macaque)	excise integrated proviral DNA in SIV-infected cells	in vivo	SIV proviral DNA in SIV-infected cells	CRISPR (SaCas9)	AAV9	none	up to 92% and 95% decrease in proviral DNA in blood and peripheral lymph nodes	56
SCD	NHP (rhesus macaque)	POC: correct point mutation in HBB that causes SCD via single base pair HDR conversion	ex vivo	HBB in HSCs	CRISPR	electroporation of CRISPR RNP + ssDNA donor template to recreate SCD point mutation via HDR	myeloablative (TBI)	17%–26% recapitulation of SCD mutation in infusion product, ∼1% long-term engraftment	57
SCD/β-thalassemia	NHP (pigtail macaque)	disrupt BCL11A in HSCs to reactivate fetal hemoglobin	ex vivo	BCL11A in HSCs	TALEN	electroporation of TALEN mRNA	myeloablative (TBI)	1.5% BCL11A editing in infusion product, 0.3%–0.4% long-term engraftment	58
SCD/β-thalassemia	NHP (rhesus macaque)	prevent BCL11A repression of fetal hemoglobin by disrupting BCL11A binding site in γ-globin promoter	ex vivo	HBG promoter in HSCs	CRISPR	electroporation of CRISPR RNPs	myeloablative (TBI)	75% editing and 39% recapitulation of HPFH mutation in infusion product, 8%–27% editing and 6%–18% HbF expression in PB cells >1 year after treatment	59
SCD/β-thalassemia	NHP (rhesus macaque)	disrupt the erythroid-specific BCL11A enhancer region to disable BCL11A in erythroid lineages and reactivate fetal hemoglobin	ex vivo	erythroid-specific BCL11A enhancer region in HSCs	CRISPR (SpCas9)	electroporation of CRISPR RNPs	myeloablative (TBI)	up to 85% editing in enhancer region in infusion product, but engraftment and γ-globin expression highly dependent on number of infused cells	60
AML	NHP (rhesus macaque)	POC: inactivate CD33 in HSPCs to establish CD33-deficient hematopoiesis and enable CD33-directed immunotherapy	ex vivo	CD33 in HSPCs	CRISPR (SpCas9)	electroporation of CRISPR RNPs	myeloablative (TBI)	<15% CD33 editing in infusion product, 2%–4% long-term engraftment	61
DMD	DeltaE50-MD dogs62	disrupt DMD exon 51 splice acceptor site to enable exon 51 skipping and restoration of dystrophin reading frame	in vivo	DMD exon 51 splice acceptor site in peripheral and cardiac muscle	CRISPR (SpCas9)	dual AAV9 to co-deliver Cas9 and gRNA	none	restoration of up to 70% and 92% of normal dystrophin in peripheral and cardiac muscles 8 weeks post-treatment	63
DMD	DMD exon 52-deficient pigs64	excise DMD exon 51 to restore dystrophin reading frame	in vivo	DMD exon 51 in peripheral and cardiac muscle	CRISPR (SpCas9)	dual AAV9 to deliver split intein Cas9 + gRNA	none	widespread expression of truncated dystrophin in cardiac and skeletal muscle, decreased fibrosis, improved cardiac function and survival	65
Hypercholesterolemia	NHP (rhesus macaque)	knock out PCSK9 to prevent degradation of LDLR and increase uptake of blood LDL-c	in vivo	PCSK9 in hepatocytes	meganuclease	AAV8	none	up to 84% reduction in serum PCSK9 and 60% LDL-c 11 months after treatment	66
Hypercholesterolemia	NHP (rhesus macaque)	knock out PCSK9 to prevent degradation of LDLR and increase uptake of blood LDL-c	in vivo	PCSK9 in hepatocytes	meganuclease	AAV8	none	sustained dose-dependent reductions in serum PCSK9 and LDL-c 3 years after treatment	67
Hypercholesterolemia	NHP (cynomolgus macaque)	introduce precise loss-of-function PCSK9 mutation to knock out PCSK9, prevent LDLR degradation, and increase uptake of blood LDL-c	in vivo	PCSK9 in hepatocytes	CRISPR adenine base editors	LNP delivery of ABE8.8 mRNA and PCSK9 gRNA	none	>60% PCSK9 editing in NHP liver, stable 90% reduction of PCSK9 and 60% reduction of LDL-c	68
Hypercholesterolemia	NHP (cynomolgus macaque)	introduce precise loss-of-function PCSK9 mutation to knock out PCSK9, prevent LDLR degradation, and increase uptake of blood LDL-c	in vivo	PCSK9 in hepatocytes	CRISPR adenine base editors	LNP delivery of ABEmax mRNA and PCSK9 gRNA	none	up to 34% PCSK9 editing in NHP liver, ∼32% reduction in PCSK9 and ∼14% reduction in LDL-c	69
Leber congenital amaurosis	NHP (cynomolgus macaque)	POC: correct aberrant splice donor created by mutation in CEP290 to restore reading frame and normal CEP290 expression	in vivo	CEP290 mutation in retinal cells	CRISPR (SaCas9)	AAV5 delivery of SaCas9 and pair of gRNA	none	up to 30% reading frame-restoring editing	70
Cone-rod dystrophy (CORD6)	NHP (cynomolgus macaque)	POC: knockout of mutant GUCY2D followed by complementation with wt GUCY2D	in vivo	GUCY2D in retinal cells	CRISPR (SaCas9)	dual AAV5 delivery of SaCas9 and gRNA	none	10%–20% editing in photoreceptor cells, up to 80% decrease in GUCY2D protein product	71