Table 2.

Minicircle applications with various viral and non-viral vector platforms

Vector platform	pUC plasmid (>2 kb pUC origin-antibiotic marker spacer region)	Minicircle (MC) retrofit (<100 bp spacer region)	Result-performance	Result-antibiotic marker gene transfer
Sleeping Beauty transposon gene integration vector	SB puroR reporter plasmid	SB puroR reporter MC	MC 2-fold increased transposition rate into established cell lines17	plasmid backbone antibiotic resistance potential expression or genome integration in T cells
	SB CD19-CAR	MC: SB CD19 CAR	MC 4-fold increased yield of CD19-CAR T cells; reduced transfection associated toxicity with nucleofection delivery34
	SB	MC: SB (218 bp MC Backbone)	MC 6- to 7-fold increased transposition into human hematopoietic stem cells; reduced transfection associated toxicity with nucleofection delivery19
CRISPR-Cas9 gene editing vectora35	3.1 kb 2A-puro/gRNA-1	0.85 kb 2A-puro/gRNA-1	MC 6-fold increased targeted integration into HeLa cells after lipofection of Cas9-2A-puro/gRNA-1	plasmid backbone antibiotic resistance potential expression or genome integration
Direct cell therapy vector36	pANGPT1	MC-ANGPT1	MC 3.7-fold greater increase in ANGPT1 protein expression after electroporation of mesenchymal stem cells	plasmid backbone antibiotic resistance potential expression or genome integration in stem cells
Direct gene therapy vector37	pRSV-hAAT-bpA pEF1α-hFIX-hGHpA	MC-RSV-hAAT-bpA MC-EF1α-hFIX-hGHpA	MC > 1 log increased extended duration expression (>20 days post hydrodynamic delivery to mouse liver) (reduced gene silencing)b	plasmid backbone antibiotic resistance marker potential expression or genome integration in liver cells

^aHomology-independent “replace editing” using non-homologous end-joining pathway.

^bLu et al.³⁷ also report that gene silencing occurs with ≥1 kb random DNA sequences, while shorter spacers ≤500 bp similarly extended duration expression as with minicircle vectors.