Progress and prospect of minicircle as a minimized non-viral DNA vector in gene therapy and regenerative medicine

Abstract

The ideal and reliable genetic treatment of human diseases relies on the use of an effective vector that does not trigger serious side effects such as immunogenicity and genotoxicity. In recent years, non-viral DNA vectors have gained significant attention due to their low cytotoxicity and minimal immune responses. Minicircles are small episomal supercoiled non-viral DNA vectors derived from conventional plasmids through in vivo recombination in bacterial cells. Numerous evidences have shown promising results in the diagnostic and therapeutic applications of minicircles due to their unique features such as small size and high ectopic expression with lower side effects. Extensive studies have shown that these minimized DNA vectors can be a superior alternative to other vectors, overcoming their technical drawbacks and clinical limitations. Based on these impressive outcomes, minicircles have remarkable potential for clinical translation in the near future. In this review, we present the characteristics, production method, advantages, and disadvantages of these vectors. We also summarize key studies that highlight the diverse and promising applications of minicircle technology as an effective tool in non-viral gene therapy and regenerative medicine.

Graphical abstract

In this study, Dormiani and colleagues provide a comprehensive review that presents the characteristics, production methods, and drawbacks of minicircles as small, episomal, non-viral DNA vectors. Additionally, the review covers several in vitro and in vivo investigations that have utilized these vectors in gene therapy and regenerative medicine.

Introduction

Somatic gene therapy is an attractive and promising approach that uses nucleic acids to regulate, repair, or replace genes in order to treat genetic disorders that are not treatable with conventional therapeutic strategies. In fact, this method aims to achieve sustained expression of a therapeutic gene at a sufficient level to ameliorate or cure symptoms of genetic disease with minimal side effects.^1,2,3 Many genes have been studied as potential candidates for gene therapy using delivery microdevices known as gene vectors to specifically shuttle therapeutic genes into cells or tissues of interest.^4,5 An increasing number of gene-based therapeutic approaches currently in clinical trials indicate the efforts to improve gene therapy by developing highly safe and efficient vectors to either treat a disease or slow its progression.⁶ Conventional viral-based vectors including adeno-associated viruses (AAVs), retroviruses, lentiviruses, and adenoviruses are widely employed for therapeutic application owing to their natural ability to infect target cells and deliver genetic cargo efficiently.^7,8 However, the production of viral vectors is expensive, time-consuming, and requires professional facilities to ensure their safety and efficacy.⁹ Furthermore, some of these vectors such as lentiviral and retroviral vectors are integrative and can cause insertional mutagenesis at integration sites.¹⁰ Adenoviral vectors generally do not integrate into the host genome but may be inactivated and can trigger strong immune responses and inflammation, limiting their systemic delivery of the transgene.^11,12 AAV vectors, on the other hand, remain episomal but may become inactive after infecting host cells, reducing transgene expression in dividing cells. Other limitations of AAV vectors include their immunogenicity and a small packaging capacity, generally less than 5 kb. These drawbacks have restricted their application for AAV-based gene therapies.^10,13,14 The side effects and potential toxic properties of viral vectors have raised safety concerns regarding their clinical applications and hindered their development.¹³ As a result, non-viral DNA vectors have been gaining more attention and making continuous progress due to their cost-effectiveness, low immunogenicity, higher gene capacity, long-term stability, and greater genomic safety resulting from their episomal maintenance.^7,15,16 The mentioned limitations of viral vectors and the advantages of non-viral DNA vectors have led to an increase in non-viral-based gene therapy trials and products.^7,17 Among non-viral DNA vectors, bacterial plasmids are the most commonly used DNA molecules in various non-viral gene transfer methods, viral particle generation, and RNA production both coding and noncoding. Moreover, plasmids can be easily produced on a large scale at a lower cost and stored for longer periods compared with viral vectors. These vectors show prolonged ectopic expression and typically have less than ${10}^{-5}$ stable integrations per transfected cell. The circular structure of plasmids allows for simple gene cloning to customize and manipulate the vector for therapeutic purposes. Despite these advantages, plasmids have limitations such as low bioavailability and unsustainable expression, which can restrict the efficiency of transgene expression in eukaryotic cells.^7,18,19 Structurally, plasmid vectors contain elements for replication and transcription. The replication elements include a bacterial origin of replication (Ori) for plasmid amplification in bacterial cells and an antibiotic resistance gene as a selection marker to prevent the propagation of plasmid-free bacteria. Transcription elements consist of a eukaryotic promoter, the gene of interest, a polyadenylation (poly(A)) signal, and other transcription termination sequences to prolong transgene expression.¹⁸ However, plasmids have some drawbacks that limit their applications as safe and effective therapeutic vectors. DNA plasmids have low bioavailability and unsustainable expression, which reduces the efficiency of transgene expression in eukaryotic cells. These vectors contain unmethylated cytosine-phosphate-guanine (CpG) motifs, which are more commonly found in bacterial DNA than mammalian DNA. These motifs trigger immunostimulatory responses via TLR-9 in mammalian host cells, interfering with gene expression and resulting in rapid transgene silencing.^7,19,20,21 Additionally, factors such as the covalent attachment of prokaryotic sequences to the transgene cassette, extragenic spacer DNAs larger than 1 kb between the 5′ and 3′ ends of the transgene cassette, and high CpG content, are considered factors that can contribute to transgene silencing.^22,23,24 On the other hand, the use of antibiotics and related resistance genes in the plasmid replication process is restricted by the Food and Drug Administration (FDA) and the European Medicines Agency due to biosafety concerns. This is because of the potential risks associated with the horizontal transfer and replication of these genes in the human microbiome.^25,26

Minicircle is a new generation of double-stranded, circular, supercoiled episomal DNA vector that has been utilized in several gene therapy studies. The promising results from these studies have confirmed the versatile potential of this minimized non-viral DNA vector for therapeutic applications.^19,27 Different methods have been proposed for the production of these minimal-size expression vectors.^19,25,28,29 However, in the most common method, the minicircle is derived from a specific larger plasmid vector, known as the parental plasmid, through a two-step recombination procedure in E. coli.^6,19 Shankar et al. have reviewed the various approaches used to produce minicircle DNA, highlighting their advantages and disadvantages.³⁰ While minicircles can be synthesized within conventional E. coli strains,³¹ a genetically modified strain, ZYCY10P3S2T, is primarily used for more efficient production of the vector. This minicircle producer strain stably expresses phiC31 integrase and I-SceI homing endonuclease, two main enzymes that play a central role in the assembly of the minicircle following L-arabinose induction. Upon induction, the expressed phiC31 integrase catalyzes a one-directional site-specific recombination between attP and attB sites in the parental plasmid. The intramolecular recombination process results in the generation of two separate supercoiled DNA molecules: a minicircle DNA that contains only the minimal expression cassette of the transgene or the sequence of interest without the deleterious bacterial backbone, and a miniplasmid that is replicative and composed of the bacterial sequences (Figure 1). The key advantage of the small size allows minicircles to be employed as complex genetic constructs to transfer multiple genes or regulatory elements.^6,32

Figure 1.

Schematic representation of minicircle vector production in bacterial cells

The parental plasmid undergoes intramolecular recombination in the ZYCY10P3S2T E. coli strain after bacterial transformation. This strain has been genetically manipulated for inducible expression from multiple copies of phiC31 and I-SceI coding sequences. After site-specific recombination, two new circular DNA molecules are generated: a miniplasmid that is digested by I-SceI due to the presence of multiple I-SceI recognition sites and is subsequently degraded by bacterial exonucleases. The second product of recombination is a minicircle, which remains intact within the bacterial host. The minicircle DNA, containing only the expression cassette encoding the gene of interest, can then be purified using a standard method for small- or large-scale production.

By using this cost-effective and relatively simple manufacturing protocol, it is possible to produce exclusive minicircle batches with 3- to 5-fold higher yield and 10-fold fewer residual contaminations compared with plasmids.^32,33,34 Recent improvements in the minicircle production protocol have enhanced the efficacy of production and purification of high-quality supercoiled minicircles.³⁵ In light of mass production, optimizing three key factors in vector manufacturing simultaneously, including antibiotic concentration, induction time, and induction temperature, can increase the yield of minicircle biosynthesis. By implementing these optimized conditions, scaling up minicircle production as a nucleic acid-based biopharmaceutical can be safer and more cost-effective than viral vectors, attracting significant attention from the biotech industry.^36,37 The main advantages of minicircle vectors over conventional plasmids include: (1) higher transfection efficiency and easier transport into the cell nucleus, as well as enhanced resistance to shearing forces, leading to prolonged in vivo expression due to their smaller size^38,39,40; (2) less DNA required to deliver an equivalent number of molecules due to their smaller size, resulting in a smaller delivery vehicle and hence reduced potential for vector toxicity²⁷; (3) long and high-level ectopic expression capability, which can be up to 1,000-fold higher in vitro and in vivo due to the lack of bacterial sequences^41,42,43,44; (4) better cell viability and a satisfactory safety profile due to the absence of evidence for random integration into the host genome^28,42,45; (5) reduced risk of potential horizontal transfer of antibiotic resistance genes to human microbiota due to the lack of a bacterial amplification unit^20,44; (6) low immunogenicity and reduced host inflammatory responses due to a lower number of unmethylated CpG motifs^6,46; and (7) large-scale manufacturing capacity for the production of high-quality-grade minicircles.^47,48 Additionally, in a review by Arévalo-Soliz et al., a summary of studies evaluating the transfection efficiency, transgene expression levels, and duration using minicircles compared with other vectors has been provided.²⁷ These characteristics make this minimized DNA vector an outstanding biopharmaceutical with great potential for therapeutic gene transfer in the treatment of genetic disorders.⁴⁹ Despite these advantages, the use of minicircles has primarily been limited to preclinical studies with only a few clinical trials reported for this vector, including NCT06411366 and NCT04499339 as listed on clinicaltrial.gov.⁵⁰ This limitation is mainly attributed to the minicircle production process, which is not yet entirely straightforward due to challenges such as a more complex production process, lower production yield, and scale-up compared with conventional plasmids, inadequate purification efficiency, and relatively high production costs.^26,48,51,52 However, some advancements such as the use of more efficient recombinases, modifications to the backbone, and innovations in fermentation approaches have increased the productivity of minicircle DNA.^{20,51,52,53,54,55,56} Furthermore, improvements in purification techniques have enabled the application of clinical-grade minicircle vectors.^57,58 In an effort to improve minicircle production, Oliynyk et al. developed the Plasmid2MC technology as a simple and cell-free method for the efficient production of high-purity, nearly endotoxin-free minicircle DNA.⁵⁹ Moreover, the development of novel delivery systems including physical methods like electroporation, chemical reagents, such as cationic polymers and cationic lipids, and biological nanoparticles like extracellular vesicles, has further enhanced the therapeutic efficacy of minicircle vectors.^60,61,62,63 The mentioned improvements and other ongoing advances in designing and producing the minicircle vectors are expected to continue paving the way for the future development of this promising technology to be applied in the clinic.^52,64,65 In this study, we provide a comprehensive review that covers several in vitro and in vivo investigations that have utilized minicircle technology for gene therapy and regenerative medicine since the studies reviewed by Gaspar et al.²⁰

Minicircle applications

After more than three decades of effort in evaluating gene therapy, this sophisticated approach is now considered a clinically effective method for treating certain genetic disorders for which there were previously no effective therapeutic options.⁶⁶ Non-viral DNA vectors, such as minicircles, are gaining attention due to their safer features, greater flexibility for various administrations, easier production at a lower cost, and longer shelf life compared with viral vectors.^7,67 The clear advantages of minicircles in achieving high-level and sustained expression of transgenes compared with other traditional vectors, have led to the implementation of these innovative vectors not only in basic research but also in diagnostic and mechanistic studies on a number of human disease models.^68,69,70,71 Minicircle-based vectors have also been widely used in preclinical studies in various fields including vaccination, infectious diseases, immunotherapy, induced pluripotent stem cell (iPSC) generation, regenerative medicine, and gene therapy for cancers and genetic disorders.^{29,72,73,74,75,76,77,78} These minimized vectors are employed ex vivo and in vivo through different gene delivery techniques to overexpress or downregulate key genes involved in a wide range of diseases, both acute and chronic in order to prevent or treat them (Figure 2).⁷⁹ Furthermore, recent studies have elucidated that using minicircle-based vectors as novel vehicles for delivering targeted therapeutic tools, such as CRISPR platforms to express the endonuclease and guide RNA or using them as the donor vector for gene targeting, has improved the speed, efficiency, and safety of targeted gene treatment.^37,79,80

Figure 2.

Schematic representation of ex vivo and in vivo gene delivery using a minicircle vector

In the in vivo method, a functional copy of the target gene is contained in the minicircle, which is administered systemically via hydrodynamic injection through the tail vein. The minicircle enters the bloodstream and efficiently penetrates hepatocytes, inducing the transfected hepatocytes to express the protein product of the transgene. This hydrodynamics-based gene delivery is a common procedure for studying gene function and investigating various gene-based therapies for liver and other organ diseases in rodent models of various disorders. In the ex vivo method, specific cells are first isolated from the target tissue of the animal model and genetically modified by delivering the minicircle containing a functional copy of the gene of interest in the laboratory. The genetically modified cells are then characterized to select the transgenic cells with desired phenotypes before their application. Finally, the modified cells with verified characteristics are transplanted back into the donor animal.

Gene therapy

Gene therapy is a method used to deliver genetic material to repair, replace, or regulate genes with the aim of treating or curing genetic disorders caused by mutations in the endogenous genes, which lead to the expression of deficient or dysfunctional proteins. Vectors designed for gene therapy are generally classified as viral or non-viral.² Viral vectors are still widely used in clinical trials due to their high transduction efficiency; however, their immunogenicity and risk of insertional mutagenesis have raised safety issues, leading to the development of non-viral DNA vectors. Plasmids are easy and low-cost non-viral alternatives with a better safety profile including low immunogenicity and non-integrative properties.^7,81 However, plasmids have insufficient delivery rates compared with viral vectors and a short duration of transgene expression, which has driven the development of novel non-viral gene transfer methods.¹⁵ Additionally, the use of antibiotic resistance genes in the plasmid structure may pose a risk of transfer and replication of these genes in the host microbiome and the environment. Residual antibiotics remaining after vector preparations may also provoke immune responses in patients.^6,7 These drawbacks along with the unfavorable effects of prokaryotic sequences on transfection efficiency, transgene expression, and biological safety, have led to increased efforts to develop new classes of non-viral DNA vectors including minicircles that only contain therapeutic sequences.^72,82 Numerous gene therapy studies using minicircles have resulted in successful therapeutic outcomes, confirming the versatility of this minimal vector for potential use in gene-based therapies. Detailed information on recent studies that utilized the minicircle system in the field of gene therapy has been summarized in Table 1.

Table 1.

Summary of the studies that were conducted on the applications of minicircle vector for somatic gene therapy

Disorder	Minicircle vector	Target cell or tissue	Mode of minicircle delivery	Animal model	Therapeutic effect/application	Reference
Blood diseases	MC-SaCas9-sgRNA	hematopoietic stem/progenitor cells	electroporation	–	significant increase in γ-globin expression and fetal hemoglobin reactivation	Ma et al.83
Retinal diseases	MC.CMV-GFP	eye retina	intravitreally or subretinally injection	male Sprague-Dawley rats	higher retinal transfection efficiency and expression rate without ocular inflammation or toxicity	Gallego et al.84
	RHOK.RHO.IRES.dsRed minicircle	eye retina	subretinal injection of minicircle-transfected rod photoreceptor precursors	female rhodopsin knockout mice (Rho−/−)	reconstruction of the functional retina that restored vision in blind Rho−/− mouse	Barnea-Cramer et al.85
Skin diseases	GCP-2 MC	excisional wounds	intradermal injection of minicircle-transfected HDF cells around the wounds	male nude mice with wounds generated by full thickness excision of skin	increasing re-epithelialization and capillary density that accelerated wound healing	Han et al.86
	MCC7	RDEB keratinocytes	in vitro transfection with highly branched multifunctional poly(β-amino ester) (HPAE)	–	high-level expression of C7 protein with no obvious cytotoxicity in RDEB keratinocytes	Zeng et al.87
		HPDFs and C7 null RDEB fibroblasts	in vitro transfection with linear-branched hybrid poly(β-amino ester) (LBPAE)	–	high-level expression of C7 protein with no obvious cytotoxicity in RDEB fibroblasts	Zeng et al.88
	MC501C7-EF1α	RDEB keratinocytes	in vitro transfection with BrPERfect	–	highly efficient and safe expression of C7 by human promoters in RDEB keratinocytes	Wang et al.89
Brain diseases	anti-α-synuclein shRNA-MC	brain	i.v. injection of RVG-exosomes	male S129D α-synuclein transgenic mice	decreasing in cellular aggregates of α-synuclein, reducing the loss of dopaminergic neurons, improving motor and behavioral abnormalities	Izco et al.90
Liver diseases	MC-TTR-miR-206 MC-TTR-miR-206-MM	liver of dietary obese mice	tail vein injection of minicircle-jetPEI complexes	dietary obese male C57Bl/6 mice	reducing the biosynthesis of lipids and glucose in hepatocytes and therapeutic effects on both hyperglycemia and hepatosteatosis	Wu et al.91
Kidney diseases	mc-Klotho	mouse liver	tail vein injection	male BALB/c mice with ischemia-reperfusion injury	decreasing kidney damage, and improving the function and histological features of acute and chronic kidney injuries	Shin et al.92
Metabolic disorders	MC.P3-hCBS	Liver	tail vein injection	Tg-I278T Cbs−/− mice	efficient delivery and liver-specific expression of functional human CBS in mouse hepatic cells resulted in the successful correction of the key metabolic and phenotypic abnormalities associated with CBS deficiency with no observed symptoms of liver damage	Lee et al.93
Hepatitis B infection	MC.IFNλ3	HepG2.2.15 hepatocyte cell line	lipofection	–	specific expression of IFNλ3 by the minicircle-activated JAK/STAT pathway that resulted in a strong in vitro anti-HBV activity in the form of repressing both viral antigen expression and DNA replication, indicating that minicircle can be used as a promising tool for gene therapy against chronic or persistent HBV infections	Kang et al.94
Self-production of therapeutic proteins	mc-anti-CD25HC mc-anti-CD25LC mc-IL-10 mc-IL-10-anti-CD25HC	immune system	tail vein injection	BALB/c mice transplanted with the skin tissue from C57BL/6 mice	improving skin allograft survival, better histological signs, and decreasing immunosuppressive side effects	Lim et al.95
	mc-anti-CD25HC mc-anti-CD25LC mc-anti-CD25HC-IL-10-CXCR3	immune system	tail vein injection	BALB/c mice transplanted with the skin tissue from C57BL/6 mice	increasing skin allograft survival, improving histological parameters and immunological regulation	Lim et al.96

Blood diseases

Among blood disorders, β-thalassemia is one of the most common monogenic defects worldwide and imposes a heavy health and economic burden on patients at risk.⁹⁷ β-Thalassemia is an autosomal recessive disorder caused by various deletion mutations in the β-globin gene, defects in β-globin transcription, mRNA maturation, mRNA translation, and instability of the β-globin chain that disrupt β-globin synthesis.⁹⁸ These defects result in reduced or absent normal adult hemoglobin, ineffective erythropoiesis, and red blood cell lysis, which manifests as severe anemia.^99,100 In recent years, various approaches have been proposed for the treatment of blood monogenic diseases such as β-thalassemia based on genetic manipulation of hematopoietic stem/progenitor cells (HSPCs).¹⁰¹ In adults, the B cell lymphoma/leukemia 11A (BCL11A) gene expresses a transcription suppressor in hematopoietic cells that plays a critical role in controlling fetal-to-adult hemoglobin switching. In fact, BCL11A protein blocks the interaction of the γ-globin promoter with the locus control region by binding to the γ-globin proximal promoter and therefore represses the production of fetal hemoglobin.^102,103 Consequently, reactivation of fetal hemoglobin is considered a potential approach for the treatment of β-thalassemia.^104,105 By improving gene editing tools such as CRISPR-Cas9, knocking out the BCL11A gene or editing the γ-globin promoter to inhibit the transcription-suppressing effect of BCL11A could be a means to reactivate γ-globin expression and thus fetal hemoglobin production.^106,107 In a recent study, Ma et al. used a minicircle vector encoding the CRISPR-Cas9 system in HSPCs to reactivate γ-globin expression as a potential therapeutic approach for β-thalassemia.⁸³ The researchers designed a minicircle to express S. aureus Cas9 (SaCas9) under the cytomegalovirus (CMV) promoter and a sgRNA under the U6 promoter (MC-SaCas9-sgRNA) for knocking out the BCL11A gene. SaCas9 was chosen because its activity is comparable with S. pyogenes Cas9 (SpCas9) for genome editing in mammalian and human cells, but it is 25% smaller than SpCas9.¹⁰⁸ Therefore, SaCas9 was applied to reduce the size of the gene editing vector. Considering that minicircles are smaller than conventional plasmids, they may be a more ideal vector for CRISPR-SaCas9 delivery. HSPCs were isolated from peripheral blood using CD34⁺ beads and then used for minicircle electroporation. Subsequently, fluorescence-activated cell sorting analysis was utilized to collect erythroblasts. The transfected HSPCs were then subjected to erythroid differentiation using a three-stage differentiation protocol. After differentiation, the expression of γ-globin was assessed in terminally differentiated erythroid HSPCs.⁸³ The results provided evidence that γ-globin expression and fetal hemoglobin reactivation were significantly increased in transfected cells compared with HSPCs transfected only with MC-SaCas9 (without sgRNA expression) as the control group. Interestingly, deep sequencing analysis confirmed that the insertion frequency of the MC-SaCas9-sgRNA vector into the genome of transfected cells was negligible. These findings indicate that SaCas9-mediated editing of the BCL11A locus using minicircle vectors is associated with minimal integration rate and genotoxicity, highlighting their potential for safe gene therapy applications.⁸³

Retinal diseases

Several blinding retinal disorders have a genetic basis including photoreceptor degeneration, which is the most common cause of blindness in several inherited retinal diseases.¹⁰⁹ Currently, there are no definitive treatments for these disorders but gene therapy appears to be a promising treatment strategy.^110,111 The most commonly used vector for gene therapy of retinopathies is the recombinant adeno-associated viral (rAAV) vector, as it is generally considered a safe and efficient vector for delivery into non-dividing cells.¹¹² Nevertheless, there are limitations to consider when using the AAV vector for gene therapy. These limitations include the size of the transgene, potential immune responses against viral proteins, the risk of oncogenesis, and the ability of the virus to spread to the systemic circulation and distant organs from the site of administration. These obstacles hinder the widespread application of this viral vector.^113,114,115 On the other hand, gene therapy based on non-viral delivery systems may also be a potential approach for the treatment of retinopathies, although poor transfection efficiency and toxicity of non-viral methods are still major barriers to achieving an efficient gene therapy method for retinal diseases.^110,116 Ex vivo gene delivery to photoreceptor cells can overcome some of the challenges of in vivo gene transfer methods for long-term expression by non-viral approaches.⁸⁵ Niosomes are a type of non-viral delivery system composed of cationic lipids that can bind to nucleic acids and form complexes with positive charges protecting the nucleic acids from enzymatic digestion. They have a stable formulation, low toxicity, and the capability of in vivo transfection.^{117,118,119,120,121} In 2019, Gallego et al. demonstrated the ability of niosomes to interact with DNA molecules, which is influenced by time and temperature. These carriers also showed the ability to protect and release different genetic materials with controlled kinetics.⁸⁴ In this study, niosomes were used to deliver DNA molecules of different sizes including the minicircle containing green fluorescent protein, MC.CMV-GFP (2.3 kb), to human ARPE-19 retinal pigment epithelium cells. The results showed that the transfection efficiency of minicircle nioplexes was double that of conventional plasmids and maintained acceptable cell viability in all cases.⁸⁴ In the preclinical study, minicircle nioplexes were intravitreally and subretinally injected into rat retina. These injections to the back of the eye resulted in a higher expression rate of the reporter without any symptoms of ocular inflammation or toxicity for minicircle nioplexes in comparison with plasmid counterparts. Therefore, considering the greater capacity of cationic niosomes carrying minicircle into retina cells, it seems that minicircle-loaded nioplexes can overcome the expression limitations of plasmids that have low transfection efficiency.⁸⁴

Barnea-Cramer et al. also used a minicircle as a functional non-viral DNA vector for the robust treatment of photoreceptors in a rhodopsin knockout mouse model. In this ex vivo gene delivery experiment, genetically modified photoreceptor precursors were transplanted with the aim of retinal reconstruction and restoration.⁸⁵ A human rhodopsin-expressing minicircle with a bicistronic expression cassette was constructed for this purpose. The vector, named RHOK.RHO.IRES.dsRed minicircle, contained a human rhodopsin kinase promoter, a human rhodopsin coding sequence, an internal ribosome entry site (IRES), and a dsRed (Discosoma sp. red) fluorescent marker.⁸⁵ The minicircle vector was transfected into primary rod photoreceptor precursors sourced from the retinas of mice with homozygous rhodopsin null mutations (Rho^−/−) using NanoJuice transfection reagent (Novagen). Transfected minicircles showed comparable long-term expression of a wild copy of the missing gene to the AAV vectors in photoreceptors in vitro. For in vivo testing, non-functional murine rod photoreceptor precursors were genetically corrected using the RHOK.RHO.IRES.dsRed minicircle firstly and then subretinally transplanted into blind Rho^−/− mice with the same genetic mutation. The successful ex vivo minicircle-based gene transfer reconstructed the functional retina and restored vision in blind animal models. Also, the assessment of gene delivery efficiency by the minicircle in comparison with recombinant AAV vector displayed no significant difference between the two vectors.⁸⁵ The successful ex vivo gene therapy through transplantation of minicircle-manipulated Rho^−/− rod precursors demonstrated efficient and sustained rhodopsin expression for 3 months post-transplantation. Additionally, the expression level of the missing gene and its duration using minicircle technology was comparable with AAV. This new approach provides reasonable insights into autologous cell-based gene therapy using a source of genetically corrected patient’s own cells through a non-viral system. It holds promise for the safe and specific treatment of hereditary retinopathies such as retinitis pigmentosa and potentially other neurodegenerative disorders.⁸⁵

Skin diseases

Effective therapies for wound healing are challenging due to the constant state of inflammation, infection, tissue hypoxia, and vascular insufficiency.¹²² Previous studies have shown that certain chemotactic cytokines, known as chemokines, play important roles in wound healing by regulating cell migration and angiogenesis.¹²³ For example, overexpression of granulocyte chemotactic protein 2 (GCP-2) from the CXC chemokine subfamily has been found to induce the expression of factors associated with wound healing such as insulin-like growth factor-1, hepatocyte growth factor, and interleukin-8 (IL-8).^123,124 In a recent study by Han et al., a GCP-2-expressing minicircle (GCP-2 MC) with CMV-GCP2 and EF1α-GFP cassettes was constructed and used to transfect human dermal fibroblast (HDF) cells by microporation.⁸⁶ Episomal overexpression of GCP-2 significantly enhanced the expression of angiogenic and epithelialization-associated genes such as epidermal growth factor, vascular endothelial growth factor A (VEGF-A), and IL-8 compared with the mock minicircle. In an in vitro test, the scratch wound healing assay also showed that the culture medium from GCP-2 MC-transfected HDF cells significantly improved the rate of wound healing and the number of tube branches compared with the mock minicircle.⁸⁶ Furthermore, in an in vivo study, HDF cells containing GCP-2 MC or mock minicircle were injected near the wounds generated by excision of full-thickness skin in male nude mice. The results showed that the overexpression of GCP-2 accelerated the healing of the wounds. Histological analysis of the treated wounds also revealed improved re-epithelialization and a larger area covered by neo-epidermis. Besides, angiogenesis in wounds injected with GCP-2 MC was significantly higher than those injected with mock minicircle or PBS. These findings confirm the therapeutic potential of GCP-2-expressing minicircles in wound treatment.⁸⁶

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe and heterogeneous congenital skin disease caused by a deficiency of type VII collagen (C7), an essential extracellular matrix protein for maintaining the integrity of the skin. This genetic condition results from various loss-of-function mutations in the COL7A1 gene, leading to a lack of C7 as a structural protein, which causes skin fragility.^125,126 Despite numerous therapeutic strategies proposed over the years to alleviate symptoms and improve patient’s quality of life, there is still no effective treatment for RDEB. In an effort to address this, Zeng et al. used a minicircle-based delivery system to restore C7 expression in cells isolated from RDEB patients. They constructed a 12 kb minicircle DNA (MCC7) containing the complete COL7A1 cDNA sequence of approximately 9 kb under the CMV promoter as a therapeutic vector.⁸⁷ Combining MCC7, which was labeled with the red fluorescent dye Cy3 for intracellular tracking with a highly branched multifunctional poly (β-amino ester), a synthetic polymer,¹²⁷ gave rise to the formation of minicircle-containing polyplexes used for transfection of RDEB keratinocytes. This method achieved a high transfection efficiency of approximately 96.4% of transfected cells, leading to a substantial increase in the COL7A1 mRNA level in the transfected cells and robust expression of recombinant C7. Additionally, the mean fluorescence intensity of the transfected RDEB keratinocytes with Cy3-labeled MCC7 minicircles was approximately 2-fold higher than that of the control cells. These findings demonstrated that the keratinocyte cells took up several copies of MCC7 with no obvious cytotoxicity, indicating the potential of the minicircle vector as a promising translational approach toward palliative therapy in RDEB.⁸⁷

In line with previous research, a multifunctional linear-branched hybrid poly (β-amino ester) (LBPAE) was used to transfect human primary dermal fibroblasts and C7 null RDEB fibroblasts with MCC7. LBPAE/MCC7 polyplexes were effectively internalized, leading to the restoration of C7 expression in both fibroblast cell types.⁸⁸ Therefore, by employing an efficient gene delivery system, minicircle has the potential to be used for successful cell-based gene therapy to correct the genetic defect in RDEB and reverse the disease phenotype.

To prevent undesirable immune responses and irritation reactions caused by repeated application of gene therapy procedure, a long-term treatment is preferred for RDEB patients similar to any genetic disorder. Therefore, therapeutic approaches that can provide sustained expression of C7 are applicable with a lower frequency and are attractive for RDEB patients with severely fragile skin.⁸⁹ Accordingly, to provide a safer and more efficient gene replacement therapy in RDEB patients, Wang et al. constructed parental plasmids expressing C7 under the human EF1α constitutive promoter and human COL7A1 tissue-specific promoter (C7P) for use in gene therapy experiments on the skin.^128,129 Although the CMV promoter is a strong promoter that has been widely used in many DNA vaccines and gene therapy experiments, it is reported that methylation of this viral promoter can result in gene silencing.^130,131 As a result, the authors believe that using the human promoter instead of the CMV promoter not only leads to persistent and sufficiently high expression of the therapeutic gene but also could minimize the risk of potential immunogenic responses or oncogenic events. Three parental plasmids with CMV, EF1α, or C7P were transfected into immortalized primary human RDEB keratinocytes, and the relative transcription of C7 by each vector was measured. The results indicated that the CMV promoter achieved the highest mRNA level compared with EF1α, while the C7P promoter resulted in the lowest transcription rate. However, western blot analysis showed no significant difference in C7 protein expression between the CMV and EF1α promoters in the cells. The lowest protein expression level observed with the C7P promoter could be attributed to the natural intracellular regulators specific to this promoter, which function similarly to regulate normal C7 protein production in healthy epidermal keratinocytes. Therefore, the C7P promoter could be a rational candidate for application in non-viral gene therapy vectors, provided that in vivo delivery of the vectors can efficiently supply physiological levels of C7.⁸⁹ As a rule, when selecting a promoter for a clinical application, it is crucial to evaluate the transfection efficiency of the vector, which is largely dependent on its size. Therefore, minicircles, as minimal vectors containing human promoters, could be utilized for highly efficient and safe non-viral gene replacement therapy to restore C7 expression and alleviate the symptoms in RDEB patients.⁸⁹

Overall, based on the studies mentioned above, it is suggested that a combination of synthetic polymers with minicircles composed of a large gene payload under the control of human promoters could be a promising non-viral, non-integrating polymeric vehicle for the topical genetic treatment of inherited skin disorders.

Brain diseases

Parkinson’s disease is considered the second most common neurodegenerative disorder worldwide, particularly affecting people over 50 years old. However, there is currently no effective disease-modifying therapy for it.^132,133 Even though the primary cause of Parkinson’s disease has not been identified in most patients, the progressive loss of dopaminergic cells in the substantia nigra is believed to be the main cause of the disease. Moreover, the accumulation of intraneuronal cytoplasmic proteinaceous inclusions and dystrophic neuritis containing α-synuclein deposits have been ascribed to the pathogenesis of the disease.¹³⁴ Several studies have also identified mutations in the α-synuclein gene (SNCA) that play a central role in the progressive presynaptic aggregation of α-synuclein protein in the brain. This aggregation may lead to neurodegeneration, motor abnormalities, and the stepwise emergence of both familial and sporadic forms of Parkinson’s disease.^135,136,137 Accordingly, it has been suggested that suppressing neuronal α-synuclein expression could potentially delay or even halt the progression of the disease.^134,138 Based on these findings, Izco et al. developed a minicircle encoding anti-α-synuclein shRNA (anti-α-synuclein shRNA-MC) capable of efficient knocking down the human and mouse SNCA genes.⁹⁰ To deliver the minicircle specifically to the brain tissue in vivo, the researchers used a transgenic mouse model that expresses human S129D α-synuclein-HA with aggregates present throughout its brain. Brain-targeting exosomes were isolated and purified from the conditioned medium of mouse bone marrow primary dendritic cells engineered to express rabies virus glycoprotein (RVG) peptides. The anti-α-synuclein shRNA-MC molecules were loaded into the exosomes with RVG peptides on their exterior surface (RVG-exosomes). Subsequently, RVG-exosomes loaded with the minicircle were then injected into the tail vein of mouse models.⁹⁰ This delivery method effectively distributed the shRNA-MC to various brain regions in parkinsonism models, leading to a sustained downregulation of the SNCA gene in those regions. The exosomal transfer of the minicircle delayed the process of neurodegeneration while maintaining sufficient protein levels to fulfill its normal function. This intervention reduced the intraneuronal aggregates of α-synuclein and reduced the loss of dopaminergic neurons in the substantia nigra without triggering immune responses. Additionally, the widespread downregulation of SNCA improved the clinical symptoms of progressive disease including motor and behavioral abnormalities.⁹⁰ The results exhibited that central nervous system (CNS)-targeted delivery of specific shRNA-encoding minicircles using RVG-exosomes as an in vivo gene silencing strategy holds immense potential for prolonged and safe treatment of Parkinson’s disease. This approach also offers significant economic and therapeutic advantages over siRNAs, because it requires less frequent administration compared with siRNAs, which have a short in vivo half-life and has the potential to be employed for treating other chronic neurodegenerative disorders as well.⁹⁰

Liver diseases

The liver plays a crucial role in various body functions including digestion, metabolism, immune activity, detoxification, and blood clotting.¹³⁹ Liver diseases are responsible for approximately two million deaths per year worldwide. Targeted gene therapy is an attractive treatment strategy that could alleviate the burden of liver diseases.¹⁴⁰ Hyperlipidemia is a major cause of atherosclerosis and cardiovascular disease (CVD).¹⁴¹ While the administration of routine cholesterol-lowering drugs such as statins has reduced the risk of coronary artery disease, concerns regarding safety, high cost, and limited efficacy on cardiovascular mortality have restricted their use.^142,143 Therefore, the need for novel and effective approaches to control hyperlipidemia and CVD has become increasingly vital. Some microRNAs are involved in lipid and lipoprotein metabolism, and their expression is altered in patients with dyslipidemia, atherosclerosis, and CVD.^144,145,146 As a result, they could be novel molecular targets for the treatment of lipid-related disorders. For example, in hepatic insulin resistance, which is observed in approximately 90% of obese patients, glucose metabolism does not respond to insulin leading to hyperglycemia and promoting hepatic lipogenesis, which can give rise to fatty liver disease (hepatosteatosis).^147,148 In this context, Wu et al. demonstrated that the overexpression of microRNA-206 in the livers of dietary obese mice had significant therapeutic impacts on hyperglycemia and hepatosteatosis. They constructed two minicircle vectors: MC-TTR-miR-206 containing the coding sequence of the miR-206 precursor, and MC-TTR-miR-206-MM, which contained a mismatched miR-206 sequence in its seed region, serving as a control vector to rule out non-specific effects of MC-TTR-miR-206. Both vectors were equipped with the transthyretin (TTR) gene promoter to ensure liver-specific expression. These minicircles were complexed with an in vivo transfection reagent and injected into male C57Bl/6 mice on a high-fat diet via the tail vein.⁹¹ The results revealed that hepatic expression of microRNA-206 significantly reduced glucose production by facilitating insulin signaling, thereby decreasing gluconeogenesis and blood glucose levels. Additionally, microRNA-206 suppressed hepatic lipogenesis, leading to a decrease in fatty liver disorder and blood cholesterol in dietary obese mice with low liver toxicity.⁹¹ Mechanistically, the authors elucidated that miR-206 targeted protein tyrosine phosphatase, non-receptor type 1 (PTPN1) mRNA, resulting in a potent downregulation of target gene expression and diminished biosynthesis of intracellular glucose and lipids in hepatocytes. These findings suggest that the microRNA-206-expressing minicircle has the potential for therapeutic applications in co-morbid disorders such as hyperglycemia and hepatosteatosis.⁹¹

Kidney diseases

Clinical studies have demonstrated that Klotho expression is significantly downregulated in human acute kidney injury (AKI) and chronic kidney disease. These findings suggest that the loss of Klotho may underlie the pathogenesis of these conditions.^149,150,151 Due to the short half-life of Klotho, the recombinant protein must be administered daily or every other day to effective.^152,153 One strategy to overcome this challenge is to utilize the minicircle system, which leverages the host cell’s protein synthesis machinery to produce a long-acting Klotho protein for therapeutic application. To achieve this, a minicircle containing the Klotho coding sequence under a CMV promoter was constructed. The resulting Klotho-encoding minicircle, named mc-Klotho was transfected into HEK293T cells, and the level of Klotho protein in the culture medium was assessed using the ELISA method. The in vitro results confirmed the efficient expression and secretion of recombinant Klotho protein by the minicircle.⁹² For in vivo gene delivery, AKI was induced by kidney ischemia-reperfusion in male BALB/c mice and mc-Klotho was hydrodynamically injected into the tail vein of renal injury mouse models as a single dose. To investigate the robust expression and secretion of exogenous Klotho protein in liver cells, the authors measured the Klotho concentration in the plasma samples of the mouse models. The data showed that the Klotho concentration in the mice injected with mc-Klotho was significantly higher than that of the control group during the experiment.⁹² The Klotho-encoding minicircle also upregulated some antioxidant genes including SOD2 and catalase, while downregulating proinflammatory genes such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), as well as profibrotic genes like collagen I and TGFβ1. Additionally, the authors indicated that a single injection of Klotho minicircle increased the concentration of functionally active Klotho in the liver cells and it was detected in the mice serum at therapeutic levels for a long time.⁹² As a result, this method reduced kidney damage and improved the function and histological features of both acute and chronic kidney injuries. In conclusion, it has been proposed that this non-viral system has some advantages for the treatment of kidney injuries over recombinant Klotho, including bypassing the complex processes of synthesis, purification, and functional characterization of recombinant protein drugs.⁹²

Metabolic disorders

Cystathionine β-synthase (CBS) deficiency, also known as classical homocystinuria, is a rare autosomal recessive disorder in the catabolic pathway of methionine. It is caused by mutations in the CBS gene, leading to impaired synthesis of cystathionine and very high levels of homocystine in the patients’ blood and urine. CBS deficiency is the most common inborn error of sulfur metabolism with various clinical symptoms.^154,155 Currently, the administration of pyridoxine along with betaine and a low-protein diet containing a limited amount of methionine as the homocysteine precursor is used as a strategy to rapidly and permanently reduce the serum homocysteine concentration in order to control CBS deficiency.^155,156,157 Gene therapy has emerged as a promising approach for treating various inherited metabolic disorders by targeting the defective gene. In a study conducted by Lee et al., minicircle technology was employed for experimental gene therapy in a transgenic mouse model of CBS deficiency, Tg-I278T Cbs^−/−, characterized by alopecia, reduced bone density, and loss of fat mass.¹⁵⁸ For the genetic treatment of CBS deficiency, a 2.3 kb minicircle vector containing human CBS cDNA driven by the P3 promoter, an artificial liver-specific promoter, was used. The minicircle named MC.P3-hCBS was introduced into mouse models by a single hydrodynamic tail vein injection.⁹³ The minicircle-encoded human CBS resulted in a significant reduction in total serum homocysteine levels from day 7 to at least 41 days after injection with sustained effects compared with saline-injected controls. Enhanced CBS expression in liver tissue was confirmed through western blot analysis, and tissue enzymatic activity measurement based on homocysteine levels, indicating a 34-fold increase in liver CBS activity in the minicircle group relative to the controls. Besides, the serum homocysteine levels were decreased with a maximum reduction of 64% compared with the control group. Long-term monitoring of young treated mice demonstrated persistent metabolic correction and significant improvement in the alopecia phenotype, indicating a broader therapeutic impact. The authors also concluded that the injection of higher amounts of MC.P3-hCBS could lead to longer and more efficient homocysteine reduction.⁹³ These findings indicate that naked MC.P3-hCBS was safe with no observed symptoms of liver damage and efficient in delivering functional human CBS into mouse hepatic cells. The vector successfully corrected the key metabolic and phenotypic abnormalities associated with CBS deficiency. This study suggests that minicircle-based gene therapy could be a practical approach in treating CBS-deficient patients.⁹³

Hepatitis B infection

Hepatitis B virus (HBV) infection is a major global health concern that can lead to severe liver complications including cirrhosis and hepatocellular carcinoma.^159,160 Currently, there is no effective curative therapy for chronic hepatitis B infection. While some nucleos(t)ide analogs can inhibit viral reverse transcriptase, they are unable to eliminate the pre-existing episomal viral cccDNA.⁹⁴ Other anti-HBV therapies involve the administration of interferons (IFNs) such as interferon alpha (IFN-α). However, the clinical use of IFN-α is limited due to its high cost, the need for repeated administration over a long treatment period, severe systemic side effects, and low therapeutic response.^161,162 Given that HBV specifically infects hepatocytes and the higher antiviral potency and low toxicity of IFNλ3,¹⁶² liver-targeted IFNλ3 gene therapy could be a novel strategy to overcome the limitations of systemic administration of other IFNs. To investigate this approach, Guo et al. constructed a liver-specific minicircle encoding IFNλ3 (MC.IFNλ3).⁶² The vector was designed to sustain the expression of IFNλ3 under the control of a hepatocyte-specific ApoE promoter and was tested for its anti-HBV activity in vitro. MC.IFNλ3 was used to transfect the HepG2.2.15 cell line as a hepatocyte-derived cell model. The authors found that the tissue-specific expression of IFNλ3 activated the interferon-stimulated gene by activating the JAK/STAT pathway in HepG2.2.15 cells. As a result, strong in vitro anti-HBV activity was observed with repression of both viral antigen expression and viral DNA replication, especially in the later stage of the infection. This study evidenced that liver-targeted IFN gene transfer using the MC.IFNλ3 vector could be a promising tool for developing a gene therapy approach for the treatment of chronic or persistent HBV infections as an alternative to IFN protein therapy.⁶²

Self-production of therapeutic proteins

The in vivo production of protein drugs is a novel strategy for biosynthesizing therapeutic proteins by the host’s own cells after in vivo transfection of an expression system. This strategy reduces treatment costs compared with the administration of recombinant protein drugs, which require complex processes for active production, purification, and efficacy testing.¹⁶³ Minicircle are a promising tool for robustly expressing the protein of interest in target cells. With a suitable signal peptide, the expressed protein can be secreted from the host cells and reach the site of action.¹⁶⁴ Based on this platform for protein drug production, Lim et al. evaluated the effect of minicircle-expressed new protein drugs on reducing the risk of allograft rejection.⁹⁵ They designed two minicircles to express an anti-CD25 antibody, which plays a role in preventing allograft rejection by blocking peripheral T cells with immunosuppressive function. To do this, they constructed separate minicircles with the coding sequences of basiliximab (anti-CD25mAb) light chain (mc-anti-CD25LC) and heavy chain (mc-anti-CD25HC). They also designed other minicircles, one encoding IL-10 (mc-IL-10) and the other encoding a fusion protein in which the IL-10 molecule was conjugated to the basiliximab heavy chains (mc-IL-10-anti-CD25HC). Conjugated IL-10 in the anti-CD25/IL-10 fusion protein (Figure 3A) can adjust the downregulatory effect of anti-CD25 on T regulatory (Treg) cells, which have a vital role in regulating immune tolerance in transplantation.^165,166 All the minicircle vectors were assembled with a CMV promoter and a sequence encoding for a signal peptide was also inserted at the beginning of each ORF to enable the secretion of the expressed proteins.⁹⁵ Successful and efficient production of intact anti-CD25 protein was confirmed by co-delivery of mc-anti-CD25HC and mc-anti-CD25LC to HEK293T cells in the culture medium. Similarly, this test was carried out by co-transfecting mc-IL-10-anti-CD25HC and mc-anti-CD25LC, resulting in the expression of the anti-CD25/IL-10 fusion protein as a dual target-directed agent (DTA).⁹⁵ For in vivo evaluation of the therapeutic efficacy of the minicircles, BALB/c mice were divided into four groups. They received mock minicircle (control), mc-anti-CD25LC and mc-anti-CD25HC (expressing anti-CD25), mc-IL-10, and the minicircles expressing anti-CD25/IL-10, respectively, by hydrodynamic delivery. The next day, all mice were transplanted with skin allografts from C57BL/6 mice as skin graft models.⁹⁵ The in vivo findings showed substantial self-production and secretion of IL-10-anti-CD25 protein that was detected in the plasma samples for 10 days after injection. Also, this antibody-cytokine fusion protein was functionally active and caused a longer survival of the allografts, better histological changes in skin allografts, and superior immunological regulation in the anti-CD25/IL-10 group compared with the other groups. Furthermore, the study showed that co-administration of IL-10-anti-CD25-encoding minicircles with tacrolimus (Tac), the first-line immunosuppressive drug, increased the mean survival time of the allografts and reduced immunosuppressive side effects in comparison with the group that only received Tac.⁹⁵

Figure 3.

Schematic representation of the structure of antibody-cytokine fusion proteins

(A) The IL-10-anti-CD25 fusion protein is a DTA, consisting of a pair of IL-10 molecules that are conjugated to the heavy chains at the end of Fab regions in an anti-CD25 antibody via short linkers. (B) The anti-CD25/IL-10/CXCR3 fusion protein is a multiple target-directed agent (MTA), composed of an anti-CD25 antibody conjugated to a pair of IL-10 molecules at the end of the Fc region, with IL-10 molecules bonded to CXCR3 molecules from the other side. Short flexible linkers fuse all the protein molecules to each other.

In a similar investigation conducted by the same group in 2022, they developed a new minicircle vector, mc-anti-CD25HC-IL-10-CXCR3, in addition to mock minicircle, mc-anti-CD25LC, and mc-anti-CD25HC. This new minicircle vector was designed for the self-biosynthesis of a multifunctional protein by host cells to reduce acute rejection of skin allografts.⁹⁶ In detail, the minicircle contained in-frame coding sequences of anti-CD25HC, IL-10, and C-X-C motif chemokine receptor 3 (CXCR3). CXCR3 is a chemokine that directs the infiltration of upregulated T cells into the allograft.¹⁶⁷ All minicircles included the EF1 promoter and a sequence encoding for a signal peptide at the beginning of their ORFs.⁹⁶ Co-transfection of mc-anti-CD25HC-IL-10-CXCR3 and mc-anti-CD25LC into HEK293T cells demonstrated the successful and efficient production of intact anti-CD25/IL-10/CXCR3 protein as an MTA. Structurally, each molecule of this fusion protein consists of an anti-CD25 antibody, two IL-10, and two CXCR3 molecules (Figure 3B).⁹⁶ The effectiveness of anti-CD25/IL-10/CXCR3 in preventing skin allograft rejection was evaluated using animal models from the previous study. The allograft mice were divided into three groups and received intravenous (i.v.) injections of mock minicircle+Tac (control), anti-CD25/IL-10-encoding minicircles+Tac, and anti-CD25/IL-10/CXCR3-encoding minicircles+Tac 1 day before receiving the skin allografts. Tac was administered with daily doses through oral gavage in all groups.⁹⁶ The results showed that the anti-CD25/IL-10/CXCR3 fusion protein was self-produced and secreted for approximately 40 days in the liver tissue. Furthermore, this antibody-cytokine fusion protein, along with Tac, elevated the survival of skin allografts and enhanced histological parameters in the skin allografts compared with the other groups. It also improved immunological regulation such as reducing proinflammatory T cells and increasing the restoration of Treg cells, confirming the influential role of CXCR3 chemokine in promoting the described immunological benefits.⁹⁶

These studies collectively demonstrate that by improving the safety and efficacy of minicircle technology, self-biosynthesis of functionally active antibody-cytokine fusion proteins is well achievable in the patient’s target cells. Therefore, this method could be considered a favorable and cost-effective strategy that robustly addresses the need for repeated administration of therapeutic proteins to prevent acute allograft rejection or for other therapeutic applications.^95,96

Regenerative medicine

Regenerative medicine combines various engineering and life science disciplines to restore or replace tissues or entire organs that have been damaged due to trauma, inherited defects, age-related disorders, and chronic or acute diseases. The advancements in this new field of medicine offer the potential to treat diseases at their source without being limited by the availability of tissue or organ donors or the risk of severe immune responses against transplanted tissues or organs.^168,169 In fact, regenerative medicine utilizes various biomedical techniques including stem cell therapy, gene therapy, tissue engineering, and biomaterials to improve tissue and organ functions and provide therapeutic options for the mentioned disorders.^170,171 One promising approach to enhance this process is through gene-directed methods. The use of non-viral DNA vectors such as minicircles, which are free of any viral and bacterial sequences inhibits innate immune responses, improves the effectiveness of therapeutic genes for safe modification of various types of stem cells, and increases the survival rate of the cells in different animal models.⁷⁵ This reasonable approach suggests that minicircle-based cell manipulation has great potential for regenerative therapy.^27,172 Detailed information regarding the recent studies that utilized the minicircle system in the field of regenerative medicine is summarized in Table 2.

Table 2.

Summary of the studies that were conducted on the applications of minicircle vector for regenerative medicine

Disorder	Minicircle vector	Target cell or tissue	Mode of minicircle delivery	Animal model	Therapeutic effect/application	Reference
Brain and spinal cord injuries	mc-GFP	neural stem cells	in vitro magnetofection with magnetic nanoparticles	–	the minicircle-nanoparticle modified NSCs showed high viability, proliferation, and maturation capacity with the potential versatility of this cell-biomaterial platform for neuroregeneration	Finch et al.173
	mC-BDNF-GFP	olfactory mucosal cells	in vitro magnetofection with iron oxide nanoparticle	–	manipulated cells functioned as biopumps for successful secretion of neurotrophic factors, which improved the function of cOMCs in the site of spinal cord injury without detectable adverse effects	Delaney et al.174
Bone and joint diseases	Bcl-2/GFP minicircle	bone	in vivo magnetofection of iron oxide-minicircle complexes incorporated into HA-PLGA scaffold	adult male nude mice with calvarial defects	increasing cellular proliferation, reducing apoptotic markers in ASCs, and augmenting bone regeneration	Brett et al.175
	mcSOX9/6 mcSOX9/6/shANG	articular cartilage	intraarticular injection of transfected ADSCs	surgically induced osteoarthritis in the knee joint of male Sprague-Dawley rats	improving chondrogenesis and suppressing inflammation in osteoarthritis that result in chondroprotective effects	Mathieu et al.176
	CMVBMP2-Adv EF1αBMP2-Adv	C2C12 myoblast precursor cell line and adipose-derived mesenchymal stem cells	in vitro transfection with Peqfect	–	improving therapeutic gene expression and protein secretion by minicircles to induce efficient osteogenic differentiation for regeneration of bone defects	Hacobian et al.177
	mc-BMP2 mc-TGFβ3	joint	lipofection	Sprague-Dawley rat with osteochondral defects in articular cartilage	minicircle-based in vitro differentiation of iPSCs into chondrogenic pellets with therapeutic application in cartilage regeneration	Rim et al.178
	mcPTHrP 1–34 + 107–139	bone	hydrodynamic injection and intraperitoneal delivery of the minicircle-transfected MSCs	12-week-old C57BL/6 female ovariectomized mice	a gene therapy-based strategy for in vivo bone regeneration and osteoporosis treatment	Kim et al.179
	mcOPG mcPTHrP	bone	tail vein injection	female ovariectomized Sprague-Dawley rats	the stepwise administration of OPG and PTHrP enhanced bone formation and suppressed bone resorption, resulting in improved bone microstructure and mineral density. This approach suggests a promising new minicircle-based gene therapy method for the treatment of osteoporosis	Eom et al.180
Lung diseases	MC-ANGPT1	lung	i.v. infusion of genetically engineered MSCs through the jugular vein	C57Bl/6J female mice	a gene-enhanced cell-based therapy for ARDS that reduces lung inflammation and vascular permeability	Florian et al.181
Ischemic diseases	MC-VEGF	MSCs	electroporation	–	improving tissue oxygenation in ischemic diseases by cell-based gene therapy with engineered bone marrow MSCs whose angiogenic activity was increased via minicircle-mediated overexpression of VEGF	Serra et al.182
	MC-VEGF	MSCs	electroporation	–	improving tissue oxygenation in ischemic diseases by cell-based gene therapy with MSCs from different sources such as adipose tissue and umbilical cord matrix whose angiogenic activity was increased via minicircle-mediated overexpression of VEGF	Serra et al.183

Brain and spinal cord injuries

One important limitation of therapeutic transplantation of neural stem cells (NSCs) into the CNS is the high rate of cell death that occurs post-transplantation. This is attributed to challenges such as mechanical shearing and clumping of the cells during transplantation using fine gauge needles.^184,185 High cell losses reduce the efficacy of the therapy and induce secondary inflammatory responses, which in turn require more cells for transplantation procedures and increase the cost of treatment.¹⁷³ Additionally, the pathological microenvironment of neural injury has been shown to limit tissue regeneration. Therefore, overexpression of regeneration enhancers such as neurotrophic factors can change the microenvironment to a pro-regenerative medium, improving the survival of transplanted cells and facilitating the tissue repair process.^186,187 Accordingly, to investigate the safety and efficacy of genetically engineered NSCs for protected cell therapy, Finch et al. transfected NSCs with a minicircle vector encoding GFP under the EF1α constitutive promoter named mc-GFP along with NeuroMag, a commercial iron oxide nanoparticle. They demonstrated that minicircle magnetofection of the NSCs is associated with lower cytotoxicity than viral vectors, can be easily scaled up for clinical development, and offers a safe method that yields augmented therapeutic cells to enhance regenerative outcomes.¹⁷³ To examine whether the nanoengineered NSCs can propagate at the site of neurosurgical transplantation, the researchers used Duragen Plus matrix as a delivery scaffold to support the incorporation and growth of NSCs. This matrix is a clinical-grade biomaterial derived from type I bovine collagen. The minicircle-nanoparticle modified NSCs showed high viability, proliferative capacity, and a normal stem cell phenotype in the Duragen Plus matrix. Moreover, maturation of NSCs to daughter astrocytes, neurons, and oligodendrocytes within the biomaterial matrix after different time points confirmed the versatility of this platform for potential neuroregeneration at the site of CNS injury and other neural transplants.¹⁷³

Spinal cord injury can lead to paralysis and a reduced quality of life. Additionally, the cost of caring for patients with this injury is substantial. Recent preclinical studies have employed the transplantation of autologous olfactory ensheathing cells (OECs), a unique population of glial cells in the olfactory nervous system at the site of injury. The results have been promising, indicating that this method could be a successful strategy for regenerating spinal cord injuries.^188,189 However, previous experiences have shown that genetic manipulation of OECs to secrete some neurotrophic factors in spinal cord injury could potentially promote regenerative outcomes.^190,191 In a separate study, minicircle technology was used for the safe genetic engineering of canine olfactory mucosal cells (cOMCs) through magnetofection of a minicircle construct encoding brain-derived neurotrophic factor (BDNF) and GFP (mC-BDNF-GFP).¹⁷⁴ BDNF is a key regenerative molecule that can stimulate local axonal sprouting and neuronal plasticity at CNS injuries.¹⁹² After magnetofection under optimal field conditions, the results showed similar numbers of viable and proliferating cOMCs with no adverse effects on cell health parameters compared with control conditions. The bioengineered cells functioned as efficient biopumps for the successful secretion of BDNF, resulting in a 3-fold increase in its mean concentration in the culture supernatant versus the control group.¹⁷⁴ Moreover, the minicircle-based expression and secretion of the neurotrophic factor enhanced the function of cOMCs at the site of spinal cord injury with an efficiency comparable with viral vectors, without any detectable adverse effects on cell health. Consequently, the authors suggested that this non-viral platform has significant translational advantages for safe application in regenerative therapy for spinal cord injuries.¹⁷⁴

Bone and joint diseases

Adipose-derived stem cells (ADSCs) are multipotent cell populations abundant in adipose tissue and capable of osteogenic differentiation. For that reason, these cells are ideal for application in cell-based regeneration of various craniofacial skeletal defects. However, it was disclosed that the fragility of progenitor cells after transplantation restricts lineage differentiation and active participation of the cells in bone regeneration due to hypoxia and a high amount of inflammatory mediators in the injury location.^193,194 Hyun et al. suggested a solution to enhance stem cell survival by modulating the apoptosis pathways in the engrafted stem cells, which would be particularly beneficial in the early days after transplantation.¹⁹⁵ In a study by Brett et al., a prefabricated scaffold with magnetic nanoparticles containing a minicircle encoding Bcl-2 and GFP (Bcl-2/GFP minicircle) through the human ubiquitin C promoter was applied for bone regeneration.¹⁷⁵ To that end, the minicircle was attached to a synthetic iron oxide nanoparticle, and minicircle-nanoparticle complexes were magnetofected into adipose-derived stromal cells (ASCs). After confirming the in vitro osteogenic capacity of modified ASCs, an in vivo test was conducted using adult male nude mice with calvarial defects in their right parietal bone.¹⁷⁵ Hydroxyapatite-coated poly(lactic-coglycolic acid) (HA-PLGA) scaffolds were prepared, and the minicircle-nanoparticle complexes were incorporated into the scaffolds. The scaffolds were then placed into the calvarial defects in nude mice, freshly harvested ASCs were seeded on the scaffolds, and then the skin over the defect was sutured. The mice were exposed to an external magnetic field over the site of the transplanted scaffold to execute in vivo magnetic-assisted transfection (Figure 4E). Through this approach, transient expression of Bcl-2 as a prosurvival protein triggered cell survival by increasing cellular proliferation and reducing apoptotic markers in transplanted human ASCs.¹⁷⁵ The highest rate of bone regeneration in mouse models was observed in magnetofected ASCs compared with a similar group that was not exposed to the magnetic field, serving as the negative control. Therefore, in vivo magnetofection of human ASCs with magnetic nanoparticles loaded with the Bcl-2-expressing minicircle attached to a pre-prepared HA-PLGA scaffold could be a promising approach for bone regeneration.¹⁷⁵

Figure 4.

Schematic representation of various applications of minicircle vectors for regenerative medicine in animal models

The figure illustrates how minicircles encoding therapeutic genes target animal cells using various delivery methods. Transplantation of the minicircle-manipulated cells resulted in increased and prolonged transgene expression. This approach provides new insights into regenerative medicine, where minicircles are used in successful cell-based gene therapy to repair or replace damaged cells or tissues. (A) In vivo magnetofection of iron oxide-minicircle complexes incorporated into HA-PLGA scaffold improved cellular proliferation of ASCs, reduced their apoptotic markers, and augmented bone regeneration. (B) Intraarticular injection of minicircle-transfected ADSCs increased chondrogenesis and suppressed inflammation in osteoarthritis, providing chondroprotective effects. (C) Hydrodynamic injection and intraperitoneal delivery of minicircle-transfected MSCs offer a gene therapy-based strategy for in vivo bone regeneration and osteoporosis treatment. (D) Minicircle systems are utilized for in vitro differentiation of iPSC-derived MSCs into chondrogenic pellets with therapeutic application in cartilage regeneration. (E) The i.v. infusion of minicircle-engineered MSCs via the jugular vein is a gene-enhanced cell-based therapy for ARDS by reducing vascular permeability and pulmonary inflammation.

Genetic engineering of ADSCs using a family of regulatory molecules can also enhance their chondrogenic capacity. These regulatory molecules include sex-determining region Y (SRY)-box 5, 6, and 9 (SOX6, 5, and 9), collectively known as the (SOX)-trio, which are transcription factors. Among them, SOX6 can act as a co-factor for SOX-9, playing a crucial role in efficient cartilage formation.^196,197,198 Angiopoietin-like 4 (ANGPTL4) is a multifunctional glycoprotein involved in lipid and glucose metabolism, angiogenesis, and vascular permeability in various tissues. Besides, this physiological mediator contributes in various pathological conditions such as tumorigenesis, chronic inflammation, rheumatoid arthritis, and osteoarthritis.^199,200 Studies have shown that suppressing ANGPTL4 can reduce the levels of cartilage-degrading enzymes including matrix metalloproteinases (MMPs) such as MMP1, MMP3 and MMP13.²⁰⁰ Jeong et al. constructed a minicircle encoding SOX6 and SOX9 (mcSOX9/6) by the CMV promoter, and another minicircle encoding SOX6 and SOX9 along with a small hairpin RNA (shANG) under the U6 promoter, targeting ANGPTL4 (mcSOX9/6/shANG).²⁰¹ These minicircles were combined with cationic dexamethasone conjugated-polyethyleneimine (_DEXPEI) and separately transfected into human ADSCs. In vitro chondrogenic differentiation of transfected ADSCs in spheroid culture with chondrogenic medium, excluding growth factors such as transforming growth factor β 3 (TGF-β3) and bone morphogenic protein 2 (BMP2), was well confirmed.²⁰¹ On the other hand, Sprague-Dawley rats with surgically induced osteoarthritis were prepared as models of the disease.²⁰² The mcSOX9/6/shANG- or mcSOX9/6-transfected ADSCs were then injected intraarticularly into the knee joints (Figure 4B). In vivo tracking revealed a significant reduction in joint destruction along with lower levels of cyclooxygenase 2 (COX-2) and MMP13 in synovial fluids versus the control group treated with untransfected ADSCs. This study demonstrated that dual-functional minicircles in nanoparticles can effectively improve the chondrogenesis ability of ADSCs in articular cartilage and alleviate inflammation in osteoarthritis, leading to reduced joint destruction.²⁰¹

Bone tissue has a remarkable ability to self-repair; however, in certain cases such as aging, diabetes, trauma, or tumor surgery, fracture healing may be delayed or incomplete.^203,204 One method to augment bone regeneration and fracture healing is the application of appropriate growth factors such as recombinant human BMP2 at the fracture site.^205,206 While BMP2 is commonly used for successful treatment of human spine injuries, there is a concern that higher concentrations of the recombinant protein could potentially lead to tumor formation.^207,208 Therefore, alternative approaches such as gene therapy can be utilized to improve bone regeneration in challenging cases that do not respond to drug-based treatments or surgical interventions.^209,210 In this regard, the development and use of a minicircle expressing BMP2 as a non-viral DNA vector have shown several advantages including safe and more efficient induction of target cells for osteogenesis compared with conventional plasmids with lower osteogenic capability.²¹¹ In a study by Hacobian et al., two minicircles were developed harboring a codon-optimized human BMP2 cDNA driven by a CMV or EF1α promoter for non-viral osteogenic gene therapy. They also inserted the sequences of a truncated artificial intron and an appropriate signal peptide into the BMP2 cDNA to enhance the efficiency and duration of BMP2 expression and secretion.¹⁷⁷ The osteogenic minicircles, _CMVBMP2-Adv, and _EF1αBMP2-Adv were evaluated for BMP2 expression activity and in vitro osteogenic differentiation by transfecting them into C2C12, a mouse myoblast precursor cell line, and rat adipose-derived mesenchymal stem cells (rADSCs) (Figure 4A). The findings showed that these optimized osteogenic minicircles exhibited a higher delivery rate, prolonged and increased expression and secretion of exogenous BMP2 compared with conventional BMP2-plasmid at the same DNA dosage. Additionally, the expression of osteogenic markers such as osteocalcin and alkaline phosphatase, as well as matrix mineralization, was more efficiently increased in cells treated with the minicircle compared with those transfected with BMP2-plasmid.¹⁷⁷ The study also revealed that rADSCs transfected with the EF1α promoter minicircle exhibited higher BMP2 expression activity and in vitro osteogenic differentiation relative to those transfected with the minicircle with CMV promoter. Given the significant remarkable osteoinductive capacity of BMP2 minicircles, the authors concluded that these highly bioactive non-viral DNA vectors hold promise as therapeutic platforms for clinical use in bone tissue engineering and regeneration.¹⁷⁷

The limited regenerative capacity of damaged cartilage, attributed to low cellularity and avascularity, has provoked the development of new strategies for cartilage regeneration using various cell sources such as autologous or in-vitro-generated chondrocytes from adult stem cells.^212,213 However, the utilization of these cells is impeded by challenges like limited sources and the loss of original characteristics after multiple passages in vitro.^214,215 As a result, pluripotent stem cells have gained significant attention for cartilage regeneration in recent years. For example, to overcome the limited proliferation ability of mesenchymal stem cells (MSCs), several studies have explored the use of iPSCs as primary cellular sources with infinite proliferation potential to differentiate into mesenchymal progenitor cells. This approach offers a new option for cell-based therapy for cartilage defects.^216,217 Furthermore, a study demonstrated that the simultaneous use of recombinant BMP2 and transforming growth factor 3 (TGFβ3) resulted in potent chondrogenic differentiation of mesenchymal cells compared with the standard approach of administering only one growth factor.²¹⁸ According to these findings, Rim et al. constructed two minicircles, one encoding human BMP2 (mcBMP2) and the other encoding human TGFβ3 (mcTGFβ3). The vectors were equipped with a CMV promoter to express propeptides of the growth factors for extracellular secretion.¹⁷⁸ To generate a chondrogenic lineage, mesenchymal-like stem cells were derived from human iPSCs using a defined induction medium. The cells showing increased expression of mesenchymal markers such as CD44 and CD73 were transfected with mcBMP2 and/or mcTGFβ3 as test vectors along with a mcMock vector. The transfected cells successfully formed chondrogenic pellets that retained the minicircles for 30 days. Additionally, chondrogenic markers such as SOX9 and ACAN were upregulated in the pellets compared with the mock group. The minicircle-based chondrogenic pellets exhibited enhanced production of extracellular matrix (ECM) and high-quality in vitro chondrogenesis. These pellets also showed the highest expression of COL2A1 and its product, collagen type II, compared with pellets generated with only one of the growth factors.¹⁷⁸ In an in vivo investigation, Sprague-Dawley rats with defects in their articular cartilage of the distal femur were used as osteochondral defect models. The cartilage regeneration capacity of the minicircle-based chondrogenic pellets was evaluated by transplanting them into the rat models (Figure 4A). Following the transplantation period, the rats were sacrificed and the recovery of osteochondral defects was assessed in the implanted joints.¹⁷⁸ Consistent with the in vitro findings, the production of ECM in the defected cartilage and joint healing were significantly improved in the models that received pellets differentiated with minicircles encoding BMP2 and TGFβ3 compared with those that received mcMock pellets. Assessment of pluripotency and tumorigenicity in minicircle-transfected mesenchymal-like stem cells and implanted chondrogenic pellets confirmed the safety of the minicircle-based regenerated cartilage tissue. This proof-of-concept study presents a novel regeneration strategy by combining iPSCs with minicircle technologies for therapeutic applications in cartilage recovery.¹⁷⁸

Osteoporosis is a metabolic bone disorder that affects the mineral density and microarchitecture of bones. It is characterized by reduced thickness and strength of bones, particularly in trabecular bones, which can result in bone fragility.²¹⁹ Estrogen deficiency during menopause disrupts normal bone metabolism, increasing bone resorption by osteoclasts without a compensatory increase in osteoblastic activity, thereby raising the risk of fractures.²²⁰ To manage postmenopausal osteoporosis, two groups of therapeutics are commonly prescribed, anti-resorptive agents and anabolic agents. Among anabolic agents that promote bone formation, the FDA has approved a number of parathyroid hormone analogs, including parathyroid hormone-related protein (PTHrP).^221,222,223 PTHrP is necessary for normal bone formation and remodeling, exerting an anti-osteoporotic effect.²²⁴ Experimental studies have shown that daily administration of PTHrP analogs in ovariectomized mice has anabolic features and improves bone formation rate and reduces bone resorption, demonstrating anabolic properties.²²⁵ Additionally, it has been found that transplantation of MSCs can enhance osteogenic differentiation and bone mineral density, impeding the progression of osteoporosis.²²⁶ Given these findings, Kim et al. developed a minicircle vector encoding a peptide consisting of two regions of PTHrP (amino acids 1–34 and 107–139) along with GFP as a reporter driven by a CMV promoter.¹⁷⁹ The expression activity of this minicircle referred to as mcPTHrP 1–34 + 107–139, was confirmed in vitro in HEK293T cells by observing the green fluorescent signal. The vector was then electroporated into bone marrow-derived human MSCs, which maintained their phenotype and was subsequently employed as a cellular delivery system. Osteoporosis was induced in female C57BL/6 mice through ovariectomy. To evaluate the therapeutic impact of PTHrP 1–34 + 107–139 on osteoporosis mouse models, two delivery approaches were employed. Firstly the naked minicircle was administered via multiple tail vein injections and, secondly, minicircle-manipulated MSCs were injected intraperitoneally as a cell-based gene transfer strategy (Figure 4D). Both approaches successfully delivered the minicircle in vivo, resulting in increased expression levels of the PTHrP peptide in various tissues for several days. Notably, the expression of the peptide was significantly higher in femur tissues of the models treated with transfected MSCs compared with the controls.¹⁷⁹ The results revealed that both minicircle delivery approaches not only increased bone mass and trabecular bone thickness but also suppressed bone resorption, leading to improved bone regeneration. This in vivo experiment highlighted the potential of minicircle technology in developing a novel gene therapy-based strategy for bone regeneration and osteoporosis treatment.¹⁷⁹

In a similar therapeutic strategy for postmenopausal osteoporosis, researchers utilized sequential administration of minicircle encoding red fluorescent protein and human osteoprotegerin (mcOPG), as well as minicircle encoding GFP and human parathyroid hormone-related peptide 1–34 + 107–139 (mcPTHrP), wherein both OPG and PTHrP were driven by the CMV promoter.¹⁸⁰ Osteoprotegerin inhibits osteoclast activity and bone resorption. The recombinant minicircles were constructed, confirmed through double digestion, and their functionality was validated by assessing the expression of fluorescent proteins in HEK293T cells using fluorescence microscopy. In vitro testing showed that these minicircles significantly promoted osteoblast differentiation from human iPSCs and increased the expression of osteogenic markers such as collagen type I alpha 1 chain and osteocalcin. Furthermore, female ovariectomized Sprague-Dawley rats were used to generate postmenopausal osteoporosis models. The rats initially received mcOPG intravenously, and 8 weeks later were injected with mcPTHrP once a week for 3 weeks. Micro-CT analysis of the femur at 24 weeks post-ovariectomy revealed that the sequential combined treatment, achieved by simultaneous overexpression of OPG and PTHrP, improved bone microarchitecture, including bone mineral density and trabecular volume, more effectively than single treatment with either minicircle alone.¹⁸⁰ This proof-of-concept study highlights the potential of stepwise treatment in the ovariectomized rat model of osteoporosis using minicircle-based gene therapy to transiently express OPG and PTHrP, suggesting a promising new approach to enhance bone formation and suppress bone resorption in osteoporosis while minimizing risks associated with prolonged protein expression.¹⁸⁰

Lung diseases

Acute lung injury and its more severe form, acute respiratory distress syndrome (ARDS), are respiratory disorders characterized by increased inflammation and permeability in the pulmonary vascular system. The mortality rate in severe ARDS is significant, and survivors still suffer from complications such as pulmonary dysfunction and skeletal muscle weakness.^227,228,229 Currently, there is no specific treatment for ARDS with a poor prognosis, and limited supportive care is available for these patients.²³⁰ Plenty of studies have demonstrated that MSCs derived from bone marrow can reduce pathological inflammation and vascular permeability in ARDS owing to their immunomodulatory and anti-inflammatory effects.^231,232 While MSC-based therapy is considered a promising method, some complementary strategies such as transient genetic modification of MSCs have been proposed to enhance the therapeutic properties of the cells including angiogenesis and immunomodulatory potency.²³³ In an investigation by Florian et al., a minicircle vector encoding human angiopoietin 1 (ANGPT1) under the CMV promoter was constructed and named MC-ANGPT1.¹⁸¹ MSCs isolated from C57Bl/6J male mice were electroporated with the minicircle and a counterpart plasmid encoding ANGPT1 to optimize the therapeutic benefits of the cells for cell-based therapy. In vitro results showed that transfection of MSCs with the minicircle led to significantly more efficient and sustained expression of ANGPT1 compared with the plasmid in conditioned medium. In animal experiments, acute lung injury was induced in female C57Bl/6J mice by intratracheal instillation of a lipopolysaccharide (LPS) solution. Following the induction of acute lung injury, the mice were i.v. infused with engineered MSCs (Figure 4C). The data illustrated that lung inflammation and vascular permeability were reduced in all mice treated with engineered MSCs compared with negative controls that received cell-free PBS. However, mice infused with MC-ANGPT1-transfected MSCs exhibited a greater reduction in alveolar neutrophil infiltration, pulmonary inflammation and vascular permeability compared with those that received cells transfected with ANGPT1-expressing plasmid or empty vector.¹⁸¹ Furthermore, overexpression of ANGPT1 in minicircle-engineered mice effectively decreased proinflammatory cytokines close to baseline levels in the control group. These findings uncovered that genetically modified MSCs using MC-ANGPT1 could be a specific tool for gene-enhanced cell-based therapy for acute lung injury including ARDS.¹⁸¹

Ischemic diseases

MSCs have shown an intrinsic angiogenic capacity by promoting revascularization in ischemic regions, making them promising potential candidates for treating ischemic conditions in diseases such as myocardial infarction and peripheral arterial disease.^234,235 This pro-angiogenic activity is attributed to the secretion of VEGF, which stimulates the growth and survival of endothelial cells, fostering vascular regeneration and remodeling.^236,237 MSCs possess unique properties, including proliferative activity, multilineage differentiation, and intrinsic immunomodulatory capacities making them suitable candidates for autologous or allogeneic cell-based therapy.²³⁸ One reliable approach to enhance the angiogenic activity of MSCs is using VEGF-containing vectors to promote the cellular production and secretion of VEGF.^236,239 These gene-enhanced MSCs can serve as effective carriers of VEGF for angiogenic therapy.^240,241 However, it has been observed that overexpression of VEGF may have deleterious effects such as abnormal blood vessel growth and abnormal blood flow, potentially resulting in hemangiomas or even malignancy.²⁴² To address this issue, the minicircle has been proposed as an outperformed non-viral DNA vector that, with transient but efficient expression of VEGF, can induce early angiogenesis in ischemic areas with limited side effects of VEGF overexpression.^182,243 In two recent studies in 2019 and 2021, Serra et al. designed and utilized a minicircle expressing VEGF driven by a CMV promoter (MC-VEGF) to enhance the angiogenic potential of human MSCs isolated from bone marrow, adipose tissue, and umbilical cord matrix from healthy donors. In these studies, the minicircle was compared with a VEGF-encoding plasmid as the control vector.^182,183 Both vectors were electroporated into isolated MSCs at early passages. The manipulated cells from all tissue sources were evaluated in light of their ability to transiently express the transgene using the culture supernatant. As expected, the rate of VEGF expression by MC-VEGF was much higher than that of the plasmid. This finding was consistent with previous studies reporting that the transgene expression level by minicircles was comparable with some viral vectors.^239,240 The MSCs engineered with the two non-viral DNA vectors were then characterized in vitro to verify the maintenance of their proliferative capability by determining the number of viable cells. Intriguingly, the results showed that the number of viable cells after electroporation with MC-VEGF was similar to the non-transfected cells and significantly higher than the cells transfected with the VEGF-encoding plasmid, highlighting the advantage of minicircles for clinical use. At the same time, the immunophenotype of transfected MSCs and their multilineage differentiation capacity were evaluated. The manipulated cells with both vectors not only maintained their phenotypic profile but also showed the capacity for in vitro differentiation into osteocytes and adipocytes.^182,183 Furthermore, the functionality of the manipulated MSCs from different sources was assessed by culturing endothelial cells in Matrigel-coated plates containing conditioned medium derived from the MSCs manipulated with both vectors and the intact cells as the control. The results signified that, in comparison with control cells, high levels of VEGF in the medium from the transfected MSCs, regardless of their source, improved the formation of vessel-like tube networks with a higher number and concentration. However, the conditioned medium from minicircle-transfected MSCs exhibited higher angiogenic activity than that of the plasmid-transfected cells. Similarly, the migration ability of endothelial cells in response to VEGF, a crucial event during angiogenesis.²⁴⁴ was evaluated, and the highest cell migration was observed in the conditioned medium of MC-VEGF-engineered MSCs.^182,183

Based on these in vitro studies, the authors believe that genetic modification with non-viral DNA vectors, combined with the intrinsic properties of MSCs, could enhance the angiogenic activity of the cells compared with unmodified cells. Among non-viral DNA vectors, minicircle technology could be an effective method for transient and safe overexpression of VEGF in MSCs from different sources, potentially improving tissue oxygenation and regeneration of damaged tissues in ischemic diseases.^182,183

Conclusion

In this review, we discuss the recent promising therapeutic and diagnostic outcomes resulting from minicircle applications in gene therapy and regenerative medicine. We specifically highlight preclinical studies that utilized minicircles to express the genes of interest for more precise targeting and correction of a variety of genetic and cell-based diseases. These studies documented the enhanced potency of this type of minimized non-viral DNA vector for novel gene-based therapies and treatments including gene therapy and regenerative medicine compared with other conventional vectors. The success of these investigations suggests that minicircles could be employed as a safe and versatile alternative to current viral and other non-viral DNA vectors in clinical settings. For instance, the vector has been successfully used for correcting or replacing mutated or missing genes, genome editing, gene silencing, and long-lasting self-production of therapeutic proteins. The preclinical assessments mentioned are crucial steps toward the potential clinical translation of the minicircle vector. The promising results of these assessments indicate that this type of vector represents a breakthrough in highly efficient and safe non-viral gene therapy, paving the way for accelerating their adoption for therapeutic purposes. However, further investigations are needed to improve minicircle production yield and purification to establish the vector’s efficiency and safety for convincing regulatory agencies to accept the minicircle vector in diverse therapeutic applications as an outperformed non-viral DNA vector.

Acknowledgments

This study was supported by the Royan Institute for Biotechnology, Isfahan, Iran. Funding bodies did not play a role in the design and writing of this manuscript.

Author contributions

P.C., K.D., and N.S.A. collected the information and wrote the original manuscript. N.R. and K.D. contributed in reviewing, editing, and final formatting. N.R., M.S., and P.C. prepared the tables and figures and sorted the references. All authors read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.