Episomal Vectors Research Articles

Generation of Induced Pluripotent Stem Cell Line iTAF15Xsk4 from Fibroblasts of a Patient with Microdeletion at Xq24

Differentiation of induced pluripotent stem (iPS) cells from patients and healthy donors allows in vitro study of genetic disorders. We have previously reported a clinical case of recurrent pregnancy loss in a patient with skewed X-chromosome inactivation in peripheral blood lymphocytes, endometrium, and buccal epithelium. We have found a 239 kb microdeletion at Xq24 that affected eight genes including UBE2A. In this work, we produced iPS cell line iTAF15Xsk4 from the patient’s skin fibroblasts using non-integrating episomal vectors. iPS cell line had a normal karyotype, expressed pluripotency markers, and upon differentiation in embryoid bodies expressed markers of all three germ layers. This cell line could be used for the UBE2A deficiency syndrome study.

Generation of the induced pluripotent stem cell line ESi108-A from a familial atrial fibrillation patient

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

CMV/AAT promoter of MAR-based episomal vector enhanced transgene expression in human hepatic cells.

The online version contains supplementary material available at 10.1007/s13205-023-03774-x.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese-hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MAR), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR-1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+ hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells. This article is protected by copyright. All rights reserved.

Generation of induced pluripotent stem cell lines from three individuals with autism spectrum disorder

Uncovering the molecular mechanisms of autism spectrum disorder (autism) necessitates development of relevant experimental models that are capable of recapitulating features of the clinical phenotype. Using non-integrative episomal vectors, peripheral blood mononuclear cells derived from three unrelated individuals diagnosed with autism were reprogrammed to induced pluripotent stem cells (iPSCs). The resultant lines exhibited the expected cellular morphology, karyotype, and evidence of pluripotency. These iPSCs constitute a valuable resource to support investigations of the underlying aetiology of autism.

Human Endometrium Derived Induced Pluripotent Stem Cells Are Amenable to Directed Erythroid Differentiation

BACKGROUND:A protocol for using human endometrium derived induced pluripotent stem cells (iPSCs) to derive hematopoietic and erythroid lineages will be elaborated, through a two-phase culture system.METHODS:Discarded endometrial tissues were obtained from women receiving hysterectomy in their 4th to 5th decade due to benign uterine conditions. pCE-Sox2, Oct4, Klf4, L-Myc and Lin28 episomal vectors were used to electrotransfect the endometrial stromal cells. The first 8 days involves commitment to hematopoietic stem cells through embryoid body with robust expansion on murine bone marrow stromal cells. The second phase involves feeder free conditions with hydrocortisone, stem cell factor, interleukin-3, and recombinant EPO. After 22 days of feeder free culture, the expression profiles of CD235a+, CD34+, CD43+ and CD 71+ were analyzed by flow cytometry and Wright-Giemsa staining for differential counting. The oxygen carrying capacity of cultured RBCs was measured using a hemoxanalyser.RESULTS:As a result of inducing these cells via co-culture with murine stromal fibroblasts, all endometrium derived iPSCs were differentiated into erythroblasts with a stable yield of approximately 80% for polychromatic and orthochromatic normoblasts. The protocol for complete induction of erythroid lineage cells starting from human endometrial tissue via iPS cells has been optimized.CONCLUSION:Successful directed erythroid differentiation has occurred from human endometrium-derived iPS cells. A comprehensive process of actually deriving iPS cells using discarded surgical hysterectomy specimens to the erythroid fate has significance in that the scope of using human iPSC cell lines for tissue regeneration could be expanded in the future.

Targeted gene deletions in the dimorphic fungal pathogen Histoplasma using an optimized episomal CRISPR/Cas9 system.

The rapid development of CRISPR/CRISPR-associated (Cas) systems has revolutionized the ability to produce genetic mutations in a desired locus, particularly in organisms with low rates of homologous recombination. Histoplasma is an important respiratory and systemic fungal pathogen that has few reverse genetic options. We describe an optimized CRISPR/Cas system for the efficient generation of mutations in desired genes. The limited requirements for CRISPR/Cas, namely a gene-targeting guide RNA (gRNA) and expression of a Cas endonuclease, enabled both the gRNA and the Streptococcus pyogenes Cas9 gene to be expressed from a single episomal vector. The gRNAs are expressed from a strong Pol(II) promoter, a critical parameter for increasing the recovery of mutated genes, and processed into the mature gRNA by ribozymes in the mRNA. Expression of dual-tandem gRNAs facilitates the generation of gene deletions at a good frequency which can be detected by PCR-based screening of pooled isolates resulting in the isolation of marker-less deletion mutants. The CRISPR/Cas system is encoded on an episomal telomeric vector facilitating curing strains of the CRISPR/Cas vector upon generation of the mutant. We demonstrate the successful application of this CRISPR/Cas system in diverse Histoplasma species and applicable for multiple genes. The optimized system shows promise for accelerating reverse genetic studies in Histoplasma spp. IMPORTANCE The ability to eliminate gene product functions is central to understanding molecular mechanisms. In the fungal pathogen Histoplasma, methods to inactivate or deplete gene products are inefficient, which hampers progress in defining Histoplasma's virulence mechanisms. We describe an efficient CRISPR/Cas-based system for generating gene deletions in Histoplasma and show its validation on multiple genes with selectable and non-selectable phenotypes.

Establishment of induced pluripotent stem cell line SDPHi003-A from a healthy male donor

Peripheral blood mononuclear cells (PBMCs) were derived from a 67-year-old Chinese male. We used non-integrating episomal vectors containing OCT4, SOX2, KLF4 and c-MYC to reprogram PBMCs into induced pluripotent stem cell (iPSCs). This iPSC line, SDPHi003-A, have a normal karyotype, expressed pluripotent markers and have the potential for trilineage differentiation. This iPSC line can be used as a control for disease modeling studies and contribute to the research exploring disease pathogenesis.

Successful large gene augmentation of USH2A with non-viral episomal vectors

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

Generation of stable integration-free pig induced pluripotent stem cells under chemically defined culture condition.

Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in pre-clinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under chemically defined culture condition previously used for generation of stable pig pre-gastrulation epiblast stem cells (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be established from pig somatic cells using episomal vectors in this culture condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Establishment of an induced pluripotent stem cell line SDQLCHi048-A from a healthy boy donor

The induced pluripotent stem cells (iPSCs) line was generated using peripheral blood mononuclear cells (PBMCs) from a healthy 1-month-old boy. Expression of pluripotency markers, deletion of free episomal vectors, preservation of normal karyotype, and potential for in vitro trilineage differentiation were demonstrated in the iPSCs line SDQLCHi048-A. This cell line may serve as a basis for disease modeling and help to further explore the molecular pathogenesis.

Identification and Construction of Strong Promoters in Yarrowia lipolytica Suitable for Glycerol-Based Bioprocesses.

Yarrowia lipolytica is a non-pathogenic aerobic yeast with numerous industrial biotechnology applications. The organism grows in a wide variety of media, industrial byproducts, and wastes. A need exists for molecular tools to improve heterologous protein expression and pathway reconstitution. In an effort to identify strong native promoters in glycerol-based media, six highly expressed genes were mined from public data, analyzed, and validated. The promoters from the three most highly expressed (H3, ACBP, and TMAL) were cloned upstream of the reporter mCherry in episomal and integrative vectors. Fluorescence was quantified by flow cytometry and promoter strength was benchmarked with known strong promoters (pFBA1in, pEXP1, and pTEF1in) in cells growing in glucose, glycerol, and synthetic glycerol media. The results show that pH3 > pTMAL > pACBP are very strong promoters, with pH3 exceeding all other tested promoters. Hybrid promoters were also constructed, linking the Upstream Activating Sequence 1B (UAS1B8) with H3(260) or TMAL(250) minimal promoters, and compared to the UAS1B8-TEF1(136) promoter. The new hybrid promoters exhibited far superior strength. The novel promoters were utilized to overexpress the lipase LIP2, achieving very high secretion levels. In conclusion, our research identified and characterized several strong Y. lipolytica promoters that expand the capacity to engineer Yarrowia strains and valorize industrial byproducts.

In vitro erythrocyte production using human-induced pluripotent stem cells: determining the best hematopoietic stem cell sources

BackgroundBlood transfusion is an essential part of medicine. However, many countries have been facing a national blood crisis. To address this ongoing blood shortage issue, there have been efforts to generate red blood cells (RBCs) in vitro, especially from human-induced pluripotent stem cells (hiPSCs). However, the best source of hiPSCs for this purpose is yet to be determined.MethodsIn this study, hiPSCs were established from three different hematopoietic stem cell sources—peripheral blood (PB), cord blood (CB) and bone marrow (BM) aspirates (n = 3 for each source)—using episomal reprogramming vectors and differentiated into functional RBCs. Various time-course studies including immunofluorescence assay, quantitative real-time PCR, flow cytometry, karyotyping, morphological analysis, oxygen binding capacity analysis, and RNA sequencing were performed to examine and compare the characteristics of hiPSCs and hiPSC-differentiated erythroid cells.ResultshiPSC lines were established from each of the three sources and were found to be pluripotent and have comparable characteristics. All hiPSCs differentiated into erythroid cells, but there were discrepancies in differentiation and maturation efficiencies: CB-derived hiPSCs matured into erythroid cells the fastest while PB-derived hiPSCs required a longer time for maturation but showed the highest degree of reproducibility. BM-derived hiPSCs gave rise to diverse types of cells and exhibited poor differentiation efficiency. Nonetheless, erythroid cells differentiated from all hiPSC lines mainly expressed fetal and/or embryonic hemoglobin, indicating that primitive erythropoiesis occurred. Their oxygen equilibrium curves were all left-shifted.ConclusionsCollectively, both PB- and CB-derived hiPSCs were favorably reliable sources for the clinical production of RBCs in vitro, despite several challenges that need to be overcome. However, owing to the limited availability and the large amount of CB required to produce hiPSCs, and the results of this study, the advantages of using PB-derived hiPSCs for RBC production in vitro may outweigh those of using CB-derived hiPSCs. We believe that our findings will facilitate the selection of optimal hiPSC lines for RBC production in vitro in the near future.

Generation of an induced pluripotent stem cell line SDPHi002-A from a patient with lung cancer

Here, an induced pluripotent stem cell (iPSC) line from a patient with lung neuroendocrine carcinoma was generated. The iPSCs were developed using nonintegrating episomal vectors carrying OCT4, SOX2, KLF4, BCL-XL and c-MYC. The established iPSCs (SDPHi002-A) had normal karyotype, excellent differentiation potential in vitro, showing pluripotent stem cell morphology and high expression of pluripotent markers.

Reprogramming of human peripheral blood mononuclear cells into induced mesenchymal stromal cells using non-integrating vectors

Mesenchymal stromal cells (MSCs) have great value in cell therapies. The MSC therapies have many challenges due to its inconsistent potency and limited quantity. Here, we report a strategy to generate induced MSCs (iMSCs) by directly reprogramming human peripheral blood mononuclear cells (PBMCs) with OCT4, SOX9, MYC, KLF4, and BCL-XL using a nonintegrating episomal vector system. While OCT4 was not required to reprogram PBMCs into iMSCs, omission of OCT4 significantly impaired iMSC functionality. The omission of OCT4 resulted in significantly downregulating MSC lineage specific and mesoderm-regulating genes, including SRPX, COL5A1, SOX4, SALL4, TWIST1. When reprogramming PBMCs in the absence of OCT4, 67 genes were significantly hypermethylated with reduced transcriptional expression. These data indicate that transient expression of OCT4 may serve as a universal reprogramming factor by increasing chromatin accessibility and promoting demethylation. Our findings represent an approach to produce functional MSCs, and aid in identifying putative function associated MSC markers.

Establishment of an induced pluripotent stem cell (SIAISi016-A) line from a 62-years old Chinese Han patient with Alzheimer's disease

A 62-years old Alzheimer's disease (AD) male patient donated his Peripheral blood mononuclear cells. The non-integrating episomal vector system used to reprogram PBMCs with Oct3/4, Klf4, Sox2 and c-Myc transcription factors. The pluripotency of transgene-free pluripotent stem cell (iPSC) was confirmed by immunocytochemistry for pluripotency markers-SOX2, NANOG, OCT3/4, SSEA4, TRA1-60, and TRA1-81. The differentiation capacity of the iPSCs into endoderm, mesoderm and ectoderm was assessed by AFP, SMA and βIII-TUBULIN, respectively. In addition, the iPSC line displayed a normal karyotype. This iPSC line might offer a good cell model to explore the pathological mechanisms and treatment strategies for AD.

Chenodeoxycholic acid rescues axonal degeneration in induced pluripotent stem cell-derived neurons from spastic paraplegia type 5 and cerebrotendinous xanthomatosis patients

BackgroundBiallelic mutations in CYP27A1 and CYP7B1, two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive.MethodsTo generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes.ResultsCTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration.ConclusionsOur data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.