TABLE 1.

Outcomes of studies comparing non-viral minimized DNA vectors to other vector systems.

Vectors used	Vector length	Sequence encoded	Transfection method	Outcomes	Ref.
Minicircle Plasmid	3,881 6,233	Firefly luciferase	Sequence-defined oligoamino amides/cationic polymer	Compact, rod-shaped polyplexes were 65–100 nm using plasmid and 35–40 nm using minicircle; all formulations of minicircle polyplexes lacked cell cycle dependence. Mini-circle transfected ~3-fold more than equal moles of plasmid. Combined, tyrosine trimer integration, combination polyplexes, and use of minicircle increased gene expression ~200-fold over an equal mass of plasmid.	[2]
Minicircle Plasmid	4,573 8,147	Mesothelin CAR	Electroporation	CAR expression, IFNγ and granzyme B secretion, and specific lysis of pancreatic cancer cell lines was significantly increased in NK cells electroporated with minicircle over plasmid. Use of minicircle resulted in increased NK cell viability after electroporation.	[3]
Minicircle Plasmid Minicircle Plasmid	3,700* 7,700* 4,000* 7,900*	Firefly luciferase TK/NTR	Microvesicles/cationic lipid	Equal moles of minicircle resulted in prolonged transgene expression in breast cancer cells. Minicircles loaded into microvesicles twice as efficiently as equal moles of plasmid but resulted in a peak bioluminescent signal 14 times higher than in cells treated with microvesicles containing plasmid. Microvesicles loaded with minicircles encoding TK/NTR led to greater activity of prodrug converting enzymes over microvesicles with equal mass of plasmid.	[4]
Minicircle Plasmid	364 8,318	Guide RNA (to inhibit PLK1)	LHNPs	Cas9 protein co-delivered in LHNPs with minicircles decreased PLK1 expression more than Cas9 protein co-delivered with plasmid or minicircle co-delivered with Cas9 DNA in vitro.	[5]
Minicircle Plasmid Minicircle Lentivirus	Unknown Unknown Unknown NA	eGFP 2nd gen. anti-CD19 CAR	Electroporation	CD34+, H9 hESCs, and T cells electroporated with minicircle encoding eGFP resulted in more and brighter eGFP+ cells, increased cell viability, and increased CFUs compared to equal mass of plasmid. T cells electroporated with CAR minicircle killed tumor cells in vitro and in mice comparably to T cells transduced with lentiviral vector.	[6]
Minivector Plasmid siRNA	400* 3,900* NA	shRNA/siRNA against GFPor ALK	Cationic lipid	Minivector and siRNA, but not plasmid, decreased GFP expression in difficult-to-transfect Jurkat cells and decreased expression of ALK in Karpas 299 cells; the three vectors were comparable in easy-to-transfect 293 FT cells. Minivector and siRNA, but not plasmid, arrested growth of ALCL cells. Minivector DNA survived human serum > 10-fold longer than plasmid or siRNA.	[9]
Minivector Plasmid	281–2,679 1,711–5,302	Multiple different1	NA	Minivectors ≤ 1,200 bp survived nebulization while longer vectors sheared faster as a function of increasing length. Negative supercoiling afforded up to 2-fold additional protection from nebulization and sonication shear forces.	[10]
Minicircle Plasmid Plasmid	2,257 3,487 5,541	GFP	Cationic lipid (niosomes)	Minicircle transfected twice as efficiently as an equal mass of plasmid. Minicircle had higher capacity to deliver to primary retinal cells and rat retinas than equal mass of plasmid.	[28]
Minicircle Plasmid Minicircle AAV	Unknown Unknown 2,500* NA	GFP Rhodopsin	Cationic lipid	Minicircle GFP expression in retinal cells was maintained for 7 days while GFP expression from an equal mass of plasmid was lost before 7 days. Gene delivery to retinal cells in vitro using AAV or minicircles encoding rhodopsin was comparable in efficiency. Cells modified ex vivo with AAV or minicircles encoding rhodopsin reconstructed functional retinal tissue and supported vision function in blind mice.	[29]
Plasmid Plasmid2 Plasmid Plasmid2	7,722 9,668 8,738 10,684	eGFP eGFP/Cre recombinase HN HNHis/Cre recombinase	Cationic lipid	Plasmids encoding genes with or without Cre recombinase were transfected into Salmonella as a platform for oral DNA vaccination against Newcastle disease virus in poultry. Plasmid containing Cre recombinase allowed for the in vivo generation of minicircle encoding either eGFP or HN. Chickens orally inoculated with Salmonella transfected with Cre/eGFP-containing plasmid contained significantly more eGFP in liver than plasmid without Cre. Chickens that received Cre/HN inoculation were protected against challenge with NDV significantly more than chickens inoculated with Salmonella containing HN plasmid alone.	[35]
Minicircle Adenovirus	Unknown NA	Bcl-2	Cationic lipid	Percentage of NSCs overexpressing Bcl-2 was comparable when using adenovirus or minicircle but minicircle-treated cells lost expression faster. NSCs treated with adenovirus or minicircle overexpressing Bcl-2 were partially rescued from transplant-associated insults.	[38]
Minicircle Plasmid Minicircle Plasmid	3,088 7,100 4,618 8,581	GFP GFP/Sox9	Cationic lipid	Percent GFP+ was increased ~10-fold in canine, equine, and rat MSCs following transfection with GFP minicircle over an equal mass of GFP plasmid. Sox9 was successfully expressed in canine MSCs after transfection with Sox9 minicircle in vitro.	[40]
Minicircle Plasmid	1,715 3,531	VEGF	Electroporation/microporation	Transfection with either plasmid or minicircle did not change expansion potential, differentiation capacity, or immunophenotype of MSCs, but transfection with minicircle led to 2.5-fold more VEGF transcripts, greater VEGF production, and improved angiogenic potential of MSCs in vitro.	[41]
Minicircle Plasmid Lentivirus	4,129 8,133 NA	hPAX7/GFP	Cationic lipid	Repeated transfection with hPAX7 minicircle generated myogenic progenitors that could terminally differentiate, but their transplantation resulted in limited engraftment. Formation of hPAX7+ myogenic progenitors using lentivirus remains the more efficient platform for generation of myogenic progenitors.	[42]
Minicircle Plasmid Minicircle Plasmid	3,400* 6,100* 2,300* 4,700*	Venus fluorescent protein SB100X transposase	Nucleofection	CD34+ HSPC electroporated with minicircle encoding Sleeping Beauty components resulted in increased cell viability, enhanced transient gene delivery, and higher rates of stable gene integration over equimolar amounts of plasmid expressing these components.	[43]

^*Italicized values are estimated lengths provided by the authors of those studies.

¹This study focused on DNA vector length.

²This study sought to use plasmids harboring Cre recombinase and the gene of interest for the in vivo production of minicircles (carrying the gene of interest without Cre recombinase) by using Cre expression to recombine the originally transfected plasmid, effectively amounting to the delivery of both plasmid and minicircle to the Salmonella cells receiving plasmid encoding Cre recombinase.

AAV: Adeno-associated virus; ALCL: Anaplastic large cell lymphoma; ALK: Anaplastic lymphoma kinase; Bcl-2: B-cell lymphoma 2; CAR: Chimeric antigen receptor; Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; CD19: Cluster of differentiation 19; CD34: Cluster of differentiation 34; CFU: Colony forming unit; Cre: Causes recombination; eGFP: Enhanced green fluorescent protein; gen.: Generation; GFP: Green fluorescent protein; hESCs: Human embryonic stem cells; His: Histidine-tagged; HN: Hemagglutinin neuraminidase; hPAX7: Human paired box 7; HSPC: Hematopoietic stem and progenitor cells; IFN: Interferon; LHNPs: Liposome-templated hydrogel nanoparticles; MSCs: Mesenchymal stem cells; NA: Not applicable; NDV: Newcastle disease virus; NK: Natural killer; NSCs: Neural stem cells; PLK1: Polo-like kinase 1; Ref.: Reference; SB100X: Sleeping Beauty 100X transposase; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; Sox: Sex-determining region Y-box transcription factor 9; TK/NTR: Thymidine kinase/nitroreductase; VEGF: Vascular endothelial growth factor.